A Novel Orally Active Small Molecule Potently Induces G1Arrest
in Primary Myeloma Cells and Prevents Tumor Growth by
Specific Inhibition of Cyclin-Dependent Kinase 4/6
Linda B. Baughn,
Malcolm A.S. Moore,
1Maurizio Di Liberto,
4and Selina Chen-Kiang
4Peter L. Toogood,
Weill Medical College of Cornell University;
3Graduate Program in Immunology and Microbial Pathogenesis,
4Memorial Sloan-Kettering Cancer Center, New York, New York;
5Pfizer Global Research and Development, Ann Arbor, Michigan
Cell cycle deregulation is central to the initiation and fatality
of multiple myeloma, the second most common hematopoietic
cancer, although impaired apoptosis plays a critical role in the
accumulation of myeloma cells in the bone marrow. The
mechanism for intermittent, unrestrained proliferation of
myeloma cells is unknown, but mutually exclusive activation
of cyclin-dependent kinase 4 (Cdk4)-cyclin D1 or Cdk6-cyclin
D2 precedes proliferation of bone marrow myeloma cells
in vivo. Here, we show that by specific inhibition of Cdk4/6,
the orally active small-molecule PD 0332991 potently induces
G1arrest in primary bone marrow myeloma cells ex vivo and
prevents tumor growth in disseminated human myeloma
xenografts. PD 0332991 inhibits Cdk4/6 proportional to the
cycling status of the cells independent of cellular transforma-
tion and acts in concert with the physiologic Cdk4/6 inhibitor
p18INK4c. Inhibition of Cdk4/6 by PD 0332991 is not accom-
panied by induction of apoptosis. However, when used in
combination with a second agent, such as dexamethasone, PD
0332991 markedly enhances the killing of myeloma cells by
dexamethasone. PD 0332991, therefore, represents the first
promising and specific inhibitor for therapeutic targeting of
Cdk4/6 in multiple myeloma and possibly other B-cell cancers.
(Cancer Res 2006; 66(15): 7661-7)
Multiple myeloma, the second most common hematopoietic
cancer, represents a clinically defined collection of plasma cell
neoplasms in which malignant plasmacytoid cells are arrested at
various stages of plasma cell differentiation (1). Unlike normal
plasma cells, which are permanently withdrawn from the cell cycle
(2, 3), multiple myeloma cells retain their self-renewing potential
(4, 5). During the stable phase of the disease, myeloma cells
accumulate in the bone marrow mainly due to impaired apoptosis
(6). However, among the noncycling cells, there are self-renewing
myeloma cells, which reenter the cell cycle and divide without
restraint during relapse and drug resistance. As multiple myeloma
is invariably fatal, it is imperative to define the mechanism of cell
cycle deregulation in multiple myeloma.
Reentry and progression through the G1phase of the cell cycle
is driven by cyclin-dependent kinases (Cdk) in cooperation with
cyclins and inhibited by Cdk inhibitors (CKI; ref. 7). Phosphor-
ylation of the retinoblastoma protein p105 (Rb) by Cdk4 or Cdk6
together with cyclin D in early G1and by Cdk2 in conjunction
with cyclin E in late G1leads to the release of E2F transcription
factors and S-phase entry (8). In turn, the Cdk4 and Cdk6
activities are attenuated by CKIs of the INK4 family, whereas that
of Cdk2 is inhibited by CKIs of the Cip/Kip family (7). Inhibition
of Cdk6 by p18INK4c(p18) of the INK4 family (9, 10) is required for
G1 arrest and differentiation of antibody-secreting, end-stage
plasma cells from antigen-activated B cells in vivo and in vitro
(2, 3). In the absence of p18, plasmacytoid cells expressing CD138
(syndecan-1), a proteoglycan present on both normal and
malignant plasma cells (11), are generated, but they continue to
cycle and are rapidly eliminated by apoptosis (3). Attenuation of
Cdk4/6 by p18, therefore, is critical for normal plasma cell
The mechanism that underlies cell cycle deregulation in
myeloma is largely unknown. Overexpression of D cyclins has
been implicated in promoting myeloma progression based on
chromosomal translocation of cyclin D1 and cyclin D3 genes to
immunoglobulin loci and elevation of D cyclin RNA in myeloma
cells by microarray analysis (12–14). Paradoxically, cyclin D1
overexpression was associated with a more favorable clinical
outcome (15–17). Analysis of Cdk4/6-specific Rb phosphorylation
as an indicator of progression through early G1in primary bone
marrow myeloma cells in vivo and ex vivo has now shown that
overexpression of cyclin D1 alone is insufficient to promote cell
cycle progression (18). Instead, aberrant coactivation and pairing
of Cdk4-cyclin D1 and Cdk6-cyclin D2 precedes proliferation of
myeloma cells and is enhanced in advanced disease, regardless of
the treatment history or the clinical heterogeneity (18). The
mutually exclusive pairing of Cdk4-cyclin D1 and Cdk6-cyclin D2
reveals a previously unappreciated specificity and complexity of
cell cycle deregulation in myeloma and identifies Cdk4 and Cdk6
as key determinants in the loss of cell cycle control in myeloma.
These findings further suggest that Cdk4/6 may be effective
targets for therapeutic intervention. To test this possibility, we
inhibited Cdk4 and Cdk6 in primary bone marrow myeloma cells
using PD 0332991, an orally active, water-soluble cell-permeable
pyridopyrimidine, which potently inhibits recombinant Cdk4 and
Cdk6 (IC50 = 0.011-0.016 Amol/L) by competing for the ATP-
binding sites (19). Unlike other Cdk4/6 inhibitors that have been
Note: Supplementary data for this article are available at Cancer Research Online
L.B. Baughn and M. Di Liberto contributed equally to this work.
Requests for reprints: Selina Chen-Kiang, Department of Pathology, Cornell
University Medical College, 1300 York Avenue, C-338, New York, NY 10021. Phone: 212-
746-6440; Fax: 212-746-7996; E-mail: email@example.com.
I2006 American Association for Cancer Research.
