Selective BCL-2 Inhibitor Research Articles

Predictors of response to venetoclax and therapeutic potential of CDK7 inhibition in multiple myeloma

AbstractVenetoclax, a selective BCL2 inhibitor, has emerged as a promising therapeutic agent for multiple myeloma (MM), particularly in patients harboring the t(11;14) translocation. In this study, we set out to identify markers of sensitivity and resistance to venetoclax in a real-world patient population, aiming to facilitate the development of personalized therapeutic strategies. Through the analysis of RNA-seq data from relapsed/refractory patients treated with venetoclax, either as a single agent or in combination with other drugs, we unveiled a novel 6-gene signature that significantly stratified patients into risk groups for relapse and further validated its clinical relevance in 2 independent clinical and ex vivo data sets. Our analysis also highlighted the negative impact of chromosome 1q gain, which harbors the MCL1 gene, on progression-free survival, even in t(11;14)-positive patients. Encouraged by the well-documented role of MCL1 in resistance to venetoclax in various malignancies and the prognostic importance of the BCL2/MCL1 ratio in our cohort, we explored Cyclin-Dependent Kinase 7 (CDK7) inhibition as a potential strategy to overcome venetoclax resistance. In vitro experiments demonstrated that CRISPR-Cas9–mediated CDK7 depletion led to decreased MCL1 levels, enhancing the sensitivity of MM cells to venetoclax. Moreover, the combination of the CDK7 inhibitor THZ1 with venetoclax markedly induced cell death in venetoclax-resistant MM cells harboring 1q gain, thus offering a rational therapeutic approach, particularly for patients with this aberration. Overall, these findings provide important insights for optimizing venetoclax-based therapeutic strategies in MM.

A systematic review of venetoclax for the treatment of unfit AML patients in real-world: is all thatglitters gold?

Acute myeloid leukemia (AML) is an aggressive hematological disease that mainly affects elderly patients. Following the randomized VIALE-A trial, current standard treatment in patients who are not candidates for intensive chemotherapy consists of the combination of venetoclax (VEN), a selective inhibitor of the anti-apoptotic protein BCL-2, with azacitidine (AZA) or decitabine (DEC). We performed a systematic review to critically assess the growing existing evidence regarding the effectiveness of the VEN-based combinations in unfit adult patients with newly diagnosed AML in the real-world setting. Following PRISMA guidelines, a systematic search of published manuscripts and conference abstracts (European Hematology Association and American Society of Hematology) was conducted (updated March 2024). Primary outcomes were composite complete remission (CRc) and median overall survival (mOS). A total of 73 studies fulfilled inclusion criteria, with a median age of 73years old. The weighted mean mOS was 10.3months among 7 138 patients, significantly lower than expected according to the VIALE-A trial (14.7months), while the weighted mean CRc rate was 58.2% among 5 831 patients, slightly lower to that reported in the VIALE-A (66.4%). Early death rates at 30 and 60days were 5% and 13%, respectively. The weighted mean percentage of subsequent allogeneic transplant was 15.4%. In conclusion, breakthrough mOS reported in the VIALE-A trial using VEN-AZA was not well reproduced in real world for unfit newly diagnosed AML patients, while CRc rates were more consistent. Strategies to optimize patient selection, dosing regimens, and supportive care are crucial to improve outcomes in real-world.

Design, Synthesis, and Evaluation of BCL-2 Targeting PROTACs.

BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other haematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.

Disarib, a Specific BCL2 Inhibitor, Induces Apoptosis in Triple-Negative Breast Cancer Cells and Impedes Tumour Progression in Xenografts by Altering Mitochondria-Associated Processes.

Targeted cancer therapy aims to disrupt the functions of proteins that regulate cancer progression, mainly by using small molecule inhibitors (SMIs). SMIs exert their effect by modulating signalling pathways, organelle integrity, chromatin components, and several biosynthetic processes essential for cell division and survival. Antiapoptotic protein BCL2 is highly upregulated in many cancers compared with normal cells, making it an ideal target for cancer therapy. Around 75% of primary breast cancers overexpress BCL2, providing an opportunity to explore BCL2 inhibitors as a therapeutic option. Disarib is an SMI that has been developed as a selective BCL2 inhibitor. Disarib works by disrupting BCL2-BAK interaction and activating intrinsic apoptotic pathways in leukemic cells while sparing normal cells. We investigated the effects of Disarib, a BCL2 specific inhibitor, on breast cancer cells and xenografts. Cytotoxicity and fluorometric assays revealed that Disarib induced cell death by increasing reactive oxygen species and activating intrinsic apoptotic pathways in Triple-Negative Breast Cancer cells (MDA-MB-231 and MDA-MB-468). Disarib also affected the colony-forming properties of these cells. MDA-MB-231- and MDA-MB-468-derived xenografts showed a significant reduction in tumours upon Disarib treatment. Through the transcriptomics approach, we also explored the influence of BCL2 inhibitors on energy metabolism, mitochondrial dynamics, and epithelial-to-mesenchymal transition (EMT). Mitochondrial dynamics and glucose metabolism mainly regulate energy metabolism. The change in energetics regulates tumour growth through epithelial-mesenchymal transition, and angiogenesis. RNA sequencing (RNAseq) analysis revealed that BCL2 inhibitors ABT-199 and Disarib maintain Oxphos levels in MDA-MB-231. However, key glycolytic genes were significantly downregulated. Mitochondrial fission genes were seen to be downregulated both in RNAseq data and semi quantitative real time polymerase chain reaction (qRTPCR) in Disarib-treated TNBC cells and xenografts. Lastly, Disarib inhibited wound healing and epithelial-to-mesenchymal transition. This study showed that Disarib disrupts mitochondrial function, activates the intrinsic apoptotic pathway in breast cancer, and inhibits epithelial-to-mesenchymal transition both in vitro and in vivo. These findings highlight Disarib's potential as a multifaceted therapeutic strategy for patients with Triple-Negative Breast Cancer.

