MCL-1 BH3 Research Articles

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.

The Use of a BH3 Mimetic Toolkit Reveals a Switch of Myeloma Patients Dependency from BCL2 at Diagnosis to MCL1 at Relapse

Targeting anti-apoptotic proteins of the BCL2 family by BH3 mimetic small molecules is a new attractive therapeutic approach in hematological malignancies. The BH3 mimetics trigger apoptosis by mimicking the BH3-only proteins and promoting the release of pro-apoptotic BCL2 family members from anti-apoptotic proteins. Thus, the specific BH3 mimetics targeting BCL2, BCLXL or MCL1 exploit the dependency on these different anti-apoptotic proteins to kill tumor cells. Multiple myeloma is considered as a disease mainly dependent on MCL1 for survival based on the MCL1 dependency observed in human myeloma cell lines (HMCL), which are derived from the extramedullary stage of the disease and not representative of the diagnosis stage. Because no study up to date has reported the dependence of primary cells, we studied the dependency on BCL2 anti-apoptotic proteins both in a large collection of HMCL and in a cohort of 24 myeloma patients. We used a BH3 mimetic toolkit that includes venetoclax (ABT-199), A1155463 and A1210477, which target BCL2, BCLXL and MCL1 respectively.Using a panel of 34 HMCLs, we demonstrated that 23% of HMCL were highly sensitive to venetoclax (LD50 <0.3 mM) all of them harboring a t(11;14) translocation and therefore dependent on BCL2, whereas only 3% of HMCL was efficiently killed by A-1155463 (LD50 <0.3 mM) showing that a minor subset of HMCL is highly BCLXL-dependent. We found that the majority of HMCLs (56%) were efficiently killed by A1210477; this result is in total agreement with the BH3 profiling performed with the MS1 peptide, specific for targeting MCL1 (r=0.74, p=0.0001), confirming the specificity of A1210477 as a specific MCL1 BH3 mimetic. Among the BCL2 dependent HMCLs, one third were also MCL1 dependent, indicating co-dependency to both molecules.We also determined the dependency of primary myeloma cells by treating “ ex-vivo” 24 consecutive bone marrow samples from myeloma patients (12 at diagnosis and 12 at relapse) with the respective BH3 mimetic for 20 hours. Patient characteristics showed that the age, sex and % of plasma cells from myeloma samples either at diagnosis or relapse were not significantly different. Analysis of patients at diagnosis revealed three stricking findings. First, a large group of patients (50%) were resistant to the three BH3 mimetics indicating that they were not dependent on any of the main anti-apoptotic proteins. Second, 41% of patients were sensitive to venetoclax, among them 20% were also sensitive to A1210477 indicating a co-dependency to BCL2/MCL1. Third only 8 % of patients showed a strict MCL1 dependency. Interestingly, at relapse 65% of myeloma patients showed a MCL1 dependency. Among them 25% were also killed by venetoclax while none of the myeloma samples presented a strict dependency to BCL2. While no dependency on BCLXL has been found at diagnosis, 24% of patients at relapse were efficiently killed by A1155463. Finally, only 16% of myeloma samples at relapse were resistant to the three BH3 mimetics compared to the 50% found at diagnosis. We hypothesize that the increase of the priming in favor of MCL1 or BCLXL at relapse could be the results of either the influence of drug treatments or a clonal selection.Our study demonstrated that the dependency of myeloma cells on a particular anti-apoptotic BCL2 molecule differs with regards to the stage of the disease; At diagnosis, patients were mainly not dependent on BCL2, BCLXL or MCL1 or BCL2 dependent, whereas at relapse they were mainly MCL1 dependent or co-dependent. Finally, our results in myeloma suggested that the vulnerability of the tumor cells to the MCL1 BH3 mimetic rises during the progression of the disease while the resistance of myeloma cells to conventional drugs increased. Overall our results open the way to the use of BH3 mimetic targeted therapies not only at myeloma relapse but also at diagnosis. DisclosuresMoreau:Celgene: Consultancy, Honoraria; Celgene, Janssen, Takeda, Novartis, Amgen, Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Onyx Pharmaceutical: Consultancy, Honoraria. Touzeau:AbbVie: Research Funding.

Unmasking BCL-2 Addiction in Synovial Sarcoma by Overcoming Low NOXA.