Cancer Res 2006; 66: (15). August 1, 2006
used in clinical trials, such as flavopiridol, R-roscovitine (CYC202
or Seliciclib), UNC-01 (7-hydrozystaurosporine), and BMS-387032
(20, 21), PD 0332991 is highly selective for Cdk4 and Cdk6, dis-
playing little or no activity against a panel of 36 additional kinases,
in particular Cdk2 (19). Inhibition of Cdk4 by PD 0332991 leads to
G1arrest in nonhematopoietic tumor cell lines in vitro (IC50= 0.06
Amol/L), growth suppression, and marked regression of several
solid tumors in xenografts (19). By contrast, treatment of human
myeloma cell lines (HMCL) with flavopiridol or R-roscovitine
mainly results in apoptosis without evidence of inhibition of spe-
cific Cdks (22, 23).
Here, we show that PD 0332991 potently induces G1arrest in
primary myeloma cells by inhibiting Cdk4/6 according to the
cycling status of the cells and nearly completely prevents tumor
growth in a disseminated xenograft human myeloma model.
Furthermore, PD 0332991 enhances the killing of myeloma cells by
dexamethasone. The ability of PD 0332991 to selectively inhibit
Cdk4/6 and cell cycle progression of primary bone marrow
myeloma cells suggests PD 0332991 as a promising candidate for
mechanism-driven therapy for multiple myeloma.
Materials and Methods
Bone marrow myeloma cells and cell lines. Bone marrow specimens
were obtained from multiple myeloma patients at the New York
Presbyterian Hospital under informed consent as part of an Institutional
Review Board–approved study. Multiple myeloma was staged according to
the Salmon-Durie classification based on the criteria of monoclonal serum
immunoglobulin levels, reciprocal depression of normal immunoglobulin,
other peripheral blood studies, renal function, and the number of lytic bone
lesions (24). Live, mononuclear cells were isolated from bone marrow
aspirates by Ficoll-Hypaque density gradient centrifugation. The CD138+
bone marrow myeloma cells were then enriched from this fraction to >95%
purity by using an automated MACS CD138 MicroBeads system (Miltenyi
Biotechnology, Inc., Auburn, CA), unless otherwise indicated. CAG and
MM1.S human multiple myeloma cell lines were kindly provided,
respectively, by Dr. J. Epstein (University of Arkansas, Little Rock, AK)
and Dr. N. Krett (Northwestern University, Chicago, IL). The HS-5 human
stromal cell line was obtained from the American Type Culture Collection
Primary CD138+multiple myeloma cells were cultured at a starting
density of 2 ? 105/mL in RPMI 1640 (Invitrogen, Carlsbad, CA)
supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone,
Logan, UT) and 2 mmol/L L-glutamine, 100 units/mL penicillin/streptomy-
cin, 4 mmol/L HEPES, 0.1 mmol/L MEM nonessential amino acid solution
(all from Invitrogen), 50 Amol/L h-mercaptoethanol (Sigma, St. Louis, MO),
recombinant human interleukin-6 (IL-6; 40 units/mL), soluble IL-6 receptor
(40 units/mL; ref. 2), insulin-like growth factor-1 (IGF-1; 100 ng/mL, R&D
Systems, Minneapolis, MN), and soluble BLyS (BAFF; 50 ng/mL; ref. 25).
Multiple myeloma cell lines were cultured in the same media at an initial
density of 3 ? 105/mL. In some cultures, PD 0332991 (19) dissolved in dH2O
or dexamethasone (Sigma) dissolved in dimethyl sulfoxide (DMSO, Sigma)
was added to the media at concentration and for the time indicated. Cell
viability was determined by trypan blue exclusion in triplicate.
In some experiments, CD138+primary bone marrow myeloma cells were
cocultured at a 2:1 ratio with HS-5 stromal cells, prelabeled with the PKH67
green fluorescent cell linker kit (Sigma), and plated overnight in the
presence or absence of PD 0332991 for the time indicated. For biochemical
analysis, primary bone marrow myeloma cells were removed from stromal
cells by gentle pipetting and rinsing with RPMI 1640. The purity of multiple
myeloma cells was assessed to be >95% by staining with a PE-anti-human
CD138 (Biosource, Camarillo, CA) antibody and flow cytometry using a
FACScan (Becton Dickinson, Franklin Lakes, NJ).
Isolation of resting and activated primary mouse B cells.
Splenocytes were isolated from p18+/+and p18?/?mice (7-8 weeks, age-
matched, either sex; ref. 26). High-density (resting B) and low-density
(activated B and plasma) cells were isolated from splenocytes from the
60% to 70% and 50% to 60% interfaces of a discontinuous Percoll gradient,
respectively, as previously described (27). Resting B cells were cultured at
4 ? 105/mL in RPMI 1640 containing 10% FBS, as previously described
(25) in the presence or absence of F(ab¶)2 anti-IgM (7.4 Ag/mL; Zymed,
South San Francisco, CA) together with recombinant soluble human BLyS
(BAFF; 50 ng/mL).
Analysis of bromodeoxyuridine uptake, DNA content, and apopto-
sis. Bromodeoxyuridine (BrdUrd; 5 Ag/mL; Sigma) was added to cultures of
CD138+bone marrow myeloma cells and HMCL cells at 3 ? 105/mL for the
time indicated. BrdUrd uptake was measured by flow cytometry as
described (27), using a FITC-anti-BrdUrd (Roche Diagnostics, Pleasanton,
CA) or an APC-anti-BrdUrd (Becton Dickinson) monoclonal antibody
(mAb). For DNA content analysis, cells were fixed in 70% ethanol at 4jC for
30 minutes and incubated in PBS containing 50 Ag/mL propidium iodide
(Sigma) and 100 units/mL RNase A (Sigma) for 30 minutes at 37jC in the
dark. The DNA content per cell was analyzed by flow cytometry.
Immunoblotting. Preparation of whole-cell lysates and immunoblotting
was as previously described (25). The following antibodies were used for
immunoblotting: mouse mAbs to human Rb, Cdk6, Cdk4, cyclin D1 (all from
Cell Signaling, Beverly, MA), mouse Rb (Becton Dickinson), or actin (Becton
Dickinson); rabbit polyclonal antibodies to pSRb780or pSRb807/811(Cell
Signaling), cyclin D2 (Santa Cruz Biotechnology, Santa Cruz, CA), and
poly(ADP-ribose) polymerase-1 (PARP; Cell Signaling). Membranes were
rinsed in TBS-T [10 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 0.02%
Tween 20], incubated with horseradish peroxidase–linked goat anti-mouse
or anti-rabbit (1:5,000) secondary antibodies for 60 minutes, and developed
with the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce
Biotechnology, Rockford, IL). The signals were determined by densitometry
Establishment of disseminated myeloma xenografts and therapy.
