Pro-survival Bcl-2 Family Proteins Research Articles

Key residues in the VDAC2-BAK complex can be targeted to modulate apoptosis.

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.

A Phase 1 Study of Venetoclax in Combination with a Pediatric-Inspired Regimen for the Treatment of Newly Diagnosed Adults with B-Cell Ph-like ALL

Background: Aberrant overexpression of pro-survival Bcl-2 family proteins is one mechanism by which leukemic cells circumvent apoptosis. BCL-2 overexpression is common in various subtypes of B-acute lymphocytic leukemia (ALL), including Philadelphia (Ph)-like ALL. Ph-like ALL is a chemotherapy-resistant disease associated with higher rates of induction failure and persistent measurable residual disease (MRD), and poor overall survival (OS) outcomes with frontline chemotherapy regimens, compared to other subtypes. Venetoclax (VEN) is a selective BCL2 inhibitor, and its addition to pediatric-type frontline regimens could improve response and long-term outcomes for B-ALL, including Ph-like subtype. Design: This is a phase 1 study with an expansion cohort investigating the safety of combining VEN with induction (IND) and consolidation (CON) cycles of the CALGB 10403 backbone regimen (NCT05157971). Post CONS therapy was per the treating physician discretion. The study enrolled newly diagnosed adults [18-54 years (yrs)] with Ph-negative B-ALL. To enrich the study for Ph-like ALL, we excluded patients (pts) with KMT2A-rearranged, TCF3::PBX1, and ETV6::RUNX1 subtypes at diagnosis. Identification of Ph-like ALL was performed using RNA-sequencing, conventional cytogenetics, fluorescence in situ hybridization, and whole genome array studies. Dose level (DL) 1 of VEN was 400 mg orally daily on days 1-14 of IND and CON, with a ramp-up dosing during IND [Day 1= 100 mg, Day 2= 200 mg, Days 3-14= 400 mg/day]. Results: We enrolled 14 pts as of July 1 st, 2023. The median age was 33 (20-53) yrs, of which 9 pts were males and 13 (93%) were Hispanics. Seven (50%) pts had Ph-like ALL (6 with CRLF2-alterations, 1 JAK2-fusion). Six pts were treated on the phase 1 part, and no dose limiting toxicity was observed during IND. Among 14 pts that completed IND, all achieved complete remission (CR), including 1 pt who required an extended IND phase, and 79% (n= 11) achieved MRD- (<0.01%) by multicolor flow (MCF) on day 28 of IND. Eleven pts completed CONS, and all achieved undetectable MRD CR or CR with incomplete count recovery (CRi) by MCF. Post CONS next-generation sequencing of MRD (clonoSEQ) was available for all 11 pts who completed CONS, and 7 (64%) were undetectable while 4 were detected at very low levels (range: 0.0007-0.0024%). Median time to neutrophil count ≥1000 and platelet count ≥100,000 were 19 (15-26) and 14 (2-27) days from starting IND, respectively. No pt died during IND or CONS cycles. Grade ≥3 adverse events at least possibly related to venetoclax and/or the C10403 regimen that occurred in ≥ 20% of evaluable pts during either IND or CONS cycles were leukopenia (100%), neutropenia (100%), lymphopenia (100%), anemia (70%), thrombocytopenia (50%), hyperbilirubinemia (30%) and hypertriglyceridemia (20%). Only 1 pt underwent consolidation with allogeneic stem cell transplant while in remission. With a median follow up for the entire cohort of 7.2 months (range: 1.9-16.1 months), only 1 pt with Ph-like ALL relapsed and he is currently alive. Conclusion: The addition of 14 days of VEN at 400 mg daily dose to a pediatric-inspired regimen during IND and CONS was safe in adults with newly diagnosed B-ALL and did not delay counts recovery. The addition of VEN led to a promising higher than expected MRD- CR rate in high-risk B-ALL, including for pts with Ph-like subtype. The study is actively enrolling on the expansion cohort, and BH3 profiling on pre-treatment and post relapse samples will be performed to determine BCL-2 dependency and correlates to early MRD- response.

Mitochondrial Functional Profiling Links Apoptotic Sensitivity to Treatment Outcomes Based on Age and Cell Lineage in Acute Leukemia

