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Supplemental Figure 3. Chemotherapy and venetoclax combination cell viability assays. Cell viability assays were performed for 72h treatment of cyclophosphamide and topotecan, venetoclax, or combination at indicated concentrations, with bliss synergy scores in (A) MYCN-amplified, and (B) MYCN-wild-type cell lines. (C) Plot of Bliss sum of cell lines in A-B, calculated by the summation of the bliss synergy scores across the doses presented.

Supplemental Figure 2. PDX ex vivo model demonstrated cleavage of PARP with corresponding NOXA upregulation (A) Western blot of COG-N-561 ex vivo cell line treated overnight with antibodies as indicated.

Supplemental Figure 2: The susceptibility of breast cancer cell lines to MCL-1 siRNA according to genotype and expression of BCL-2 family members.

Since gaining approval for the treatment of chronic lymphocytic leukemia (CLL), the BCL-2 inhibitor venetoclax has transformed the treatment of this and other blood-related cancers. Reflecting the large and hydrophobic BH3-binding groove within BCL-2, venetoclax has significantly higher molecular weight and lipophilicity than most orally administered drugs, along with negligible water solubility. Although a technology-enabled formulation successfully achieves oral absorption in humans, venetoclax tablets have limited drug loading and therefore can present a substantial pill burden for patients in high-dose indications. We therefore generated a phosphate prodrug (3, ABBV-167) that confers significantly increased water solubility to venetoclax and, upon oral administration to healthy volunteers either as a solution or high drug-load immediate release tablet, extensively converts to the parent drug. Additionally, ABBV-167 demonstrated a lower food effect with respect to venetoclax tablets. These data indicate that beyond-rule-of-5 molecules can be successfully delivered to humans via a solubility-enhancing prodrug moiety to afford robust exposures of the parent drug following oral dosing.

BCL2, BCL2L1 and MCL1 mRNA in CD138 primary MM patient samples and sensitivity to veneteclax ex vivo

Legends for Tables S1 to S3 and Figures S1 to S5

Caspase-dependent degradation of MCL-1 in NCI-H929 HMCL following treatment with bortezomib treatment

1. Measured pKa values of ABBV-167 (Table S1. Ionization constants of ABBV-167) 2. Preparation of ABBV-167 on large scale (Scheme S1: Synopsis of GMP synthesis of ABBV-167) 3. Preparation of clinical ABBV-167 drug supply 4. Animal pharmacokinetics (Table S2. Single dose pharmacokinetics of prodrug 3 and parent venetoclax in mouse and dog after IV dosing of prodrug 3. Table S3. Single dose pharmacokinetics of 3 after oral dosing in mouse and dog. Table S4. Single dose pharmacokinetics of parent venetoclax in mouse and dog after oral dosing of 3.) 5. Clinical study (Study design, Pharmacokinetic and statistical analysis, Safety and tolerability assessments, Clinical pharmacokinetic results, Table S5. Effect of prodrug and food on venetoclax bioavailability, Safety results) 6. Crystallography methods (Protein preparation. BCL-2 ABBV-167 complex crystallization. X-ray structure determination. Table S6. Diffraction data collection and refinement statistics for BCL-2 in complex with ABBV-167 (PDB code 7LHB))

Supplemental Figure 3: MCL-1 dependency dictates cellular response to the CDK9 inhibitor flavopiridol and correlates with kinetics of death resulting from MCL-1 siRNA treatment.

BCL-2 and BCL-XL IHC correlates with qPCR and immunobot analysis of HMCLs

BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL–selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2High/BCL-XLLow. In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132–44. ©2016 AACR.

Supplemental Figure 4: BCL-XL but not BCL-2 gene silencing sensitize HCC-1806 cells to A-1210477-induced apoptosis.

List of Supplementary Figure Legends. Figure legend information.

Supplemental Figure 5: Flavopiridol synergizes with navitoclax in breast cancer cell lines in vitro.

Correlation of mRNA expression of BCL2 family members and sensitivity to venetoclax or navitoclax in HMCLs

Supplemental Table 1: Viability of breast cancer cell lines treated with PBS, scrambled siRNA (Sc; 20nMol) or MCL-1 siRNA (20nMol) for 72hrs compared to non-treated cells with associated genomic info.

Supplemental Figure 1: Validation of MCL-1 siRNA.

