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Activation of apoptosis in malignant cells is an established strategy for controlling cancer and is potentially curative. To assess the impact of concurrently inducing the extrinsic and intrinsic apoptosis-signaling pathways in acute myeloid leukemia (AML), we evaluated activity of the TRAIL receptor agonistic fusion protein eftozanermin alfa (eftoza; ABBV-621) in combination with the B-cell lymphoma protein-2 selective inhibitor venetoclax in preclinical models and human patients. Simultaneously stimulating intrinsic and extrinsic apoptosis-signaling pathways with venetoclax and eftoza, respectively, enhanced their activities in AML cell lines and patient-derived ex vivo/in vivo models. Eftoza activity alone or plus venetoclax required death receptor 4/5 (DR4/DR5) expression on the plasma membrane but was independent of TP53 or FLT3-ITD status. The safety/tolerability of eftoza as monotherapy and in combination with venetoclax was demonstrated in patients with relapsed/refractory AML in a phase 1 clinical trial. Treatment-related adverse events were reported in 2 of 4 (50%) patients treated with eftoza monotherapy and 18 of 23 (78%) treated with eftoza plus venetoclax. An overall response rate of 30% (7/23; 4 complete responses [CRs], 2 CRs with incomplete hematologic recovery, and 1 morphologic leukemia-free state) was reported in patients who received treatment with eftoza plus venetoclax and 67% (4/6) in patients with myoblasts positive for DR4/DR5 expression; no tumor responses were observed with eftoza monotherapy. These data indicate that combination therapy with eftoza plus venetoclax to simultaneously activate the extrinsic and intrinsic apoptosis-signaling pathways may improve clinical benefit compared with venetoclax monotherapy in relapsed/refractory AML with an acceptable toxicity profile. This trial was registered at www.clinicaltrials.gov as #NCT03082209.

Dose-dependent induction of G1 arrest and senescence by ABBV-075.

Figure S2. ABBV-744 retains strong transcription regulatory activities in AML cells and it did not alter the baseline expression of representative IFN-r or PMA responsive genes.

Characterization of MDV_R cell line with acquired resistance to Enzalutamide.

Primary patient sample information

Suppl. Figure 1. Caspase inhibitor prevents caspase 3/7 activity; Suppl. Figure 2. Levels of BIM, BAD, BAX and BAK silencing by siRNA; Suppl. Figure 3. mRNA and protein expression of BCL2 and BCLxl upon treatment with ABBV-075.

Supplementary figure and table legends

AR-dependent prostate cancer cell lines are dependent on BET.

Suppl. Figure 1. Dependency of human melanoma cell lines on MITF; Suppl. Figure 2. A-485 treatment reduces viability of primary melanoma cell line; Suppl. Figure 3. A-485 treatment does not induce apoptosis in melanoma cells; Suppl. Figure 4. A-485 treatment does not increase invasiveness in melanoma cells; Suppl. Figure 5. MITF gene expression and copy number is associated with A-485-induced senescence

Dual bromodomain BET inhibitors that bind with similar affinities to the first and second bromodomains across BRD2, BRD3, BRD4, and BRDT have displayed modest activity as monotherapy in clinical trials. Thrombocytopenia, closely followed by symptoms characteristic of gastrointestinal toxicity, have presented as dose-limiting adverse events that may have prevented escalation to higher dose levels required for more robust efficacy. ABBV-744 is a highly selective inhibitor for the second bromodomain of the four BET family proteins. In contrast to the broad antiproliferative activities observed with dual bromodomain BET inhibitors, ABBV-744 displayed significant antiproliferative activities largely although not exclusively in cancer cell lines derived from acute myeloid leukemia and androgen receptor positive prostate cancer. Studies in acute myeloid leukemia xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable with the pan-BET inhibitor ABBV-075 but with an improved therapeutic index. Enhanced antitumor efficacy was also observed with the combination of ABBV-744 and the BCL-2 inhibitor, venetoclax compared with monotherapies of either agent alone. These results collectively support the clinical evaluation of ABBV-744 in AML (Clinical Trials.gov identifier: NCT03360006).

Supplementary Figures S1-S10

290 genes that were modulated by MS417 in both NCI-H1299 and MiaPaCa-2 cells.

