Progression after First-Line Cyclin-Dependent Kinase 4/6 Inhibitor Treatment: Analysis of Molecular Mechanisms and Clinical Data

Abstract

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6iss) are widely used in first-line metastatic breast cancer. For patients with progression under CDK4/6is, there is currently no standard treatment recommended at the category 1 level in international guidelines. The purpose of this article is to review the cellular mechanisms underlying the resistance to CDK4/6is, as well as treatment strategies and the clinical data about the efficacy of subsequent treatments after CDK4/6is-based therapy. In the first part, this review mainly discusses cell-cycle-specific and cell-cycle-non-specific resistance to CDK4/6is, with a focus on early and late progression. In the second part, this review analyzes potential therapeutic approaches and the available clinical data on them: switching to other CDK4/6is, to another single hormonal therapy, to other target therapies (PI3K, mTOR and AKT) and to chemotherapy.

Full text

Citation: Villa, F.; Crippa, A.;
Pelizzoni, D.; Ardizzoia, A.;
Scartabellati, G.; Corbetta, C.;
Cipriani, E.; Lavitrano, M.; Ardizzoia,
A. Progression after First-Line
Cyclin-Dependent Kinase 4/6
Inhibitor Treatment: Analysis of
Molecular Mechanisms and Clinical
Data. Int. J. Mol. Sci. 2023,24, 14427.
https://doi.org/10.3390/
ijms241914427
Academic Editors: Monica Iorfida
and Nicoletta Cordani
Received: 31 July 2023
Revised: 31 August 2023
Accepted: 8 September 2023
Published: 22 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Review
Progression after First-Line Cyclin-Dependent Kinase 4/6
Inhibitor Treatment: Analysis of Molecular Mechanisms and
Clinical Data
Federica Villa 1,*, Alessandra Crippa 1, Davide Pelizzoni 1, Alessandra Ardizzoia 2, Giulia Scartabellati 3,4 ,
Cristina Corbetta 1, Eleonora Cipriani 1, Marialuisa Lavitrano 2and Antonio Ardizzoia 1
1Medical Oncology, Oncology Department ASST Lecco, 23900 Lecco, Italy; a.crippa@asst-lecco.it (A.C.);
d.pelizzoni@asst-lecco.it (D.P.); c.corbetta@asst-lecco.it (C.C.); e.cipriani@asst-lecco.it (E.C.);
a.ardizzoia@asst-lecco.it (A.A.)
2School of Medicine and Surgery, University of Milano-Bicocca, 20126 Milano, Italy;
a.ardizzoia@campus.unimib.it (A.A.); marialuisa.lavitrano@unimib.it (M.L.)
3
Medical Oncology, Fondazione IRCCS San Gerardo dei Tintori, 20900 Monza, Italy; g.scartabellati001@unibs.it
4
Department of Medical and Surgical Specialties, Medical Oncology, University of Brescia, 25121 Brescia, Italy
*Correspondence: fe.villa@asst-lecco.it
Abstract:
Cyclin-dependent kinase 4/6 inhibitors (CDK4/6iss) are widely used in first-line metastatic
breast cancer. For patients with progression under CDK4/6is, there is currently no standard treatment
recommended at the category 1 level in international guidelines. The purpose of this article is
to review the cellular mechanisms underlying the resistance to CDK4/6is, as well as treatment
strategies and the clinical data about the efficacy of subsequent treatments after CDK4/6is-based
therapy. In the first part, this review mainly discusses cell-cycle-specific and cell-cycle-non-specific
resistance to CDK4/6is, with a focus on early and late progression. In the second part, this review
analyzes potential therapeutic approaches and the available clinical data on them: switching to other
CDK4/6is, to another single hormonal therapy, to other target therapies (PI3K, mTOR and AKT) and
to chemotherapy.
Keywords:
metastatic breast cancer; CDK 4/6 inhibitors; cell-cycle-specific and non-specific resistance
1. Introduction
Breast cancer (BC) is the most commonly diagnosed cancer in women worldwide [
1
].
An estimated 5–10% of patients are diagnosed at the metastatic stage of the disease [
2
].
Some studies have found that approximately 20–30% of patients with early BC may recur
with metastatic BC (mBC), although the data are limited [3,4]. Up to 70% of mBC patients
have luminal BC, which is defined by estrogen receptor (ER) positive (+) and human
epidermal growth factor 2 (HER2) negative (
−
) expression [
5
], with a median overall
survival (OS) of as long as 57 months [
6
]. Endocrine therapy (ET) is a preferred option for
the treatment of these patients [7].
Cyclin-dependent kinase (CDK4/6), as well as its target protein, cyclin D1, is involved
in cell cycle regulation and has been implicated in the pathogenesis of BC and the po-
tential development of endocrine resistance [
8
]. Several large, randomized trials have
demonstrated substantial clinical benefit from the use of CDK4/6 inhibitors (CDK4/6is)
(palbociclib, ribociclib and abemaciclib) in the first-line setting for metastatic hormone re-
ceptor HR+/HER2
−
disease, with a substantial improvement in progression-free survival
(PFS) [
9
–
12
]. OS benefit has also been demonstrated for ribociclib plus letrozole/fulvestrant,
with a median OS of greater than 5 years in patients receiving CDK4/6is [
13
]. Abemaci-
clib, in the MONARCH 3 study at the second ad interim analysis, showed a numerical
improvement in the OS (an increase in the median OS by >12 months with the addition of
abemaciclib to aromatase inhibitor (AI)) but without reaching statistical significance [
14
]. A
Int. J. Mol. Sci. 2023,24, 14427. https://doi.org/10.3390/ijms241914427 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023,24, 14427 2 of 25
follow-up is ongoing for the final OS analysis. Palbociclib failed to demonstrate an overall
survival benefit in comparison to ET alone [
15
]. Whether this is related to the differences
in drug efficacy or in patient populations or due to patients being lost to survival follow-
up remains unclear. Most patients, however, develop resistance to CDK4/6is in about
2–3 years. For patients with progression under CDK4/6is, there is currently no standard
treatment recommended at the category 1 level in international guidelines. Reasonable
options include switching to another ET monotherapy, cytotoxic chemotherapy and ET
with everolimus (a mammalian target of rapamycin (mTOR) inhibitor), talazoparib or
olaparib (poly (ADP-ribose) polymerase (PARP) inhibitors) for patients with germline
BRCA mutations, or alpelisib (a phosphoinositide 3-kinase (PI3K) inhibitor) for patients
with somatic PIK3CA mutations. Whether or not the CKD4/6i treatment should be contin-
ued after the initial disease progression is currently unknown. The median OS of about
5 years achieved with CDK4/6is in the first-line setting with a median PFS of 25–28 months
suggests that there is limited efficacy in subsequent antineoplastic treatments. The data on
subsequent treatment options and their efficacy are limited. Moreover, most of these data
were obtained prior to the widespread use of a CDK4/6is. Understanding the molecular
mechanisms of CDK4/6is resistance is crucial to develop prospective trials.
This article will review the cellular mechanisms underlying the resistance to CDK4/6is,
as well as treatment strategies and clinical data about the efficacy of subsequent treatments
after CDK4/6is-based therapy.
2. CDK4/6 Inhibitors’ Resistance Mechanisms
Cyclin-dependent kinase (CDK) 4 and its functional homolog CDK6 are two struc-
turally related kinases with biochemical and biological similarities. Despite having few
differences in some of their activities, these enzymes are constantly expressed throughout
the cell cycle and, with their partners, D-cyclins, are fundamental for integrating mito-
genic and antimitogenic extracellular signals, among which stimulating factors, cytokines,
cell–cell contacts and other factors are included, representing a boundary between the
environment and the cell cycle machinery [
16
,
17
]. The cyclin D-CDK4/6 complex is a
driving force that controls the transition from the G1 to the S phases. Also, the INK4 (the
cyclin D-CDK4/6 inhibitor molecule) retinoblastoma protein (pRb) pathway regulates
cellular proliferation by controlling the G1 to the S cell cycle checkpoint. The dysregulation
of this pathway is frequently observed in cancer and contributes to cell cycle progression
and persistent growth. CDK4/6 mediates the transition from the G1 phase to the S phase by
associating with D-type cyclins and regulating the phosphorylation state of pRb [
18
]. Un-
phosphorylated pRb binds and represses the functions of the E2 family (E2F) transcription
factors; upon phosphorylation, pRb dissociates from the E2F transcription factors, freeing
them to be able to participate in DNA replication and cell division [19].
For these reasons, CDK4/6 has become a treatment target. However, it is common to
develop resistance and for the disease to progress during CDK 4/6i treatment. Here, we
summarized the various cell-cycle-specific and cell-cycle-non-specific resistance mecha-
nisms depending on the biological determinant of resistance, and in mechanisms condi-
tioning early or late progression, based on the timing of the onset of resistance.
2.1. Cell-Cycle-Specific Mechanisms
Multiple factors involved in the regulation of the cell cycle are associated with a
resistance to CDK4/6is, the loss of drug target genes, and the overexpression of other genes
involved in the progression of the cell cycle (Figure 1).

Int. J. Mol. Sci. 2023,24, 14427 3 of 25
Int. J. Mol. Sci. 2023, 23, x FOR PEER REVIEW 3 of 26
Figure 1. Cell-cycle-specific mechanisms that could be associated with CDK4/6is resistance. Sharp
arrows (→) indicate stimulation, blunt arrows (┴) indicate inhibition.
2.1.1. pRb Loss or Mutations
pRb is a tumor suppressor protein coded by the RB transcriptional corepressor 1
(RB1) that plays a pivotal role as a cell cycle checkpoint factor. In fact, it is involved in the
control of the CDK4/6 pathway, one of the main targets of the CDK4/6is drugs and whose
loss, most frequently caused by inactivating mutations of RB1, is the main reason for the
resistance to CDK4/6is [20,21]. In this case, in spite of this loss, the cell cycle progresses
through other molecular pathways, such as the E2F and the cyclin E-CDK2 axis, thus
bypassing its dependence on CDK4/6 and causing resistance to CDK4/6is [22]. It has been
suggested that administration of cyclin E-CDK2 inhibitors with CDK4/6is may be a valid
solution to overcoming resistance [23].
2.1.2. p16 Amplification
p16 is a tumor suppressor protein belonging to the INK4 family involved in cell cy-
cle regulation if pRb is functional. It is an inhibitor of CDK4, and it has been reported to
be an accurate biomarker of pRb loss in different tumors [24,25]. p16 amplification is
frequently found in BC, and this leads to lower levels of CDK4, thus representing the loss
of a target of CDK4/6is. Moreover, p16 amplification is frequently caused by the loss of
pRb, thus leading again to resistance to CDK4/6is ex vivo [26]. Palafox et al. suggested
that high p16 protein levels and heterozygous RB1 loss-of-function mutations could be
predictive in order to identify the CDK4/6is resistance in BC. Moreover, they demon-
strated that targeting the p16-CDK4/6 interaction sensitizes p16-overexpressing tumor
cells to CDK4/6is [27].
2.1.3. CDK2 Amplification
Figure 1.
Cell-cycle-specific mechanisms that could be associated with CDK4/6is resistance. Sharp
arrows (→) indicate stimulation, blunt arrows (⊥) indicate inhibition.
2.1.1. pRb Loss or Mutations
pRb is a tumor suppressor protein coded by the RB transcriptional corepressor 1 (RB1)
that plays a pivotal role as a cell cycle checkpoint factor. In fact, it is involved in the
control of the CDK4/6 pathway, one of the main targets of the CDK4/6is drugs and whose
loss, most frequently caused by inactivating mutations of RB1, is the main reason for the
resistance to CDK4/6is [
20
,
21
]. In this case, in spite of this loss, the cell cycle progresses
through other molecular pathways, such as the E2F and the cyclin E-CDK2 axis, thus
bypassing its dependence on CDK4/6 and causing resistance to CDK4/6is [
22
]. It has been
suggested that administration of cyclin E-CDK2 inhibitors with CDK4/6is may be a valid
solution to overcoming resistance [23].
2.1.2. p16 Amplification
p16 is a tumor suppressor protein belonging to the INK4 family involved in cell cycle
regulation if pRb is functional. It is an inhibitor of CDK4, and it has been reported to be an
accurate biomarker of pRb loss in different tumors [
24
,
25
]. p16 amplification is frequently
found in BC, and this leads to lower levels of CDK4, thus representing the loss of a target
of CDK4/6is. Moreover, p16 amplification is frequently caused by the loss of pRb, thus
leading again to resistance to CDK4/6is ex vivo [
26
]. Palafox et al. suggested that high p16
protein levels and heterozygous RB1 loss-of-function mutations could be predictive in order
to identify the CDK4/6is resistance in BC. Moreover, they demonstrated that targeting the
p16-CDK4/6 interaction sensitizes p16-overexpressing tumor cells to CDK4/6is [27].
2.1.3. CDK2 Amplification
Cyclin E is encoded by the CCNE1 gene and its association with CDK2 is involved in
cell cycle progression from phase G1 to S. Its main role is the pRb phosphorylation, resulting
in complete release of E2F [
28
]. It has been widely reported that CCNE1 overexpression

Int. J. Mol. Sci. 2023,24, 14427 4 of 25
is responsible for the resistance to CDK4/6is. In fact, cells losing their dependence on
CDK4/6 use bypass mechanisms, such as CDK2 amplification, to keep up with cell cycle
progression [
29
,
30
]. Moreover, there is a study that reports that the CDK2 upregulation
in ER+ breast cancer is also controlled by TROJAN. TROJAN is a noncoding RNA that
can bind to NKRF (NF-Kappa-B-repressing factor) and inhibit its interaction with RELA.
This binding leads to CDK2 overexpression and, as aforementioned, leads to CDK4/6is
resistance [31].
2.1.4. E2F Amplification
As a pRb transcription factor, E2F plays an important role in cell cycle regulation. As
aforementioned, D-CDK4/6 phosphorylates the pRb leading to E2F release with concurrent
transcription of proteins (such as cyclin E) necessary for cell cycle progression. At the same
time, cyclin E transcription leads to cyclin E-CDK2 formation, which further phosphorylates
pRb. For these reasons, the E2F amplification leads cells to evade CDK4/6 cell cycle
regulation thus, driving resistance to their inhibitors [18,20,21,28].
2.1.5. CDK7 Overexpression
CDK7 regulates the cell cycle by activating the CDK-activating kinase (CAK) and
participates in G1 and G2 phases [
32
]. Martin et al. reported that its overexpression is
involved in CDK4/6is resistance [
33
]. However, details of its involvement in CDK4/6is
resistance are not clear, and further studies are needed to better understand its role.
2.1.6. CDK6 Amplification
The CDK6, together with the CDK4, plays a pivotal role in the cell cycle progression,
and its function is mainly kinase-dependent. However, it also upregulates the transcription
of p16 in the presence of STAT3 and cyclin D in a kinase-independent way [
34
]. Moreover,
together with c-Jun, CDK6 upregulates the vascular endothelial growth factor A (VEGF-A)
which is responsible for cancer progression and drug resistance due to its ability to induce
angiogenesis [
35
]. It was also demonstrated that a particular drug that specifically degrades
CDK6 was able to overcome the CDK4/6is resistance [27].
2.1.7. WEE1 Overexpression
WEE1 is a serine/threonine kinase involved in guaranteeing an accurate DNA repli-
cation and, in coordination with CDK1, it is responsible for inhibiting DNA-damaged
cells from entering mitosis. Most importantly, it plays a key role in the transition from
G2 to M phase of the cell cycle. Its role in the CDK4/6is resistance is not clear; however,
it was reported that its inhibition in resistant cells could partially restore the CDK4/6
sensitivity [36].
2.1.8. MDM2 Overexpression
Mouse double minute 2 homolog (MDM2) is a negative regulator of p53 which is
involved in the activation of p21 (a CDK inhibitor), thus leading to cell cycle
arrest [37,38]
.
MDM2 overexpression leads to interruption of cell senescence and hence CDK4/6is resis-
tance. For this reason, MDM2 inhibitors may be useful in treating patients’ resistant to
CDK4/6is, despite further studies being needed [39].
2.1.9. HDACs Activation
Histone deacetylases (HDACs) are responsible for removing the acetyl group from
the histones’
ε
-N-acetyl lysins, playing a crucial role in gene expression regulation [
40
].
Moreover, they inhibit p21, which interacts with cyclin D [
41
]. Recently, it has been reported
that the CDK4/6i palbociclib resistance may be conferred by HDAC5 depletion through
disruption of palbociclib-induced histone deacetylation and suppression of oncogenic gene
expression. Thus, the HDAC5 deficiency in cancer cells could be useful to drive the clinical
use of CDK4/6is [42].

