Bcl-xL Inhibitor Research Articles

Abstract P1-13-02: Autophagy inhibition and senolytics to improve the response to fulvestrant + palbociclib in ER positive breast cancer

Abstract While anti-estrogens or aromatase inhibitors in combination with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are the current standard of care for estrogen receptor-positive (ER+) Her-2 negative metastatic breast cancer, relapse after remission continues to be a serious limitation to the effectiveness of these therapies. Although these combination therapies prolong progression-free survival compared to endocrine therapy alone, the growth-arrested state of residual tumor cells is clearly transient. Tumor cells that escape what might be considered a dormant or quiescent state and regain proliferative capacity often acquire resistance to further therapies. Our studies are based upon the observation that breast tumor cells arrested by Fulvestrant + Palbociclib enter into a state of autophagy/senescence from which a subpopulation ultimately escapes, potentially contributing to disease recurrence. ER+ MCF-7 breast tumor cells that were induced into autophagy/senescence by Fulvestrant + Palbociclib in vitro were exposed to the autophagy inhibitors, chloroquine and bafilomycin, prior to as well as after growth arrest. However, autophagy inhibition only moderately enhanced the response to therapy and, at best, delayed proliferative recovery. These findings were confirmed by genetic inhibition of autophagy. Despite clear entry into a state of senescence, the Bcl-xl inhibitor and senolytic ABT-263 (navitoclax) failed to promote tumor cell death; this is likely a consequence of high levels of survivin in the MCF-7 breast tumor cell line. However, the BET inhibitor, ARV-825, eliminated a large fraction of the senescent cell population and significantly delayed proliferative recovery. Cell death did not appear to be a consequence of increased apoptosis. Additionally, analysis of three database sets demonstrated that higher breast cancer expression of BRD4, a primary target of ARV-825, correlated with lower recurrence-free survival compared to patients with cancers expressing lower levels of BRD4. These studies indicate that administration of BET inhibitors, which are currently being investigated in multiple clinical trials, may potentially improve standard of care therapy in metastatic ER+ breast cancer patients by prolonging progression-free survival. Citation Format: Ryan Michael Finnegan. Autophagy inhibition and senolytics to improve the response to fulvestrant + palbociclib in ER positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-13-02.

BCL-XL inhibition induces an FGFR4-mediated rescue response in colorectal cancer.

The heterogeneous therapy response observed in colorectal cancer is in part due to cancer stem cells (CSCs) that resist chemotherapeutic insults. The anti-apoptotic protein BCL-XL plays a critical role in protecting CSCs from cell death, where its inhibition with high doses of BH3 mimetics can induce apoptosis. Here, we screen a compound library for synergy with low-dose BCL-XL inhibitor A-1155463 to identify pathways that regulate sensitivity to BCL-XL inhibition and reveal that fibroblast growth factor receptor (FGFR)4 inhibition effectively sensitizes to A-1155463 both invitro and invivo. Mechanistically, we identify a rescue response that is activated upon BCL-XL inhibition and leads to rapid FGF2 secretion and subsequent FGFR4-mediated post-translational stabilization of MCL-1. FGFR4 inhibition prevents MCL-1 upregulation and thereby sensitizes CSCs to BCL-XL inhibition. Altogether, our findings suggest a cell transferable induction of a FGF2/FGFR4 rescue response in CRC that is induced upon BCL-XL inhibition and leads to MCL-1 upregulation.

Targeting entry into mitochondria for increased anticancer efficacy of BCL-XL-selective inhibitors in lung cancer

The BCL-XL-selective inhibitors exhibit potential clinical application value when combined with chemotherapeutic drugs for the treatment of solid tumors. However, their efficacy in these settings is still low when treated with BCL-XL inhibitors alone in solid tumors. The mechanism responsible for the poor efficacy remains unclear. We show here that unable to interact with target of BCL-XL-selective inhibitors caused by invalid entry into mitochondria is essential for their inefficacy in solid tumors. We demonstrated in non-small-cell lung cancer (NSCLC) cells that the instability of A-1155463 in cells as well as invalid entry into mitochondria of A-1331852, two BCL-XL-selective inhibitors, accounted for their off-target problems. Furthermore, we found that a mitochondria-targeted, non-toxic small molecule NA-2a improved the on-target effect of A-1331852 to enhance its apoptotic regulatory activity, thereby increasing its anticancer activity in NSCLC. Our results indicated that NA-2a was selectively enriched in mitochondria transported by organic-anion-transporting polypeptide (OATP) transporters, which altered the permeability of the mitochondrial membrane, thereby promoting the entrance of A-1331852 to mitochondria and enhancing its disruption of the BIM-BCL-XL complex, which finally led to the increased anticancer activity in vitro and in vivo. Collectively, our data provided overwhelming evidence that the combination of NA-2a and A-1331852 could be used as a promising synergistic therapeutic agent in NSCLC therapy.

Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma

Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors.

Pediatric multicellular tumor spheroid models illustrate a therapeutic potential by combining BH3 mimetics with Natural Killer (NK) cell-based immunotherapy

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

3093 – SUBTYPE-SPECIFIC INDUCTION OF MITOCHONDRIAL APOPTOSIS AS A NOVEL THERAPEUTIC OPTION IN HIGH-FATALITY PEDIATRIC LEUKEMIA

A rare subtype of acute myeloid leukemia (AML) is acute megakaryoblastic leukemia (AMKL). Recurrent and mutually exclusive oncogenic fusions are detected in AMKL and are considered the transforming event in this disease. AMKL usually affects children below 3 years of age and is associated with less than 40% cure rates. In addition, the development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material. Our research group has established synthetic human models of high-fatality pediatric AMKL, driven by distinct oncogenic fusions, which phenocopy the disease in a patho-physiological context in mice. Inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, recently entered the therapeutic arena in pediatric leukemia with promising results. Therefore, we set out to investigate the potential of Venetoclax (inhibitor of BCL-2) and Navitoclax (inhibitor of BCL-2, BCL-XL and BCL-W) as novel therapeutic options as a bridge to curative bone marrow transplantation in this high-fatality infant leukemia. Our research demonstrates that genetic and pharmacological inhibition of the pro-survival BCL-2 family member BCL-XL results in significant induction of apoptosis in our models of AMKL. In contrast, inhibition of BCL-2 does not induce apoptosis in our models, which is currently the most investigated BCL-family member in the clinic for other subtypes of AML (Venetoclax). In addition, Navitoclax showed promising in vivo activity with a significant decrease in infiltrating leukemic cells in bone-marrow and spleen after drug treatment in comparison to vehicle-only controls. In summary, this project will bring mechanistic insight into the role of apoptotic pathways in AMKL and greatly accelerate the identification of novel therapeutic options in high-fatality infant AML. A rare subtype of acute myeloid leukemia (AML) is acute megakaryoblastic leukemia (AMKL). Recurrent and mutually exclusive oncogenic fusions are detected in AMKL and are considered the transforming event in this disease. AMKL usually affects children below 3 years of age and is associated with less than 40% cure rates. In addition, the development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material. Our research group has established synthetic human models of high-fatality pediatric AMKL, driven by distinct oncogenic fusions, which phenocopy the disease in a patho-physiological context in mice. Inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, recently entered the therapeutic arena in pediatric leukemia with promising results. Therefore, we set out to investigate the potential of Venetoclax (inhibitor of BCL-2) and Navitoclax (inhibitor of BCL-2, BCL-XL and BCL-W) as novel therapeutic options as a bridge to curative bone marrow transplantation in this high-fatality infant leukemia. Our research demonstrates that genetic and pharmacological inhibition of the pro-survival BCL-2 family member BCL-XL results in significant induction of apoptosis in our models of AMKL. In contrast, inhibition of BCL-2 does not induce apoptosis in our models, which is currently the most investigated BCL-family member in the clinic for other subtypes of AML (Venetoclax). In addition, Navitoclax showed promising in vivo activity with a significant decrease in infiltrating leukemic cells in bone-marrow and spleen after drug treatment in comparison to vehicle-only controls. In summary, this project will bring mechanistic insight into the role of apoptotic pathways in AMKL and greatly accelerate the identification of novel therapeutic options in high-fatality infant AML.

MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma

Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein−protein interactions are critical in MCL1’s nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53−/− CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53−/− CRC via exploitation of this unique MCL1-based chemoresistance mechanism.

MDM2-BCL-XL PROTACs enable degradation of BCL-XL and stabilization of p53.

Inhibition or degradation of anti-apoptotic protein BCL-XL is a viable strategy for cancer treatment. Despite the recent development of PROTACs for degradation of BCL-XL, the E3 ligases are confined to the commonly used VHL and CRBN. Herein we report the development of MDM2-BCL-XL PROTACs using MDM2 as E3 ligase for degradation of BCL-XL. Three MDM2-BCL-XL PROTACs derived from MDM2 inhibitor Nutlin-3, which can also upregulate p53, and BCL-2/BCL-XL inhibitor ABT-263 with different linker length were designed, synthesized, and evaluated in vitro. We found BMM4 exhibited potent, selective degradation activity against BCL-XL and stabilized tumor suppressor p53 in U87, A549 and MV-4-11 cancer cell lines. Moreover, combination of BMM4 and BCL-2 inhibitor ABT-199 showed synergistic antiproliferative activity. The unique dual-functional PROTACs offers an alternative strategy for targeted protein degradation.

