BH3 Mimetics Research Articles

Abstract 4098: Chimeric antigen receptor (CAR) T cells overexpressing Bcl-xL increase proliferation and antitumor activity alone and in combination with BH3 mimetics

Abstract Background: Chimeric antigen receptor (CAR) T cells have become a well-established treatment option for patients, with six products approved for different hematologic diseases and new approvals allowing for their therapeutic use as early as second line. However, relapse rates of around 50% have been observed in all patient subsets, with one major mechanism associated with CAR T failure being cancer cell resistance to apoptosis. A form of cancer therapeutic named BH3-mimetics has been designed to inhibit members of the anti-apoptotic B cell lymphoma-2 (Bcl-2) family and, therefore, directly activate the apoptotic machinery in malignant cells. We hypothesized that integration of these anti-apoptotic molecules into CAR T cells would induce resistance towards the BH3 mimetics and allow combinational therapeutic approaches. Methods: 4-1BB and CD28 CAR constructs were designed to overexpress one of four anti-apoptotic proteins: wildtype Bcl-2, a Venetoclax-resistant Bcl-2 variant (G101V), B cell extra-large (Bcl-xL), or myeloid cell leukemia-1 (Mcl-1). CAR T cells made from these constructs were tested against leukemia (Nalm6) and lymphoma (JeKo-1) cell lines in combination with three different BH-3 mimetics: Venetoclax (ABT-199, an FDA-approved Bcl-2 inhibitor), Navitoclax (ABT-263, a Bcl-2/Bcl-xL inhibitor), and AZD5991 (an Mcl-1 inhibitor). Results: CAR T cells with a 4-1BB costimulatory domain tended to have increased killing over CARs with CD28 and were less susceptible to apoptosis; therefore, 4-1BB CARs were used for all further testing. Integration of Bcl-2 family proteins into CAR T cells didn’t impair or even increase tumor cell clearance in vitro; however, in combination with Venetoclax, Navitoclax, or AZD5991, killing capacity significantly increased compared to control CAR T cells. Even without combination with drugs, CAR T cells overexpressing Bcl-xL and Bcl-2 (both wildtype and mutant) provided higher anti-tumor activity and prolonged survival against JeKo-1 cells in vivo, whereas only Bcl-xL overexpression showed increased tumor control compared to regular 4-1BB CARs against Nalm6 cells. Conclusion: Of the tested antiapoptotic proteins, Bcl-xL overexpressing CAR T cells proved superior, having higher proliferation and increased anti-tumor activity in combination with or without BH3 mimetics, providing a new strategy to optimize CAR T cell function for the treatment of leukemia and lymphoma. Citation Format: Felix Korell, Michael Olson, Diego Salas-Benito, Mark B. Leick, Rebecca C. Larson, Harrison Silva, Alessandro Gasparetto, Trisha R. Berger, Amanda Bouffard, Michael C. Kann, Markus Mergen, Tamina Kienka, Marc Wehrli, Stefanie R. Bailey, Anthony Letai, Marcela V. Maus. Chimeric antigen receptor (CAR) T cells overexpressing Bcl-xL increase proliferation and antitumor activity alone and in combination with BH3 mimetics. [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 4098.

Abstract 2530: DELE1 loss and dysfunctional integrated stress signaling in TP53 mutated AML is a novel pathway for venetoclax resistance

Abstract 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.

Abstract 5731: Apoptosis Inducing Agent 1 enhances cancer therapy-induced apoptosis by direct interaction with BAX and BAK

Abstract Many of the most effective anti-cancer therapies induce apoptosis in cancer cells by damaging DNA. Even when effective at eradicating primary cancers, DNA-damaging anti-cancer agents cause mutations that can lead to the development of secondary cancers. Furthermore, DNA damage responses are dependent on wild-type p53, which is mutated in over 50% of cancers and causes resistance to therapies. Fortunately, many p53 mutant cancers continue to express high levels of the pro-apoptotic, pore-forming proteins BAX and BAK and are consequently “primed for apoptosis.” Primed cells are highly sensitive to pro-apoptotic signals and this vulnerability can be exploited using inhibitors of pro-survival BCL-2 family proteins such as the BCL-2 inhibitor ABT-199, which has been successful as therapy for chronic lymphocytic and acute myeloid leukemias (AML). Furthermore, most irreplaceable cells within healthy tissues are apoptosis resistant and express low levels of BAX and BAK. We therefore hypothesized that direct activators of BAX or BAK could be effective single-agent therapies for primed cancers and chemo-/radio-sensitizers for unprimed cancers. Using WT versus BAX -/- BAK -/- HeLa cells, we performed a high-throughput screen of nearly 100,000 compounds to identify small molecules that could directly activate BAX or BAK. 196 compounds were identified as hits and multiple orthogonal validation studies identified Apoptosis Inducing Agent 1 (AIA1) as the most specific and potent putative activator of BAX and BAK. Further validation studies demonstrated that AIA1 directly induces cytochrome c release from mitochondria in a BAX/BAK dependent manner and strongly enhances BIM-mediated activation of BAX and BAK and permeabilization of liposomes. Single-agent AIA treatment triggers apoptotic cell death in a broad panel of cancer cell lines and also sensitizes cancer cells to chemotherapeutic agents and especially BH3 mimetics targeting pro-survival BCL-2 family proteins. Importantly, AIA1 sensitizes ovarian cancer cells to BCL-XL inhibitors and AML cells to BCL-2 inhibitors regardless of p53 status. In vivo, AIA1 suppresses tumor growth and prolongs overall survival in both ovarian cancer and AML xenograft models without causing weight loss, thrombocytopenia or leukopenia. Notably, unprimed cancer cells that don’t immediately undergo apoptosis in response to single-agent AIA1 treatment upregulate expression of BAX and BAK, pro-survival proteins (BCL-XL, BCL-2, MCL-1) and pro-apoptotic proteins (BIM, BID, PUMA, Noxa) to produce a more highly primed apoptosis pathway and setting the stage for increased sensitivity to BH3 mimetics. Based on these findings, AIA1 may exploit and induce apoptotic vulnerabilities in cancers in a p53-independent manner and represent a safer strategy to target primed cancer cells while eliminating the potential for secondary malignancies and p53-mediated resistance. Citation Format: Xingping Qin, Cameron Fraser, Adam Presser, Johan KE Spetz, Stacey J. Yu, Gary A. Bradshaw, Marian Kalocsay, Bo R. Rueda, Tudor Moldoveanu, Kristopher A. Sarosiek. Apoptosis Inducing Agent 1 enhances cancer therapy-induced apoptosis by direct interaction with BAX and BAK. [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 5731.