Establishment of the disseminated xenograft nonobese diabetic/severe
combined immunodeficient (NOD/SCID) mouse multiple myeloma model
has been described (28). In brief, 10 ? 106CAG cells stably expressing the
HSV-TK-eGFP-luciferase fusion protein (28) were injected i.v. via tail vein to
NOD/SCID (NOD/LTSZPrko/J) mice (The Jackson Laboratory, Bar Harbor,
ME) at 8 to 9 weeks of age. The tumor distribution was followed by serial
whole-body noninvasive imaging of visible light emitted by luciferase-
expressing myeloma cells upon injection of mice with luciferin. Seven days
after tumor injection, a group of NOD/SCID mice with established
disseminated multiple myeloma were divided into two cohorts, with
statistically equivalent tumor burden between cohorts (as evaluated by
bioluminescence imaging). PD 0332991 was dissolved in sodium lactate
buffer (50 mmol/L, pH 4) and was given daily at 150 mg/kg by gavage. The
control mice received the same amount of sodium lactate buffer solution
through the same route. The overall survival of mice was defined as the
time between injection of tumor cells and death, or sacrifice upon
development of hind leg paralysis, and was compared with the treatment
group with Kaplan-Meier survival analysis.
Statistical analysis. All analyses were done using the Stata 7.0 statistical
software (Stata Corp., College Station, TX), with P < 0.05 considered to be
significant. For comparing tumor-associated variables, the nonpaired
Student’s t test was used unless otherwise specified. Log-rank tests were
used to calculate the statistical significance of difference of Kaplan-Meier
Luciferase assay. Luciferase activity was measured in HSV-TK-eGFP-
luciferase+CAG cells using the Dual-Glo luciferase assay system according
to manufacturer’s instructions (Promega, Madison, WI). Luciferase activity
was measured using the LMax (Molecular Devices, Sunnyvale, CA)
microplate reader and the Softmax Pro Software (Molecular Devices).
Immunohistochemistry. Immunohistochemistry was done on 4-Am
sections of paraffin-embedded vertebrae from the CAG xenograft using a
TechMate500 BioTek automated immunostainer (Ventana Medical Systems,
Tucson, AZ) according to the manufacturer’s specification. CD138+CAG
cells were detected using an anti-CD138 mAb (Serotec, Oxford, England)
and a red chromogen. The nuclei were visualized by counterstaining with
Cancer Res 2006; 66: (15). August 1, 2006
hematoxylin (blue). Simultaneous expression of other proteins was detected
with mAbs to Rb (Cell Signaling), polyclonal rabbit antibodies to pSRb807/811
of Rb and cleaved caspase-3 (Cell Signaling). Controls include pretreating
tissue sections with calyculin A (Cell Signaling) to prevent dephosphory-
lation during or after antigen retrieval or with calf intestine phosphatase
(Cell Signaling) to verify phosphorylation. Simultaneous detection of
apoptosis of CD138+cells by the terminal deoxynucleotidyl transferase–
mediated nick-end labeling (TUNEL) assay was done using the ApopTag kit
(Intergen, Purchase, NY) and a brown chromogen.
PD 0332991 potently inhibits Cdk4/6 and induces G1arrest
in primary bone marrow myeloma cells. The ability of PD
0332991 to inhibit Cdk4/6 was examined in freshly isolated CD138+
(a cell surface marker for normal and malignant plasma cells) bone
marrow myeloma cells by detecting Cdk4/6-specific phosphoryla-
tion of Rb on Ser780(pSRb780) and Ser807/811(pSRb807/811; refs. 29,
30). Primary bone marrow CD138+myeloma cells retain their
cycling capacity ex vivo only within the initial hour of isolation.
However, even in the absence of continuous cycling, PD 0332991
inhibited Cdk4/6 phosphorylation regardless of the disease stage
or treatment history in all multiple myeloma cases tested (n = 14;
Fig. 1A; Supplementary Table S1), because deregulation of Cdk4/6
is a frequent event in multiple myeloma (18). Consistent with
previous observations in nonhematopoietic cell lines (19), PD
0332991 inhibits Cdk4/6 within 4 to 6 hours in primary bone mar-
row myeloma cells (Supplementary Table S1; data not shown).
These findings show directly, for the first time, that PD 0332991
potently inhibits Cdk4/6 in primary myeloma cells.
To study the inhibition of Cdk4/6 by PD 0332991 in cycling
primary myeloma cells, we optimized a coculture system with
human HS-5 bone marrow stromal cells (BMSC; ref. 31), in which
proliferation of primary CD138+myeloma cells can be prolonged
for 1 to 2 days ex vivo in the presence of IL-6, IGF-1, and BLyS
(BAFF).6PD 0332991 potently inhibited Cdk4/6-specific phosphor-
ylation of Rb (IC50< 0.1 Amol/L) in cycling CD138+primary bone
marrow myeloma cells (Fig. 1B). This led to G1arrest, as evidenced
by the reduction in BrdUrd uptake in the presence of PD 0332991,
at as low as 0.3 Amol/L (Fig. 1C).
Cdk4 was coactivated with cyclin D1 (Pt 1), and Cdk6 was
coactivated with cyclin D2 (Pt 2 and Pt 3; Fig. 1A and B), further
corroborating that Rb is phosphorylated by mutually exclusive
Cdk4-cyclin D1 and Cdk6-cyclin D2 pairs in individual multiple
myeloma cases (18). Moreover, inhibition of Cdk4/6 by PD 0332991
was more effective in cycling primary bone marrow myeloma cells
in BMSC cocultures (Pt 3) than in those cultured in media, which
are no longer cycling (Pt 1 and Pt 2). Thus, PD 0332991 inhibits
Cdk4/6 and induces G1arrest proportional to the activation status
of primary bone marrow myeloma cells.