Simple SummarySynovial sarcoma is a soft-tissue sarcoma that lacks effective systemic therapy and carries poor prognosis due to frequent late local recurrence and metastases. The cancer is known to be driven in part by increased expression of the pro-survival protein BCL-2. Surprisingly, synovial sarcoma proved resistant to BCL-2 inhibitors in pre-clinical trials. We identified increased activity of a second pro-survival protein, MCL-1, as responsible for this resistance. We showed that co-targeting both BCL-2 and MCL-1 proves to be an effective therapeutic approach both in cell culture and animal models of synovial sarcoma, supporting translation into clinical trials.Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.

Binding mode of Pyridoclax to myeloid cell leukemia-1 (Mcl-1) revealed by nuclear magnetic resonance spectroscopy, docking and molecular dynamics approaches

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family proteins. Its amplification is one of the most frequent genetic aberrations found in human cancers. Pyridoclax, a promising BH3 mimetic inhibitor, interacts directly with Mcl-1 and induces massive apoptosis at a concentration of 15 µM in combination with anti-Bcl-xL strategies in chemo-resistant ovarian cancer cell lines. In this study, a combined experimental and theoretical approach was used to investigate the binding mode of Pyridoclax to Mcl-1. The representative poses generated from dynamics simulations compared with NMR data revealed: (i) Pyridoclax bound to P1 and P2 pockets of Mcl-1 BH3 binding groove through its styryl and methyl groups establishing mainly hydrophobic contacts, (ii) one of the ending pyridines interacts through electrostatic interaction with K234 side chain, a negatively charged residue present only in this position in Mcl-1. Communicated by Ramaswamy H. Sarma

A short term biomarker-based assay predicts apoptotic response to combined BCL-2/MCL-1 BH3 mimetic targeting in Myeloma cells

Multiple myeloma is an incurable haematological malignancy characterised by molecular complexity and clonal heterogeneity. Although both MCL-1 and BCL-2 are necessary for myeloma survival, most myelomas are dependent on MCL-1 such that BCL-2 inhibition alone only yields significant cytotoxicity in a minority of cases. Cooperative inhibition of both proteins may be beneficial. In the era of novel medicines and targeted molecular therapies, there is a shortfall of rapid and reliable assays that can predict patient response to new agents.

PS1351 CRITICAL ANALYSIS OF THE STRINGENT COMPLETE RESPONSE IN MULTIPLE MYELOMA. AN UPDATED OF THE ANALYSIS BASED ON THE PETHEMA/GEM2012MENOS65 PHASE III CLINICAL TRIAL

Background: Multiple myeloma (MM) is a heterogenous hematological cancer that remains largely incurable. Apoptosis dysregulation by overexpression of BCL2 family anti-apoptotic proteins (BCL2, MCL1, BCLXL) is involved in MM cell survival and resistance to chemotherapy. This particularity makes MM cells vulnerable to BH3 mimetics that trigger apoptosis by specifically disrupting complexes between anti-apoptotic and pro-apoptotic proteins. BH3 mimetics targeting either BCL2 (Venetoclax) or MCL1 (S63845, AMG176 and AZD5991) are currently tested in clinic for MM treatment. BCL2 targeting was shown to be effective in a minority of patients, mainly t(11;14), while MCL1 targeting is widely effective, particularly at relapse. However, some patients remain resistant to BCL2 and MCL1 BH3 mimetics and could benefit from a dual targeting. Aims: The objective of this study was to highlight the interest of combining two clinically available BH3 mimetics, Venetoclax and S63845, targeting respectively BCL2 and MCL1. We also intended to decipher the mechanism of action of this combination and to identify resistance factors. Methods: For this study, we treated a panel of 25 Human Myeloma cell lines (HMCLs) and a cohort of more than 40 myeloma patient samples with Venetoclax and S63845 to identify those resistant to both agents. This group of resistant HMCLs and patient samples was treated with the combination S63845/Venetoclax at low doses. siRNA assays were performed to identify the role of effectors BAX and BAK and BH3-only proteins BIM and NOXA in combination-induced apoptosis. To understand the mechanism of action of this combination, we also studied the activation of BAX and BAK using specific antibodies. Finally, the role playing by BCLXL in the resistance to the S63845/Venetoclax combination was studied by both silencing and overexpression experiments. Results: One third of our cell line collection as well as our patient cohort was resistant to each BH3 mimetic. The combination was highly effective and synergic at low doses in three fourth of these cells. We demonstrated that the efficiency of the combination was equally dependent on BAX and BAK and that the simultaneous silencing of both effectors completely abrogated the combination effect. Conversely, the response to S63845 was mainly dependent on BAK. Interestingly, we also showed that the combination-induced apoptosis was associated with a hetero-complex formation between BAX and BAK. The BH3-only protein BIM, but not NOXA, was also shown to play a role in the combination response. Finally, BCLXL remained a resistance factor to the S63845/Venetoclax combination in agreement with its role in the resistance to both MCL1 and BCL2 BH3 mimetics alone. Summary/Conclusion: Our work demonstrates the interest of using the combination of two BH3 mimetics targeting both MCL1 and BCL2 in MM cells otherwise resistant to each inhibitor alone. Using low doses, this therapeutic strategy could potentially minimize side effects. We also demonstrated the crucial role of BAX and BAK in this BH3 mimetic combination-induced cell death. This is in agreement with the hetero-complex formation of BAX and BAK observed during myeloma cell apoptosis resulting from the combination treatment.