Molecular mechanisms of extracellular-ATP-mediated colorectal cancer progression: Implication of purinergic receptors-mediated nucleocytoplasmic shuttling of HuR.

One of the leading causes of cancer-related deaths worldwide is colorectal cancer (CRC). Extracellular ATP (e-ATP) and purinergic receptors (P2R) play a central role in CRC proliferation and progression. Human antigen R (HuR) is becoming more and more understood to be essential for the expression of genes linked to cancer. The current study demonstrates that ATP can mediate CRC (Caco-2 cells) progression via induction of HuR nucleocytoplasmic shuttling and subsequent expression of cancer-related genes, a consequence mostly mediated via the P2R receptor. It was also noted that suppression of HuR activity by using dihydrotanshinone I (DHTS) prevents cancer-related gene expression and subsequent CRC (Caco-2 cells) progression induced by ATP. The expression of cyclin A2/cyclin-dependent kinase 2 (CDK2), Bcl-2, ProT-α, hypoxia-inducible factor1-α (HIF1-α), vascular endothelial growth factor A (VEGF-A), transforming growth factor-β (TGF-β) and matrix metallopeptidase 9 (MMP-9) induced by ATP were highly reduced in the presence of either PPADS (non-selective P2R antagonist) or DHTS. In addition, e-ATP-induced Caco-2 cell proliferation as well as cell survival were highly reduced in the presence of either PPADS or DHTS or selective CDK-2 inhibitor (Roscovitine) or selective Bcl-2 inhibitor (ABT-263). Furthermore, it was found that MMP-9 is critical for Caco-2 cells migration induced by e-ATP as demonstrated by a clear reduction in cells migration in the presence of a selective MMP-9 inhibitor (Marimastat). Collectively, these data demonstrate that ATP through P2R activation can induce HuR nucleocytoplasmic shuttling that could be translated into an increase in cancer-related genes expression and subsequent, cell proliferation and progression.

Discovery of the Clinical Candidate Sonrotoclax (BGB-11417), a Highly Potent and Selective Inhibitor for Both WT and G101V Mutant Bcl-2.

The approval of venetoclax, a B-cell lymphoma-2 (Bcl-2) selective inhibitor, for the treatment of chronic lymphocytic leukemia demonstrated that the antiapoptotic protein Bcl-2 is a druggable target for B-cell malignancies. However, venetoclax's limited potency cannot produce a strong, durable clinical benefit in other Bcl-2-mediated malignancies (e.g., diffuse large B-cell lymphomas) and multiple recurrent Bcl-2 mutations (e.g., G101V) have been reported to mediate resistance to venetoclax after long-term treatment. Herein, we described novel Bcl-2 inhibitors with increased potency for both wild-type (WT) and mutant Bcl-2. Comprehensive structure optimization led to the clinical candidate BGB-11417 (compound 12e, sonrotoclax), which exhibits strong in vitro and in vivo inhibitory activity against both WT Bcl-2 and the G101V mutant, as well as excellent selectivity over Bcl-xL without obvious cytochrome P450 inhibition. Currently, BGB-11417 is undergoing phase II/III clinical assessments as monotherapy and combination treatment.

A phase 3b study of venetoclax and azacitidine or decitabine in an outpatient setting in patients with acute myeloid leukemia.

