Preclinical Anticancer Efficacy of BET Bromodomain Inhibitors Is Determined by the Apoptotic Response

Published OnlineFirst January 12, 2016; DOI: 10.1158/0008-5472.CAN-15-1458 Cancer Priority Report Research

Preclinical Anticancer Efﬁcacy of BET Bromodomain Inhibitors Is Determined by the Apoptotic Response Andrew R. Conery1, Richard C. Centore1, Kerry L. Spillane1, Nicole E. Follmer1, Archana Bommi-Reddy1, Charlie Hatton1, Barbara M. Bryant1, Patricia Greninger2, Arnaud Amzallag2, Cyril H. Benes2, Jennifer A. Mertz1, and Robert J. Sims III1

Abstract

Small-molecule inhibitors of the bromodomain and extra- acquired tolerance to BETi is driven by the robustness of the terminal (BET) family of proteins are being tested in clinical apoptotic response, and that genetic or pharmacologic manip- trials for a variety of cancers, but patient selection strategies ulation of the apoptotic signaling network can modify the remain limited. This challenge is partly attributed to the het- phenotypic response to BETi. We further reveal that the expres- erogeneous responses elicited by BET inhibition (BETi), includ- sion signatures of the apoptotic genes BCL2, BCL2L1,andBAD ing cellular differentiation, senescence, and death. In this study, significantly predict response to BETi. Taken together, our we performed phenotypic and gene-expression analyses of findings highlight the apoptotic program as a determinant treatment-naive and engineered tolerant cell lines representing of response to BETi, and provide a molecular basis for human melanoma and leukemia to elucidate the dominant patient stratification and combination therapy development. features defining response to BETi. We found that de novo and Cancer Res; 76(6); 1–7. 2016 AACR.

Introduction its cell lines by SNP genotyping. CA46, CHL1, CHP212, Colo783, Colo829, Daudi, H929, HCC1143, HCT116, HCT15, HL-60, HT, The development and progression of tumors is driven by HT29, K562, Kasumi-1, KOPN8, LP-1, MC116, MDAMB231, altered transcriptional programs and underlying tumor-specific Mewo, MOLT4, MV411, Namalwa, OPM-2, Raji, Ramos, REH, chromatin states. BET proteins are highly enriched and function- RPMI8226, SKMEL28, SKNAS, SKNDZ, ST486, SU-DHL-4, SU- ally essential at gene control elements that drive the expression of DHL-5, SU-DHL-6, SU-DHL-8, SW48, SW480, SW620, THP-1, critical oncogenes such as MYC (1). As such, BET inhibition affects U266, WSU-DLCL2, and Z-138 were obtained from ATCC. AMO- many pathways critical for cancer cell growth, leading to broad 1, DOHH2, IGR1, IGR39, JJN-3, KARPAS422, KMS-12PE, Melho, efficacy in pre-clinical disease models (2–5). However, at the ML2, MOLM-13, MOLP-8, MOLT16, NB4, OCI-AML2, OCI- molecular and phenotypic level, response to BETi can be quite AML3, OCI-AML5, OCI-LY-19, PL21, RC-K8, RL, Set-2, U-2932, heterogeneous, making it unclear what the critical drivers are for and U2940 were obtained from DSMZ. KMM-1, KMS-26, KMS- defining patient stratification strategies. A deeper understanding 20, KMS-27, KMS-34, and KMS-28PE were obtained from HSRRB/ of the pathways responsible for maximal response to BET inhi- JCRB. All cell banks authenticate with STR genotyping. Cell lines bition will, therefore, refine these strategies and allow for effective were used within 1 to 2 months of thawing from original stocks patient selection. and were not further authenticated.