Computational design of an apoptogenic protein that binds BCL-xL and MCL-1 simultaneously and potently

One of the hallmarks of cancer cells is their ability to evade apoptosis, which confers survival advantages and resistance to anti-cancer drugs. Cancers often exhibit overexpression of anti-apoptotic BCL-2 proteins, the loss of which triggers apoptosis. In particular, the inhibition of both BCL-xL and MCL-1, but neither one individually, synergistically enhances apoptotic cell death. Here, we report computational design to produce a protein that inhibits both BCL-xL and MCL-1 simultaneously. To a reported artificial three-helix bundle whose second helix was designed to bind MCL-1, we added a fourth helix and designed it to bind BCL-xL. After structural validation of the design and further structure-based sequence design, we produced a dual-binding protein that interacts with both BCL-xL and MCL-1 with apparent dissociation constants of 820 pM and 196 pM, respectively. Expression of this dual binder in a subset of cancer cells induced apoptotic cell death at levels significantly higher than those induced by the pro-apoptotic BIM protein. With a genetic fusion of a mitochondria-targeting sequence or the BH3 sequence of BIM, the activity of the dual binder was enhanced even further. These data suggest that targeted delivery of this dual binder alone or as a part of a modular protein to cancers in the form of protein, mRNA, or DNA may be an effective way to induce cancer cell apoptosis.

Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors.

Simple SummaryApoptosis is one of the best-known types of programmed cell death. This process is regulated by a number of genes and proteins, among which the Bcl-2 protein family plays a key role. This family includes anti- and proapoptotic proteins. Cancer cell resistance to apoptosis is commonly associated with overexpression of the antiapoptotic members of Bcl-2 family proteins, in particular, Bcl-2, Bcl-xL, and Mcl-1. Subsequently, these proteins represent perspective targets for anticancer therapy. Here, using an inhibitory approach, we found that Bak and Bcl-xL regulate sensitivity of cancer cells to Mcl-1 inhibition.BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.

A computational perspective for tailor-made selective Mcl-1 and Bcl-XL inhibitors

In view of the crucial role of Mcl-1 in tumor survival and resistance as well as the importance of Bcl-XL in platelets homeostasis, an appropriate tuning of binding selectivity between Mcl-1 and Bcl-XL is crucial to discover safer anticancer candidates. In this respect, comprehensive computational approaches such as protein comparison, molecular docking, molecular dynamics simulations, mutagenesis, and density functional theory (DFT) calculation were applied to elucidate the different binding modes between highly selective Mcl-1 and Bcl-XL inhibitors. It was found that although Mcl-1 and Bcl-XL shared high sequence homology in their active pockets, the P2 pocket of Mcl-1 merged with P1 and P3, whereas the P3 pocket bridged through the P2 and P4 pocket of Bcl-XL. Accordingly, the key residues that determined the selectivity between Mcl-1 and Bcl-XL included Asn260, Arg263, and Phe270 in Mcl-1, and Glu96, Ser106, Leu108, Asn136, and Arg139 in Bcl-XL. In particular, Phe270 served as a major contributor to the π-π stacking interactions between Mcl-1 and its selective inhibitor, whereas Tyr195 was the key residue establishing the π−π stacking between Bcl-XL and its selective inhibitor. Collectively, these data shed promising light in depicting the selective mechanisms between Mcl-1 and Bcl-XL, which would lay a solid foundation for the future designing of the selective inhibitors in the discovery and optimization of a compound on the path toward developing future anticancer drugs.

Artificial intelligence-based identification of octenidine as a Bcl-xL inhibitor

Apoptosis plays an essential role in maintaining cellular homeostasis and preventing cancer progression. Bcl-xL, an anti-apoptotic protein, is an important modulator of the mitochondrial apoptosis pathway and is a promising target for anticancer therapy. In this study, we identified octenidine as a novel Bcl-xL inhibitor through structural feature-based deep learning and molecular docking from a library of approved drugs. The NMR experiments demonstrated that octenidine binds to the Bcl-2 homology 3 (BH3) domain-binding hydrophobic region that consists of the BH1, BH2, and BH3 domains in Bcl-xL. A structural model of the Bcl-xL/octenidine complex revealed that octenidine binds to Bcl-xL in a similar manner to that of the well-known Bcl-2 family protein antagonist ABT-737. Using the NanoBiT protein–protein interaction system, we confirmed that the interaction between Bcl-xL and Bak-BH3 domains within cells was inhibited by octenidine. Furthermore, octenidine inhibited the proliferation of MCF-7 breast and H1299 lung cancer cells by promoting apoptosis. Taken together, our results shed light on a novel mechanism in which octenidine directly targets anti-apoptotic Bcl-xL to trigger mitochondrial apoptosis in cancer cells.

Mcl-1 and Bcl-xL levels predict responsiveness to dual MEK/Bcl-2 inhibition in B-cell malignancies.