PD 0332991 potently inhibits Cdk4/6 and induces G1arrest
in HMCLs. We next investigated the mechanism of PD 0332991
action in MM1.S (IgG secreting) and CAG (IgA secreting) cell lines
ex vivo. Both HMCLs were established from end-stage multiple
myeloma patients, when myeloma cells expanded without restraint
independent of stromal support. They coexpressed high levels of
cyclin D2 and Cdk6, along with Cdk4 but not cyclin D1 (Fig. 2A),
as is the case with most other 25 HMCLs tested (data not shown).
PD 0332991 potently and rapidly inhibited Cdk4/6-specific phos-
phorylation of Rb (IC50= 0.06-0.07 Amol/L; Fig. 2A; data not shown)
in both MM1.S and CAG cells.
This led to G1cell cycle arrest, as evidenced by the profound
reduction in the proportion of MM1.S cells that took up BrdUrd in
a 30-minute pulse following treatment with 0.1 Amol/L PD
0332991, from 36% to 18% by 8 hours and to 7% by 12 hours
(Fig. 2B). This reduction was not enhanced by higher PD 0332991
concentrations, suggesting that induction of G1 arrest by PD
0332991 was saturated at 0.1 Amol/L (IC50 f 0.05 Amol/L).
Although the uptake of BrdUrd was also not reduced further by
24 hours of treatment with 0.1 Amol/L PD 0332991, presumably
due to depletion of PD 0332991, it was abolished by treatment
with 0.25 Amol/L PD 0332991 at this time (Fig. 2B). The reduction
of BrdUrd in response to PD 0332991 was corroborated by the
increases in cells in G1, as determined by fluorescence-activated
cell sorting analysis of DNA content (Supplementary Fig. S1).
Similar results were obtained with the CAG cells (data not shown).
PD 0332991, therefore, effectively induces G1arrest in a time- and
dose-dependent manner in cycling HMCL cells.
PD 0332991 did not induce apoptosis at concentrations that spe-
cifically inhibit Cdk4/6 because at concentrations below 5 Amol/L,
PD 0332991 did not increase the cleavage of PARP, indicative of
caspase activation, or Annexin V–binding activity (Fig. 2C, top; data
not shown). However, the expansion of total live myeloma cells
was reduced with increasing concentrations of PD 0332991, as
a consequence of induction of apoptosis in addition to G1arrest
(Fig. 2C, bottom). Taken together, these results show that at
concentrations that specifically inhibit Cdk4/6, PD 0332991
potently induces G1arrest without inducing apoptosis in cycling
Figure 1. PD 0332991 inhibits Cdk4/6 and progression through G1in primary
CD138+bone marrow myeloma cells. A, immunoblotting of pSRb807/811in
CD138+bone marrow myeloma cells freshly isolated from multiple myeloma
patients (Pt 1, stage III, treated; Pt 2, stage II, untreated) and cultured ex vivo
with PD 0332991 (PD) for 12 hours. Similar results were obtained with PD
0332991 treatment for 4 to 6 hours. B, bone marrow myeloma cells from multiple
myeloma patient (Pt 3, stage III, untreated) were cocultured ex vivo with HS-5
stromal cells and PD 0332991 for 24 hours. The ratios of pSRb to total Rb were
determined by densitometry analysis. C, fluorescence-activated cell sorting
analysis of BrdUrd (BrdU) uptake in freshly isolated CD138+bone marrow
myeloma cells (Pt 4, stage III, untreated) cocultured with PKH67 green-labeled
HS-5 human stromal cells and PD 0332991 for 13 hours, with BrdUrd present in
the last 10 hours. BrdUrd uptake was measured using an APC-anti-BrdUrd
antibody. Representative of 14 experiments (A and B). Representative of three
6M. Di Liberto, R. Gottschalk, and S. Chen-Kiang, unpublished.
PD 0332991 Prevents Myeloma Tumor Growth
Cancer Res 2006; 66: (15). August 1, 2006
PD 0332991 inhibits Cdk4/6 proportional to the activation
status of the cells in concert with p18INK4c. The ability of PD
0332991 to inhibit Cdk4/6 in proportion to the cycling status in
primary myeloma cells suggests that PD 0332991 may preferentially
target myeloma cells, particularly during relapse. To address this
possibility further, we asked whether PD 0332991 also inhibits
Cdk4/6 proportional to the cell activation status in nontrans-
formed primary B cells. Resting (G0-G1) mouse splenic B cells were
isolated and treated with increasing concentrations of PD 0332991
before or after activation ex vivo by anti-IgM, which functions as a
surrogate antigen to induce cell cycle reentry and progression. In
resting B cells, the levels of Cdk4/6-specific phosphorylation of Rb,
Cdk4, and cyclin D2 were all below detection by immunoblotting,
as reported previously (25). However, albeit extremely low, pSRb
and a gradual diminution with increasing amounts of PD 0332991
were seen following prolonged exposure of the same blot (1 hour;
Fig. 3, *). By contrast, in anti-IgM–activated B cells, Rb was highly
phosphorylated as a consequence of elevated Cdk4 and cyclin D2
synthesis. PD 0332991 potently inhibited Cdk4 phosphorylation
of Rb (IC50f 0.05 Amol/L), with an efficiency comparable with
that in cycling HMCLs (Fig. 2) and cycling primary bone marrow
myeloma cells (Fig. 1). PD 0332991, therefore, inhibits Cdk4
proportional to the activation status of the cells independent of
The physiologic Cdk4/6 inhibitor p18INK4cmay have a role in
myeloma pathogenesis as it is deleted in some HMCLs (32) and
is not detectable by immunohistochemistry in primary CD138+
bone marrow myeloma cells in 37% of multiple myeloma cases
(n = 245).7To further investigate the mechanism of PD 0332991
action, we asked whether PD 0332991 inhibits Cdk4/6 independent
of p18INK4c. In resting B cells, the absence of p18 seemed to lower
the threshold of Cdk4 activation because Cdk4 phosphorylation of
Rb in p18?/?cells (detected in a 5-minute exposure of the same
blot, *) was substantially higher than in their normal counterparts
(Fig. 3), although it had no effect on the expression of Cdk4 or
cyclin D2. Importantly, following activation of the cell cycle by anti-
IgM, inhibition of Cdk4 phosphorylation of Rb by PD 0332991 was
compromised in p18?/?B cells (IC50f 0.12 Amol/L) compared
with their normal counterparts (Fig. 3). PD 0332991, therefore,
inhibits Cdk4 in concert with the physiologic Cdk4/6 inhibitor p18
in nontransformed B cells.