Abstract 6612: Data-driven hallmarks of cancer as a new paradigm for precision medicine: multi-omics and functional profiling in acute myeloid leukemia

Abstract The cancer hallmark concept defines biological properties that play a key role in cancer development and progression. The hallmarks reflect generic properties across all cancer types, and are not directly quantifiable nor applicable in cancer diagnostics. Here, we applied a data-driven approach to identify quantifiable hallmarks to be used as a basis for precision cancer medicine (PCM) in Acute Myeloid Leukemia (AML). We applied deep exome, transcriptome, DNA methylation and proteome profiling as well as ex-vivo functional testing of 525 drugs to 118 AML patient samples. Unsupervised multi-omic dimensionality reduction defined 11 independent axes of biological variability that we interpreted to reflect data-driven hallmarks (DDHM) of AML. Each DDHM integrates and ranks different data types and features, pinpointing those molecular features that were most informative for each hallmark. We calculated values for the 11 DDHM for each patient, constructing a DDHM-based precision medicine approach for AML diagnosis and therapy assignment. Most DDHMs were driven by other data types than genomics. For several DDHMs, different cytogenetic and mutational drivers converge on the same hallmarks and specific drug vulnerabilities. We also see how DDHM predictors of poor prognosis and high-risk AML are distinct from those that dictate specific drug response vulnerabilities. DDHM 2 reflected the cell differentiation path leading towards resistance to BH3 mimetics, ACK-inhibitors, and anthracyclines, and sensitivity to MEK inhibitors and TLR8 agonists. DDHM 2 derived a strong impact from protein expression of GATA2, MLLT11, DNMT3B and from RNA expression of WT1 and SOX4 as well as from multiple markers of different monocytic subtypes. DDHM 5 depicted cell cycle regulation and the Megakaryocytic-erythrocyte progenitor cell state that was enriched in AML patients with antecedent hematological disease. Interestingly, DDHM5 was linked to the sensitivity to purine analogs and vinca alkaloids. Moreover, DDHMs 1 and 8 captured clinical risk groups, prognosis, and responsiveness to hypomethylating agents. These DDHMs also revealed the phenotypic and functional biology that distinguished patients with high versus low FLT3-ITD mutant allele frequency. Validation of the DDHMs is realized through profiling and analysis of prospective AML samples, AML cell lines and previously published datasets. In summary, we present a data-driven approach for defining hallmarks in AML. The application of the DDHMs in AML provides a new paradigm for PCM and an opportunity for combinatorial therapeutic targeting. Each patient is characterized by a combination of independent and potentially druggable hallmarks, as opposed to traditional stratification in PCM, where each patient is assigned to one specific subgroup defined by genetic or other biomarkers. Citation Format: Tom Erkers, Nona Struyf, Tojo James, Francesco Marabita, Mattias Vesterlund, Nghia Vu, Cornelia Arnroth, Albin Österroos, Anna Bohlin, Sofia Bengtzén, Matthias Stahl, Rozbeh Jafari, Lukas Orre, Yudi Pawitan, Brinton Seashore-Ludlow, Janne Lehtiö, Sören Lehmann, Päivi Östling, Olli Kallioniemi. Data-driven hallmarks of cancer as a new paradigm for precision medicine: multi-omics and functional profiling in acute myeloid 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 6612.

Abstract 6130: Cancer sensitivity to therapy is constrained by apoptosis regulation in cells of origin