Venetoclax, a highly selective BCL-2 inhibitor, combined with hypomethylating agents (HMAs) azacitidine or decitabine, is approved for the treatment of newly diagnosed acute myeloid leukemia (ND AML) in patients who are ineligible to receive intensive chemotherapy. Previous clinical studies initiated venetoclax plus HMA in an inpatient setting owing to concerns of tumor lysis syndrome (TLS). This study (NCT03941964) evaluated the efficacy and safety of venetoclax plus HMA in a United States community-based outpatient setting in patients with ND AML (N=60) who were treatment naïve for AML, ineligible to receive intensive chemotherapy, had no evidence of spontaneous TLS at screening, and were deemed as appropriate candidates for outpatient initiation of venetoclax plus HMA by the investigator. Patients received venetoclax in combination with azacitidine (75mg/m2) or decitabine (20mg/m2) for up to 6 cycles during the study. With a median time on study of 18.3weeks, the best response rate of composite complete remission was 66.7%, and the overall post-baseline red blood cell (RBC) and platelet transfusion independence rate was 55.0%, consistent with results of studies in which treatment was initiated in an inpatient setting. Key adverse events included nausea, anemia, thrombocytopenia, neutropenia, and white blood cell count decrease of any grade (≥50% of patients). The observed safety profile was generally consistent with that of venetoclax plus HMA observed in inpatient AML studies. With close monitoring, 2 cases of TLS were identified, appropriately managed, and the patients were able to continue study treatment. CLINICAL TRIALS REGISTRATION: This study is registered at ClinicalTrials.gov. The registration identification number is NCT03941964.

Abstract LB021: BAU-243: A novel BCL-2 inhibitor inducing autophagic cell death in glioblastoma, unveiled through multistep virtual screening, comprehensive in vitro and in vivo animal model evaluation

Abstract The excessive expression of antiapoptotic members in the B-cell leukemia/lymphoma-2 (BCL-2) family proteins emerges as a noteworthy oncogenic target in cancer cells. Employing a multistep virtual screening and filtering approach that integrates structure and ligand-based drug design, we aimed to identify novel and potent BCL-2 inhibitors from a sizable small molecule database, comprising over 210,000 compounds. Subsequently, seven selected molecules underwent rigorous biological activity tests in human cancer cell lines and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays. Four of these compounds exhibited notable inhibitory effects on cancer cell proliferation and led to an increase in apoptotic cell fractions. Among the identified compounds, BAU-243 displayed a substantial affinity for Bcl-2, demonstrating efficacy in reducing glioblastoma (GBM) cell proliferation, including cancer-initiating cells. Notably, in contrast to the selective Bcl-2 inhibitor ABT-199, BAU-243 induced autophagic cell death in GBM cells. In vivo investigations using orthotopic brain tumor models revealed a significant reduction in tumor growth with BAU-243 compared to both the vehicle group and animals treated with ABT-199. Computational modeling simulations, in concordance with in vitro findings, proposed that BAU-243 activates autophagic cell death by disrupting the Beclin 1:Bcl-2 complex. This positions BAU-243 as a promising small molecule for the treatment of GBM. Citation Format: Seyma Calis, Berna Dogan, Asuman Celebi, Ozlem Yapicier, Turker Kilic, Eda Tahir Turanli, Timucin Avsar, Serdar Durdagi. BAU-243: A novel BCL-2 inhibitor inducing autophagic cell death in glioblastoma, unveiled through multistep virtual screening, comprehensive in vitro and in vivo animal model evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB021.

492 Novel BH3 Mimetic Bcl-2 Inhibitor Promotes Autophagic Cell Death and Reduces in Vivo Glioblastoma Tumor Growth

INTRODUCTION: Anti-apoptotic members of the Bcl-2 family proteins play central roles in the regulation of cell death in glioblastoma, the most malignant type of brain tumor. Despite the advances in GBM treatment, there is still an urgent need for new therapeutic approaches. METHODS: In silico studies resulted in 500,000 molecules being screened with computer-aided drug design methods and the molecule with the highest binding affinity was selected and named BAU-243. Cell viability, IC50 value, tumor formation potential, tumor spheroid formation potential, tumor invasion studies were investigated following BAU-243 application. The molecular changes caused by Bcl-2 inhibition were demonstrated by quantitative PCR, western blot and immunofluorescence methods. In in vivo studies, glioma tumors were transplanted intracranially into Nod Scid Gamma(NSG) mice using the mouse xenograft model. RESULTS: Here, we report a novel 4-thiazolidinone derivative BH3 mimetic, BAU-243 that binds to Bcl-2 with a high affinity. BAU-243 effectively reduced overall glioblastoma cell proliferation including a subpopulation of cancer-initiating cells in contrast to the selective Bcl-2 inhibitor ABT-199. While ABT-199 successfully induces apoptosis in high BCL2-expressing neuroblastoma SHSY-5Y cells, BAU-243 triggered autophagic cell death rather than apoptosis in A172 cells, indicated by the upregulation of BECN1, ATG5, and MAP1LC3B expression. Lc3b-II, a potent autophagy marker, was significantly upregulated following BAU-243 treatment. Moreover, BAU-243 significantly reduced tumor growth in vivo in orthotopic brain tumor models when compared to the vehicle group, and ABT-199 treated animals. To elucidate the molecular mechanisms of action of BAU-243, we performed computational modeling simulations that were consistent with in vitro results. CONCLUSIONS: Results indicated that BAU-243 activates autophagic cell death by disrupting Beclin 1:Bcl-2 complex, and may serve as a potential small molecule for the treatment of glioblastoma.