Most patients with chronic lymphocytic leukemia (CLL) initially respond to targeted therapies, but eventually relapse and develop resistance. Novel treatment strategies are therefore needed to improve patient outcomes. Here, we performed direct drug testing on primary CLL cells and identified synergy between eight different mitogen‐activated protein kinase kinase (MEK) inhibitors and the B‐cell lymphoma 2 (Bcl‐2) antagonist venetoclax. Drug sensitivity was independent of immunoglobulin heavy‐chain gene variable region (IGVH) and tumor protein p53 (TP53) mutational status, and CLL cells from idelalisib‐resistant patients remained sensitive to the treatment. This suggests that combined MEK/Bcl‐2 inhibition may be an option for high‐risk CLL. To test whether sensitivity could be detected in other B‐cell malignancies, we performed drug testing on cell line models of CLL (n = 4), multiple myeloma (MM; n = 8), and mantle cell lymphoma (MCL; n = 7). Like CLL, MM cells were sensitive to the MEK inhibitor trametinib, and synergy was observed with venetoclax. In contrast, MCL cells were unresponsive to MEK inhibition. To investigate the underlying mechanisms of the disease‐specific drug sensitivities, we performed flow cytometry‐based high‐throughput profiling of 31 signaling proteins and regulators of apoptosis in the 19 cell lines. We found that high expression of the antiapoptotic proteins myeloid cell leukemia‐1 (Mcl‐1) or B‐cell lymphoma‐extra large (Bcl‐xL) predicted low sensitivity to trametinib + venetoclax. The low sensitivity could be overcome by combined treatment with an Mcl‐1 or Bcl‐xL inhibitor. Our findings suggest that MEK/Bcl‐2 inhibition has therapeutic potential in leukemia and myeloma, and demonstrate that protein expression levels can serve as predictive biomarkers for treatment sensitivities.

Tenofovir disoproxil fumarate directly ameliorates liver fibrosis by inducing hepatic stellate cell apoptosis via downregulation of PI3K/Akt/mTOR signaling pathway.

BackgroundAntifibrotic agent for the treatment of liver fibrosis has not been developed so far. Long term treatment of chronic hepatitis B patients with antiviral drugs tenofovir disoproxil fumarate (TDF) and entecavir (ETV) results in the regression of liver fibrosis, but the underlying mechanism has not been clarified. Therefore, we aimed to investigate the direct impact of TDF and ETV on liver fibrosis.MethodsActivated hepatic stellate cell (HSC) cell lines were used to evaluate the effects of TDF and ETV. After treatment with each antiviral agent, cell viability, morphology, apoptotic features, autophagy and antifibrosis signalling pathways were examined. Then, collagen deposition, fibrosis markers and activated HSCs were measured in liver tissues of the liver fibrosis model mice.ResultsAfter TDF treatment, the viabilities of LX2 and HSC-T6 cells were decreased, and the cells exhibited apoptotic features, but ETV did not induce these effects. Cleavage of PARP and Caspase-3 and the inhibition of the antiapoptotic gene Bcl-xl indicated activated HSC apoptosis following TDF treatment. TDF simultaneously increased autophagy, which also regulated apoptosis through crosstalk. TDF inactivated the PI3K/Akt/mTOR signalling pathway, which was associated with the activation of both apoptosis and autophagy. In the liver fibrosis mouse model, the fibrotic area and activated HSC markers were decreased by TDF but not ETV treatment. Additionally, apoptotic cells were concentrated in the periportal fibrotic area after TDF treatment, which indicated the specific antifibrotic effect of TDF.ConclusionsTDF directly ameliorates liver fibrosis by downregulating the PI3K/Akt/mTOR signalling pathway, which results in the apoptosis of activated HSCs. The antifibrotic effects of TDF indicate that it may be a therapeutic agent for the treatment of liver fibrosis.

Functional apoptosis profiling identifies MCL-1 and BCL-xL as prognostic markers and therapeutic targets in advanced thymomas and thymic carcinomas

BackgroundMulti-omics studies have shown a high and lack of common driver mutations in most thymomas (TH) and thymic carcinomas (TC) that hamper the development of novel treatment approaches. However, deregulation of apoptosis has been proposed as a common hallmark of TH and TC. BH3 profiling can be utilized to study the readiness of living cancer cells to undergo apoptosis and their dependency on pro-survival BCL-2 family proteins.MethodsWe screened a cohort of 62 TH and TC patient samples for expression of BCL-2 family proteins and used the TC cell line 1889c and native TH for dynamic BH3 profiling and treatment with BH3 mimetics.ResultsImmunohistochemical overexpression of MCL-1 and BCL-xL was a strong prognostic marker of TH and TC, and BH3 profiling indicated a strong dependency on MCL-1 and BCL-xL in TH. Single inhibition of MCL-1 resulted in increased binding of BIM to BCL-xL as an escape mechanism that the combined inhibition of both factors could overcome. Indeed, the inhibition of MCL-1 and BCL-xL in combination induced apoptosis in a caspase-dependent manner in untreated and MCL-1-resistant 1889c cells.ConclusionTH and TC are exquisitely dependent on the pro-survival factors MCL-1 and BCL-xL, making them ideal candidates for co-inhibition by BH3 mimetics. Since TH show a heterogeneous dependency on BCL-2 family proteins, upfront BH3 profiling could select patients and tailor the optimal therapy with the least possible toxicity.

