BH3 Mimetics Research Articles

Co-operation of MCL-1 and BCL-XL anti-apoptotic proteins in stromal protection of MM cells from carfilzomib mediated cytotoxicity

IntroductionBCL-2 family proteins are important for tumour cell survival and drug resistance in multiple myeloma (MM). Although proteasome inhibitors are effective anti-myeloma drugs, some patients are resistant and almost all eventually relapse. We examined the function of BCL-2 family proteins in stromal-mediated resistance to carfilzomib-induced cytotoxicity in MM cells.MethodsCo-cultures employing HS5 stromal cells were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1-hour pulse method. The expression of BCL-2 family proteins was assessed by flow cytometry and WB. Pro-survival proteins: MCL-1, BCL-2 and BCL-XL were inhibited using S63845, ABT-199 and A-1331852 respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB.ResultsCFZ induced dose-dependent cell death of MM cells, primarily mediated by apoptosis. Culture of MM cells on HS-5 stromal cells resulted in reduced cytotoxicity to CFZ in a cell contact-dependent manner, upregulated expression of MCL-1 and increased dependency on BCL-XL. Inhibiting BCL-XL or MCL-1 with BH-3 mimetics abrogated stromal-mediated protection only at high doses, which may not be achievable in vivo. However, combining BH-3 mimetics at sub-therapeutic doses, which alone were without effect, significantly enhanced CFZ-mediated cytotoxicity even in the presence of stroma. Furthermore, MCL-1 inhibition led to enhanced binding between BCL-XL and BIM, while blocking BCL-XL increased MCL-1/BIM complex formation, indicating the cooperative role of these proteins.ConclusionStromal interactions alter the dependence on BCL-2 family members, providing a rationale for dual inhibition to abrogate the protective effect of stroma and restore sensitivity to CFZ.

A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells.

BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.

AMPK inhibition sensitizes acute leukemia cells to BH3 mimetic-induced cell death.

BH3 mimetics, including the BCL2/BCLXL/BCLw inhibitor navitoclax and MCL1 inhibitors S64315 and tapotoclax, have undergone clinical testing for a variety of neoplasms. Because of toxicities, including thrombocytopenia after BCLXL inhibition as well as hematopoietic, hepatic and possible cardiac toxicities after MCL1 inhibition, there is substantial interest in finding agents that can safely sensitize neoplastic cells to these BH3 mimetics. Building on the observation that BH3 mimetic monotherapy induces AMP kinase (AMPK) activation in multiple acute leukemia cell lines, we report that the AMPK inhibitors (AMPKis) dorsomorphin and BAY-3827 sensitize these cells to navitoclax or MCL1 inhibitors. Cell fractionation and phosphoproteomic analyses suggest that sensitization by dorsomorphin involves dephosphorylation of the proapoptotic BCL2 family member BAD at Ser75 and Ser99, leading BAD to translocate to mitochondria and inhibit BCLXL. Consistent with these results, BAD knockout or mutation to BAD S75E/S99E abolishes the sensitizing effects of dorsomorphin. Conversely, dorsomorphin synergizes with navitoclax or the MCL1 inhibitor S63845 to induce cell death in primary acute leukemia samples ex vivo and increases the antitumor effects of navitoclax or S63845 in several xenograft models in vivo with little or no increase in toxicity in normal tissues. These results suggest that AMPK inhibition can sensitize acute leukemia to multiple BH3 mimetics, potentially allowing administration of lower doses while inducing similar antineoplastic effects.

Abstract 690: Investigation of BH-3 mimetics as radiosensitizing agents in glioblastoma

Abstract Glioblastoma (GBM) is a highly aggressive primary brain tumor. Treatment consists of maximal safe tumor resection, chemotherapy (temozolomide) and radiotherapy; however, this aggressive treatment offers only a modest improvement in outcomes, with average life expectancy of approximately 12 months. Consequently, novel, effective and easily translatable therapies are urgently required. While radiotherapy is an effective treatment for many solid tumors, GBM displays an innate radio-resistance that contributes to treatment failure. Therefore, there is a need to identify chemotherapeutics that can be used as radiosensitizers to increase efficacy. Notably, a key pathway involved in therapeutic resistance is apoptotic evasion, which is driven by upregulation of pro-survival BCL-2 proteins that inhibit the pro-apoptotic proteins necessary for the apoptotic response. Recent advances in drug development have produced a range of BH3 mimetics - novel chemotherapeutics with potential to reinstate apoptosis by targeting specific pro-survival molecules. We hypothesized that the radiation sensitivity of GBM cells would be enhanced by using BH3 mimetics to inhibit pro-survival molecules and release the apoptotic block. A panel of BH3 mimetics was tested in combination with radiotherapy across three patient-derived GBM cell lines (G7, E2 and R15), using a 3D clonogenic culture system that recapitulates in vivo growth patterns and responses. We found that the BCL-XL inhibitor, A1331852, significantly radiosensitized all 3 GBM lines. In addition, Western blot analysis revealed A1331852 to induce PARP1 cleavage in G7 cells when used as a single agent. Subsequent in vivo studies were conducted in our well characterized orthotopic G7 xenograft murine model of GBM. Immunohistochemical studies showed that the pro-survival proteins MCL-1 and BCL-XL were upregulated in irradiated tumors. Furthermore, a pharmacokinetic study in the same orthotopic murine model, allowing for drug delivery evaluation, and optimized treatment scheduling. Importantly, A1331852 was found to be blood brain barrier penetrant at 25 mg/kg, with increased drug detected in tumor vs normal brain tissue. From these data, a survival study investigating A1331852 as a radiosensitizing agent was carried out in the same G7 GBM orthotopic xenograft model. Kaplan-Meier analysis showed an increase in survival of mice treated with A133852 plus radiotherapy compared with the radiotherapy only group (p = 0.0309). Overall, we have demonstrated that dependence on BCL-2 family proteins is a vulnerability that can be targeted to improve radiotherapy response in GBM. Furthermore, we have identified a promising BH3 mimetic, A1331852, to use in combination with RT and have completed a proof-of-concept in vivo study that confirms the potential of BH3 mimetics to improve outcomes for patients with GBM. Citation Format: Louise R. Dutton, Clara Mullen, Anna L. Koessinger, Mark Jackson, Katrina Stevenson, Anthony J. Chalmers, Stephen Tait, Kirsteen J. Campbell, Joanna L. Birch. Investigation of BH-3 mimetics as radiosensitizing agents in glioblastoma [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 690.