Abstract Many chemotherapeutic agents target cellular components or processes that are present in all cancers, yet clinical responses to these agents vary greatly between cancer types and even patient age - the basis for these broad-scale differences are unclear. The vast majority of targeted and cytotoxic cancer therapies including ionizing radiation produce pro-apoptotic signaling in exposed cells, suggesting that the mitochondrial apoptosis sensitivity of cancer cells could act as a central signaling “node” to broadly impact therapy outcomes. To test this, we used BH3 profiling and complementary chemosensitivity assays to analyze hundreds of primary cancer specimens across twelve major cancer types. We find that cancers with typically favorable outcomes including certain hematologic malignancies, testicular cancer, and some pediatric cancers contain mitochondria that are highly primed for apoptosis, which renders them hypersensitive to cytotoxic as well as targeted agents and radiation therapy. Priming levels in many epithelial cancers including ovarian cancer and non-small cell lung cancer are highly heterogeneous, mirroring their variability in clinical outcomes. Finally, many tumor types that are typically chemoresistant including adult soft tissue sarcomas, hepatocellular carcinoma and pancreatic cancer are almost completely resistant to pro-apoptotic signaling. By analyzing in vitro and in vivo pancreatic, ovarian, hepatocellular and sarcoma tumorigenesis models, we find that apoptotic priming generally increases during neoplastic transformation, in part due to consistent upregulation of pro-apoptotic proteins BAX and BAK. However, the level of apoptotic priming in cancer cells is constrained by the baseline apoptosis sensitivity of normal cells prior to transformation. Remarkably, we find that apoptotic priming is dynamically regulated by cell lineage and differentiation state but can also be modulated by oncogenes. For instance, Myc activation typically increases apoptotic priming while activation of mutant Ras signaling decreases it - these changes in priming alter the chemosensitivity of cancer cells. Finally, we use inducible mouse tumor models to demonstrate that neoplastic transformation of cells from developmentally immature tissues yields pediatric tumors that are more primed for apoptosis than equivalent tumors arising in adults. This difference in priming causes pediatric tumors to be more sensitive to front-line therapies and BH3 mimetics targeting pro-survival BCL-2 family proteins in vitro and in vivo. Thus, lineage-determined regulation of apoptosis prior to and during neoplastic transformation leads to broad-scale differences in cancer cell chemosensitivity and can be exploited therapeutically by targeting BCL-2 family proteins. Citation Format: Cameron Fraser, Xingping Qin, Kenichi Shimada, Johan Spetz, Mary Heather Florido, Rumani Singh, Stacey Yu, Adam Presser, Zintis Inde, Gaurav Joshi, Jennifer Guerriero, Francisco Sanchez-Rivera, Alison Karst, Omar Lopez, Chendi Li, Peter Winter, Ying Yue, Peter Sorger, Jingwei Cheng, Izidore Lossos, Aaron Hata, Ronny Drapkin, Adam Palmer, James Decaprio, Manisha Thakuria, Charles Yoon, Ursula Matulonis, Matthew Meyerson, Elizabeth Stover, Diana Cardona, Kris Wood, Shayna Sarosiek, David Kirsch, Joseph Mancias, Andrew Cherniack, Anthony Letai, Kristopher Sarosiek. Cancer sensitivity to therapy is constrained by apoptosis regulation in cells of origin. [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 6130.

The mtDNA-STING pathway plays an important role in both navitoclax- and S63845-induced autophagy and enhances cell death.

Targeting BCL2 family proteins to induce cancer cell death has been successful in the treatment of cancer. BH3 mimetics such as ABT-737 not only induce cell death, but also activate autophagy. The molecular mechanism by which the BH3 mimetics induce autophagy is still controversial. In this study, we show that the BCL2/BCLXL/BCLw inhibitor navitoclax and the MCL1 inhibitor S63845 induce both apoptosis and autophagy in mouse embryonic fibroblasts (MEFs) and leukemia cell lines, while autophagy induced by navticlax and S63845 in leukemia cell lines requires the inhibition of caspase activities. Further experiments demonstrate that the autophagy induced by navitoclax or S63845 does not depend on Beclin 1, but downstream of Bax/Bak. Moreover, both navitoclax and S63845 treatment induce mtDNA release in MEFs, which activates STING and thereby induces autophagy, while STING KO inhibits both navitoclax- and S63845-induced autophagy. Furthermore, STING KO diminishes navitoclax- or S63845-induced apoptosis, suggesting that STING activation enhances rather than inhibits apoptosis. Thus, our findings provide new insights into the regulations of navitoclax- or S63845-induced autophagy and cell death.

8P Targeting antiapoptotic Bcl-2 proteins with highly specific BH3 mimetics in solid tumors

8P Targeting antiapoptotic Bcl-2 proteins with highly specific BH3 mimetics in solid tumors

Characterizing the consensus residue specificity and surface of BCL-2 binding to BH3 ligands using the Knob-Socket model.

Cancer cells bypass cell death by changing the expression of the BCL-2 family of proteins, which are apoptotic pathway regulators. Upregulation of pro-survival BCL-2 proteins or downregulation of cell death effectors BAX and BAK interferes with the initiation of the intrinsic apoptotic pathway. In normal cells, apoptosis can occur through pro-apoptotic BH3-only proteins interacting and inhibiting pro-survival BCL-2 proteins. When cancer cells over-express pro-survival BCL-2 proteins, a potential remedy is the sequestration of these pro-survival proteins through a class of anti-cancer drugs called BH3 mimetics that bind in the hydrophobic groove of pro-survival BCL-2 proteins. To improve the design of these BH3 mimetics, the packing interface between BH3 domain ligands and pro-survival BCL-2 proteins was analyzed using the Knob-Socket model to identify the amino acid residues responsible for interaction affinity and specificity. A Knob-Socket analysis organizes all the residues in a binding interface into simple 4 residue units: 3-residue sockets defining surfaces on a protein that pack a 4th residue knob from the other protein. In this way, the position and composition of the knobs packing into sockets across the BH3/BCL-2 interface can be classified. A Knob-Socket analysis of 19 BCL-2 protein and BH3 helix co-crystals reveal multiple conserved binding patterns across protein paralogs. Conserved knob residues such as a Gly, Leu, Ala and Glu most likely define binding specificity in the BH3/BCL-2 interface, whereas other residues such as Asp, Asn, and Val are important for forming surface sockets that bind these knobs. These findings can be used to inform the design of BH3 mimetics that are specific to pro-survival BCL-2 proteins for cancer therapeutics.

IFN-γ Signaling Sensitizes Melanoma Cells to BH3 Mimetics

Immunotherapy targeting PD-1 and/or CTLA4 leads to durable responses in a proportion of patients with melanoma. However, many patients will not respond to these immune checkpoint inhibitors, and up to 60% of responding patients will develop treatment resistance. We describe a vulnerability in melanoma driven by immune cell activity that provides a pathway towards additional treatment options. This study evaluated short-term melanoma cell lines (referred to as PD1 PROG cells) derived from melanoma metastases that progressed on PD-1 inhibitor-based therapy. We show that the cytokine IFN-γ primes melanoma cells for apoptosis by promoting changes in the accumulation and interactions of apoptotic regulators MCL-1, NOXA, and BAK. The addition of pro-apoptotic BH3 mimetic drugs sensitized PD1 PROG melanoma cells to apoptosis in response to IFN-γ or autologous immune cell activation. These findings provide translatable strategies for combination therapies in melanoma.