CBIO-27. INTEGRATED FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF THE INTRINSIC APOPTOTIC MACHINERY IDENTIFIES THERAPEUTIC VULNERABILITIES IN GLIOBLASTOMA

Abstract Molecular profiling frequently fails to predict therapeutic outcomes in cancer. This is in part due to the myriad of genetic alterations comprising a tumor which enable rapid rewiring of cancer signaling pathways. Functional profiling ascertains signaling dynamics and has emerged as an alternative method to predict drug responses. It is unknown whether incorporating molecular with functional profiling offers superior insight into specific tumor dependencies and consequently therapeutic susceptibilities. Here we performed integrated molecular and functional characterization (via BH3 profiling) of the intrinsic apoptotic machinery in 50 GBM patient specimens. We found that treatment with standard of care (e.g., temozolomide or radiation) rewired the apoptotic machinery in a p53 dependent manner, eliciting an exclusive survival dependency on BCL-xL in a genetically defined subset of GBM tumors. However, functional BH3 profiling of ‘apoptotic priming’ was required to predict response to combined IR/TMZ and BCL-xL inhibition in p53 WT GBM tumors. Accordingly, a composite of genetic and functional profiling predicted the response of patient derived GBM models to IR/TMZ with a novel GBM specific BCLxL antagonist. Collectively, these studies identify the mechanisms of intrinsic apoptosis resistance in GBM and demonstrate how functional and molecular data can be complementary to robustly predict therapy-induced cell death.

Venetoclax and Epigenetic Modifiers: Promising Novel Combinations for the Treatment of Multiple Myeloma

Multiple Myeloma (MM) is a haematological malignancy characterised by clonal proliferation of plasma cells within the bone marrow (Landgren and Weiss, 2009). Unfortunately, despite the major improvements to the treatment of MM within the last decade, it remains an incurable disease. Therefore, novel innovative combinations of less toxic therapies are warranted, especially for elderly patients with relapsed/refractory disease.The anti-apoptotic BCL-2 family of proteins (BCL-2, BCL-XL and MCL-1) are critical regulators of the intrinsic apoptotic pathway and determine the survival of human MM cells (Letai et al., 2004, Del Gaizo Moore et al., 2008). Recently, Venetoclax, a selective BCL-2 inhibitor, was FDA approved for the treatment of chronic lymphocytic leukaemia (CLL) and acute mylogenous leukemia (AML), in combination with demethylating agents (Roberts et al., 2016, DiNardo et al., 2018, DiNardo et al., 2019) It is known that some MM patients with t(11;14) have a good response to combination treatment with venetoclax, however certain patients who do not have t(11;14) also respond to venetoclax. Therefore, a biomarker for response is urgently required in MM, as it has heterogeneous anti-apoptotic dependencies. In AML, venetoclax is combined with the epigenetic modifier 5-azacytidine. Highlighting, that screening for epigenetic modifier's, maybe a useful approach to identify synergistic combination of treatments with venetoclax in MM.Methods:To assess anti-apoptotic protein dependence in MM cell lines (JJN3, RPMI-8226, KMS18, MM1S, U266) and primary patient samples, BH3 profiling was used. Briefly, cells are exposed to a series of BH3-only peptides (20-23 mer in length) following gentle permeabilisation of cell membrane with low concentrations of digitonin. The loss of mitochondrial potential (JC-1) or the release of cytochrome c (cytochrome-c-FITC antibody) was assessed by plate reader or by flow cytometry. Cell death was assessed by Annexin V/ propidium iodide staining by flow cytometry. Using primary patient samples, CD138 + cells were isolated using the Miltenyi MAC sorter. For the epigenetic screen cell viability was assessed by CellTiter-Glo® and death was then confirmed by Annexin V/Pi staining.ResultsBH3 profiling was used to assess anti-apoptotic dependence in a panel of five MM cell lines. It is a functional assay that interrogates BCL-2 protein interactions using synthetic BH3 peptides to measure the loss of mitochondrial membrane potential. The BH3 profiling was correlated to the response of the cell lines to a series of BH3 mimetics : venetoclax (selective BCL-2 inhibitor), ABT-263 (BCL-2, BCL-XL and BCL-W inhibitor), WEHI-539 (BCL-XL inhibitor) and AMG-176 (MCL-1 inhibitor). This data highlighted that BH3 profiling is a powerful tool for identifying anti-apoptotic dependnece in MM. It also showed and that there is a diverse anti-apoptotic dependence in MM cell lines and primary patient samples. Remarkably, one patient with plasma cell leukemia showed BCL-2 dependence by BH3 profiling, a t(11:14) translocation and a sustained in-vivo single agent response to venetoclax (Glavey et al., 2020)..Next, we performed an unbiased epigenetic modifier screen in two MM cell lines JJN3 (MCL-1 dependent cell lines) and KMS-18 (mixed anti-apoptotic dependence) to induce BCL-2 dependence and sensitivity to venetoclax. The screen included the following classes of epigenetic modifiers: histone deacetylase inhibitors, histone methyltransferase inhibitors, DNA methyltransferase inhibitors and histone acetylase inhibitors. Interestingly, two classes of epigenetic drugs were synergistic with venetoclax in three different MM cell lines (CI <0.8). The combination was also validated by dynamic BH3 profiling in cell lines MM patient samples ex-vivo. Further work is required, in the form of a clinical trial, to validate our hypothesis that this provides a highly effective novel combination therapy option for myeloma patients.Conclusion.BH3 profiling is a powerful tool to identify the anti-apoptotic dependence in MM cell lines and MM patient samples, which we can exploit pharmacologically to kill MM cells in a personalised medicine approach. Combining epigenetic modifiers with venetoclax induces BCL-2 dependence in MM and enhances response to treatment. [Display omitted] DisclosuresFlanagan: AbbVie: Research Funding. O'Dwyer: Bristol Myers Squibb: Research Funding; ONK Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Quinn: Takeda: Honoraria. Glavey: Janssen: Honoraria, Research Funding; Celgene and BMS company: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding. Ni Chonghaile: AbbVie: Research Funding.