Int. J. Mol. Sci. 2023,24, 14427 5 of 25
2.1.10. FZR1 Loss
Fizzy and the cell division cycle 20-related 1 (FZR1) is a co-activator of the ubiquitin-
ligase APC/C that, once activated, can interact with pRb during the cell cycle phase G1 [
43
].
Ruijtenberg et al. showed that FZR1 is a substrate of cyclin D-CDK4/6 and that, once
phosphorylated, it loses its ability to activate APC/C. They further demonstrated that
knockdown of both FZR1 and APC/C leads cells to bypass their dependence on cyclin
D-CDK4/6 for the progression of the cell cycle [
44
]. The involvement of the APC/C-FZR1
complex in the downregulation of CDK2/4/6 is particularly interesting because it can
also upregulate p27 (a CDK inhibitor) through S-phase kinase-associated protein 2 (SKP2)
degradation [45]. For all these reasons, the FZR1 loss is linked to resistance to CDK4/6is.
2.1.11. TK1 Activation
Thymidine kinase 1 (TK1) is an enzyme important for thymidine metabolism during
the synthesis of DNA. In resting cells, TK1 activity is low and it increases gradually until
it reaches its peak during the S phase. However, it was reported to be continuously
overexpressed in patients with different malignancies [
46
]. It has been demonstrated that
TK1 activity is associated with OS and PFS in patients with advanced BC [
47
]. Moreover,
Del Re et al. analyzed exosomal mRNA expression of TK1 and CDK9 in patients with
ER+/HER2
−
mBC enrolled in the ECLIPS biomarker study, showing that higher mRNA
expression levels of both of them were linked to palbociclib resistance [48].
2.1.12. miRNAs Expression
In recent years, microRNA (miRNAs) expression in cancer has been widely studied,
due to its important role in cancer progression [
49
]. Among the different miRNAs identified,
some were found to be linked to CDK4/6is resistance, such as miR-432-5p, miR-223, and
miR-106b [
50
]. In particular, miR-432-5p was reported to induce resistance through CDK6
overexpression [
51
]. Moreover, the oncogene c-Myc could reduce the inhibitory effect
of miR-29b-3p on CDK6 by downregulating miR-29b-3p, thus inducing BC resistance to
palbociclib [52].
2.1.13. S6K1 Amplification
Ribosomal protein S6 kinase beta-1 (S6K1) is a serine/threonine protein kinase that
acts downstream of the mTORC1 complex and regulates cell size, protein translation and
cell proliferation [
53
]. Recently, it has been reported that S6K1 amplification is responsible
for primary resistance to CDK4/6is, mainly linked to c-Myc overexpression that induces
hyperactivation of cyclins/CDKs. The authors also suggested the use of mTOR inhibitors
combined with CDK4/6is to overcoming resistance [54].
2.2. Cell-Cycle-Non-Specific Mechanisms
Cell-cycle-non-specific mechanisms include the overexpression of factors that are
upstream of the cell cycle, such as FGFR and PI3K/AKT/mTOR, resulting in decreased
efficacy of CDK4/6is (Figure 2).
2.2.1. FGFR Pathway Activation
The fibroblast growth factor receptor 1 (FGFR1) is a tyrosine kinase involved mostly in
cell proliferation, differentiation and survival [
55
]. Mutations in FGFR1 have been widely
reported in different cancers, including BC [
56
]. Different studies report that FGFR1 and
FGFR2 are linked to CDK4/6is resistance. In particular, it was reported that this resistance
was caused by the amplification of FGFR1, which led to the activation of PI3K/AKT
and RAS/MEK/ERK signaling pathways [
57
]. In addition, a recent study demonstrated
that giving a specific FGFR1 tyrosine kinase-inhibitor to resistant cells could revert the
resistance [
58
]. Finally, it was shown that the FGFR2-activating mutation in ER+ BC could
contribute to palbociclib resistance and, when the cells were given a high dose of FGFR

Int. J. Mol. Sci. 2023,24, 14427 6 of 25
inhibitors, they could be completely resensitised to the drug [
59
]. These studies suggest a
potential therapeutic approach to overcoming such resistance.
Int. J. Mol. Sci. 2023, 23, x FOR PEER REVIEW 6 of 26
Figure 2. Cell-cycle-non-specific mechanisms that could be associated with CDK4/6is resistance.
Sharp arrows (→) indicate stimulation, blunt arrows (┴) indicate inhibition.
2.2.1. FGFR Pathway Activation
The fibroblast growth factor receptor 1 (FGFR1) is a tyrosine kinase involved mostly
in cell proliferation, differentiation and survival [55]. Mutations in FGFR1 have been
widely reported in different cancers, including BC [56]. Different studies report that
FGFR1 and FGFR2 are linked to CDK4/6is resistance. In particular, it was reported that
this resistance was caused by the amplification of FGFR1, which led to the activation of
PI3K/AKT and RAS/MEK/ERK signaling pathways [57]. In addition, a recent study
demonstrated that giving a specific FGFR1 tyrosine kinase-inhibitor to resistant cells
could revert the resistance [58]. Finally, it was shown that the FGFR2-activating mutation
in ER+ BC could contribute to palbociclib resistance and, when the cells were given a high
dose of FGFR inhibitors, they could be completely resensitised to the drug. [59]. These
studies suggest a potential therapeutic approach to overcoming such resistance.
2.2.2. PI3K/AKT/mTOR Pathway Activation
Many studies have reported a main role of the PI3K/AKT/mTOR activation in
CDK4/6is resistance. In particular, high levels of pyruvate dehydrogenase kinase 1
(PDK1, a protein kinase that acts downstream of PI3K) and activation of the AKT path-
way (phospho-S477/T479 AKT) were reported in ribociclib-resistant BC cells. Moreover,
inhibition of PDK1 led to higher sensitivity to ribociclib in these cells [60]. Another study
revealed that mTORC1/2 may also be involved in CDK4/6is resistance: inhibition of
mTOR in ER+ BC results in a decrease in cyclin D1 protein, pRb phosphorylation, and so
E2F mediated transcription. In addition, they found that even if cells resistant to
CDK4/6is reactivated the CDK-pRb-E2F pathway, they still were sensitive to mTORC1/2
inhibitors [61]. Also, the protein phosphatase and tensin homolog (PTEN) plays a pivotal
role in the regulation of the AKT/mTOR pathway. Costa et al. showed that its loss caused
a CDK4/6i (ribociclib) resistance by AKT activation. Its loss translated into p27 down-
regulation which, again, turned into activation of CDK2 and CDK4 [62]. Moreover,
Figure 2.
Cell-cycle-non-specific mechanisms that could be associated with CDK4/6is resistance.
Sharp arrows (→) indicate stimulation, blunt arrows (⊥) indicate inhibition.
2.2.2. PI3K/AKT/mTOR Pathway Activation
Many studies have reported a main role of the PI3K/AKT/mTOR activation in
CDK4/6is resistance. In particular, high levels of pyruvate dehydrogenase kinase 1 (PDK1, a
protein kinase that acts downstream of PI3K) and activation of the AKT pathway (phospho-
S477/T479 AKT) were reported in ribociclib-resistant BC cells. Moreover, inhibition of
PDK1 led to higher sensitivity to ribociclib in these cells [
60
]. Another study revealed
that mTORC1/2 may also be involved in CDK4/6is resistance: inhibition of mTOR in ER+
BC results in a decrease in cyclin D1 protein, pRb phosphorylation, and so E2F mediated
transcription. In addition, they found that even if cells resistant to CDK4/6is reactivated
the CDK-pRb-E2F pathway, they still were sensitive to mTORC1/2 inhibitors [
61
]. Also,
the protein phosphatase and tensin homolog (PTEN) plays a pivotal role in the regulation
of the AKT/mTOR pathway. Costa et al. showed that its loss caused a CDK4/6i (ribo-
ciclib) resistance by AKT activation. Its loss translated into p27 downregulation which,
again, turned into activation of CDK2 and CDK4 [
62
]. Moreover, knockdown of PTEN in
CDK4/6is-sensitive cell lines led to the upregulation of CDK6 and resistance to abemaci-
clib [
63
]. These data suggest a wide range of drugs that could be used in combination with
CDK4/6is in order to prevent/revert resistance.
2.2.3. MAPK Pathway Activation
The mitogen-activated protein kinase (MAPK) pathway (RAS/RAF/MEK/ERK) is
one of the main pathways downstream of FGFR1. It has been reported that selumetinib, a
MEK1/2 inhibitor, in association with fulvestrant and palbociclib could inhibit BC prolifera-
tion in patients resistant to CDK4/6is [
64
]. Moreover, it was shown how the overexpression

Int. J. Mol. Sci. 2023,24, 14427 7 of 25
of KRAS, a member of the RAS family, was involved in palbociclib and fulvestrant resis-
tance [65].
2.2.4. Hippo Pathway Inhibition by FAT1
The Hippo pathway is a pathway reported to have a tumor-suppressive role [
66
].
FAT atypical cadherin 1 (FAT1) is a cadherin that interacts both with the Hippo pathway
and
β
-catenin, and it is reported to act as a tumor suppressor gene [
67
]. Interestingly, a
study of 348 patients treated with CDK4/6is showed loss of FAT1 in patients who became
resistant to CDK4/6is. The resistance was probably caused by the fact that FAT1 loss led to
Hippo pathway inhibition, which turned into CDK6 overexpression [
68
]. However, another
study reported that more genetic alterations are needed besides FAT1 loss to have CDK6
overexpression [
63
]. This suggests that more in-depth studies need to be performed to
clarify this pathway and to understand whether its targeting could have a therapeutic effect.
2.2.5. Epithelial–Mesenchymal Transition
Epithelial–mesenchymal transition (EMT) consists of the transition of cells from an
epithelial to a mesenchymal phenotype. EMT is linked with tissue morphogenesis. How-
ever, it has been widely reported to be linked also with tissue invasion, drug resistance
and metastasis of cancer [
69
]. It has been reported that inhibition of the CDK4/6 can
induce EMT by activating TGF-
β
-Smad and PI3K/AKT/mTOR pathways. Once acti-
vated, TGF-
β
can phosphorylate and so activate Smad2 and Smad3. These can create
a complex with Smad4 and activate EMT transcription factors [
70
]. In addition, Smad3
inhibition leads to CDK4/6is resistance, probably because it is no longer able to block
E2F from the pRb-E2F complex [
71
,
72
]. Moreover, TGF-
β
can lead to EMT through activa-
tion of the PI3K/AKT/mTOR pathway [
73
]. Recently, it has also been reported that the
fibronectin/DHPS/SLC3A2 signaling axis may be involved in the resistance to CDK4/6is.
Galler et al. showed that the targeting of this signaling axis improved palbociclib sensitivity
in pRb-negative BC cells [74].
2.2.6. Apoptosis Failure
The combination of ET with CDK4/6 inhibition has a predominantly cytostatic effect,
thus leading to reduced apoptosis [
75
]. For this reason, it has been suggested that a
combination therapy using CDK4/6is and BCL2 inhibitors could inhibit proliferation and
induce apoptosis of cancer cells. Moreover, this approach reduced the proliferation of
regulatory T cells in the tumor microenvironment [76].
2.2.7. Stemness Properties
In the last few years, many studies have reported a central role of cancer stem cells
(CSCs) in drug resistance. CSCs are a population of cells capable of self-renewal and
differentiation potential. Moreover, they show many alterations in different pathways, and
for different reasons, and at dates not completely clear, they also play a main role in drug
resistance [
77
]. Interestingly, Wang et al. found that ER+ BC cells treated with palbociclib
developed resistance by showing a senescence-like phenotype, which went on to promote
stemness. Particularly, PFKFB4 played a major role in this transformation by enhancing
glycolysis, and its downregulation resulted in higher palbociclib sensitivity [
78
]. Thus, even
if more studies are needed, targeting CSCs could be a promising therapeutic approach.
3. Short- and Long-Term Adaptation to CDK4/6 Inhibitors
CDK4/6 inhibition in BC cells is limited by the inability to induce complete and
durable cell-cycle arrest. The mechanisms involved are categorized into two classes: short-
term/de novo and long-term adaptation. This distinction is important for the clinic to
distinguish the onset of the resistance [79].

Int. J. Mol. Sci. 2023,24, 14427 8 of 25
3.1. Short-Term Adaptation to CDK4/6 Inhibitors
Early adaptation can be mediated by persistent G1-S-phase cyclin expression and
CDK2 signaling. Research led by Herrera-Abreu et al. found that early adaptation to
CDK4/6 blockade is mediated by the non-canonical cyclin D1/CDK2 complex promot-
ing pRb phosphorylation recovery. In fact, in response to chronic CDK4/6 inhibition,
there is a PI3K-dependent upregulation of cyclin D1 along with CDK2-dependent pRb
phosphorylation and S-phase entry [30].
A second mechanism that could lead to short-term adaptation to CDK4/6 inhibitors is
the promotion of a proinflammatory, senescence-associated secretory phenotype (SASP)
in the stroma [
80
]. These patients have a poor prognosis. Early identification is necessary
for cases of de novo or primary resistance, characterized by disease progression within 6
months of initiating treatment [79].
3.2. Long-Term Acquisition of CDK4/6 Inhibitors Resistance
On the other side, acquired resistance is a widespread phenomenon, prompting
vigorous exploration of various targets, agents, and treatment strategies [79].
Prolonged exposure to CDK4/6is eventually leads to the creation of cell populations
that show different resistance mechanisms, like the loss or mutation of pRb, which are
the most frequently observed changes in CDK4/6is-resistant cells [
78
,
79
]. While this
was first observed in preclinical data [
30
], examples of RB1-inactivating mutations in
ctDNA, acquired during the treatment with CDK4/6 inhibitors in patients, have begun to
emerge [81,82].
A second known long-term mechanism of resistance is the upregulation of CDK6. Its
kinase function is essential for resistance, proven by the correlation between the dose of the
kinase inhibitor necessary to cause the pRb loss and the dose that blocks cell proliferation.
CDK4 expression, on the other hand, can be reduced in resistant cells since the partner cyclin
or other components of the complex influence inhibitor response [
83
]. The overexpression
of CDK2 and CDK4 are amenable to a drug holiday, leading to re-sensitization to palbociclib
in vitro
and
in vivo
. Notably, the palbociclib-resistant cells that retain pRb expression are
sensitive to abemaciclib, possibly because of increased expression of CDK4 acting as a
compensatory mechanism [
33
]. Other resistance mechanisms found in treatment with
CDK4/6is are high levels of cyclins D1 and D2 [
60
]. At last, CDK6 with c-Jun upregulates
VEGF-A, inducing tumor angiogenesis, thus promoting cancer progression and drug
resistance [35,84].
In non-cancerous cells, cyclin E-CDK2 (cyclin E1-CDK2 or cyclin E2-CDK2) complexes
phosphorylate pRb after a first phosphorylation by cyclin D-CDK4/6 as part of a second
phase of signaling. CDK4/6is have multiple effects on the CDK2 action. Without the phos-
phorylation of the pRb by D-CDK4/6, CDK2 cannot efficiently phosphorylate pRb to release
transcription factors like E1 [
85
]. Different studies have demonstrated an upregulation of cy-
clin E1, cyclin E2 and CDK2 in CDK4/6is resistance models [
30
,
33
,
83
]. Etemadmoghadam
et al. showed how CDK2 inhibitors reduced the growth of cells overexpressing cyclin
E1 [29].
In addition to cell-cycle regulation mechanisms, several studies have found that a
number of growth factor signaling pathways could determine the insurgence of resistance
while in treatment with CDK4/6is. For example, Jansen et al. found that PDK1, the PI3K
pathway kinase, was upregulated in cells resistant to ribociclib [
60
]. Also, in endocrine-
resistant cancers, the expansion of FGFR1 activates the PI3K/AKT and RAS/MEK/ERK
pathways, both associated with the CDK4/6 resistance [
23
,
64
]. PI3K/AKT/mTOR signaling
pathway is also involved in the resistance mechanisms: it promotes the stability of the
CDK4/6 complex, thus reversing the effects of CDK4/6 inhibition [86].
The detection of acquired resistance allows to explore targets feasible of target therapy,
broadening the set of possible therapeutic strategies [79].