Abstract 1993: Cytosolic BAX dimerization as a mechanism of resistance to apoptosis and a novel target to potentiate BAX activation and apoptosis

Abstract BCL-2 family proteins are master regulators of cellular life and death and have become drug targets for the development of therapeutic agents to overcome the apoptotic blockade of cancer. BAX is a pro-apoptotic BCL-2 member that is a crucial mediator of apoptosis induced by diverse stimuli including various chemotherapeutic and targeted agents. BAX predominantly resides in the cytosol in a quiescent state and upon stress, it undergoes conformational activation and mitochondrial translocation leading to mitochondrial outer membrane permeabilization, a critical event in apoptosis execution. While cytosolic BAX is typically considered an inactive monomer, we previously reported structural and cell-based evidence of a cytosolic BAX dimer. However, it remains unclear how this regulates BAX. Moreover, we found that specific BAX mutants such as P168G and G67R formed inactive BAX dimers and mutations of P168A and G67R have been identified in AML patients and found to have impaired apoptotic activity and notably the BAX P168A mutant appeared in AML patients acquiring resistance to the BCL-2 inhibitor Venetoclax. Here, we investigated the role of the inactive BAX dimer in regulating apoptosis and pro-apoptotic drug treatments in various hematologic and solid tumor cancer cells. Surprisingly, cancer cell lines express cytosolic inactive BAX dimers and/or monomers. Expression of inactive dimers, results in reduced BAX activation, translocation and apoptosis upon pro-apoptotic drug treatments such as BH3 mimetics and chemotherapy drugs. Our findings support the notion that formation of the inactive BAX dimer is a mechanism adopted by cancer cells to further suppress BAX activation and gain survival advantage. Using the inactive BAX dimer crystal structure, we designed a pharmacophore-based drug screen, and identified a small-molecule modulator, BDM19 that binds and activates cytosolic BAX dimers and prompts cells to apoptosis alone or in combination with BCL-2/BCL-XL inhibitors. Moreover, we validated interactions of BDM19 and BAX, using structural, biochemical and cellular methods, critical for BAX activation. Our findings provide mechanistic insights into the regulation of BAX and resistance to apoptosis in cancer through inactive BAX dimerization. Further, we demonstrate a rational strategy to target BAX dimer for therapeutic modulation of BAX and overcoming resistance to apoptosis by pro-apoptotic agents. Citation Format: Nadege Gitego, Bogos Agianian, Oi Wei Mak, Vasantha Kumar MV, Emily Cheng, Evripidis Gavathiotis. Cytosolic BAX dimerization as a mechanism of resistance to apoptosis and a novel target to potentiate BAX activation and apoptosis [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 1993.

Abstract 2532: Deciphering multiple protein complexes in BCL2 family to measure apoptotic dependencies in acute myeloid leukemia

Abstract The BCL2 protein family controls apoptosis by shifting the dynamic equilibrium state of complex formation. BH3 mimetics disrupt this balance by disturbing the interaction of a specific anti-apoptotic protein, leading to the initiation of apoptosis. However, relying solely on molecular data for the specific anti-apoptotic protein can be challenging due to the functional redundancy within the BCL2 protein family and their complex interaction network. Therefore, comprehensive profiling information considering protein complexes is essential to determine apoptotic dependencies, particularly in a clinical context. In this study, we adapted a Single-molecule Protein Interaction Detection (SPID) platform that can assess levels of protein complexes in a quantitative manner. Out of the 47 metrics included in our Apoptosis Signaling 47 panel, we utilized 22 assays to measure apoptosis dependencies in acute myeloid leukemia (AML) cells obtained from 32 patients. By combining these metrics, we developed a predictive model for the ex vivo response to ABT-199 treatment, focusing on three key biomarkers: BCL2-BAX complex, BCL2-BIM binding potential, and BCLxl-BAK level. From a receiver operating curve (ROC) analysis, the model exhibited high accuracy with a value of 0.94 area under the curve (AUC). We further validated our model by comparing the predicted response to the therapeutic outcomes in 10 AML patients according to the 2017 ELN guidelines, and achieved 100% sensitivity (4 out of 4 identified correctly) and 83.3% specificity (5 out of 6 identified correctly). Overall, our platform provides an effective method to explore apoptosis dependencies in clinical samples based on the information regarding protein complexes within the BCL2 protein family. Three key molecular biomarkers can be translated into a gauge for predicting therapeutic outcomes of BH3 mimetics, suggesting an improvement of precision medicine in AML treatment. Citation Format: Hongwon Lee, Changju Chun, Byungsan Choi, Saem Hong, Yunseo Lee, Youngil Koh, Tae-Young Yoon. Deciphering multiple protein complexes in BCL2 family to measure apoptotic dependencies in acute myeloid leukemia [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 2532.