BH3 mimetics and TKI combined therapy for Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) was considered for a long time one of the most hostile leukemia that was incurable for most of the patients, predominantly due to the extreme resistance to chemotherapy. Part of the resistance to cell death (apoptosis) is the result of increased levels of anti-apoptotic and decreased levels of pro-apoptotic member of the BCL-2 family induced by the BCR-ABL1 oncoprotein. BCR-ABL1 is a constitutively active tyrosine kinase responsible for initiating multiple and oncogenic signaling pathways. With the development of specific BCR-ABL1 tyrosine kinase inhibitors (TKIs) CML became a much more tractable disease. Nevertheless, TKIs do not cure CML patients and a substantial number of them develop intolerance or become resistant to the treatment. Therefore, novel anti-cancer strategies must be developed to treat CML patients independently or in combination with TKIs. Here, we will discuss the mechanisms of BCR-ABL1-dependent and -independent resistance to TKIs and the use of BH3-mimetics as a potential tool to fight CML.

Characterization of the Second Apoptosis Inhibitor Encoded by Guinea Pig Cytomegalovirus.

Despite the usefulness of guinea pig cytomegalovirus (GPCMV) for studies on congenital CMV infection, its viral mechanisms for the evasion of host defense strategies have not been fully elucidated. We reported previously that GPCMV gp38.1 functions as a viral mitochondria-localized inhibitor of apoptosis-like function, and its weak activity suggested the presence of an additional inhibitory molecule(s). Here, we identified gp38.3-2, a 42-amino-acid (aa) reading frame embedded within the gp38.3 gene that encodes a positional homolog of murine CMV (MCMV) m41. Characterization of gp38.3-2 resulted in the following findings: (i) the aa sequence of gp38.3-2 shows some similarity to that of MCMV m41.1, a viral inhibitor of oligomerization of a member of Bcl-2 family protein BAK, but there is no correspondence in their predicted secondary structures; (ii) gp38.3-2, but not gp38.3, showed inhibitory activities against staurosporine-induced apoptosis; (iii) three-dimensional protein complex prediction suggests that the N-terminal α-helix of gp38.3-2 interacts with residues in the BH3 and BH1 motifs of BAK, and analysis of gp38.3-2 and BAK mutants supported this model; (iv) guinea pig fibroblast cells infected with gp38.3-2-deficient GPCMV strain Δ38.3-2 died earlier than cells infected with rescued strain r38.3-2, resulting in lower yields of Δ38.3-2; (v) Δ38.3-2 exhibited a partial but significant decrease in monocyte and macrophage infection in comparison with r38.3-2; and, however, (vi) little difference in the viral infection of guinea pigs was observed between these two strains. Therefore, we hypothesize that gp38.3-2 contributes little to the evasion of host defense mechanisms under the experimental conditions used. IMPORTANCE Although GPCMV provides a useful animal model for studies on the pathogenesis of congenital CMV infection and the development of CMV vaccine strategies, our understanding of the viral mechanisms by which it evades apoptosis of infected cells has been limited in comparison with those of murine and human CMVs. Here, we report a second GPCMV apoptosis inhibitor (42 amino acids in length) that interacts with BAK, a Bcl-2 family proapoptotic protein. Three-dimensional structural prediction indicated a unique BAK recognition by gp38.3-2 via the BH3 and BH1 motif sequences. Our findings suggest the potential development of BH3 mimetics that can regulate inhibition or induction of apoptosis based on short ~40-amino-acid peptide molecules as with GPCMV.

Killing in self-defense: proapoptotic drugs to eliminate intracellular pathogens.

Intracellular infections rely on host cell survival for replication and have evolved several mechanisms to prevent infected cells from dying. Drugs that promote apoptosis, a noninflammatory form of cell death, can dysregulate these survival mechanisms to kill infected cells via a mechanism that resists the evolution of drug resistance. Two such drug classes, known as SMAC mimetics and BH3 mimetics, have shown preclinical efficacy at mediating clearance of liver-stage malaria and chronic infections such as hepatitis-B virus and Mycobacterium tuberculosis. Emerging toxicity and efficacy data have reinforced the broad applicability of these drugs and form the foundations for preclinical and clinical studies into their various usage cases.

Bcl-2 Family Members Bcl-xL and Bax Cooperatively Contribute to Bortezomib Resistance in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma with poor prognosis, due to the inevitable development of drug resistance. Despite being the first-in-class proteasome inhibitor for relapsed/refractory MCL, resistance to bortezomib (BTZ) in MCL patients remains a major hurdle of effective therapy, and relapse following BTZ is frequent. Understanding the mechanisms underlying BTZ resistance is, therefore, important for improving the clinical outcome and developing novel therapeutic strategies. Here, we established de novo BTZ-resistant human MCL-derived cells with the highest resistance index of 300-fold compared to parental cells. We provided compelling evidence that both Bcl-xL and Bax are key mediators in determining BTZ sensitivity in MCL cells. Overexpression of antiapoptotic Bcl-xL and depletion of proapoptotic Bax cooperatively protected MCL cells against BTZ-induced apoptosis, causing acquired BTZ resistance, likely by tilting the balance of Bcl-2 family proteins toward antiapoptotic signaling. Bioinformatics analyses suggested that high BCL2L1 (encoded Bcl-xL) and low BAX were, in part, associated with poor prognosis of MCL patients, e.g., when combined with low OGT, which regulates cellular O-GlcNAcylation. Our findings support recent strategies in small molecule drug discovery co-targeting antiapoptotic Bcl-2 family proteins using BH3 mimetics and Bax using Bax activators to overcome cancer drug resistance.