Int. J. Mol. Sci. 2023,24, 14427 9 of 25
4. Types of Subsequent Therapies after Randomized Controlled Trial (RCT) and in the
Real World
CDK4/6is treatment in first-line therapy was investigated in five RCTs (MONALEESA-
2/7, MONARCH-3, PALOMA-1/2): the first subsequent antineoplastic therapy was ET
in 65% of cases (min–max 48–83%), chemotherapy in 44% (min–max 32–73%) of cases,
CDK4/6 inhibitors up to 38% of cases (on average in 18% of cases) and mTOR inhibitors in
17% of cases (min–max 14–24%) [9–11,22,87].
When CDK4/6is was used in second-line therapy (MONARCH-2 and PALOMA-3
RCTs), ET was administered on average in 55% of cases (min–max 37–71%), chemotherapy
in 66% of cases (min–max 56–76%), CDK4/6is in 9% of cases (min–max 2–21%) and mTOR
inhibitors in 24% of cases (min–max 15–33%) [88,89].
In MONALEESA-3, patients treated with fulvestrant and the CDK4/6/placebo in-
hibitor in first- and second-line therapy received ET (55%), chemotherapy (43%), CDK4/6
inhibitor (20%) and mTOR inhibitor (30%) [90].
Real-world studies suggest that chemotherapy and ET are the preferred second-line
options. In a population-based study, second-line regimens were ET in 38%, chemotherapy
in 35.6%, everolimus-based (mTOR inhibitor) in 14.4% and CDKi-based in 9.4% [
91
]. In a
retrospective real-world analysis, cytotoxic chemotherapy was the most commonly chosen
second-line therapy (29.7%), followed by endocrine monotherapy (12.4%). The 36.0% of
patients continued a CDK4/6is in the second-line treatment setting, either alone or in
combination with ET. Other targeted therapies used were everolimus (11.7%), alpelisib
(1.9%) and a PARP inhibitor (0.5%) [92].
Little data about the preferred regimen of chemotherapy are available. In a multicenter
analysis, taxanes were the most used agents followed by capecitabine and vinorelbine [
93
].
5. Progression after CDK4/6is
Previous case studies reported a rapid, secondary disease progression after the treat-
ment with CDK4/6is: in four patients treated at MD Anderson Cancer Center, a median
time-to-progression of 2.35 months (range 1.46–2.8) was reported [
94
]. In a retrospective
monocentric review (abstract presented in ASCO 2021) the 4-month incidence of post CDKi
progression or death was 31% [95].
These initial suggestions were not further confirmed.
Progression-free survival 2 (PFS2), time from randomization to second disease pro-
gression or death, is recommended by the EMA as a surrogate for OS, and to assess the
effect of maintenance therapy or the impact of treatment on the efficacy of a subsequent
line of therapy [96].
In the systematic review by Munzone et al., a PFS2 benefit was observed in patients
who received CDK4/6is plus ET (pooled hazard ratio (HR) 0.68, 95%, confidence interval
(CI) 0.62–0.74). A delay in subsequent time to chemotherapy (TTC) (pooled HR 0.65, 95%
CI 0.60–0.71) was reported as well [97].
6. Potential Therapeutic Approaches
6.1. Continuation of CDK4/6is
The role of CDK4/6is persistance after initial progression was investigated in several
retrospective and a few prospective trials. Four single-institution experiences and one
multicenter experience showed an interesting clinical benefit of this strategy [
98
–
102
].
Several studies explored the use of abemaciclib after initial progression to palbociclib.
The Taussig Cancer Institute followed 30 patients who continued CDK4/6is beyond
the first progression. Most patients received palbociclib plus letrozole as initial therapy
(67%), followed by palbociclib plus fulvestrant (23%) and palbociclib plus anastrozole
(10%). At progression, most patients received palbociclib plus fulvestrant (56.7%) followed
by palbociclib plus AI (23.3%), palbociclb plus tamoxifene (13.3%) and abemaciclib with
either fulvestrant or letrozole (6.6%). The estimated median PFS for continued CDK4/6is
use beyond the first PD was 11.8 months (95% CI, 5.34–13.13 months) [99].

Int. J. Mol. Sci. 2023,24, 14427 10 of 25
In another larger analysis by Martin et al., 839 patients received a second-line therapy
after progression of first-line CDK4/6is treatment, and 308 patients (36%) continued a
CDK4/6is therapy [
92
]. Palbociclib was the most commonly used CKD4/6i in the first-line
setting (88.2%), followed by ribociclib (7.2%) and abemaciclib (4.6%). The most interesting
data show that 74.4% of patients in the second-line treatment received the same CDK4/6i
of the first-line treatment; in particular, 78.2% of patients who received palbociclib in the
first-line treatment received the same CDK4/6i compared to 60.9% of patients who started
with ribociclib and 45.8% who started with abemaciclib. The PFS and OS of patients who
continued CDK4/6is in the second-line treatment were 8.25 months and 35.7 months,
respectively, which are significantly improved compared to chemotherapy (HR PFS 0.48,
95% CI 0.43–0.53, p< 0.0001; HR OS 0.30, 95% CI 0.26–0.35, p< 0.0001).
The prospective trials produced conflicting results (Table 1).
In the MAINTAIN trial (NCT02632045), ET plus ribociclib demonstrated a longer
PFS (5.29 months vs. 2.76 months) than ET plus placebo after progression of a first-line
treatment with CDK4/6is [
103
]. Notably, 84% of the patients had previously received
palbociclib and more than two-thirds had previously received CDK4/6is for more than
12 months.
However, in the phase II PACE trial (NCT03147287), the median PFS of patients treated
with fulvestrant (4.8 months) was the same as in patients treated with both fulvestrant and
palbociclib (4.6 months) [
104
]. The PACE trial randomized patients, after progression on
CDK4/6is, to palbociclib plus fulvestrant versus placebo plus fulvestrant versus palbociclib
plus fulvestrant plus avelumab. Notably, more than 90% of patients received palbociclib as
the initial CDK4/6i and 76% received it for more than 12 months.
In the PALMIRA trial (NCT03809988), presented at ASCO 2023, 198 patients showing
a clinical benefit from the first-line with palbociclib plus ET for at least 6 months were
randomized at progression to receive another ET different from the first-line treatment plus
palbociclib or placebo: median investigator-assessed PFS was 4.2 months (95% CI 3.5–5.8)
in the palbociclib arm vs. 3.6 months (95% CI 2.7–4.2) in the ET arm (HR 0.8, 95% CI 0.6–1.1,
p= 0.206). The authors stated that maintaining palbociclib with a second-line ET beyond
progression of prior palbociclib-based therapy did not significantly improve PFS compared
with second-line ET alone [105].
The benefits of continuing CDK4/6is after initial progression remain unclear. Whether
the results of MAINTAIN, PACE and PALMIRA are related to differences in drug efficacy
(palbociclib and ribociclib) or in patient populations (in the PACE and PALMIRA trials,
all patients had to be on a CDK4/6i first-line therapy for at least 6 months, while in the
MAINTAIN trial there are no limitations) or due to switch to a different CDK4/6i (in PACE
and PALMIRA, a rechallenge of palbociclib was performed, while in MAINTAIN, a switch
to ribociclib was carried out) remains unclear as well (Table 1).
The BioPER trial (NCT03184090) evaluated the antitumor activity, the safety and the
predictive biomarkers of palbociclib rechallenge in 33 patients with confirmed progressive
disease after having achieved a clinical benefit on immediately prior palbociclib plus
endocrine therapy regimen. The clinical benefit rate was 34.4%, but the median PFS
was modest (2.6 months). The analysis of biomarkers revealed that low Rb score, high
cyclin E1 score and ESR1 mutations were associated with worse outcomes than palbociclib
rechallenge [106].
In the future, new prospective clinical trials and more data could clarify if continuing
CDK4/6is after progression is a valid option to overcoming resistance, particularly the
following:
PostMONARCH (NCT05169567) is a randomized, double-blind, placebo-controlled,
phase III trial that compares the efficacy of abemaciclib plus fulvestrant to placebo plus
fulvestrant in patients with HR+/HER2
−
, advanced or metastatic BC following progression
on CDK4/6is and ET (recruiting);
Serena-6 (NCT04964934) is a phase III, double-blind, randomized trial that could
assess switching to AZD9833 (a next-generation oral SERD) plus CDK4/6is (palbociclib or

Int. J. Mol. Sci. 2023,24, 14427 11 of 25
abemaciclib) vs. continuing AI (letrozole or anastrozole) plus CDK4/6is in HR+/HER2
−
metastatic BC patients with detectable ESR1 mutation without disease progression during
first-line treatment with IA plus CDK4/6is (recruiting).
Table 1. Summary of the trials focused on continuing CDK4/6is after progression.
Trial Number of
Patients Population Prior CDK4/6is Subsequent ET Subsequent
CDK4/6is Efficacy PFS (Months)
MAINTAIN
[103]119 Progression on
CDK4/6is + ET
•Palbociclib
86.5%
•Ribociclib
11.7%
•Fulvestrant
83.2%
•Exemestane
16.8%
Ribociclib •RR 20%
•CBR 43%
•ET +
Ribociclib
5.29
•ET + placebo
2.76
PACE [104]220
Progression on
CDK4/6is + ET
after at least 6
months of therapy
•Palbociclib
90.9%
•Ribocliclib
4.5%
•Abemaciclib
4.1%
Fulvestrant Palbociclib
•RR ET 10.8%
•RR palbociclib
+ ET 3.8%
•RR Triplet
17.9%
•ET 4.8
•Palbociclib +
ET 4.6
•Triplet 8.1
PALMIRA
[105]198 CB during
palbociclib Palbociclib 100% •Fulvestrant
•Letrozole Palbociclib 6-months CBR 41.9%
•ET +
Palbociclib
4.9
•ET 3.6
BIOPER
[106]33
•CB during
palbociclib
•Palbociclib
as last
treatment
Palbociclib 100%
•Fulvestrant
56%
•Letrozole
28%
•Others 15.6%
Palbociclib CBR 34.4% 2.6
Abbreviation: ET = endocrine therapy; CB = clinical benefit; RR = response rate; CBR = clinical benefit rate; PFS =
progression-free survival.
6.2. Endocrine Therapy
In some cases, de-escalation to only ET after progression of CDK4/6is is a possible
strategy; as shown by Karacin C. et al., PFS from patients receiving ET after progression on
CDK4/6is was not different compared to those receiving chemotherapy [
107
]. ET could be
fulvestrant, AI or tamoxifen.
Fulvestrant is more effective than AI, as demonstrated in the FALCON trial (NCT01602380);
PFS was significantly longer in the fulvestrant group than in the anastrozole group (HR
0.797, 95% CI 0.637–0.999, p= 0.0486), and mPFS was 16.6 months (95% CI 13.83–20.99) in
the fulvestrant group versus 13.8 months (11.99–16.59) in the anastrozole group [108].
However, in these trials, fulvestrant was given as a first-line therapy. Nevertheless, in
the second-line treatment after progression of CDK4/6is, the PFS of fulvestrant has been
demonstrated to be much lower (EFECT trial, NCT00065325) [109].
In the phase III EMERALD trial (NCT03778931), 477 patients pretreated with a
CDK4/6is and
≤
1 chemotherapy were randomized to elacestrant, a new oral selective ER
degrader (SERD), or standard of care (fulvestrant). Elacestrant had a superior PFS rate com-
pared to fulvestrant, and the HR improved in the subgroup of patients with ESR1-mutated
tumors. The mPFS in the intention-to-treat (ITT) population on elacestrant was 2.8 months,
compared to 1.9 months for fulvestrant monotherapy [
110
]. In this study, patients with
ESR1 mutation had a higher benefit with a median PFS of 3.8 months with elacestrant and
6- and 12-month PFS rates of 41% and 27%. Particularly, patients who had remained on a
prior CDK4/6 inhibitor for
≥
12 months showed a PFS benefit of 6 months (median 8.6 vs.
1.91 months, hazard ratio 0.41, 0.26–0.63), indicating that this might be the ideal setting for
elacestrant [111].
In a phase II SERENA-2 trial (NCT03616587), another SERD, camizestrant, demon-
strated an advantage in PFS compared to fulvestrant (7.7 months vs. 3.7 months). Patients
were eligible if they had received
≥
1 line of ET and no more than 1 prior chemotherapy
regimen. A total of 50% of patients had a prior CDK4/6i, 42% of patients had prior SERM,
and 37% of patients had an ESR1 mutation [112].
These data suggested that the use of fulvestrant after progression on the first-line
treatment of CDK4/6is could be exploited in the case of limited progression because of

Int. J. Mol. Sci. 2023,24, 14427 12 of 25
low PFS. A more viable alternative to ET is the use of SERD, as it is more effective than
fulvestrant.
6.3. Switch to Combined Target Therapies
As mentioned above, many studies have reported a main role of PI3K/AKT/mTOR
activation in the CDK4/6is resistance: targeting these specific pathways could be another
option to overcoming the resistance (Table 2).
6.3.1. ET Combined with PI3K Inhibitors
Approximately 40% of patients with HR+/HER2
−
advanced BC have PIK3CA
(phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit
α
) mutations. These
mutations can promote endocrine resistance through the activation of the PI3K pathway,
which is associated with a poor prognosis [113].
In the SOLAR-1 study (NCT02437318), the combination of fulvestrant and alpelisib,
an
α
-selective PI3K inhibitor, was compared with fulvestrant and placebo for patients with
HR+/HER2
−
advanced BC with PIK3CA mutations who progressed during or after the
treatment with AI. The PFS time of alpelisib plus fulvestrant group was prolonged by
5.3 months (11 vs. 5.7 months; p= 0.00065) and the mOS time was prolonged by 7.9 months
(39.3 vs. 31.4 months) [
114
]. According to these results, alpelisib has been approved by
the FDA for marketing. However, <10% of the patients enrolled in the SOLAR-1 study
received CDK4/6is treatment, so the drug was approved by EMA only with the specific
indication “after disease progression following endocrine therapy as monotherapy” [115].
The BYLieve study (NCT03056755) investigated the question of whether alpelisib
plays the same role in patients who progress after treatment with CDK4/6is [
116
]. Patients
with PIK3CA mutations who progressed after first-line treatment were enrolled in two
different cohorts: cohort A for patients who received AI combined with CDK4/6is as first-
line treatment and received alpelisib combined with fulvestrant as second-line treatment;
cohort B for patients who were treated with fulvestrant combined with CDK4/6is as first-
line treatment and received alpelisib combined with letrozole as second-line treatment.
Updated data results were presented at the ASCO 2022 meeting: mPFS time was reported
as 8.2 months in cohort A and 5.6 months in cohort B [
117
], showing a potential clinical
benefit of alpelisib plus endocrine therapy also after a CDK4/6is. Some issues remain about
the toxicity profile of the drug: in this trial, about 26% of patients had grade >3 side effects
including hyperglycemia, rash and diarrhea.
Other PI3K inhibitors (such as inavolisib, LOXO-783 [
118
] and RLY-2608 [
119
]) are
currently being studied in this setting in order to maintain the activity of this class of drugs
while reducing off-target toxicities.
6.3.2. ET Combined with mTOR Inhibitors
Everolimus is the mTOR inhibitor most commonly used in BC. Its effect has been
confirmed in the BOLERO-2 study (NCT00863655): for patients who progressed after NSAI
treatment, the everolimus plus exemestane (SAI) group had significantly longer mPFS
and mOS than the placebo plus exemestane group (mPFS: 6.93 vs. 2.83 months; HR, 0.43;
p< 0.0001; mOS: 31.0 vs. 26.6 months; HR, 0.89; p= 0.14) [120].
In real-world studies, the combination of everolimus plus exemestane was largely used
as second-line therapy after CDK4/6is. One study from the US analyzed 525 patients who
received systemic therapy after a CDKi-based line: the second-line regimen (n= 208) after
CDK4/6is was everolimus-based in 14.4% of patients [
91
]. Another recent study analyzed
609 patients and calculated the PFS of subsequent treatments (chemotherapy, n:434 or ET,
n:175) after CDKi. Patients were evaluated as three groups: those who received CDKi in
first-line therapy (group A, n: 202), second-line therapy (group B, n: 153) and
≥
third-line
therapy (group C, n: 254). PFS was compared according to the use of ET and chemotherapy.
In addition, patients who received ET after CDKi were compared as those who received
everolimus-based combination therapy versus those who received ET monotherapy: the

