MCL-1 Dependence Research Articles

Preclinical assessment of combined BCL-2 and MCL-1 inhibition in high-risk neuroblastoma

BackgroundNeuroblastoma is the most common extracranial pediatric solid malignancy with high-risk patients showing survival rates less than 50 %. These tumors frequently escape apoptosis through upregulation of the anti-apoptotic protein BCL-2. Unfortunately, monotherapy with the BCL-2 inhibitor venetoclax leads to therapy-induced resistance mediated by upregulation of MCL-1, thereby indicating that tumors could be responsive to dual inhibition of BCL-2 and MCL-1. MethodsIn vitro efficacy of venetoclax and multiple MCL-1 inhibitors (MIK665, S63845, AMG-176, AZD-5991) was studied in neuroblastoma cell lines with BCL-2 and/or MCL-1 dependency. Synergy assays were performed to identify the MCL-1 inhibitor with best performance in combination with venetoclax, followed by examining if dual BCL-2 and MCL-1 inhibition protects against resistance. Lastly, the combination was analyzed in two patient-derived xenograft (PDX) models. ResultsVenetoclax showed highest efficacy in neuroblastoma cell lines with high BCL-2 mRNA expression, BCL-2 protein levels or BIM:BCL-2 complex formation, while MCL-1 inhibitor efficacy was best correlated with BIM:MCL-1 complex levels. Most promising anti-tumor effects were observed following combination therapy with venetoclax and MIK665. Targeting BCL-2 and MCL-1 led to strong synergy and efficacy at low concentrations, induced apoptosis and prevented resistance. PDX validation studies combining venetoclax with MIK665 showed improved responses compared to monotherapies. ConclusionDual inhibition of BCL-2 and MCL-1 in neuroblastoma cells showed highly efficacious and synergistic responses in vitro, but only led to mild additive effects in vivo. Hence, better understanding of in vivo efficacy and side effects of this combination treatment are warranted prior to clinical translation.

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.

Abstract 5966: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies

Abstract CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer cell growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. Specifically, we show that brief treatment with PRT2527 (6h) potently induces apoptosis and inhibits proliferation in DLBCL, MCL and CLL cell lines. We also show that the addition of BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) potentiates the apoptosis response induced by PRT2527 in vitro and leads to more robust and durable responses including complete tumor regressions in DLBCL CDX and PDX models in vivo. In all studies, the combination of PRT2527 with BTKi or BCL2i is well tolerated. Mechanistically, we show that BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency in TMD-8 cells. Concurrent depletion of MCL1 and BFL1 with PRT2527 leads to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In similar fashion, Venetoclax-driven BCL2 inhibition also potentiates the anti-tumor activity of PRT2527. Consistent with these findings in vitro, we observe reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrate potent induction of apoptosis in peripheral blood mononuclear cells isolated from patients with both treatment-naïve and relapse/refractory CLL following brief treatment with PRT2527 (6h). Co-treatment with BTKi or BCL2i further enhances the apoptotic effect of PRT2527, similarly suggesting combinatorial benefit. Additionally, we characterize the activity of PRT2527 in models of BTKi relapsed/refractory MCL where increased CDK9 dependency has previously been described. In an Ibrutinib-resistant Mino CDX model, we observe increased tumor growth inhibition following treatment with PRT2527 compared to tumors derived from parental Mino cells, suggesting PRT2527 is efficacious in BTKi-resistant MCL. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory lymphoid malignancies. Citation Format: Norman Fultang, Ashley M. Schwab, Sophia McAneny-Droz, Diane Heiser, Peggy Scherle, Neha Bhagwat. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies [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 5966.

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.

PRT2527, a Novel Highly Selective Cyclin-Dependent Kinase 9 (CDK9) Inhibitor, Has Potent Antitumor Activity in Combination with BTK and BCL2 Inhibition in Various Lymphoid Malignancies

CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. We also show that combination of PRT2527 with BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) leads to more robust and durable responses in multiple pre-clinical models. In these studies, the combination of PRT2527 with BTKi or BCL2i was well tolerated. Mechanistically, we show that in cell lines, BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency. Concurrent depletion of MCL1 and BFL1 with PRT2527 led to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In a similar fashion, Venetoclax-driven BCL2 inhibition also potentiated the anti-tumor activity of PRT2527. Consistent with our findings in vitro, we confirmed potent combinatorial inhibition of tumor growth by PRT2527, administered once weekly, and BTKi or BCL2i, administered daily, in CDX and PDX in vivo models of DLBCL and MCL. We observed reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrated enhanced induction of apoptosis by PRT2527 in primary CLL patient samples simultaneously treated with BTKi or BCL2i. Additionally, we demonstrated that PRT2527 potently inhibits growth in Ibrutinib-resistant MCL cell lines. Further work characterizing PRT2527 as monotherapy and in combination with BTKi and BCL2i in in vivo models of BTKi-resistant MCL and CLL is currently ongoing. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory hematologic malignancies.