Abstract 562: Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor that predominantly occurs in children, adolescents, and young adults. The treatment for OS that combines surgery with chemotherapy, which consists of a four-drug combination of adriamycin (DOX), cisplatin (CDDP), high-dose methotrexate (MTX), and ifosfamide, was established in 1970s, and it is still used as a standard therapy. Oncogenic driver mutations and fusion genes common to OS have not been identified, which is one of the reasons for the lack of success in drug development for OS. Herein, to explore the characteristic vulnerability in OS, molecular targeted drugs were screened against OS cell lines and patient-derived cells; obatoclax, a pan-BH3 mimetic, and a concomitant drug that enhances obatoclax-induced apoptosis, OSI906, a dual inhibitor of IGF1R and IR, were found. Fluorescence in situ hybridization (FISH) analysis revealed that copy number gains of MCL1, an antiapoptotic Bcl-2 family protein, were observed in 46.3% of formalin-fixed paraffin-embedded (FFPE) specimens from patients with OS (N = 41) and in 45.5% of OS cell lines (N = 11), thereby causing vulnerability to BH3 mimetics. IHC staining also revealed high expression of Mcl-1 protein in specimens with MCL1-amplification in FISH analysis. Pharmacological or genetic inhibition of Mcl-1 induced potent apoptosis in MCL1-amplified OS cells, which was enhanced by the co-inhibition with Bcl-xL. Moreover, chromosome 1q21.2-3, where MCL1 is located, contains several IGF1R/PI3K pathway-related genes, including PIP5K1A, TARS2, OUTD7B, and ENSA, whose copy numbers were also increased in MCL1-amplified OS cells, suggesting that 1q21.2-3-amplified OS cells readily activate downstream pathways via IGF1R. IGF signaling activation in 1q21.2-3-amplified OS cells negated the obatoclax-induced apoptosis, which was overcome by OSI906 treatment. Combined obatoclax and OSI906 administration remarkably suppressed MCL1-amplified OS tumor growth in the xenograft mouse model and resulted in tumor regression; however, none of the treatments reduced tumor growth of OS without MCL1 amplification. These results suggest that combining obatoclax with OSI906 could be a potential therapeutic intervention for 1q21.2-3-amplified OS, and that prior stratification of OS based on the 1q21.2-3 amplification status would be important for the success of this combination therapy. Thus, we elucidated the mechanism of action of drugs and their potential as a novel therapeutic strategy for approximately 50% of patients with OS. Moreover, a FISH analysis that can detect copy number gains of MCL1 in FFPE specimens from patients with OS was established to stratify patients eligible for this therapy. Citation Format: Satoshi Takagi, Ryohei Katayama. Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma [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 562.

MCL1 inhibition targets Myeloid Derived Suppressors Cells, promotes antitumor immunity and enhances the efficacy of immune checkpoint blockade

Immune checkpoint inhibitors (ICIs) are now the first-line treatment for patients with advanced melanoma. Despite promising clinical results, many patients fail to respond to these therapies. BH3 mimetics, a novel class of small molecule inhibitors that bind and inhibit anti-apoptotic members of the BCL2 family proteins such as BCL2 or MCL1, have been very successful in treating hematologic malignancies. However, there are limited studies on the immunomodulatory role of the BH3 mimetics. Several factors contribute to ICI resistance including myeloid-derived suppressor cells (MDSCs) that exert immunosuppressive effects through direct and indirect inhibition of antitumor immunity. Thus, targeting MDSCs to enhance antitumor immunity has the potential to enhance the efficacy of ICIs. In this study, we show that the MCL1 inhibitor S64315 reduces melanoma tumor growth in an immune cell-dependent manner in mice. Specifically, S64315 enhances antitumor immunity by reducing MDSC frequency and by promoting the activity of CD8+T cells. Additionally, human MDSCs are 10 times more sensitive to S64315 than cutaneous melanoma lines. Further, we found that a higher expression of MCL1 is associated with poor survival for patients treated with anti-PD-1. Finally, combining S64315 and anti-PD-1 significantly slowed tumor growth compared to either agent alone. Together, this proof-of-concept study demonstrates the potential of combining an MCL1 inhibitor with anti-PD-1 in the treatment of melanoma. It justifies the further development of next generation MCL1 inhibitors to improve efficacy of ICIs in treating malignant melanoma.

Delineating functional and molecular impact of ex vivo sample handling in precision medicine

Consistent handling of samples is crucial for achieving reproducible molecular and functional testing results in translational research. Here, we used 229 acute myeloid leukemia (AML) patient samples to assess the impact of sample handling on high-throughput functional drug testing, mass spectrometry-based proteomics, and flow cytometry. Our data revealed novel and previously described changes in cell phenotype and drug response dependent on sample biobanking. Specifically, myeloid cells with a CD117 (c-KIT) positive phenotype decreased after biobanking, potentially distorting cell population representations and affecting drugs targeting these cells. Additionally, highly granular AML cell numbers decreased after freezing. Secondly, protein expression levels, as well as sensitivity to drugs targeting cell proliferation, metabolism, tyrosine kinases (e.g., JAK, KIT, FLT3), and BH3 mimetics were notably affected by biobanking. Moreover, drug response profiles of paired fresh and frozen samples showed that freezing samples can lead to systematic errors in drug sensitivity scores. While a high correlation between fresh and frozen for the entire drug library was observed, freezing cells had a considerable impact at an individual level, which could influence outcomes in translational studies. Our study highlights conditions where standardization is needed to improve reproducibility, and where validation of data generated from biobanked cohorts may be particularly important.

The heart of VIALE-A: Cardiac complications of hypomethylating agents and venetoclax in acute myeloid leukaemia.