Int. J. Mol. Sci. 2023,24, 14427 13 of 25
median PFS in groups A, B, and C was 11.0 vs. 5.9 (p= 0.047), 6.7 vs. 5.0 (p= 0.164), 6.7 vs.
3.9 (p= 0.763) months, suggesting a role for everolimus in achieving better PFS [107].
At the ASCO 2019 meeting, the TRINITI-1 clinical study (NCT02732119) reported
enrolled patients who progressed after treatment with CDK4/6is [
121
]. After receiving
exemestane, everolimus and ribociclib combination therapy, the clinical benefit rate at 24
weeks reached 41%, which exceeds the pre-defined primary endpoint threshold (>10%).
The mPFS time of the overall population reached 5.7 months [122].
An ongoing phase III study (evERA, NCT05306340) is currently evaluating the combi-
nation of the oral SERD giredestrant and everolimus compared to everolimus and exemes-
tane in patients who have previously progressed onto CDK4/6is [123].
The combination of CDK4/6is with PI3K or mTOR inhibitors is currently being tested
in clinical trials because synergistic activity for these drugs has been observed [
124
], but
the studies evaluating these combinations showed severe toxicity. The trial with the
combination of palbociclib plus everolimus plus exemestane was limited by frequent
high-grade mucositis and neutropenia and also showed limited efficacy [
125
]. Similar
toxicity concerns were observed in another study with the combination ribociclib plus
everolimus plus exemestane [
126
]. Other ongoing phase I trials are investigating the safety
and efficacy of combinations including abemaciclib and xentuzumab (an IGF-neutralizing
antibody) [
127
], gedatolisib (an mTOR/PI3K inhibitor) in combination with palbociclib
and either letrozole or fulvestrant [
128
], fulvestrant, palbociclib and erdafitinib (a newer
generation FGFR inhibitor) [129].
6.3.3. ET Combined with Other Targeted Drugs
AKT acts as a bridge connecting the PI3K and mTOR signaling pathways. The phase Ib
TAKTIC study (NCT03959891) evaluated the efficacy of the AKT-1 inhibitor ipatasertib plus
endocrine therapy plus or not palbociclib in patients with HR+/HER2
−
advanced BC. The
results showed that in some patients (8/12) who had failed previous CDK4/6is treatment,
the combination of AKT inhibitor plus palbociclib plus endocrine therapy achieved good
clinical effects and was well tolerated [130]. More data remain to be seen.
In a recent phase 3, randomized, double-blind trial (CAPItello-291, NCT04305496), 708
patients with HR+/HER2
−
advanced BC, who had had a relapse or disease progression
during or after treatment with an IA, with or without previous CDK4/6is therapy, were
randomly assigned in a 1:1 ratio to receive capivasertib (AKT inhibitor) plus fulvestrant or
placebo plus fulvestrant. The PFS was 7.2 months in the capivasertib–fulvestrant group as
compared with 3.6 months in the placebo–fulvestrant group [
131
], suggesting an interesting
clinical activity of this combination.
HDAC inhibitors are another target therapy of interest. In the ACE study (NCT02482753),
the HDAC inhibitor tucidinostat (formerly known as chidamide) combined with exemes-
tane showed longer mPFS time compared with placebo and exemestane (7.4 vs. 3.8 months;
p= 0.033) [
132
]. The ENCORE 301 (NCT00676663), a phase II randomized, placebo-
controlled study, demonstrated a significant improvement in PFS and OS with the addition
of entinostat to exemestane in patients with HR+ advanced BC with disease progression af-
ter prior non-steroidal AI. The subsequent phase III E2112 registration trial (NCT02115282)
did not confirm the results, with an mPFS of 3.3 months in the entinostat-exemestane
group versus 3.1 months in the placebo–exemestane group; the mOS was 23.4 months
(entinostat–exemestane) versus 21.7 months (placebo–exemestane); the objective response
rate was 5.8% (entinostat–exemestane) and 5.6% (placebo–exemestane). In the E2112 study,
35% of the patients were previously treated with CDK4/6is [
133
]. Probably, this class of
drugs is not so efficient in overcoming CDK4/6is resistance.
Another interesting target is the B-cell lymphoma-2 (BCL2) gene, an estrogen-responsive
gene coding for an antiapoptotic protein overexpressed in approximately 80% of patients
with HR+ BC [
134
]. Venetoclax, a potent and selective BCL2 inhibitor, combined with ta-
moxifen had a tolerable safety profile and significant activity in patients with ER+/HER2
−
advanced BC that overexpresses BCL2 in a phase I clinical trial [
135
]. The VERONICA trial

Int. J. Mol. Sci. 2023,24, 14427 14 of 25
(NCT03584009) evaluated venetoclax plus fulvestrant vs. fulvestrant in women with locally
advanced ER+/HER2
−
or mBC, who received
≤
2 prior lines of ET and no prior chemother-
apy in the locally advanced or mBC setting and experienced disease recurrence/progression
during/after CDK4/6is therapy. Preliminary results from the primary analysis did not
show a significant difference in PFS time between the combination and monotherapy
groups (2.69 vs. 1.94 months; p= 0.7853) [
136
]. An ongoing phase I clinical trial, PALVEN
(NCT03900884) [
137
] is evaluating the safety and efficacy of AI plus CDK4/6is plus vene-
toclax triple therapy as a first-line treatment for patients with ER+/HER2
−
advanced BC
with BCL2 overexpression, and we are waiting for the results.
6.3.4. CDK4/6 Inhibitors plus Other Targeted Drugs
The combination of CDK4/6is with immune checkpoint inhibitors is under investi-
gation because of the demonstrated activity of CDK4/6is not only in inducing the tumor
cell cycle arrest, but also in promoting anti-tumor immunity [138]. CDK4/6is have effects
both on tumor cells and on regulatory T cells: they markedly suppress the proliferation
of regulatory T cells and indirectly stimulate the production of type III interferons and
hence enhance tumor antigen presentation. These events promote cytotoxic T-cell-mediated
clearance of tumor cells, which is further enhanced by the addition of immune checkpoint
blockade.
The PACE trial, already discussed above, had a third arm with avelumab, fulvestrant
and palbociclib. This triplet regimen increased PFS and OS compared with the other two
groups, although neither difference achieved statistical significance. The median PFS was
8.1 months (HR 0.75 vs. fulvestrant, 95% CI 0.47–1.20), and the median OS was 42.5 months
(HR 0.68 vs. fulvestrant, 95% CI 0.4–1.15). No major toxicity signals were identified. The
comparison of a triplet arm versus fulvestrant was a secondary endpoint. The numerical
superiority of PFS and OS with avelumab deserves further study [93,104].
An ongoing phase IB clinical trial, JPCE (NCT02779751), is evaluating the efficacy and
safety of abemaciclib in combination with pembrolizumab (a PD-1 inhibitor) in the first-line
treatment of HR+/HER2−mBC.
Table 2. Summary of the perspective trials focused on target therapies (TT).
Target Therapy Trial Number of
Patients Population Prior CDK4/6is Subsequent TT Efficacy PFS
(Months)
PI3K inhibitor
SOLAR-1 [114]572
•Progression on AI.
•Stratification by
prior treatment with
CDK4/6is
Any Fulvestrant +
Alpelisib NA 7.3
BYLieve [116]127
•Progression on no
more than two
previous anticancer
therapies and no
more than one
previous
chemotherapy
regimen.
•Confirmed PIK3CA
mutation
a. CDK4/6i + AI
b. CDK4/6i +
fulvestrant
a. Fulvestrant +
Alpelisib
b. Letrozole +
Alpelisib
•PR 17%
•SD 45% a. 8.2
b. 5.6
MTOR inhibitor TRINITI-1
[122]104
Progression on a CDK4/6i
after ≥4 months of
therapy as the last prior
treatment regimen
Any Exemestane +
Everolimus +
Ribociclib CBR 41% 5.7
AKT inhibitor CAPItello-291
[131]708
Progression during or
after treatment with an IA,
with or without previous
CDK4/6is
Any (69.1% of pts)
a. Capivasertib +
fulvestrant
b. Placebo +
fulvestrant
a. ORR 22.9%
b. ORR 12.2% a. 7.2
b. 3.6

Int. J. Mol. Sci. 2023,24, 14427 15 of 25
Table 2. Cont.
Target Therapy Trial Number of
Patients Population Prior CDK4/6is Subsequent TT Efficacy PFS
(Months)
HDAC inhibitor
ACE [132]365 Progression after at least
one endocrine therapy
Palbociclib (>1%
pts)
a. Tucidinostat +
exemestane
b. Placebo
+exemestane
a. PR 18%
SD 56%
b. PR 9%
SD 54%
a. 7.4
b. 3.8
E2112 phase
III [133]608
Progression on AI in the
adjuvant or metastatic
setting
Any (35% of pts)
a. Etinostat +
exemestane
b. Placebo +
exemestane
a. 5.8%
b. 5.6% a. 3.3
b. 3.1
BCL-2 inhibitors VERONICA
[136]103
≤2 prior lines of ET and
prior chemotherapy in the
locally advanced/mBC
setting and progression
during/after CDK4/6is
Any a. Venetoclax +
fulvestrant
b. Fulvestrant
a. CBR 11.8%
b. 13.7% a. 2.69
b. 1.94
Immune
checkpoint
inhibitors
PACE [104]200
Prior response to and
subsequent progression
on CDK4/6is and ET
•Palbociclib
90%
•Ribociclib
4.5%
•Abemaciclib
4.1%
•Palbociclib +
Ribociclib
1.4%
a. Avelumab +
Fulvestrant +
Palbociclib
b. Fulvestrant +
Palbociclib
c. Fuvestrant
a. 17.9%
b. 13.8%
c. 10.8%
a. 8.1
b. 4.6
c. 4.8
Abbreviation: TT = target therapy; ORR = objective response rate; CBR = clinical benefit rate; PR = partial response;
SD = stable disease; PFS = progression-free survival.
6.3.5. PARP Inhibitors
Two randomized phase III studies, OlympiAD (NCT02000622) and EMBRACA
(NCT01945775), demonstrated the efficacy of PARP inhibitors (olaparib and talazoparib) in
germline-BRCA-mutated mBC patients: they both showed an improvement in the overall
response rate and PFS (median improvement of 3 months) but failed to demonstrate an
improvement in OS [
139
–
141
]. These trials were designed before the CDK4/6is era, so
there are very little data about the efficacy of PARP inhibitors after CDK4/6is. However, for
selected patients with the germline BRCA mutation, PARP inhibitors could be a reasonable
alternative to chemotherapy beyond CDK4/6is in the endocrine refractory setting [142].
6.4. Switch to Chemotherapy
Chemotherapy is also a good option for patients with HR+/HER2
−
advanced BC
who are resistant to ET plus CDK4/6is [
143
]. As mentioned above, considering the
five randomized clinical trials with CDK4/6is in a first-line setting (MONALEESA-2/7,
MONARCH-3, PALOMA-1/2, NCT01958021, NCT02278120, NCT02246621, NCT00721409
and NCT01740427), patients received chemotherapy as the first subsequent antineoplastic
therapy after study discontinuation in an average of 44% (min–max 32–73%) of cases. In
MONARCH-2 and PALOMA-3 (NCT02107703 and NCT01942135, respectively) chemother-
apy was administered in 66% of cases (min–max 56–76%); patients enrolled in MONALEESA-
3 (NCT02422615) treated with fulvestrant and the CDK4/6/placebo inhibitor in the first-
and second-line settings subsequently received chemotherapy in 43% of cases [97].
Many real-world studies have been published in the past years (Table 3). One still-
mentioned study from the US showed that out of 525 patients who progressed after
receiving CDK4/6is treatment, more than a third of the patients received subsequent
chemotherapy, and the drugs used were capecitabine and taxanes [
91
]. Another American
study analyzed 1210 patients with HR+/HER2
−
mBC who were treated in a first-line
setting with a CDK4/6i from 2015 to 2020; a total of 839 patients received a documented
second-line therapy after progression of first-line CDK4/6is treatment. Chemotherapy was
chosen for 29.7% of patients, and the use of chemotherapy decreased over time [
92
]. Eribu-
lin, a tubulin polymerization inhibitor, demonstrated efficacy after CDK4/6is treatment in
a Russian study [
144
], with an mPFS of 10.0 months; the 6-month, 1-year, and 2-year PFSs
were 79.5%, 44.8% and 26.5%, respectively.

Int. J. Mol. Sci. 2023,24, 14427 16 of 25
Trastuzumab–deruxtecan (T-DXd) is an antibody–drug conjugate consisting of mono-
clonal antibody trastuzumab with a topoisomerase I inhibitor payload. In the DESTINY-
Breast04 trial (NCT03734029), a group of HR+ patients with endocrine-resistant disease
was randomized to T-DXd versus the physician’s choice of chemotherapy. The percentage
of patients who had a prior CDK4/6i was 78% in the investigational arm and 81% in the
control arm. In this HR+ group, patients had an mPFS of 10.1 months on T-DXd versus
5.4 months in the control arm (HR 0.5164, 95% CI 0.40–0.64, p< 0.0001). Patients also had
a statistically significant improvement in mOS of 6.4 months (HR 0.64, 95% CI 0.48–0.86,
p= 0.0028
). In the T-DXd arm, OS was 23.9 months versus 17.5 months in the control arm.
Patients with HR+ disease had an overall response rate of 52.6% and a clinical benefit rate
(CBR) of 71.2%, establishing T-DXd as a possible new standard of care post-CDK4/6is [
145
],
especially in patients with rapidly progressive disease. In DESTINY-Breast04, all patients
received one or two lines of previous chemotherapy. It will be very interesting to analyze
the results of the DESTINY-Breast06 trial, which is evaluating the role of trastuzumab
deruxtecan before chemotherapy.
An exploratory analysis of CDK4/6is resistance marker signatures, performed in
DESTINY-Breast04, was recently reported [
146
]. The signature is not yet clinically validated
and includes analysis of CCND1, CCNE1, CDK6, and FGFR1/2 amplification and RB1,
PTEN, RAS, AKT1, ERBB2, and FAT1 mutations [
147
]. Notably, improved ORR for T-DXd
over TPC was observed regardless of CDK4/6is resistant markers. CDK4/6is resistance
markers were also assessed in patients with or without prior CDK4/6is therapy and a
longer median PFS was observed in the T-DXd arm compared to the TPC arm regardless of
the presence of these markers.
Sacituzumab–govitecan (SG) is an antibody–drug conjugate that targets human tro-
phoblast cell surface antigen 2 (TROP-2) and is designed to effectively deliver a chemother-
apeutic agent. It initially received FDA approval for patients with triple-negative mBC, but
TROP-2 is overexpressed also in other breast subtypes and has been studied in patients
with HR+, HER- mBC. In the multicenter phase III TROPiCs-02 study (NCT03901339),
patients with HR+/HER2
−
advanced BC were randomized 1:1 into SG versus physicians’
choice of chemotherapy. Patients were heavily pretreated because they were required
to have progressed onto a CDK4/6i and at least two chemotherapy agents, including a
taxane. The study demonstrated statistically significant improvement in PFS on SG of 5.5
months versus 4.0 months on physicians’ choice chemotherapy (HR 0.66; 95% CI, 0.53–0.83;
p= 0.0003
). Patients also had an improved overall response rate of 21% versus 14% on SG,
as well as an improved CBR of 34% versus 22% [
31
]. In the second interim analysis, an OS
benefit of 3.2 months was seen (HR 0.79, CI 0.65–0.96, p= 0.02). Patients on SG had an OS
of 14.4 months compared to 11.2 months for patients on the treatment of their physician’s
choice [
148
], suggesting an interesting role for this drug, maybe after trastuzumab derux-
tecan, considering its 6.4 months benefit in OS in the DESTINY-Breast04 trial mentioned
above (Table 3).
There are ongoing clinical trials evaluating the efficacy of chemotherapy after CDK4/6
inhibition. In particular, there is the TATEN trial (NCT04251169), investigating pem-
brolizumab plus paclitaxel in HR+/HER2
−
non-luminal (by PAM50) advanced BC af-
ter CDK4/6is progression, and a phase I study (NCT04134884) to test the safety of a
combination of ASTX727 with talazoparib in patients with triple-negative BC or HER2
−
mBC [149,150].