PD 0332991 prevents tumor growth in a disseminated
xenograft human myeloma model. Our finding that PD
0332991 inhibits Cdk4/6 according to the cell activation status
further supports investigating the antitumor effect of PD 0332991
in vivo. We addressed this possibility in a newly developed NOD/
SCID xenograft human multiple myeloma model in which
disseminated tumors develop aggressively following injection of
the HMCL CAG cells stably expressing the HSV-TK-eGFP-luciferase
fusion protein. The tumor distribution can be quantified within
days of tumor induction by serial whole-body noninvasive
bioluminescence imaging of visible light emitted by luciferase-
expressing multiple myeloma cells upon luciferin injection (28).
PD 0332991 rapidly inhibited myeloma tumor growth to 2.1%
(dorsal) and 3.9% (ventral) by the end of the 12-day treatment
(Fig. 4A). As a control, the luciferase activity in HSV-TK-eGFP-
luciferase+CAG cells was sustained following overnight incubation
with PD 0332991 in vitro (Supplementary Fig. S2). Kaplan-Meier
survival curves (P = 0.0007) further showed that the untreated mice
Figure 2. PD 0332991 selectively inhibits Cdk4/6 and induces G1arrest in
HMCLs. A, immunoblotting of pSRb807/811in MM1.S and CAG cells treated
with PD 0332991 for 12 hours. The ratios of pSRb to total Rb were determined
by densitometry analysis. B, fluorescence-activated cell sorting analysis of
BrdUrd uptake in MM1.S cells treated with PD 0332991 for time indicated, with
BrdUrd present in the last 30 minutes before cell harvesting. C, immunoblotting
of intact (116 kDa) and cleaved (89 kDa) PARP in MM1.S cells cultured with
PD 0332991 for 12 hours (top). Live CAG and MM1.S cells cultured in the
presence of PD 0332991 was determined by trypan blue exclusion and
expressed as the percentage of the number of live cells at 0 hour (bottom). Three
independent cell counts. Representative of three experiments.
Figure 3. PD 0332991 inhibits Cdk4 according to the activation status of primary
mouse B cells. Immunoblotting of pSRb807/811of freshly isolated p18+/+and
p18?/?resting B cells cultured with PD 0332991 for 8 hours (Resting), or
cultured in the presence of anti-IgM and BLyS (BAFF) for 36 hours and an
additional 8 hours in the presence of PD 0332991 (Activated ex vivo).
* designates extended exposure of pSRb in resting cells. The ratios of pSRb to
total Rb were determined by densitometry analysis. Representative of six
7S. Ely and S. Chen-Kiang, unpublished.
Cancer Res 2006; 66: (15). August 1, 2006
died of tumor burden around 35 days after tumor induction,
whereas the PD 0332991-treated mice survived (Fig. 4B). They also
recovered completely from the loss of body weight (f15%) during
treatment (Fig. 4C). However, tumor growth resumed after dis-
continuation of PD 0332991 treatment, and the treated mice suc-
cumbed to death f12 days later than the untreated mice (Fig. 4B).
Confirming that PD 0332991 rapidly inhibits the Cdk4/6
activities in vivo, treatment of tumor-induced mice with PD
0332991 overnight was sufficient to drastically reduce Cdk4/6-
specific Rb phosphorylation in CD138+CAG cells present in the
vertebrae (Fig. 4D). PD 0332991 did not induce apoptosis of CAG
cells in vivo based on the absence of TUNEL and caspase-3 activity
(Fig. 4D; data not shown). There were also no overt changes in the
spleen architecture (Fig. 4D), consistent with the absence of liver
toxicity or other gross side effects following long-term PD 0332991
treatment (Fig. 4D; data not shown). Thus, through specific
inhibition of Cdk4/6, PD 0332991 nearly completely prevents
myeloma tumor growth in the aggressive, disseminated NOD/SCID
xenograft human myeloma model.
PD 0332991 enhances killing of myeloma cells by dexa-
methasone. As a therapeutic agent, PD 0332991 can be used either
at low concentrations to specifically and potently inhibit Cdk4/6
without induction of apoptosis, or at higher doses (>5 Amol/L) to
induce apoptosis (Fig. 2C). These features suggest that PD 0332991
is an ideal molecule to specifically target Cdk4/6 and induce G1
arrest in combination with a second cytotoxic agent. This may lead
to synergistic killing of myeloma cells, with a lower concentration
of the cytotoxic agent required to bring about the same level of
killing, or, with the same dose of each agent, a greater level of
killing. We chose as the second agent dexamethasone, a
glucocorticoid that is currently used as an antitumor agent in
the treatment of myeloma as well as other cancers (33).
Induction of G1arrest in MM1.S myeloma cells by treatment
with 2 Amol/L PD 0332991 for 24 hours did not induce apoptosis
as anticipated (Fig. 5A and B). However, when this was followed
by treatment with dexamethasone for 48 hours, both the
proportion and the total number of live cells were markedly
reduced. The dose of dexamethasone required to achieve the same
level of killing was 10 times lower when used in combination with
PD 0332991 than alone (Fig. 5B, top, compare 0.01 Amol/L
dexamethasone/PD 0332991 with 0.1 Amol/L dexamethasone
Figure 5. PD 0332991 enhances killing of myeloma cells by dexamethasone.
A, MM1.S cells were incubated in the presence or absence of 2 Amol/L PD
0332991 for 24 hours before addition of dexamethasone. Analysis of total live
cells by trypan blue staining (B), and DNA content per cell by propidium iodide
analysis (C) was done at 48 hours after addition of dexamethasone.
Representative of three independent experiments.
Figure 4. PD 0332991 prevents tumor
growth in a disseminated NOD/SCID
xenograft human myeloma model.
A, HSV-TK-eGFP-luciferase–positive CAG
cells were injected in NOD/SCID mice;
150 mg/kg of PD 0332991 was given on
day 7 after tumor induction, and the tumor
mass (photons/s/cm2/steradian) was
evaluated. % Ventral and dorsal tumor
burden relative to that of vehicle-treated
(untreated) mice. Columns, tumor burden
from six individual mice. B, Kaplan-Meier
survival curves of six PD 0332991–treated
mice and six vehicle-treated (untreated)
mice. C, % body weight of PD 0332991–
treated and untreated mice relative
to the start of PD 0332991 treatment.
