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Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia


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Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.
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Clonal dynamics towards the development of
venetoclax resistance in chronic lymphocytic
Carmen D. Herling1, Nima Abedpour2,3, Jonathan Weiss1, Anna Schmitt1,3,4, Ron Daniel Jachimowicz1,3,4,
Olaf Merkel1,4, Maria Cartolano2,3, Sebastian Oberbeck1,3,4,5, Petra Mayer1,3,4,5, Valeska Berg1,4,
Daniel Thomalla1,4, Nadine Kutsch1, Marius Stiefelhagen1, Paula Cramer1, Clemens-Martin Wendtner6,
Thorsten Persigehl7, Andreas Saleh8, Janine Altmüller3,9, Peter Nürnberg3,4,9, Christian Pallasch1,3,4,
Viktor Achter10, Ulrich Lang10,11, Barbara Eichhorst1, Roberta Castiglione12, Stephan C. Schäfer12,
Reinhard Büttner12, Karl-Anton Kreuzer1, Hans Christian Reinhardt1,3,4, Michael Hallek1,3,4, Lukas P. Frenzel1,4 &
Martin Peifer 2,3
Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to
understand the mechanisms that lead to treatment resistance. To this end, we analyzed
whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that
developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent muta-
tions in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that
developed during treatment, as well as a mutation in BRAF and a high-level focal amplication
of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further
therapeutic interventions.
DOI: 10.1038/s41467-018-03170-7 OPEN
1Department of Internal Medicine I, Center of Integrated Oncology Cologne-Bonn, University of Cologne, 50937 Cologne, Germany. 2Department of
Translational Genomics, Center of Integrated Oncology Cologne-Bonn, Medical Faculty, University of Cologne, 50931 Cologne, Germany. 3Center for
Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany. 4Cologne Excellence Cluster on Cellular Stress Response in Aging-
Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany. 5Laboratory of Lymphocyte Signaling and Oncoproteom, University of
Cologne, 50931 Cologne, Germany. 6Department of Hematology, Oncology, Immunology, Palliative Care, Infectious Diseases and Tropical Medicine,
Klinikum Schwabing, 80804 Munich, Germany. 7Department of Radiology, Cologne University Hospital, 50937 Cologne, Germany. 8Department of
Diagnostic and Interventional Radiology and Pediatric Radiology, Städtisches Klinikum München Schwabing, 80804 Munich, Germany. 9Cologne Center for
Genomics (CCG), University of Cologne, 50931 Cologne, Germany. 10 Computing Center, University of Cologne, 50931 Cologne, Germany. 11 Department of
Informatics, University of Cologne, 50931 Cologne, Germany. 12 Department of Pathology, University of Cologne, 50937 Cologne, Germany. Carmen D.
Herling, Nima Abedpour, Lukas P. Frenzel, and Martin Peifer contributed equally to this work. Correspondence and requests for materials should be
addressed to M.P. (email:
NATURE COMMUNICATIONS | (2018) 9:727 |DOI: 10.1038/s41467-018-03170-7 | 1
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Chronic lymphocytic leukemia (CLL) is the most common
leukemia in the western world with a diverse clinical
course and a substantial degree of inter- and intra-patient
heterogeneity18. Ongoing clonal evolution is sought to be a key
mechanism for the development of treatment-resistant or
-refractory CLL3,4,9and is, therefore, limiting treatment success
and duration. Data from whole-exome or genome sequencing can
be used to reconstruct the clonal evolution of cancer specimens
and has led to a deeper understanding of underlying principles10
13. In CLL, studies of clonal evolution under treatment with the
Bruton tyrosine kinase (BTK) inhibitor ibrutinib have provided
novel insights into changes of the clonal architecture towards
treatment resistance3,14.
Recently, treatment with the BCL2-inhibitor venetoclax has
demonstrated substantial efcacy, even in high-risk relapsed and
chemotherapy-refractory CLL patients with alterations in
TP532,15,16. This group of patients has a particularly poor prog-
nosis2. In contrast to ibrutinib, genetic causes underlying resis-
tance to the treatment with venetoclax have not been determined,
so far. To this end, we conducted whole-exome sequencing and
methylation proling of serial CLL samples obtained from eight
individuals before initiation of venetoclax treatment and at the
manifestation of resistance. From these data, we inferred the
clonal evolution under venetoclax therapy and identied recur-
rent genome alterations that developed during treatment.
Samples and clinical data. All patients were previously treated
(18 previous lines of treatment) and harbored either genomic
losses (del(17p)) or protein-damaging point mutations in TP53 in
all samples. The median time between the initiation of venetoclax
treatment to clinical progression/relapse was 15.4 months (Sup-
plementary Table 1). Partial remission as best response to vene-
toclax was achieved in six patients (Fig. 1a, Supplementary Fig. 1).
Patient C586 presented a stable disease with disappearance of
lymphocytosis, but only a reduction of nodal mass by 41%
(Fig. 1a), and patient C548 showed a stable disease, due to per-
sisting splenomegaly. Time to progressive disease ranged between
4 and 22 months. Half of the patients developed a Richters
transformation (RT) during venetoclax treatment (Fig. 1b, Sup-
plementary Table 1), histologically presenting as diffuse large B-
cell lymphoma.
Accumulation of genome alterations during treatment.On
average, we detected a total of 25.5 exonic mutations (including
synonymous and non-synonymous point mutations, insertions,
and deletions) prior to venetoclax therapy, in accordance with
previous CLL exome-sequencing efforts3. As expected for ongo-
ing evolutionary processes, copy number changes and the number
of point mutations increased during venetoclax therapy (Fig. 1b).
Remarkably, for patient C651 we did not detect any copy number
change before venetoclax treatment, but 12.5% of the genome
showed losses or gains at the occurrence of venetoclax resistance
(Fig. 1b). At time of relapse, only lymph node specimens with a
lower purity were available for most patients, in contrast to the
generally pure pre-treatment samples derived from peripheral
blood (Fig. 1b, Supplementary Table 2). This hampers a robust
assessment of treatment-specic changes in the methylation
proles, since most of the variability seen in the methylation
patterns within each patient is likely due to differences in the cell
type composition of the compartments analyzed (Supplementary
Fig. 2).
Alterations in cancer-related genes. We next selected somatic
genome alterations in cancer-related genes that showed clonal
dynamics during venetoclax therapy. Recurrent non-synonymous
mutations were seen in: TP53,NOTCH1,BTG1, whereas BRAF,
SF3B1,RB1,BIRC3, and MLL3 were non-synonymously affected
in single patients only (Fig. 1b). These mutations were validated
by either dideoxy sequencing or digital droplet PCR (except for
TP53 mutations that were previously assessed at study inclusion)
16 (Supplementary Fig. 3). Note that the mutation in BIRC3 could
not be validated, due to a lack of genomic material after whole-
exome sequencing. Allelic fractions between digital droplet PCR
and whole-exome sequencing were highly comparable for single-
nucleotide mutations (Supplementary Fig. 3a). Furthermore, we
observed homozygous deletions of CDKN2A/B and a high-level
focal amplication containing CD274 encoding for the immune-
checkpoint ligand PD-L1 (Fig. 1b). Copy numbers from exome
sequencing were validated by methylation arrays (Supplementary
Fig. 4).
In line with previous ndings15,16 patients responded to
venetoclax therapy, even if TP53 was initially mutated in a bi-
allelic fashion (5/8 patients). Two patients showed genome
alterations that might qualify for further therapeutic options after,
or in combination with venetoclax therapy: (1) patient C548
harbored a BRAF (p.K601E) mutation that was shown to be
oncogenic and can be targeted by, e.g., MEK inhibitors17, and (2)
the CD274 amplication, which was paralleled by high CD274
protein expression levels and a prominent inltrate of CD3-
positive T cells (Fig. 1c) may be susceptible to immune-
checkpoint blockade18 in patient C811. High-level and focal
amplications of CD274 have been described in a variety of
human cancer entities19,20, but so far not in CLL. In contrast, a
pooled analysis of two major CLL-sequencing studies3,8revealed
that mutations in BRAF occur at a frequency of 3.8% (n=559) in
treatment-naive samples, and can, therefore, be considered as a
potential driver alteration in CLL.