Increased MCL1 dependency leads to new applications of BH3-mimetics in drug-resistant neuroblastoma

BackgroundNeuroblastoma is a paediatric cancer that is characterised by poor prognosis for chemoresistant disease, highlighting the need for better treatment options. Here, we asked whether BH3-mimetics inhibiting BCL2 proteins may eliminate chemoresistant neuroblastoma cells.MethodsWe utilised cisplatin-adapted neuroblastoma cell lines as well as patient tissues before and after relapse to study alterations of BCL2 proteins upon chemoresistance.ResultsIn a direct comparison of cisplatin-resistant cells we identified a prominent loss of sensitivity to BCL2/BCL-XL inhibitors that is associated with an increase in MCL1 dependency and high expression of MCL1 in patient tumour tissues. Screening of FDA-approved anti-cancer drugs in chemoresistant cells identified therapeutics that may be beneficial in combination with the clinically tested BH3-mimetic ABT263, but no synergistic drug interactions with the selective MCL1 inhibitor S63845. Further exploration of potential treatment options for chemoresistant neuroblastoma identified immunotherapy based on NK cells as highly promising, since NK cells are able to efficiently kill both parental and chemoresistant cells.ConclusionsThese data highlight that the application of BH3-mimetics may differ between first line treatment and relapsed disease. Combination of NK cell-based immunotherapy with BH3-mimetics may further increase killing of chemoresistant neuroblastoma, outlining a new treatment strategy for relapsed neuroblastoma.

Development of a Robust BH3 Profiling Toolkit for Precision Medicine in Hematologic Malignancies

INTRODUCTION. BCL2 homology 3 (BH3) mimetics represent a novel class of pro-apoptotic anti Hematologic malignancies (HM) drugs directly targeting apoptosis, even in chemoresistant cells. Each disease is heterogenous which makes it hard to provide standardized treatment for each patient and highlight the need for new diagnosis tools to deliver precision medicine. In the other hand, a technic called BH3 profiling has been developed, allowing to measure the apoptosis priming of the cells as well as their dependencies on specific anti-apoptotic proteins. Using specific BH3 peptides, this assay measures the percentage of cytochrome c release for each anti-apoptotic protein, mainly Bcl2, Mcl1 and Bcl-xl. BH3 toolkit, a variation of this technic, was developed using BH3 mimetics instead of BH3 peptides. Those small molecules mimic the effect of BH3 sensitizer protein by competing for anti-apoptotic protein catalytic pocket. Using these drugs allows to avoid cell permeabilization and thus reduce variability in the assay. A dynamic BH3 toolkit is also possible, measuring early drug-induced changes in net proapoptotic signaling, to identify modification of antiapoptotic dependencies. The aim of the present work is to develop a standardize and reproducible BH3 toolkit (baseline and dynamic), with internal control, to allow an absolute measurement of specific anti-apoptotic dependencies in HM cell lines or primary tumor cells, as well as identifying optimal therapeutic combination. MATERIAL AND METHODS. We used different HM cell lines as well as primary cells from patients with CLL. Cells were seeded right after thawing at the concentration of 200,000 cells/mL in a 96 well plate, using a robotic liquid handler. Primary cells were cocultured with stromal cells (Resto-6 cell line). Cells are then incubated for 20h in a 5% CO2 and 37°C incubator. Next, we treated them with BH3 mimetics (or vehicle): Venetoclax (VEN, Bcl2 inhibitor), Navitoclax (Bcl2 and Bcl-xl inhibitor), AZD-5991 (AZD, Mcl1 inhibitor), A-1155463 (Bcl-xl inhibitor) for 4h. Then, cells were stained using Annexin V and 7AAD and analyzed by flow cytometry. Our gating strategy focuses on live cells to measure annexin V flipflop as an apoptotic marker. This assay is paired with a cell viability measurement at 24h, using CellTiterGlo assay. For dynamic BH3 toolkit, the influence of the tested drug on antiapoptotic dependencies is measured by adding this drug (or vehicle) at the same time as cells of interest. RESULTS. First, we wanted to optimize the protocol on cell lines with known anti-apoptotic dependencies. We used OCI-Ly1 and LP1 cell lines which are known for their exclusive Bcl2 and Mcl1 dependencies, respectively. As expected, OCI-Ly1 cells showed a significant increase in annexin V staining (reaching 48.2% (P=0.0003) at maximum concentration) upon VEN treatment, whereas it does not change significantly with Mcl1 or Bcl-xl inhibitors (Figure A). Likewise, we detected an increase of 15.35% (P=0.005) in Annexin V staining only upon AZD in LP1 cells, but no significant modification with other inhibitors. Consistent with these data, we saw a significant and specific decrease in cell viability (CTG) with VEN and AZD on OCI-Ly1 and LP1 respectively (data no shown). These 2 cell lines are used as internal controls for the other analyses. Next, we optimized the assay on CLL primary cells. Figure A shows that VEN induces an increase in Annexin V staining by 62.7 % (P=0.0003), demonstrating a significant Bcl2 dependency. Finally, we optimized the dynamic part of the assay (Figure B). We used ironomycin, a lysosomal iron-targeting small molecule that has been studied by our team in HM (Devin et al. 2022). We found that ironomycin increased significantly Bcl2 dependency in OCI-Ly1 cells by 8.55% (P=0.02) with VEN. Interestingly, we observed the induction of a MCL1 dependency in those cells, with an annexin V staining increase by 8.21% (P=0.02). CONCLUSION. We were therefore able to measure the baseline dependencies using a BH3 toolkit that avoid cell permeabilization. We were also able to measure changes in dependencies induced by an innovative therapy. We believe that our results show the robustness of the BH3 toolkit and its ability to detect potential new therapeutic strategies in HM cell lines and primary cells. Detailed results will be presented at the meeting, as well as data on primary lymphoma and myeloma cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Integrating High-Throughput Dynamic BH3 Profiling and Molecular Phenotyping to Identify Therapeutic Vulnerabilities in CLL