D, immunohistochemical analysis of
simultaneous expression of CD138 (red)
and Rb (brown), pSRb807/811(brown),
or TUNEL (brown) in vertebrae of
tumor-induced mice 12 hours after
administration of PD 0332991 or vehicle.
% CD138+cells expressing total Rb or
pSRb807/811. As a control, spleen sections
from the same mice were stained with
hematoxylin. Representative of two
PD 0332991 Prevents Myeloma Tumor Growth
Cancer Res 2006; 66: (15). August 1, 2006
DNA content analysis indicated that dexamethasone treatment
alone resulted in an increase in dead cells (<2N) and a corre-
sponding reduction of cells in S and G2-M phases but not in G1
(Fig. 5C, top). These results were corroborated with the reduction
in viability and loss of total live cells (Fig. 5B), confirming that
dexamethasone acts mainly as a cytotoxic agent. Consistent with
specific inhibition of Cdk4/6, PD 0332991 treatment alone led to a
time-dependent increase in the proportion of MM1.S cells in G1,
from 55% to f80% by 72 hours, and a corresponding reduction of
cells in S and G2-M phases (Fig. 5C, bottom). The subsequent
combination with dexamethasone treatment resulted in a striking
dose-dependent increase in the proportion of dead cells (<2N;
Fig. 5C). The killing of MM1.S cells by 0.01 Amol/L dexamethasone
in combination with PD 0332991 was comparable to that by
0.1 Amol/L dexamethasone alone. With increasing dexamethasone
concentrations, the killing was further augmented by combining
with PD 0332991.
Thus, as a proof of concept, sequential treatment with PD
0332991 and dexamethasone leads to synergistic killing of myeloma
cells. This finding strongly suggests that by combining the ability of
PD 0332991 to specifically inhibit Cdk4/6 with a second cytotoxic
agent, a PD 0332991–based combination therapy represents a novel
and effective therapeutic strategy for myeloma.
Inhibition of Cdk4/6 by PD 0332991 prevents myeloma
tumor progression. PD 0332991, a newly developed small-
molecule inhibitor of Cdk4 and Cdk6, is superior to other Cdk4
or Cdk6 inhibitors that have been used in clinical trials in its oral
activity, high potency, and specificity (19, 34). Treatment of HMCLs
or myeloma cells in vitro with the Cdk inhibitor flavopiridol, which
also inhibits Cdk2 (35), or R-roscovitine, which poorly inhibits Cdk4
and Cdk6 (IC50> 100 Amol/L; ref. 36), mainly leads to apoptosis
(22, 23). It is unclear whether either compound inhibits Cdk4 and
Cdk6 in multiple myeloma cells selectively or has antitumor
activity in vivo. We showed in this study that PD 0332991 potently
inhibits Cdk4/6 in primary bone marrow myeloma cells (Fig. 1) and
nearly completely prevents the growth of human myeloma tumors
in aggressive, disseminated xenografts without gross side effects
Moreover, the high specificity of PD 0332991 action lies in part in
its ability to inhibit Cdk4 and Cdk6 according to the activation
status of the cells independent of cellular transformation. PD
0332991 inhibits Cdk4/6 much more effectively in cycling primary
bone marrow myeloma cells and in HMCL cells than in noncycling
primary bone marrow myeloma cells (Figs. 1-2) and in activated
primary mouse B cells more than in resting B cells (Fig. 3).
Of interest, PD 0332991 inhibits Cdk4/6 in concert with the
physiologic CKI p18 (Fig. 3). p18 inhibits Cdk4/6 mainly by forming
a stable inactive binary complex, thereby precluding the activation
of Cdk4/6 by D cyclins (9). It can also form a ternary complex with
Cdk4/6-cyclin D and inhibit Cdk4/6 (37). PD 0332991 inhibits
Cdk4/6 by competing for the ATP-binding sites on Cdk4 and Cdk6,
even when they are in complex with the D cyclin (19). Whether the
partial dependence on p18 for PD 0332991 inhibition of Cdk4/6
stems from cooperative binding of p18 and PD 0332991 to distal
sites on the same kinase, or from differential inhibition of free
Cdk4/6 and cyclin D–bound Cdk4/6 remains to be determined.
In either case, p18 protein expression is below detection by
immunohistochemistry in primary bone marrow myeloma cells
in f37% of multiple myeloma cases.7The genes encoding p18 (32)
and other INK4 family CKIs have been reported to be deleted or
inactivated by hypermethylation in myeloma cell lines (38, 39).
Understanding the mechanism by which PD 0332991 and INK4
CDK inhibitors, in particular p18, cooperate in inhibiting Cdk4/6
should provide novel insights into targeting Cdk4/6 in myeloma.
PD 0332991 as a novel mechanism-driven therapeutic stra-
tegy for myeloma. PD 0332991 is orally active, water soluble, and
seems to selectively inhibit Cdk4/6 without inducing drug
resistance in solid tumor xenografts (19). These are favorable fea-
tures for therapeutic application. Cdk4 and Cdk6 are dispensable
for development (40). We showed that PD 0332991 inhibits Cdk4/6
according to the activation status of the cells, in particular in cycl-
ing primary bone marrow myeloma cells freshly isolated from both
new and relapsed patients (Fig. 1). These findings provide a strong
rationale for targeting myeloma progression with PD 0332991.
In support of this possibility, we showed by noninvasive
bioimaging in our aggressive, disseminated CAG-NOD/SCID xeno-
graft human myeloma model (28) that PD 0332991 rapidly and
effectively prevents myeloma tumor growth, with reversible weight
loss and without discernible side effects (Fig. 4). When PD 0332991
was similarly given in rats, the maximum concentration of PD
0332991 in plasma was estimated to be f5 Amol/L (34). Assuming
that the pharmacokinetics of PD 0332991 in mice is comparable
with that in rats, this plasma concentration would allow PD 0332991
to act as a specific Cdk4/6 inhibitor without inducing cell death
in vivo, as observed in vitro (Figs. 1-2). Consistent with this possi-
bility, we found that PD 0332991 prevented tumor progression in
xenografts without causing overt side effects (Fig. 4). Together, these
observations support the potential to inhibit Cdk4/6 with specificity
by PD 0332991 in myeloma therapy.