Abstract 7218: Venetoclax triggers apoptosis and augments the effectiveness of doxorubicin against acute myeloid leukemia cells

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the clonal proliferation of immature blast cells in the peripheral blood and bone marrow. AML is the second most common type of childhood leukemia, and 50% to 60% of relapses occur within the first year of diagnosis. Chemotherapy side effects and resistance are becoming major clinical issues. Although anthracyclines are one of the most commonly used induction regimens for children with AML in the United States, clinicians avoid them due to adverse effects and clinical resistance issues. To address this clinical issue, we are looking for a novel AML approach to boost the usage of anthracyclines. Since Bcl-2 protein overexpression in AML is related to uncontrolled growth, poor prognosis, and chemoresistance. Multiple clinical trials have suggested combining venetoclax (a selective Bcl-2 inhibitor) with well-established medicines to boost efficacy and overcome resistance. We suggest that inhibiting Bcl-2 is a feasible strategy for increasing the use of anthracycline medicines. We proposed combining doxorubicin and venetoclax as an alternative approach for AML patients. In a panel of three AML cell lines, Kusami-1, U937, and THP-1, our western blot revealed that Kusami-1 had the highest expression of Bcl-2 proteins. Surprisingly, venetoclax causes Kusami-1 cell death and increases the efficacy of doxorubicin in a dose-dependent manner, suggesting that utilizing a lower clinical standard dose may avoid major adverse effects such as cardiotoxicity. Furthermore, the Annexin V Apoptosis Detection Kit indicated that venetoclax coupled with doxorubicin synergistically promoted total apoptosis in Kusami-1 cells. The combination medication shown promising and synergistic in vitro anti-leukemic action against AML cell lines and represents a potentially viable treatment option for AML patients. Future research will use the combination approach in AML cell lines, as well as in patient samples and in vivo experiments. Citation Format: Osama Aloudat, Omar S. Al Odat, Tulin Budak-Alpdogan, Subash Jonnalagadda, Yong Chen, Manoj Pandey. Venetoclax triggers apoptosis and augments the effectiveness of doxorubicin against acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7218.

Abstract 7598: The novel eIF4A inhibitor potently synergizes with BCL2 inhibitor in high grade B-cell lymphoma with MYC and BCL2 rearrangements through regulating UPR

Abstract Double hit lymphoma(DHL) is defined to be a chromosomal break with MYC gene combined with BCL2 or BCL6. MYC is an oncogene which proliferation the cells and BCL2 inhibit cell death. These combinations could be a cause of highly aggressive clinical features and poor survival of DLBCL. The WHO-HAEM5 renames the entity DLBCL/high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Despite the dismal prognosis, no widely accepted standard treatment exists to the management of DHL. Therefore, new therapeutic approaches for these patients are urgently needed. Previously, we looked at the eIf4A inhibition was shown to downregulate MYC expression in lymphoma cell lines. Venetoclax, a highly selective inhibitor of BCL2, shows clinical activity for patients with DLBCL although there are concerns about increasing toxicity. Our hypothesis in the following: If eIF4A inhibitor preferentially inhibit the translation of oncogenes such as cMyc and venetoclax inhibit the BCL2 in DHL, then the combination of eIF4A inhibitor and BCL2 inhibitor will be highly effective for DHL and potentially safe for animals and humans. We tested the anti-lymphoma effect of CR-1-31-B, in combination with Venetoclax in lymphoma cell lines using flow cytometry. We performed cell apoptosis assays in increasing concentrations of Venetoclax, CR-1-31-B and a combination of the two drugs at 48 hours. To better understand molecular mechanisms underlying the observed synergistic effect in DLBCL-DHL, RNA-seq profiling was performed on cells treated with single drugs and their combinations, respectively. Venetoclax and CR1-31-B exhibited concentration and time dependent cytotoxicity in lymphoma cell lines at low nanomolar concentrations. Overall, the combination of eIF4A inhibitor and BCL2 inhibitor showed synergy in all kinds of DLBCL cell lines. Especially DHL cell lines were more sensitive to the drug combination and showed enhanced synergy compared to the non-DHL cell lines(p<0.0001). We analyzed RNA-seq profiling of cells treated with single drugs and their combinations and found that the transcriptional signature of the synergistic combination was unique relative to that of either constituent monotherapy. Gene set enrichment analysis was performed in the DHL and non-DHL group. The analysis demonstrates that known Unfolded protein response(UPR) part and metabolism, are enriched in DHL groups. This suggests that the UPR and metabolism pathway are closely related to the combination effect in DHL. These findings suggest that the combination of eIF4A inhibitor and BCL2 inhibitor have potent pharmacological activity in DHL lines, it was closely related with UPR. These combination is promising and novel treatment for high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Further pre-clinical investigations of this combination for these patients are needed. Citation Format: So-Young Seol, Chung Hyun Park, Soo Jeong Kim, Haerim Chung, Hyunsoo Cho, Jin Seok Kim, Yu Ri Kim. The novel eIF4A inhibitor potently synergizes with BCL2 inhibitor in high grade B-cell lymphoma with MYC and BCL2 rearrangements through regulating UPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7598.