Supplementary Table 1

Expression change of 25 potential BET inhibitor responsive genes in NCI-H1299, MiaPaCa-2, and MX-1 cells treated with DMSO or 0.5 μM MS417 for 6 hours.

ABBV-075 inhibits AR-dependent transcription and growth.

ABBV-075 inhibits ETS family member ETV1 target gene transcription.

Metastatic melanoma is responsible for approximately 80% of deaths from skin cancer. Microphthalmia-associated transcription factor (MITF) is a melanocyte-specific transcription factor that plays an important role in the differentiation, proliferation, and survival of melanocytes as well as in melanoma oncogenesis. MITF is amplified in approximately 15% of patients with metastatic melanoma. However, no small-molecule inhibitors of MITF currently exist. MITF was shown to associate with p300/CBP, members of the KAT3 family of histone acetyltransferase. p300 and CREB-binding protein (p300/CBP) regulate a wide range of cellular events such as senescence, apoptosis, cell cycle, DNA damage response, and cellular differentiation. p300/CBP act as transcriptional coactivators for multiple proteins in cancers, including oncogenic transcription factors such as MITF. In this study, we showed that our novel p300/CBP catalytic inhibitor, A-485, induces senescence in multiple melanoma cell lines, similar to silencing expression of EP300 (encodes p300) or MITF. We did not observe apoptosis and increase invasiveness upon A-485 treatment. A-485 regulates the expression of MITF and its downstream signature genes in melanoma cell lines undergoing senescence. In addition, expression and copy number of MITF is significantly higher in melanoma cell lines that undergo A-485–induced senescence than resistant cell lines. Finally, we showed that A-485 inhibits histone-H3 acetylation but did not displace p300 at promoters of MITF and its putative downstream genes. Taken together, we provide evidence that p300/CBP inhibition suppressed the melanoma-driven transcription factor, MITF, and could be further exploited as a potential therapy for treating melanoma.

Supplemental Figure 1. Chemical structure and biochemical potency of BET inhibitors used in this study. Supplemental Figure 2. Rapid reversal of PD marker responses upon BET inhibitor washout in OPM-2 cells. Supplemental Figure 3. In vivo antitumor efficacies of ABBV-075 in the OPM-2 model at the 0.25 mg/kg, qdx21 and 1 mg/kg, qdx21 dose levels. Supplemental Figure 4. Identification of potential PD markers in skin. Supplemental Table 2. Common pathways regulated by MS417 in NCI-H1299 and MiaPaCa-2 cells Supplemental Table 4.Correlation of EC50s of MS417 and ABBV-075 in regulating PD makers vs. their anti-proliferative EC50s in OPM-2 cells. Supplemental Table 5. Identification of potential PD markers in whole blood.

The antiapoptotic protein BCL2 plays critical roles in regulating lymphocyte development and immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL2 is critical for antitumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL2, would influence the antitumor activity of immune checkpoint inhibitors (ICI). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the antitumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T-cell function in response to antigen stimuli in vitro and did not antagonize T-cell activation induced by anti–PD-1. Furthermore, we demonstrate that the antiapoptotic family member BCL-XL provides a survival advantage in effector T cells following inhibition of BCL2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy.Significance:The antiapoptotic oncoprotein BCL2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development, and immune system regulation. Here we demonstrate that venetoclax, the first FDA/European Medicines Agency–approved BCL2 inhibitor, unexpectedly can be combined preclinically with immune checkpoint inhibitors to enhance anticancer immunotherapy, warranting clinical evaluation of these combinations.This article is highlighted in the In This Issue feature, p. 1

AR-dependent recruitment of BRD4 to ARE-enhancers.