PS1350 STRONG PRE‐CLINICAL RATIONAL FOR THE USE OF MCL1/BCL2 BH3 MIMETIC COMBINATION TO EFFECTIVELY KILL PRIMARY MYELOMA CELLS INSENSITIVE TO EACH BH3 MIMETIC ALONE

Background:Multiple myeloma (MM) is a heterogenous hematological cancer that remains largely incurable. Apoptosis dysregulation by overexpression of BCL2 family anti‐apoptotic proteins (BCL2, MCL1, BCLXL) is involved in MM cell survival and resistance to chemotherapy. This particularity makes MM cells vulnerable to BH3 mimetics that trigger apoptosis by specifically disrupting complexes between anti‐apoptotic and pro‐apoptotic proteins. BH3 mimetics targeting either BCL2 (Venetoclax) or MCL1 (S63845, AMG176 and AZD5991) are currently tested in clinic for MM treatment. BCL2 targeting was shown to be effective in a minority of patients, mainly t(11;14), while MCL1 targeting is widely effective, particularly at relapse. However, some patients remain resistant to BCL2 and MCL1 BH3 mimetics and could benefit from a dual targeting.Aims:The objective of this study was to highlight the interest of combining two clinically available BH3 mimetics, Venetoclax and S63845, targeting respectively BCL2 and MCL1. We also intended to decipher the mechanism of action of this combination and to identify resistance factors.Methods:For this study, we treated a panel of 25 Human Myeloma cell lines (HMCLs) and a cohort of more than 40 myeloma patient samples with Venetoclax and S63845 to identify those resistant to both agents. This group of resistant HMCLs and patient samples was treated with the combination S63845/Venetoclax at low doses. siRNA assays were performed to identify the role of effectors BAX and BAK and BH3‐only proteins BIM and NOXA in combination‐induced apoptosis. To understand the mechanism of action of this combination, we also studied the activation of BAX and BAK using specific antibodies. Finally, the role playing by BCLXL in the resistance to the S63845/Venetoclax combination was studied by both silencing and overexpression experiments.Results:One third of our cell line collection as well as our patient cohort was resistant to each BH3 mimetic. The combination was highly effective and synergic at low doses in three fourth of these cells. We demonstrated that the efficiency of the combination was equally dependent on BAX and BAK and that the simultaneous silencing of both effectors completely abrogated the combination effect. Conversely, the response to S63845 was mainly dependent on BAK. Interestingly, we also showed that the combination‐induced apoptosis was associated with a hetero‐complex formation between BAX and BAK. The BH3‐only protein BIM, but not NOXA, was also shown to play a role in the combination response. Finally, BCLXL remained a resistance factor to the S63845/Venetoclax combination in agreement with its role in the resistance to both MCL1 and BCL2 BH3 mimetics alone.Summary/Conclusion:Our work demonstrates the interest of using the combination of two BH3 mimetics targeting both MCL1 and BCL2 in MM cells otherwise resistant to each inhibitor alone. Using low doses, this therapeutic strategy could potentially minimize side effects. We also demonstrated the crucial role of BAX and BAK in this BH3 mimetic combination‐induced cell death. This is in agreement with the hetero‐complex formation of BAX and BAK observed during myeloma cell apoptosis resulting from the combination treatment.