Conceptual workflow for establishing BCL-2 family IHC cut-offs for analysis of MM patient tumor samples

Supplemental Figure 4. Body Weight of Mice for in vivo treatments. (A) Plot of percent change in body weight from pre-treatment body weight in all in-vivo treatments.

qPCR primer sequences for Apopanel of BCL2 family members

Supplemental Table 2: Navitoclax EC50s in breast cancer cell lines in the presence or absence of MCL-1 siRNA.

Supplemental Figure 1. NVP-CGM097 and venetoclax combination cell viability assays. Cell viability assays were performed for 16h treatment of NVP-CGM097, venetoclax, or combination at indicated concentrations, with bliss synergy scores in (A) MYCN-wild-type, p53-wild-type, (B) MYCN-amplified, p53-mutant, and (C) MYCN-wild-type, p53-mutant NB cell lines. Red indicates synergy, blue indicates antagonism. (D) Plot of Bliss sum of cell lines in A-C along with cell lines in Fig. 1B, calculated by the summation of the bliss synergy scores across the doses presented.

Supplemental Table 1 shows venetoclax screen data (AUC and IC50) across 791 solid tumor cell lines.

EWS-FLI1 increases BCL-2 expression in EWFT.

Sup. Table 2. Top 3 functional pathways from ingenuity pathways analysis (IPA) in DTC.

Sup. Table 1. Genes analyzed by RNA-seq with >1 absolute Log2 fold change between PC9 parental and PC9-DTC.

Purpose: EGFR inhibitors (EGFRi) are effective against EGFR-mutant lung cancers. The efficacy of these drugs, however, is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M. We recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells [referred to as drug-tolerant cells (DTC)] prior to acquiring secondary mutations like T790M.Experimental Design: We have developed DTCs to EGFRi in EGFR-mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR-mutant lung cancer tumors in vivo.Results: We demonstrate surviving EGFR-mutant lung cancer cells upregulate the antiapoptotic protein MCL-1 in response to short-term EGFRi treatment. Mechanistically, DTCs undergo a protein biosynthesis enrichment resulting in increased mTORC1-mediated mRNA translation of MCL-1, revealing a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors. Moreover, MCL-1 is a key molecule governing the emergence of early EGFR-mutant DTCs to EGFRi, and we demonstrate it can be effectively cotargeted with clinically emerging MCL-1 inhibitors both in vitro and in vivo.Conclusions: Altogether, these data reveal that this novel therapeutic combination may delay the acquisition of secondary mutations, therefore prolonging therapy efficacy. Clin Cancer Res; 24(22); 5658–72. ©2018 AACR.

Olaparib sensitivity is modulated by BCL2 family expression

Supplemental Figure 1. Venetoclax sensitivity and BCL-2 mRNA correlation analysis; Supplemental Figure 2. Cell viability assays of border line venetoclax-sensitive and - resistant SCLC cell lines and combination of venetoclax with chemotherapy; Supplemental Figure 3. Correlation analysis of BCL-2 family members to venetoclax sensitivity in SCLC cell lines; Supplemental Figure 4. Comparison of BCL-2 family members to venetoclax sensitivity in SCLC cell lines; Supplemental Figure 5. BCL-2 mRNA expression is increased in SCLC compared to other cancer types; Supplemental Figure 6. Cell viability of venetoclax-sensitive and -resistant SCLC cell lines; Supplemental Figure 7. Weight profiles of xenograft and PDX models treated with venetoclax; Supplemental Figure 8. The PDX models from this study express high levels of BCL-2.

A chemotherapy naive and chemotherapy resistant cell line pair respond differently to olaparib.