Supplemental figure legends

Progress in understanding tumor stromal biology has been constrained in part because cancer-associated fibroblasts (CAF) are a heterogeneous population with limited cell-type–specific protein markers. Using RNA expression profiling, we identified the membrane protein leucine-rich repeat containing 15 (LRRC15) as highly expressed in multiple solid tumor indications with limited normal tissue expression. LRRC15 was expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 expression was induced by TGFβ on activated fibroblasts (αSMA+) and on mesenchymal stem cells. These collective findings suggested LRRC15 as a novel CAF and mesenchymal marker with utility as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody–drug conjugate (ADC) directed against LRRC15, and it demonstrated robust preclinical efficacy against LRRC15 stromal-positive/cancer-negative, and LRRC15 cancer-positive models as a monotherapy, or in combination with standard-of-care therapies. ABBV-085′s unique mechanism of action relied upon the cell-permeable properties of MMAE to preferentially kill cancer cells over LRRC15-positive CAF while also increasing immune infiltrate (e.g., F4/80+ macrophages) in the tumor microenvironment. In summary, these findings validate LRRC15 as a novel therapeutic target in multiple solid tumor indications and support the ongoing clinical development of the LRRC15-targeted ADC ABBV-085.Significance: These findings identify LRRC15 as a new marker of cancer-associated fibroblasts and cancers of mesenchymal origin and provide preclinical evidence for the efficacy of an antibody-drug conjugate targeting the tumor stroma. Cancer Res; 78(14); 4059–72. ©2018 AACR.

High level of concomitant Bim and Bcl-2 expression correlates with sensitivity to ABBV-075-induced apoptosis

Cross species binding and tolerability of ABBV-085.

Figures S1-S7 show LRRC15 RNA and protein expression in cancer, and demonstrate the anti-tumor efficacy of the LRRC15-targeted ADC ABBV-085 in vitro and in vivo as a monotherapy and in combination with standard-of-care therapies.

Apoptosis induction by MS417 across cancer cell lines

Restoration of Myc expression under BET inhibitor treatment failed to rescue cells from apoptosis or cell cycle arrest

ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro/in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976–89. ©2017 AACR.

ABBV-075 inhibits Bcl-XL transcription and displace BRD4 from Bcl-XL regulatory regions

Apoptosis induction by ABBV-075 across cancer cell lines

Induction of apoptosis across cancer cell lines and CD34 normal cells

Anti-proliferative activity of ABBV-075 across cancer cell lines

BET inhibitor causes cell cycle arrest and triggers senescence in solid tumor cells

Supplementary Figure Legends

supplemental figure legends

Expression of exogenous Bcl-XL rescues ABBV-075 induced apoptosis but not cell cycle arrest

Down regulation of Bcl-XL by MS417 and ABBV-075

ABBV-075 inhibits genes that are important for cell cycle

Supplementary Figure 2 from Genetic Alterations in Mouse Medulloblastomas and Generation of Tumors De novo from Primary Cerebellar Granule Neuron Precursors

Mice lacking p53 and one or two alleles of the cyclin D–dependent kinase inhibitor p18Ink4c are prone to medulloblastoma development. The tumor frequency is increased by exposing postnatal animals to ionizing radiation at a time when their cerebella are developing. In irradiated mice engineered to express a floxed p53 allele and a Nestin-Cre transgene, tumor development can be restricted to the brain. Analysis of these animals indicated that inactivation of one or both Ink4c alleles did not affect the time of medulloblastoma onset but increased tumor invasiveness. All such tumors exhibited complete loss of function of the Patched 1 (Ptc1) gene encoding the receptor for sonic hedgehog, and many exhibited other recurrent genetic alterations, including trisomy of chromosome 6, amplification of N-Myc, modest increases in copy number of the Ccnd1 gene encoding cyclin D1, and other complex chromosomal rearrangements. In contrast, medulloblastomas arising in Ptc1+/− mice lacking one or both Ink4c alleles retained p53 function and exhibited only limited genomic instability. Nonetheless, complete inactivation of the wild-type Ptc1 allele was a universal event, and trisomy of chromosome 6 was again frequent. The enforced expression of N-Myc or cyclin D1 in primary cerebellar granule neuron precursors isolated from Ink4c−/−, p53−/− mice enabled the cells to initiate medulloblastomas when injected back into the brains of immunocompromised recipient animals. These “engineered” tumors exhibited gene expression profiles indistinguishable from those of medulloblastomas that arose spontaneously. These results underscore the functional interplay between a network of specific genes that recurrently contribute to medulloblastoma formation. [Cancer Res 2007;67(6):2676–84]

Supplementary Figure 1 from Genetic Alterations in Mouse Medulloblastomas and Generation of Tumors De novo from Primary Cerebellar Granule Neuron Precursors

Supplementary Figure Legends 1-2 from Genetic Alterations in Mouse Medulloblastomas and Generation of Tumors De novo from Primary Cerebellar Granule Neuron Precursors