Abstract 5991: Improved venetoclax therapy by a novel conjugate with artemisinin by NOXA-mediated reduction of Mcl-1 and cyclin D1 protein in myeloid leukemia cells

Abstract Evasion of apoptosis is crucial for the growth, survival and chemoresistance of myeloid leukemia. The Bcl-2 selective inhibitor venetoclax is the only apoptosis inducer approved for clinical use. Venetoclax in combination with hypomethylating agents or low-dose cytarabine has now become the standard care for elderly AML patients. However, a sizeable fraction of patients are either refractory to venetoclax combination therapy or ultimately relapse. High or induced expression of Mcl-1 and Bcl-xL mediates venetoclax resistance. Previously we reported that artemisinin enhances venetoclax apoptosis induction by degrading Mcl-1 through NOXA induction. We designed a novel conjugate A1 of venetoclax with dihydroartemisinin. A1 shows dual functions of inhibiting Bcl-2 and inducing NOXA. A1 treatment releases Bim from both Bcl-2 and Mcl-1, further degrades Mcl-1 protein, and presents ten-fold stronger apoptotic induction ability in venetoclax insensitive cells. Acquired increase of Mcl-1 protein was observed in venetoclax resistant MOLM-13/VEN cells, which are sensitive to A1-induced apoptosis. In Bcl-xL expressing venetoclax refractory leukemia cells A1 obtains a new mechanism of inhibiting cell cycling by downregulating cyclin D1 through a new NOXA/cyclin D1 cascade, mediated through the endoperoxide moiety of artemisinin and intracellular iron, independent of Bcl-2 inhibition. We reveal a new compound A1 overcoming venetoclax resistance by NOXA-mediated degradation of Mcl-1 and cyclin D1 protein. Citation Format: Jingyi Zhang, Zhenwei Zhang, Samuel Waxman, Linxiang Zhao, Yongkui Jing. Improved venetoclax therapy by a novel conjugate with artemisinin by NOXA-mediated reduction of Mcl-1 and cyclin D1 protein in myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5991.

Abstract P02: Uncovering Tailored Therapies for Acute Myeloid Leukemia in Children with Down Syndrome

Abstract P02: Uncovering Tailored Therapies for Acute Myeloid Leukemia in Children with Down Syndrome

Abstract P29: Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth

Abstract New targeted therapies such as the selective and potent BCL-2 inhibitor Venetoclax have revolutionized the treatment of acute myeloid leukemia (AML). However, due to the cellular heterogeneity of the disease, drug-resistant cell types often emerge and drive relapse. We found that the eukaryotic translation initiation factor 4A (EIF4A) is highly expressed in multiple primitive AML cell types compared to their healthy counterparts, suggesting its potential as a therapeutic target. By unwinding complex structures in the 5’ UTR of transcripts, eIF4A promotes the translation of oncogene transcripts. A first-in-class drug, Zotatifin, inhibits the helicase activity of eIF4A and curtails tumor growth in receptor tyrosine kinase-driven tumors. Zotatifin is currently being evaluated in phase 1/2 trials across multiple solid tumors (NCT04092673). However, its effectiveness in blood cancers such as AML remains unexplored, and it is unknown whether Zotatifin may work synergistically with Venetoclax. Here, we treated AML cell lines in vitro and primary healthy or AML bone marrow ex vivo with Zotatifin or Venetoclax alone and in combination, and demonstrate synergistic killing of AML cells by the combination therapy (ZIP synergy score = 57.0 in MOLM-13, 19.3 in MV4-11, P < 0.0001). Mechanistically, we show that Zotatifin decreases eIF4A and AKT expression, and its combination with Venetoclax drives increased BAK and cleaved BAK expression, as well as increased cleavage of MCL-1 and BCL-2, consistent with drug synergy by convergence on the apoptotic pathway. In pilot colony-forming unit (CFU) assays, we found the combination to be tolerable in healthy CD34+ human hematopoietic stem and progenitor cells (HSPCs). To test drug efficacy in vivo, we generated a patient-derived xenograft (PDX) AML model from a patient who experienced relapse with complex cytogenetics, FLT3 and NPM1 mutations. Only the combination of Zotatifin and Venetoclax but not monotherapies effectively suppressed tumor burden as assessed by human CD45 engraftment in the peripheral blood. Further, the combination of Zotatifin and Venetoclax significantly prolonged survival of xenografted mice (median survival after treatment: vehicle 11 days, Venetoclax 13 days, Zotatifin 15 days, combination 42 days, P = 0.0002). Together, our data indicates that Zotatifin is synergistic with Venetoclax and may represent an effective treatment option for patients who are either primary refractory or have developed resistance to existing Venetoclax-containing regimens. Citation Format: Yoke Seng Lee, Jonathan Good, Noha Awais, Benjamin Eschle, Jenny Noel, Jean Acosta, Erica DePasquale, David Cucchi, Prafulla Gokhale, Fadi Najm, Peter van Galen. Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P29.