Int. J. Mol. Sci. 2023,24, 14427 17 of 25
Table 3. Summary of the trials focused on chemotherapies (CT).
Trial Number of
Patients Population Prior CDK4/6is Subsequent CT Efficacy PFS
(Months)
1-US study [91]525 Progression on
CDK4/6is Any Capecitabine or
Taxanes (35.6%) NA NA
2-US study [92]1210
Progression on
first-line
CDK4/6is
•Palbociclib
88.2%
•Ribociclib
7.2%
•Abemaciclib
4.6%
NA NA 3.71
Russian study
[144]54 Progression on
CDK4/6is
•Palbociclib
75.9%
•Ribociclib
22.2%
•Both 1.9%
•Eribulin
61.1%
•Others 38.9 %
•PR 24.4%
•SD 66.7% 10.0
DESTINY-
Breast04 (HR+
cohort) [145]494 Progression on
CT or OT
Any (about 70% of
pts)
T-DXd vs. TPC
(eribulin,
capecitabine,
nab-paclitaxel,
gemcitabine or
paclitaxel)
52.3% T-DXd vs.
16.3% TPC 10.1 T-DXd
vs. 5.4 TPC
TROPiCS-02 [
148
]
543
Progression after
CDK4/6is and at
least 2 chemother-
apeutic agents
(including a
taxane)
Any (98% of pts)
SG vs. TPC
(eribulin,
capecitabine,
gemcitabine or
vinorelbine)
21% SG vs. 14%
TPC 5.5 SG vs. 4.0
TPC
Abbreviation: CT = chemotherapy; OT = ormonal therapy; PR = partial response; SD = stable disease;
PFS = progression-free survival; TPC = Treatment of Physician’s Choice; T-DXd = trastuzumab deruxtecan;
SG = sacituzumab govitecan.
7. Conclusions
The widespread use of CDK4/6is in metastatic BC resulted in a question: what to do
next? There is currently no standard treatment after CDK4/6is.
Molecular mechanisms underlying CDK4/6is resistance are very complicated; thus,
their clarification is crucial in determining the next treatment plan. According to the
different resistance mechanisms, targeted drugs that could be chosen after resistance
should also be different.
Several factors are currently considered to select the best treatment after CDK4/6is: the
presence or absence of driver mutations (ESR1, PIK3CA, germline BRCA1-2), the duration
of exposure to CDK4/6is, the burden and sites of metastatic disease, comorbidities, patient
preference and the availability of clinical trials.
In ESR1 mutant patients, especially in those with prolonged exposure to prior CDK4/6is,
elacestrant seems to be the best choice.
In PI3KCA-mutated tumors, the combination of fulvestrant plus alpelisib should
be evaluated, being aware of the side effects. A less toxic alternative in this setting is
capivasertib plus fulvestrant as shown in CAPItello-291 study.
For patients with germline BRCA mutation, a PARP inhibitor could be considered
particularly as an alternative to chemotherapy. Another option, notably in patients without
driver mutations, could be everolimus plus exemestane.
Continuation of CK4/6i beyond progression remains controversial.
Finally, in patients with rapidly progressive disease, chemotherapy is the most rea-
sonable choice: nevertheless, which agent is the best remains unknown. The ongoing
DESTINY-breast06 trial will contribute to answering this question.
In third-line treatment, trastuzumab deruxtecan and sacituzumab govitecan are valid
options.

Int. J. Mol. Sci. 2023,24, 14427 18 of 25
Some novel agents (like BCL2 inhbitors, HDAC inhibitors or AKT-1 inhibitors) or
novel combinations (like immune checkpoint inhibitors with CDK4/6is) are also promising.
For these reasons, patient enrollment in clinical trials should be encouraged.
Author Contributions:
Conceptualization, F.V.; methodology, F.V., C.C. and E.C.; investigation, F.V.,
A.C., D.P., M.L., A.A. (Antonio Ardizzoia), G.S. and A.A. (Alessandra Ardizzoia); resources, F.V.,
A.C., D.P., A.A. (Alessandra Ardizzoia) and G.S.; data curation, F.V., A.C., D.P., M.L., A.A. (Antonio
Ardizzoia), G.S., C.C., E.C. and A.A. (Alessandra Ardizzoia); writing—original draft preparation,
F.V., A.C., D.P., A.A. (Alessandra Ardizzoia) and G.S.; writing—review and editing, all authors. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors would like to acknowledge Gabriella Di Luca for linguistic revision.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global Cancer Statistics 2018: GLOBOCAN Estimates of
Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2018,68, 394–424. [CrossRef] [PubMed]
2.
Cardoso, F.; Costa, A.; Norton, L.; Cameron, D.; Cufer, T.; Fallowfield, L.; Francis, P.; Gligorov, J.; Kyriakides, S.; Lin, N.; et al. 1st
International Consensus Guidelines for Advanced Breast Cancer (ABC 1). Breast 2012,21, 242–252. [CrossRef] [PubMed]
3.
O’Shaughnessy, J. Extending Survival with Chemotherapy in Metastatic Breast Cancer. Oncologist
2005
,10 (Suppl. S3), 20–29.
[CrossRef]
4.
Bradley, R.; Burrett, J.; Clarke, M.; Davies, C.; Duane, F.; Evans, V.; Gettins, L.; Godwin, J.; Gray, R.; Liu, H.; et al. Aromatase
Inhibitors versus Tamoxifen in Early Breast Cancer: Patient-Level Meta-Analysis of the Randomised Trials. Lancet
2015
,386,
1341–1352. [CrossRef]
5.
Lobbezoo, D.J.A.; Van Kampen, R.J.W.; Voogd, A.C.; Dercksen, M.W.; Van Den Berkmortel, F.; Smilde, T.J.; Van De Wouw,
A.J.; Peters, F.P.J.; Van Riel, J.M.G.H.; Peters, N.A.J.B.; et al. Prognosis of Metastatic Breast Cancer Subtypes: The Hormone
Receptor/HER2-Positive Subtype Is Associated with the Most Favorable Outcome. Breast Cancer Res. Treat.
2013
,141, 507–514.
[CrossRef]
6.
Caswell-Jin, J.L.; Plevritis, S.K.; Tian, L.; Cadham, C.J.; Xu, C.; Stout, N.K.; Sledge, G.W.; Mandelblatt, J.S.; Kurian, A.W. Change in
Survival in Metastatic Breast Cancer with Treatment Advances: Meta-Analysis and Systematic Review. JNCI Cancer Spectr.
2018
,
2, pky062. [CrossRef]
7.
Carlson, R.W.; Allred, D.C.; Anderson, B.O.; Burstein, H.J.; Edge, S.B.; Farrar, W.B.; Forero, A.; Giordano, S.H.; Goldstein, L.J.;
Gradishar, W.J.; et al. Metastatic Breast Cancer, Version 1.2012: Featured Updates to the NCCN Guidelines. J. Natl. Compr. Cancer
Netw. 2012,10, 821–829. [CrossRef] [PubMed]
8.
VanArsdale, T.; Boshoff, C.; Arndt, K.T.; Abraham, R.T. Molecular Pathways: Targeting the Cyclin D-CDK4/6 Axis for Cancer
Treatment. Clin. Cancer Res. 2015,21, 2905–2910. [CrossRef] [PubMed]
9.
Finn, R.S.; Martin, M.; Rugo, H.S.; Jones, S.; Im, S.-A.; Gelmon, K.; Harbeck, N.; Lipatov, O.N.; Walshe, J.M.; Moulder, S.; et al.
Palbociclib and Letrozole in Advanced Breast Cancer. N. Engl. J. Med. 2016,375, 1925–1936. [CrossRef]
10.
Hortobagyi, G.N.; Stemmer, S.M.; Burris, H.A.; Yap, Y.-S.; Sonke, G.S.; Paluch-Shimon, S.; Campone, M.; Blackwell, K.L.; André, F.;
Winer, E.P.; et al. Ribociclib as First-Line Therapy for HR-Positive, Advanced Breast Cancer. N. Engl. J. Med.
2016
,375, 1738–1748.
[CrossRef]
11.
Goetz, M.P.; Toi, M.; Campone, M.; Trédan, O.; Bourayou, N.; Sohn, J.; Park, I.H.; Paluch-Shimon, S.; Huober, J.; Chen, S.C.; et al.
MONARCH 3: Abemaciclib as Initial Therapy for Advanced Breast Cancer. J. Clin. Oncol. 2017,35, 3638–3646. [CrossRef]
12.
Im, S.-A.; Lu, Y.-S.; Bardia, A.; Harbeck, N.; Colleoni, M.; Franke, F.; Chow, L.; Sohn, J.; Lee, K.-S.; Campos-Gomez, S.; et al.
Overall Survival with Ribociclib plus Endocrine Therapy in Breast Cancer. N. Engl. J. Med. 2019,381, 307–316. [CrossRef]
13.
Hortobagyi, G.N.; Stemmer, S.M.; Burris, H.A.; Yap, Y.-S.; Sonke, G.S.; Hart, L.; Campone, M.; Petrakova, K.; Winer, E.P.; Janni, W.;
et al. Overall Survival with Ribociclib plus Letrozole in Advanced Breast Cancer. N. Engl. J. Med.
2022
,386, 942–950. [CrossRef]
14.
Goetz, M.P.; Toi, M.; Huober, J.; Sohn, J.; Tredan, O.; Park, I.H.; Campone, M.; Chen, S.C.; Sanchez, L.M.M.; Paluch-Shimon, S.;
et al. LBA15—MONARCH 3: Interim Overall Survival (OS) Results of Abemaciclib plus a Nonsteroidal Aromatase Inhibitor
(NSAI) in Patients (Pts) with HR+, HER2-Advanced Breast Cancer (ABC). Ann. Oncol. 2022,33, S1384. [CrossRef]

Int. J. Mol. Sci. 2023,24, 14427 19 of 25
15.
Finn, R.S.; Rugo, H.S.; Dieras, V.C.; Harbeck, N.; Im, S.-A.; Gelmon, K.A.; Walshe, J.M.; Martin, M.; Chavez Mac Gregor, M.;
Bananis, E.; et al. Overall Survival (OS) with First-Line Palbociclib plus Letrozole (PAL+LET) versus Placebo plus Letrozole
(PBO+LET) in Women with Estrogen Receptor–Positive/Human Epidermal Growth Factor Receptor 2–Negative Advanced
Breast Cancer (ER+/HER2−ABC): Analyses from PALOMA-2. J. Clin. Oncol. 2022,40, LBA1003. [CrossRef]
16.
Bockstaele, L.; Bisteau, X.; Paternot, S.; Roger, P.P. Differential Regulation of Cyclin-Dependent Kinase 4 (CDK4) and CDK6,
Evidence That CDK4 Might Not Be Activated by CDK7, and Design of a CDK6 Activating Mutation. Mol. Cell. Biol.
2009
,29,
4188–4200. [CrossRef] [PubMed]
17.
Fassl, A.; Geng, Y.; Sicinski, P. CDK4 and CDK6 Kinases: From Basic Science to Cancer Therapy. Science
2022
,375, eabc1495.
[CrossRef] [PubMed]
18. Diehl, J.A. Cycling to Cancer with Cyclin D1. Cancer Biol. Ther. 2002,1, 226–231. [CrossRef] [PubMed]
19. Hamilton, E.; Infante, J.R. Targeting CDK4/6 in Patients with Cancer. Cancer Treat. Rev. 2016,45, 129–138. [CrossRef]
20.
Malumbres, M.; Sotillo, R.; Santamaría, D.; Galán, J.; Cerezo, A.; Ortega, S.; Dubus, P.; Barbacid, M. Mammalian Cells Cycle
without the D-Type Cyclin-Dependent Kinases Cdk4 and Cdk6. Cell 2004,118, 493–504. [CrossRef]
21.
O’Leary, B.; Finn, R.S.; Turner, N.C. Treating Cancer with Selective CDK4/6 Inhibitors. Nat. Rev. Clin. Oncol.
2016
,13, 417–430.
[CrossRef] [PubMed]
22.
Finn, R.S.; Crown, J.P.; Lang, I.; Boer, K.; Bondarenko, I.M.; Kulyk, S.O.; Ettl, J.; Patel, R.; Pinter, T.; Schmidt, M.; et al. The
Cyclin-Dependent Kinase 4/6 Inhibitor Palbociclib in Combination with Letrozole versus Letrozole Alone as First-Line Treatment
of Oestrogen Receptor-Positive, HER2-Negative, Advanced Breast Cancer (PALOMA-1/TRIO-18): A Randomised Phase 2 Study.
Lancet Oncol. 2015,16, 25–35. [CrossRef] [PubMed]
23.
Huang, J.; Zheng, L.; Sun, Z.; Li, J. CDK4/6 Inhibitor Resistance Mechanisms and Treatment Strategies (Review). Int. J. Mol. Med.
2022,50, 128. [CrossRef] [PubMed]
24.
Shapiro, G.I.; Edwards, C.D.; Kobzik, L.; Godleski, J.; Richards, W.; Sugarbaker, D.J.; Rollins, B.J. Reciprocal Rb Inactivation and
P16INK4 Expression in Primary Lung Cancers and Cell Lines. Cancer Res. 1995,55, 505–509.
25.
Lukas, J.; Parry, D.; Aagaard, L.; Mann, D.J.; Bartkova, J.; Strauss, M.; Peters, G.; Bartek, J. Retinoblastoma-Protein-Dependent
Cell-Cycle Inhibition by the Tumour Suppressor P16. Nature 1995,375, 503–506. [CrossRef]
26.
Dean, J.L.; McClendon, A.K.; Hickey, T.E.; Butler, L.M.; Tilley, W.D.; Witkiewicz, A.K.; Knudsen, E.S. Therapeutic Response to
CDK4/6 Inhibition in Breast Cancer Defined by Ex Vivo Analyses of Human Tumors. Cell Cycle 2012,11, 2756–2761. [CrossRef]
27.
Palafox, M.; Monserrat, L.; Bellet, M.; Villacampa, G.; Gonzalez-Perez, A.; Oliveira, M.; Brasó-Maristany, F.; Ibrahimi, N.; Kannan,
S.; Mina, L.; et al. High P16 Expression and Heterozygous RB1 Loss Are Biomarkers for CDK4/6 Inhibitor Resistance in ER+
Breast Cancer. Nat. Commun. 2022,13, 5258. [CrossRef]
28.
Gladden, A.B.; Diehl, J.A. Cell Cycle Progression without Cyclin E/CDK2: Breaking down the Walls of Dogma. Cancer Cell
2003
,
4, 160–162. [CrossRef]
29.
Etemadmoghadam, D.; Au-Yeung, G.; Wall, M.; Mitchell, C.; Kansara, M.; Loehrer, E.; Batzios, C.; George, J.; Ftouni, S.; Weir, B.A.;
et al. Resistance to CDK2 Inhibitors Is Associated with Selection of Polyploid Cells in CCNE1-Amplified Ovarian Cancer. Clin.
Cancer Res. 2013,19, 5960–5971. [CrossRef]
30.
Herrera-Abreu, M.T.; Palafox, M.; Asghar, U.; Rivas, M.A.; Cutts, R.J.; Garcia-Murillas, I.; Pearson, A.; Guzman, M.; Rodriguez,
O.; Grueso, J.; et al. Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer.
Cancer Res. 2016,76, 2301–2313. [CrossRef]
31.
Jin, X.; Ge, L.P.; Li, D.Q.; Shao, Z.M.; Di, G.H.; Xu, X.E.; Jiang, Y.Z. LncRNA TROJAN Promotes Proliferation and Resistance to
CDK4/6 Inhibitor via CDK2 Transcriptional Activation in ER+ Breast Cancer. Mol. Cancer 2020,19, 87. [CrossRef] [PubMed]
32.
Schachter, M.M.; Merrick, K.A.; Larochelle, S.; Hirschi, A.; Zhang, C.; Shokat, K.M.; Rubin, S.M.; Fisher, R.P. A Cdk7-Cdk4 T-Loop
Phosphorylation Cascade Promotes G1 Progression. Mol. Cell 2013,50, 250–260. [CrossRef] [PubMed]
33.
Martin, L.-A.; Pancholi, S.; Ribas, R.; Gao, Q.; Simigdala, N.; Nikitorowicz-Buniak, J.; Johnston, S.; Dowsett, M. Abstract P3-03-09:
Resistance to Palbociclib Depends on Multiple Targetable Mechanisms Highlighting the Potential of Drug Holidays and Drug
Switching to Improve Therapeutic Outcome. Cancer Res. 2017,77 (Suppl. S4). [CrossRef]
34.
Tigan, A.S.; Bellutti, F.; Kollmann, K.; Tebb, G.; Sexl, V. CDK6-a Review of the Past and a Glimpse into the Future: From Cell-Cycle
Control to Transcriptional Regulation. Oncogene 2016,35, 3083–3091. [CrossRef] [PubMed]
35.
Kollmann, K.; Heller, G.; Schneckenleithner, C.; Warsch, W.; Scheicher, R.; Ott, R.G.; Schäfer, M.; Fajmann, S.; Schlederer, M.;
Schiefer, A.I.; et al. A Kinase-Independent Function of CDK6 Links the Cell Cycle to Tumor Angiogenesis. Cancer Cell
2016
,30,
359–360. [CrossRef]
36. Matheson, C.J.; Backos, D.S.; Reigan, P. Targeting WEE1 Kinase in Cancer. Trends Pharmacol. Sci. 2016,37, 872–881. [CrossRef]
37.
Efeyan, A.; Ortega-Molina, A.; Velasco-Miguel, S.; Herranz, D.; Vassilev, L.T.; Serrano, M. Induction of P53-Dependent Senescence
by the MDM2 Antagonist Nutlin-3a in Mouse Cells of Fibroblast Origin. Cancer Res. 2007,67, 7350–7357. [CrossRef]
38.
Cox, L.S. Multiple Pathways Control Cell Growth and Transformation: Overlapping and Independent Activities of P53 and
P21Cip1/Waf1/Sdi1.J. Pathol 1997,183, 134–140. [CrossRef]
39.
Portman, N.; Chen, J.; Lim, E. MDM2 as a Rational Target for Intervention in CDK4/6 Inhibitor Resistant, Hormone Receptor
Positive Breast Cancer. Front. Oncol. 2021,11, 777867. [CrossRef]