Despite recent advances in chronic lymphocytic leukemia (CLL) therapy, such as the use of targeted agents including, Bruton's tyrosine kinase (BTK) inhibitor ibrutinib and the potent BCL-2 antagonist venetoclax, this disease remains incurable for most patients, who are refractory or become resistant to the novel agents. Thus, identifying new treatment regimens for CLL and building a precision medicine framework that can match CLL patients to the appropriate drugs are of high priority. To address this challenge, we optimized high-throughput dynamic BH3 profiling (HT-DBP), a functional assay that rapidly measures the initiation of apoptotic signaling after ex vivo exposure to drugs, for interrogation of CLL samples. The main advantages of this assay over other ex vivo assays are: (i) rapidity - under 24 hours, which is especially important in CLL, where cell viability substantially decreases after 24 hours; (ii) miniaturization - a very limited number of primary cells are required; and (iii) scalability - allowing to conduct hundreds of drug response tests in parallel on one 384-well plate. These features collectively maximize the information yield from a given patient sample. We recently identified prognostic genetic alterations and molecular subtypes of CLL, based on multi-omics profiling of >1100 CLL samples (Knisbacher et al., Nat Genet, in press). To determine if these molecular findings were associated with novel therapeutic vulnerabilities in CLL, we performed HT-DBP on 65 primary CLL samples previously characterized by exome, transcriptome and methylome profiling, using 42 FDA approved drugs that were selected for potential relevance to CLL biology. We isolated peripheral blood mononuclear cells (PBMCs) and cultured them in conditioned media derived from stroma cells to reduce spontaneous apoptosis. Target cells were treated with a drug for 20 hours followed by BH3 peptide exposure. Mitochondrial outer membrane permeabilization (MOMP) was then measured on digitonin-permeabilized cells in response to BH3-only synthetic peptides that mimic pro-apoptotic BCL-2 family proteins. Mitochondrial cytochrome c release was quantified as a measure of MOMP by flow cytometry, gating on CD19+ and CD5+ cells. This assay measures if the cell has moved closer to the threshold of apoptosis after drug treatment and thereby identifies drugs that enhance apoptosis priming. The peptides used in each experiment were derived from BIM or PUMA to measure increases in overall apoptotic priming, or BAD and MS1 peptides that identified BCL-2 and MCL-1 dependence, respectively. Our screen revealed differential drug-induced apoptotic priming for various drugs. As expected, venetoclax and ibrutinib were highly effective across CLL. Other drugs that demonstrated high priming included navitoclax (BCL-XL/BCL-2), nutlin-3 (MDM2), abexinostat (HDAC), gandotinib (JAK2), duvelisib (PI3K δ/γ), idelalisib (PI3Kδ) and cerdulatinib (SYK/JAK). The assay was robust, as indicated by an 0.92 median Pearson correlation across replicates. Additionally, the majority of drugs had greater effect on CLL samples than on healthy PBMCs (p<0.001, paired t-test), supporting their specificity. We focused our analysis on the differential drug effects among molecular subtypes of CLL. First, we found that IGHV-mutated CLLs (M-CLLs) became more primed to apoptosis than IGHV-unmutated CLLs (U-CLLs) across the panel of drugs (p<0.001, paired t-test) and significantly in response to fludarabine and umbralisib (FDR<0.1, t-test). Second, we compared drug-induced apoptotic priming among 8 CLL subtypes (i.e. RNA expression clusters (ECs); Knisbacher et al.). Notable among the many drug priming-EC relationships was that within M-CLL ECs, venetoclax was most effective in EC-m3 (high IL-10 expression) and least effective in EC-m2 (low IL-10 and enriched in trisomy 12). Interestingly, EC-m1, associated with high TFEC expression and poor outcome, was most sensitive to nutlin-3. For U-CLLs, EC-u1 was most sensitive to gandotinib and EC-u2 to navitoclax. EC-i, associated with the intermediate methylation subtype of CLL, was the most resistant EC to ibrutinib but was very sensitive to navitoclax, more than to any other drug. Altogether, we present a framework that links ex-vivo drug response with molecular features including expression subtypes to highlight new therapeutic opportunities in CLL.

Combinatorial Effects of BH3 Mimetics and CAR T Cells Targeting T-Cell Lymphomas

T-cell lymphomas (TCL) represent a significant unmet need for improved therapeutic strategies. Recent advances in disease modeling and molecular characterization have greatly increased the potential for advancement. We previously showed that a subset of TCL cell lines, patient-derived xenografts (PDXs), and primary samples express high and uniform levels of the tetraspanin CD37 (PMID: 30089630). Selective expression on malignant T cells distinguishes CD37 from other CAR T cell targets under development against TCL and led TCL inclusion in our phase 1 clinical trial of autologous CD37-directed CAR T cells (CAR-37; NCT04136275). CAR-37 T cells specifically lysed CD37+ TCL cell lines in vitro. In an aggressive T cell prolymphocytic leukemia PDX model, we observed >100-fold reduction in disease burden in vivo with CAR-37 T cells, but all mice eventually relapsed. We transplanted the relapsed tumor cells and re-treated with CAR-37 T cells. All recipients experienced a second response, with relapse in only a subset. Thus, a third dose of CAR-37 T cells was administered, but only a subset of mice underwent a third response. At the time of relapse/progression, CD37 expression was detected on tumor cells from each mouse. In addition, a small percentage of CAR-37 T cells were present in the tumor. Based on these results, we sought to evaluate combinatorial strategies that potentiate the tumoricidal activity of CAR-37 T cells and increase the rate of complete disease eradication. CAR T cells kill primarily through driving the intrinsic pathway of apoptosis. We previously showed that BH3 mimetics of BCL2, BCL-xL, and MCL1 can selectively induce apoptosis in TCLs based on predicted BH3 protein dependencies (PMID: 30498064). Thus, to define novel therapeutic combinations with CAR-37 T cells, we performed BH3 profiling on CD37-expressing TCL cell lines. Corresponding to the predicted dependencies via BH3 profiling, FEPD and SMZ1 were most sensitive to the MCL1 inhibitor AZD5991, whereas HUT78 was most sensitive to the BCL-2/BCL-xL inhibitor navitoclax. We observed concordant sensitivity patterns to the BH3 mimetics by viability/apoptosis assay and decreased active mitochondrial membrane potential. Importantly, CD37 antigen density was unaffected by exposure to BH3 mimetics. To evaluate the feasibility of combining BH3 mimetics and CAR T cells, we determined the impact of BH3 mimetics on CAR-37 T cell phenotype. First, we did not observe a reduction in CAR-37 T cell viability when cultured in vitro in the presence of TCL target cells and either venetoclax 0.5μM, navitoclax 0.5μM, or AZD5991 0.1μM for 48 hours. The same culture conditions also did not affect the differentiation or subset distribution of CAR-37 T cells. Nearly 60% of the CAR-37 T cells had an effector memory phenotype, whereas approximately 20% had a central memory phenotype and the remaining were either effector memory T cells re-expressing CD45RA or naïve. In addition, cytokine production (GMCSF and IFN-γ) and expression of T-cell exhaustion markers (PD-1, TIM3, and LAG3) was not affected by co-culture with BH3 mimetics. Since BH3 mimetics did not alter CAR-37 T cell activity, we evaluated the cytotoxicity effect of their combination against target TCL cell lines. Using a luminescence based killing assay and xCELLigence RTCA (ACEA), we found that all 3 BH3 mimetics in combination with CAR-37 T cells resulted in greater specific lysis of FEPD tumor cells. The most significant increase occurred in combination with AZD5991, which corresponds to the predicted MCL1 dependence of FEPD cells. Next, we evaluated the combination of CAR-37 T cells and AZD5991 in NSG mice engrafted with FEPD. CAR-37 T cells were administered upon a total flux (photons/s) ≥106 and the combination cohort subsequently received AZD5991 (100 mg/kg) on days 1 and 2, 8 and 9, and 15 and 16 post-CAR T cells. We observed significantly reduced tumor burden and prolonged survival in the combination cohort (median 42 days) compared to CAR-37 T cell (median 18 days) or AZD5991 (median 31 days) alone. Together, our findings demonstrate that BH3 mimetics with predicted activity against individual TCLs can enhance the cytotoxic activity of CAR-37 T cells. These results could be used to nominate and/or deprioritize future combination strategies both for TCL and for other patients being treated with effector T cells for a range of hematologic malignancies. AZD5991 was kindly provided by AstraZeneca.

ASXL1 Mutations Are Associated with a Response to the Combination of Alvocidib and 5-Azacytidine in Higher-Risk Myelodysplastic Syndromes

Introduction: Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are perspective therapeutic approaches in acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (MDS). Inhibitors of BCL-2 family proteins previously showed potent activity against AML and higher-risk MDS. Alvocidib (Alv), a cyclin-dependent kinase 9 inhibitor and indirect transcriptional repressor of the anti-apoptotic BCL-2 family member MCL-1, has previously shown clinical activity in AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and HMAs 5-Azacytidine (5-AZA) or decitabine in higher-risk MDS patients was recently completed (NCT03593915). However, biomarkers of response to 5-AZA + Alv combination are insufficiently characterized. In order to possibly identify biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-AZA effects in a cohort of n=45 higher-risk MDS patients. Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) aspirates using positive selection with MACS microbeads. CD34+ cells of aged-matched healthy controls (HC, n=11) were obtained from bone specimen after femur endoprosthesis surgery. HSPCs were expanded for four days in StemSpan SFEM II medium containing StemSpan Myeloid Expansion Supplement (Stem Cell Technologies) and treated with 5-AZA for 48h, Alv for 24h or their sequential combination (5-AZA for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of primary MDS HSPCs was assessed using NOXA-derived T-MS1 peptide. MDS recurrent mutations in BM mononuclear cells were assessed using myeloid NGS panel deep sequencing containing 67 genes. For in vivo tumor transfer experiments, splenic cells from CD45.2+Asxl1Y588X transgenic (Tg) mice were injected intravenously into sublethally irradiated CD45.1+ recipients through tail veins. Engrafted mice were treated with 1 mg/kg 5-AZA intraperitoneally (i.p.) on days 1-3 (qdx) every other week. Alv was administered i.p. at a dose of 2.5 mg/kg on day 5 (qw) in the weeks when 5-AZA was applied. Mice were sacrificed 11 weeks after the transplantation and the percentage of CD45.2+ cells in the BM was assessed using flow cytometric analysis. Results: The combination of 5-AZA+Alv showed an additive cytotoxic effect on MDS cells in CTG cell viability assays (median cell viability was 74%, 74% and 55% for 5-AZA, Alv and combination, respectively, p<0.0001). In annexin-V apoptotic assays, MDS cells were more sensitive to the cytotoxic effect of the combination treatment compared to HC (median percent of apoptotic and dead cells was 37% for MDS vs 26% for HC, p=0.0256). In analogy to in vitro studies, accelerated clearance of xenografted MDS-L cells from the peripheral blood of NSGS mice was observed after 5-AZA+Alv treatment compared to single agents. MCL-1 dependency of primary MDS samples did not significantly correlate with cytotoxic effects of Alv and combination treatment. However, the presence of ASXL1 mutations was significantly associated with higher cytotoxic activity of 5-AZA+Alv combination in univariable (p=0.0044) and multivariable (p=0.042) analyses. Increased sensitivity of BM cells to the cytotoxic effects of 5-AZA+Alv was also observed in Asxl1Y588XTg mice. The combination treatment facilitated clearance of CD45.2+Asxl1Y588X mutant leukemic cells from CD45.1+ recipients compared to 5-AZA monotherapy (p=0.0156). Histological analysis of BM cells after 5-AZA+Alv treatment in CD45.1+ recipients showed decrease in the percentage of immature blasts and improved development of mature BM cells when compared to 5-AZA monotherapy (p=0.0391). Improved cytotoxic effects of Alv and 5-AZA+Alv in ASXL1 mutant compared to ASXL1 wild type MDS samples were associated with higher expression of pro-apoptotic MCL-1 antagonist NOXA (p=0.0017). Conclusions: We provided a pre-clinical rationale for the use of the 5-AZA+Alv combination for higher risks MDS and proposed ASXL1 mutations as potential genetic biomarkers of response in prospective clinical trials.

Exploiting endogenous and therapy-induced apoptotic vulnerabilities in immunoglobulin light chain amyloidosis with BH3 mimetics

Immunoglobulin light chain (AL) amyloidosis is an incurable hematologic disorder typically characterized by the production of amyloidogenic light chains by clonal plasma cells. These light chains misfold and aggregate in healthy tissues as amyloid fibrils, leading to life-threatening multi-organ dysfunction. Here we show that the clonal plasma cells in AL amyloidosis are highly primed to undergo apoptosis and dependent on pro-survival proteins MCL-1 and BCL-2. Notably, this MCL-1 dependency is indirectly targeted by the proteasome inhibitor bortezomib, currently the standard of care for this disease and the related plasma cell disorder multiple myeloma, due to upregulation of pro-apoptotic Noxa and its inhibitory binding to MCL-1. BCL-2 inhibitors sensitize clonal plasma cells to multiple front-line therapies including bortezomib, dexamethasone and lenalidomide. Strikingly, in mice bearing AL amyloidosis cell line xenografts, single agent treatment with the BCL-2 inhibitor ABT-199 (venetoclax) produces deeper remissions than bortezomib and triples median survival. Mass spectrometry-based proteomic analysis reveals rewiring of signaling pathways regulating apoptosis, proliferation and mitochondrial metabolism between isogenic AL amyloidosis and multiple myeloma cells that divergently alter their sensitivity to therapies. These findings provide a roadmap for the use of BH3 mimetics to exploit endogenous and induced apoptotic vulnerabilities in AL amyloidosis.

C-Myc plays a critical role in the antileukemic activity of the Mcl-1-selective inhibitor AZD5991 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a low 5-year overall survival rate of 29.5%. Thus, more effective therapies are in need to prolong survival of AML patients. Mcl-1 is overexpressed in AML and is associated with poor prognosis, representing a promising therapeutic target. The oncoprotein c-Myc is also overexpressed in AML and is a significant prognostic factor. In addition, Mcl-1 is required for c-Myc induced AML, indicating that c-Myc-driven AML harbors a Mcl-1 dependency and co-targeting of Mcl-1 and c-Myc represents a promising strategy to eradicate AML. In this study, we investigated the role of c-Myc in the antileukemic activity of Mcl-1 selective inhibitor AZD5991 and the antileukemic activity of co-targeting of Mcl-1 and c-Myc in preclinical models of AML. We found that c-Myc protein levels negatively correlated with AZD5991 EC50s in AML cell lines and primary patient samples. AZD5991 combined with inhibition of c-Myc synergistically induced apoptosis in AML cell lines and primary patient samples, and cooperatively targeted leukemia progenitor cells. AML cells with acquired resistance to AZD5991 were resensitized to AZD5991 when c-Myc was inhibited. The combination also showed promising and synergistic antileukemic activity in vitro against AML cell lines with acquired resistance to the main chemotherapeutic drug AraC and primary AML cells derived from a patient at relapse post chemotherapy. The oncoprotein c-Myc represents a potential biomarker of AZD5991 sensitivity and inhibition of c-Myc synergistically enhances the antileukemic activity of AZD5991 against AML.

Abstract 6257: Mutations in the ASXL1 and ZRSR2 genes are associated with the response to the combination of alvocidib and 5-azacytidine in higher-risk myelodysplastic syndromes

Abstract The hypomethylating agent 5-azacytidine (5-Aza) is a standard-of-care for patients with higher-risk myelodysplastic syndromes (MDS). Although an initial response is induced in approximately 50% of 5-Aza treated patients, subsequent relapse is almost certain. Recently, inhibitors of anti-apoptotic BCL-2 protein family members have shown therapeutic potential in acute myeloid leukemia (AML) and higher-risk MDS. Alvocidib (Alv), a CDK9 inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has shown anti-leukemic effects in a phase 1 study of patients with AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and 5-Aza or decitabine in higher-risk patients with MDS was recently completed (NCT03593915); however, biomarkers for response to the Alv and 5-Aza combination are not well characterized. To identify potential biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-Aza combination using a clinically well-characterized cohort of n=45 higher-risk patients with MDS and n=11 healthy controls (HC). CD34+ cells were purified from bone marrow (BM) aspirates using positive selection with MACS beads. CD34+ cells of HC were obtained from femur head replacement surgery bone specimens. After 4 days of expansion in SFEM II medium containing StemSpan Myeloid Expansion Supplement, cells were treated with 5-Aza for 48h, Alv for 24h or their combination (5-Aza for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of MDS samples was assessed using MS1 peptide-based assay. Recurrent myeloid neoplasia mutations in 67 genes were assessed in BM mononuclear cells using NGS panel deep sequencing. The combination of 5-Aza+Alv had an additive cytotoxic effect on CD34+ MDS cells in CTG assay (median cell viability = 74%, 73.8% and 55% for 5-Aza, Alv and combination respectively, p&amp;lt;0.0001). In Annexin-V apoptosis assay, MDS samples were more sensitive to the combination treatment compared to HC (median % of apoptotic and dead cells = 36.6% for MDS vs 25.6% for HC, p=0.0288). MCL-1 dependency inversely and not significantly correlated with CD34+ cell viability in CTG assays (Spearman r=-0.37, p=0.1119). In contrast, we found significant associations between ASXL1 and ZRSR2 mutations and higher sensitivity of MDS samples to 5-Aza+Alv combination (p=0.008 and p=0.0005 in univariable analysis respectively). ZRSR2 mutations also retained an independent impact on cell viability in multivariable analysis (p=0.035). Overall, we provide pre-clinical support for the use of 5-Aza+Alv combination for higher-risk MDS and identified ASXL1 and ZRSR2 mutations as potential genetic biomarkers of augmented response. Citation Format: Vladimir Ryabov, Nanni Schmitt, Qingyu Xu, Alexander Streuer, Johann-Christoph Jann, Alina Wein, Eva Altrock, Verena Nowak, Nadine Weimer, Julia Obländer, Iris Palme, Ahmed Jawhar, Ali Darwich, Patrick Wuchter, Christel Weiss, Georgia Metzgeroth, Jason M. Foulks, Laurenz Steiner, Mohamad Jawhar, Wolf-Karsten Hofmann, Daniel Nowak. Mutations in the ASXL1 and ZRSR2 genes are associated with the response to the combination of alvocidib and 5-azacytidine in higher-risk myelodysplastic syndromes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6257.

Abstract 2150: LKB1 loss rewires JNK-induced apoptotic protein dynamics through NUAKs and sensitizes KRAS-mutant non-small cell lung cancers to combined KRAS G12C + MCL-1 blockade

Abstract The recent approval of the KRAS G12C inhibitor sotorasib (AMG 510) for non-small cell lung cancer (NSCLC) marked a milestone in the development of targeted therapies for KRAS mutant cancers. While sotorasib and other KRAS G12C inhibitors have demonstrated rapid and durable responses in the clinic, some patients do not achieve responses. The identification of specific vulnerabilities conferred by recurrent co-occurring mutations may enable the development of biomarker-driven combination therapies with enhanced activity in distinct subsets of patients. We screened a panel of KRAS-mutant NSCLC cell lines as well as patient-derived xenograft (PDX) mouse models and observed that loss of the tumor suppressor STK11/LKB1 is associated with increased sensitivity to combined MAPK (either the KRAS G12C inhibitor sotorasib or MEK inhibitor trametinib) and MCL-1 inhibition (AMG 176). Restoration of LKB1 expression in LKB1-deficient cell lines and PDX tumors blunted the apoptotic response to MAPK + MCL-1 inhibition; conversely, deletion of LKB1 in LKB1 wild-type models increased sensitivity. Mitochondrial apoptotic cell death is regulated by interactions between pro- (e.g., BIM) and anti-apoptotic (e.g., MCL-1, BCL-XL) BCL-2 family members. MAPK inhibition increases BIM, while MCL-1 inhibition prevents BIM sequestration by MCL-1, resulting in apoptosis. LKB1 deficient cells exhibit increased association of BIM and MCL-1 upon MAPK inhibition, effectively priming cells for death upon inhibition of MCL-1. Mechanistically, LKB1 deficiency and associated loss of NUAK phosphorylation leads to hyperactivation of the JNK phospho-kinase network. JNK phosphorylates MCL-1 at S64 and T163, which enhances BIM: MCL-1 protein-protein interaction. Conversely, JNK phosphorylates BCL-XL at S62 and prevents sequestration of BIM. This series of phosphorylation events increases MCL-1 dependence and creates a specific vulnerability of KRAS-LKB1 tumors to MAPK + MCL-1 inhibition. Consistent with this mechanism, ex vivo treatment of tumor tissue from a KRAS-LKB1 mutant NSCLC patient with sotorasib or trametinib increased MCL-1 dependent priming. These results reveal a novel link between LKB1 and the regulation of BCL-2 family proteins and provide preclinical rationale for evaluation of combined KRAS G12C + MCL-1 inhibitors for KRAS-LKB1 mutant NSCLC. Citation Format: Chendi Li, Mohammed Usman Syed, Yi Shen, Audris Oh, Cameron Fraser, Johannes Kreuzer, Christopher Nabel, Kaitlyn Webster, Robert Morris, Sean Caenepeel, Anne Y. Saiki, Karen Rex, J. Russell Lipford, Wilhelm Hass, Kristopher Sarosiek, Paul E. Hughes, Aaron Hata. LKB1 loss rewires JNK-induced apoptotic protein dynamics through NUAKs and sensitizes KRAS-mutant non-small cell lung cancers to combined KRAS G12C + MCL-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2150.

Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia

AbstractInducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R–mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Elongator Complex Regulates MCL1 Dependency Via IRE1-XBP1 Axis of the ER Stress Response Pathway in Multiple Myeloma

Elongator Complex Regulates MCL1 Dependency Via IRE1-XBP1 Axis of the ER Stress Response Pathway in Multiple Myeloma

Targeting BCL-XL and BCL-2 By Protac 753B Effectively Eliminates AML Cells and Enhances Efficacy of Chemotherapy By Targeting Senescent Cells

Targeting BCL-XL and BCL-2 By Protac 753B Effectively Eliminates AML Cells and Enhances Efficacy of Chemotherapy By Targeting Senescent Cells

Maturity State and MCL-1 Dependence Predetermines Response to NOTCH1 Inhibition in T-ALL

Introduction:Acute T cell lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and young adults that frequently becomes treatment-refractory and relapses. The Notch1 pathway is a key oncogenic driver in T-ALL and is aberrantly activated in more than 50% of the cases. Despite promising pre-clinical data using gamma secretase inhibitors such as DBZ to target NOTCH1, resistance is rapidly occurring in vivo. As molecular heterogeneity has been linked to treatment escape, we focused our study on defining transcriptional cell states driving resistance to NOTCH inhibition and understanding their relation to mitochondrial priming.Methods:5 primary T-ALLs harboring NOTCH activating mutations were engrafted in NSG (NOD-scidIL2Rgnull) mice. Upon reaching ~ 10% of human CD45+ positive leukemic blasts in the peripheral blood, randomized groups of 8 mice per primary T-ALL were treated with DBZ (Dipenzazepine; 10 μM/kg every other day through tail vein) or vehicle (VEH). 3 mice per group were sacrificed after one week of treatment to assess short-term effect of DBZ, while the remaining 5 mice were weekly monitored for disease progression, leukemic blasts were collected from lymphoid organs and overall survival was determined.Full-length transcriptome analysis of 3188 blasts present in the blood of 20 sensitive and 22 refractory mice was performed by Smart-Seq2. Based on scRNA features, ‘scVelo’ and ‘CytoTRACE’ were used to identify developmental potential and differentiation trajectories. Cell fate and transcriptional regulatory networks were defined and reconstructed using ‘SCENIC’.Assessment of mitochondrial priming as measured by BH3 profiling was used to identify anti-apoptotic vulnerabilities present in these PDX models.Results:Upon DBZ, short or long-term disease control was observed in two strains, while rapid resistance occurred in three strains, thus establishing two sensitive and three refractories to NOTCH inhibition PDX models. Immunohistochemical analysis showed decreased expression of active NOTCH1 in spleen biopsies of all strains, validating the efficacy of DBZ and suggesting a mechanism of resistance independent of ICN1.Single cell transcriptional profiling showed enrichment of immature hematopoietic signatures and co-expression of lymphoid and myeloid progenitor programs in refractory models. Interestingly, pre-existing cells harboring refractory-like transcriptional circuits within the untreated sensitive population were identified. Upon treatment, despite increased differentiation in all models, lineage promiscuity was maintained in refractory strains, suggesting that cellular plasticity mediates treatment escape.Next, we characterized cell states driving treatment refraction. RNA velocity projections identified two distinct immature states differing in cell cycle and oncogenic signaling. Clustering of untreated, sensitive leukemic cells in immature state imply that aberrant lineage commitment can predict response to NOTCH inhibition in vivo. These observations were further confirmed by differentiation state analysis, where prior to treatment, high developmental potential was correlated to treatment escape. Surprisingly, in addition to early lineage differentiation drivers such as BCL11A, state-specific regulons analysis associated immature states with BCLAF1 a transcriptional regulator of apoptosis.We postulated that these transcriptional circuits lead to differential apoptotic priming, therefore the dependence on individual anti-apoptotic proteins was evaluated. Mitochondrial priming at baseline revealed BCL-2 dependence in sensitive strains whereas MCL1-dependence was observed in refractory ones. Upon DBZ treatment, while dependency profiles in refractory strains remained unchanged, a functional switch from BCL-2 to MCL1-dependency occurred in sensitive models.Conclusion:Our results suggest that response to NOTCH inhibition is predetermined by cell maturity states and their associated transcriptional circuits responsible for differential sensitivity to apoptotic priming via BCL2 and MCL1. These data suggest that combining BH3 and lineage commitment profiling may predict drug responses in vivo. Moreover, our findings highlight the importance of targeting co-existing cell states to overcome transcriptional heterogeneity as a driver of treatment escape. DisclosuresLetai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Weinstock: Daiichi Sankyo: Consultancy, Research Funding; Verastem: Research Funding; Abcuro: Research Funding; Bantam: Consultancy; ASELL: Consultancy; SecuraBio: Consultancy; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.