BH3-mimetic toolkit guides the respective use of BCL2 and MCL1 BH3-mimetics in myeloma treatment

AbstractBH3 mimetics are promising drugs for hematologic malignancies that trigger cell death by promoting the release of proapoptotic BCL2 family members from antiapoptotic proteins. Multiple myeloma is considered to be a disease dependent mainly on MCL1 for survival, based mostly on studies using cell lines. We used a BH3-mimetic toolkit to study the dependency on BCL2, BCLXL, or MCL1 in malignant plasma cells from 60 patients. Dependencies were analyzed using an unbiased BH3 mimetics cell-death clustering by k-means. In the whole cohort of patients, BCL2 dependency was mostly found in the CCND1 subgroup (83%). Of note, MCL1 dependence significantly increased from 33% at diagnosis to 69% at relapse, suggesting a plasticity of the cellular dependency favoring MCL1 dependencies at relapse. In addition, 35% of overall patient samples showed codependencies on either BCL2/MCL1 or BCLXL/MCL1. Finally, we identified a group of patients not targeted by any of the BH3 mimetics, predominantly at diagnosis in patients not presenting the common recurrent translocations. Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection from cell death observed by BAK knock-down, as well as the complete and early disruption of MCL1/BAK complexes on A1210477 treatment. Interestingly, this complex was also dissociated in A1210477-resistant cells, but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. In conclusion, our study opens the way to rationally use venetoclax and/or MCL1 BH3 mimetics for clinical evaluation in myeloma at both diagnosis and relapse.

Respective Place of Venetoclax and MCL1 BH3 Mimetics in Multiple Myeloma Treatment

Targeting anti-apoptotic proteins of the BCL2 family by BH3 mimetics is a new promising therapeutic approach in multiple myeloma (MM). The specific BH3 mimetic targeting BCL2, BCLXL or MCL1 trigger apoptosis and exploit the dependency on these different anti-apoptotic proteins to kill tumor cells. Because MM is mostly considered as dependent on MCL1, the recent clinical availability of MCL1 BH3 mimetics underlines an urgent need to better define patients that would benefit from a MCL1 targeted therapy. In the present study, we used a BH3 mimetic toolkit that includes venetoclax, A1155463 and A1210477, which target BCL2, BCLXL and MCL1 respectively to define dependencies/co-dependencies in a large cohort of 60 myeloma patients (21 at diagnosis and 39 at relapse). Alternatively, MCL1 dependency was confirmed using the S63845 MCL1 inhibitor in MM patient samples. Mononuclear bone marrow/blood cells were treated overnight with the respective BH3 mimetic and cell death was specifically measured in the tumor cell population. Primary MM cells dependencies were stratified using PCA analysis in three groups as highly dependent, intermediately dependent or not dependent. Our study demonstrated that half of patients at diagnosis were BCL2 dependent while only 10% were BCLXL dependent. The dependence on BCL2 or BCLXL was not significantly different between samples at diagnosis and relapse. Strikingly, we found that the MCL1 dependency was 33% at diagnosis while it was 69% at relapse, suggesting a significant increase in MCL1 dependency during the disease progression (p=0.01). Besides, 36% of overall patients showed co-dependencies on BCL2/MCL1. We also identified primary MM cells that did not depend on any of the three pro-survival molecules, both at diagnosis and relapse.Among this cohort of MM patients, 47 samples were further analyzed for the presence of recurrent translocations (t(11;14), t(6;14), t(4;14) and t(14;16)) allowing the analysis of dependencies in the different subgroups; these recurrent translocations lead to the overexpression of CCND1, CCND3, MMSET and MAF oncogenes, respectively. We found that BCL2 dependency was significantly higher in CCND1 subgroup (83%) compared to all other subgroups (20%, p=0.008). We also confirmed the BCL2/BCLXL mRNA ratio as a valuable biomarker to define BCL2 dependence (p=0.0001). At diagnosis, MCL1 dependency was absent in patients not harboring the common recurrent translocations while at relapse 6 out 9 patients not harboring the recurrent translocations were MCL1 dependent, indicating an increase of MCL1 dependency at relapse in this subgroup (p=0.03).Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection of cell death observed by BAK knock-down and the complete disruption of MCL1/BAK complexes upon A1210477 treatment, observed in MM cell lines and also in a patient sample. Interestingly, this complex was also dissociated in A1210477 resistant cells but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. Thus, BCLXL may act as a sink to bind freed pro-apoptotic proteins from MCL1 and limits MM cell death triggered by the specific targeting of MCL1.In conclusion, our study highlights the potential clinical use of BH3 mimetics in MM treatment guided by the practical ex-vivo testing of myeloma cell dependencies using the BH3 toolkit. This strategy could be used to identify the respective and tailored use of venetoclax, MCL1 BH3 mimetics or their combination in myeloma treatment. DisclosuresMoreau:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Statins Enhance the Efficacy of BH3 Mimetics in Multiple Myeloma