Materials and Methods Generation of BETi-tolerant cell lines Cell line information A375 cells (ATCC) were cultured in 1 mmol/L CPI203 for Viability analysis was carried out at the MGH Center for approximately 90 days. NOMO-1 cells (DSMZ) were cultured Molecular Therapeutics (245 cell line panel), which authenticates with CPI203 (increasing concentrations) for approximately 9 months. Live cells were periodically enriched by centrifugation over Ficoll-Paque (GE Healthcare). Following selection, cells were 1 Constellation Pharmaceuticals, Inc., Cambridge, Massachusetts. maintained in 1 mmol/L CPI203. Parental and BETi-tolerant cells 2Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts. were authenticated using SNP genotyping with RNA-sequencing data (Supplementary Tables S3 and S4). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Analysis of gene expression in BETi-sensitive and -insensitive A.R. Conery and R.C. Centore contributed equally to this article. cell lines Corresponding Author: Robert J. Sims III, Constellation Pharmaceuticals, 215 Cells were treated with CPI203 (3–4 days) and viability was First Street, Suite 200, Cambridge, MA 02142. Phone: 617-714-0543; assessed [CellTiter Glo (Promega) or resazurin (Sigma)]. Expres- Fax: 617-577-0472; E-mail: [email protected] sion values were obtained from the Cancer Cell Line Encyclopedia doi: 10.1158/0008-5472.CAN-15-1458 (6). For qRT-PCR, DCt values for BCL2 and BCL2L1 were obtained 2016 American Association for Cancer Research. by subtracting Ct PPIB from Ct BCL2 or BCL2L1. DDCt values were

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Figure 1. BETi-tolerant cells display an abrogated apoptotic response to BETi and increased expression of BCL2 family genes. A, viability was measured by ﬂow cytometry after 11 days (A375, left) or 4 days (NOMO-1, right) and normalized to cell number in DMSO (A375 and A375-t1) or to parental cell number in DMSO (NOMO-1 and NOMO-1-t). Values are the mean, n ¼ 3 (A375) or n ¼ 6 (NOMO-1), and SEM. B, cells were treated with 1 mmol/L of CPI203 for 10 days (A375) or 4 days (NOMO-1). Values are the mean, n ¼ 3 (A375) or n ¼ 6 (NOMO-1), and SEM. C, expression heatmap of the indicated genes ( FC, log2 fold change) in tolerant cells relative to parental cells (CPI203 treated).

calculated by subtracting the median DCt across all cell lines BH3 profiling (Supplementary Data) from the DCt value in each cell line, and BH3 profiling was performed as described previously (7). BH3 DDCt log2 fold change was calculated as log2 (2 ). peptides were obtained from Anaspec (BIM: cat#62439, BAD: cat. #64082) or Abgent (HRK: cat. #SP1016a). Cell cycle, viability, and apoptosis analyses Cells were analyzed for cell-cycle distribution and viable cell Statistical analysis number as described previously (5). GraphPad Prism was used for all statistical calculations. Unless otherwise noted, P values were calculated by parametric, unpaired fi Gene-expression pro ling two-tailed t tests. P values are indicated as , 0.01–0.05; , 0.001– fi Total RNA was puri ed as described previously (5) and RNA 0.01; , 0.0001–0.001; and , <0.0001. For linear regression sequencing and alignments were performed at Ocean Ridge Bio- R2 is the square of the Pearson coefficient r, and the P value sciences. RNA-seq data have been deposited at GEO as GSE69383. indicates the probability that the slope does not differ from zero.

Comparison of apoptotic and cell-cycle arrest response with Compounds GI50 CPI203 has been described previously (8). ABT-737 and ABT- Cell lines were treated with CPI203 (4 days) and viability 199 were obtained from Selleck Chemicals. assessed (resazurin; Sigma). The median of the values for %subG1 Additional details can be found in Supplemental Information. and %G1 increase (relative to DMSO) across all cell lines was calculated; Z-scores were deﬁned as the number of SDs of the Results and Discussion values in each cell line from the median. Alteration of the apoptotic response in models of acquired BETi tolerance Lentiviral shRNA transduction Long-term culture of A375 (melanoma) and NOMO-1 (acute Lentiviral shRNA constructs targeting BCL2L1 and BCL2 were myelogenous leukemia) in the BET inhibitor CPI203 generated obtained from Sigma and transduced according to the manufac- cells that could proliferate in either CPI203 or the distinct BET turer's instructions. inhibitor JQ1. Two BETi-tolerantA375clones,A375-t1and

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Figure 2. Increased expression of BCL2 family members mediates BETi tolerance. A and B, shRNA-transduced cells were treated with 1 mmol/L CPI203 (A, A375-t1 for 11 days; B, NOMO-1-t for 4 days) before assessing viability and the percentage of sub-G1. Values represent the mean and SEM [A, n ¼ 2; B, n ¼ 2 (shLuc and shBCL2) or n ¼ 5 (shBCL2L1)]. P values are relative to shLuc. , P < 0.05; , P < 0.01; , P < 0.0001 by parametric, unpaired two-tailed t tests. C, cells were cotreated with ABT-737 and 1 mmol/L CPI203 or DMSO (11 days). The percentage of growth in CPI203 is indicated relative to DMSO. Values represent the mean and SEM (n ¼ 3, P values are relative to 0 mmol/L ABT-737). , P < 0.05; , P < 0.01, , P < 0.001 by parametric, unpaired two-tailed t tests. D, cells were treated with 0.25 mmol/L CPI203 and the indicated concentration of ABT-737 (4 days). The percentage of growth in ABT-737 is relative to CPI203-treated cells. Values represent the mean and SEM (n ¼ 6, P values are relative to 0 mmol/L ABT-737). , P < 0.0001 by parametric, unpaired two-tailed t tests.