Combinatorial Effects of BH3 Mimetics and CAR T Cells Targeting T-Cell Lymphomas

T-cell lymphomas (TCL) represent a significant unmet need for improved therapeutic strategies. Recent advances in disease modeling and molecular characterization have greatly increased the potential for advancement. We previously showed that a subset of TCL cell lines, patient-derived xenografts (PDXs), and primary samples express high and uniform levels of the tetraspanin CD37 (PMID: 30089630). Selective expression on malignant T cells distinguishes CD37 from other CAR T cell targets under development against TCL and led TCL inclusion in our phase 1 clinical trial of autologous CD37-directed CAR T cells (CAR-37; NCT04136275). CAR-37 T cells specifically lysed CD37+ TCL cell lines in vitro. In an aggressive T cell prolymphocytic leukemia PDX model, we observed >100-fold reduction in disease burden in vivo with CAR-37 T cells, but all mice eventually relapsed. We transplanted the relapsed tumor cells and re-treated with CAR-37 T cells. All recipients experienced a second response, with relapse in only a subset. Thus, a third dose of CAR-37 T cells was administered, but only a subset of mice underwent a third response. At the time of relapse/progression, CD37 expression was detected on tumor cells from each mouse. In addition, a small percentage of CAR-37 T cells were present in the tumor. Based on these results, we sought to evaluate combinatorial strategies that potentiate the tumoricidal activity of CAR-37 T cells and increase the rate of complete disease eradication. CAR T cells kill primarily through driving the intrinsic pathway of apoptosis. We previously showed that BH3 mimetics of BCL2, BCL-xL, and MCL1 can selectively induce apoptosis in TCLs based on predicted BH3 protein dependencies (PMID: 30498064). Thus, to define novel therapeutic combinations with CAR-37 T cells, we performed BH3 profiling on CD37-expressing TCL cell lines. Corresponding to the predicted dependencies via BH3 profiling, FEPD and SMZ1 were most sensitive to the MCL1 inhibitor AZD5991, whereas HUT78 was most sensitive to the BCL-2/BCL-xL inhibitor navitoclax. We observed concordant sensitivity patterns to the BH3 mimetics by viability/apoptosis assay and decreased active mitochondrial membrane potential. Importantly, CD37 antigen density was unaffected by exposure to BH3 mimetics. To evaluate the feasibility of combining BH3 mimetics and CAR T cells, we determined the impact of BH3 mimetics on CAR-37 T cell phenotype. First, we did not observe a reduction in CAR-37 T cell viability when cultured in vitro in the presence of TCL target cells and either venetoclax 0.5μM, navitoclax 0.5μM, or AZD5991 0.1μM for 48 hours. The same culture conditions also did not affect the differentiation or subset distribution of CAR-37 T cells. Nearly 60% of the CAR-37 T cells had an effector memory phenotype, whereas approximately 20% had a central memory phenotype and the remaining were either effector memory T cells re-expressing CD45RA or naïve. In addition, cytokine production (GMCSF and IFN-γ) and expression of T-cell exhaustion markers (PD-1, TIM3, and LAG3) was not affected by co-culture with BH3 mimetics. Since BH3 mimetics did not alter CAR-37 T cell activity, we evaluated the cytotoxicity effect of their combination against target TCL cell lines. Using a luminescence based killing assay and xCELLigence RTCA (ACEA), we found that all 3 BH3 mimetics in combination with CAR-37 T cells resulted in greater specific lysis of FEPD tumor cells. The most significant increase occurred in combination with AZD5991, which corresponds to the predicted MCL1 dependence of FEPD cells. Next, we evaluated the combination of CAR-37 T cells and AZD5991 in NSG mice engrafted with FEPD. CAR-37 T cells were administered upon a total flux (photons/s) ≥106 and the combination cohort subsequently received AZD5991 (100 mg/kg) on days 1 and 2, 8 and 9, and 15 and 16 post-CAR T cells. We observed significantly reduced tumor burden and prolonged survival in the combination cohort (median 42 days) compared to CAR-37 T cell (median 18 days) or AZD5991 (median 31 days) alone. Together, our findings demonstrate that BH3 mimetics with predicted activity against individual TCLs can enhance the cytotoxic activity of CAR-37 T cells. These results could be used to nominate and/or deprioritize future combination strategies both for TCL and for other patients being treated with effector T cells for a range of hematologic malignancies. AZD5991 was kindly provided by AstraZeneca.

BH3 mimetics in combination with nilotinib or ponatinib represent a promising therapeutic strategy in blast phase chronic myeloid leukemia

Dysregulation of the BCL-2 family is implicated in protecting chronic myeloid leukemia (CML) cells from intracellular damage and BCR::ABL1-inhibition with tyrosine kinase inhibitors (TKIs) and may be a viable therapeutic target in blast phase (BP-)CML, for which treatment options are limited. BH3 mimetics, a class of small molecule inhibitors with high-specificity against the prosurvival members of the BCL-2 family, have displayed clinical promise in the treatment of chronic lymphocytic and acute myeloid leukemia as single agents and in combination with standard-of-care therapies. Here we present the first comparison of inhibition of BCL-2 prosurvival proteins BCL-2, BCL-xL and MCL-1 in combination with a second or third generation TKI, crucially with comparisons drawn between myeloid and lymphoid BP-CML samples. Co-treatment of four BP-CML cell lines with the TKIs nilotinib or ponatinib and either BCL-2 (venetoclax), MCL-1 (S63845) or BCL-xL (A-1331852) inhibitors resulted in a synergistic reduction in cell viability and increase in phosphatidylserine (PS) presentation. Nilotinib with BH3 mimetic combinations in myeloid BP-CML patient samples triggered increased induction of apoptosis over nilotinib alone, and a reduction in colony-forming capacity and CD34+ fraction, while this was not the case for lymphoid BP-CML samples tested. While some heterogeneity in apoptotic response was observed between cell lines and BP-CML patient samples, the combination of BCL-xL and BCR::ABL1 inhibition was consistently effective in inducing substantial apoptosis. Further, while BH3 mimetics showed little efficacy as single agents, dual-inhibition of BCL-2 prosurvival proteins dramatically induced apoptosis in all cell lines tested and in myeloid BP-CML patient samples compared to healthy donor samples. Gene expression and protein level analysis suggests a protective upregulation of alternative BCL-2 prosurvival proteins in response to BH3 mimetic single-treatment in BP-CML. Our results suggest that BH3 mimetics represent an interesting avenue for further exploration in myeloid BP-CML, for which alternative treatment options are desperately sought.