Evolved recurrent somatic alterations. Recurrent genomic
changes that evolved during venetoclax treatment were homo-
zygous deletions affecting CDKN2A/B in three patients (C548,
C577, C586) and BTG1 missense mutations in two cases (C577: p.
Q36H; C789: p.E46K). BTG1 has been shown to counteract cell
proliferation and to be regulated downstream of BCL2 and
CDKN2A/B (p16Ink4a/p14Arf)21,22. Thus, aside the abrogation of
cell cycle control by loss of CDKN2A/B, damaging mutations in
BTG1 may provide a survival advantage to CLL cells under tar-
geted BCL2-inhibition. Non-synonymous mutations affecting
BTG1 have been rarely detected in untreated CLL patients (only
in 3 of 559 samples)3,8. Therefore, the probability that the BTG1
mutations developed spontaneously and are not due to a selection
process by the venetoclax treatment is 7.8 × 104. To assess the
frequency of homozygous CDKN2A/B deletions, we queried
125 samples for which copy number data were publically avail-
able8. This analysis revealed that homozygous deletions of
CDKN2A/B were not detected in these treatment-naive CLL
samples. However, homozygous loss of CDKN2A/B has been
associated with CLL cases that have undergone RT23,24. Intrigu-
ingly, two out of the three cases that developed homozygous
CDKN2A/B deletions in our series had undergone RT at relapse
(Fig. 1b). Furthermore, all patients with homozygous deletions of
CDKN2A/B also harbored other cancer-related mutations
(Fig. 1b).
Clonal dynamics under venetoclax treatment. To gain insight
into the clonal evolution towards therapy resistance, we inferred
subclonal populations and reconstructed phylogenetic trees (see
Methods). Intriguingly, we observed a wide spectrum of evolu-
tionary dynamics, including linear, divergent, and convergent
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evolution (Fig. 2and Supplementary Data). Patient C789 showed
a linear evolution, where the clone harboring the BTG1 (p.E46K)
mutation was selected as the dominant clone in the sample at
relapse (Fig. 2a). On the contrary, the other BTG1-mutated
sample (C577) exhibited a branching evolution into three mole-
cularly distinct lineages (Fig. 2b). The branch composed of sub-
population C1 and C2 was detected only in the pre-treatment
sample (T0-peripheral blood [PB]). The sample harvested during
treatment (T1-lymph node [LN]) only contained the branch of
subclones C3 and C4. However, the branch carrying the BTG1 (p.
Q36H) mutation was only seen in the relapse sample (T2-LN). In
addition, this branch also harbored a homozygous loss of
CDKN2A/B. Taken together, the evolutionary trajectory of the
clones carrying BTG1 mutations (Fig. 2a, b) is in favor of an
involvement of BTG1 in the resistance to venetoclax treatment.
Patient C548 showed a divergent evolutionary path of two
branches (Fig. 2c). One branch (subclone C3 and C4) was selected
during venetoclax therapy. This branch harbored a homozygous
loss of CDKN2A/B, and mutations in BRAF (p.K601E) and MLL3
(p.S321fs), which rst appeared in subclone C3 and were retained
in its descendent C4. Therefore, all those alterations appear to be
clonal in the relapse sample (T1-PB). In contrast, the branch
composed of subclone C1 and C2 was suppressed by venetoclax,
even though it contained a frameshift deletion in the known CLL
driver gene BIRC3 (p.Q547fs).
Finally, case C586 showed a remarkable pattern of convergent
evolution (Fig. 2d). We found two SF3B1 mutations (c.1996A > C;
p.K666Q and c.1997A > C; p.K666T) affecting the same codon,
but evolved in two independent clones during venetoclax
exposure (Supplementary Fig. 5). Both mutations were not
detected in the pre-treatment sample by whole-exome sequen-
cing. Digital droplet PCR, however, revealed that the SF3B1
mutations were already present in extremely small subclones
before treatment (T0-PB): a cancer cell fraction of 0.04% for p.
K666Q and 0.033% for p.K666T. This shows that the convergent
evolution of both SF3B1 mutations occurred before the initiation
of venetoclax therapy. Although both subclones carrying the
SF3B1 mutation were selected during treatment, they only
reached a combined cancer cell fraction of 62% in the peripheral
blood (T2-PB) and 96% in the bone marrow (T4-BM) sample at
relapse. This suggests that the SF3B1 mutations were not the
single cause of venetoclax resistance in this patient. In contrast,
Absolute lymphocyte counts (×1000/μl)
Lymphadenopathy (%)
change from baseline
–100 0 100 200 300 400 500 600 700
Bone marrow
Bone infiltrate
Lymph node
Peripheral blood
Number of
Richter's trans.
Giemsa Ki67 CD3 CD274
Homozygous loss
Hemizygous loss
Frame shift deletion
Frame shift insertion
Nonsense mutation
Splice site mutation
Missense mutation
Silent mutation
Fig. 1 Patient and their related matched pre-treatment and relapse samples characteristics. aAbsolute lymphocyte counts and lymphadenopathy of the
patients during venetoclax therapy. Day zero marks the start of the venetoclax treatment. Green lines show the time points of sample collection. Computer
tomography (CT) scans for staging were performed at the time points marked by red lines. bResults from whole-exome sequencing are shown, including:
number of somatic mutations, sample ploidy, percent of the genome undergoing copy number alterations (blue for losses and red for gains), and cancer-
related gene mutations with pronounced clonal dynamics during therapy. Genomic alterations are annotated according to the color panel below the image.
Sample type/compartment and the status if a patient has undergone a Richters transformation are additionally indicated. Pre-treatment samples (T0) are
shown in red. cGiemsa, Ki67, CD3, and CD274 stains from lymph node material of patient C811 after relapse from venetoclax. High protein levels of
CD274 are consistent with the genomic amplication of the locus containing CD274. Scale bar, 100 µm
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the rapidly proliferating (Ki67-score of 90%) extranodal disease
manifestation inside the right radial bone (T3-BI) was completely
dominated by the single subpopulation C7, which carried a
homozygous loss of CDKN2A/B.
Functional analyses. In order to assess if oncogenic BRAF sig-
naling may induce venetoclax resistance, we overexpressed
mutated BRAF (p.V600E) in a venetoclax-sensitive cell line OCI-
LY19 (Fig. 3a). Exome sequencing of this cell line revealed a
nonsense mutation in CDKN2A/B (p.W110*), as well as a geno-
mic loss of one allele of TP53 and a splice site mutation on the
other allele. Therefore, this cell line presents a similar damage of
key cancer-related genes, compared to patient C548. We found
that the BRAFV600E-transduced cell line exhibited a pronounced
venetoclax resistance with a half-maximal growth inhibitory
concentration (GI
) larger than 10 μM in contrast to the empty
vector control: GI
=0.43 μM (Fig. 3b). In another venetoclax-
sensitive cell line (U-2932) overexpression of the mutated
BRAFV600E led only to a slight increase of the GI
value (from
0.72 μM for the empty vector control to 0.99 μM; Supplementary
Fig. 6a). A reanalysis of published whole-exome-sequencing
data25 of U-2932 showed mutated TP53 together with a loss of
17p, but no alterations in CDKN2A/B. In both cases, the
expression of mutant BRAFV600E is paralleled by an increase in
MCL1 protein levels. Next, we tested if a loss of CDKN2A/B alone
can lead to venetoclax resistance and deleted the gene in the cell
line OSU26, using the CRISPR/Cas9 system. We found no dif-
ference in venetoclax sensitivity between OSU wild-type and
TP53 p.G245S
* hem-del
TP53 p.R213*
NOTCH1 p.P2514fs
MLL3 p.S321fs
BRAF p.K601E
TP53 p.S261fs
C7 C6
Fig. 2 Heterogeneous clonal evolutions under venetoclax therapy. Phylogenetic trees at the left side of each panel demonstrate the clonal evolution of the
reconstructed cell populations for each patient. Highlighted mutations that occurred during tumor evolution are present in all descendent clones. Therefore,
mutations in the most common ancestor population (C0) are present in all analyzed samples at a clonal level. The second type trees (right-bottom of each
panel) demonstrate the phylogenetic relations of the matched pre-treatment and relapse samples from a patient, as commonly used in other cancer
evolution studies10,13. Clonal composition of the samples (top-right of each panel) provides a link between both types of phylogenetic trees. We inferred
diverse evolutionary paths across the patients: alinear evolution (C789), bbranching evolution into three lineages (C577), cdivergent evolution of two
branches (C548), and dconvergent evolution (C586). Pre-treatment sample names are displayed in red. Notable gene alterations are shown in the context
of the ancestral relation of the clones
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CDKN2A/B knockout cells (Supplementary Fig. 6b). This result
may explain why the CDKN2A/B homozygous deletions co-
occured in all cases with other genomic alterations that developed
during treatment.