Purpose:It was recently demonstrated that the EWSR1-FLI1 t(11;22)(q24;12) translocation contributes to the hypersensitivity of Ewing sarcoma to PARP inhibitors, prompting clinical evaluation of olaparib in a cohort of heavily pretreated Ewing sarcoma tumors. Unfortunately, olaparib activity was disappointing, suggesting an underappreciated resistance mechanism to PARP inhibition in patients with Ewing sarcoma. We sought to elucidate the resistance factors to PARP inhibitor therapy in Ewing sarcoma and identify a rational drug combination capable of rescuing PARP inhibitor activity.Experimental Design:We employed a pair of cell lines derived from the same patient with Ewing sarcoma prior to and following chemotherapy, a panel of Ewing sarcoma cell lines, and several patient-derived xenograft (PDX) and cell line xenograft models.Results:We found olaparib sensitivity was diminished following chemotherapy. The matched cell line pair revealed increased expression of the antiapoptotic protein BCL-2 in the chemotherapy-resistant cells, conferring apoptotic resistance to olaparib. Resistance to olaparib was maintained in this chemotherapy-resistant model in vivo, whereas the addition of the BCL-2/XL inhibitor navitoclax led to tumor growth inhibition. In 2 PDXs, olaparib and navitoclax were minimally effective as monotherapy, yet induced dramatic tumor growth inhibition when dosed in combination. We found that EWS-FLI1 increases BCL-2 expression; however, inhibition of BCL-2 alone by venetoclax is insufficient to sensitize Ewing sarcoma cells to olaparib, revealing a dual necessity for BCL-2 and BCL-XL in Ewing sarcoma survival.Conclusions:These data reveal BCL-2 and BCL-XL act together to drive olaparib resistance in Ewing sarcoma and reveal a novel, rational combination therapy that may be put forward for clinical trial testing.

Sup. Table 3. Fold change of FBX7 from RNA-seq analysis.

DNA damage is increased with the combination of olaparib and navitoclax

Figure legends for Supplementary Figures S1-S8.

A chemotherapy naive and chemotherapy resistant cell line pair respond differently to chemotherapeutics

Olaparib induces similar levels of DNA damage in CHLA9 and CHLA10

Figure S1. DTCs upregulate TORC1-mediated MCL-1 translation. Figure S2. High-content cell imaging assay. Figure S3. Genetic inhibition of MCL-1 is sufficient to induce apoptosis following EGFR inhibitor treatment. Figure S4. EGFR mutant tumors (PC9) respond to EGFR inhibitor/MCL-1 inhibitor (S63845) combination therapy in vivo. Figure S5. EGFR mutant tumors (HCC827) respond to EGFR inhibitor/MCL-1 inhibitor (S63845) combination therapy in vivo. Figure S6. Upregulation of MCL-1 in EGFR mutant cell lines occurs with other insults.

Supplementary figure legends

Figure legends for Supplementary Figures S1-S8.

Purpose: Small-cell lung cancer (SCLC) is an often-fatal neuroendocrine carcinoma usually presenting as extensive disease, carrying a 3% 5-year survival. Despite notable advances in SCLC genomics, new therapies remain elusive, largely due to a lack of druggable targets.Experimental Design: We used a high-throughput drug screen to identify a venetoclax-sensitive SCLC subpopulation and validated the findings with multiple patient-derived xenografts of SCLC.Results: Our drug screen consisting of a very large collection of cell lines demonstrated that venetoclax, an FDA-approved BCL-2 inhibitor, was found to be active in a substantial fraction of SCLC cell lines. Venetoclax induced BIM-dependent apoptosis in vitro and blocked tumor growth and induced tumor regressions in mice bearing high BCL-2–expressing SCLC tumors in vivo. BCL-2 expression was a predictive biomarker for sensitivity in SCLC cell lines and was highly expressed in a subset of SCLC cell lines and tumors, suggesting that a substantial fraction of patients with SCLC could benefit from venetoclax. Mechanistically, we uncover a novel role for gene methylation that helped discriminate high BCL-2–expressing SCLCs.Conclusions: Altogether, our findings identify venetoclax as a promising new therapy for high BCL-2–expressing SCLCs. Clin Cancer Res; 24(2); 360–9. ©2017 AACR.