Inhibition of PRMT5 Increases Sensitivity to BH3 Mimetics in Aggressive B Cell Malignancies

Introduction: Protein Arginine Methyltransferase 5 (PRMT5) is a type II methyltransferase that catalyzes symmetric dimethylation of arginine. PRMT5 is highly expressed in several types of aggressive B cell malignancies, and increased PRMT5 expression has been associated with poor disease prognosis. Several small molecule inhibitors targeting PRMT5 have been developed and showed promising anti-tumor effect in pre-clinical studies including in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). JNJ-64619178, a potent and specific PRMT5 inhibitor, exhibited broad antiproliferative activity in cancer cell lines from multiple cancer types and cell lines derived xenograft mouse model (Brehmer et al., 2021). In a phase 1 clinical trial of JNJ-64619178 (NCT03573310), patients with advanced solid tumors and non-Hodgkin lymphoma demonstrated manageable toxicity and preliminary evidence of antitumor activity at selected dose levels (Villar et al., 2020). In addition, in patients with lower-risk myelodysplastic syndromes, JNJ-64619178 had manageable hematologic toxicity at the selected expansion dose, but did not have significant clinical activity (Haque et al., 2021). Inhibition of PRMT5 alone could induce cell cycle arrest instead of apoptosis depending on the types of cancer (Zhu et al., 2019), therefore we hypothesized that the combination of PRMT5i and pro-apoptotic BH3 mimetics therapies could be synergistic in aggressive B cell malignancies. Methods: We used dynamic BH3 profiling (DBP) to measure the net pro-apoptotic signaling induced by ex vivo drug treatments (as per Montero et al., 2015). Other standard techniques included shRNA mediated gene knockdown, Western blot, cell viability assays including PI/Annexin V and CellTiter-Glo, and reactive oxygen species (ROS) measurement by flow cytometry with dihydroethidium (DHE) staining. The DLBCL cell lines (HBL1, TMD8, RI-1, OCI-Ly1, Karpas 422, SUDHL4, Toledo), double hit lymphoma (DHL) patient-derived xenograft (PDX) cell lines (DW19), mantle cell lymphoma (MCL) cell lines (Mino, Jeko-1), T cell acute leukemia (T-ALL) cell line (Jurkat) and Burkitt lymphoma (BL) cell line (Raji) were used to investigate the in vitro anti-cancer activity of JNJ-64619178 (Janssen Pharmaceuticals) and BH3 mimetics (venetoclax [BCL2i], S63845 [MCL-1i] and A1331852 [BCL-xLi], Selleckchem). SynergyFinder was used to calculate drug synergism. Results: In all 7 DLBCL cell lines and the DHL-PDX cell line, we found synergistic induction of apoptosis with JNJ-64619178 plus venetoclax (synergy score, 2.82~51.42). The lowest level of synergy was observed in OCI-Ly1 cells, which are intrinsically highly sensitive to venetoclax monotherapy. The combination of JNJ-64619178 with S63845 synergistically induced apoptosis in 5 out of 7 cell lines (synergy score, -1.91~35.64). While the combination of JNJ-64619178 with A1331852 synergistically induced apoptosis in all 7 cell lines (synergy score 1.88~46.73), 4 of them had weak synergy score (1.88~9.4). To confirm the effects we observed with pharmacologic inhibition of PRMT5, we performed shRNA mediated knockdown of PRMT5, which similarly revealed increased sensitivity to venetoclax. In MCL cell lines, combining JNJ-64619178 with venetoclax or A1331852 was more synergistic in inducing cell death than combining with S63845. The T-ALL Jurkat cell line was resistant to JNJ-64619178 and single BH3 mimetic-induced apoptosis. Combining JNJ-64619178 with A1331852 but not venetoclax or S63845 strongly induced apoptosis. The Burkitt cell line Raji was resistant to JNJ-64619178, venetoclax and A1331852, but was sensitive to S63845. The addition of JNJ-64619178 and a single BH3 mimetic in these Raji cells had only a minor synergistic effect. The synergy scores are summarized in Figure 1. Using DBP, JNJ-64619178 was found to increase overall apoptotic priming of DLBCL and MCL cell lines (> 15% increase in cytochrome c loss). By measuring ROS levels, we found increased ROS production (> 2-fold increase in median fluorescent intensity) in response to JNJ-64619178 before the onset of apoptosis, suggesting a possible mechanism by which JNJ-64619178 increases apoptotic priming. Conclusions: The combination of a PRMT5 inhibitor with BH3 mimetics, especially venetoclax, is worthy of further exploration as a potential therapeutic strategy for DLBCL and MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Apoptosis with Novel BH3 Mimetics in T-Lineage Acute Lymphoblastic Leukemia

Targeting Apoptosis with Novel BH3 Mimetics in T-Lineage Acute Lymphoblastic Leukemia

Ironomycin Induces Mantle Cell Lymphoma Cell Death By Targeting Iron Metabolism Addiction