Dual BCL-2/BCL-XL Inhibitor Pelcitoclax (APG-1252) Overcomes Intrinsic and Acquired Resistance to Venetoclax in Multiple Myeloma Cells

Multiple myeloma (MM) is marked by several genetic abnormalities, including chromosome translocation t(11;14). Overexpression of anti-apoptotic BCL-2 in t(11;14) MM promotes disease progression, prompting clinical use of the BH3 mimetic and BCL-2 inhibitor venetoclax in combination with proteasome inhibitor therapy. Despite high initial response rates and prolonged progression-free survival, patients commonly relapse.To delineate mechanisms contributing to acquired drug resistance we modeled responses to venetoclax in two highly sensitive MM cell lines (KMS27 and KMS-12PE). Colonies generated from a surviving cell were cultured in high-dose venetoclax to generate monoclonal drug-tolerant expanded persister (DTEP) clones. To determine whether venetoclax resistance in DTEP clones is mediated by transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprogramming, we conducted whole-genome sequencing (WGS) and RNA-seq of the clones. WGS analysis did not show significant differences between parental and resistant clones, but transcriptomic analysis showed shared and unique transcriptome signatures in DTEP clones. Gene set enrichment analysis of the common significantly modulated genes in resistant clones revealed that PKA-ERK-CREB and K-Ras pathway genes were significantly upregulated, whereas apoptotic genes were downregulated in resistant clones compared to parental cells. Importantly, ectopically expressed ERK in venetoclax-sensitive cells conferred a resistant phenotype that was rescued using two specific ERK inhibitors in DTEP clones. These data confirm a key role for ERK activation in acquired venetoclax resistance.Resistant clones were further characterized by reduced mitochondrial priming assessed by dynamic BH3 profiling, with altered expression of anti-apoptotic regulators including MCL-1, BCL-xL, and BCL-W and the replaced BCL-2: BIM complex by both MCL-1 and BCL-xL. Because these data suggested a functional substitution between anti-apoptotic BCL-2 family members in cells with acquired resistance to venetoclax, we next evaluated if MCL-1 or BCL-xL are codependent in MM cells that are insensitive or resistant to venetoclax. Simultaneous inhibition of MCL-1 (via S63845) or BCL-xL (via A155463) and BCL-2 (via venetoclax) increased BIM release and enhanced cell death in resistant clones (vs single agents), with combination index values < 0.3 in all doses. Upregulation of BCL-xL or MCL-1 in MM cells also mediated primary venetoclax resistance independent of genetic hallmarks (e.g. t [11;14]-translocated cells). Thus, simultaneous inhibition of MCL-1 or BCL-xL and BCL-2 triggered synergistic cytotoxicity in MM cell lines intrinsically resistant to venetoclax.These data suggest that combined inhibition of BCL-2 and BCL-xL may overcome venetoclax resistance. However, the dependence of BCL-xL in mature platelets had triggered thrombocytopenia for patients under therapy using BCL-xL inhibitor. To further explore the potential clinical application of targeting BCL-xL, we employed novel BCl-2/BCL-xL dual inhibitor, BH3 mimetic pelcitoclax (APG-1252). Using pro-drug strategy for design, pelcitoclax has limited cell permeability during circulation, and was converted to a more potent metabolite APG-1252-M1 in tumors/tissues. APG-1252-M1 was thus used for in vitro cell based assays. We discovered that APG-1252-M1 induced cytotoxicity in MM cell lines intrinsically resistant to venetoclax (regardless of genetic background or BCL-2:BCL-xL ratio) and also significantly reduced MM cell viability in clones with acquired venetoclax resistance, overcoming ERK activation and decreasing BIM sequestration by BCL-xL. In vivo study using pelcitoclax is ongoing and will be presented at the meeting.In conclusion, we report that venetoclax resistance in MM evolves from outgrowth of persister clones displaying activation of the ERK pathway and a shift in mitochondrial dependency towards BCL-xL, which can potentially be effectively targeted via the novel BCL-2/BCL-xL inhibitor pelcitoclax (APG-1252), which is currently in clinical investigation for solid tumors (NCT03080311). DisclosuresDeng: Ascentage Pharma Group: Current Employment. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Novartis: Consultancy; Janssen: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Karyopharm: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Amgen: Consultancy; Abbvie: Consultancy; Legend: Consultancy; Pfizer: Consultancy.