Selective pharmacologic targeting of CTPS1 shows single-agent activity and synergizes with BCL2 inhibition in aggressive mantle cell lymphoma.

Innovative therapeutic strategies have emerged over the past decade to improve outcomes for most lymphoma patients. Nevertheless, the aggressive presentation seen in high-risk mantle cell lymphoma (MCL) patients remains an unmet medical need. The highly proliferative cells that characterize these tumors depend on nucleotide synthesis to ensure high DNA replication and RNA synthesis. To take advantage of this vulnerability, STP-B, a clinically available small molecule selectively targeting CTP synthase 1 (CTPS1) has been recently developed. CTPS1 is a key enzyme of the pyrimidine synthesis pathway mediated through its unique ability to provide enough CTP in highly proliferating cells. Herein, we demonstrated that CTPS1 was expressed in all MCL cells, and that its high expression was associated with unfavorable outcomes for patients treated with chemotherapy. Using aggressive MCL models characterized by blastoid morphology, TP53 mutation or polyresistance to targeted therapies, we showed that STP-B was highly effective at nanomolar concentrations in vitro and in vivo, irrespective of these high-risk features. Inhibition of CTPS1 rapidly leads to cell cycle arrest in early S-phase accompanied by inhibition of translation, including of the anti-apoptotic protein MCL1. Consequently, CTPS1 inhibition induced synergistic cell death in combination with the selective BCL2 inhibitor venetoclax, both in vitro and in vivo. Overall, our study identified CTPS1 as a promising target for MCL patients and provided a mechanism-based combination with the BCL2 inhibitor venetoclax for the design of future chemotherapy-free treatment regimens to overcome resistance.

Use of venetoclax in t(11;14) positive relapsed/refractory multiple myeloma: A systematic review.

The plasma cell malignancy, multiple myeloma (MM), remains incurable despite advanced treatment protocols. Overexpression of Bcl-2 (an anti-apoptotic protein), in MM harboring the translocation (11;14), contributes to resistance to prior therapy. Venetoclax, a selective oral inhibitor of BCL-2 is a novel agent that shows promise as a therapeutic agent. The objective of this systematic review is to address how the use of venetoclax, alone or as a combination regimen, contributed to the treatment of patients with t(11:14) positive relapsed/refractory multiple myeloma (RRMM). This systematic review was conducted in accordance to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and was done on 5th June 2022. A literature search was conducted on PubMed and Scopus, 145 articles were screened and 10 studies were included. Risk of bias assessment was performed using the Methodological Index for Non-Randomized Studies (MINORS) criteria. Across the studies reviewed, a total of 311 patients were identified with t(11;14) positive RRMM. The overall response rate achieved ranged between 33% and 95.5%. Furthermore, the use of venetoclax has exhibited a favorable adverse effect profile. Side effects included hematological side effects, nausea, vomiting, and diarrhea. Venetoclax demonstrates promising results. When given with drugs like dexamethasone, daratumumab and carfilzomib, a synergistic effect is seen in treating translocation (11:14) positive relapsed/refractory MM. The use of venetoclax in clinical practice can potentially improve outcomes and quality of life in RRMM patients, and future research should continue to explore this promising treatment option.

The BCL-2 inhibitor APG-2575 resets tumor-associated macrophages toward the M1 phenotype, promoting a favorable response to anti-PD-1 therapy via NLRP3 activation

The main challenges in the use of immune checkpoint inhibitors (ICIs) are ascribed to the immunosuppressive tumor microenvironment and the lack of sufficient infiltration of activated CD8+ T cells. Transforming the tumor microenvironment (TME) from “cold” to “hot” and thus more likely to potentiate the effects of ICIs is a promising strategy for cancer treatment. We found that the selective BCL-2 inhibitor APG-2575 can enhance the antitumor efficacy of anti-PD-1 therapy in syngeneic and humanized CD34+ mouse models. Using single-cell RNA sequencing, we found that APG-2575 polarized M2-like immunosuppressive macrophages toward the M1-like immunostimulatory phenotype with increased CCL5 and CXCL10 secretion, restoring T-cell function and promoting a favorable immunotherapy response. Mechanistically, we demonstrated that APG-2575 directly binds to NF-κB p65 to activate NLRP3 signaling, thereby mediating macrophage repolarization and the activation of proinflammatory caspases and subsequently increasing CCL5 and CXCL10 chemokine production. As a result, APG-2575-induced macrophage repolarization could remodel the tumor immune microenvironment, thus improving tumor immunosuppression and further enhancing antitumor T-cell immunity. Multiplex immunohistochemistry confirmed that patients with better immunotherapeutic efficacy had higher CD86, p-NF-κB p65 and NLRP3 levels, accompanied by lower CD206 expression on macrophages. Collectively, these data provide evidence that further study on APG-2575 in combination with immunotherapy for tumor treatment is required.