Background Survival outcomes in paediatric T and B cell acute lymphoblastic leukemia (ALL) are far superior to acute myeloid leukemia (AML). In addition, children with ALL and AML have better response rates than adults. While the basis for these differences in therapy outcomes is multifactorial, it is poorly understood. The majority of anticancer therapies activate apoptosis signalling to target cancer cells. The balance between pro-survival (BCL-2, BCL-XL and MCL-1) and pro-apoptotic proteins as well as evasion of apoptosis, plays a key role in cancers. Dependencies to these anti-apoptotic pro-survival proteins is a hallmark of haematopoietic malignancies and can be exploited with novel agents especially by using Venetoclax (BCL-2 inhibitor) and Navitoclax (BCL-2 and BCL-XL inhibitor) that are now available in the clinic. We hypothesized that difference in apoptosis threshold (priming) based on cell lineage and patient age offers key functional insights into the differences in outcomes as well as gives us an in vitro window to assess inherent BCL-2 or BCL-XL dependencies that can be targeted. Methods We prospectively and retrospectively examined functional regulation of apoptosis by performing a BH3 profiling assay on leukemia patient specimens from two tertiary cancer centres in Singapore. Since BCL-2 family consists of different anti-apoptotic proteins, we asked whether protection from specific pro-survival BCL-2 family proteins such as BCL-2, BCL-XL or MCL-1, predicted by sensitivity to BAD, HRK and MS-1 peptide, respectively, contribute to overall difference in apoptotic sensitivity between lymphoid and myeloid precursors. We also aimed to determine dominant factors that govern differences in clinical chemosensitivity to lymphoid compared to myeloid tumours. Blasts isolated via flow cytometry from bone-marrow aspirate (BMA) or peripheral blood were exposed to a variety of pro-apoptotic BH3 peptides to measure mitochondrial outer membrane permeabilization (cytochrome c release). The percentage of cytochrome c loss with each BH3 peptide at different concentrations was normalized to the cytochrome c release from negative control DMSO wells. Assays were performed in triplicate. Alamethacin was used as positive control. Flow cytometric data was acquired with a BD analyser, analysed with FlowJo and plotted in GraphPad Prism. Results Initial analysis of seventy one patient samples are presented here (40 AML, 17 T-ALL, 12 B-ALL and 2 MPAL). Lymphoblasts showed significantly increased apoptotic sensitivity as measured by Bim response compared to myeloblasts (p<0.001). We found lymphoblasts to be significantly more dependent on BCL-2 and BCL-XL compared to myeloblasts (p<0.001). Higher MCL-1 dependence was also noted in B-ALL cells as compared to AML (p<0.05). We next asked if apoptotic sensitivity measured by mitochondrial priming could explain higher chemosensitivity of pediatric over adult patients. Indeed we noted increase in overall priming in paediatric (N=26) as compared to adults (N=36) irrespective of cell of origin (P<0.001). Within the AML cohort, we found pediatric AML (N=8) to be more sensitive to Bim peptide compared to adults (N=32), as well as showed closer proximity to apoptosis (p<0.05). Of note there were no differences in overall priming or selective dependencies between diagnosis and relapse/refractory (R/R) patients in AML or ALL cohorts. By using blasts of AML and +ALL patients (N=15), we found that increased mitochondrial priming response to HRK+MS1 peptides (which infer BCL-XL and MCL-1 dependence respectively) inversely correlated with achievement of remission with venetoclax based therapy (p<0.001). Mitochondrial sensitivity to MS-1 peptide, increased from pre-treatment to on treatment to relapse (N=3/3 patients) in sequential samples for three patients suggesting an opportunity for the use of MCL-1 inhibition in relapse setting. Conclusion Our study shows increased overall priming in lymphoblast as compared with myeloblast and in pediatric as compared to blasts from adult leukaemia specimens. These findings offer a functional window to further explain differences in outcomes in leukaemia in different ages as well as lineages. Innovative trial designs incorporating cellular vulnerabilities to anti-apoptotic proteins using functional BH3 profiling as an accessory biomarker will enable improved treatment outcomes with reduced toxicity.

Anti-apoptotic protein BCL-XL as a therapeutic vulnerability in gastric cancer.