Navitoclax sensitizes EWFT to olaparib

Therapeutic outcomes achieved in head and neck squamous cell carcinoma (HNSCC) patients by concurrent cisplatin-based chemoradiotherapy initially reflect both tumor regression and tumor stasis. However, local and distant metastasis and disease relapse are common in HNSCC patients. In the current work, we demonstrate that cisplatin treatment induces senescence in both p53 wild-type HN30 and p53 mutant HN12 head and neck cancer models. We also show that tumor cells can escape from senescence both in vitro and in vivo. We further establish the effectiveness of the senolytic, ABT-263 (Navitoclax), in elimination of senescent tumor cells after cisplatin treatment. Navitoclax increased apoptosis by 3.3-fold (P ≤ 0.05) at day 7 compared with monotherapy by cisplatin. Additionally, we show that ABT-263 interferes with the interaction between B-cell lymphoma-x large (BCL-X(L)) and BAX, anti- and pro-apoptotic proteins, respectively, followed by BAX activation, suggesting that ABT-263-induced apoptotic cell death is mediated through BAX. Our in vivo studies also confirm senescence induction in tumor cells by cisplatin, and the promotion of apoptosis coupled with a significant delay of tumor growth after sequential treatment with ABT-263. Sequential treatment with cisplatin followed by ABT-263 extended the humane endpoint to ∼130 days compared with cisplatin alone, where mice survived ∼75 days. These results support the premise that senolytic agents could be used to eliminate residual senescent tumor cells after chemotherapy and thereby potentially delay disease recurrence in head and neck cancer patients. SIGNIFICANCE STATEMENT: Disease recurrence is the most common cause of death in head and neck cancer patients. B-cell lymphoma-x large inhibitors such as ABT-263 (Navitoclax) have the capacity to be used in combination with cisplatin in head and neck cancer patients to eliminate senescent cells and possibly prevent disease relapse.

[Image: see text] Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase involved in the regulation of transcription elongation. An inhibition of CDK9 downregulates a number of short-lived proteins responsible for tumor maintenance and survival, including the antiapoptotic BCL-2 family member MCL-1. As pan-CDK inhibitors under development have faced dosing and toxicity challenges in the clinical setting, we generated selective CDK9 inhibitors that could be amenable to an oral administration. Here, we report the lead optimization of a series of azaindole-based inhibitors. To overcome early challenges with promiscuity and cardiovascular toxicity, carboxylates were introduced into the pharmacophore en route to compounds such as 14 and 16. These CDK9 inhibitors demonstrated a reduced toxicity, adequate pharmacokinetic properties, and a robust in vivo efficacy in mice upon oral dosing.

Venetoclax is a small molecule inhibitor of the prosurvival protein BCL-2 that has gained market approval in BCL-2–dependent hematologic cancers including chronic lymphocytic leukemia and acute myeloid leukemia. Neuroblastoma is a heterogenous pediatric cancer with a 5-year survival rate of less than 50% for high-risk patients, which includes nearly all cases with amplified MYCN. We previously demonstrated that venetoclax is active in MYCN-amplified neuroblastoma but has limited single-agent activity in most models, presumably the result of other pro-survival BCL-2 family protein expression or insufficient prodeath protein mobilization. As the relative tolerability of venetoclax makes it amenable to combining with other therapies, we evaluated the sensitivity of MYCN-amplified neuroblastoma models to rational combinations of venetoclax with agents that have both mechanistic complementarity and active clinical programs. First, the MDM2 inhibitor NVP-CGM097 increases the prodeath BH3-only protein NOXA to sensitize p53-wild-type, MYCN-amplified neuroblastomas to venetoclax. Second, the MCL-1 inhibitor S63845 sensitizes MYCN-amplified neuroblastoma through neutralization of MCL-1, inducing synergistic cell killing when combined with venetoclax. Finally, the standard-of-care drug cocktail cyclophosphamide and topotecan reduces the apoptotic threshold of neuroblastoma, thus setting the stage for robust combination efficacy with venetoclax. In all cases, these rational combinations translated to in vivo tumor regressions in MYCN-amplified patient-derived xenograft models. Venetoclax is currently being evaluated in pediatric patients in the clinic, including neuroblastoma (NCT03236857). Although establishment of safety is still ongoing, the data disclosed herein indicate rational and clinically actionable combination strategies that could potentiate the activity of venetoclax in patients with amplified MYCN with neuroblastoma.

BCL-XL, an antiapoptotic member of the BCL-2 family of proteins, drives tumor survival and maintenance and thus represents a key target for cancer treatment. Herein we report the rational design of a novel series of selective BCL-XL inhibitors exemplified by A-1293102. This molecule contains structural elements of selective BCL-XL inhibitor A-1155463 and the dual BCL-XL/BCL-2 inhibitors ABT-737 and navitoclax, while representing a distinct pharmacophore as assessed by an objective cheminformatic evaluation. A-1293102 exhibited picomolar binding affinity to BCL-XL and both efficiently and selectively killed BCL-XL-dependent tumor cells. X-ray crystallographic analysis demonstrated a key hydrogen bonding network in the P2 binding pocket of BCL-XL, while the bent-back moiety achieved efficient occupancy of the P4 pocket in a manner similar to that of navitoclax. A-1293102 represents one of the few distinct structural series of selective BCL-XL inhibitors, and thus serves as a useful tool for biological studies as well as a lead compound for further optimization.