CLL with dysfunctional p53, especially in the situation of relapse
or refractory disease, has a particularly poor prognosis, and there
are only few effective treatment options available for this high-
risk category of patients. The BCL2 antagonist venetoclax has
recently been approved for treatment of patients p53-decient
CLL (del(17p) or TP53 mutations), who have failed or are not
suitable for B-cell receptor (BCR) pathway inhibition with ibru-
tinib or idelalisib. In addition, the drug can be used in patients,
who have failed both chemo-immunotherapy and BCR pathway
inhibition. Therefore, deciphering resistance mechanisms that
arise during venetoclax therapy is of high clinical relevance.
We analyzed whole-exome-sequencing data of CLL specimens
from eight patients before the initiation of venetoclax therapy and
at the time of venetoclax resistance. All patients had shown a
signicant clinical response to venetoclax, before occurrence of
disease progression or relapse. Our sequencing effort revealed
intriguingly diverse patterns of clonal dynamics, such as linear,
convergent, and divergent evolution under venetoclax treatment.
In addition, all patients demonstrated signs of accumulating
genomic instability, as demonstrated by an increasing number of
acquired copy number alterations or aneuploidy. In most patient
samples, we were able to detect alterations in cancer-related genes
(i.e., BRAF,CD274,NOTCH1,RB1,SF3B1, and TP53) that
evolved during venetoclax treatment. Surprisingly, genetic
alterations in BCL2 or functionally connected genes, such as BAX
and BAK were not identied. Furthermore, we detected muta-
tions in BTG1 and homozygous deletions of CDKN2A/B as
recurrent genomic events at the time of relapse under venetoclax
exposure. BTG1 might underlie an increased selection pressure
under venetoclax therapy, as it seems to be required to maintain
cell proliferation and its expression is regulated by BCL221,22.
Furthermore, our data suggests that complete loss of CDKN2A/B
alone is not sufcient to induce venetoclax resistance.
Overexpression of oncogenic BRAF in lymphoma cell lines
showed that it rendered lymphoma cells against venetoclax
resistance, in vitro. Further functional analyses, however, point to
a context-dependent interplay of genetically altered driver genes.
Given the diverse genomic dynamics in our patient set investi-
gated, it is likely that there are other/additional cellular
mechanisms involved in multiple molecular patterns, which
ultimately lead to venetoclax resistance in CLL cells. Once large
collectives of venetoclax-resistant CLL samples are available,
further studies are required to elucidate these mechanisms.
Notably, our sequencing effort was able to identify genome
alterations at relapse (BRAF,CD274) that might qualify for fur-
ther therapeutic options. Finally, comprehensive molecular test-
ing is a suitable methodology to decipher mechanisms of
venetoclax resistance and should be incorporated into future
protocols to create molecularly-targeted salvage strategies for CLL
patients under venetoclax treatment.
Patient sampling and nucleic acid extraction. Between February 2014 and
February 2016, we collected peripheral blood, bone marrow and tissue samples
from eight patients, who presented with relapsed/refractory CLL upon oral vene-
toclax therapy. The study was approved by the ethical review board of the Uni-
versity of Cologne and performed according to the Declaration of Helsinki. All
participants provided written informed consent.
Seven patients had received oral venetoclax as single agent therapy within the
M13-982 2012-004027-20 trial16 (NCT01889186). One patient (C789) had been
treated with two cycles of bendamustine and obinutuzumab debulking
(bendamustine 70 mg/m² day 1/2, q28d; obinutuzuma b 100 mg/900 mg day 1/2,
1000 mg d8/15 in cycle 1, 1000 mg d1/8/15 in cycle 26, and q84d thereafter) with
oral venetoclax added on day 1 of cycle 2 and maintained daily thereafter (CLL2-
BAG study of the German CLL study group, EudraCT: 2014-000580-40,
NCT02401503). After a weekly ramp-up schedule starting with 20 mg qod, all
patients had achieved maximum dosing of venetoclax at 400 mg qod.
Baseline (T0) peripheral blood samples were obtained at a median of 12 days
(range, 089) prior to the rst dose of venetoclax. At this time all patients had CLL
disease in peripheral blood detectable by ow cytometry according to the
International Workshop on Chronic Lymphocytic Leukemia guidelines 200827,
however, four patients presented a B lymphocyte count of <5.000/μl. Follow-up
samples from peripheral blood, bone marrow or tissue were collected whenever the
patient presented with signs of refractory or relapsing CLL disease and leftover
material was available from diagnostic procedures. High purity (95%) CLL cells
were separated from peripheral blood using a negative-selection of CD19-B-cells
and column-based magnetic cell separation (Miltenyi, Bergisch-Gladbach,
Germany). The CD19-negative non-B cells were collected as a matched non-cancer
cell fraction (normal) in each patient. At the time of refractory/progressive
disease only in 3 of the 8 patients CLL-B cells could be sufciently enriched from
peripheral blood, as most patients did not present with a peripheral blood
lymphocytosis at this time. In one case, a full lymph node biopsy (C577, T2-LN)
was obtained at disease progression and CLL-B cells were selected via uorescent
labeling of CD5/19-positive cells (Biolegend, San Diego, CA, USA) and ow
cytometry based cell sorting. In another case (C586, T2-BM) a bone marrow
aspirate was available at relapse and CLL cells were enriched via positive selection
of CD19-B cells (Miltenyi, Bergisch-Gladbach, Germany). In seven patients at least
one punch biopsy of a lymph node or an extranodal disease lesion was obtained
and used for nucleic acid extraction without additional cell separation due to the
limited volume of tissue. Genomic DNA was puried from CLL-B-cell and non-B-
cell fractions using standard columns (Qiagen, Hilden, Germany). All DNA
specimens were conrmed to be of high molecular weight (>10 kb) by agarose gel
Concentration of venetoclax [μM]
0.1 0.5
GI50=0.43 μM
87 kDa
40 kDa
42 kDa
Fig. 3 Overexpression of oncogenic BRAF in the OCI-LY19 cell line. aWestern blot analysis of BRAF and MCL1 in the BRAFV600E overexpressing OCI-LY19
cell line vs. its empty vector control. bGrowth inhibition of BRAFV600E transfected OCI-LY19 cells and the empty vector control is shown as a function of
the concentration of venetoclax
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Whole-exome sequencing. Whole-exome sequencing was performed from
genomic DNA, using the SureSelect Human All Exon V6 (Agilent, Santa Clara, CA,
USA) or the NimbleGen v2 target enrichment (Supplementary Table 2) according
to the manufacturers instructions. Obtained exome libraries were paired-end
sequenced on either a HiSeq2000 (2 × 100 bp) or a HiSeq4000 (2 × 75 bp) platform
(Illumina, San Diego, CA, USA). We achieved an average sequencing depth of
144X in the tumors and 90X in the normals.
Analysis of whole-exome-sequencing data. Raw sequencing reads were aligned
to the human reference genome (NCBI build 37/hg19) using the BWA mem
aligner (version 0.7.13-r1126). Concordant read pairs were masked as possible PCR
duplicates and areas of overlapping read pairs were excluded from analysis in one
read. Furthermore, we compared coverage differences between the two enrichment
kits and ltered genomic partitions out that showed no sufcient coverage (<10X)
in one of the kits to avoid kit-specic biases in the mutation calls. Somatic sub-
stitutions, insertions, deletions, copy numbers, and cellularity estimates were
determined by our in-house cancer genome analysis pipeline2830.