Identifying Mechanisms Associated with Venetoclax Resistance in Multiple Myeloma (MM)

Venetoclax (VTX) is a selective small-molecule inhibitor of BCL-2 that exhibits antitumoral activity against MM cells presenting lymphoid features and those with translocation t(11;14). Despite its impressive clinical activity, VTX therapy for a prolonged duration can lead to drug resistance. Therefore, it is important to understand the underlying mechanisms of resistance in order to develop strategies to prevent or overcome resistance. In the present study, we established four VTX resistant human myeloma cell lines (HMCLs) from four sensitive HMCLs, including three with t(11;14), in culture with a stepwise increase in treatment dose with VTX. To identify the molecular basis of acquired VTX resistance, whole exon sequencing (WES), mRNA-sequencing (mRNAseq), and protein expression assays were performed in the four isogenic VTX-sensitive/resistant HMCLs and three MM patients with samples collected before VTX administration and after clinical resistance to the drug. Compared with sensitive cell lines and patient samples collected before VTX administration, mRNAseq analysis identified downregulation of BIM and upregulation of BCLXL in both resistant cell lines and MM cells from relapse patients. Other transcriptional changes detected included upregulation of AURKA, BIRC3, BIRC5, and IL32. Enrichment analysis of differentially expressed genes suggested involvement of PI3K and MAPK signaling, likely associated with cytokines, growth factors (EGF, FGF and IGF family members), and receptor tyrosine kinase (EGF and FGF). Western blot analysis was performed to compare BCL2 family expression in resistant cell lines versus sensitive cell lines and it showed upregulation of BCL2 survival members (such as MCL-1 and BCLXL), and downregulation of pro-apoptotic BH3 members (such as BIM and PUMA). BIM expression was completely lost in one resistant cell line, and introduction of exogenous BIM into this cell line enhanced VTX sensitivity. Interestingly, BCL2 was upregulated in some resistant cell lines generated after a long-term treatment with VTX, suggesting BCL2 expression level may not be suitable as a marker of VTX sensitivity for acquired resistance. Unlike in CLL, BCL2 mutations were not identified through WES in any resistant cell lines or primary patient sample harvested after relapse. While 8 genes were mutated in two resistant samples , no clear mutational pattern emerged .Based on the above, we further tested some specific inhibitors in in vitro or ex vivo cell models to help understanding resistant mechanism and identify strategies to overcome VTX resistance. We found that inhibition of MCL-1, with the compound S68345, substantially enhanced VTX sensitivity in three resistant HMCLs and in primary cells from one relapsed MM patient. A BCLXL inhibitor (A155463) only significantly enhanced VTX sensitivity in one resistant cell line after co-treatment with VTX. Co-treatment of the other three resistant cell lines with VTX, S68345 and A155463 resulted in the most synergistic anti-myeloma activity, suggesting those cell lines are co-dependent on MCL-1, BCLXL, and BCL2 for survival, although they are more dependent on MCL-1. We also found that inhibition of PI3K signaling, IGF1, RTK (EGF and FGF) and AURKA significantly increased VTX sensitivity, partially through downregulation of MCL-1, and BCLXL, and upregulation of BIM. Conventional anti-MM drugs such as dexamethasone, bortezomib and lenalidomide, were shown to have little activity on augmenting VTX sensitivity in most resistant cell lines.In summary, we find that acquired resistance to VTX in MM is largely associated with BCL2 family regulation, including upregulation of survival members such as MCL-1, BCLXL, BCL2, and downregulation of pro-apoptotic members, especially BIM. Our study also indicates that upstream signaling involved in BCL2 family regulation during acquired resistance is likely related to cytokine, growth factor, and/or RTK-induced cell signaling such as PI3K. Co-inhibition of MCL-1, or BCLXL, as well as the upstream PI3K, RTK (FGF and EGF), IGF-1 mediated signaling were effective in overcoming VTX resistance. DisclosuresFonseca: Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Juno: Consultancy; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; AbbVie: Consultancy; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees.