Int. J. Mol. Sci. 2023,24, 14427 20 of 25
40.
Sabnis, G.J.; Goloubeva, O.; Chumsri, S.; Nguyen, N.; Sukumar, S.; Brodie, A.M.H. Functional Activation of the Estrogen
Receptor-
α
and Aromatase by the HDAC Inhibitor Entinostat Sensitizes ER-Negative Tumors to Letrozole. Cancer Res.
2011
,71,
1893–1903. [CrossRef]
41.
Dash, B.C.; El-Deiry, W.S. Phosphorylation of P21 in G2/M Promotes Cyclin B-Cdc2 Kinase Activity. Mol. Cell. Biol.
2005
,25,
3364–3387. [CrossRef] [PubMed]
42.
Zhou, Y.; Jin, X.; Ma, J.; Ding, D.; Huang, Z.; Sheng, H.; Yan, Y.; Pan, Y.; Wei, T.; Wang, L.; et al. HDAC5 Loss Impairs RB
Repression of Pro-Oncogenic Genes and Confers CDK4/6 Inhibitor Resistance in Cancer. Cancer Res.
2021
,81, 1486–1499.
[CrossRef] [PubMed]
43.
Ramanujan, A.; Tiwari, S. APC/C and Retinoblastoma Interaction: Cross-Talk of Retinoblastoma Protein with the Ubiquitin
Proteasome Pathway. Biosci. Rep. 2016,36, e00377. [CrossRef] [PubMed]
44.
The, I.; Ruijtenberg, S.; Bouchet, B.P.; Cristobal, A.; Prinsen, M.B.W.; Van Mourik, T.; Koreth, J.; Xu, H.; Heck, A.J.R.; Akhmanova,
A.; et al. Rb and FZR1/Cdh1 Determine CDK4/6-Cyclin D Requirement in C. Elegans and Human Cancer Cells. Nat. Commun.
2015,6, 5906. [CrossRef] [PubMed]
45.
Fujita, T.; Liu, W.; Doihara, H.; Wan, Y. Regulation of Skp2-P27 Axis by the Cdh1/Anaphase-Promoting Complex Pathway in
Colorectal Tumorigenesis. Am. J. Pathol. 2008,173, 217–228. [CrossRef]
46.
He, Q.; Zou, L.; Zhang, P.A.; Lui, J.X.; Skog, S.; Fornander, T. The Clinical Significance of Thymidine Kinase 1 Measurement in
Serum of Breast Cancer Patients Using Anti-TK1 Antibody. Int. J. Biol. Markers 2000,15, 139–146. [CrossRef]
47.
Bjöhle, J.; Bergqvist, J.; Gronowitz, J.S.; Johansson, H.; Carlsson, L.; Einbeigi, Z.; Linderholm, B.; Loman, N.; Malmberg, M.;
Söderberg, M.; et al. Serum Thymidine Kinase Activity Compared with CA 15-3 in Locally Advanced and Metastatic Breast
Cancer within a Randomized Trial. Breast Cancer Res. Treat. 2013,139, 751–758. [CrossRef]
48.
Del Re, M.; Bertolini, I.; Crucitta, S.; Fontanelli, L.; Rofi, E.; De Angelis, C.; Diodati, L.; Cavallero, D.; Gianfilippo, G.; Salvadori, B.;
et al. Overexpression of TK1 and CDK9 in Plasma-Derived Exosomes Is Associated with Clinical Resistance to CDK4/6 Inhibitors
in Metastatic Breast Cancer Patients. Breast Cancer Res. Treat. 2019,178, 57–62. [CrossRef]
49.
Lin, S.L.; Chang, D.C.; Ying, S.Y.; Leu, D.; Wu, D.T.S. MicroRNA MiR-302 Inhibits the Tumorigenecity of Human Pluripotent Stem
Cells by Coordinate Suppression of the CDK2 and CDK4/6 Cell Cycle Pathways. Cancer Res. 2010,70, 9473–9482. [CrossRef]
50.
Pulito, C.; Cristaudo, A.; La Porta, C.; Zapperi, S.; Blandino, G.; Morrone, A.; Strano, S. Oral Mucositis: The Hidden Side of
Cancer Therapy. J. Exp. Clin. Cancer Res. 2020,39, 210. [CrossRef]
51.
Cornell, L.; Wander, S.A.; Visal, T.; Wagle, N.; Shapiro, G.I. MicroRNA-Mediated Suppression of the TGF-
β
Pathway Confers
Transmissible and Reversible CDK4/6 Inhibitor Resistance. Cell Rep. 2019,26, 2667–2680.e7. [CrossRef]
52.
Ji, W.; Zhang, W.; Wang, X.; Shi, Y.; Yang, F.; Xie, H.; Zhou, W.; Wang, S.; Guan, X. C-Myc Regulates the Sensitivity of Breast
Cancer Cells to Palbociclib via c-Myc/MiR-29b-3p/CDK6 Axis. Cell Death Dis. 2020,11, 760. [CrossRef] [PubMed]
53.
Fingar, D.C.; Blenis, J. Target of Rapamycin (TOR): An Integrator of Nutrient and Growth Factor Signals and Coordinator of Cell
Growth and Cell Cycle Progression. Oncogene 2004,23, 3151–3171. [CrossRef] [PubMed]
54.
Mo, H.; Liu, X.; Xue, Y.; Chen, H.; Guo, S.; Li, Z.; Wang, S.; Li, C.; Han, J.; Fu, M.; et al. S6K1 Amplification Confers Innate
Resistance to CDK4/6 Inhibitors through Activating c-Myc Pathway in Patients with Estrogen Receptor-Positive Breast Cancer.
Mol. Cancer 2022,21, 171. [CrossRef] [PubMed]
55.
Turner, N.; Grose, R. Fibroblast Growth Factor Signalling: From Development to Cancer. Nat. Rev. Cancer
2010
,10, 116–129.
[CrossRef]
56.
Luqmani, Y.A.; Graham, M.; Coombes, R.C. Expression of Basic Fibroblast Growth Factor, FGFR1 and FGFR2 in Normal and
Malignant Human Breast, and Comparison with Other Normal Tissues. Br. J. Cancer 1992,66, 273–280. [CrossRef]
57.
Turner, N.; Pearson, A.; Sharpe, R.; Lambros, M.; Geyer, F.; Lopez-Garcia, M.A.; Natrajan, R.; Marchio, C.; Iorns, E.; Mackay, A.;
et al. FGFR1 Amplification Drives Endocrine Therapy Resistance and Is a Therapeutic Target in Breast Cancer. Cancer Res.
2010
,
70, 2085–2094. [CrossRef]
58.
Sobhani, N.; Fassl, A.; Mondani, G.; Generali, D.; Otto, T. Targeting Aberrant FGFR Signaling to Overcome CDK4/6 Inhibitor
Resistance in Breast Cancer. Cells 2021,10, 293. [CrossRef]
59.
Mao, P.; Kusiel, J.; Cohen, O.; Wagle, N. Abstract PD4-01: The Role of FGF/FGFR Axis in Resistance to SERDs and CDK4/6
Inhibitors in ER+ Breast Cancer. Cancer Res. 2018,78 (Suppl. S4), PD4-01. [CrossRef]
60.
Jansen, V.M.; Bhola, N.E.; Bauer, J.A.; Formisano, L.; Lee, K.M.; Hutchinson, K.E.; Witkiewicz, A.K.; Moore, P.D.; Estrada, M.V.;
Sánchez, V.; et al. Kinome-Wide RNA Interference Screen Reveals a Role for PDK1 in Acquired Resistance to CDK4/6 Inhibition
in ER-Positive Breast Cancer. Cancer Res. 2017,77, 2488–2499. [CrossRef]
61.
Michaloglou, C.; Crafter, C.; Siersbaek, R.; Delpuech, O.; Curwen, J.O.; Carnevalli, L.S.; Staniszewska, A.D.; Polanska, U.M.;
Cheraghchi-Bashi, A.; Lawson, M.; et al. Combined Inhibition of MTOR and CDK4/6 Is Required for Optimal Blockade of E2F
Function and Long-Term Growth Inhibition in Estrogen Receptor-Positive Breast Cancer. Mol. Cancer Ther.
2018
,17, 908–920.
[CrossRef] [PubMed]
62.
Costa, C.; Ye, W.; Ly, A.; Hosono, Y.; Murchi, E.; Walmsley, C.S.; Huynh, T.; Healy, C.; Peterson, R.; Yanase, S.; et al. PTEN Loss
Mediates Clinical Cross-Resistance to CDK4/6 and PI3K
α
Inhibitors in Breast Cancer. Cancer Discov.
2020
,10, 72–85. [CrossRef]
[PubMed]
63.
Li, Q.; Jiang, B.; Guo, J.; Shao, H.; Del Priore, I.S.; Chang, Q.; Kudo, R.; Li, Z.; Razavi, P.; Liu, B.; et al. INK4 Tumor Suppressor
Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors. Cancer Discov. 2022,12, 356–371. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2023,24, 14427 21 of 25
64.
Formisano, L.; Lu, Y.; Servetto, A.; Hanker, A.B.; Jansen, V.M.; Bauer, J.A.; Sudhan, D.R.; Guerrero-Zotano, A.L.; Croessmann, S.;
Guo, Y.; et al. Aberrant FGFR Signaling Mediates Resistance to CDK4/6 Inhibitors in ER+ Breast Cancer. Nat. Commun.
2019
,10,
1373. [CrossRef] [PubMed]
65.
Raimondi, L.; Raimondi, F.M.; Pietranera, M.; Di Rocco, A.; Di Benedetto, L.; Miele, E.; Lazzeroni, R.; Cimino, G.; Spinelli, G.P.
Assessment of Resistance Mechanisms and Clinical Implications in Patients with KRAS Mutated-Metastatic Breast Cancer and
Resistance to CDK4/6 Inhibitors. Cancers 2021,13, 1928. [CrossRef]
66.
Dey, A.; Varelas, X.; Guan, K.L. Targeting the Hippo Pathway in Cancer, Fibrosis, Wound Healing and Regenerative Medicine.
Nat. Rev. Drug Discov. 2020,19, 480–494. [CrossRef]
67.
Finn, R.S.; Crown, J.P.; Ettl, J.; Schmidt, M.; Bondarenko, I.M.; Lang, I.; Pinter, T.; Boer, K.; Patel, R.; Randolph, S.; et al. Efficacy
and Safety of Palbociclib in Combination with Letrozole as First-Line Treatment of ER-Positive, HER2-Negative, Advanced Breast
Cancer: Expanded Analyses of Subgroups from the Randomized Pivotal Trial PALOMA-1/TRIO-18. Breast Cancer Res.
2016
,18,
67. [CrossRef]
68.
Li, Z.; Razavi, P.; Li, Q.; Toy, W.; Liu, B.; Ping, C.; Hsieh, W.; Sanchez-Vega, F.; Brown, D.N.; Da Cruz Paula, A.F.; et al. Loss of
the FAT1 Tumor Suppressor Promotes Resistance to CDK4/6 Inhibitors via the Hippo Pathway. Cancer Cell
2018
,34, 893–905.e8.
[CrossRef]
69.
Bae, S.N.; Arand, G.; Azzam, H.; Pavasant, P.; Torri, J.; Frandsen, T.L.; Thompson, E.W. Molecular and Cellular Analysis of
Basement Membrane Invasion by Human Breast Cancer Cells in Matrigel-Based in Vitro Assays. Breast Cancer Res. Treat.
1993
,24,
241–255. [CrossRef]
70.
Lamouille, S.; Xu, J.; Derynck, R. Molecular Mechanisms of Epithelial-Mesenchymal Transition. Nat. Rev. Mol. Cell Biol.
2014
,15,
178–196. [CrossRef]
71.
Zelivianski, S.; Cooley, A.; Kall, R.; Jeruss, J.S. Cyclin-Dependent Kinase 4-Mediated Phosphorylation Inhibits Smad3 Activity in
Cyclin D-Overexpressing Breast Cancer Cells. Mol. Cancer Res. 2010,8, 1375–1387. [CrossRef]
72.
Yang, J.; Song, K.; Krebs, T.L.; Jackson, M.W.; Danielpour, D. Rb/E2F4 and Smad2/3 Link Survivin to TGF-Beta-Induced Apoptosis
and Tumor Progression. Oncogene 2008,27, 5326–5338. [CrossRef]
73.
Du, B.; Shim, J.S. Targeting Epithelial-Mesenchymal Transition (EMT) to Overcome Drug Resistance in Cancer. Molecules
2016
,21,
965. [CrossRef] [PubMed]
74.
Geller, C.; Maddela, J.; Tuplano, R.; Runa, F.; Adamian, Y.; Güth, R.; Soto, G.O.; Tomaneng, L.; Cantor, J.; Kelber, J.A. Fibronectin,
DHPS and SLC3A2 Signaling Cooperate to Control Tumor Spheroid Growth, Subcellular EIF5A1/2 Distribution and CDK4/6
Inhibitor Resistance. bioRxiv 2023. [CrossRef]
75.
Johnston, S.; Puhalla, S.; Wheatley, D.; Ring, A.; Barry, P.; Holcombe, C.; Boileau, J.F.; Provencher, L.; Robidoux, A.; Rimawi,
M.; et al. Randomized Phase II Study Evaluating Palbociclib in Addition to Letrozole as Neoadjuvant Therapy in Estrogen
Receptor-Positive Early Breast Cancer: PALLET Trial. J. Clin. Oncol. 2019,37, 178–189. [CrossRef] [PubMed]
76.
Whittle, J.R.; Vaillant, F.; Surgenor, E.; Policheni, A.N.; Giner, G.; Capaldo, B.D.; Chen, H.R.; Liu, H.K.; Dekkers, J.F.; Sachs, N.;
et al. Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor-Positive Breast Cancer.
Clin. Cancer Res. 2020,26, 4120–4134. [CrossRef]
77.
Zeng, X.; Liu, C.; Yao, J.; Wan, H.; Wan, G.; Li, Y.; Chen, N. Breast Cancer Stem Cells, Heterogeneity, Targeting Therapies and
Therapeutic Implications. Pharmacol. Res. 2021,163, 105320. [CrossRef]
78.
Wang, S.; Bei, Y.; Tian, Q.; He, J.; Wang, R.; Wang, Q.; Sun, L.; Ke, J.; Xie, C.; Shen, P. PFKFB4 Facilitates Palbociclib Resistance in
Oestrogen Receptor-Positive Breast Cancer by Enhancing Stemness. Cell Prolif. 2023,56, e13337. [CrossRef]
79.
Cetin, B.; Wabl, C.A.; Gumusay, O. CDK4/6 Inhibitors: Mechanisms of Resistance and Potential Biomarkers of Responsiveness in
Breast Cancer. Future Oncol. 2022,18, 1143–1157. [CrossRef]
80.
Guan, X.; LaPak, K.M.; Hennessey, R.C.; Yu, C.Y.; Shakya, R.; Zhang, J.; Burd, C.E. Stromal Senescence By Prolonged CDK4/6
Inhibition Potentiates Tumor Growth. Mol. Cancer Res. 2017,15, 237–249. [CrossRef]
81.
Xu, B.; Krie, A.; De, P.; Williams, C.; Elsey, R.; Klein, J.; Leyland-Jones, B. Utilizing Tumor and Plasma Liquid Biopsy in Treatment
Decision Making for an Estrogen Receptor-Positive Advanced Breast Cancer Patient. Cureus
2017
,9, e1408. [CrossRef] [PubMed]
82.
Condorelli, R.; Spring, L.; O’Shaughnessy, J.; Lacroix, L.; Bailleux, C.; Scott, V.; Dubois, J.; Nagy, R.J.; Lanman, R.B.; Iafrate, A.J.;
et al. Polyclonal RB1 Mutations and Acquired Resistance to CDK 4/6 Inhibitors in Patients with Metastatic Breast Cancer. Ann.
Oncol. 2018,29, 640–645. [CrossRef] [PubMed]
83.
Yang, C.; Li, Z.; Bhatt, T.; Dickler, M.; Giri, D.; Scaltriti, M.; Baselga, J.; Rosen, N.; Chandarlapaty, S. Acquired CDK6 Amplification
Promotes Breast Cancer Resistance to CDK4/6 Inhibitors and Loss of ER Signaling and Dependence. Oncogene
2017
,36, 2255–2264.
[CrossRef] [PubMed]
84.
Gacche, R.N.; Assaraf, Y.G. Redundant Angiogenic Signaling and Tumor Drug Resistance. Drug Resist. Updat.
2018
,36, 47–76.
[CrossRef]
85.
Caldon, C.E.; Sergio, C.M.; Schütte, J.; Boersma, M.N.; Sutherland, R.L.; Carroll, J.S.; Musgrove, E.A. Estrogen Regulation of
Cyclin E2 Requires Cyclin D1 but Not C-Myc. Mol. Cell. Biol. 2009,29, 4623–4639. [CrossRef]
86.
Papadimitriou, M.C.; Pazaiti, A.; Iliakopoulos, K.; Markouli, M.; Michalaki, V.; Papadimitriou, C.A. Resistance to CDK4/6
Inhibition: Mechanisms and Strategies to Overcome a Therapeutic Problem in the Treatment of Hormone Receptor-Positive
Metastatic Breast Cancer. Biochim. Biophys. Acta Mol. Cell Res. 2022,1869, 119346. [CrossRef]