As we commemorate 50 years since the introduction of classical 7 + 3 induction chemotherapy for acute myeloid leukaemia (AML), we also embark upon new territory with the advent of novel targeted therapeutics, including BH3 mimetics. To date, we do not have much large-scale longitudinal data regarding the toxicities of such novel therapies. Johnson etal. perform a comprehensive analysis of cardiac toxicities with hypomethylating agents and venetoclax and offer valuable insight into risk-benefit analysis when considering front-line therapy for AML. Commentary on: Johnson etal. Cardiac events in newly diagnosed acute myeloid leukaemia during treatment with venetoclax + hypomethylating agents. Br J Haematol 2024;204:1232-1237.

Abstract IA26: Exploring breast cancer vulnerabilities through apoptotic pathways

Abstract Combined CDK4/6 inhibitor and endocrine therapy has rapidly become standard therapy for patients with early line relapsed estrogen receptor (ER)-positive, HER2-negative breast cancer. Although this combination has been shown to significantly improve progression free and overall survival, relapse is virtually inevitable. This may in part be due to the profound cytostatic effect of CDK4/6 inhibitors on tumors, which is associated with reduced apoptotic cell death. It seems likely that growth arrested and/or senescent tumor cells acquire resistance to therapy, leading to relapse. BH3 mimetics are an emerging class of drug that target pro-survival proteins such as BCL2, BCL-xL and MCL1. Venetoclax, a BH3 mimetic that has potent and specific activity against BCL2, has shown considerable promise in hematopoietic tumors such as CLL, but has yet to be fully explored in solid tumors. We have previously demonstrated safety and efficacy of combining tamoxifen with venetoclax for patients with metastatic ER-positive BCL2-positive disease using pre-clinical PDX models and a dose-finding phase 1 study [mBEP study, ISRCTN98335443], where most patients were CDK4/6 inhibitor therapy-naïve. In the post-CDK4/6 inhibitor setting, however, venetoclax did not improve clinical benefit rate or progression free survival when added to fulvestrant in the phase 2 study VERONICA [NCT03584009]. In that study a minority of tumours exhibited strong BCL2 expression and exploratory biomarker analysis revealed a trend towards improved progression free survival for patients with tumors exhibiting high BCL2 expression (IHC 3+), a BCL2:BCL-xL Histoscore ratio ≥1, or with PIK3CA wild-type status. Using pre-clinical models including patient derived xenografts of ER-positive breast cancer, we have shown that the addition of venetoclax to dual therapy comprising fulvestrant and the CDK4/6 inhibitor palbociclib elicits a superior and more durable tumor response, accompanied by increased apoptosis. These findings have formed the basis of a phase 1 clinical trial of palbociclib, letrozole and venetoclax [PALVEN, NCT03900884] in patients with early line relapsed ER and BCL2-positive metastatic breast cancer. In parallel, pre-clinical studies are underway to determine the effect of dual targeting BCL-xL and MCL1, as well as dual targeting of AKT and with a BH3 mimetic in CDK4/6 inhibitor refractory breast cancer cells. Citation Format: James R Whittle, François Vaillant, Elliot Surgenor, Luuk Heitink, Christine Muttiah, Jane E Visvader, Geoffrey J Lindeman. Exploring breast cancer vulnerabilities through apoptotic pathways [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA26.

Abstract B070: Myc synthetic lethality as a therapeutic strategy for resistance: Insights into the targetability of mitochondrial complex I in Myc-high TNBC

Abstract Patients with advanced triple-negative breast cancer (TNBC) frequently develop chemoresistance, emphasizing an urgent need for novel therapeutic strategies able to eradicate treatment resistant tumor cells. Recent studies have highlighted MYC as a common oncogenic alteration and a driver of stem-like TNBC phenotype, which commonly features high mitochondrial respiratory activity instated through OXPHOS system and chemoresistance. Interestingly, emerging evidence suggests that residual tumors from chemo treatment remain susceptible to OXPHOS inhibition through inhibition of respiratory complex I. Our previous studies have revealed a specific MYC-dependent and tumor suppressive synthetic lethal action for combination of metformin and BH3 mimetics in cells, ex vivo and in vivo models of aggressive breast cancer (PMID: 30728358). Metformin is a systemic and local metabolic modulator, which acts as a weak complex I inhibitor and promoter of AMPK activation. Here, we have explored the role of MYC in OXPHOS regulation, to address the question if metformin specifically and lethally targets MYC-modulated OXPHOS in MYC-high TNBC. We found that a selective c-MYC inhibitor (EN4) leads to a significant decrease in complex I activity despite only a modest drop in complex I mass in endogenously MYC-high TNBC cell lines. Conversely, engineered MYC overexpression in these cells upregulated complex I activity. Our current work explores transcriptomic, proteomic, and functional approaches to the mechanisms through which MYC regulates complex I activity, structure, and dynamics, ultimately hoping to define a biological rationale for therapeutic targeting of OXPHOS in Myc-high TNBC. Citation Format: Antti O Hiltunen, Linda J Id, Juha Klefström. Myc synthetic lethality as a therapeutic strategy for resistance: Insights into the targetability of mitochondrial complex I in Myc-high TNBC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B070.

A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals a major role of BAX in the response to BH3 mimetics

AbstractTo establish a strict p53-dependent gene-expression profile, TP53−/− clones were derived from TP53+/+ and TP53−/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53−/− clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation–coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.

Statin-induced Mitochondrial Priming Sensitizes Multiple Myeloma Cells to BCL2 and MCL-1 Inhibitors.

BH3 mimetics including venetoclax hold promise for treatment of multiple myeloma but rational combinations are needed to broaden efficacy. This study presents mechanistic and clinical data to support addition of pitavastatin to venetoclax regimens in myeloma. The results open a new avenue for repurposing statins in blood cancer.