Homoharringtonine and Mebendazole Are Preferentially Lethal Against Models of Myeloid Malignancy Associated with Germline Mutant RUNX1

RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Majority of mono-allelic germline mutations in RUNX1 are missense, large deletions or truncation mutations, behaving mostly as loss of function (LOF) mutations. They are ~40%-penetrant and cause Familial Platelet Disorder (RUNX1-FPD) that has a propensity to evolve into myeloid malignancy (FPD-MM), i.e., MDS or AML. FPD-MM harbors co-mutations, most commonly on the second allele of RUNX1, and on BCOR, PHF6, K-RAS, WT1 or TET2, which confer relative resistance to standard therapy for MDS or AML. Although curative in some patients with FPD-MM, allogeneic transplantation from matched, un-related donors carries risk of graft versus host disease and frequent AML relapse. This creates a strong rationale and an unmet need to develop novel targeted therapies for FPD-MM. We previously reported on utilizing the RNA-Seq signature of RUNX1 knockdown, which exerted more lethality in AML cells with mutant (mt) RUNX1 compared to AML harboring two copies of wild-type RUNX1, for conducting LINCS1000-CMap analysis. This identified several expression mimickers (EMs), including the protein synthesis inhibitor homoharringtonine (HHT or omacetaxine) and anthelmintic fenbendazole (analog of mebendazole). Present studies demonstrate that treatment with HHT or mebendazole (MB) dose-dependently induced significantly greater loss of viability in four patient-derived (PD) bone marrow aspirate (BMA) samples of FPD-MM (3 AML and 1 MDS) compared to RUNX1-FPD (3 samples) or in normal CD34+ progenitor cells. In a patient with RUNX1-FPD (expressing mtRUNX1 K194N), who developed FPD-MM, following co-mutations were documented by NGS: BCOR A1437fs, PHF6 L324fs, SF3B1 D781G and SRSF2 P95R/L. From BMA of this patient, we successfully established the first ever, continuously cultured cell line (GMR-AML1) expressing the same germline mtRUNX1. GMR-AML1 cells were cytogenetically diploid and lacked MYC or MLL1 rearrangement, or any copy number gains or losses on array CGH. However whole exome sequencing (WES) identified additional mutations in TP53 (P72R), AIM2 (K340fs), NELFB (L523F), CEP152 (Y370X), SUGP2 (H23L), RRM2B (R71fs), TADA3 (T27R), SPDYE6 (G292C) and PRDM9 (S814R) with % VAF ranging between 33 to 55%. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL3R), CD86 and CD33, but without expression of CD34, CD14, CD11b, MPO or CD135 (FLT3). Compared to the AML OCI-AML5 cells with somatic mtRUNX1, GMR-AML1 cells demonstrated markedly reduced protein expression of RUNX1, RUNX2, PU.1, c-Myb, GFI1, GFI1B, FLT3, MEIS1 and CEBPα (p42), but much higher protein expression of RUNX3 and NOTCH (p120). CRISPR-Cas9 knockout of RUNX3 in GMR-AML1 cells restored RUNX1 expression, while significantly increasing % of differentiated cells. Although dose-dependently sensitive to daunorubicin, etoposide, cytarabine and panobinostat (class I and II HDAC inhibitor), GMR-AML1 cells were relatively insensitive to venetoclax, A1155463 (Bcl-xL inhibitor), AZD-5991 (MCL1 inhibitor), azacytidine or decitabine. Notably, treatment with HHT or MB dose-dependently induced loss of viability of GMR-AML1 cells (LD50: 40 and 330 nM, respectively). Additionally, co-treatment with HHT and venetoclax synergistically induced apoptosis in GMR-AML1 cells, as determined by the SynergyFinder algorithm. This synergy in GMR-AML1 cells was associated with abrogation of venetoclax-induced increase in MCL1 and Bcl-xL levels, as well as greater decline in levels of RUNX3, PU.1, c-Myb, c-Myc, MPL and CDK4/6. Tail vein infusion and engraftment of luciferase-transduced GMR-AML1 (10 6 cells) caused marked splenomegaly and 100% mortality of NSG mice by day-18, post-infusion. We will present at the ASH meeting findings of ongoing in vivo studies determining effects of treatment with HHT and/or venetoclax, versus vehicle control, on AML burden and overall survival of NSG mice engrafted with GMR-AML1 cells. Overall, preclinical findings presented here highlight the molecular and genetic features associated with progression of RUNX1-FPD to FPD-MM, especially in the newly established GMR-AML1 cell line. They also demonstrate that HHT or MB are preferentially more lethal against FPD-MM versus RUNX1-FPD cells and exert synergistic lethality with venetoclax against GMR-AML1 cells. DisclosuresDiNardo: GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; ImmuneOnc: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Takahashi: GSK: Consultancy; Celgene/BMS: Consultancy; Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Khoury: Stemline Therapeutics: Research Funding; Angle: Research Funding; Kiromic: Research Funding.