New therapeutic targets are needed to improve the outcomes for gastric cancer (GC) patients with advanced disease. Evasion of programmed cell death (apoptosis) is a hallmark of cancer cells and direct induction of apoptosis by targeting the pro-survival BCL2 family proteins represents a promising therapeutic strategy for cancer treatment. Therefore, understanding the molecular mechanisms underpinning cancer cell survival could provide a molecular basis for potential therapeutic interventions. Here we explored the role of BCL2L1 and the encoded anti-apoptotic BCL-XL in GC. Using Droplet Digital PCR (ddPCR) technology to investigate the DNA amplification of BCL2L1 in GC samples and GC cell lines, the sensitivity of GC cell lines to selective BCL-XL inhibitors A1155463 and A1331852, pan-inhibitor ABT-263, and VHL-based PROTAC-BCL-XL was analyzed using (CellTiter-Glo) CTG assay in vitro. Western Blot (WB) was used to detect the protein expression of BCL2 family members in GC cell lines and the manner in which PROTAC-BCL-XL kills GC cells. Co-immunoprecipitation (Co-IP) was used to investigate the mechanism of A1331852 and ABT-263 kills GC cell lines. DDPCR, WB, and real-time PCR (RTPCR) were used to investigate the correlation between DNA, RNA, protein levels, and drug activity. The functional assay showed that a subset of GC cell lines relies on BCL-XL for survival. In gastric cancer cell lines, BCL-XL inhibitors A1155463 and A1331852 are more sensitive than the pan BCL2 family inhibitor ABT-263, indicating that ABT-263 is not an optimal inhibitor of BCL-XL. VHL-based PROTAC-BCL-XL DT2216 appears to be active in GC cells. DT2216 induces apoptosis of gastric cancer cells in a time- and dose-dependent manner through the proteasome pathway. Statistical analysis showed that the BCL-XL protein level predicts the response of GC cells to BCL-XL targeting therapy and BCL2L1 gene CNVs do not reliably predict BCL-XL expression. We identified BCL-XL as a promising therapeutic target in a subset of GC cases with high levels of BCL-XL protein expression. Functionally, we demonstrated that both selective BCL-XL inhibitors and VHL-based PROTAC BCL-XL can potently kill GC cells that are reliant on BCL-XL for survival. However, we found that BCL2L1 copy number variations (CNVs) cannot reliably predict BCL-XL expression, but the BCL-XL protein level serves as a useful biomarker for predicting the sensitivity of GC cells to BCL-XL-targeting compounds. Taken together, our study pinpointed BCL-XL as potential druggable target for specific subsets of GC.

Abstract 6155: Lurbinectedin exhibits combinatorial activity with BCL2/BCL2L1 inhibitors in vitro and in vivo by modulation of MCL1 expression

Abstract Lurbinectedin (Zepzelca) is a marine-derived anti-tumor agent currently approved for metastatic small cell lung cancer (SCLC). Lurbinectedin has been previously shown to exert its anti-tumor effect by binding to the minor groove of DNA and inhibition of nucleotide excision repair. In addition, lurbinectedin inhibits transcription by inducing degradation of RNA polymerase II. While these activities hint towards a general mechanism, specific molecular targets for lurbinectedin remain obscure. We demonstrate that in vitro, Lurbinectedin specifically induced downregulation of MCL1 protein, a member of the BCL2 family of pro-survival proteins, in a dose- (0-30 nM) and time-dependent manner (0-24 h) by Western analysis. Expression of other pro-survival proteins BCL2 and BCL2L1 were not affected. The downregulation of MCL1 was consistent in all nine tumor cell lines tested regardless of indication. Increased activity or expression of MCL1 is a known compensatory mechanism to offset loss of BCL2 or BCL2L1. We therefore hypothesized that lurbinectedin induced MCL1 loss, along with inhibition of the pro-survival proteins BCL2 and BCL2L1 would lead to an enhancement in cell killing. Consistent with the dominant pro-survival role of BCL2 in SCLC, the combination of lurbinectedin with a specific BCL2 inhibitor (venetoclax) in vitro induced synergistic cytotoxicity (measured by Bliss Sum analysis) in a panel of small cell lung cancer lines. In solid tumor lines other than SCLC, the combination of lurbinectedin with a specific BCL2L1 inhibitor (A-1331852) was also synergistic. Correspondingly in vivo, enhancement in the efficacy of lurbinectedin was observed in combination with venetoclax in SCLC models, while in solid tumor models other than SCLC the combination with a specific BCL2L1 inhibitor enhanced efficacy. Collectively, these data identify MCL1 as a specific lurbinectedin target and suggest that combinations that target other pro-survival proteins may represent a viable strategy to enhance the anti-tumor activity of lurbinectedin. Citation Format: Kedar S. Vaidya, Aparna Gupta, Robert Hauptschein, Robin C. Humphreys. Lurbinectedin exhibits combinatorial activity with BCL2/BCL2L1 inhibitors in vitro and in vivo by modulation of MCL1 expression [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 6155.

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 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.

Lymphoma cells lacking pro-apoptotic BAX are highly resistant to BH3-mimetics targeting pro-survival MCL-1 but retain sensitivity to conventional DNA-damaging drugs