Novel Drug to Target HK2 Overcomes Therapeutic Resistance in Preclinical B-Cell Lymphoma Models

Background: The Warburg's effect is a hallmark in cancer. Hexokinase II (HK2) is a glycolic kinase to phosphorylate glucose to glucose-6-phosphate, which can be utilized as the energy source for cells. HK2 is predominantly overexpressed in cancer cells and has been associated with poor clinical outcomes in cancer patients. Previously, we demonstrated that HK2 mRNA and protein levels were increased in diffuse large B-cell lymphoma patients, and were significantly associated with inferior progression free survival (PFS) and overall survival (OS). In our rituximab and chemotherapy-resistant cell lines (TRCL), we demonstrated an upregulation of HK2 expression level is association with voltage-dependent anion channel (VDAC) resulting in a) repress mitochondrial potential and b) global therapy resistance. Knocking out HK2 overcame the chemo-resistance phenotype observed in TRCL. Here, we designed a novel specific inhibitor of HK2 and tested its activity in lymphoma pre-clinical models. Material and Methods: A HK2 specific inhibitor (HK2-001) was designed and generated according to antisense peptide technology, CABS-docking system and ROSWTTA analyzing. A panel of B-cell lymphoma cell lines including rituximab/chemo-sensitive and resistant cell lines (Raji, Raji 4RH, RL and RL 4RH), diffuse large B-cell lymphoma (DLBCL) (DHL4, DOHH2, TMD8 and U2932) and mantle cell lymphoma (MCL) (HBL2, Granta, Mino and Z138) were used. In addition, primary tumor cells isolated from chronic lymphocytic lymphoma (CLL) or other B-cell lymphoma patients were used. Briefly, cell lines or primary tumor cells were exposed to escalating doses of HK2-001 alone or in combination with different chemotherapy agents (doxorubicin, dexamethasone), targeted agents (i.e. burton tyrosine kinase inhibitors, BH3 mimetics) or monoclonal anti-CD20 antibodies. Cell viability was evaluated using the Presto Blue assay in experiments testing pharmacological interactions between HK2-001 and chemotherapy or targeted agents. All experiments were performed in triplicates in three separate experiments. The coefficient of synergy was calculated using CalcuSyn. Results: In vitro exposure of lymphoma cell lines to HK2-001 induced cell death in a panel of B-cell lymphoma cell lines representing rituximab/chemo-sensitive and resistant lymphoma. In addition, synergistic activity was observed between HK2-001 and doxorubicin, ibrutinib, and Venetoclax. These effects were also observed in primary tumor cells isolated from B-cell lymphoma patients. In addition, HK2-001 potentiated the anti-tumor activity of Rituximab and Obinutuzumab ex vivo. Conclusion: We developed a novel peptide targeting HK2. Pre-clinical studies suggested anti-tumor activity as a single agent. More importantly, HK2-001 enhanced the anti-tumor activity of chemotherapy agents, BTK inhibitors, BH3 mimetics and monoclonal antibodies targeting CD20. HK2-001 is a promising novel therapeutic strategy for against B-cell lymphoma, especially in refractory or relapsed patients. Future studies aimed to evaluate the effects of HKII-001 drug combinations in lymphoma animal models are planned and to evaluate its effects on Chimeric antigen receptor T-cells (CART) or bi-specific antibodies.

New Suitable Eµ-Tcl1 Mouse Model for Research on BH3-Mimetics Therapy Response In Vivo

Introduction: Generation of appropriate mouse models to study clinically active compounds is inevitable. Currently, the commonly used mouse model for CLL is the TCL1 transgenic model, which shows an expression of the human TCL1 oncogene in B cells. While this model resembles many features of human CLL, it is resistant to venetoclax, a BCL2 inhibitor which represents an important treatment option for CLL and other hematological malignancies. The lack of therapeutic response to venetoclax is an important limitation of the TCL1 mouse model. As a result, the effectiveness, the mechanisms, and the development of venetoclax resistance and other BH3 mimetics cannot be investigated in the TCL1 transgenic setting. Methods and results: To overcome the stated limitations, a novel mouse model was generated expressing human BCL2 in B cells under the control of the TCL1 promoter. We established a new mouse model (TBC) by crossbreeding Eµ-Tcl1 tg/wt mice with mice containing a B cell specific conditional Bcl-2 Rosa26/wt; Cd19Cre Cre/wt overexpression (control group Eµ-Tcl1 tg/wt; Cd19Cre Cre/wt: TC). Initial data show a strong leukocytosis ( p<0,0001) and splenomegaly in TBC mice leading to a significantly shortened overall survival compared to TC mice (log-rank: p=0,0028). Immunophenotyping revealed a population of class-switched IgM - B cells within TBC mice and only a small population of Cd19 +/Cd5 dim cells. Transcriptomics followed by gene enrichment analysis showed a development of GCB-type DLBCL in TBC mice compared to TC mice. Apoptosis assays revealed high sensitivity towards BH3 mimetics, especially BCL2 inhibitor venetoclax. In vitro experiments demonstrated that B cells from TBC mice are 10 times more sensitive towards venetoclax treatment. Based on this fact, we treated 5 TBC mice for 4 weeks with venetoclax. Even in this short treatment time frame, there was a strong loss of leukocytes in peripheral blood of about 60%, leading to normal ranges of leukocytes in TBC mice after even 14 days of treatment with venetoclax. Interestingly, we observed a massive change of Cd4 +/Cd8 + ratio towards Cd4 + cells due to venetoclax treatment, indicating that our novel model resembles human settings. Conclusions: We were able to show a strong therapy response towards treatment with venetoclax in vivo in TBC mice leading to a loss of leukocytes, a gain of CLL typical Cd19 +/Cd5 dim cells and a strong effect on tumormicroenvironment with a significantly changed Cd4 +/Cd8 + ratio. This makes the new mouse model very suitable for testing and observing venetoclax therapy response, its side effects and outcome in vivo, to provide our patients with the best possible therapy.