Supplementary Table 1 from Genetic Alterations in Mouse Medulloblastomas and Generation of Tumors De novo from Primary Cerebellar Granule Neuron Precursors

Background: Protein tyrosine phosphatase non-receptor type 2 and type 1 (PTPN2/1) antagonists inhibit PTPN2/1-mediated negative regulation of immune response pathways. They may promote antitumor activity by increasing function of immune cells such as T cells and dendritic cells, inducing cytokine production, increasing antigen presentation, and amplifying interferon gamma-mediated tumor growth arrest. ABBV-CLS-484 and ABBV-CLS-579 are potent, orally bioavailable, first-in-class PTPN2/1 inhibitors that showed promising antitumor activity and were well tolerated in preclinical models. Preclinical data also indicated that PTPN2/1 inhibitors have improved efficacy when combined with PD-1-targeting agents (eg, pembrolizumab) or vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs) in multiple tumor models. Here, we describe 2 first-in-human trials of ABBV-CLS-484 or ABBV-CLS-579 as monotherapy and in combination with pembrolizumab or VEGFR TKI in patients with locally advanced/metastatic tumors. Methods: These phase 1, open-label, multicenter, non-randomized trials (ABBV-CLS-484: NCT04777994; ABBV-CLS-579: NCT04417465) comprise dose-escalation (ESC) and dose-expansion (EXP) phases. ESC is guided by a Bayesian optimal interval design based on dose-limiting toxicities. ESC data are used to inform evaluation of EXP cohorts in select tumor types including relapsed/refractory (R/R) head and neck squamous cell carcinoma, R/R non-small cell lung cancer, advanced renal cell carcinoma, and microsatellite instability-high tumors. In both ESC and EXP phases, the study drug (ABBV-CLS-484 or ABBV-CLS-579) is administered orally either alone or in combination with pembrolizumab (200 mg intravenously once every 3 weeks) and is slated to also be administered in combination with a VEGFR TKI. Eligible patients have locally advanced/metastatic tumors for which no effective standard therapy exists (or has failed), and Eastern Cooperative Oncology Group performance status ≤2. Patients (≥18 years) must have received ≥1 prior systemic anticancer therapy for the indication being considered, have relapsed or be refractory to ≥1 prior anti-PD-1/PD-L1 therapy (EXP monotherapy and pembrolizumab combination), or have relapsed after ≤1 (ABBV-CLS-484) or ≥1 (ABBV-CLS-579) prior VEGFR TKI therapy (EXP VEGFR TKI combination). Primary objectives are to assess safety, tolerability, pharmacokinetics, and to determine the recommended expansion dose and/or maximum tolerated dose of ABBV-CLS-484 or ABBV-CLS-579, both as monotherapy and in combination. Evaluation of objective response rate (RECIST v1.1) is a primary objective in EXP phase and a secondary objective in ESC phase. Both studies are active, and as of 30 Nov 2022, 30 (ABBV-CLS-484) and 45 (ABBV-CLS-579) patients had been enrolled in ESC phase. Citation Format: Patricia M. LoRusso, Emiliano Calvo Aller, Noboru Yamamoto, Chia-Chi Lin, Thaddeus Beck, David Sommerhalder, Do-Youn Oh, John D. Powderly, Talia Golan, Linu Sara Abraham, Joel S. Hayflick, Tamar Uziel, Priya Brunsdon, Wijith Munasinghe, Marcia Paddock, Cathleen Brdlik Hartman, Jonathan Powell, Bruno Medeiros, Martha R. Neagu, Benedito A. Carneiro. First-in-human phase 1 studies of PTPN2/1 inhibitors ABBV-CLS-484 and ABBV-CLS-579 in locally advanced or metastatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT257.