BH3-mimetic drugs are an anti-cancer therapy that can induce apoptosis in malignant cells by directly binding and inhibiting pro-survival proteins of the BCL-2 family. The BH3-mimetic drug venetoclax, which targets BCL-2, has been approved for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia by regulatory authorities worldwide. However, while most patients initially respond well, resistance and relapse while on this drug is an emerging and critical issue in the clinic. Though some studies have begun uncovering the factors involved in resistance to BCL-2-targeting BH3-mimetic drugs, little focus has been applied to pre-emptively tackle resistance for the next generation of BH3-mimetic drugs targeting MCL-1, which are now in clinical trials for diverse blood cancers. Therefore, using pre-clinical mouse and human models of aggressive lymphoma, we sought to predict factors likely to contribute to the development of resistance in patients receiving MCL-1-targeting BH3-mimetic drugs. First, we performed multiple whole genome CRISPR/Cas9 KO screens and identified that loss of the pro-apoptotic effector protein BAX, but not its close relative BAK, could confer resistance to MCL-1-targeting BH3-mimetic drugs in both short-term and long-term treatment regimens, even in lymphoma cells lacking the tumour suppressor TRP53. Furthermore, we found that mouse Eµ-Myc lymphoma cells selected for loss of BAX, as well as upregulation of the untargeted pro-survival BCL-2 family proteins BCL-XL and A1, when made naturally resistant to MCL-1 inhibitors by culturing them in increasing doses of drug over time, a situation mimicking the clinical application of these drugs. Finally, we identified therapeutic approaches which could overcome these two methods of resistance: the use of chemotherapeutic drugs or combined BH3-mimetic treatment, respectively. Collectively, these results uncover some key factors likely to cause resistance to MCL-1 inhibition in the clinic and suggest rational therapeutic strategies to overcome resistance that should be investigated further.

Structural basis of the specificity and interaction mechanism of Bmf binding to pro-survival Bcl-2 family proteins

The apoptotic pathway is regulated by protein-protein interactions between members of the Bcl-2 family. Pro-survival Bcl-2 family proteins act as cell guardians and protect cells against death. Selective binding and neutralization of BH3-only proteins with pro-survival Bcl-2 family proteins is critical for initiating apoptosis. In this study, the binding assay shows that the BH3 peptide derived from the BH3-only protein Bmf has a high affinity for the pro-survival proteins Bcl-2 and Bcl-xL, but a much lower affinity for Mcl-1. The complex structures of Bmf BH3 with Bcl-2, Bcl-xL and Mcl-1 reveal that the α-helical Bmf BH3 accommodates into the canonical groove of these pro-survival proteins, but the conformational changes and some interactions are different among the three complexes. Bmf BH3 forms conserved hydrophobic and salt bridge interactions with Bcl-2 and Bcl-xL, and also establishes several hydrogen bonds to support their binding. However, the highly conserved Asp-Arg salt bridge is not formed in the Mcl-1/Bmf BH3 complex, and few hydrogen bonds are observed. Furthermore, mutational analysis shows that substitutions of less-conserved residues in the α2-α3 region of these pro-survival Bcl-2 family proteins, as well as the highly conserved Arg, lead to significant changes in their binding affinity to Bmf BH3, while substitutions of less-conserved residues in Bmf BH3 have a more dramatic effect on its affinity to Mcl-1. This study provides structural insight into the specificity and interaction mechanism of Bmf BH3 binding to pro-survival Bcl-2 family proteins, and helps guide the design of BH3 mimics targeting pro-survival Bcl-2 family proteins.

TET2 Loss Conspires with MYC to Foster Lymphomagenesis

Programmed DNA methylation patterning serves the generation and maintenance of cell identity, and represents a major barrier against transformation. TET dioxygenases (TET1-3) facilitate DNA demethylation at enhancers, promoting chromatin accessibility and cell type-specific gene expression patterns. Work from our and other labs had shown that TET enzymes settle B lymphocyte identity and warrant antibody-mediated immunity. Due to the prevalence of TET2 mutations in hematological and immune cell tumors, often identified as loss-of-function (LOF) versions, TET2 LOF is considered a cancer driver. Intriguingly, although showing a predisposition for blood cancer, healthy humans carrying TET2 mutations in hematopoietic stem cells can remain cancer-free for decades despite increasing clonal dominance of the affected cells and their offspring, a condition termed clonal hematopoiesis. Furthermore, mice deficient for TET2 only rarely develop tumors. This scenario suggests roles for TET2 LOF as cancer facilitator, rather than driver. In B lymphocytes, the MYC oncoprotein is a potent driver of malignancy. However, therapeutic targeting of MYC is far from reaching the clinic. Thus, establishing functional links between MYC and dependencies imposed by TET2 LOF should provide information on the molecular mechanisms of predisposition, and guide clinical practice and targeted therapy in the future. To address whether TET2 LOF conspires with MYC, we employed the prototypic EμMYC transgenic mouse model, where enforced MYC overexpression early in the B lymphocyte lineage drives lymphomas, but additional hits are required for full transformation. Confirming a tumor suppressor role for TET2, partial or complete loss of the protein accelerated lymphomagenesis, and facilitated specifically the outgrowth of IgM+ lymphomas. The premalignant IgM+ population contained a higher fraction of cells with double strand breaks (EμMYC 2,5% vs. EμMYCTet2+/- or EμMYCTet2-/- 6%) and in the S/G2/M cell cycle phases (EμMYC 15% vs. EμMYCTet2+/- or EμMYCTet2-/- 25%), along with higher spontaneous apoptosis rates (EμMYC 4% vs. EμMYCTet2+/- or EμMYCTet2-/- 7%). Mechanistically, in the context of MYC overexpression, TET2 LOF associates with enhanced JAK/STAT signaling and selective upregulation of the pro-survival BCL2 family protein BCLX. To recapitulate alternative scenarios of TET2 LOF in diffuse large B cell lymphoma (DLBCL), a tumor that originates from germinal center B lymphocytes, we established two preclinical mouse models, (1) either with TET2 LOF in the germline to mimic the scenario of clonal hematopoiesis along with ectopic MYC expression restricted to germinal center B lymphocytes, or (2) using a fully conditional approach where we synchronize ectopic MYC expression and TET2 LOF specifically in germinal center B lymphocytes. In line with the results from the EμMYC model, germline heterozygous Tet2 loss (scenario (1)) sufficed to boost MYC-driven lymphomagenesis, a phenotype that is strongly enhanced by the absence of both Tet2 alleles. Germinal center-specific elimination of Tet2 in scenario (2) also produced lymphomas, however, heterozygous Tet2 loss in germinal center B lymphocytes was not sufficient to unleash MYC-driven lymphomagenesis. Our data from three powerful MYC-driven mouse models suggest (i) that TET2 loss in B lymphocytes nudges MYC-mediated transformation, (ii) that TET2 loss, early on in hematopoietic stem cells, potentiates the transformative potential later on in B lymphocytes, and (iii) that TET2 loss dampens apoptosis during transformation and in established lymphomas, potentially via JAK/STAT signaling and BCLX. Altogether, we provide novel in vivo evidence that TET2 prevents MYC-mediated transformation in B lymphocytes, and our results produced testable dependencies. Beyond MYC-driven blood cancer, TET2 LOF in hematopoietic stem cells predisposes to all-cause mortality in humans. Thus, identifying targetable vulnerabilities of TET2 LOF will serve healthy aging.