Targeting ADSS2 Enhances BH3 Mimetics Treatment Induced Mitochondrial Apoptosis in AML Cells

Recently, the BH3 mimetic-Bcl2 inhibitor venetoclax (VEN), has been approved by FDA in 2018 for the treatment of patients with AML in combination with a hypomethylating agent (HMA) such as azacytidine (AZA). This synergy is likely due to HMA-induced mitochondrial vulnerability associated with cytoplasmic RNA metabolism. Nonetheless, most AML patients treated with the combination eventually relapse, possibly due to persistence of tumor cells not dependent on antiapoptotic proteins (Bcl2, Mcl1). There is a pressing need to develop targeted therapeutics sensitizing AML cells to mitochondrial apoptosis, particularly for those patients failed from VEN associated treatment. To achieve the goal, we performed a CRISPR/Cas9 Knockout (KO) screen (using Bim peptide-based dynamic ΔBH3 profiling as read-out) targeting genes (n=534) involved in cytosolic nucleotides metabolism pathways to increase mitochondrial vulnerability in a BH3 mimetic-refractory AML cell line (HEL). That search identified ADSS2, which catalyzes the first committed/rate-limiting step of AMP biosynthesis from IMP, emerged as the top hit, exhibiting highest ΔBH3 priming. Subsequent BH3 priming assays validated that ADSS2 KO significantly enhanced sensitivity to BH3 mimetics across various AML cell lines, including MOLM13, SKM1, OCI-AML3, and HEL cells. ADSS2 KO also led to a noteworthy reduction in the IC50 values of both VEN and the Mcl1 inhibitor S63845 in these cell lines. Additionally, the synergistic effect between sgRNA targeting ADSS2 and VEN or S63845 was also confirmed in Cas9 expressing cells from two AML patient-derived-xenograft (PDX) samples. We next transplanted AML cells (form one PDX samples) with or without ADSS2-KO into NGS mice, then treated mice with VEN. Notably, VEN treatment (100 mg/kg, QDx14, PO) significantly reduced tumor burden in ADSS2-KO AML mice compared to WT AML mice. Additionally, ADSS2 KO combined with VEN treatment group displayed a survival advantage compared to single VEN treatment group. Further mechanistic investigations revealed that ADSS2 KO led to a remarkable depletion of AMP rather than ATP, and a robust repression of AMPK activity in HEL cells. To assess whether AMPK inhibition mimics the effect of ADSS2 KO, AMPKα-deficient (Mx1-Cre/Prkaa1 fl/fl/ Prkaa2 fl/fl) MLL-AF9 (MA9) transformed murine AML cells were assessed. Indeed, AMPKα depletion substantially enhanced VEN treatment efficiency ex-vivo. Notably, enforced overexpression of a constitutively active AMPKa2 (T172D) construct in a VEN sensitive cell line SKM1, promoted cell resistance to mitochondrial apoptosis by VEN or S63845. Moreover, T172D expression can also abolish ADSS2-KO mediated mitochondrial vulnerability to VEN or S63845 treatment, indicating the pivotal role of AMPK signaling in mediating ADSS2 function. There are no reported ADSS2 inhibitors. To explore ADSS inhibitor, we conducted a fragment-based virtual screen of 8,000,000 compounds (MolPort) library and requested the top-ranking hits for ADSS2 catalysis evaluation by ELISA. Notably, the top two hits AS104 and AS71 exhibited the most potent inhibitory effects (AS104 IC50=2.43µM, AS71 IC50=3.85µM). NMR-STD analysis was conducted in a cell-free system, which unequivocally confirmed the physical binding of inhibitors and the ADSS2 protein. To further investigate the interactions between AS71 and AS104 with ADSS2 in a cellular context, both compounds were subjected to a cellular thermal shift assay (CETSA). Notably, treatment with AS71 and AS104 led to substantial shifts in ADSS2 protein thermal stability, providing evidence of their interactions with ADSS2 within the cellular environment. In both HEL cells and a primary VEN-resistant AML samples, treatment with AS71 and AS104 significantly increased ΔBH3 priming. Collectively, our observation revealed that targeting ADSS2 is critical for sensitizing AML cells to BH3 mimetics. We are now conducting preclinical assessments of ADSS2 inhibitors.

Venetoclax Pre-Treatment of CLL/SLL Patients Enhances Autologous CAR T-Cells Efficacy