Mantle-cell lymphoma (MCL) accounts for approximately 5 to 7% of all lymphomas, and in North America and Europe. MCL remains an aggressive and incurable cancer with existing therapies, presenting a significant unmet clinical need. Accumulating evidence reveals that abnormal iron metabolism plays an important role in tumorigenesis and in cancer progression of many tumors. In cancer cells, the demand for iron increases in response to sustained deregulation of cell proliferation and DNA synthesis. Based on these data, we searched to identify alterations of iron homeostasis in MCL that could be exploited to develop novel therapeutic strategies. Analysis of the iron metabolism gene expression profile of a cohort of patients with MCL (n=71) established the iron score (IS), a gene expression-based risk score enabling identification of patients with MCL with a poor outcome who might benefit from a suitable targeted therapy. We analyzed the therapeutic interest of ironomycin, a new promising iron depleting molecule. Ironomycin is known to sequester iron in the lysosome and to induce ferroptosis (Mai TT et al. Nature Chemistry 2017). Preclinical studies in Acute Myeloid Leukemia (Garciaz et al., 2022 Cancer Discovery) and Diffuse Large B-Cell Lymphoma (Devin et al., 2022 Can Res) have revealed the therapeutic interest of ironomycin in treating hematological malignancies. In a panel of 6 MCL cell lines, ironomycin inhibited MCL cell growth at nanomolar concentrations compared with typical iron chelators. Then, we investigated if ironomycin induces cell death through apoptosis. Flow cytometry analysis revealed that ironomycin treatment resulted in AnnV+/7AAD+ and AnnV+/7AAD- death profiles in MCL. This effect was partially inhibited by the pan-caspase inhibitor Q-VD-Oph. Ironomycin also induced caspases 8 and 9 cleavage, and the activation of caspases 3/7. Furthermore, we found that ironomycin induces ferroptosis with lipid peroxidation and the production of ROS monitored by the fluorescence probe CM-H2DCFDA. Ironomycin induced cell death is inhibited by the ferroptosis inhibitor, ferrostatin-1. The treatment did not induce ferritinophagy, as there was no significant change on the expression of LC3B-II, ferritin and the transferrin receptor (CD71).Taken together, these data indicate that ironomycin kills MCL cells by apoptosis mediated by caspase activation and ferroptosis. Ironomycin significantly decreased cell proliferation rate, with a reduced percentage of cells in S-phase together with Ki67 and Cyclin D1 downregulation. Western blot analysis showed an increase of Chk1 and Chk2 phosphorylation, two central factors of DNA damage response, and an increase of the phosphorylation of γ-H2AX, indicative of DNA damage. With major importance, we validated the therapeutic interest of ironomycin in primary MCL cells of patients (n=5). Ironomycin (20 nM) demonstrated a significant higher toxicity in MCL cells compared to normal cells from the microenvironment. Furthermore, ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment. We tested the therapeutic interest to combine ironomycin with conventional chemotherapy used in MCL. Interestingly, we identified a synergistic effect when ironomycin is combined with Ibrutinib BTK inhibitor. Furthermore, Significant synergistic effects were also observed by combining ironomycin with BH3 mimetics or Doxorubicin. Altogether, these data underline that MCL patients my benefit from targeting iron homeostasis using ironomycin alone or in combination with conventional MCL treatments.

Effect of Mcl-1 Inhibitor, AMG-176, on T Cell Quality and Function

Introduction AMG-176 belongs to a class of small molecule inhibitors known as BH3 mimetics, which mimic the role of BH3-only proteins and inhibit pro-survival Bcl-2 family proteins. AMG-176 specifically inhibits the pro-survival Mcl-1 protein. Overexpression of anti-apoptotic protein Mcl-1 has been extensively reported in hematological malignancies including mantle cell lymphomas (MCL), diffuse large B cell lymphomas (DLBCL), and multiple myeloma (MM). Chromosomal aberration of Mcl1 was identified as one of the most important survival genes for MM, follicular lymphoma (FL), MCL, and DLBCL, and is associated with shorter patient survival. These hematological malignancies are targetable by FDA-approved chimeric antigen receptor (CAR) T cell therapies with varying efficacies as single agent regimens. The role of Mcl-1 in T cell biology could be further elucidated to improve therapeutic outcomes of malignancies characterized by Mcl-1 overexpression. Here, our team explores the effect of Mcl-1 inhibitor, AMG-176, on T cell quality and function. Methods Our lab obtained healthy human donor blood from consenting adults, then isolated pan T cells using microbead negative selection (Miltenyi Biotec). Following isolation, T cells were cryopreserved in cryopreservative medium consisting of 90% fetal bovine serum (Corning) and 10% dimethyl sulfoxide (Sigma-Aldrich). Upon thaw, T cells were cultured in complete RPMI supplemented with [10ng/mL] h-IL-7 (Peprotech) and [100IU/mL] h-IL-2, and activated for 24 hours using αCD3/αCD28 coated beads (Thermofisher). At 24 hours post-activation, cells were exposed to various concentrations of AMG-176 (MedChem Express). Following 72-hour exposure, T cells were harvested and assessed for ATP production, viability, proliferation, immunophenotype, and effector function. To determine ATP production, we used a commercially available luminescence-based assay (Promega) and obtained our readout using a BioTek Synergy HTX plate reader, T cell immunophenotype was identified following 72-hour exposure to AMG-176 at 0nM, 10nM, 100nM, 500nM, and 1uM concentrations. Samples from each cohort were stained with Live/dead yellow viability dye, CD3, CD4, CD8, CD69, CD25, and CD127, then acquired using a BD LSRII flow cytometer. Flow cytometry data was analyzed using FCS Express analytical software (De Novo Software). Intracellular cytokine staining was performed using markers for Granzyme B, Perforin, and IFN-γ. Results T cells exposed in vitro to 10nM and 100nM concentrations of AMG-176 exhibited greater ATP production (Fig 1.), viability, and proliferation (Fig. 2) than the unexposed control group. T cells exposed to 500nM or 1uM concentrations of AMG-176 exhibited unchanged or decreased ATP production, viability, and proliferation relative to the unexposed control group. T cells exposed to AMG-176 significantly increased CD8+ T cell frequency at 10nM and 100nM concentrations (Fig. 3), without affecting viability of CD3+ Pan T cells. Our flow data also indicates dose-dependent increase in expression of early activation marker CD69+. Conclusion Exposure of human T cells to Mcl-1 inhibitor, AMG-176, provides an increase in ATP production, viability, proliferation, and activation. These results shed light on the role of Mcl-1 in T cell biology. Furthermore, increases in human T cell viability, proliferation, and function as facilitated by AMG-176 could offer insight into the advancement of effector cell-based therapies, such as chimeric antigen receptor (CAR) therapy. We may employ AMG-176 alongside CAR T therapy in order to achieve more effective clinical outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TP53 CRISPR/Cas9 Reveals Strict BAX Dependence in the Response to BH3 Mimetic Targeting MCL1