Multiple Myeloma (MM) is a disease of malignant plasma cells for which if left untreated, patients face a 6 month median survival (Osgood, 1960). New agents and combinations continue to improve MM outcomes, extending the median survival to 5 years; however, patients will ultimately succumb to the disease after exhausting treatment options (Fonseca et al., 2017). A novel class of drugs, BH3 mimetics, specifically inhibit the gatekeepers of apoptosis known as pro-survival BCL2 family members BCL2, MCL1 and BCL-xL (Figure 1A). BCL-2 selective BH3 mimetic, venetoclax (ABT-199), has shown promise in a subset of MM patients; however, combinations are required to improve the depth of response. (Moreau et al., 2017)Our lab is pioneering the novel combination of apoptosis-promoting BH3 mimetics with the cancer-killing effect of statins in blood cancers (Lee et al., 2018). Statins are FDA-approved for their ability to lower cholesterol by blocking the synthesis of a key precursor: mevalonate, a major biosynthetic substrate (Figure 1B). Inhibition of the mevalonate pathway by statins stops the synthesis of intermediates necessary for small GTPase function, providing an attractive avenue for targeting these “undruggable” oncogenes (Berndt, Hamilton, & Sebti, 2011; ten Klooster & Hordijk, 2007). Though this potential has been long recognized, previous work on repurposing statins reported effective concentrations that are well above the plasma levels typically achieved in patients receiving standard doses for hypercholesterolemia (Ahmed, Hayslip, & Leggas, 2013; Björkhem-Bergman, Lindh, & Bergman, 2011; van der Spek et al., 2006). Furthermore, previous studies provided insufficient rationale for mechanism-based combination strategies. We have achieved a breakthrough towards this goal by combining statins with the BCL2-specific BH3 mimetic, venetoclax. Our retrospective analysis of chronic lymphocytic leukemia (CLL) clinical trial data revealed that subjects taking statins at the FDA-approved dose had significantly higher frequency of complete remissions following venetoclax treatment (Lee et al., 2018). Through mechanistic studies we showed that statins increase expression of the pro-apoptotic, BH3-only sensitizer PUMA, thereby synergizing with venetoclax to induce apoptosis in various leukemia and lymphoma cell types.Our current work leverages these findings to treat MM. Retrospective analysis of clinical trials of venetoclax in combination with bortezomib and dexamethasone in the venetoclax-sensitive subset of MM demonstrates a similar trend as in the CLL clinical trials in support of background doses of statins (Roberts et al., 2017). Pre-clinical results of MCL-1 BH3 mimetics in development support their future use in MM (Kotschy et al., 2016). Based on these data, we tested two BH3 mimetics, venetoclax and a pre-clinical MCL-1 inhibitor, S63845. Using a panel of 9 human MM cell lines, the data demonstrate that statins potentiate BH3 mimetic-induced apoptosis as measured by reductions in BH3 mimetic IC50. Sensitivity to the combination was maintained in both MM1R and MM1S, MM lines from the same origin that model glucocorticoid resistance and sensitivity respectively, while venetoclax combination with dexamethasone was lost in MM1R cells (Greenstein et al., 2003; Matulis et al., 2016). Additionally, combination-sensitive cell lines corroborate a p53-independent induction of PUMA expression. These data suggest that BH3 mimetic and statin combination may be useful in the clinical settings of dexamethasone resistance and high risk p53-null (17p deleted) patients. To further guide the appropriate application of BH3 mimetics and statins, we are validating that dynamic BH3 profiling can predict efficacy of statins in combination-sensitive versus non-sensitive MM lines. Moreover, preliminary ex vivo studies on freshly isolated CD138+ MM cells from patient bone-marrow aspirates demonstrate combined killing with BH3 mimetics and statins. Ongoing studies will investigate the dependence on upstream GTPase signaling on survival and PUMA upregulation. [Display omitted] DisclosuresBrem:Bayer: Membership on an entity's Board of Directors or advisory committees; Genetech: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharamcyclics: Membership on an entity's Board of Directors or advisory committees.

Increased Synthesis of MCL-1 Protein Underlies Initial Survival of EGFR-Mutant Lung Cancer to EGFR Inhibitors and Provides a Novel Drug Target.