MS-553, a Selective PKC-ß Inhibitor Inhibits BCR Signaling and Synergizes with Venetoclax for the Treatment of High Risk CLL

Inhibitors of Bruton's tyrosine kinase (BTK) have been transformative in treating patients with chronic lymphocytic leukemia (CLL) due to their ability to inhibit B-Cell Receptor (BCR) signaling. However, despite durable remissions in many patients, complete responses are uncommon and acquired mutations in BTK and its downstream target phospholipase Cγ2 (PLCγ2) lead to relapses and poor survival. In parallel, genomic high-risk features such as aberrations in TP53 represent an independent risk factor that favor progression on BTK inhibitors (BTKi). TP53 aberrations comprise of deletions in chromosome 17 (del17p) which result in the loss of one allele of TP53 and point mutations in the TP53 gene. CLL patients with del17p acquire clonal mutations that affect the other p53 allele in 82% of cases and largely express only mutant TP53 protein. Therefore, there is an urgent need to identify therapies for patients who progress on BTKi. We have observed over-expression of protein kinase C-β (PKC-β) in primary CLL patient samples including in those that have mutations in TP53. PKC-β is a downstream of the BCR and functions downstream of BTK. PKC-β is required for CLL cell survival and proliferation and therefore can be a potential target for patients with mutations in p53 that do not respond to other targeted therapies. We hypothesized that targeting PKC-β will inhibit BCR signaling downstream of BTK and may offer an effective therapeutic strategy for patients who relapse on ibrutinib. Mechanistically, we found that CLL cells (n=6 patients) in contact with stromal support upregulated their levels of PKC-β. Pre-exposure of stromal cells to MS-553, a selective PKC-β inhibitor prevented the stromal cell contact-induced increase in PKC-β within the CLL cells establishing that stromal contact was an important source of PKC-β in CLL cells. We have shown in primary CLL patient samples that exposure to MS-553 decreases activation of PKC-β and its downstream effectors GSK3-β and ERK in a dose-dependent manner regardless of mutations in p53 both in the presence and absence of stromal support. In parallel, MS-553 also decreased the levels of BCL-xL, a key pro-survival BCL2 family protein via downregulation of β-catenin. Using dynamic BH3 profiling we show that CLL samples (both WT and mutant TP53) exposed to MS-553 (5µM, 24h) increase their dependence on BCL-2 and BCL-xL for survival. This finding suggested that MS-553 could synergize with the BCL-2 inhibitor venetoclax to eliminate CLL cells. Correspondingly, cells exposed to a combination of MS-553 and venetoclax released more cytochrome c (Cyt c), used as a measure of mitochondrial dysfunction, compared to each single agent alone and led to a synergistic increase in apoptosis as measured by Annexin V positivity. These data provide strong rationale for an ongoing Phase I/II trial that evaluates MS-553 as a single agent and in combination with targeted agents such as venetoclax. Early interim data indicate that MS-553 is safe, tolerable and effective both as a single agent and in combination with venetoclax.