A375-t2, revealed a modest shift in the GI50 values for BETi treated with CPI203 (Fig. 1C; Supplementary Fig. S2A–S2C). In (Fig. 1A, left; Supplementary Fig. S1A and Supplementary Fig. BETi-treated NOMO-1-t cells, BCL2 was also upregulated com- S1C, left). BETi-tolerant NOMO-1-t cells proliferated at con- pared with BETi-treated parental cells (Fig. 1C; Supplementary centrations of BETi approximately 30-fold higher than their Fig. S2C). MCL1 and BCL2A1 were reduced in both tolerant A375 original GI50 concentrations (Fig. 1A, right; and Supplementary and NOMO-1 cells, highlighting the selective alteration of specific Fig. S1C, right) and surprisingly grew best in the presence of BCL2 family members. In contrast with recent work pointing to CPI203. Both BETi-tolerant cell lines A375-t1 and NOMO-1-t the Wnt pathway as a determinant of response to BETi (9, 10), we displayed a blunted apoptotic response to BETi relative to did not observe increased expression of Wnt target genes in either parental cells (Fig. 1B; Supplementary Fig. S1B and S1D). A375 or NOMO-1 BETi resistance models. Notably, A375-t1 and NOMO-1-t were equally or more sensitive than parental cells to both cytarabine and doxorubicin, BCL2L1 and BCL2 are critical mediators of BETi sensitivity and which suggests that they are not resistant to all apoptosis- resistance inducing agents and points toward the use of BETi in combi- Given the expression changes noted above, we focused on nation with cytotoxic chemotherapies (Supplementary Fig. the functional roles of BCL2L1 in A375-t1 and NOMO-1-t S1E–S1F). cells, and BCL2 in NOMO-1-t cells given that BCL2 was Consistent with these phenotypes, through gene-expression expressed at very low levels in A375-t1 (Fig. 1C; Supplemen- profiling we observed increased expression of the BCL2 family tary Fig. S2B). Knockdown of BCL2L1 in A375-t1 cells, and of member BCL2L1 (encoding BCL2L1, formerly known as BCL-xL) BCL2 or BCL2L1 in NOMO-1-t cells, led to dramatically in both A375-t and NOMO-1-t compared with parental cells reduced proliferation and significantly increased apoptosis in

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Figure 3. Robust apoptosis correlates with BETi sensitivity. A, 51 cell lines (Supplementary Data) were treated with CPI203 (4 days) and GI50, apoptosis and G0–G1 cell-cycle arrest were measured. Sensitive, GI50 <0.25 mmol/L. Values represent the mean and SEM. B, cell lines were treated with CPI203 (2 mmol/L) for 24 hours. Top, expression of the indicated genes (qRT-PCR). Bottom, composite gene score (see Supplemental Methods) was plotted against the z-score for sub-G1 increase calculated in A. C, mitochondrial depolarization (JC-1 ﬂuorescence) resulting from BIM peptide in cells treated overnight with DMSO or CPI203 (1 mmol/L). Values shown are the mean n ¼ 12 (n ¼ 8 for Kasumi-1) and SEM (P values for CPI203 are relative to DMSO at each BIM peptide concentration). , P < 0.05; , P < 0.001 by parametric, unpaired two-tailed t tests; ns, nonsigniﬁcant.