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

Molecular dynamics study of segment peptides of Bax, Bim, and Mcl-1 BH3 domain of the apoptosis-regulating proteins bound to the anti-apoptotic Mcl-1 protein

Mcl-1 has emerged as a potential therapeutic target in the treatment of several malignancies. Peptides representing BH3 region of pro-apoptotic proteins have been shown to bind the hydrophobic cleft of anti-apoptotic Mcl-1 and this segment is responsible for modulating the apoptotic pathways in living cells. Understanding the molecular basis of protein–peptide interaction is required to develop potent inhibitors specific for Mcl-1. Molecular dynamics simulations were performed for Mcl-1 in complex with three different BH3 peptides derived from Mcl-1, Bax, and Bim. Accordingly, the calculated binding free energies using MM-PBSA method are obtained and comparison with the experimentally determined binding free energies is made. The interactions involving two conserved charged residues (Aspi, and Arg/Lysi-4) and three upstream conserved hydrophobic residues (Leui-5, Ile/Vali-2, and Glyi-1, respectively) of BH3 peptides play the important roles in the structural stability of the complexes. The calculated results exhibit that the interactions of Bim BH3 peptides to Mcl-1 is stronger than the complex with Bax 19BH3 peptides. The hydrophobic residues (position i − 9, i − 8 and i + 2) of BH3 peptides can be involved in their inhibitory specificity. The calculated results can be used for designing more effective MCL-1 inhibitors.

A Competitive Stapled Peptide Screen Identifies a Selective Small Molecule that Overcomes MCL-1-Dependent Leukemia Cell Survival

Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochemically by an antiapoptotic surface groove that neutralizes the proapoptotic BH3 α helix of death proteins. Antiapoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Although targeting the BCL-2 antiapoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of molecules tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor molecule that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochemical lock-hold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence.

Abstract 4851: Selective small molecule inhibition of anti-apoptotic MCL-1

Abstract BCL-2 family proteins are key regulators of the mitochondrial apoptotic pathway in health and disease. Anti-apoptotic members such as BCL-2, BCL-XL, and MCL-1 have been implicated in the initiation, progression, and chemoresistance of human cancer. The anti-apoptotic BCL-2/BCL-XL groove that binds and sequesters pro-apoptotic BH3 death helices has been successfully targeted by small molecules and peptides. Such compounds induce tumor cell apoptosis and are being advanced in clinical trials as promising next-generation cancer therapeutics. Notably, selective antagonists such as ABT-737 are highly effective at inducing apoptosis in BCL-2/BCL-XL-dependent cancers, but are rendered inactive by overexpression of MCL-1, a formidable chemoresistance protein that lies outside the molecule's binding spectrum. By screening a library of Stabilized Alpha-Helix of BCL-2 domains (SAHBs), we previously discovered that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor (Stewart et al., Nat Chem Biol, 2010). Here, we deployed this natural peptidic inhibitor of MCL-1 in a competitive binding screen to identify an MCL-1 inhibitor molecule (MIM) that displays exquisite selectivity in in vitro binding and functional assays. NMR analysis documented that MIM engages the canonical BH3-binding pocket of MCL-1. Importantly, MIM selectively triggers caspase 3/7 activation and apoptosis in a cancer cell line that is dependent on induced overexpression of MCL-1, but showed no activity in the isogenic cell line that is driven instead by overexpressed BCL-XL. Conversely, ABT-737 manifested activity in the BCL-XL-overexpressing cancer cells but had no effect on the corresponding MCL-1-dependent cells. Thus, we report the application of MCL-1 SAHB to identify a selective small molecule inhibitor of MCL-1 that exhibits pro-apoptotic activity in the specific context of MCL-1 dependence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4851. doi:1538-7445.AM2012-4851

The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer

The development of selective inhibitors for discrete anti-apoptotic BCL-2 family proteins implicated in pathologic cell survival remains a formidable but pressing challenge. Precisely tailored compounds would serve as molecular probes and targeted therapies to study and treat human diseases driven by specific anti-apoptotic blockades. In particular, MCL-1 has emerged as a major resistance factor in human cancer. By screening a library of Stabilized Alpha-Helix of BCL-2 domains (SAHBs), we determined that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor. X-ray crystallography and mutagenesis studies defined key binding and specificity determinants, including the capacity to harness the hydrocarbon staple to optimize affinity while preserving selectivity. MCL-1 SAHB directly targets MCL-1, neutralizes its inhibitory interaction with pro-apoptotic BAK, and sensitizes cancer cells to caspase-dependent apoptosis. By leveraging nature’s solution to ligand selectivity, we generated an MCL-1-specific agent that defines the structural and functional features of targeted MCL-1 inhibition.