Reconstruction of clonal evolution. Two different types of phylogenetic trees
have been presented in this work. The rst type of phylogenetic tree demonstrates
the clonal evolution of reconstructed cell populations across cancer samples. The
direction of evolution is shown from bottom to top and is rooted from the com-
mon ancestor clone. In each tree, nodes represent different clones that have existed
during the evolution of the disease. Edges represent the ancestral relationships
between clones. Mutations that distinguish the child clone from the mother clone
are assigned to each edge. Here, phylogenetic tree reconstruction is performed by a
novel algorithm, which is an extension of our previous approach28. The procedure
consists of two steps: (1) inferring the mutation clusters that represent an evolu-
tionary populations and (2) tree reconstruction based on these clusters. To infer
mutation clusters based on cancer cell fractions of the mutations, we used a two-
dimensional version of our mutation clustering method28. To reconstruct the tree,
we rst assume that the tumor samples are monoclonal, which means all cancer
cells within a tumor are decedents of a single cell. We also assume that the
mutations satisfy the innite sites assumption, which states that a mutation occurs
only once at a specic locus during evolution of the cancer. The owing rules are
considered to infer the tree from the cancer cell fractions of the mutation clusters:
(1) connectivity rule (2) sum rule. The connectivity rule enforces that each node is
connected to one mother node except the common ancestor (root) node. The sum
rule ensures that the sum of cancer cell fractions over all child nodes is less than or
equal to the cancer cell fraction of the mother node. Under these constraints our
method automatically builds phylogenetic trees using linear integer programming.
The second type of trees are constructed based on maximum parsimony
assumption using the Pars module form PHYLIP package31 as it has been done by
others13. Each tree shows the phylogenetic relations of the matched pre-treatment
and relapse samples of a patient rooted from the normal cells of the patient. Leafs
of the tree are different tumor samples from the same patient. Branches show the
phylogenetic relations based on the somatic mutations difference of the samples.
Length of each branch is proportional to the number of mutations assigned to the
branch. The innite sites assumption may not be fullled in the reconstruction of
the second type trees.
Methylation arrays. DNA methylation was quantied using the HumanMethy-
lationEPIC (EPIC) BeadChip (Illumina, CA, USA) according to the manufacturers
instruction. Raw IDAT les were processed in R (3.3.1) using the Bioconductor
package RnBeads 1.6.132. Methylation β-values were normalized using the bmiq
method in order to correct for potential bias in DNA methylation measurements
between Type I and Type II probes33. Principle component analysis was performed
in R using the default values of the built-in function prcomp. Copy number proles
were derived by adapting our analysis tool for SNP 6.0 arrays34 to methylation
Digital droplet PCR. Digital Droplet PCR was performed using the Bio-Rad
QX200 ddPCR system (Bio-Rad, Hercules, CA, USA). The ddPCR probe mas-
termix and primers targeting distinct mutation sites or wild-type genes of interest
were purchased from Bio-Rad. Obtained PCR raw data were processed using
QuantaSoft v.1.6 (Bio-Rad).
Production of retroviruses for BRAFV600E overexpression. HEK293T cells
(ATCC, Manassas, VA, USA) were plated into 10 cm dishes in Iscoves Modied
Dulbeccos Medium (IMDM) +20% fetal calf serum (FCS) +50 mM beta-
mercaptoethanol or RPMI +20% FCS and incubated over night at 37 °C. Cells
were co-transfected with pMDLg/pRRE und pMD2.G packaging plasmids and
pBABEpuro expression plasmids encoding for BRAF V600E or empty vector using
a standard calcium phosphate transfection protocol. The next day, medium was
changed and cell culture supernatants were collected after 24, 48, and 72 h, cen-
trifuged at 200 × gfor 5 min and sterile ltered. Using these viral supernatants
human OCI-LY19 cells or human U-2932 cells (both kindly provided by Louis
Staudt, National Cancer Institute, Bethesda, MD, USA) were transduced in the
presence of 4 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 24
h and selected with puromycin (Sigma-Aldrich, Munich, Germany). Finally,
overexpression efciency of BRAF and MCL1 expression was conrmed by
immunoblotting. All cell lines used in these experiments were conrmed to be
mycoplasma negative and authenticated by short-tandem repeat proling.
Cell viability measurement. OCI-LY19 or U-2932 cells were plated into sterile 96-
well plates at 10,000 cells per well. Twenty-four hours after seeding, cells were
treated with various concentrations of venetoclax (Selleckchem, Munich, Germany)
for 72 h. After completion of drug exposure, relative cell viability was determined
by measuring the ATP content in each well (CellTiter-Glo®; Promega, Madison,
WI, USA) and is normalized to a control treated with the vehicle solution.
Cell culture: CDKN2A CRISPR screen. HEK293 cells (DSMZ, Braunschweig,
conrmed mycoplasma negative, not further authenticated) were cultured in
Dulbeccos modied Eagles medium (DMEM; Gibco, Thermo Fisher Scientic,
Waltham, MA, USA) supplemented with 10% FCS (Gibco) 1%
penicillinstreptomycin (v/v) (Gibco). CLL derived OSU cells (kindly provided by
John Byrd, Ohio State University Comprehensive Cancer Center, Columbus, OH,
USA, conrmed mycoplasma negative, not further authenticated) were cultured in
RPMI 1640 (Gibco) supplemented with 10% FCS and 1% penicillin-streptomycin
(v/v) (Gibco). Cells were incubated at 37 °C in a humidied 5% CO
DNA constructs. Eight 20-nt DNA sequences (see below) in exons 1 and 2 in the
human genomic CDKN2A locus were selected for producing single-guide RNA for
CRISPR-associated DNA endonuclease targets by using a publically available
resource ( The lentiCRISPR v2 vector35 was purchased from
Addgene (Cat. #52961). The oligos were annealed with bottom oligos and cloned
into lentiCRISPR v2 restricted by BsmB1 (#R0580S; New England Biolabs, Boston,
MA, USA). All generated constructs were analyzed by DNA sequencing using a
primer specic to the U6 promoter.
Sequence Target site
Lentivirus production and transduction. The lenticCRISPR v2 inserted with
single-quide RNA, together with psPAX2 packaging plasmid (Addgene, Cam-
bridge, MA, USA Cat. #12260) and pMD2.G envelope plasmid (Addgene Cat.
#12259) DNA were combined together and transfected into HEK293 cells by using
the Lipofectamine 2000 reagent (Cat. #11668-019, Invitrogen, Thermo Fisher
Scientic, Waltham, MA, USA). The transfection solution was added dropwise to
the cells and incubated for 6 h at 37 °C in a humidied 5% CO
cell culture
incubator. Six hours post transfection the medium was replaced by adding DMEM
supplemented with 5% FCS and 1% penicillinstreptomycin and incubated for 24
h. Eighteen hours post medium replacement, sodium butyrate (Cat. #B5887;
Sigma-Aldrich) was added to the culture medium at a nal concentration of 1 mM.
The viral harvest was performed for two times in 24-h intervals. After each col-
lection, the virus-containing medium was cleared by centrifugation at 300 × gfor 5
min at 4 °C, ltered through a 0.45 μm lter unit (Cat. # 10462100; Whatman, GE
Healthcare Life Sciences, Buckinghamshire, UK) and stored at 80 °C until use.
Lentivirus-containing medium was used to infect OSU cells. For this purpose, 107
cells were resuspended in viral supernatant supplemented with 2 μg/ml polybrene
and centrif uged at 800 × gfor 2 h and 32 °C. After spinoculation the cell pellet was
resuspended and cells were incubated for further 24 h under viral containing
conditions at 37 °C in a humidied 5% CO
atmosphere. After incubation, cells
were washed with PBS, centrifuged and resuspended in cell culture growing
medium. Cells were cultivated for 4 to 5 days until start of selection by 0.5 μg/ml
puromycin (Cat. #ant-pr-1; InvivoGen, San Diego, CA, USA) for 72 h. Subse-
quently 85 cells were resuspended in 20 ml of medium and distributed by pipetting
200 μl of this cell suspension into 96 wells. After outgrowth of single-cell clones,
these clones were lysed and prepared for western blotting.