Background In multiple myeloma (MM), 1q gain/amplification (1q+) and deletion of the short arm of chromosome 17 (del17p), encompassing MCL1 and TP53, respectively, as well as the t(4;14) translocation are adverse events that reduce both event-free survival and overall survival of patients. The genome gatekeeper p53 is well known for its multiple roles in the regulation of mitochondrial apoptosis: p53 induces early direct transactivation of the BH3-only protein PUMA (BBC3) and the death effector BAX (BAX) and late direct-to-indirect transactivation of NOXA (PMAIP1) and BAK (BAK1). To investigate the direct impact of loss of p53 function in myeloma cells, we generated TP53 CRISPR/Cas9 isogenic cells from cell lines harboring t(4;14) and 1q+. Materials and Methods To knockout the expression of all p53 isoforms in two TP53wt t(4;14)+ 1q+ myeloma cell lines, NCI-H929 and XG7 cells were sequentially transduced with 2 lentiviruses expressing Cas9 and a doxycycline-inducible TP53-specific sgRNA targeting exon 5. TP53-/-clones (n=3) and control clones (n=3) were derived for each cell line by limiting dilution. Results RNAseq analysis was performed in triplicate in the 12 clones of the two MM cell lines: analysis of the 6 TP53-/-clones versus the 6 control clones showed significant changes (log2FC>0.4, p>0.05) in the expression of 92 genes under steady-state conditions (and of more than 3000 upon nutlin3a-induced p53 accumulation) : 27% of the genes were known to be direct p53 target genes, 39% were known to be indirectly regulated, and 35% of the genes were not identified as p53 targets in solid cancer cell lines (Andrysik Z Genome Res 2017). Among the 51 downregulated genes, only 2 belonged to apoptosis pathways, namely BAX and TNFRSF10B, both direct early targets of p53. Of note, the expression of BAK1, PMAIP1 or BBC3 was not significantly modified. To directly assess the impact of BAX decrease in the 6 TP53-/- clones, we used 3 BH3 mimetics specific for BCL2, Venetoclax, MCL1, S63845, and BCLXL, A1155463. The XG7 and NCI-H929 cell lines were predominantly resistant to Venetoclax and to A1155463, and TP53 knockout did not alter responses. In contrast, as compared to control clones, the response to S63845 was decreased 5.7 to 7.4-fold in NCI-H929 TP53-/- (LD50 median values increased from 7 to 40nM, p<0.0001) and XG7 TP53-/- clones (LD50 median values increased from 25 to 186nM, p=0.0007), respectively. This result suggested a major role of BAX in the response to S63845. Indeed, BAX silencing significantly inhibited S63845-induced cell death while BAK silencing had no protective effect, demonstrating the BAX dependence of the S63845 response. Of note, this strong dependence on p53-dependent BAX expression was not limited to XG7 or NCI-H929 : in 21 TP53abnormal myeloma cell lines, BAX expression (GEP) was 1.7-fold lower than in 11 TP53wt cell lines (LD50 median values 34 vs. 59, p=0.0004), and sensitivity to S63845 was also significantly lower in TP53abn MM cell lines compared to MM TP53wt (LD50 median values 75 nM vs. 12 nM, p=0.0296). The resistance of TP53-/- cells to S63845 was overcome by the addition of Venetoclax, although the death remained lower in TP53-/- clones compared to control clones (p=0.001). BAX and BAK activation and MCL1 co-immunoprecipitation assays further demonstrated the central role of MCL1/BAX expression and complexes in the response to S63845. In 63 consecutive independent samples from patients with MM (23 at diagnosis, 40 at relapse), 33 1q+ and 30 1q-, we show that 1q gain/amplification favored response to 25 nM S63845 alone (death median 16% in 29 1q- vs 30% in 34 1q+, p=0.0132) and had a trend in combination with 300 nM Venetoclax (80% vs 89%, p=0.152). Finally, we show that 1q was sufficient to induce responses to S63845 plus Venetoclax regardless of del17p status (death median 30% in 26 1q+ del17p- vs. 40% in 6 1q+ del17p+ samples). To analyze the response to the combination of S63845 and Venetoclax at the single cell level, we performed scRNASeq analysis in t(4;14)+ 1q+ del17p+/- patients and determined the transcriptomic profile of cells surviving the combination: we show that BH3 mimetics combination heterogeneously induced death in and between patient's samples and we identified a resistance signature that is under evaluation. All these results support the S63845 / Venetoclax combination for patients with double (1q+, del17p) to triple (t(4;14), 1q+, del17p) hit. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal