BCL-XL Dependence Research Articles

Abstract 675: Targeting Bcl-2/Bcl-xl for small cell lung cancer treatment with LP-118

Abstract Small cell lung cancer (SCLC) is an aggressive form neuroendocrine carcinoma with limited treatment options and poor prognosis, and novel approaches are urgently needed for the treatment of this disease. Bcl-2 and Bcl-xl are aberrantly expressed in 60-70% SCLC and have been considered as promising therapeutic targets for SCLC. However, navitoclax (ABT-263), a Bcl-2/Bcl-xl dual inhibitor, demonstrated limited efficacy in SCLC while induced sever adverse events, such as thrombocytopenia due to the platelet toxicity mediated by Bcl-xl inhibition. Therefore, agents with novel mechanisms of action and safety profile are to be developed for the treatment of SCLC with Bcl-2/Bcl-xl dependency. LP-118 is a new generation and super potent Bcl-2 inhibitor with moderate Bcl-xl activity, and it is being evaluated in Phase1 clinical trials in solid tumors and hematological cancers (NCT05025358, NCT04771572). In this preclinical study, we aimed to evaluate the single agent activity of LP-118 in a panel of 9 SCLC cell lines and to further determine if LP-118 can enhance the efficacies of Standard of Cares (SOC) in vitro and in vivo. The results show that LP-118 has high in vitro activity in a subset of SCLC cell lines, and it is particularly potent in Bcl-2-depended cell lines, such as NCI-H889, with the IC50 of 0.15 nM. LP-118 is also very potent in NCI-H1963, a Bcl-xl dependent cell line (IC50: 5nM), and NCI-H526, a Bcl-2/Bcl-xl co-dependent cell line (IC50: 4-5.1 nM). Overall, LP-118 is more potent than ABT-263 in Bcl-2 or Bcl-2/Bcl-xl co-dependent cell lines, and it is relatively less active in Bcl-2/Bcl-xl/Mcl-1 co-dependent cell lines, such as NCI-H69 (IC50: 169.7 nM). In addition, combinations of LP-118 and SN-38 (the active metabolite of irinotecan), topotecan, or etoposide, induced strong synergist effects in both NCI-H69 and NCI-H146 cell lines, suggesting that LP-118 could enhance the cytotoxicity of these drugs. Consistent with the in vitro data, treatment with LP-118 alone induced complete tumor remissions in NCI-H889-derived xenograft models (10/10), whereas no complete tumor remission was observed in the ABT-263-treated group. Moreover, LP-118 plus carboplatin or topotecan dramatically inhibited tumor growth of NCI-H526 xenograft models and has significantly better efficacy than either carboplatin or topotecan alone. Importantly, no significant body weight loss was observed in mice treated with LP-118 single agent or the combinations, indicating such regimens were well tolerated. In summary, LP-118, a new generation Bcl-2/Bcl-xl dual inhibitor, is highly active, as single agent or in combination with SOCs, in a large subset of SCLC cell lines. Clinical trials are ongoing to further evaluate the safety and efficacy of LP-118 in hematological cancers and solid tumors, including SCLC. Additional studies are also underway to develop predictive biomarkers for facilitating patient stratification in the clinic. Citation Format: Ling-Ling Lee, Huan Chen, Cong Zhu, Yao Chen, Brianna Feldmeier, Yue Shen, Yu Chen, Stephen P. Anthony, Fenlai Tan, Yi Chen, Bing Dai. Targeting Bcl-2/Bcl-xl for small cell lung cancer treatment with LP-118 [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 675.

Abstract 4605: AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-cell lymphoma models

Abstract Peripheral T-cell Lymphoma (pTCL) makes up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor prognosis. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E. Here we examine the efficacy of CDK9 inhibition for the treatment of pTCL. CDK9 regulates transcription through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor; acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are often overexpressed in hematologic tumors. It has been shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression of BCL-2 family members is highly heterogeneous in pTCL, with MCL-1 being the most universally expressed. Using MCL-1 inhibitor AZD5991, we showed statistically significant benefit in survival when combined with CHOP in MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition, we have previously reported the importance of the anti-apoptotic protein BFL-1 in cell survival in NHL, including a subset of pTCL (Boiko et al 2021). AZD4573 treatment showed a range of activity across the panel of 18 human pTCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, with EC50 values less than 100 nM, and 13 reached a max caspase activation of over 40%. pTCL subtypes that responded to AZD4573 treatment included ALCL, NK-TCL, and PTCL-NOS and CTCL. Acute AZD4573 treatment resulted in decreased pSer2-RNAP2 and reduced protein levels of c-MYC and MCL-1. To determine what was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 for viability after KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that efficacy is mediated through MCL-1 in ALCL pTCL cell lines assessed. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and measured viability after 24-72 hours using RealTime-Glo after 24hr exposure to CHOP with AZD4573 added for the last 6hrs. Adding AZD4573 to CHOP in vitro increased the efficacy in pTCL ALCL cell lines with MCL-1 dependency, but not in CTCL cell line with BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels but not MCL-1, suggesting that combination benefit may be driven through c-MYC. This data suggests that treatment with AZD4573 as a monotherapy or in combination with CHOP regimen would be an effective therapeutic strategy in pTCL. Citation Format: Danielle Sinead Potter, India Ott, Jamal Saeh, Richard Olsson, Stephen Fawell, Lisa Drew, Justine Roderick. AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-cell lymphoma models [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 4605.

Combination drug screen identifies synergistic drug interaction of BCL-XL and class I histone deacetylase inhibitors in MYC-amplified medulloblastoma cells

PurposePatients with MYC-amplified Group 3 medulloblastoma (MB) (subtype II) show poor progression-free survival rates. Class I histone deacetylase inhibitors (HDACi) are highly effective for the treatment of MYC-amplified MB in vitro and in vivo. Drug combination regimens including class I HDACi may represent an urgently needed novel treatment approach for this high risk disease.MethodsA medium-throughput in vitro combination drug screen was performed in three MYC-amplified and one non-MYC-amplified MB cell line testing 75 clinically relevant drugs alone and in combination with entinostat. The drug sensitivity score (DSS) was calculated based on metabolic inhibition quantified by CellTiter-Glo. The six top synergistic combination hits were evaluated in a 5 × 5 combination matrix and a seven-ray design. Synergy was validated and characterized by cell counts, caspase-3-like-activity and poly-(ADP-ribose)-polymerase-(PARP)-cleavage. On-target activity of drugs was validated by immunoprecipitation and western blot. BCL-XL dependency of the observed effect was explored with siRNA mediated knockdown of BCL2L1, and selective inhibition with targeted compounds (A-1331852, A-1155463).Results20/75 drugs effectively reduced metabolic activity in combination with entinostat in all three MYC-amplified cell lines (DSS ≥ 10). The combination entinostat and navitoclax showed the strongest synergistic interaction across all MYC-amplified cell lines. siRNA mediated knockdown of BCL2L1, as well as targeted inhibition with selective inhibitors showed BCL-XL dependency of the observed effect. Increased cell death was associated with increased caspase-3-like-activity.ConclusionOur study identifies the combination of class I HDACi and BCL-XL inhibitors as a potential new approach for the treatment of MYC-amplified MB cells.Graphical abstractGraphical abstract created with BioRender.com, illustrating the workflow and summarizing main results.

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

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

Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia

T-Cell acute lymphoblastic leukemia (T-ALL) is a disease caused by the malignant transformation of T-cell lineage progenitors. With the use of intensive chemotherapies survival rates have improved, but outcomes particularly of relapsed patients remain poor. In addition, currently used intensive chemotherapies are associated with high rates of treatment-related morbidity and mortality, emphasizing the need to develop new and improved therapies. The intrinsic apoptosis pathway, one of the key pathways controlling cell death, is dysregulated in many cancers and contributes to leukemogenesis and treatment failure. The main steps of this pathway are controlled by proteins of the B-cell lymphoma (BCL-2) family at the outer mitochondrial membrane. Hence, targeting this pathway by BH3-mimetics has emerged as an effective new treatment strategy in different cancers. Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity. The aims of this study are to evaluate susceptibilities of T-ALL to inhibitors of BCL-2 family proteins, to identify markers of response, to elucidate mechanisms of action and to assess combination effects. First, we analyzed the anti-leukemia activities of inhibitors targeting BCL-2 (venetoclax), BCL-XL (A-1331852) and MCL-1 (AZD5991) and of the dual BCL-2/BCL-XL inhibitor AZD4320 in T-ALL cell lines (N=6) and a series of patient-derived xenograft (PDX) samples (N=8). Inhibition of MCL-1 alone was not effective in the cell lines tested (EC50 >1 µM), but heterogeneous sensitivity was found in PDX samples (EC50 <1µM in 3/8 samples). In cell lines, sensitivity for BCL-2 inhibition was exclusively found in a cell line with an early T-cell precursor ALL phenotype (Loucy). Similar to cell lines, we found insensitivity for BCL-2 inhibition in all PDX samples tested (N=8). For inhibition of BCL-XL, we found heterogeneous sensitivities with 4/6 cell lines and 5/8 PDX samples showing an EC 50 <1 µM. Interestingly, BH3-profiling confirmed BCL-2 dependence in Loucy and BCL-XL dependence in the other cell lines. Importantly, dual inhibition of BCL-2 and BCL-XL showed clear responses in cell lines and PDX samples (median EC 50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (r s=0.89, p=0.03), providing a marker of response. We next sought to elucidate the molecular mechanisms by which different BH3-mimetics induce cell death in T-ALL. Analyzing protein interactions of BCL-2 family members by co-immunoprecipitation studies, we found that AZD4230 acts on-target by releasing BIM from BCL-2 and BCL-XL, but with compensatory increased protein complexes of BIM and MCL-1. Accordingly, combined inhibition of BCL-2 and BCL-XL with inhibition of MCL-1 leads to release of BIM from all three anti-apoptotic proteins enabling downstream apoptosis signaling. These findings suggest that combined inhibition of BCL-2 and BCL-XL together with MCL-1 may provide synergistic benefits. To further validate these data, we applied dynamic BH3-profiling and determined dependencies of T-ALL samples on BCL-2 family members in response to BCL-2/BCL-XL inhibition. Here, we found that the MCL-1 dependence strongly increases upon BCL-2/BCL-XL inhibition, suggesting MCL-1 as a target for combined inhibition. Based on these results, we performed dose-response matrix analyses to test the effects of combined BCL-2/BCL-XL and MCL-1 inhibition. Most importantly, combined inhibition showed synergism in all cell lines and PDX samples tested (Bliss scores >10), including leukemias being insensitive to single compounds. Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.

AZD4573 in Combination with CHOP Increases Combination Benefit in Preclinical Peripheral T-Cell Lymphomas Models

Peripheral T-cell Lymphoma (pTCL) make up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor outcomes to standard of care therapy. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E and for CD30-postive pTCLs, brentuximab vedotin is approved. CDK9 regulates transcription elongation through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor, acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are frequently overexpressed in haematologic tumours. It has been previously shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression and dependencies of the anti-apoptotic BCL-2 family members is highly heterogeneous in pTCL; however MCL-1 is the most universally expressed BCL-2 family member. Using the MCL-1 inhibitor AZD5991, we have shown statistically significant benefit in survival when combined with CHOP in 2 MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition to MCL-1, we have previously reported the importance of the anti-apoptotic protein BFL-1 in mediating cell survival in NHL, including a subset of pTCL (Boiko et al 2021). Consistent with these findings, AZD4573 treatment exhibited a range of activity across the panel of 18 human TCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, having EC 50 values < 100 nM, and 13 reached a max caspase-3/7 activation of greater than 40%. pTCL cell lines that responded to AZD4573 treatment including ALCL, NK-TCL, and PTCL-NOS and CTCL subtypes. Acute AZD4573 treatment of pTCL cell lines resulted in decreased p-SerRNApoll II and reduced levels of c-MYC and MCL-1 protein levels. To determine if MCL-1 or c-MYC was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 after long term KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that AZD4573 efficacy is mediated through MCL-1, in ALCL pTCL cell lines tested. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and assessed for viability after 24-72 hours using RealTime-Glo after 18hr exposure to CHOP and AZD4573 added for the last 6hrs. Adding AZD4573 treatment to CHOP schedule in vitro increased the combination benefit in pTCL ALCL cell lines that show MCL-1 dependency, but not in CTCL cell line which show a BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels at 24-48 hours, but not MCL-1 suggesting that combination benefit may be driven through decreased c-MYC levels, as KO of c-MYC resulted is loss of cell viability in these models. These data suggested that treatment with AD4573 either as a monotherapy or in combination with CHOP, would be an effective therapeutic strategy in pTCL. AZD4573 monotherapy is currently being evaluated in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK in patients with relapsed/refractory pTCL.

Erythroid/megakaryocytic differentiation confers BCL-XL dependency and venetoclax resistance in acute myeloid leukemia

AbstractMyeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL–selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

Functional Anti-Apoptotic Protein Dependence and Its Relation to Genomic Prognostic Markers and Response to Treatment in Chronic Lymphocytic Leukemia

Introduction Due to its heterogenous biology, chronic lymphocytic leukemia (CLL) is a well-suited disease for identifying new relationships between genomic and functional biomarkers for drug response. Here, we combined a multi-omics approach and ex vivo drug-treatment data with functional characterization of apoptotic sensitivity to correlate genetic features and drug sensitivity with specific anti-apoptotic signaling patterns measured by BH3 profiling in primary CLL samples. Methods BH3 profiling is a functional assay to measure the proximity of cells to undergo apoptosis via overall priming as well as to identify specific anti-apoptotic proteins the cells are dependent on for survival. BH3 peptides and mimetic such as BIM and PUMA were used to identify overall priming or proximity to apoptosis, BAD and ABT199 were used to identify BCL-2 dependence, and FS1, MS1 and HRK were used to identify BFL-1, MCL-1 and BCL-xL dependences respectively (Ryan et al, Biol Chem, 2016). Results We analyzed primary CLL samples from 73 patients and using BH3 profiling identified the specific associations between anti-apoptotic protein dependence and genetic features (such as IGHV status, trisomy-12, del(17p) and TP53 mutation. Our univariate models demonstrated that BCL-2 dependence, as measured by ABT199 drug (p=0.001) and BAD (p=0.004) BH3 peptide, are highly correlated with mutated IGHV status, a favorable prognostic marker in CLL (Figure 1A). Using ex vivo treatment with multiple classes of small molecule inhibitors, we further identified several specific drug response or sensitivity patterns correlating with anti-apoptotic protein dependence as well as CLL prognostic factors (Figure 1B). In particular, our multivariate models (LASSO regularized) demonstrated that viability following ex vivo treatment with the MDM-2 inhibitor Nutlin-3 was positively correlated with TP53 mutation/deletion, but inversely correlated with BFL-1 (FS1 peptide) and BCL-xL (HRK peptide) dependence as well as trisomy-12 (Figure 1B), though the inverse correlation between Nutlin-3 and BFL-1 or BCL-xL dependence is relatively weak. CLL cell viability following ex vivo treatment with the dual BCL-2/BCL-xL inhibitor navitoclax was negatively correlated with BCL-2 (BAD peptide) and BCL-xL (HRK peptide) dependence, though primarily BCL-2 dependence, suggesting that sensitivity to navitoclax is highly dependent on BCL-2 (Figure 1B). At the transcriptomic level, CLL cell dependence on BCL-2 positively correlated with NOXA expression, NFκB signaling, and reduced intracellular oxidative level, various pathways that could potentially maintain BCL-2 dependence. Finally, we utilized BH3 profiling to retrospectively predict in vivo drug response, with high BCL-2 dependence (p=0.016) correlating with greater absolute lymphocyte count decrease. Conclusion Functional characterization of apoptotic signaling in CLL demonstrated that patients with mutated IGHV are more dependent on BCL-2 for survival, and that BH3 profiling could retrospectively predict in vivo sensitivity to BCL-2 inhibition. This functional precision medicine approach may help to stratify patients with CLL beyond genetic markers and should be further explored for correlation with clinical outcomes in prospective clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BCL-XL Blockage with A1155463 Significantly Increases Efficacy of Venetoclax in Mantle Cell Lymphoma in Vitro and In Vivo

Mantle cell lymphoma (MCL) is a rare chronically relapsing subtype of aggressive B-cell non-Hodgkin lymphoma characterized by the canonical chromosomal translocation t (11;14) and other molecular cytogenetic aberrations including overexpression of BCL2 protein. Venetoclax (VEN), a BCL2 inhibitor, has demonstrated activity in MCL both as a monotherapy and in combination with other targeted agents. Treatment with single agent venetoclax is, however, hampered by frequent development of drug resistance caused in large part by adaptive upregulation of other anti-apoptotic BCL2 family members. Immunodeficient NOD-SCID-gamma mice were xenografted subcutaneously with MCL cell lines and PDX cells established from patients with relapsed and refractory MCL. The therapy was given for two weeks. VEN was administered by oral gavage (100 mg/kg/d), A1155463 was administered intraperitoneally (10 mg/kg/d). Western blotting was implemented to evaluate expression levels of BCL2 proteins, immunoprecipitation was used to analyze the levels of BIM bound to BCL2 and BCL-XL before and after exposure to VEN. CRISPR-Cas9 was employed to derive clones with BCL-XL knock-out. Blood cell counts in mice on A1155463 +/- VEN therapy were analyzed using the Mindray BC-5300 Auto Hematology Analyzer. In this study, we analyzed molecular mechanisms of in vivo acquired resistance to VEN using a panel of several murine cell line-based xenografts (CDX) and patient-derived xenografts (PDX) of MCL. First, the mice xenotransplanted with VEN-sensitive MCL cells were subject to monotherapy with VEN until development of resistant tumors (VEN-R). Western blot analysis of VEN-R tumors revealed upregulation of BCL-XL in majority of VEN-R tumors compared to controls. Other changes included upregulation of MCL1 and downregulation of BIM proteins in several models. Immunoprecipitation experiments confirmed that BCL-XL indeed serves as a buffer for BIM released from BCL2 after exposure to VEN thereby blocking VEN-triggered apoptosis. Importantly, we demonstrated that the upregulation of BCL-XL caused not only VEN resistance, but also led to BCL-XL-specific pro-apoptotic priming of VEN-R lymphoma cells. In vitro, the combination of VEN and A1155463, a specific BCL-XL inhibitor, induced cytotoxic synergy on a panel of MCL cell lines and primary cells. CRISPR-Cas9-mediated BCL-XL knock-out resulted in marked sensitization to VEN-induced apoptosis in MCL cell lines. In vivo, pharmacological blockage of BCL-XL strongly increased sensitivity to VEN. Despite that A1155463 exerted limited anti-lymphoma activity as monotherapy on tested CDX and PDX models, its combination with VEN was synthetically lethal and exerted significantly enhanced anti-lymphoma activity. The efficacy of the VEN and A1155463 combination was highly effective even in mice bearing VEN-R tumors (tumors with acquired VEN resistance, Figure 1). It has been published that mechanisms responsible for BCL-XL upregulation include activation of NFkappaB signaling via CD40 and hypoxia. Upregulation of BCL-XL induced by these factors in turn caused VEN resistance in vivo. Indeed, we confirmed that upregulation of BCL-XL was observed already after engraftment of VEN-sensitive MCL cells in immunodeficient mice. MCL cells isolated ex vivo from the established CDX tumors were significantly more sensitive to BCL-XL inhibition compared to the corresponding in vitro growing cell lines. Our results thus confirmed increased BCL-XL dependence of MCL cells in vivo compared to in vitro. We suggest that these microenvironmental factors were also critical for the selection of VEN-resistant clones during the therapy with VEN. Historically, experimental therapy of patients with chronic lymphocytic leukemia with navitoclax, a combined inhibitor of BCL2 and BCL-XL, was rater disappointing. The plausible reasons included both low efficacy of navitoclax due to insufficient BCL2 inhibition, and a dose-limiting thrombocytopenia. We demonstrated that the thrombocytopenia associated with continued therapy with A1155463 could be managed by 4 days on / 3 days off treatment strategy, which cannot be applied in case of fixed dual BCL2/BCL-XL inhibitors like navitoclax, or AZD4320. In summary, the combined inhibition of BCL2 and BCL-XL with VEN and A1155463 is a highly effective experimental treatment strategy for R/R MCL with a potential translation to the clinical grounds. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Erythroid/Megakaryocytic Differentiation Confers BCL-XL Dependency and Venetoclax Resistance in Acute Myeloid Leukemia

Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia (PEL), myelodysplastic syndrome (MDS) with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Hypothesizing that the distinct lineage of acute myeloid leukemia (AML) with erythroid or megakaryocytic differentiation may confer specific vulnerabilities, we sought to identify selective dependencies in these rare subtypes in comparison with other AML types. We show that AML cells with erythroid or megakaryocytic differentiation depend on the anti-apoptotic protein BCL-XL, rather than BCL-2, using a combination of ex vivo drug sensitivity and resistance testing, genetic perturbation, transcriptomic profiling and mouse xenograft experiments. To identify druggable dependencies in erythroid and megakaryoblastic leukemias, we screened over 500 drugs across a 10,000-fold concentration range in four erythroid and two megakaryoblastic AML cell lines and used other AML cell lines as controls (Figure A). The high-throughput screens identified BCL-XL-selective inhibitors including A-1331852 as highly effective against erythroid and megakaryoblastic leukemia cell lines in comparison with 15 cell lines representing other AML types (Figure B). In contrast, the erythroid/megakaryoblastic AML subtypes were resistant to the BCL-2 inhibitor venetoclax used clinically in the treatment of AML (Figure A). To confirm the on-target mechanism of BCL-XL inhibition, we next evaluated the genetic dependencies of erythroid and megakaryoblastic leukemias using public genome-scale CRISPR-Cas9 and RNAi screening data from 5 erythroid, 3 megakaryoblastic, and 15 other AML cell lines. This analysis demonstrated striking essentiality of BCL2L1 encoding BCL-XL, but not BCL2 or MCL1, for the survival of erythroid and megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared to other subtypes of AML and other hematological malignancies, providing a basis for the observed functional dependencies. BCL-XL inhibition effectively killed bone marrow cells of 5 out of 8 AML patient samples with erythroid or megakaryocytic differentiation ex vivo, with IC50 ranging from 0.6 to 55 nM. In contrast, only one out of 40 other AML patient samples was sensitive (IC50 < 60 nM) to BCL-XL inhibition. To better understand the drug sensitivity and gene expression profiles of the blasts, we performed integrated single-cell transcriptomics and ex vivo flow cytometry-based drug sensitivity profiling on mononuclear cells from two patients with erythroid/megakaryocytic AML. The blasts showed enriched BCL2L1 expression compared to other cell populations, providing a basis for the increased sensitivity of the blasts to BCL-XL inhibition and venetoclax resistance observed also at the single-cell resolution. In a mouse xenograft model using the HEL erythroleukemia cell line, treatment with the BCL-XL inhibitor A-1331852 effectively reduced tumor burden. To find treatment regimens enabling long-term disease control, we explored potential combination treatments by testing 6 other compounds with the BCL-XL inhibitor A-1331852 in 8x8 concentration matrices. Combining BCL-XL inhibition with the JAK inhibitor ruxolitinib, the BCL-2 inhibitor venetoclax, or the hypomethylating agent azacitidine showed potential for more durable responses both in a 3-day ex vivo assay as well as long-term treatments spanning over a month. Collectively, our results suggest targeting BCL-XL as a potential therapy option in erythroid and megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Selected Articles from This Issue

α-FR is overexpressed in a variety of solid tumors, including high-grade serous ovarian cancer, triple-negative breast cancer, and non–small cell lung cancer. CT900 is a small molecule thymidylate synthase (TS) inhibitor that binds to and is taken up by α-FR. Banerjee and colleagues report results from the first clinical trial to show reproducible single agent responses caused by a small molecule in α-FR-overexpressing cancers. The toxicity profile did not show classical TS inhibitor toxicity such as myelosuppression suggesting differential α-FR mediated uptake in tumor tissue. This trial is proof of concept for developing α-FR targeting small molecules, which are selectively taken up in α-FR-overexpressing tumors for patient benefit and are an alternate approach to antibody drug conjugates that already target α-FR.Immune checkpoint inhibitors (ICI) have modest efficacy in patients with mucosal melanoma. Although PD-1/PD-L1 monotherapy has limited benefit, combined therapy with CTLA4 and PD-1 inhibitors improves response rates but increases toxicity. VEGF is highly expressed in melanoma and contributes to progression. Combined treatment with ICI and VEGF inhibitors has shown positive outcomes across various cancer types. Here, Mao and colleagues present the results of a phase II study evaluating the PD-L1 inhibitor atezolizumab and the VEGF inhibitor bevacizumab as first-line therapy in Chinese patients with advanced mucosal melanoma. Over a median follow-up of 13.4 months, the objective response rate was 45.0% in 40 evaluable patients. Atezolizumab in combination with bevacizumab showed promising efficacy and manageable safety. This study sheds light on the dual strategy of VEGF and PD-L1 inhibition in patients with this rare melanoma subtype, for whom prognosis is otherwise generally poor.Defining the immune mechanisms responsible for early control of tumors is critical for guiding treatment strategies. Ferguson and colleagues applied imaging mass cytometry to map the immune landscape and identify differences in immune populations and their specific interactions between high-risk primary cSCC tumors that did not progress and those that developed metastases. The study also reveals that the immunosuppressive state alters the cellular composition of primary tumors. The ability to predict metastatic tumor progression at the time of initial diagnosis of primary cSCC will aid personalized medical care and better disease surveillance.Using publicly available genome-wide genetic dependency maps, Grubb and colleagues sought lineage-enriched anticancer targets. In kidney cancer, this strategy identified BCL2L1 which encodes the antiapoptotic BCL-XL protein as a candidate. Relying on cell-based apoptosis assays, in vivo studies, and clinical validations, as well as using genetic and pharmacological approaches, these studies revealed that a tumor's mesenchymal cell state conferred BCL-XL dependence. The findings identify BCL-XL as a novel target in kidney cancer and present potential biomarkers to stratify renal tumors based on their reliance on BCL-XL.

DIPG-46. Radiation induced senescence in diffuse intrinsic pontine glioma cells reveals selective vulnerability to Bcl-XL inhibition

Diffuse intrinsic pontine glioma remains a devastating condition with a dismal five year survival rate less than 5%. New approaches for treating this aggressive disease are critical to driving progress. Conventional radiotherapy remains the cornerstone of treatment, with no chemotherapeutic agent found to improve survival. However, radiotherapy is often delivered as a palliative treatment, and disease often recurs 3-6 months after. Radiation causes DNA damage and oxidative stress yielding a senescent state of replicative arrest in susceptible cells. However, increasing evidence demonstrates malignant cells can escape senescence leading to tumour recurrence. Targeted ablation of non-replicating senescent tumour cells following radiation could negate tumour recurrence. It remains unknown whether DIPG undergoes senescence following radiation, and furthermore, whether senolytics can be utilised to target senescent DIPG cells. We employed radiation to induce a senescent state in primary human DIPG cell lines. Senescence was confirmed using SA-β-gal staining, lack of EdU incorporation and qRT-PCR to characterise the SASP in three primary human DIPG cell lines. RNA-sequencing on DIPG cells following radiation revealed senescence and SASP signatures. Likewise, expression of senescence markers has been detected in human tumours. Viable cells that survive radiation were then utilised to screen candidate senolytic drugs, only Bcl-XL inhibitors demonstrated reproducible senolytic activity in radiation treated DIPG cells. In addition, Bcl-XL degradation using PROTACs (proteolysis targeting chimeras) resulted in a significant increase in senolysis of susceptible tumour cells. Conversely, Bcl-2 inhibitors failed to show any consistent senolytic activity. We are currently performing preclinical studies in the mouse to test the efficiency of senolytics against DIPG. These results demonstrate future possibilities of targeting radiation induced senescence in DIPG, using novel senolytic therapies and highlight Bcl-XL dependency as a potential vulnerability of surviving DIPG cells following exposure to radiation.

Selective Vulnerability of Senescent Glioblastoma Cells to BCL-XL Inhibition.

Glioblastoma (GBM) is a rapidly fatal malignancy typically treated with radiation and temozolomide (TMZ), an alkylating chemotherapeutic. These cytotoxic therapies cause oxidative stress and DNA damage, yielding a senescent-like state of replicative arrest in surviving tumor cells. Unfortunately, recurrence is inevitable and may be driven by surviving tumor cells eventually escaping senescence. A growing number of so-called “senolytic” drugs have been recently identified that are defined by their ability to selectively eliminate senescent cells. A growing inventory of senolytic drugs is under consideration for several diseases associated with aging, inflammation, DNA damage, as well as cancer. Ablation of senescent tumor cells after radiation and chemotherapy could help mitigate recurrence by decreasing the burden of residual tumor cells at risk of recurrence. This strategy has not been previously explored for GBM. We evaluated a panel of 10 previously described senolytic drugs to determine whether any could exhibit selective activity against human GBM persisting after exposure to radiation or TMZ. Three of the 10 drugs have known activity against BCL-XL and preferentially induced apoptosis in radiated or TMZ-treated glioma. This senolytic activity was observed in 12 of 12 human GBM cell lines. Efficacy could not be replicated with BCL-2 inhibition or senolytic agents acting against other putative senolytic targets. Knockdown of BCL-XL decreased survival of radiated GBM cells, whereas knockdown of BCL-2 or BCL-W yielded no senolytic effect.

Characterization of LP-118, a Novel Small Molecule Inhibitor of Bcl-2 and Bcl-Xl in Chronic Lymphocytic Leukemia Resistant to Venetoclax

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia, which is characterized by the accumulation of mature CD19+CD5+ B cells that evade apoptosis by upregulating anti-apoptotic BH3 protein, B cell lymphoma protein 2 (Bcl-2). Venetoclax, a first-in-class Bcl-2 inhibitor has transformed therapy for CLL. However, with continuous administration of venetoclax, patients often acquire resistance via mutations at or near the drug binding pocket at G101 in Bcl-2 (Blombery et al, Cancer Discov 2019, Lucas et al, Blood, 2020). In some patients, the acquisition of a mutation in tumor suppressor protein TP53 or the upregulation of anti-apoptotic family members including Bcl-xL has been shown to drive resistance to venetoclax. In particular, a switch to Bcl-xL dependency provides the rationale for dual inhibition of Bcl-2/-xL.We performed BH3 profiling on treatment naïve and venetoclax relapsed/refractory (R/R) primary CLL cells to investigate the dependency of several anti-apoptotic proteins and their contributions to cell survival. Primary CLL cells were incubated with Bcl-xL specific HRK peptides, Mcl-1 specific MS-1 peptides, Bcl-2 interacting BAD peptides, or BIM peptides which interact with all anti-apoptotic members. We observed that the majority of these cells responded to both BIM and BAD peptides indicating the dependency on anti-apoptotic protein Bcl-2. However, in some samples, including venetoclax R/R primary CLL cells, showed dependency on other anti-apoptotic proteins including Bcl-xL as indicated by a depolarization from the HRK peptide.We subsequently performed pre-clinical investigations of LP-118, a novel small molecule inhibitor of Bcl-2, more potent than venetoclax and with added advantage of selectively binding to Bcl-xL while minimizing the platelet toxicity. LP-118 was rationally designed to have an enzymatic IC 50 for Bcl-xL at 10.1 nM which was in-between venetoclax (62.2 nM) and navitoclax (2.9 nM) to prevent the on-target effects of platelet toxicity. Since most venetoclax- R/R patients have mutation in Bcl-2 and/or have increased dependency on Bcl-xL, we hypothesized that these patients would benefit from the additional inhibition of Bcl-xL.To test this hypothesis, treatment naïve and venetoclax R/R primary CLL cells were incubated with LP-118, venetoclax, or navitoclax continuously for 18 hours at concentrations ranging from 0.1 nM to 10 nM. Apoptosis was measured through Annexin V/Tetramethylrhodamine methyl ester perchlorate (TMRM) staining followed by flow cytometry. We found that treatment naive CLL cells were more sensitive to LP-118 than venetoclax and navitoclax with IC 50 values at 0.5nM, 10 nM, and >10 nM, respectively. Venetoclax R/R CLL cells were 50% killed by 1 nM LP-118 and were not sensitive to either venetoclax or navitoclax. To investigate the mechanism of LP-118 induced cell death, we used intracellular flow cytometry and observed pore-forming pro-apoptotic protein Bak transforming to an active conformation as early as 8 hours after treatment in venetoclax R/R and treatment naive CLL cells. Furthermore, after 12 hours of treatment we observed a release of cytochrome c indicating that cells were going through apoptosis.To determine whether cells harboring the Bcl-2 Gly101Val mutation respond to LP-118, we transfected RS4; 11 cells with plasmids containing the mutation. We treated these mutant cells for 72 hours and found that while these mutant cells were not sensitive to venetoclax, they responded to LP-118 with IC 50 of 20 nM. Furthermore, LP-118 induced quicker and earlier bak transformation and cytochrome C release in these mutant cells than venetoclax. To further evaluate the efficacy of LP-118, we tested this compound in a RS4; 11 xenograft model. We treated mice daily via oral gavage and observe that 100 mpk of LP-118 significantly decreases overall tumor growth and increases survival compared to 100 mpk of venetoclax (p=0.0004).Collectively, our in vitro studies demonstrated preclinical efficacies of LP-118 in primary CLL samples and Gly101Val mutant cells. LP-118 is highly potent in venetoclax resistant patient samples and cell lines. Additional in vivo work has further confirmed the efficacy of LP-118 in mutant cell lines. This work justifies continued preclinical and clinical work with this agent, and a phase 1 first in human study will begin soon in patients with relapsed hematologic malignancies (NCT04771572). DisclosuresTan: Guangzhou Lupeng Pharmaceutical Company Ltd.,: Current Employment. Chen: Newave Pharmaceutical Inc.: Current Employment. Anthony: Newave Pharmaceutical Inc.: Current Employment. Chen: Newave Pharmaceutical Inc.: Current Employment. Shen: Guangzhou Lupeng Pharmaceutical Company Co. Ltd.,: Current Employment. Byrd: Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria. Woyach: AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; Gilead Sciences Inc: Other: Data & Safety.

Dynamic BH3 Profiling to Assess the Effects of Novel Agents on Anti-Apoptotic Protein Dependence of CLL Cells

IntroductionOur group previously developed dynamic BH3 profiling (DBP) (Montero el al, 2015), which measures early changes in net pro-apoptotic signaling of mitochondria in response to various drug combinations. The degree of change in the propensity to undergo apoptosis is known as “delta priming”. We recently used DBP to demonstrate that BTK inhibition with ibrutinib leads to increased BCL-2 dependence and enhanced sensitivity to the BCL-2 inhibitor venetoclax in CLL cells (Deng et al., 2017). The effects of other novel agents on BCL-2 dependency in CLL cells relative to other anti-apoptotic family members such as MCL-1 and BCL-XL have yet to be systematically explored.Pathways that are recurrently mutated in CLL represent a logical next area in which to explore the potential of DBP to study the effects of novel agents on the BCL-2 family. One such gene is splicing factor 3B subunit 1 (SF3B1), which is recurrently mutated in a subset of patients with CLL, and confers an adverse prognosis. Here, we treated CLL cells ex vivo with a panel of novel agents including the splice modulator E7107, and utilized DBP to evaluate the functional effects of drug treatments on BCL-2 family proteins.MethodsMononuclear cells were isolated from 13 CLL patients and treated ex vivo with E7107 (H3 Biomedicine) and/or one of several other drugs (all obtained commercially). Cells were co-cultured with the human stroma NKTert cell line (1:10 ratio) with 20-hour drug treatments. DBP was performed as previously reported by measuring the release of cytochrome C (cyto-C) in gently permeabilized CLL cells in response to BH3-only peptides using a BD FACS Fortessa. Cyto-C release in response to BAD BH3 peptide was used as a measure of BCL-2 dependence, while cyto-C release in response to HRK and MS-1 BH3 peptide was used as a measure of BCL-XL and MCL-1 dependence, respectively. Western blot analyses were performed by standard protocols with signals captured by an LAS 4000 imager. Statistical analyses were by Welch t-test and one-sample t-test, with a two-tailed nominal p≤0.05 considered as significant.ResultsAs an initial screen, we treated cells from two different CLL patients in duplicate with a panel of 13 drugs and found that 5 agents resulted in increased BCL-2 dependence (defined as at least 35% cyto C release in both patients) (Fig. 1). These included E7107, ibrutinib, fludarabine, LY2784544 (JAK2 inhibitor), and AZD8055 (AKT inhibitor). As the greatest increase in BCL-2 dependence was observed with E7107, we next explored its effects on 11 additional CLL patient samples (6 SF3B1mut and 5 SF3B1wt). Significantly higher baseline BCL-2 dependence was observed in untreated SF3B1mut cells compared to SF3B1wt (p=0.02) (Fig. 2A). Treatment with E7107 alone or in combination with venetoclax increased BCL-2 dependence of CLL cells irrespective of SF3B1 mutation status (p values between 0.01-0.02). Venetoclax treatment alone had no effect on BCL-2 dependence in either group (p=0.39, p=0.68). In all samples, E7107 treatment made CLL cells significantly more BCL-2 dependent (BAD BH3 peptide, p=0.0009) and less MCL-1 dependent (MS-1 BH3 peptide, p<0.0001), but did not affect BCL-XL dependence (HRK BH3 peptide, p=0.11) (Fig. 2B). Consistent with these findings, CLL cells treated with E7107 had uniformly decreased MCL-1 protein expression on Western blot compared to untreated samples across all patient samples.ConclusionWe demonstrate the feasibility of using DBP to examine the effects of a panel of novel agents on BCL-2 family anti-apoptotic protein dependence of CLL cells. We identified several candidate drugs to explore in future studies, including the splice modulator E7107, which consistently increased BCL-2 dependence while reciprocally decreasing MCL-1 dependence. These preliminary results support the exploration of DBP in a high throughput format on a larger panel of drugs across more patient samples, with a goal of prioritizing the combinations that appear most promising to explore in clinical trials, to develop more personalized treatment for patients with CLL. [Display omitted] DisclosuresValentin:Abbvie: Other: Travel reimbursement; Roche: Other: Travel reimbursement. Buonamici:H3 Biomedicine, Inc.: Employment. Neuberg:Synta Pharmaceuticals: Other: Stock shares. Wu:Neon Therapeutics: Consultancy. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Davids:Merck: Consultancy; InCyte: Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Research Funding; Celgene: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy.

Dynamic BH3 Profiling Reveals Novel Therapeutic Strategies for the Treatment of Double-Hit Lymphoma

Introduction: Double-hit lymphomas (DHL), a subset of diffuse large B-cell lymphoma (DLBCL) harboring both a MYC and BCL-2 or BCL-6 gene rearrangement, are associated with rapidly progressive disease that is often refractory to aggressive therapy. Given these poor outcomes, novel therapeutic strategies and drug combinations are desperately needed.BCL-2 family proteins, consisting of both pro- and anti-apoptotic members, are the key mediators of the intrinsic pathway of apoptosis. BH3 profiling is a functional assay that uses synthetic peptides that mimic the BH3 domains of pro-apoptotic BCL-2 family members to measure mitochondrial “priming”, or how close a cell is to its apoptotic threshold. This tool is also extremely powerful in that it can identify which anti-apoptotic proteins (i.e. BCL-2, BCL-XL, MCL-1) the cell relies on for survival. Dynamic BH3 profiling (DPB) further demonstrates whether ex vivo drug treatment enhances priming, which is an effective predictor of in vivo response, or anti-apoptotic dependencies, which can identify synergistic partners to use with BH3 mimetics such as venetoclax.Methods: We utilized five DHL (Dogkit, DHL-4, DHL-6, OCI-Ly8, OCI-Ly18) and three non-DHL (OCI-Ly7, Toledo, and Pfeiffer) cell lines as well as two DHL patient-derived xenograft (PDX) models (available at http://www.PRoXe.org). We performed BH3 profiling in order to functionally measure overall priming as well as dependence on specific anti-apoptotic proteins. DBP was performed by treating cells with various concentrations of drugs for short intervals (16-24 hours) and subsequently performing BH3 profiling. We screened nine drugs, including conventional chemotherapy and targeted agents with suspected activity in lymphoma. Delta priming represents the change in cyctochrome c release following exposure to BH3 peptides in treated cells as compared to untreated cells. We assessed cell viability using annexin/PI staining and measured protein expression levels with immunoblotting.Results: Baseline BH3 profiling of DHL cell lines and cell lines derived from PDX models revealed that cells rely on multiple simultaneous anti-apoptotic proteins for survival. There was a universal dependency on BCL-2, as well as in some cases, MCL-1 and BCL-XL.Conventional chemotherapy, including doxorubicin, vincristine, mafosfamide and etoposide, had a variable effect on priming and BCL-2 dependency in DHL cell lines. Doxorubicin for example, significantly enhanced priming and BCL-2 dependency in the cell line Dogkit, but not in DHL-4 or DHL-6. In vivo treatment of two DHL PDX models with chemotherapy, including doxorubicin and cyclophosphamide, enhanced priming and dependency on the anti-apoptotic proteins, BCL-2, BCL-XL and MCL-1.DBP was also performed using a variety of targeted agents, including lenalidomide, ibrutinib, azacitidine, bortezomib and EPZ-6438 (an EZH2 inhibitor). Interestingly, treatment with the hypomethylating agent, azacitidine, consistently enhanced priming and BCL-2 dependency across DHL cell lines and cell lines derived from PDX mice. Azacitidine treatment resulted in enhanced cell death when combined with venetoclax in cell viability assays and was associated with increased protein expression of Bim and Bad. Treatment with bortezomib also enhanced priming, though non-specifically enhanced all anti-apoptotic dependencies.Drug treatment in non-DHL cell lines less reliably increased dependency on BCL-2 for survival, but also enhanced BCL-XL, MCL-1 and BFL-1 dependency, depending on the cell line.Conclusions: BH3 profiling can be used to identify targetable dependencies on BCL-2, BCL-XL and MCL-1 in DHL cells lines and cells derived from DHL PDX mice. DBP additionally provides a novel approach to rationally identify drug combinations with venetoclax and other emerging inhibitors of apoptotic proteins. This strategy can identify which chemotherapy would be most efficacious for a specific cell type, especially in combination with BH3 mimetics. Furthermore, we have identified azacitidine as a promising combination therapy to use with venetoclax for the treatment of DHL. Further exploration of the role of azacitidine in combination with venetoclax for the treatment of DHL is warranted. [Display omitted] DisclosuresDavids:Celgene: Consultancy; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Merck: Consultancy; InCyte: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding.

HGG-07. RADIATION INDUCED SENESCENCE IN DIFFUSE INTRINSIC PONTINE GLIOMA CELLS REVEALS SELECTIVE VULNERABILITY TO BCL-XL INHIBITION

Diffuse intrinsic pontine glioma remains a devastating condition with a dismal five year survival rate less than 5%. New approaches for treating this aggressive disease are critical to driving progress. Conventional radiotherapy remains the cornerstone of treatment, with no chemotherapeutic agent found to improve survival. However, radiotherapy is often delivered as a palliative treatment, and disease often recurs 3–6 months after. Radiation causes DNA damage and oxidative stress yielding a senescent state of replicative arrest in susceptible cells. However, increasing evidence demonstrates malignant cells can escape senescence leading to tumour recurrence. Targeted ablation of non-replicating senescent tumour cells following radiation could negate tumour recurrence. It remains unknown whether DIPG undergoes senescence following radiation, and furthermore, whether senolytics can be utilised to target senescent DIPG cells. We employed radiation to induce a senescent state in primary human DIPG cell lines. Senescence was confirmed using SA-β-gal staining, lack of EdU incorporation and qRT-PCR to characterise the SASP in three primary human DIPG cell lines. RNA-sequencing on DIPG cells following radiation revealed senescence and SASP signatures. Viable cells that survive radiation were then utilised to screen candidate senolytic drugs, only Bcl-XL inhibitors demonstrated reproducible senolytic activity in radiation treated DIPG cells. Conversely, Bcl-2 inhibitors failed to show any consistent senolytic activity. These results demonstrate future possibilities of targeting radiation induced senescence in DIPG, using novel senolytic therapies and highlight Bcl-XL dependency as a potential vulnerability of surviving DIPG cells following exposure to radiation.

CBIO-11. NOVEL THERAPY TO TARGET PR-RECURRENT GLIOMA

Abstract BACKGROUND Glioblastoma is a fatal infiltrative primary brain tumor, and standard care includes maximal safe surgical resection followed by radiation and Temozolomide (TMZ). Therapy-resistant residual cells persist in a latent state a long time before inevitable recurrence. Conventional radiation and Temozolomide (TMZ) treatment cause oxidative stress and DNA damage resulting senescent-like state of cell-cycle arrest. However, increasing evidence demonstrates escaping senescence leads to tumor recurrence. Thus, the ablation of senescent tumor cells after chemoradiation may be an avenue to limit tumor recurrence. METHODS 100uM TMZ for 7days or 10-20Gy radiation (cesium gamma radiator) was used for senescence induction in human glioblastoma in vitro and confirmed by SA-Beta gal staining and PCR. Replication arrest assessed by automated quantification of cellular confluence (Thermo Scientific Series 8000 WJ Incubator). We evaluated the IC50 for several senolytics targeting multiple SCAPs, including Dasatinib, Quercetin, AMG-232, Fisetin, Onalespib, Navitoclax, and A1331852, and in senescent vs. proliferating cells. RESULTS Among the senolytic tested, the Bcl-XL inhibitors A1331852 and Navitoclax both shown senolytic effect by selectively killing radiated, senescent tumor cells at lower concentrations as compared to 0Gy treated non-senescent cells. Across 12 GBM cell lines, IC50 for senescent cells was 6–500 times lower than non-senescent GBM(p&amp;lt; 0.005). Such differential sensitivity to Bcl-XL inhibition after radiation has also observed by BCL-XL knockdown in radiated glioma. CONCLUSION These findings suggest the potential to harness radiation-induced biology to ablate surviving quiescent cells and demonstrate Bcl-XL dependency as a potential vulnerability of surviving tumor cells after exposure to chemoradiation.

Abstract A73: BCL-XL dependency in a subset of ovarian clear-cell carcinoma cell lines

Abstract Clear-cell ovarian carcinoma is a rare histologic subtype of epithelial ovarian cancers. Clear-cell ovarian cancers tend to be less responsive to platinum-based chemotherapy compared to high-grade serous ovarian cancers. In a dataset of large-scale CRISPR knockout and RNAi screens of cancer cell lines (depmap.org), a subset of ovarian clear-cell carcinoma cell lines were sensitive to knockout or knockdown of BCL2L1, encoding antiapoptotic protein BCL-XL. Most clear-cell ovarian cancer cell lines also express BCL2L1 mRNA and protein. We therefore tested a panel of ovarian cancer cell lines for response to BH3 mimetic drugs, which inhibit different members of the BCL-2 family of antiapoptotic proteins. Initial data showed that a subset of ovarian clear-cell carcinoma cell lines was sensitive to single-agent BCL-XL inhibition with A1331852 and dual BCL-2/BCL-XL inhibition with ABT-263. In contrast, these cell lines were relatively insensitive to single-agent BCL-2 inhibition or MCL1 inhibition. We are currently applying BH3 profiling, a functional assay for apoptotic priming, to test the response of ovarian clear-cell carcinoma cell lines to BCL-XL inhibition using a BCL-XL specific peptide, HRK. Collectively, these studies suggest that ovarian clear-cell carcinomas may be sensitive to BCL-XL inhibition, either alone or in combination with chemotherapy. Citation Format: Elizabeth H. Stover, Maya K. Gelbard, Ursula A. Matulonis, Matthew Meyerson. BCL-XL dependency in a subset of ovarian clear-cell carcinoma cell lines [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A73.

Mesothelioma Cells Depend on the Antiapoptotic Protein Bcl-xL for Survival and Are Sensitized to Ionizing Radiation by BH3-Mimetics

The incidence of mesothelioma continues to rise and prognosis remains dismal owing to resistance to conventional therapies and few novel treatment options. Failure to activate apoptotic cell death is a resistance mechanism that may be overcome by inhibition of antiapoptotic Bcl-2 proteins using BH3-mimetic drugs. We investigated the role of antiapoptotic proteins in the radioresistance of mesothelioma, identifying clinically relevant targets for radiosensitization and evaluating the activity of BH3-mimetics alone and in combination with radiation therapy in preclinical models. Mesothelioma cell lines 211H, H2052, and H226 exposed to BH3-mimetics demonstrated Bcl-xL dependence that correlated with protein expression and was confirmed by genetic knockdown. The Bcl-xL inhibitor A1331852 exhibited cytotoxic (EC50, 0.13-1.42 μmol/L) and radiosensitizing activities (sensitizer enhancement ratios, 1.3-1.8). Cytotoxicity was associated with induction of mitochondrial outer membrane permeabilization and caspase-3/7 activation. Efficacy was maintained in a 3-dimensional model in which combination therapy completely eradicated mesothelioma spheroids. Clinical applicability was confirmed by immunohistochemical analysis of Bcl-2 proteins in patient samples and radiosensitizing activity of A1331852 in primary patient-derived mesothelioma cells. Mesothelioma cells exhibit addiction to the antiapoptotic protein Bcl-xL, and their intrinsic radioresistance can be overcome by small molecule inhibition of this novel therapeutic target.