Flow cytometry. Apoptosis was determined by ow cytometry using AnnexinV-
FITC (Immuno Tools, Friesoythe, Germany)/7AAD (eBioscience, San Diego, CA,
USA) staining in AnnexinV staining buffer (BD, Heidelberg, Germany). Mea-
surement was carried out by uorescence-activated cell sorting (FACS) Canto ow
cytometer (BD). Double negatives were considered as viable. Data was analyzed by
FACS DIVA Software (BD).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03170-7
6NATURE COMMUNICATIONS | (2018) 9:727 |DOI: 10.1038/s41467-018-03170-7 |
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Western blot and immunodetection. After harvesting, cells were washed in ice-
cold PBS, subsequently cells were lysed with RIPA III buffer (50 mM TRIS-HCl pH
8, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0,5% DOC, 1% NP-40)
supplemented with protease inhibitor (#P8340, Sigma-Aldrich) and phosphatase
inhibitor cocktail (#P5726; #P0044, Sigma-Aldrich). Protein content was deter-
mined by Roti-Quant (#K.015.1, Roth, Karlsruhe, Germany). Cells were mixed with
SDS-loading buffer (60 m M Tris-HCl pH 6.8, 3.3% SDS, 20 mM Dithiothreitol
(DTT), 0.01% bromphenol blue), then mixt ure was heated to 95 °C for 5 min. In
the following protein samples were loaded on a NuPAGE 512% Bis-Tris gel
(Thermo Fisher Scientic), separated by gel electrophoresis at 80 V for 3 h and then
transferred to a Protran nitrocellulose membrane (0.45 μM pore size, Whatman,
Maidstone, UK) by tank-blotting at 80 V for 1 h. Afterwards the blotting mem-
brane was blocked with 5% non-fat dry milk in tris-buf fered saline for 1 h. Blots
were probed with the following antibodies against: CDKN2A (#ab81278, Abcam,
Cambridge, UK, 1:1000), BRAF (sc-5285, Santa Cruz, 1:500), and MCL1 (CST-
5453, Cell Signaling, Danvers, MA, USA, 1:1000). β-actin (#A5316, Sigma-Aldrich,
1:10,000) was used as loading control. Detection of primary antibodies was
achieved via specic IRDye secondary antibodies (LI-COR Biosciences, Lincoln,
NE, USA, 1:20,0001:10,000). Protein bands were detected by LI-COR reader (LI-
COR) and quantied by LI-COR Software (LI-COR). Full-length images of the
most important western blots (Fig. 3and Supplementary Fig. 6b) are shown in
Supplementary Fig. 7.
Data availability. Sharing of the exome-sequencing data of this study outside our
institution is not permitted due to restrictions in our informed patient consent.
Compatible with our patient consent is to release a data set that contains infor-
mation of somatic mutations. Therefore, we provide tables of all somatic mutation
calls and our entire copy number analysis in the Supplementary Data. Mini bam-
les around the positions of somatic mutations are uploaded to the European
Nucleotide Archive ( under accession number:
PRJEB24344. These data sets are sufcient to reproduce our ndings and offer the
possibility to apply, e.g., alternative phylogenetic reconstruction methods. Mutation
data of untreated CLL patients were downloaded from Landau et al.3,Nature, 2015
(doi: 10.1038/nature15395) and Puente et al.8,Nature, 2015 (doi: 10.1038/
Received: 12 May 2017 Accepted: 24 January 2018
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We are indebted to our patients for making available the specimens that were analyzed
within this study. We also like to thank William Pao for fruitful discussions and Laura
Wilden for her contribution to collect and prepare samples for sequencing. This work
was supported by the Deutsche Forschungsgemeinschaft (DFG, CRU-286) to C.D.H., K.-
A.K., H.C.R., M.H., L.P.F., M.P., and (DFG, JA 2439/1-1) to R.D.J., the German Ministry
of Science and Education (BMBF) as part of the e:Med initiative (grant no. 01ZX1303A
to H.C.R., U.L., and M.P. and grant no. 01ZX1406 to M.P.), the Volkswagenstiftung
(Lichtenberg Program, H.C.R.), the Else Kröner-Fresenius Stiftung (EKFS-2014-A06, H.
C.R.), the Deutsche Krebshilfe (111724, H.C.R.), German José Carreras Leukemia
Foundation (grant no. DJCLS R 13/33, K.-A.K. and DJCLS R12/26, L.P.F and H.C.R.),
Roche research funding (L.P.F. and C.D.H.), the Stiftung Kölner Krebsforschung (L.P.F.
and H.C.R.), the Gusyk family support program at the University of Cologne (C.D.H.),
and the Center for Molecular Medicine Cologne (CMMC).
Author contributions
Conception and design: C.D.H., N.A., H.C.R., M.H., L.P.F., M.P. Provision of study
materials and patients: C.D.H., P.M., N.K., M.S., P.C., C.-M.W., T.P., A.S., C.P., B.E., R.
B., K.-A.K. Conduct of the experiments: C.D.H., J.W., A.S., R.D.J., O.M., S.O., P.M., V.B.,
D.T., J.A., P.N., R.C., S.C.S. Data analysis, and interpretation: C.D.H., N.A., M.C., V.A.,
U.L., H.C.R., L.P.F., M.P. Manuscript writing: C.D.H., N.A., L.P.F., M.P. All authors read
and approved the nal manuscript.
Additional information
Supplementary Information accompanies this paper at
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03170-7 ARTICLE
NATURE COMMUNICATIONS | (2018) 9:727 |DOI: 10.1038/s41467-018-03170-7 | 7
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Competing interests: C.D.H, C.-M.W., B.E., K.-A.K., M.H., and L.P.F. received research
funding from Hofmann-La Roche. Research support was also provided by AbbVie to
P.C., C.-M.W., B.E., K.-A.K., and M.H. P.C., C.-M.W., B.E., K.-A.K., H.C.R., M.H., and L.
P.F. obtained consulting and/or speakers honoraria from AbbVie. P.C., C.-M.W., B.E.,
K.-A.K., and M.H. received consulting and/or speakers honoraria from Hofmann-La
Roche. AbbVie provided travel support to P.C., N.K., and L.P.F. The remaining authors
declare no competing nancial interests.
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... 11,12 Recurrent mutations/deletions in the cell cycle regulators BTG1 and CDKN2A contribute to resistance particularly in CLL, where resistance occurs earlier and is associated with a more aggressive phenotype. 13,14 Beside mutations, especially upregulated MCL1 due to amplification of chromosome 1 (amp[1q]) confers resistance toward VEN. [15][16][17][18][19] MCL1, an antiapoptotic protein, can be targeted pharmacologically. ...
... The material of 6 CLL patients from the M13 982 trial were investigated as reported earlier. 13 T-cell prolymphocytic leukemia (T-PLL) patients were diagnosed according to World Health Organization criteria. Samples were obtained from patients under institutional review board-approved protocols following written informed consent. ...
... Whole-exome sequencing (WES) of genomic DNA from VEN-/ S63845-sensitive and -resistant B-cell lymphoma cell lines was performed as reported earlier. 13 Details on DNA extraction are given in supplemental Methods. ...
Full-text available
The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Since there is no common genetic alteration causing resistance to venetoclax in CLL and B cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole exome sequencing, methylated DNA immunoprecipitation sequencing and genome wide CRISPR/Cas9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter which is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity towards venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher OXPHOS and ATP production, resembling the metabolic phenotype that is seen upon venetoclax resistance. While PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity towards both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive DLBCL in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.
... RT sometimes occurs within the first months after treatment initiation [7][8][9] , suggesting selection of pre-existing subclones 10 . Nonetheless, the genomic/epigenomic mechanisms driving RT after CIT [11][12][13][14][15][16][17] or targeted agents [18][19][20][21] are not well known. The aims of the present study were to reconstruct the evolutionary history of RT and to reveal the molecular processes underlying this transformation. ...
... No major differences were seen among RT occurring after different therapies ( Fig. 1b and Extended Data Fig. 2b). We discovered new driver genes and mechanisms in RT, expanding previous observations [12][13][14][15][16][17][18][21][22][23][24] The main alterations involved cell-cycle regulators (17 of 19, 89%), chromatin modifiers (79%), MYC (74%), nuclear factor (NF)-κB (74%) and NOTCH (32%) pathways. These aberrations were simultaneously present in most cases but alterations in MYC and NOTCH pathways only co-occurred in 2 of 19 cases (Fig. 1c). ...