Introduction Venetoclax is an oral targeted therapy drug that is FDA-approved for the treatment of chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL) in both the frontline and relapsed/refractory (R/R) settings. While Venetoclax's dominant mechanism of action is the inhibition of Bcl-2 and thus, priming malignant CLL/SLL cells for apoptosis, there are emerging data highlighting the effects of Venetoclax on T-cell fitness/function. T-cell populations in CLL/SLL are known to be functionally impaired, in part due to abnormal expansion of T-regulatory cell (Treg) populations that blunt normal anti-tumoral cellular immune responses by CD8 + T-cells, Th1 Helper T-cells, and NK-cells and allows CLL/SLL cells to proliferate. Treg cell frequency has also been shown to directly correlate with CLL/SLL progression, and treatment of CLL/SLL results in lower circulating Treg populations. The unfavorable and generally anti-inflammatory tumoral microenvironment fostered by CLL/SLL cells is a major obstacle to the development of effective chimeric antigen receptor (CAR) T-cell options for this disease, for which there are currently no FDA-approved products despite more than a decade of investigation. Objective: In our current work, we studied the impact of Venetoclax on T-cell composition and function in serially collected specimens from patients with CLL/SLL; and evaluated the function of CAR T-cells manufactured from Venetoclax treated patients. Methods Peripheral blood mononuclear cells (PMBCs) and plasma was collected from CLL/SLL patients (N=5) who were treated with Venetoclax using the standard ramp up dosing-protocol. All procedures were approved by our IRB under BDR 164122. Samples were collected at pre-treatment (baseline) and several post-Venetoclax dosing timepoints (Day +1 day, day +7, and day +30). B-cells were isolated by negative selection using magnetic beads. CLL cells were the B-cells were exposed Venetoclax alone or in combination with several chemotherapy agents. Cell viability was determined by Cell Titer glo after 48hrs of drug exposure. In addition, PBMCs samples were analyzed using multicolor parameter flow cytometry to determine T-cell subsets composition including memory phenotype/stemness and others at each time point. Thorough serum cytokine (IL-15, IL-6, Il-2 and IL-10) analysis was conducted at each time point. Using baseline and 1-week post Venetoclax PBMCs samples, T-cells were activated, and autologous second generation CD19-directed CAR T-cells were manufactured. The efficacy of CARs were then tested at a 1:1 effector to target ratio against Raji and Raji 4RH cells for 24 hours and analyzed for cytotoxicity via flow cytometry. Results As expected Venetoclax was active in all samples tested. In addition, synergistic activity was observed when combined with chemotherapy agents or targeted agents (i.e. BRD-4 inhibitors). Regarding T-cell function, flow cytometry analysis at day +30 of Venetoclax therapy demonstrated that T-helper and T-cytotoxic T-cells displayed less exhaustion markers when compared to pre-Venetoclax samples (Baseline). Of interest, autologous αCD19 CAR T-cells manufactured 1 week following Venetoclax therapy induced higher cytotoxicity against Raji (rituximab chemotherapy sensitive cell lines) or Raji 4RH (rituximab-chemotherapy resistant cell lines) cells when compared to CAR T-cells generated from Venetoclax pre-treatment T-cells. Conclusion Our data suggests that Venetoclax has anti-tumor activities beyond direct CLL cytotoxicity. Venetoclax therapy appears to repair T-cell subsets in CLL patients. Perhaps by decreasing exhaustion markers in CD4/CD8 T-cells, Venetoclax potentiate the cytotoxicity of anti-CD19 CAR T cells against B-cell lymphoma. Further work is needed to fully elucidate the mechanism(s) by which Venetoclax modifies the fitness and function of T-cell subsets. These efforts may guide the development of combination strategies using BH-3 mimetics and T-cell engagers based therapeutic options for patients with CLL/SLL.

Targeting Mitochondrial Apoptotic Priming State to Personalize Therapy for Relapsed Acute Myeloid Leukemia

Despite of remarkable changes in therapeutic landscape of AML in past 5 years, relapsed AML patients still succumb to disease. In majority of patients, the common cause of death ultimately is the emergence multi-drug resistance at relapse. Using patient-derived xenograft (PDX) as our models, we investigated the basis of multi-drug resistance and methods for identifying drug sensitivity in relapsed AML. We hypothesized that there is differential activation of drug-induced mitochondrial apoptotic signaling between treatment-naïve and relapsed myeloblasts. To study mechanisms of drug resistance in AML, we developed PDX models (n=18 models) of in vivo acquired resistance to single agents (BCL-2/MCL-1 inhibitor, FLT3 inhibitor, SMAC mimetics and BRD-4 inhibitor) of distinct mechanisms, which caused board chemoresistance across all models. We tested whether multidrug resistance is associated with reduction in apoptotic signaling, using BH3 profiling, a method for measurement of mitochondrial sensitivity to apoptosis via exposure to synthetic BH3 domain peptides, on matched pre- and post-resistant PDXs. Using BH3 profiling, we found that a reduction in mitochondrial apoptotic priming consistently accompanied the acquisition of multidrug resistance irrespective of the mechanism of drug action and genetic status of PDX models. Next we created pharmacologic sensitivity landscape for 22 AML PDX models by exposing splenic myeloblasts isolated after confirmed engraftment (>70% circulating hCD45+ blasts) to drug a drug panel (n=40) for 14 hours. We first demonstrate that ex vivo drug-induced mitochondrial sensitivity singling measured via dynamic BH3 profiling (DBP) segregates PDXs according to prior treatment. By using unsupervised hierarchical clustering we found that AML PDXs developed from treatment naïve patients clustered together and showed greater apoptotic priming responses to a larger number of targeted agents than PDXs from R/R patients, which also formed discrete clusters. We replicated drug-response patterns from PDX models in primary and relapsed AML patients by analyzing ex vivo sensitivity responses to >500 drugs across relapse and diagnosis patients (Malani et al, 2021 and Tyner et al, 2018). We show that human AML patients at relapse also broadly lose sensitivity to agents in a similar pan-resistant manner as PDX models. Using unbiased whole transcriptomic analysis of naïve and resistant PDXs (n=78 samples), we found common pathways of resistance where positive enrichment was observed for cytokine-cytokine receptor interactions, xenobiotics biodegradation, metabolic pathways, Hippo signaling, and Ras signaling in resistant compared to treatment-naïve PDXs. Conversely, mechanisms relating to DNA repair and replication, such as base excision repair, mismatch repair, and homologous recombination, were negatively enriched. Moreover, pathways associated with specific drug resistance were also observed. One of the known multi-drug resistance mechanisms to chemotherapy drugs in AML is increased expression of efflux pumps from the superfamily of ATP binding cassette. We found enrichment in ABC transporter family pathway signatures in all 3 PDX models, DFAM-61786, DFAM-15354, and DFAM-61345, resistant to quizartinib, birinapant, and JQ-1 and validated higher drug-efflux activity in quizartinib and birinapant resistant model using calcein-AM efflux assay. We finally tested whether DBP could predict in vivo response of patient myeloblasts in PDX models. We tested in vivo sensitivity to 5 drugs of disparate mechanisms of action: birinapant and LCL-161 (SMAC mimetics), JQ-1 (BRD-4 inhibitor), venetoclax (BCL-2 antagonist), and quizartinib (FLT-3 inhibitor) in 4-9 different PDX models each. Using ex vivo DBP assay, we identified drugs that prime not only treatment naïve PDXs but also R/R PDXs. We identified persistent in vivo activity in a drug-specific manner, with BH3 mimetics and HDAC inhibitors showing overlapping activity across different drug-resistant models. Overall, we demonstrate that acquired resistance to targeted therapy in AML is accompanied by common mechanism of reduction in mitochondrial priming along with drug-specific resistance mechanisms. Further, we find that, even in the context of a multiply-resistant PDX model, DBP can still identify therapeutic vulnerabilities that can be efficaciously exploited in vivo.