the presence of CPI203 (Fig. 2A and B and Supplementary Fig. Engagement of the apoptotic program is associated with robust S3B–S3C). In addition, overexpression of BCL2L1 in parental phenotypic response to BET inhibition A375 cells resulted in a blunted apoptotic response to BETi Next, we tested whether the apoptotic program correlates with (Supplementary Fig. S3A). de novo sensitivity to BET inhibitors by profiling 51 hematologic Treatment of A375-t1 and NOMO-1-t with ABT-737, an inhib- cell lines that we termed "sensitive" or "insensitive" based on a itor of BCL2, BCL2L1, and BCL2L2 (11), concurrently with GI50 cutoff value of 0.25 mmol/L CPI203. As shown in Fig. 3A, the CPI203 led to growth suppression and induction of apoptosis, magnitude of apoptosis, but not the cytostatic response, is highly while affecting parental cells to a lesser extent (Fig. 2C and D; correlated with sensitivity to BET inhibition. This is consistent Supplementary Fig. S3D–S3E). Consistent with their sustained with previous work showing that the induction of apoptosis is expression of BCL2, NOMO-1-t cells also showed growth inhi- often associated with preclinical efficacy (2, 3, 5, 14–16). bition with the BCL2-specific inhibitor, ABT-199 (Supplementary Given that BETi regulates the expression of antiapoptotic Fig. S3F; ref. 12). In both models, the degree of apoptosis induc- factors like BCL2 (2), we tested whether apoptosis is associated tion in tolerant cells cotreated with CPI203 and ABT-737 was with a change in the expression of key anti- and proapoptotic comparable with parental cells treated with BETi alone, suggesting factors after CPI203 treatment. A clear trend toward downregula- that pharmacologic inhibition of BCL2 family members can tion of antiapoptotic factors and upregulation of proapoptotic abrogate tolerance to BETi. Notably, combination of the BET factors was observed in cell lines that undergo apoptosis (Fig. 3B, inhibitor JQ1 with ABT-199 showed synergistic effects on growth top), and a more robust transcriptional response is significantly inhibition (13), highlighting the potential of combining BETi correlated with the magnitude of the apoptotic response to with BH3 peptidomimetics. CPI203 (Fig. 3B, bottom). To functionally test whether these

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Figure 4. Basal expression of apoptotic factors predicts BETi response. A, expression (RMA) is shown relative to BETi sensitivity (GI50 <0.25 mmol/L CPI203). B, mitochondrial depolarization (JC-1 fluorescence) from the indicated cell lines treated with a BAD BH3 peptide (50 mmol/L). Values are the mean of n ¼ 12 (n ¼ 8for SUDHL4); error is shown in Supplementary Fig. S4B. C, high or low was defined as the expression in the top or bottom third of all cell lines. Dashed line, the overall response rate (28%). Values represent the mean and SEM (P values were determined by two-tailed Fisher exact t test). , P < 0.0001 by parametric, unpaired two-tailed t test. D, expression of BCL2 and BCL2L1 (qRT-PCR) are predictive of phenotypic response to BETi. High or low expression was defined as above or below the median (P ¼ 0.002 by two-tailed Fisher exact t test). The overall response rate is indicated in the graph by a dashed line (66%). , P < 0.01 by parametric, unpaired two-tailed t tests. changes in gene expression were associated with increased apo- cell lines with a minimal apoptotic response (Daudi and ptotic signaling, we used BH3 profiling with the BIM BH3 peptide SUDHL5; Fig. 3C; Supplementary Fig. S4A). to assess mitochondrial "priming" (7, 17). In two cell lines that The results reported above suggest that in highly sensitive cells, show a robust apoptotic response to BET inhibition (MV411 and BETi treatment disproportionately modulates apoptotic signaling Kasumi-1), preincubation with CPI203 induced measurable by altering the expression of anti- and proapoptotic genes. Wheth- increases in mitochondrial priming, which was not observed in er these changes in gene expression are the direct result of release