Full-text available
Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high–B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT. Single-cell genomic and transcriptomic analyses of longitudinal samples of patients with Richter syndrome reveal the presence and dynamics of clones driving transformation from chronic lymphocytic leukemia years before clinical manifestation
... Secondary resistance to VEN (VEN-relapse) is a major clinical problem, 2,9,10 but resistance mechanisms are beginning to be described and are unexpectedly heterogeneous. 2,[11][12][13][14][15] To date, research into clinical VEN resistance has largely focused on studies with bulk DNA sequencing, which detects coding mutations (eg, in BCL2 13 ) or gene amplification (eg, of MCL1 12 ). These approaches do not clarify what else might drive resistance (eg, other than BCL2 mutations) or give an accurate indication of potential tumor heterogeneity. ...
... The expression (mRNA and protein) of the key drivers of cellular proliferation was unaltered, and we could not confirm the loss of CDKN2A/B previously reported. 11 Taken together ( Figure 7C), our data indicate that in patients with CLL, the leukemic cells intrinsically adapt in multiple ways to selection pressure on cell survival exerted by ongoing VEN treatment. Future prospective studies are needed to resolve the potential role of the microenvironment in providing extrinsic signals to cause VEN resistance. ...
Full-text available
Venetoclax inhibits the pro-survival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL, but with eventual loss of efficacy. A spectrum of sub-clonal genetic changes associated with venetoclax resistance have now been described. To fully understand clinical resistance to venetoclax, we combined single-cell short- and long‑read RNA‑sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired venetoclax resistance. These appear to be multi-layered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the pro-survival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but heterogeneous across and within individual patient's leukemias. Changes in the pro-apoptotic genes are notably uncommon, except for single cases with sub-clonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF‑kB activation which persisted in circulating cells during venetoclax therapy. We discovered that MCL1 could be a direct transcriptional target of NF‑kB. Both the switch to alternative pro-survival factors and NF‑kB activation largely dissipate following venetoclax discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade venetoclax-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent venetoclax resistance and provide a specific biological justification for the strategy of venetoclax discontinuation once maximal response is achieved rather than maintaining long-term selective pressure with the drug.
... The best characterized biological mechanisms of venetoclax resistance in CLL/SLL are acquisition of BCL2 resistance mutations and upregulation of alternative pro-survival proteins such as BCL-X L and MCL1. Expansion of clones with genomic instability and other genetic mutations have been described among patients with early progression and RT on venetoclax, although their precise role in resistant disease biology requires further study [139,140]. The first described BCL2 mutation, Gly101Val, was detected among patients with prolonged venetoclax exposure (median 36 months) and impairs venetoclax binding to the alpha-helical groove of BCL2 without compromising its affinity for BH3-only proteins [48]. ...
... Other described resistance mechanisms in patients with CLL/ SLL include chromosome 1q23 amplification and overexpression of MCL1, with associated changes to mitochondrial metabolism [139]. Among eight patients with early PD and frequent RT, whole-exome sequencing of identified recurrent mutations in BTG1 and homozygous deletions of CDKN2A/B, PD-L1 amplification in one case, and a BRAF mutation postulated to augment MCL1 expression [140]. Emergence of TP53 aberrations or cytogenetically complex disease has been frequently observed in patients with PD on venetoclax, further implicating genomic instability in the development of resistance [147,148]. ...
Full-text available
BH3-mimetics are a novel drug class of small molecule inhibitors of BCL2 family proteins which restore apoptosis in malignant cells. The only currently approved BH3-mimetic, the selective BCL2 inhibitor venetoclax, is highly efficacious in chronic lymphocytic leukemia and has rapidly advanced to an approved standard of care in frontline and relapsed disease in combination with anti-CD20 monoclonal antibodies. In this context, tumour lysis syndrome and myelosuppression are the most commonly encountered toxicities and are readily manageable with established protocols. Venetoclax is active in other lymphoid malignancies including several B cell non-Hodgkin lymphomas, acute lymphoblastic leukemia and multiple myeloma, with the highest intrinsic sensitivity observed in mantle cell lymphoma and Waldenstrom macroglobulinemia. Venetoclax combination with standard regimens in follicular lymphoma, multiple myeloma and aggressive B cell neoplasms has shown some promise, but further studies are required to optimize dose and scheduling to mitigate increased myelosuppression and infection risk, and to find validated biomarkers of venetoclax sensitivity. Future research will focus on overcoming venetoclax resistance, targeting other BCL2 family members and the rational design of synergistic combinations.
... Other studies looking at patients with R/R CLL identified many genetics aberrations in cancer-related genes that conferred resistance to treatment. These included: mutations of BTG1 and BRAF, deletions in CDKN2A/B, and amplification of PD-L1 expressionsuggesting multiple mechanisms of resistance (79). Combination treatment strategies have been developed to improve the clinical efficacy and studies have shown improved response rates with venetoclax in combination with various agents including cytarabine, ibrutinib, rituximab, or bendamustine (98). ...
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The therapeutic landscape for lymphomas is quite diverse and includes active surveillance, chemotherapy, immunotherapy, radiation therapy, and even stem cell transplant. Advances in the field have led to the development of targeted therapies, agents that specifically act against a specific component within the critical molecular pathway involved in tumorigenesis. There are currently numerous targeted therapies that are currently Food and Drug Administration (FDA) approved to treat certain lymphoproliferative disorders. Of many, some of the targeted agents include rituximab, brentuximab vedotin, polatuzumab vedotin, nivolumab, pembrolizumab, mogamulizumab, vemurafenib, crizotinib, ibrutinib, cerdulatinib, idelalisib, copanlisib, venetoclax, tazemetostat, and chimeric antigen receptor (CAR) T-cells. Although these agents have shown strong efficacy in treating lymphoproliferative disorders, the complex biology of the tumors have allowed for the malignant cells to develop various mechanisms of resistance to the targeted therapies. Some of the mechanisms of resistance include downregulation of the target, antigen escape, increased PD-L1 expression and T-cell exhaustion, mutations altering the signaling pathway, and agent binding site mutations. In this manuscript, we discuss and highlight the mechanism of action of the above listed agents as well as the different mechanisms of resistance to these agents as seen in lymphoproliferative disorders.
... However, BCL2 mutations are presented at rather low allele frequencies and it remains unclear whether these infrequent subclones might render the whole malignant population resistant. These mutations are not found in large genomic analyses of biopsies from MCL and CLL patients relapsed from venetoclax plus ibrutinib or venetoclax single-therapy respectively, probably due to too low-resolution sequencing [5,11,15,16]. But these studies did reveal several other genetic aberrations in relapsed patients, involving CDKN2A/B, CCND1, TP53, NOTCH1/2, ATM, KMT2D and SMARCA2/4, indicating a role for cell cycle regulation and chromatin remodeling. ...
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Mantle cell lymphoma (MCL), an aggressive, but incurable B-cell lymphoma, is genetically characterized by the t(11;14) translocation, resulting in the overexpression of Cyclin D1. In addition, deregulation of the B-cell lymphoma-2 (BCL-2) family proteins BCL-2, B-cell lymphoma-extra large (BCL-XL), and myeloid cell leukemia-1 (MCL-1) is highly common in MCL. This renders these BCL-2 family members attractive targets for therapy; indeed, the BCL-2 inhibitor venetoclax (ABT-199), which already received FDA approval for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), shows promising results in early clinical trials for MCL. However, a significant subset of patients show primary resistance or will develop resistance upon prolonged treatment. Here, we describe the underlying mechanisms of venetoclax resistance in MCL, such as upregulation of BCL-XL or MCL-1, and the recent (clinical) progress in the development of inhibitors for these BCL-2 family members, followed by the transcriptional and (post-)translational (dys)regulation of the BCL-2 family proteins, including the role of the lymphoid organ microenvironment. Based upon these insights, we discuss how rational combinations of venetoclax with other therapies can be exploited to prevent or overcome venetoclax resistance and improve MCL patient outcome.