Background: Venetoclax (ven) in combination with hypomethylating agents or low dose cytarabine leads to rapid and durable remission in patients (pts) with acute myeloid leukemia (AML) unfit for intensive chemotherapy (IC), however, pts with TP53 mutations (TP53mut) exhibit adverse prognosis. Here, we identify dysregulation of the integrated stress response (ISR) pathway in pts with TP53mut, specifically through the actions of DAP3 binding cell death enhancer 1 (DELE1) and its activating protease OMA1. Methods: RNA sequencing was performed on pre-treatment bone marrow-derived mononuclear cells (BMMCs) from pts with AML ineligible for IC across four clinical trials (NCT02993523, NCT02203773, NCT03069352, and NCT02287233). CRISPR-Cas9 editing of TP53-intact AML cell lines was used to generate TP53-/-, TP53mut, DELE1-/-, OMA1-/- and TP53/DELE1 double deficient cell lines. Quantification of BCL2 and Myeloid cell leukemia 1 (MCL1) complexes with BCL2 interacting mediator (BIM) was performed using chemiluminescence assays. A drug screen to assess viability and gene expression was performed in cells treated with ven in combination with 63 drugs. Results: In total 401 pts were included in the analyses, of which 17% harbored TP53mut and 83% were TP53wt. Analysis of genes differentially expressed (DE) between pts with TP53mut and TP53wt AML revealed 19 DE genes shared across all trials. DELE1, an ISR adaptor, was associated with genes mapping to the ISR-related eukaryotic initiation factor-α (eIF2α) in TP53mut AML BMMCs. Deletion of DELE1 or OMA1 in AML cell lines blocked eIF2α activation and induction of the transcription factor ATF4, a critical ISR effector, in response to the mitochondrial stressor FCCP, ven, and azacitidine, and resulted in ven resistance similar to that of TP53 deficient cells. All modified AML cell lines exhibited a concomitant decrease in the pro-apoptotic regulator PMAIP1, encoding NOXA, and a 14-18-fold increase in MCL1-BIM complexes following ven treatment, as well as increased MCL1 expression, compared to parental lines. Further screening of these cell lines for ven sensitizing activity revealed the BH3 mimetic S63845, which targets MCL1, as the top hit, suggesting that combined BH3 mimetics may overcome ven resistance during ISR pathway defects. As DELE1 is located on chromosome 5 (CH); the frequent loss of CH 5q among TP53mut AML may contribute to low DELE1 expression. Conclusions: These data suggest that defective ISR signaling may be a factor in TP53mut AML treatment outcome and point to DELE1 dysregulation as a driver of ISR inactivation in pts with TP53mut AML. The ISR induces the expression of NOXA, which displaces MCL1 from BIM and lowers the apoptotic threshold. Our data identify p53, DELE1, and OMA1 as regulators of NOXA expression post ven treatment and indicate that co-targeting MCL1 in pts with TP53mut AML may be beneficial to overcome ven resistance. Citation Format: David Sharon, Paul Jung, Yan Sun, Weiguo Feng, Ziping Yang, Valerie Robinson, Diya Mitra, Wei Liu, Pingping Zheng, Tamar Uziel, Lloyd Lam, Mark D. Minden, Jeremy Ross, Wellington Mendes, Jalaja Potluri, Andrew H. Wei, Marina Konopleva, Monique Dail, Brenda Chyla, PK Epling-Burnette. DELE1 loss and dysfunctional integrated stress signaling in TP53 mutated AML is a novel pathway for venetoclax resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2530.

The antiapoptotic protein BCL2 plays critical roles in regulating lymphocyte development and immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL2 is critical for antitumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL2, would influence the antitumor activity of immune checkpoint inhibitors (ICI). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the antitumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T-cell function in response to antigen stimuli in vitro and did not antagonize T-cell activation induced by anti–PD-1. Furthermore, we demonstrate that the antiapoptotic family member BCL-XL provides a survival advantage in effector T cells following inhibition of BCL2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy. Significance: The antiapoptotic oncoprotein BCL2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development, and immune system regulation. Here we demonstrate that venetoclax, the first FDA/European Medicines Agency–approved BCL2 inhibitor, unexpectedly can be combined preclinically with immune checkpoint inhibitors to enhance anticancer immunotherapy, warranting clinical evaluation of these combinations. This article is highlighted in the In This Issue feature, p. 1

Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clinical activity in monotherapy cancer trials. A reduced number of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi1–5. Given that similar haematological and gastrointestinal defects were observed after genetic silencing of Brd4 in mice6, the platelet and gastrointestinal toxicities may represent on-target activities associated with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions7–9 and different cellular phenotypes after pharmacological inhibition of one or both bromodomains have been reported10,11, suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compounds that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment10–13. Here we carried out a medicinal chemistry campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukaemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-07514. Analyses of RNA expression and chromatin immunoprecipitation followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-containing super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy. ABBV-744, a selective inhibitor of the BD2 domains of BET family proteins, is effective against prostate cancer in mouse xenograft models, with lower toxicities than the dual-bromodomain BET inhibitor ABBV-075.