Int. J. Mol. Sci. 2023,24, 14427 22 of 25
87.
Tripathy, D.; Im, S.A.; Colleoni, M.; Franke, F.; Bardia, A.; Harbeck, N.; Hurvitz, S.A.; Chow, L.; Sohn, J.; Lee, K.S.; et al. Ribociclib
plus Endocrine Therapy for Premenopausal Women with Hormone-Receptor-Positive, Advanced Breast Cancer (MONALEESA-7):
A Randomised Phase 3 Trial. Lancet Oncol. 2018,19, 904–915. [CrossRef]
88.
Sledge, G.W.; Toi, M.; Neven, P.; Sohn, J.; Inoue, K.; Pivot, X.; Burdaeva, O.; Okera, M.; Masuda, N.; Kaufman, P.A.; et al.
MONARCH 2: Abemaciclib in Combination with Fulvestrant in Women with HR+/HER2
−
Advanced Breast Cancer Who Had
Progressed While Receiving Endocrine Therapy. J. Clin. Oncol. 2017,35, 2875–2884. [CrossRef]
89.
Cristofanilli, M.; Turner, N.C.; Bondarenko, I.; Ro, J.; Im, S.A.; Masuda, N.; Colleoni, M.; DeMichele, A.; Loi, S.; Verma, S.;
et al. Fulvestrant plus Palbociclib versus Fulvestrant plus Placebo for Treatment of Hormone-Receptor-Positive, HER2-Negative
Metastatic Breast Cancer That Progressed on Previous Endocrine Therapy (PALOMA-3): Final Analysis of the Multicentre,
Double-Blind, Phase 3 Randomised Controlled Trial. Lancet Oncol. 2016,17, 425–439. [CrossRef]
90.
Slamon, D.J.; Neven, P.; Chia, S.; Fasching, P.A.; De Laurentiis, M.; Im, S.A.; Petrakova, K.; Val Bianchi, G.; Esteva, F.J.; Martín, M.;
et al. Phase III Randomized Study of Ribociclib and Fulvestrant in Hormone Receptor-Positive, Human Epidermal Growth Factor
Receptor 2-Negative Advanced Breast Cancer: MONALEESA-3. J. Clin. Oncol. 2018,36, 2465–2472. [CrossRef]
91.
Princic, N.; Aizer, A.; Tang, D.H.; Smith, D.M.; Johnson, W.; Bardia, A. Predictors of Systemic Therapy Sequences Following a
CDK 4/6 Inhibitor-Based Regimen in Post-Menopausal Women with Hormone Receptor Positive, HEGFR-2 Negative Metastatic
Breast Cancer. Curr. Med. Res. Opin. 2019,35, 73–80. [CrossRef] [PubMed]
92.
Martin, J.M.; Handorf, E.A.; Montero, A.J.; Goldstein, L.J. Systemic Therapies Following Progression on First-Line CDK4/6-
Inhibitor Treatment: Analysis of Real-World Data. Oncologist 2022,27, 441–446. [CrossRef] [PubMed]
93.
Li, Y.; Li, W.; Gong, C.; Zheng, Y.; Ouyang, Q.; Xie, N.; Qu, Q.; Ge, R.; Wang, B. A Multicenter Analysis of Treatment Patterns and
Clinical Outcomes of Subsequent Therapies after Progression on Palbociclib in HR+/HER2
−
Metastatic Breast Cancer. Ther. Adv.
Med Oncol. 2021,13, 1–11. [CrossRef] [PubMed]
94.
Bashour, S.I.; Doostan, I.; Keyomarsi, K.; Valero, V.; Ueno, N.T.; Brown, P.H.; Litton, J.K.; Koenig, K.B.; Karuturi, M.; Abouharb, S.;
et al. Rapid Breast Cancer Disease Progression Following Cyclin Dependent Kinase 4 and 6 Inhibitor Discontinuation. J. Cancer
2017,8, 2004–2009. [CrossRef] [PubMed]
95.
West, M.; Kaempf, A.; Goodyear, S.; Kartika, T.; Ribkoff, J.; Mitri, Z.I. Real-World Analysis of Disease Progression after CDK 4/6
Inhibitor (CDKi) Therapy in Patients with Hormone Receptor Positive (HR+)/HER2
−
Metastatic Breast Cancer (MBC). J. Clin.
Oncol. 2021,39, e13030. [CrossRef]
96.
Evaluation of Anticancer Medicinal Products in Man—Scientific Guideline | European Medicines Agency. Available online:
https://www.ema.europa.eu/en/evaluation-anticancer-medicinal-products-man-scientific- guideline (accessed on 11 July 2023).
97.
Munzone, E.; Pagan, E.; Bagnardi, V.; Montagna, E.; Cancello, G.; Dellapasqua, S.; Iorfida, M.; Mazza, M.; Colleoni, M. Systematic
Review and Meta-Analysis of Post-Progression Outcomes in ER+/HER2
−
Metastatic Breast Cancer after CDK4/6 Inhibitors
within Randomized Clinical Trials. ESMO Open 2021,6, 100332. [CrossRef]
98.
Mariotti, V.; Khong, H.T.; Soliman, H.H.; Costa, R.L.; Fisher, S.; Boulware, D.; Han, H.S. Efficacy of Abemaciclib (Abema) after
Palbociclib (Palbo) in Patients (Pts) with Metastatic Breast Cancer (MBC). J. Clin. Oncol. 2019,37, e12521. [CrossRef]
99.
Eziokwu, A.S.; Varella, L.; Kruse, M.L.; Jia, X.; Moore, H.C.F.; Budd, G.T.; Abraham, J.; Montero, A.J. Real-World Evidence
Evaluating Continuation of CDK4/6 Inhibitors beyond First Progression in Hormone Receptor-Positive (HR+) Metastatic Breast
Cancer. J. Clin. Oncol. 2019,37, e12538. [CrossRef]
100.
Tamragouri, K.; Cobleigh, M.A.; Rao, R.D. Abemaciclib with or without Fulvestrant for the Treatment of Hormone Receptor-
Positive and HER2-Negative Metastatic Breast Cancer with Disease Progression Following Prior Treatment with Palbociclib. J.
Clin. Oncol. 2019,37, e12533. [CrossRef]
101.
dos Anjos, C.H.; Razavi, P.; Herbert, J.; Colon, J.; Gill, K.; Modi, S.; Bromberg, J.; Dang, C.T.; Liu, D.; Norton, L.; et al. A Large
Retrospective Analysis of CDK 4/6 Inhibitor Retreatment in ER+ Metastatic Breast Cancer (MBC). J. Clin. Oncol.
2019
,37, 1053.
[CrossRef]
102.
Wander, S.A.; Zangardi, M.; Niemierko, A.; Kambadakone, A.; Kim, L.S.; Xi, J.; Pandey, A.K.; Spring, L.; Stein, C.; Juric, D.;
et al. A Multicenter Analysis of Abemaciclib after Progression on Palbociclib in Patients (Pts) with Hormone Receptor-Positive
(HR+)/HER2−Metastatic Breast Cancer (MBC). J. Clin. Oncol. 2019,37, 1057. [CrossRef]
103.
Kalinsky, K.; Accordino, M.K.; Chiuzan, C.; Mundi, P.S.; Sakach, E.; Sathe, C.; Ahn, H.; Trivedi, M.S.; Novik, Y.; Tiersten, A.; et al.
Randomized Phase II Trial of Endocrine Therapy with or without Ribociclib After Progression on Cyclin-Dependent Kinase
4/6 Inhibition in Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Metastatic Breast Cancer:
MAINTAIN Trial. J. Clin. Oncol. 2023,41, JCO2202392. [CrossRef] [PubMed]
104.
Mayer, E.L.; Wander, S.A.; Regan, M.M.; DeMichele, A.; Forero-Torres, A.; Rimawi, M.F.; Ma, C.X.; Cristofanilli, M.; Anders,
C.K.; Bartlett, C.H.; et al. Palbociclib after CDK and Endocrine Therapy (PACE): A Randomized Phase II Study of Fulvestrant,
Palbociclib, and Avelumab for Endocrine Pre-Treated ER+/HER2
−
Metastatic Breast Cancer. J. Clin. Oncol.
2018
,36, TPS1104.
[CrossRef]
105.
Llombart-Cussac, A.; Harper-Wynne, C.; Perello, A.; Hennequin, A.; Fernandez, A.; Colleoni, M.; Carañana, V.; Quiroga, V.;
Medioni, J.; Iranzo, V.; et al. Second-Line Endocrine Therapy (ET) with or without Palbociclib (P) Maintenance in Patients (Pts)
with Hormone Receptor-Positive (HR[+])/Human Epidermal Growth Factor Receptor 2-Negative (HER2[-]) Advanced Breast
Cancer (ABC): PALMIRA Trial. J. Clin. Oncol. 2023,41, 1001. [CrossRef]