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

P1271: ELUCIDATING TUMOR SUPPRESSOR FUNCTIONS OF TET2 IN B CELL LYMPHOMAS

Background: Remodeling of the epigenetic landscape via DNA cytosine methylation directs cell lineage specification, establishing and maintaining gene expression programs that uniquely define given cell types. During tumorigenesis the epigenome becomes corrupted, impairing lineage-specific gene expression programs. TET2, an enzyme that contributes to the erasure of cytosine methylation via oxidation of the 5’ methyl group, is among the most frequent somatically mutated genes in human hematological tumors, commonly identified as loss-of-function versions. In mouse models, TET2 loss on its own is only weakly tumorigenic, but rather facilitates leukemic transformation driven by prototypic oncogenes such as AML-ETO or FLT3ITD. However, TET2 LOF is not limited to leukemias. In B cell malignancies, TET2 is found mutated in a fraction of diffuse large B cell lymphomas (DLBCL), which display a distinct gene expression profile. Aims: Having established a role for TET2 loss-of-function in enhancing MYC-driven lymphomas, our overall aim is to understand how TET2 loss conspires with MYC to overcome the barriers that maintain cell identity and function. Methods: Using pre-clinical mouse models and in vitro culture systems, we establish that TET2 loss boosts the formation of B cell lymphomas driven by the MYC oncoprotein. Results: Thus, (1) TET2 deficiency specifically in B cells facilitates MYC-mediated transformation in a dose dependent manner, and (2) TET2 deficiency early on in hematopoietic stem and progenitor cells enhances the transformative potential later on in antigen-activated B cells. Our data suggest that TET2 may exert tumor suppressor functions via controlling the expression of DNA repair genes, or via direct functions of oxidized cytosine species. In addition, TET2-deficient lymphomas select for high expression of the pro-survival BCL2 family protein BCLXL, potentially providing protection from MYC-driven apoptosis. Summary/Conclusion: Altogether, our data concerning the impact of TET2 loss-of-function on DNA repair and apoptosis mechanisms creates understanding of how TET2 deficiency nudges transformation, and reveals testable therapeutic vulnerabilities.

MEK and MCL-1 sequential inhibition synergize to enhance rhabdomyosarcoma treatment

Targeted agents have emerged as promising molecules for cancer treatment, but most of them fail to achieve complete tumor regression or attain durable remissions due to tumor adaptations. We used dynamic BH3 profiling to identify targeted agents effectiveness and anti-apoptotic adaptations upon targeted treatment in rhabdomyosarcoma. We focused on studying the use of BH3 mimetics to specifically inhibit pro-survival BCL-2 family proteins, overwhelm resistance to therapy and prevent relapse. We observed that the MEK1/2 inhibitor trametinib rapidly depleted the pro-apoptotic protein NOXA, thus increasing MCL-1 availability. Indeed, we found that the MCL-1 inhibitor S63845 synergistically enhanced trametinib cytotoxicity in rhabdomyosarcoma cells in vitro and in vivo. In conclusion, our findings indicate that the combination of a BH3 mimetic targeting MCL-1 with trametinib improves efficiency on rhabdomyosarcoma by blocking tumor adaptation to treatment.

The manipulation of apoptosis for cancer therapy using BH3-mimetic drugs.

Apoptosis is a form of programmed cell death that is regulated by the balance between prosurvival and proapoptotic BCL-2 protein family members. Evasion of apoptosis is a hallmark of cancer that arises when this balance is tipped in favour of survival. One form of anticancer therapeutic, termed 'BH3-mimetic drugs', has been developed to directly activate the apoptosis machinery in malignant cells. These drugs bind to and inhibit specific prosurvival BCL-2 family proteins, thereby mimicking their interaction with the BH3 domains of proapoptotic BCL-2 family proteins. The BCL-2-specific inhibitor venetoclax is approved by the US Food and Drug Administration and many regulatory authorities worldwide for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. BH3-mimetic drugs targeting other BCL-2 prosurvival proteins have been tested in preclinical models of cancer, and drugs targeting MCL-1 or BCL-XL have advanced into phase I clinical trials for certain cancers. As with all therapeutics, efficacy and tolerability need to be carefully balanced to achieve a therapeutic window whereby there is significant anticancer activity with an acceptable safety profile. In this Review, we outline the current state of BH3-mimetic drugs targeting various prosurvival BCL-2 family proteins and discuss emerging data regarding primary and acquired resistance to these agents and approaches that may overcome this. We highlight issues that need to be addressed to further advance the clinical application of BH3-mimetic drugs, both alone and in combination with additional anticancer agents (for example, standard chemotherapeutic drugs or inhibitors of oncogenic kinases), for improved responses in patients with cancer.