Inhibition of Cdk4/6 by PD 0332991 in combination therapy.
There has been renewed interest in the development of a successful
Cdk4/6 inhibitor in cancer therapy. Two recently published studies
showed a role for Cdk4-cyclin D1 kinase activity in ErbB-2-induced
mammary tumors (41, 42). Because cyclin overexpression and Cdk
activation is a common feature in breast tumors and other cancers,
including myeloma, the development of a successful Cdk inhibitor
could have significant benefit in cancer treatment.
Here, we show that killing of myeloma cells by dexamethasone
can be augmented by inhibition of Cdk4/6 and induction of G1cell
cycle arrest with PD 0332991 (Fig. 5). How a steroid, such as dexa-
methasone, functions as a cytotoxic agent is unknown. However,
recent evidence suggests that it may involve the phosphatidylino-
sitol 3-kinase (PI3K)/Akt pathway because inhibition of this
pathway seems to enhance the killing of a human follicular
lymphoma cell line by dexamethasone (43). Consistent with this
notion, prolonged treatment of MM1.S cells with 2 Amol/L PD
0332991 (48 hours) led to a modest reduction in the phosphory-
lation of Akt.8It is tempting to speculate that as a secondary effect
of prolonged G1arrest in response to PD 0332991, reduction of the
PI3K/Akt pathway may contribute to enhanced killing of myeloma
cells by dexamethasone. This possibility awaits future exploration.
Although as a proof of concept we have presented evidence here
that PD 0332991 can enhance the killing of myeloma cells by
dexamethasone, it may also be possible to achieve greater synergy
by combining PD 0332991 with other cytotoxic agents. The pro-
teasome inhibitor Bortezomib (44) is one attractive possibility.
8L.B. Baughn and S. Chen-Kiang, unpublished.
Cancer Res 2006; 66: (15). August 1, 2006
Although Bortezomib is widely used in the treatment of myeloma Download full-text
(45), only 30% of multiple myeloma patients respond (44), and the
basis for Bortezomib resistance remains unknown. Multiple mye-
loma remains incurable in part due to a lack of a mechanism-
defined combination therapy that will not only suppress tumor
progression but also prevent residual disease. The functional
evidence presented in this study strongly suggests that in
combination with a second agent, PD 0332991 is a novel candidate
for mechanism-defined combination therapy in myeloma and also
possibly other B-cell malignancies.
Received 3/24/2006; revised 5/30/2006; accepted 6/1/2006.
Grant support: NIH T32 postdoctoral fellowship (L.B. Baughn), NIH grant RO1 AR
49436 (S. Chen-Kiang), Translational Research Program grants (S. Chen-Kiang and
M.A.S. Moore), and a Specialized Center of Research grant from the Leukemia and
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Josefina Garcia for advice on the studies of primary mouse B cells;
Dr. Stephen Eck for helpful comments and suggestions; David Jayabalan, Kang Zhang,
Yi-Fang Liu, and Plinio Silva for technical assistance; and members of the Chen-Kiang
laboratory for stimulating discussions.
PD 0332991 Prevents Myeloma Tumor Growth
Cancer Res 2006; 66: (15). August 1, 2006
1. Chen-Kiang S. Cell-cycle control of plasma cell
differentiation and tumorigenesis. Immunol Rev 2003;
2. Morse L, Chen D, Franklin D, Xiong Y, Chen-Kiang S.
Induction of cell cycle arrest and B cell terminal
differentiation by CDK inhibitor p18(INK4c) and IL-6.
3. Tourigny MR, Ursini-Siegel J, Lee H, et al. CDK
inhibitor p18(INK4c) is required for the generation of
functional plasma cells. Immunity 2002;17:179–89.
4. Robillard N, Pellat-Deceunynck C, Bataille R. Pheno-
typic characterization of the human myeloma cell
growth fraction. Blood 2005;105:4845–8.
5. Matsui W, Huff CA, Wang Q, et al. Characterization of
clonogenic multiple myeloma cells. Blood 2004;103:
6. Greipp PR, Witzig TE, Gonchoroff NJ, et al. Immuno-
fluorescence labeling indices in myeloma and related
monoclonal gammopathies. Mayo Clin Proc 1987;62:
7. Sherr CJ, Roberts JM. CDK inhibitors: positive and
negative regulators of G1-phase progression. Genes Dev
8. Harbour JW, Dean DC. The Rb/E2F pathway: expand-
ing roles and emerging paradigms. Genes Dev 2000;14:
9. Guan KL, Jenkins CW, Li Y, et al. Growth suppression
by p18, a p16INK4/MTS1- and p14INK4B/MTS2-related
CDK6 inhibitor, correlates with wild-type pRb function.
Genes Dev 1994;8:2939–52.
10. Hirai H, Roussel MF, Kato JY, Ashmun RA, Sherr CJ.
Novel INK4 proteins, p19 and p18, are specific inhibitors
of the cyclin D-dependent kinases CDK4 and CDK6. Mol
Cell Biol 1995;15:2672–81.
11. Sanderson RD, Lalor P, Bernfield M. B lymphocytes
express and lose syndecan at specific stages of
differentiation. Cell Regul 1989;1:27–35.
12. Chesi M, Bergsagel PL, Brents LA, Smith CM,
Gerhard DS, Kuehl WM. Dysregulation of cyclin D1 by
translocation into an IgH gamma switch region in two
multiple myeloma cell lines. Blood 1996;88:674–81.
13. Shaughnessy J, Jr., Gabrea A, Qi Y, et al. Cyclin D3 at
6p21 is dysregulated by recurrent chromosomal trans-
locations to immunoglobulin loci in multiple myeloma.
14. Zhan F, Tian E, Bumm K, Smith R, Barlogie B,
Shaughnessy J, Jr. Gene expression profiling of human
plasma cell differentiation and classification of multiple
myeloma based on similarities to distinct stages of late-
stage B-cell development. Blood 2003;101:1128–40.
15. Fonseca R, Blood EA, Oken MM, et al. Myeloma and
the t(11;14)(q13;q32); evidence for a biologically defined
unique subset of patients. Blood 2002;99:3735–41.
16. Moreau P, Facon T, Leleu X, et al. Recurrent 14q32
translocations determine the prognosis of multiple
myeloma, especially in patients receiving intensive
chemotherapy. Blood 2002;100:1579–83.