Int. J. Mol. Sci. 2023,24, 14427 23 of 25
106.
Albanell, J.; Pérez-García, J.M.; Gil-Gil, M.; Curigliano, G.; Ruíz-Borrego, M.; Comerma, L.; Gibert, J.; Bellet, M.; Bermejo, B.;
Calvo, L.; et al. Palbociclib Rechallenge for Hormone Receptor-Positive/HER-Negative Advanced Breast Cancer: Findings from
the Phase II BioPER Trial. Clin. Cancer Res. 2023,29, 67–80. [CrossRef]
107.
Karacin, C.; Oksuzoglu, B.; Demirci, A.; Keskinkılıç, M.; Baytemür, N.K.; Yılmaz, F.; Selvi, O.; Erdem, D.; Av¸sar, E.; Paksoy,
N.; et al. Efficacy of Subsequent Treatments in Patients with Hormone-Positive Advanced Breast Cancer Who Had Disease
Progression under CDK 4/6 Inhibitor Therapy. BMC Cancer 2023,23, 136. [CrossRef]
108.
Robertson, J.F.R.; Bondarenko, I.M.; Trishkina, E.; Dvorkin, M.; Panasci, L.; Manikhas, A.; Shparyk, Y.; Cardona-Huerta, S.;
Cheung, K.L.; Philco-Salas, M.J.; et al. Fulvestrant 500 Mg versus Anastrozole 1 Mg for Hormone Receptor-Positive Advanced
Breast Cancer (FALCON): An International, Randomised, Double-Blind, Phase 3 Trial. Lancet 2016,388, 2997–3005. [CrossRef]
109.
Chia, S.; Gradishar, W.; Mauriac, L.; Bines, J.; Amant, F.; Federico, M.; Fein, L.; Romieu, G.; Buzdar, A.; Robertson, J.F.R.;
et al. Double-Blind, Randomized Placebo Controlled Trial of Fulvestrant Compared with Exemestane after Prior Nonsteroidal
Aromatase Inhibitor Therapy in Postmenopausal Women with Hormone Receptor-Positive, Advanced Breast Cancer: Results
from EFECT. J. Clin. Oncol. 2008,26, 1664–1670. [CrossRef]
110.
Bidard, F.C.; Kaklamani, V.G.; Neven, P.; Streich, G.; Montero, A.J.; Forget, F.; Mouret-Reynier, M.A.; Sohn, J.H.; Taylor, D.;
Harnden, K.K.; et al. Elacestrant (Oral Selective Estrogen Receptor Degrader) Versus Standard Endocrine Therapy for Estrogen
Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Advanced Breast Cancer: Results from the Randomized
Phase III EMERALD Trial. J. Clin. Oncol. 2022,40, 3246–3256. [CrossRef]
111.
Bardia, A.; Bidard, F.-C.; Neven, P.; Streich, G.; Montero, A.J.; Forget, F.; Mouret-Reynier, M.-A.; Sohn, J.H.; Taylor, D.; Harnden,
K.K.; et al. Abstract GS3-01: GS3-01 EMERALD Phase 3 Trial of Elacestrant versus Standard of Care Endocrine Therapy in
Patients with ER+/HER2
−
Metastatic Breast Cancer: Updated Results by Duration of Prior CDK4/6is in Metastatic Setting.
Cancer Res. 2023,83, GS3-01. [CrossRef]
112.
Oliveira, M.; Hamilton, E.P.; Incorvati, J.; de la Heras, B.B.; Calvo, E.; García-Corbacho, J.; Ruiz-Borrego, M.; Vaklavas, C.; Turner,
N.C.; Ciruelos, E.M.; et al. Serena-1: Updated Analyses from a Phase 1 Study (Parts C/D) of the next-Generation Oral SERD
Camizestrant (AZD9833) in Combination with Palbociclib, in Women with ER-Positive, HER2-Negative Advanced Breast Cancer.
J. Clin. Oncol. 2022,40, 1032. [CrossRef]
113.
Mosele, F.; Stefanovska, B.; Lusque, A.; Tran Dien, A.; Garberis, I.; Droin, N.; Le Tourneau, C.; Sablin, M.P.; Lacroix, L.; Enrico,
D.; et al. Outcome and Molecular Landscape of Patients with PIK3CA-Mutated Metastatic Breast Cancer. Ann. Oncol.
2020
,31,
377–386. [CrossRef] [PubMed]
114.
André, F.; Ciruelos, E.M.; Juric, D.; Loibl, S.; Campone, M.; Mayer, I.A.; Rubovszky, G.; Yamashita, T.; Kaufman, B.; Lu, Y.S.;
et al. Alpelisib plus Fulvestrant for PIK3CA-Mutated, Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor-2-
Negative Advanced Breast Cancer: Final Overall Survival Results from SOLAR-1. Ann. Oncol.
2021
,32, 208–217. [CrossRef]
[PubMed]
115.
CHMP Piqray—Annex I Summary of Product Characteristics. Available online: https://www.ema.europa.eu/en/documents/
product-information/piqray-epar-product-information_en.pdf (accessed on 11 July 2023).
116.
Rugo, H.S.; Lerebours, F.; Ciruelos, E.; Drullinsky, P.; Borrego, M.R.; Neven, P.; Park, Y.H.; Prat, A.; Bachelot, T.; Juric, D.;
et al. Alpelisib (ALP) + Fulvestrant (FUL) in Patients (Pts) with PIK3CA-Mutated (Mut) Hormone Receptor-Positive (HR+),
Human Epidermal Growth Factor Receptor 2-Negative (HER2–) Advanced Breast Cancer (ABC) Previously Treated with Cyclin-
Dependent Kinase 4/6 Inhibitor (CDKi) + Aromatase Inhibitor (AI): BYLieve Study Results. J. Clin. Oncol.
2020
,38, 1006.
[CrossRef]
117.
Juric, D.; Rugo, H.S.; Chia, S.K.L.; Lerebours, F.; Ruiz-Borrego, M.; Drullinsky, P.; Ciruelos, E.M.; Neven, P.; Park, Y.H.; Arce, C.H.;
et al. Alpelisib (ALP) + Endocrine Therapy (ET) in Patients (Pts) with Hormone Receptor-Positive (HR+), Human Epidermal
Growth Factor Receptor 2-Negative (HER2–), PIK3CA-Mutated (Mut) Advanced Breast Cancer (ABC): Baseline Biomarker
Analysis and Progression-Free Survival (PFS) by Duration of Prior Cyclin-Dependent Kinase 4/6 Inhibitor (CDK4/6i) Therapy in
the BYLieve Study. J. Clin. Oncol. 2022,40, 1018. [CrossRef]
118.
A Study of LOXO-783 in Patients with Breast Cancer/Other Solid Tumors. Available online: https://classic.clinicaltrials.gov/ct2
/show/NCT05307705 (accessed on 27 July 2023).
119.
First-in-Human Study of Mutant-Selective PI3K
α
Inhibitor, RLY-2608, as a Single Agent in Advanced Solid Tumor Patients and in
Combination with Fulvestrant in Patients with Advanced Breast Cancer. Available online: https://classic.clinicaltrials.gov/ct2
/show/NCT05216432 (accessed on 11 July 2023).
120. Piccart, M.; Hortobagyi, G.N.; Campone, M.; Pritchard, K.I.; Lebrun, F.; Ito, Y.; Noguchi, S.; Perez, A.; Rugo, H.S.; Deleu, I.; et al.
Everolimus plus Exemestane for Hormone-Receptor-Positive, Human Epidermal Growth Factor Receptor-2-Negative Advanced
Breast Cancer: Overall Survival Results from BOLERO-2. Ann. Oncol. 2014,25, 2357–2362. [CrossRef] [PubMed]
121.
Bardia, A.; Hurvitz, S.A.; DeMichele, A.; Clark, A.S.; Zelnak, A.B.; Yardley, D.A.; Karuturi, M.S.; Sanft, T.B.; Blau, S.; Hart, L.L.;
et al. Triplet Therapy (Continuous Ribociclib, Everolimus, Exemestane) in HR+/HER2
−
Advanced Breast Cancer Postprogression
of a CDK4/6 Inhibitor (TRINITI-1): Efficacy, Safety, and Biomarker Results. J. Clin. Oncol. 2019,37, 1016. [CrossRef]
122.
Bardia, A.; Hurvitz, S.A.; DeMichele, A.; Clark, A.S.; Zelnak, A.; Yardley, D.A.; Karuturi, M.; Sanft, T.; Blau, S.; Hart, L.; et al. Phase
I/II Trial of Exemestane, Ribociclib, and Everolimus in Women with HR+/HER2
−
Advanced Breast Cancer after Progression on
CDK4/6 Inhibitors (TRINITI-1). Clin. Cancer Res. 2021,27, 4177–4185. [CrossRef]

Int. J. Mol. Sci. 2023,24, 14427 24 of 25
123.
A Study Evaluating the Efficacy and Safety of Giredestrant Plus Everolimus Compared with the Physician’s Choice of Endocrine
Therapy Plus Everolimus in Participants with Estrogen Receptor-Positive, HER2-Negative, Locally Advanced or Metastatic Breast
Cancer (EvERA Breast Cancer). Available online: https://classic.clinicaltrials.gov/ct2/show/NCT05306340 (accessed on 11 July
2023).
124.
Korotchkina, L.G.; Leontieva, O.V.; Bukreeva, E.I.; Demidenko, Z.N.; Gudkov, A.V.; Blagosklonny, M.V. The Choice between
P53-Induced Senescence and Quiescence Is Determined in Part by the MTOR Pathway. Aging 2010,2, 344–352. [CrossRef]
125.
Palbociclib with Everolimus + Exemestane in BC. Available online: https://classic.clinicaltrials.gov/ct2/show/NCT02871791
(accessed on 11 July 2023).
126.
CDK 4/6 Inhibitor, Ribociclib, with Adjuvant Endocrine Therapy for ER-Positive Breast Cancer. Available online: https:
//classic.clinicaltrials.gov/ct2/show/NCT03285412 (accessed on 11 July 2023).
127.
This Study in Patients with Different Types of Cancer (Solid Tumours) Aims to Find a Safe Dose of Xentuzumab in Combination
with Abemaciclib with or without Hormonal Therapies. The Study Also Tests How Effective These Medicines Are in Patients
with Lung and Breast Cancer. Available online: https://classic.clinicaltrials.gov/ct2/show/NCT03099174 (accessed on 11 July
2023).
128.
A Study to Assess the Tolerability and Clinical Activity of Gedatolisib in Combination with Palbociclib/Letrozole or Palboci-
clib/Fulvestrant in Women with Metastatic Breast Cancer. Available online: https://classic.clinicaltrials.gov/ct2/show/NCT026
84032 (accessed on 11 July 2023).
129.
Fulvestrant, Palbociclib and Erdafitinib in ER+/HER2
−
/FGFR-Amplified Metastatic Breast Cancer. Available online: https:
//classic.clinicaltrials.gov/ct2/show/NCT03238196 (accessed on 11 July 2023).
130.
Wander, S.A.; Juric, D.; Supko, J.G.; Micalizzi, D.S.; Spring, L.; Vidula, N.; Beeler, M.; Habin, K.R.; Viscosi, E.; Fitzgerald, D.M.;
et al. Phase Ib Trial to Evaluate Safety and Anti-Tumor Activity of the AKT Inhibitor, Ipatasertib, in Combination with Endocrine
Therapy and a CDK4/6 Inhibitor for Patients with Hormone Receptor Positive (HR+)/HER2 Negative Metastatic Breast Cancer
(MBC) (TAKTIC). J. Clin. Oncol. 2020,38, 1066. [CrossRef]
131.
Turner, N.C.; Oliveira, M.; Howell, S.J.; Dalenc, F.; Cortes, J.; Gomez Moreno, H.L.; Hu, X.; Jhaveri, K.; Krivorotko, P.; Loibl, S.;
et al. Capivasertib in Hormone Receptor–Positive Advanced Breast Cancer. N. Engl. J. Med.
2023
,388, 2058–2070. [CrossRef]
[PubMed]
132.
Jiang, Z.; Li, W.; Hu, X.; Zhang, Q.; Sun, T.; Cui, S.; Wang, S.; Ouyang, Q.; Yin, Y.; Geng, C.; et al. Tucidinostat plus Exemestane
for Postmenopausal Patients with Advanced, Hormone Receptor-Positive Breast Cancer (ACE): A Randomised, Double-Blind,
Placebo-Controlled, Phase 3 Trial. Lancet Oncol. 2019,20, 806–815. [CrossRef] [PubMed]
133.
Connolly, R.M.; Zhao, F.; Miller, K.D.; Lee, M.J.; Piekarz, R.L.; Smith, K.L.; Brown-Glaberman, U.A.; Winn, J.S.; Faller, B.A.; Onitilo,
A.A.; et al. E2112: Randomized Phase III Trial of Endocrine Therapy Plus Entinostat or Placebo in Hormone Receptor-Positive
Advanced Breast Cancer. A Trial of the ECOG-ACRIN Cancer Research Group. J. Clin. Oncol.
2021
,39, 3171–3181. [CrossRef]
[PubMed]
134.
Martin, L.A.; Dowsett, M. BCL-2: A New Therapeutic Target in Estrogen Receptor-Positive Breast Cancer? Cancer Cell
2013
,24,
7–9. [CrossRef]
135.
Lok, S.W.; Whittle, J.R.; Vaillant, F.; Teh, C.E.; Lo, L.L.; Policheni, A.N.; Bergin, A.R.T.; Desai, J.; Ftouni, S.; Gandolfo, L.C.;
et al. A Phase Ib Dose-Escalation and Expansion Study of the BCL2 Inhibitor Venetoclax Combined with Tamoxifen in ER and
BCL2-Positive Metastatic Breast Cancer. Cancer Discov. 2019,9, 354–369. [CrossRef]
136.
Lindeman, G.J.; Bowen, R.; Jerzak, K.J.; Song, X.; Decker, T.; Boyle, F.M.; McCune, S.L.; Armstrong, A.; Shannon, C.M.; Bertelli, G.;
et al. Results from VERONICA: A Randomized, Phase II Study of Second-/Third-Line Venetoclax (VEN) + Fulvestrant (F) versus
F Alone in Estrogen Receptor (ER)-Positive, HER2-Negative, Locally Advanced, or Metastatic Breast Cancer (LA/MBC). J. Clin.
Oncol. 2021,39, 1004. [CrossRef]
137.
Muttiah, C.; Whittle, J.R.; Oakman, C.; Lindeman, G.J. PALVEN: Phase Ib Trial of Palbociclib, Letrozole and Venetoclax in Estrogen
Receptor- and BCL2-Positive Advanced Breast Cancer. Future Oncol. 2022,18, 1805–1816. [CrossRef]
138.
Goel, S.; Decristo, M.J.; Watt, A.C.; Brinjones, H.; Sceneay, J.; Li, B.B.; Khan, N.; Ubellacker, J.M.; Xie, S.; Metzger-Filho, O.; et al.
CDK4/6 Inhibition Triggers Anti-Tumour Immunity. Nature 2017,548, 471–475. [CrossRef]
139.
Robson, M.; Im, S.-A.; Senkus, E.; Xu, B.; Domchek, S.M.; Masuda, N.; Delaloge, S.; Li, W.; Tung, N.; Armstrong, A.; et al. Olaparib
for Metastatic Breast Cancer in Patients with a Germline BRCA Mutation. N. Engl. J. Med. 2017,377, 523–533. [CrossRef]
140.
Litton, J.K.; Rugo, H.S.; Ettl, J.; Hurvitz, S.A.; Gonçalves, A.; Lee, K.-H.; Fehrenbacher, L.; Yerushalmi, R.; Mina, L.A.; Martin, M.;
et al. Talazoparib in Patients with Advanced Breast Cancer and a Germline BRCA Mutation. N. Engl. J. Med.
2018
,379, 753–763.
[CrossRef]
141.
Litton, J.K.; Hurvitz, S.A.; Mina, L.A.; Rugo, H.S.; Lee, K.H.; Gonçalves, A.; Diab, S.; Woodward, N.; Goodwin, A.; Yerushalmi, R.;
et al. Talazoparib versus Chemotherapy in Patients with Germline BRCA1/2-Mutated HER2-Negative Advanced Breast Cancer:
Final Overall Survival Results from the EMBRACA Trial. Ann. Oncol. 2020,31, 1526–1535. [CrossRef]
142.
Sammons, S.; Shastry, M.; Dent, S.; Anders, C.; Hamilton, E. Practical Treatment Strategies and Future Directions After Progression
While Receiving CDK4/6 Inhibition and Endocrine Therapy in Advanced HR+/HER2
−
Breast Cancer. Clin. Breast Cancer
2020
,
20, 1–11. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2023,24, 14427 25 of 25
143.
Ogata, R.; Kishino, E.; Saitoh, W.; Koike, Y.; Kurebayashi, J. Resistance to Cyclin-Dependent Kinase (CDK) 4/6 Inhibitors Confers
Cross-Resistance to Other CDK Inhibitors but Not to Chemotherapeutic Agents in Breast Cancer Cells. Breast Cancer
2021
,28,
206–215. [CrossRef] [PubMed]
144.
Kolyadina, I.V.; Bolotina, L.; Zhukova, L.; Vladimirova, L.Y.; Sultanbaev, A.; Karabina, E.; Ganshina, I.; Ovchinnikova, E.;
Kolyadina, I.V.; Antonova, G.; et al. The Effectiveness and Safety of Eribulin Therapy in HR-Positive HER2-Negative Metastatic
Breast Cancer Post-CDK4/6 Inhibitor Therapy in Russian Clinical Practice. J. Clin. Oncol. 2021,39, e13035. [CrossRef]
145.
Modi, S.; Jacot, W.; Yamashita, T.; Sohn, J.; Vidal, M.; Tokunaga, E.; Tsurutani, J.; Ueno, N.T.; Prat, A.; Chae, Y.S.; et al. Trastuzumab
Deruxtecan in Previously Treated HER2-Low Advanced Breast Cancer. N. Engl. J. Med. 2022,387, 9–20. [CrossRef]
146.
Modi, S.; Niikura, N.; Yamashita, T.; Jacot, W.; Sohn, J.; Tokunaga, E.; Vidal, M.J.; Park, Y.H.; Lee, K.S.; Chae, Y.; et al. Trastuzumab
Deruxtecan (T-DXd) vs. Treatment of Physician’s Choice (TPC) in Patients (Pts) with HER2-Low, Hormone Receptor-Positive
(HR+) Unresectable and/or Metastatic Breast Cancer (MBC): Exploratory Biomarker Analysis of DESTINY-Breast04. J. Clin. Oncol.
2023,41, 1020. [CrossRef]
147.
Lloyd, M.R.; Spring, L.M.; Bardia, A.; Wander, S.A. Mechanisms of Resistance to CDK4/6 Blockade in Advanced Hormone
Receptor-Positive, HER2-Negative Breast Cancer and Emerging Therapeutic Opportunities. Clin. Cancer Res.
2022
,28, 821–830.
[CrossRef]
148.
Rugo, H.S.; Bardia, A.; Marmé, F.; Cortés, J.; Schmid, P.; Loirat, D.; Tredan, O.; Ciruelos, E.M.; Dalenc, F.; Gomez Pardo, P.;
et al. LBA76 Overall Survival (OS) Results from the Phase III TROPiCS-02 Study of Sacituzumab Govitecan (SG) vs Treatment
of Physician’s Choice (TPC) in Patients (Pts) with HR+/HER2
−
Metastatic Breast Cancer (MBC). Ann. Oncol.
2022
,33, S1386.
[CrossRef]
149.
Pembrolizumab + Paclitaxel in Hormone Receptor-Positive (HR+)/Human Epidermal Growth Factor Receptor 2-Negative
(HER2
−
) Non-Luminal (by PAM50) Advanced Breast Cancer After Cyclin-Dependent Kinase 4/6 (CDK4/6) Inhibitors Progres-
sion. Available online: https://classic.clinicaltrials.gov/ct2/show/NCT04251169 (accessed on 27 July 2023).
150.
Study of ASTX727 Plus Talazoparib in Patients with Triple Negative or Hormone Resistant/HER2-Negative Metastatic Breast
Cancer. Available online: https://classic.clinicaltrials.gov/ct2/show/NCT04134884 (accessed on 27 July 2023).
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