... In addition, mutations in BTG1 and homozygous deletions in CDKN2A/B were detected. These changes may be a major cause of resistance to venetoclax in CLL patients [63]. In AML patients, reconstructed existing mutations, such as expansion of FLT3-ITD, are main reasons for the poor therapeutic effect of venetoclax [40,64,65]. ...
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Venetoclax is a new type of BH3 mimetic compound that can target the binding site in the BCL-2 protein and induce apoptosis in cancer cells by stimulating the mitochondrial apoptotic pathway. Venetoclax is especially used to treat haematological malignancies. However, with the recent expansion in the applications of venetoclax, some cases of venetoclax resistance have appeared, posing a major problem in clinical treatment. In this article, we explored several common mechanisms of venetoclax resistance. Increased expression of the antiapoptotic proteins MCL-1 and BCL-XL plays a key role in conferring cellular resistance to venetoclax. These proteins can bind to the released BIM in the context of venetoclax binding to BCL-2 and thus continue to inhibit mitochondrial apoptosis. Structural mutations in BCL-2 family proteins caused by genetic instability lead to decreased affinity for venetoclax and inhibit the intrinsic apoptosis pathway. Mutation or deletion of the BAX gene renders the BAX protein unable to anchor to the outer mitochondrial membrane to form pores. In addition to changes in BCL-2 family genes, mutations in other oncogenes can also confer resistance to apoptosis induced by venetoclax. TP53 mutations and the expansion of FLT3-ITD promote the expression of antiapoptotic proteins MCL-1 and BCL-XL through multiple signalling pathways, and interfere with venetoclax-mediated apoptosis processes depending on their affinity for BH3-only proteins. Finally, the level of mitochondrial oxidative phosphorylation in venetoclax-resistant leukaemia stem cells is highly abnormal. Not only the metabolic pathways but also the levels of important metabolic components are changed, and all of these alterations antagonize the venetoclax-mediated inhibition of energy metabolism and promote the survival and proliferation of leukaemia stem cells. In addition, venetoclax can change mitochondrial morphology independent of the BCL-2 protein family, leading to mitochondrial dysfunction. However, mitochondria resistant to venetoclax antagonize this effect, forming tighter mitochondrial cristae, which provide more energy for cell survival.
Genomic profiling revealed the identity of at least 5 subtypes of DLBCL, including the MCD/C5 cluster characterized by aberrations in MYD88, BCL2, PRDM1 and/or SPIB. We generated mouse models harboring B cell-specific Prdm1 or Spib aberrations on the background of oncogenic Myd88 and Bcl2 lesions. We deployed whole exome sequencing, transcriptome, flow- and mass cytometry analyses to demonstrate that Prdm1- or Spib-altered lymphomas display molecular features consistent with pre-memory B cells and light zone B cells, whereas lymphomas lacking these alterations were enriched for late light-zone and plasmablast-associated gene sets. Consistent with the phenotypic evidence for increased B cell receptor signaling activity in Prdm1-altered lymphomas, we demonstrate that combined BTK/BCL2 inhibition displays therapeutic activity in mice and in five out of six relapsed/refractory DLBCL patients. Moreover, Prdm1-altered lymphomas were immunogenic upon transplantation into immuno-competent hosts, displayed an actionable PD-L1 surface expression and were sensitive to anti-murine-CD19-CAR-T cell therapy, in vivo.
Chronic lymphocytic leukaemia (CLL) constitutively overexpresses B-cell lymphoma 2 (BCL2) with consequent dysregulation of intrinsic apoptosis leading to abnormal cellular survival. Therapeutic use of BCL2 inhibitors (BCL2i, eg, venetoclax) in CLL, as both continuous monotherapy or in fixed duration combination, has translated scientific rationale into clinical benefit with significant rates of complete responses, including those without detectable minimal residual disease. Unlike with chemotherapy, response rates to venetoclax do not appear to be influenced by pre-existing chromosomal abnormalities or somatic mutations present, although the duration of response observed remains shorter for those with traditional higher risk genetic aberrations. This review seeks to describe both the disease factors that influence primary venetoclax sensitivity/resistance and those resistance mechanisms that may be acquired secondary to BCL2i therapy in CLL. Baseline venetoclax-sensitivity or -resistance is influenced by the expression of BCL2 relative to other BCL2 family member proteins, microenvironmental factors including nodal T-cell stimulation, and tumoral heterogeneity. With selection pressure applied by continuous venetoclax exposure, secondary resistance mechanisms develop in oligoclonal fashion. Those mechanisms described include acquisition of BCL2 variants, dynamic aberrations of alternative BCL2 family proteins, and mutations affecting both BAX and other BH3 proteins. In view of the resistance described, this review also proposes future applications of BCL2i therapy in CLL and potential means by which BCL2i-resistance may be abrogated.
Les leucémies aiguës myéloïdes (LAM) sont la classe d'hémopathies malignes la plus courante. Elles sont caractérisées par un défaut de l'hématopoïèse normale qui conduit à l'accumulation de cellules sanguines immatures, appelées blastes. Des traitements efficaces existent depuis quelques décennies, mais, mis à part l'allogreffe de moelle osseuse saine, aucun traitement n'est curatif. Cela est dû à la présence et l'apparition de cellules dites chimiorésistantes, qui adaptent leur métabolisme cellulaire pour gérer les stress rencontrés. Même si ces adaptations sont très bien connues à l'échelle de la cellule leucémique, le rôle et l'impact du microenvironnement médullaire est important et encore peu étudié. De même, à l'échelle systémique, de nombreuses reprogrammations ont lieu, dont certaines sont dues à la présence d'une maladie extra-médullaire caractérisée par des cellules leucémiques colonisant des organes distants. Ce phénomène n'est très souvent documenté qu'au moment de l'évaluation de la rémission. Cela concerne un quart des patients, qui pourraient constituer un nouveau sous-groupe d'étude. Afin d'évaluer ces adaptations aux niveaux tissulaires et systémique, j'ai pu mettre en place des méthodes de métabolomique in vivo, basées sur des méthodes de spectrométrie de masse. Cela m'a permis de caractériser les profils métaboliques des tissus naïfs de ma souris d'étude : la souris NSG, de caractériser les modulations de ces profils suite à une xénogreffe de deux lignées cellulaires de LAM (MOLM14 et U937), et suite à la réponse au traitement à la cytarabine. Enfin, j'ai pu identifier une signature plasmatique qui discrimine les cellules injectées en fonction de leur statut métabolique, ce qui pourra mener à l'identification de nouveau biomarqueurs précoces de la chimiorésistance. Ces méthodes et ces analyses ouvrent la voie à de futures études des métabolismes tissulaires et systémiques, dans le cadre de certains sous-groupes de patients et de certains traitements ciblés. Ces recherches d'identification de biomarqueurs permettront ainsi d'offrir la meilleure option aux patients.
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Background Among patients with non–small-cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early-stage NSCLC. Methods In this prospective cohort study, we performed multiregion whole-exome sequencing on 100 early-stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence-free survival. Results We observed widespread intratumor heterogeneity for both somatic copy-number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy-number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy-number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10⁻⁴), which remained significant in multivariate analysis. Conclusions Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx number, NCT01888601.)
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Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3′ region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.
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Genetic heterogeneity though common in tumors has been rarely documented in cell lines. To examine how often B-lymphoma cell lines are comprised of subclones, we performed immunoglobulin (IG) heavy chain hypermutation analysis. Revealing that subclones are not rare in B-cell lymphoma cell lines, 6/49 IG hypermutated cell lines (12%) consisted of subclones with individual IG mutations. Subclones were also identified in 2/284 leukemia/lymphoma cell lines exhibiting bimodal CD marker expression. We successfully isolated 10 subclones from four cell lines (HG3, SU-DHL-5, TMD-8, U-2932). Whole exome sequencing was performed to molecularly characterize these subclones. We describe in detail the clonal structure of cell line HG3, derived from chronic lymphocytic leukemia. HG3 consists of three subclones each bearing clone-specific aberrations, gene expression and DNA methylation patterns. While donor patient leukemic cells were CD5+, two of three HG3 subclones had independently lost this marker. CD5 on HG3 cells was regulated by epigenetic/transcriptional mechanisms rather than by alternative splicing as reported hitherto. In conclusion, we show that the presence of subclones in cell lines carrying individual mutations and characterized by sets of differentially expressed genes is not uncommon. We show also that these subclones can be useful isogenic models for regulatory and functional studies.