17. Soverini S, Cavo M, Cellini C, et al. Cyclin D1
overexpression is a favorable prognostic variable for
newly diagnosed multiple myeloma patients treated
with high-dose chemotherapy and single or double
autologous transplantation. Blood 2003;102:1588–94.
18. Ely* SA, Di Liberto* M, Niesvizky R, et al. Mutually
exclusive Cdk4-Cyclin D1 and Cdk6-Cyclin D2 pairing
inactivates Rb and promotes cell cycle dysregulation in
multiple myeloma (*equal contribution). Cancer Res
19. Fry DW, Harvey PJ, Keller PR, et al. Specific inhibition
of cyclin-dependent kinase 4/6 by PD 0332991 and
associated antitumor activity in human tumor xeno-
grafts. Mol Cancer Ther 2004;3:1427–38.
20. Sausville EA. Cyclin-dependent kinase modulators
studied at the NCI: pre-clinical and clinical studies. Curr
Med Chem Anti-Canc Agents 2003;3:47–56.
21. Senderowicz AM. Novel small molecule cyclin-
dependent kinases modulators in human clinical trials.
Cancer Biol Ther 2003;2:S84–95.
22. Raje N, Kumar S, Hideshima T, et al. Seliciclib
(CYC202 or R-roscovitine), a small-molecule cyclin-
dependent kinase inhibitor, mediates activity via down-
regulation of Mcl-1 in multiple myeloma. Blood 2005;
23. Gojo I, Zhang B, Fenton RG. The cyclin-dependent
kinase inhibitor flavopiridol induces apoptosis in
multiple myeloma cells through transcriptional repres-
sion and down-regulation of Mcl-1. Clin Cancer Res
24. Durie BG. Staging and kinetics of multiple myeloma.
Semin Oncol 1986;13:300–9.
25. Huang X, Di Liberto M, Cunningham AF, et al.
Homeostatic cell-cycle control by BLyS: induction of
cell-cycle entry but not G1/S transition in opposition to
p18INK4cand p27Kip1. Proc Natl Acad Sci U S A 2004;101:
26. Franklin DS, Godfrey VL, Lee H, et al. CDK inhibitors
p18(INK4c) and p27(Kip1) mediate two separate path-
ways to collaboratively suppress pituitary tumorigene-
sis. Genes Dev 1998;12:2899–911.
27. Do RK, Hatada E, Lee H, Tourigny MR, Hilbert D,
Chen-Kiang S. Attenuation of apoptosis underlies B
lymphocyte stimulator enhancement of humoral im-
mune response. J Exp Med 2000;192:953–64.
28. Wu KD, Cho YS, Katz J, et al. Investigation of
antitumor effects of synthetic epothilone analogs in
human myeloma models in vitro and in vivo. Proc Natl
Acad Sci U S A 2005;102:10640–5.
29. Knudsen ES, Wang JY. Differential regulation of
retinoblastoma protein function by specific Cdk phos-
phorylation sites. J Biol Chem 1996;271:8313–20.
30. Zarkowska T, Mittnacht S. Differential phosphoryla-
tion of the retinoblastoma protein by G1/S cyclin-
dependent kinases. J Biol Chem 1997;272:12738–46.
31. Roecklein BA, Torok-Storb B. Functionally distinct
human marrow stromal cell lines immortalized by
transduction with the human papilloma virus E6/E7
genes. Blood 1995;85:997–1005.
32. Kulkarni MS, Daggett JL, Bender TP, Kuehl WM,
Bergsagel PL, Williams ME. Frequent inactivation of the
cyclin-dependent kinase inhibitor p18 by homozygous
deletion in multiple myeloma cell lines: ectopic p18
expression inhibits growth and induces apoptosis.
33. Alexanian R, Dimopoulos MA, Delasalle K, Barlogie
B. Primary dexamethasone treatment of multiple
myeloma. Blood 1992;80:887–90.
34. Toogood PL, Harvey PJ, Repine JT, et al. Discovery of
a potent and selective inhibitor of cyclin-dependent
kinase 4/6. J Med Chem 2005;48:2388–406.
35. Carlson BA, Dubay MM, Sausville EA, Brizuela L,
Worland PJ. Flavopiridol induces G1 arrest with
inhibition of cyclin-dependent kinase (CDK) 2 and
CDK4 in human breast carcinoma cells. Cancer Res
36. Meijer L, Borgne A, Mulner O, et al. Biochemical and
cellular effects of roscovitine, a potent and selective
inhibitor of the cyclin-dependent kinases cdc2, cdk2 and
cdk5. Eur J Biochem 1997;243:527–36.
37. Jeffrey PD, Tong L, Pavletich NP. Structural basis of
inhibition of CDK-cyclin complexes by INK4 inhibitors.
Genes Dev 2000;14:3115–25.
38. Ng MH, Chung YF, Lo KW, Wickham NW, Lee JC,
Huang DP. Frequent hypermethylation of p16 and p15
genes in multiple myeloma. Blood 1997;89:2500–6.
39. Tasaka T, Asou H, Munker R, et al. Methylation of the
p16INK4Agene in multiple myeloma. Br J Haematol 1998;
40. Malumbres M, Sotillo R, Santamaria D, et al. Mammal-
ian cells cycle without the D-type cyclin-dependent
kinases Cdk4 and Cdk6. Cell 2004;118:493–504.
41. Landis MW, Pawlyk BS, Li T, Sicinski P, Hinds PW.
Cyclin D1-dependent kinase activity in murine develop-
ment and mammary tumorigenesis. Cancer Cell 2006;9:
42. Yu Q, Sicinska E, Geng Y, et al. Requirement for
CDK4 kinase function in breast cancer. Cancer Cell
43. Nuutinen U, Postila V, Matto M, et al. Inhibition of
PI3-kinase-Akt pathway enhances dexamethasone-in-
duced apoptosis in a human follicular lymphoma cell
line. Exp Cell Res 2006;312:322–30.
44. Adams J, Kauffman M. Development of the protea-
some inhibitor Velcade (bortezomib). Cancer Invest
45. Richardson PG, Barlogie B, Berenson J, et al. A phase
2 study of bortezomib in relapsed, refractory myeloma.
N Engl J Med 2003;348:2609–17.