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of BET family proteins from chromatin is not clear. Recent work biomarkers selected for the most sensitive tumor subtypes, which has reported that highly BRD4-occupied gene control elements may point to indications beyond hematologic malignancies that define loci that are highly BET dependent, which includes BCL2 will benefit from BETi (Supplementary Fig. S4D). Finally, the family genes (1). However, we have found that high BRD4 enrichment of phenotypically sensitive cell lines was observed occupancy and release upon BETi treatment are not universal when gene expression was measured by qRT-PCR or Western predictors of downregulation of any given gene (data not shown). blotting (Fig. 4D; Supplementary Fig. S6), indicating that these It is likely that identification of predictive features of BET tran- criteria may be used with clinically relevant methods. scriptional regulation will facilitate our ability to forecast a robust Although they have tremendous promise, BET inhibitors also apoptotic response to BET inhibition. bring new challenges for selecting patient populations and for determining optimal drug combinations. This work identifies Basal expression levels of BCL2, BCL2L1, and BAD serve as the apoptotic signaling network and the expression of factors predictive biomarkers for BETi treatment such as BCL2, BCL2L1,andBAD as patient selection biomarkers Given our functional data, we tested whether the baseline that will help BET inhibitors to reach their full therapeutic mRNA expression level of apoptotic factors (Supplementary Table potential. S1) could predict phenotypic response to BETi in a panel of 245 cell lines (Supplementary Table S2; Supplementary Information). Disclosure of Potential Conflicts of Interest We found that five genes, BCL2, BCL2L2, BCL2L1, BAD, and N.E. Follmer is a senior scientist in Merck & Co., Inc. B.M. Bryant has BCLAF1, were differentially expressed with high significance, with ownership interest (including patents) in Constellation Pharmaceuticals. BCL2 being the most differentially expressed gene (Supplemen- P. Greninger is a head of research and has ownership interest (including patents) tary Table S1, Fig. 4A). We reasoned that BETi may selectively in Constellation Pharmaceuticals. J.A. Mertz has ownership interest (including target cells that are dependent on BCL2, and tested for BCL2 patents) in Constellation Pharmaceuticals. R.J. Sims III is a vice president of research, reports receiving other commercial research support, and has owner- dependence in a subset of cell lines using mitochondrial exposure ship interest (including patents) in Constellation Pharmaceuticals. No potential to the BAD BH3 peptide (18). We observed that mitochondrial conflicts of interest were disclosed by other authors. depolarization in response to BAD peptide is correlated with the BCL2 expression of in a set of 6 cell lines (Fig. 4B, top), consistent Authors' Contributions with published data (18), and that the degree of mitochondrial Conception and design: A.R. Conery, R.C. Centore, N.E. Follmer, P. Greninger, depolarization was inversely correlated with the GI50 of BETi (Fig. C.H. Benes, J.A. Mertz, R.J. Sims III 4B, bottom). There was no significant mitochondrial depolariza- Development of methodology: A.R. Conery, R.C. Centore, N.E. Follmer, tion in response to the HRK BH3 peptide, arguing against any R.J. Sims III contribution of BCL2L1 to the BAD depolarization response Acquisition of data (provided animals, acquired and managed patients, (Supplementary Fig. S4B). As both inhibitors seem to preferen- provided facilities, etc.): A.R. Conery, R.C. Centore, K.L. Spillane, N.E. Follmer, A. Bommi-Reddy, C.H. Benes tially target cell lines that are dependent on BCL2 for survival, we Analysis and interpretation of data (e.g., statistical analysis, biostatistics, fi noted that the GI50 values of CPI203 and ABT-199 are signi cantly computational analysis): A.R. Conery, R.C. Centore, K.L. Spillane, correlated (Supplementary Fig. S4C; ref. 12). N.E. Follmer, C. Hatton, B.M. Bryant, P. Greninger, A. Amzallag, C.H. Benes, We next tested whether the expression levels of these apoptotic J.A. Mertz, R.J. Sims III mediators could predict a robust response to BET inhibition. Writing, review, and/or revision of the manuscript: A.R. Conery, R.C. Centore, Selection based on the expression of a one or two genes signif- P. Greninger, C.H. Benes, J.A. Mertz, R.J. Sims III Administrative, technical, or material support (i.e., reporting or organizing icantly increased the response rate, with selection of cells with data, constructing databases): C. Hatton, B.M. Bryant, P. Greninger, R.J. Sims III BCL2 BCL2L1 high expression and low expression of either or Study supervision: P. Greninger, J.A. Mertz, R.J. Sims III BAD improving the response rate to 65% or 75%, respectively (Fig. 4C and Supplementary Fig. S5A). Importantly, selection Acknowledgments based on these factors was not merely enriching for cell lines of The authors thank the many Constellation employees in their support of hematologic origin, as these selection criteria increased the these studies. response rate of isolated hematologic or solid tumor cell lines, and of acute lymphoblastic leukemia cell lines (Supplementary Received June 2, 2015; revised December 4, 2015; accepted December 28, Fig. S5B–S5D). We also noted that selection based on these 2015; published OnlineFirst January 12, 2016.

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Preclinical Anticancer Efficacy of BET Bromodomain Inhibitors Is Determined by the Apoptotic Response

Andrew R. Conery, Richard C. Centore, Kerry L. Spillane, et al.

Cancer Res Published OnlineFirst January 12, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-1458

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2016/01/12/0008-5472.CAN-15-1458.DC1

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