Combination Therapy of Navitoclax with Chemotherapeutic Agents in Solid Tumors and Blood Cancer: A Review of Current Evidence

Combination therapy emerges as a fundamental scheme in cancer. Many targeted therapeutic agents are developed to be used with chemotherapy or radiation therapy to enhance drug efficacy and reduce toxicity effects. ABT-263, known as navitoclax, mimics the BH3-only proteins of the BCL-2 family and has a high affinity towards pro-survival BCL-2 family proteins (i.e., BCL-XL, BCL-2, BCL-W) to induce cell apoptosis effectively. A single navitoclax action potently ameliorates several tumor progressions, including blood and bone marrow cancer, as well as small cell lung carcinoma. Not only that, but navitoclax alone also therapeutically affects fibrotic disease. Nevertheless, outcomes from the clinical trial of a single navitoclax agent in patients with advanced and relapsed small cell lung cancer demonstrated a limited anti-cancer activity. This brings accumulating evidence of navitoclax to be used concomitantly with other chemotherapeutic agents in several solid and non-solid tumors that are therapeutically benefiting from navitoclax treatment in preclinical studies. Initially, we justify the anti-cancer role of navitoclax in combination therapy. Then, we evaluate the current evidence of navitoclax in combination with the chemotherapeutic agents comprehensively to indicate the primary regulator of this combination strategy in order to produce a therapeutic effect.

Dual Sphingosine Kinase and Bcl-2 Inhibition Exhibits Synergistic Cell Death in Acute Myeloid Leukemia

Background:Pro-survival Bcl-2 family proteins such as Mcl-1 and Bcl-2 have garnered significant interest as therapeutic targets due to their ability to suppress the apoptotic potential of pro-apoptotic Bax and Bak. In response to cell stress, BH3-only proteins sequester Bcl-2 family members, promoting apoptosis. Small molecule inhibitors such as the Bcl-2 inhibitor Venetoclax have demonstrated poor single agent efficacy in AML. This poor efficacy is a result of AML cells being highly dependent on the pro-survival Bcl-2 family member Mcl-1. Sphingosine kinase 1 (SK1) is a signalling enzyme with established roles in oncogenesis and has recently emerged as a potential therapeutic target in leukaemia. SK1 promotes oncogenic signalling through the production of sphingosine-1-phopshate and subsequent activation of G-protein coupled receptors. In addition, SK1 has been reported to modulate Mcl-1 expression in a number of haematological malignancies including AML. Thus we sought to investigate the efficacy of targeting SK1 to sensitise AML cells to Venetoclax.Results:To determine how SK1 inhibition influences Mcl-1 levels we utilised a short hairpin RNA (shRNA) approach targeting members of the BH3 only protein family (Bid, Bim, Bad, Puma and Noxa). Targeting of Noxa significantly attenuated the cytotoxic effects of the SK1 inhibitor, MP-A08. Immunoprecipitation of Mcl-1 in response to MP-A08 treatment revealed increased association of BH3 only protein, Noxa and ubiquitin-ligase FBW7 suggesting this triggers Mcl-1 degradation in agreeance with previous studies. No interaction was observed between Mcl-1 and Bim further supporting the importance of Noxa in MP-A08 induced Mcl-1 degradation.Canonical pathway analysis of MP-A08 treated cells has previously shown to enrich for genes associated with the unfolded protein response (UPR) (Powell et al. Blood 2017). Western blot analysis recapitulated these findings with activation of the PERK arm, typified by eIF2a phosphorylation and ATF4 upregulation. ATF4 has previously been shown to bind within the Noxa promoter and drive transcription in response to ER stress (Wang et al PNAS 2009). Maintaining global translation with eIF2B agonist, ISRIB blocked Noxa transcription in response to MP-A08 suggesting ATF4 drives Noxa mediated Mcl-1 degradation.Given the Noxa dependent Mcl-1 degradation in response to MP-A08 treatment, we sought to assess the efficacy of combined treatment with the Bcl-2 inhibitor, Venetoclax. Combinational therapies with sub-cytotoxic doses of MP-A08 and Venetoclax induced synergistic cell death in AML cell lines, primary patient AML blasts and leukemic stem and progenitor cells. Synergy could be observed as early as two hours, in agreeance with Noxa upregulation and binding Mcl-1, implicating this as key for synergistic cell death.Conclusions:We have demonstrated SK1 inhibition invokes a pro-apoptotic UPR response via ATF4 driven Noxa upregulation and Mcl-1 degradation. Nullification of the pro-survival function of Mcl-1 by Noxa renders AML cells vulnerable to Bcl-2 targeting strategies such as Venetoclax providing a novel therapeutic angle to enhance patient response to Venetoclax. Current work is underway assessing MP-A08 and Venetoclax using patient-derived xenograft models. DisclosuresNo relevant conflicts of interest to declare.