Dual bromodomain BET inhibitors that bind with similar affinities to the first and second bromodomains across BRD2, BRD3, BRD4, and BRDT have displayed modest activity as monotherapy in clinical trials. Thrombocytopenia, closely followed by symptoms characteristic of gastrointestinal toxicity, have presented as dose-limiting adverse events that may have prevented escalation to higher dose levels required for more robust efficacy. ABBV-744 is a highly selective inhibitor for the second bromodomain of the four BET family proteins. In contrast to the broad antiproliferative activities observed with dual bromodomain BET inhibitors, ABBV-744 displayed significant antiproliferative activities largely although not exclusively in cancer cell lines derived from acute myeloid leukemia and androgen receptor positive prostate cancer. Studies in acute myeloid leukemia xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable with the pan-BET inhibitor ABBV-075 but with an improved therapeutic index. Enhanced antitumor efficacy was also observed with the combination of ABBV-744 and the BCL-2 inhibitor, venetoclax compared with monotherapies of either agent alone. These results collectively support the clinical evaluation of ABBV-744 in AML (Clinical Trials.gov identifier: NCT03360006).

8577 Background: Predicting variable therapeutic responses that are driven by the genetic and transcriptomic heterogeneity of Small Cell Lung Cancer (SCLC) offers an opportunity for implementing precision therapies within the cancer cells and tumor microenvironment (TME). MERU was a Phase III study of Rovalpituzumab Tesirine (Rova-T) as maintenance therapy following first-line platinum-based chemotherapy in participants with extensive stage SCLC. In this study, we comprehensively analyzed the baseline genomic data in the MERU cohort to interrogate SCLC’s heterogeneous TME and tumor-intrinsic molecular and genetic drivers for therapeutic vulnerabilities. Methods: RNA-seq and Whole-Exome-Sequencing (WES) data were collected from archival tumor samples of 306 of 740 subjects enrolled in MERU. RNA-seq reads were aligned with STAR and quantified for gene expression by HTSeq. WES reads were analyzed for somatic mutation and copy number variation using AbbVie’s tumor-only WES pipeline. TME heterogeneity was evaluated using gene-set variation analysis of pan-cancer TME gene signatures. SCLC subtyping was performed using expression of 4 transcriptional factors (TF): ASCL1, NEUROD1, POU2F3, and YAP1. A computational framework of mapping MERU transcriptome expression profile to Cancer Dependency Map (DepMap) dataset was developed to synthetically screen MERU samples’ drug sensitivity. Results: TF subtyping reveals high prevalence of SCLC-A and -N subtypes in the MERU cohort, consistent with high expression of DLL3 in these two neuroendocrine subtypes. Correlation of TME gene signature scores identified two distinct clusters in the MERU cohort with correspondingly polarized immune-suppressive and -inflamed phenotypes. The pro-inflammatory score combining IFN-gamma and TGF-beta signatures, predicted prognosis in the MERU cohort better than the previously reported SCLC-I signature (Hazard Ratio: 0.71 [95% CI 0.38-1.3] vs. 1.29 [95% CI 0.65-2.6]). WES analysis identified high prevalence of TP53 and RB1 mutations, in line with the reported prevalence of these genetic drivers in SCLC. The clinical characteristics including gender, smoking status, and prior treatment, are not significantly associated with either TF or TME subgroup. The computational drug screen framework maps 83% of MERU samples to DepMap SCLC cell lines. The correlation of subtype TF expression with drug sensitivity was highly concordant between MERU and DepMap. Collectively, this approach demonstrated that molecular subtyping can be leveraged to broadly predict drug response in SCLC patients. Conclusions: Our comprehensive genomic analysis of the MERU cohort provides new insights into SCLC heterogeneity from both tumor-intrinsic and tumor-immune interaction perspectives and shall contribute to the development of predictive biomarkers and therapeutic opportunities for SCLC.