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Resistance to the Brutonâ € s tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-Targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.
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Background New treatments have improved outcomes for patients with relapsed chronic lymphocytic leukemia (CLL), but complete remissions remain uncommon. Venetoclax has a distinct mechanism of action; it targets BCL2, a protein central to the survival of CLL cells. Methods We conducted a phase 1 dose-escalation study of daily oral venetoclax in patients with relapsed or refractory CLL or small lymphocytic lymphoma (SLL) to assess safety, pharmacokinetic profile, and efficacy. In the dose-escalation phase, 56 patients received active treatment in one of eight dose groups that ranged from 150 to 1200 mg per day. In an expansion cohort, 60 additional patients were treated with a weekly stepwise ramp-up in doses as high as 400 mg per day. Results The majority of the study patients had received multiple previous treatments, and 89% had poor prognostic clinical or genetic features. Venetoclax was active at all dose levels. Clinical tumor lysis syndrome occurred in 3 of 56 patients in the dose-escalation cohort, with one death. After adjustments to the dose-escalation schedule, clinical tumor lysis syndrome did not occur in any of the 60 patients in the expansion cohort. Other toxic effects included mild diarrhea (in 52% of the patients), upper respiratory tract infection (in 48%), nausea (in 47%), and grade 3 or 4 neutropenia (in 41%). A maximum tolerated dose was not identified. Among the 116 patients who received venetoclax, 92 (79%) had a response. Response rates ranged from 71 to 79% among patients in subgroups with an adverse prognosis, including those with resistance to fludarabine, those with chromosome 17p deletions (deletion 17p CLL), and those with unmutated IGHV. Complete remissions occurred in 20% of the patients, including 5% who had no minimal residual disease on flow cytometry. The 15-month progression-free survival estimate for the 400-mg dose groups was 69%. Conclusions Selective targeting of BCL2 with venetoclax had a manageable safety profile and induced substantial responses in patients with relapsed CLL or SLL, including those with poor prognostic features. (Funded by AbbVie and Genentech; number, NCT01328626.)
Background: Programmed death ligand-1 (PD-L1), encoded by the CD274 gene, is a target for immune checkpoint blockade; however, little is known about genomic CD274 alterations. A subset of small cell lung cancer (SCLC) exhibits increased copy number of chromosome 9p24, on which CD274 resides; however, most SCLCs show low expression of PD-L1. We therefore examined, whether CD274 is a target of recurrent genomic alterations. Methods: We examined somatic copy number alterations in two patient cohorts by quantitative real-time PCR in 72 human SCLC cases (cohort 1), and SNP array analysis in 138 human SCLC cases (cohort 2). Whole genome sequencing revealed the detailed genomic structure underlying focal amplification. PD-L1 expression in amplified cases from cohorts 1 and 2 was further examined by transcriptome sequencing and immunohistochemical (IHC) staining. Results: By examining somatic copy number alterations in two cohorts of primary human SCLC specimens, we observed 9p24 copy number gains (where CD274 resides) and focal, high-level amplification of CD274. We found evidence for genomic targeting of CD274 suggesting selection during oncogenic transformation. CD274 amplification was caused by genomic rearrangements not affecting the open reading frame, thus leading to massively increased CD274 transcripts and high level expression of PD-L1. Conclusions: A subset (4/210, 1.9%) of human SCLC patient cases exhibits massive expression of PD-L1 caused by focal amplification of CD274. Such tumors may be particularly susceptible to immune checkpoint blockade.
The Illumina Infinium 450 k DNA Methylation Beadchip is a prime candidate technology for Epigenome-Wide Association Studies (EWAS). However, a difficulty associated with these beadarrays is that probes come in two different designs, characterized by widely different DNA methylation distributions and dynamic range, which may bias downstream analyses. A key statistical issue is therefore how best to adjust for the two different probe designs.
Background: Deletion of chromosome 17p (del[17p]) in patients with chronic lymphocytic leukaemia confers very poor prognosis when treated with standard chemo-immunotherapy. Venetoclax is an oral small-molecule BCL2 inhibitor that induces chronic lymphocytic leukaemia cell apoptosis. In a previous first-in-human study of venetoclax, 77% of patients with relapsed or refractory chronic lymphocytic leukaemia achieved an overall response. Here we aimed to assess the activity and safety of venetoclax monotherapy in patients with relapsed or refractory del(17p) chronic lymphocytic leukaemia. Methods: In this phase 2, single-arm, multicentre study, we recruited patients aged 18 years and older with del(17p) relapsed or refractory chronic lymphocytic leukaemia (as defined by 2008 Modified International Workshop on Chronic Lymphocytic Leukemia guidelines) from 31 centres in the USA, Canada, UK, Germany, Poland, and Australia. Patients started once daily venetoclax with a weekly dose ramp-up schedule (20, 50, 100, 200, 400 mg) over 4-5 weeks. Patients were then given daily 400 mg continuous dosing until disease progression or discontinuation for another reason. The primary endpoint was the proportion of patients achieving an overall response, assessed by an independent review committee. Activity and safety analyses included all patients who received at least one dose of study drug (per protocol). This study is registered with, number NCT01889186. Follow-up is ongoing, and patients are still receiving treatment. Findings: Between May 27, 2013, and June 27, 2014, 107 patients were enrolled into the study. At a median follow-up of 12·1 months (IQR 10·1-14·2), an overall response by independent review was achieved in 85 (79·4%; 95% CI 70·5-86·6) of 107 patients. The most common grade 3-4 adverse events were neutropenia (43 [40%]), infection (21 [20%]), anaemia (19 [18%]), and thrombocytopenia (16 [15%]). Serious adverse events occurred in 59 (55%) patients, irrespective of their relationship to treatment, with the most common (≥5% of patients) being pyrexia and autoimmune haemolytic anaemia (seven [7%] each), pneumonia (six [6%]), and febrile neutropenia (five [5%]). 11 patients died in the study within 30 days of the last dose of venetoclax; seven due to disease progression and four from an adverse event (none assessed as treatment related). Interpretation: Results of this trial show that venetoclax monotherapy is active and well tolerated in patients with relapsed or refractory del(17p) chronic lymphocytic leukaemia, providing a new therapeutic option for this very poor prognosis population. Additionally, in view of the distinct mechanism-of-action of venetoclax, combinations or sequencing with other novel targeted agents should be investigated to further advance treatment of del(17p) chronic lymphocytic leukaemia. Funding: AbbVie and Genentech.
Inhibition of the PD-L1 (CD274) - PD-1 axis has emerged as a powerful cancer therapy that prevents evasion of tumor cells from the immune system. While immunohistochemical detection of PD-L1 was introduced as a predictive biomarker with variable power, much less is known about copy number alterations (CNA) affecting PD-L1 and their associations with expression levels, mutational load and survival. To gain insight, we employed The Cancer Genome Atlas (TCGA) datasets to comprehensively analyze 22 major cancer types for PD-L1 CNAs. We observed a diverse landscape of PD-L1 CNAs, which affected focal regions, chromosome 9p or the entire chromosome 9. Deletions of PD-L1 were more frequent than gains (31% vs. 12%) with deletions being most prevalent in melanoma and non-small cell lung cancer. Copy number gains most frequently occurred in ovarian cancer, head and neck cancer, bladder cancer, cervical and endocervical cancer, sarcomas, and colorectal cancers. Fine-mapping of the genetic architecture revealed specific recurrently amplified and deleted regions across cancers with putative biological and clinical consequences. We noted a strong correlation between PD-L1 CNAs and mRNA expression levels for most cancers and found tumors with PD-L1 gains to harbor significantly higher mutational loads compared to non-amplified cases (median: 78 non-synonymous mutations vs. 40, p=7.1e-69). Moreover, we observed that, in general, both PD-L1 amplifications and deletions were associated with dismal prognosis. In conclusion, PD-L1 CNAs, in particular PD-L1 copy number gains, represent frequent genetic alterations across many cancers, which influence PD-L1 expression levels, are associated with higher mutational loads, and may be exploitable as predictive biomarker for immunotherapy regimens. This article is protected by copyright. All rights reserved.