Deubiquitinase Inhibitor WP1130 Blocks FLT3-ITD to Induce Apoptosis in Leukemic Cells

FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD) is the most frequent kinase mutation seen in acute myeloid leukemia (AML). The ITD mutation promotes ligand-independent constitutive activation of FLT3 kinase, resulting in altered downstream signal pathway activation to enhance cell proliferation and prevent cell death. AML with FLT3-ITD is associated with poor prognosis, which neither high-dose chemotherapy nor allogeneic hematopoietic stem cell transplantation can overcome. Several tyrosine kinase inhibitors targeting FLT3 have been developed and tested in clinical trials, but their efficacies are limited so far partly due to resistance to kinase inhibition. Therefore, development of novel treatment strategies with distinct mechanisms to block FLT3 is warranted.Deubiquitinating enzymes (DUBs) are proteases that remove ubiquitin from target proteins, resulting in changes in protein stability and signaling. WP1130 is an inhibitor for DUBs, which has been reported to inhibit BCR/ABL not through blocking the kinase activity but through promoting ubiquitination and translocation of BCR/ABL protein into perinuclear aggresomes. WP1130 also downregulates JAK2 tyrosine kinase in the same manner. However, the effect of WP1130 against FLT3-ITD has not been reported.Here we demonstrate that WP1130 exerts anti-leukemic activity against leukemic cell lines and primary AML cells harboring FLT3-ITD. WP1130 efficiently downregulated activated form of FLT3-ITD through translocation of this mutant into aggresomes and activated the mitochondria-mediated intrinsic apoptotic pathway at least partly through accumulation of intracellular reactive oxygen species (ROS).First, we examined the anti-proliferative effect of WP1130 and found that proliferation of the FLT3-ITD-harboring AML cell line MV4-11 was suppressed in a dose dependent manner with IC50 of 1.3 μM. It was notable that MV4-11 was more susceptible to WP1130 compared with K562 (IC50 = 3.3 μM) and HEL (IC50 = 3.4 μM), leukemic cell lines harboring BCR/ABL and JAK2-V617F aberrant kinases, respectively.Next, we assessed the effect of WP1130 against FLT3-ITD in MV4-11 cells. WP1130 rapidly downregulated FLT3-ITD to suppress downstream signaling pathways. The aggresomal translocation of FLT3-ITD was confirmed by confocal microscopy and western blot analysis of detergent-insoluble fraction of cellular proteins. Importantly, the downregulation was prominently observed for the activated form of FLT3-ITD autophosphorylated on Y591. Consistent with this, inhibition of FLT3 autophosphorylation by AC220 (quizartinib) resulted in attenuation of WP1130-induced FLT3-ITD downregulation.Importantly, WP1130 induced apoptosis in MV4-11 cells much more efficiently than in other leukemic cell lines that we examined, including K562 and HEL. WP1130 activated the mitochondria-mediated intrinsic apoptotic pathway as demonstrated by Bax conformation change and loss of mitochondrial membrane potential (MMP). Forced overexpression of Bcl-xL, a member of pro-survival Bcl-2 family proteins that preserve MMP, blocked WP1130-induced apoptosis. Moreover, the apoptosis was synergistically enhanced by inhibition of Bcl-xL using ABT-737 or navitoclax, further supporting the involvement of Bcl-xL in WP1130-induced apoptosis. Interestingly, the apoptosis induction was also enhanced by 17-AAG, an HSP90 inhibitor that we previously demonstrated to disrupt the chaperone association of HSP90 with FLT3-ITD resulting in enhanced ubiquitination of FLT3-ITD. FLT3-ITD down regulation and apoptosis induction by WP1130 were also confirmed using other cell lines expressing FLT3-ITD and primary cells derived from FLT3-ITD positive AML patients.Finally, since FLT3-ITD has been reported to increase intracellular ROS, we examined if ROS was involved in the anti-leukemic effect of WP1130. Treatment with WP1130 increased ROS levels in MV4-11 cells. Antioxidants N-acetyl-cysteine (NAC) and tert-butylhydroquinone (tBHQ) both attenuated WP1130-induced apoptosis, suggesting that intracellular ROS accumulation may play a role in induction of apoptosis.Further studies are in progress to elucidate the exact molecular mechanisms involved in WP1130-mediated effects and to develop novel strategies to enhance its anti-leukemic effects in synergistic manners. DisclosuresNo relevant conflicts of interest to declare.

Venetoclax-based Rational Combinations are Effective in Models of MYCN-amplified Neuroblastoma.

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.

Targeting Bfl-1 via acute CDK9 inhibition overcomes intrinsic BH3-mimetic resistance in lymphomas

AbstractBH3 mimetics like venetoclax target prosurvival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate that endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2 inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1–expressing lymphomas, highlighting the need for targeting additional prosurvival proteins in this context. Importantly, we demonstrated that cyclin-dependent kinase 9 (CDK9) inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3-mimetic–resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in diffuse large B-cell lymphoma patient-derived xenograft models expressing Bfl-1. These data underscore the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.