BAX Mutated Clonal Hematopoiesis Arises Following Treatment with the BCL2 Inhibitor Class of Therapeutics across a Range of Hematological and Non-Hematological Neoplasms

Clonal hematopoiesis (CH) is an age-related phenomenon characterized by the expansion of genomically mutated hematopoietic clones with a selective fitness advantage. Venetoclax, a selective BCL2 inhibitor, has established efficacy in hematological malignancies such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Our previous studies described the emergence of deleterious BAX variants within the myeloid compartment of patients (pts) receiving venetoclax for CLL (Blombery, Blood2022). Whether this phenomenon is specific to venetoclax and the CLL disease context is currently unknown. We therefore aimed to investigate the prevalence and pattern of BAX-mutatedCH across different hematological and non-hematological neoplasms in response to BCL2 inhibitor therapy (both venetoclax and BGB-11417). We first examined pts with AML in complete morphologic remission after venetoclax exposure in both the newly diagnosed (CAVEAT; Chua, JCO 2020) and early relapse (VALDAC; Tiong, ASH 2022) settings using unique molecular index-based error-corrected next generation sequencing with a sensitivity of 0.5% (QIAseq targeted DNA panel). BAX variants were detected in remission samples in 11/20 (55%) and 9/26 (35%) pts in these cohorts, respectively, after a median of 9- and 4-months post venetoclax. This represented a significant higher incidence compared to remission samples from pts with newly diagnosed AML post conventional cytotoxic chemotherapy without venetoclax (6/87 [7%]; adjusted p<0.01 for both pairwise comparisons). Having observed the increased frequency of BAX-mutated CH in remission in myeloid (AML) and lymphoid (CLL) neoplasms, we then investigated whether BAX variants may be acquired in response to BCL2 inhibitor therapy outside the context of hematological neoplasm. We assessed a cohort of pts with metastatic breast cancer who received venetoclax in combination with tamoxifen (mBEP; Lok, Cancer Discover 2019), and detected BAX variants from peripheral blood samples in 5/16 (31%) pts (median age 66) after a median duration of 20 (range 13-44) months on venetoclax. In contrast, no BAX variants were detected in a similarly aged control cohort of 50 pts (median age 62) with breast cancer without exposure to venetoclax (p=0.01). The rates of any CH-related mutation were 75% and 50% pts, respectively (p=0.09). No pt developed MDS or AML during follow up in the mBEP cohort. Finally, to explore whether emergence of BAX-mutated CH is specific to venetoclax as an agent, we studied a cohort of 9 pts with CLL who had received BGB-11417, a more potent and selective BCL2 inhibitor; median treatment duration 13 months. Notably, we observed the emergence of BAX variants in 2/9 (22%) pts in this cohort. The characteristics of the BAX variants (n=88) in 28 pts (AML, CLL or breast cancer) are summarized in Figure 1; 3 variants (in 3 pts) were detectable in pre-treatment samples. Over half of the missense (n=22) or in-frame variants (n=1) were clustered in the C-terminus of the BAX protein, while truncating variants (n=65) were observed throughout the gene. The median number of BAX variants per pt was 2, with 32% of pts having ≥3 variants (max=13). The median VAF was 1.4%, and only 16% of variants had a VAF >5%. The number and VAF of BAX variants did not significantly correlate with the duration of therapy. Among 12 pts with AML who had serial testing (median inter-test interval 10 months), BAX variants (n=24) did not significantly change in VAF (median Δ 0.7%) with time after initial acquisition (Pearson's coefficient 0.17). To clarify the compartments affected by BAX variants we performed single-cell combined DNA/protein sequencing (Tapestri) of a sample with multiple BAX variants and demonstrated their presence in multiple independent clones with myeloid-biased lineage but also within the natural killer cell compartment ( Figure 2). In summary, the emergence of BAX-mutated CH appears to be a generalized phenomenon of adaptive hematopoiesis that occurs across both hematological and non-hematological malignancy settings as well being observed with other BCL2 inhibitors consistent with a therapeutic class effect. Further research is needed to understand the contribution of this phenomenon to the subsequent development of clinically relevant hematological abnormalities and its implications for future therapeutic resistance.

The PP2A-B56α Heterocomplex Regulates Response to Venetoclax Plus Azacitidine Treatment in AML