Tumor cells undergo senescence in response to both conventional and targeted cancer therapies. The induction of senescence in response to cancer therapy can contribute to unfavorable patient outcomes, potentially including disease relapse. This possibiliy is supported by our findings that tumor cells induced into senescence by doxorubicin or etoposide can give rise to viable tumors in vivo. We further demonstrate sensitivity of these senescent tumor cells to the senolytic ABT‐263 (navitoclax), therefore providing a “two‐hit” approach to eliminate senescent tumor cells that persist after exposure to chemotherapy or radiation. The sequential combination of therapy‐induced senescence and ABT‐263 could shift the response to therapy toward apoptosis by interfering with the interaction between BCL‐XL and BAX. The administration of ABT‐263 after either etoposide or doxorubicin also resulted in marked, prolonged tumor suppression in tumor‐bearing animals. These findings support the premise that senolytic therapy following conventional cancer therapy may improve therapeutic outcomes and delay disease recurrence.

Mantle cell lymphoma (MCL) is an aggressive and largely incurable subtype of non-Hodgkin's lymphoma. Venetoclax has demonstrated efficacy in MCL patients with relapsed or refractory disease, however response is variable and less durable than CLL. This may be the result of co-expression of other anti-apoptotic proteins such as MCL-1, which is associated with both intrinsic and acquired resistance to venetoclax in B-cell malignancies. One strategy for neutralizing MCL-1 and other short-lived survival factors is to inhibit CDK9, which plays a key role in transcription. Here, we report the response of MCL cell lines and primary patient samples to the combination of venetoclax and novel CDK9 inhibitors. Primary samples represented de novo patients and relapsed disease, including relapse after ibrutinib failure. Despite the diverse responses to each single agent, possibly due to variable expression of the BCL-2 family members, venetoclax plus CDK9 inhibitors synergistically induced apoptosis in MCL cells. The synergistic effect was also confirmed via venetoclax plus a direct MCL-1 inhibitor. Murine xenograft studies demonstrated potent in vivo efficacy of venetoclax plus CDK9 inhibitor that was superior to each agent alone. Together, this study supports clinical investigation of this combination in MCL, including in patients who have progressed on ibrutinib.

Introduction: Specific B cell malignancies, including CLL and the aggressive non-GCB subtype of DLBCL, are driven by constitutive activation of the B cell receptor (BCR) pathway and the transcription factor NF-κB. Pharmacological inhibition of MALT1 protease, a key mediator of the BCR/NF-κB signal transduction pathway, may therefore provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Experimental Procedures: A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results: Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-κB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton’s tyrosine kinase (BTK) inhibitors. NF-κB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-κB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with B cell malignancies. Citation Format: Joshua P. Plotnik, Raghuveer S. Mali, Velitchka Bontcheva, Colleen Dowell, Jun Chen, Adam E. Richardson, Ryan A. McClure, Paul Jung, Lan Pham, Andrew J. Souers, Jonathan A. Meulbroek, William N. Pappano. MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia [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 6156.

MCL-1 is one of the most frequently amplified genes in cancer, facilitating tumor initiation and maintenance and enabling resistance to anti-tumorigenic agents including the BCL-2 selective inhibitor venetoclax. The expression of MCL-1 is maintained via P-TEFb-mediated transcription, where the kinase CDK9 is a critical component. Consequently, we developed a series of potent small-molecule inhibitors of CDK9, exemplified by the orally active A-1592668, with CDK selectivity profiles that are distinct from related molecules that have been extensively studied clinically. Short-term treatment with A-1592668 rapidly downregulates RNA pol-II (Ser 2) phosphorylation resulting in the loss of MCL-1 protein and apoptosis in MCL-1-dependent hematologic tumor cell lines. This cell death could be attenuated by either inhibiting caspases or overexpressing BCL-2 protein. Synergistic cell killing was also observed between A-1592668 or the related analog A-1467729, and venetoclax in a number of hematologic cell lines and primary NHL patient samples. Importantly, the CDK9 inhibitor plus venetoclax combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in mouse models of lymphoma and AML. These data indicate that CDK9 inhibitors could be highly efficacious in tumors that depend on MCL-1 for survival or when used in combination with venetoclax in malignancies dependent on MCL-1 and BCL-2.