Combining BCL-XL Inhibition with Brentuximab Vedotin to Overcome Chemoresistance in EBV-Related T/NK Lymphoma

Introduction Epstein Barr Virus (EBV) related T and NK malignancies, such as extra-nodal NK/T lymphoma (ENKTL) or chronic active EBV (CAEBV), are intrinsically chemoresistant. Conventionally, this is overcome in the clinic by using high-intensity treatment protocols. However, this makes therapy difficult to deliver, especially in patients with co-morbidities, and there are few options for those with relapsed or refractory disease. Intrinsic resistance to apoptosis, driven primarily by the anti-apoptotic BH3 protein BCL-XL, appears to play a significant part in treatment resistance (Sejic, Blood Advances 2020). However, inhibition of anti-apoptotic pathways alone cannot kill all malignant cells in vitro and is not curative in animal models. Identification of drugs that function synergistically with BCL-XL inhibition is critical to unlocking the chemoresistance. We investigated the combination of BH3 inhibition with the promotion of apoptosis using the antibody-drug conjugate (ADC) Brentuximab Vedotin (BV). BV delivers an MMAE payload to CD30-positive cells triggering apoptotic cell death through cell cycle arrest and DNA damage. We identified synergism between BV and a BCL-XL BH3 mimetic that dramatically increased cell death at concentrations that should limit on-target toxicity. Methods A diverse panel of EBV+ T/NK lymphoma cell lines including SNK1, MECO4, SNK6, SNT8, SNK10, SNT15 and SNT16 were evaluated for sensitivity to MMAE alone and with specific BH3 family inhibitors including A1331852, inhibiting BCL-XL, venetoclax inhibiting and BCL-2 and the MCL-1 inhibitor S63845 (Generon). Drug sensitivity was assessed by staining with PI/Annexin (eBioscience) and then analysing on an Accuri C6 flow cytometer. Cell surface expression of CD30 was assessed by flow cytometry (eBiosience). We then tested the effect of BV in combination with A1331852 and assessed for the effect on viability using CellTitre Glo (Promega). Results Single-agent MMAE was toxic to all cell lines tested with an LD50 ranging from 0.6-33 nM and a maximal killing effect between 10%-45%.PI/Annexin staining suggested the mode of cell killing was apoptotic. As a single agent, A1331852 showed efficacy across all cell lines, apart from MEC04 which exhibited reduced susceptibility. In combination with MMAE, A1331852 enhanced both the sensitivity and maximal killing effect seen. Using a 1:50 fixed dose ratio then analysis in CompuSyn the effect of both agents was synergistic with a fraction affect (0.5) between 0.158 and 0.177 in the cell lines tested (Figure1) . Single-agent inhibition of BCL-2 or MCL-1 showed only limited efficacy and no synergy with MMAE. High levels of CD30 expression was evident on all cell lines making ENKTL a viable target for BV. While BV showed efficacy as a single agent, cell viability at a clinically achievable 100 ug/ml dose was between 30% and 78% after 72h. However, in combination with A1331852 at a low 4 nM dose maximal viability was between 1% and 25%, figure 2 shows results for the SNT8 cell line. In most resistant lines, SNK6 and MECO4, sensitivity to BV could be increased using doses of A1331852 up to 500 nM, leading to 5-6% viability at 72h. Based on murine PK data this should still be clinically achievable. Discussion Combining targeted delivery of MMAE via BV with the BCL-XL inhibitor A1331852 is highly toxic to a panel of CAEBV and ENKTL-derived cell lines. These cells are intrinsically resistant to apoptosis but are sensitive to BCL-XL inhibition. The use of BCL-XL inhibitors has been limited by on-target thrombocytopenia and the combination of BH3 mimetics with chemotherapy can cause marked haematological toxicity. Therefore, using a targeted agent such BV in combination with BCL-XL inhibition has the potential to enhance efficacy without triggering excessive toxicity. Additionally, the synergistic nature of the combination allows the use of low-dose A1331852. We are working to better understand the cell line-specific differences in resistance to combination therapy, which does not appear to relate to either baseline BCL-XL protein levels, CD30 expression or the rate of cell division. These data show the potential of BV with BCL-XL inhibition to be an effective and tolerable treatment for ENKTL. We are now planning a detailed preclinical study to better understand the efficacy and safety of this combination in vivo and progress towards a future clinical trial.