Venetoclax (VEN), a selective inhibitor of BCL2, combined with azacitidine (Aza) has emerged as the new standard of care for patients with acute myeloid leukemia (AML) ineligible for intensive chemotherapy. However, the long-term medical benefit is modest, and relapse appears to be unavoidable. We have recently reported that PP2A activators enhance the efficacy of VEN and VEN+Aza in primary samples and pre-clinical models of AML, and that the PP2A-B56α complex drives the synergistic pro-apoptotic activity observed. Our aim here is to further decipher the role of PP2A-B56α in VEN+Aza treatment response in AML. We used CRISPR-Cas9 to generate HL-60 sublines lacking the B56α or B55α PP2A regulatory subunits. Interestingly, while there were no significant differences in their baseline rate of proliferation, HL-60 B56α knockdown (KD) clones were significantly more resistant to VEN and VEN+Aza than wild type (WT) cells. In contrast, HL-60 B55α KD clones were sensitive to VEN+Aza, pointing out a specific role for specific PP2A heterotrimers, in this case the PP2A-B56α complex in the combined treatment response. Protein expression analysis of known substrates of PP2A-B56α demonstrated that pSer62-MYC and total MYC decreased after VEN+Aza treatment in WT cells but not in KD cells. It is known that the PP2A-B56α complex directly dephosphorylates MYC-Ser62, inducing MYC ubiquitin-mediated proteasomal degradation. Moreover, p21/CDKN1A, that is transcriptionally activated by p53 and inhibited by MYC, was up-regulated at both the mRNA and protein level after VEN+Aza treatment in WT cells but not in KD cells. HL-60 is a p53 null cell line; therefore, p21 up-regulation upon the combined treatment might be MYC-dependent. Of note, treatment of HL-60 B56α KD cells with the MYC inhibitor MYCi975 rescued the VEN+Aza synergy seen in these cells, confirming the relevance of MYC degradation to VEN+Aza response. Interestingly, VEN+MYCi975 had synergistic anti-leukemic effects in these same cell lines. Furthermore, CDKN1A expression was also rescued upon the triple therapy consisting of VEN+Aza+MYCi975. Combined, our results suggest that the PP2A-B56α complex is involved in the regulation of VEN+Aza response through changes in MYC expression. Interestingly, a novel small molecular glue (PMG) developed by our group, which specifically stabilizes the PP2A-B56α complex, enhanced VEN plus Aza response in vitro; however, this triple therapy did not work in HL-60 B56α KD cells and we observed a clearly decrease in MYC protein expression upon the triple therapy only in HL-60 B56α WT cells, supporting the rationale to translate these novel PMGs into the clinic. Altogether, our data suggest that PP2A-B56α might have an important role in VEN plus Aza treatment response through the regulation of MYC degradation.

Combinatorial Optimization of Chemotherapeutic Agents on T-Cell Acute Lymphoblastic Leukemia

Introduction: Up to now, treatment options for T-cell acute lymphoblastic leukemia (T-ALL) remain quite limited. This study aims to assess the efficacy of monotherapies and combination of chemotherapeutic agents for T-ALL. Jurkat, CCRF-CEM are late T-cell progenitor (LTP) cell lines, while Loucy is early T-cell progenitor (ETP) cell line characterized by an immature phenotype. The drugs under evaluation include venetoclax, a selective BCL-2 inhibitor; cytarabine, a cytosine analog with established antineoplastic activity; bendamustine, an alkylating agent; and azacitidine, a DNA methyltransferase inhibitor. Given the notable tumor heterogeneity observed in T-ALL, the comprehensive assessment of these therapeutic agents on Jurkat, CCRF-CEM, and Loucy may render valuable insights for treatment strategies in T-ALL. Methods: Cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2. Venetoclax, bendamustine, and azacitidine were prepared as stock solutions in DMSO, while cytarabine was prepared in distilled water. Monotherapies (venetoclax, cytarabine, bendamustine and azacitidine) and combination therapies (venetoclax plus cytarabine, venetoclax plus bendamustine and venetoclax plus azacitidine) were administered. A control group received DMSO or distilled water only. Cell viability was assessed after 48 hours by using Trypan blue and colorimetric method (MTS assay) on cells treated with various drug concentrations. Statistical analysis was conducted using the SynergyFinder Plus and drc R package, with significance set at p < 0.05. Results: Our study demonstrated that T-ALL cell lines displayed a different pattern of drug sensitivities according to combination of chemotherapeutic agents. Loucy showed greater sensitivity to venetoclax while Jurkat and CCRF-CEM exhibited higher sensitivity to cytarabine, bendamustine, and azacitidine. When conducting drug combinations on Jurkat and CCRF-CEM, the addition of venetoclax to cytarabine, bendamustine, or azacitidine resulted in an additive effect, with Loewe synergic scores ranging from -10 to 10 for each combination. On the other hand, Loucy demonstrated a distinct pattern of response; the combination of venetoclax with cytarabine showed additivity (Loewe synergic score: 8.45 and 5.82 with MTS and trypan blue assays, respectively), while venetoclax combined with bendamustine or azacitidine showed a synergistic effect (Loewe synergic score > 10 with MTS assay). Notably, among these combination treatments on Loucy, the pairing of venetoclax with bendamustine showed the most significant synergistic effects, with a Loewe score of 16.6. Conclusion: Venetoclax-based chemotherapeutic combinations exhibited various responses in T-ALL cell lines. Of note, the combination of venetoclax plus bendamustine showed potent synergistic effects in all T-ALL cell lines. These findings warrant further study to develop fundamentally curative therapeutic strategies in T-ALL.