Bcl-xL Dual Inhibitor 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.

Enriched Signalling Pathways in Venetoclax-Relapsed Chronic Lymphocytic Leukemia (CLL) Cells and Targeting Using a Protac-Based Bcl-2/Bcl-Xl Degrader

Venetoclax is a specific inhibitor of Bcl-2, the key protein which protects CLL cells from intrinsic apoptosis, whereas the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib kills CLL cells via blockade of B-cell receptor (BCR) signalling. Unfortunately, some patients develop treatment resistance after receiving these agents in combination or sequentially. We aimed to evaluate whether CLL cells collected upon venetoclax and ibrutinib relapse retain survival dependency on alternative targets of the BCR pathway and Bcl-2 family, and to propose new vulnerabilities based on gene expression changes. Using single cell RNA sequencing (sc-RNA-seq), we analyzed paired samples from a CLL patient collected at baseline and at relapse, which occurred after cessation of fixed-duration treatment with venetoclax and ibrutinib. The proportion of cells in each CLL cluster was vastly altered at relapse. Cluster 3 represented 4% of CLL cells at baseline versus 36% at relapse and had a high BCR score, including high expression of MAP3K8 FOSB8 and NFKB1 genes. Moreover, at relapse, it contained more CLL cells expressing transcripts from the Bcl-2 family of anti-apoptotic proteins, including Bcl-2 and Bcl-xL. We also integrated sc-RNA-seq data from three primary CLL samples collected at relapse after venetoclax and ibrutinib treatment. The four clusters of CLL cells identified were enriched for BCR, interleukin-mediated, ErbB and the Fc epsilon receptor I signalling pathways. Importantly, most of the relapsed CLL cells which expressed high levels of Mcl-1 or Bcl-xL also expressed Bcl-2, suggesting that these resistant CLL cells became dependent on various anti-apoptotic proteins for survival. These observations led us to test Bcl-2/Bcl-xL targeting PROTACs PZ18753b and WH25244 in venetoclax-resistant CLL. These PROTACs were derived from navitoclax (Bcl-2/Bcl-xL dual inhibitor) linked to a VHL E3 ligase ligand to target Bcl-2 and Bcl-xL for degradation, with improved specificity to cancer cells while sparing platelets. We used three models of venetoclax-resistant CLL: (1) OSU-CLL cells expressing mutant Bcl-2 (G101V, F104L, R107-110dup or A113G), (2) treatment-naïve primary CLL cells co-cultured with NK.Tert stromal cells for 24h (CLL+stroma), and (3) primary CLL cells collected at venetoclax relapse. CLL cells were treated with venetoclax, navitoclax, PZ18753b, WH25244, or negative controls (NC) lacking VHL recruitment, PZ18753b-NC or WH25244-NC. Protein degradation was evaluated by Western Blot; viability, by Cell Titer Glo; and apoptosis, by flow cytometric detection of active BAX and BAK, mitochondrial cytochrome C, and AnnexinV binding. WH25244 was superior to venetoclax at targeting OSU-CLL cells expressing either wildtype or mutant Bcl-2, upon 72h treatment. Cell death by WH25244 was preceded by degradation of Bcl-xL and partial degradation of Bcl-2, despite presence of Bcl-2 mutations. In treatment-naïve primary CLL cells, Bcl-xL degradation by PZ18753b and WH25244 proceeded in a dose-, time- and VHL-dependent manner. The potency of these PROTACs fell in between that of venetoclax and navitoclax, with mitochondrial apoptosis occurring at low nanomolar concentrations known to spare human platelets. Stepwise, activation of BAK and BAX, and cytochrome C release were evident at 14h of treatment, followed by phosphatidyl serine exposure at 18h of treatment. A rightward shift in the dose-response curves of PZ18753b (16-fold) and WH25244 (8-fold) was noted for CLL+stroma cells relative to paired CLL cells treated in suspension. This reduction in potency was associated with (1) uptake of the PROTACs by NK.Tert cells, since Bcl-xL degradation was noted in stromal cells yet without evident toxicity, and (2) up to 2.5-fold increased levels of Bcl-xL in CLL+stroma, which shifted the dose-dependent degradation of Bcl-xL. Finally, 2 out of 3 venetoclax-relapsed primary CLL samples showed more active BAK and cytochrome C release upon treatment with WH25244 than venetoclax, evaluated at 10 nM. In conclusion, WH25244 is a PROTAC-based Bcl-2/Bcl-xL degrader with the potential to overcome venetoclax-resistant CLL dependent on Bcl-xL and mutant Bcl-2. Relative to its precursor, navitoclax, it shows increased potency against CLL cells and decreased toxicity against platelets in vitro, due to its VHL-dependent activity and minimal expression of VHL in platelets.

Targeting BCL-XL with a Novel VHL-Based BCL-XL Degrader DT-2216 in Pre-Clinical JAK2-Mutant AML Post-MPN Models

INTRODUCTION Outcomes of acute Myeloid Leukemia (AML) progressed from JAK2-mutant ( JAK2-mut) myeloproliferative neoplasms (MPNs) (post-MPN AML) are dismal, with limited responsiveness to cytotoxic therapies, hypomethylating agents, BCL-2 inhibitor or JAK2 inhibitor, with a median overall survival of less than 1 year ( Dunbar, et al. Blood, 2020). BCL-xL has been found to be overexpressed in cells from JAK2-mut MPN patients ( Petiti et al. J Cell Mol Med, 2020), thus making it a potential therapeutic target. However, the clinical utility of navitoclax, a BCL-xL and BCL-2 dual inhibitor, as demonstrated in combination with JAK2 inhibitor ruxolitinib in patients with myelofibrosis ( Harrison et al. J Clin Oncol, 2022), has been notable for an on-target thrombocytopenia due to the essential role of BCL-xL in platelet survival. In contrast, DT2216, a VHL-recruiting BCL-xL PROTAC, exhibits potent degradation of BCL-xL in leukemia cells while sparing platelets due to minimal expression of VHL in platelets ( Khan et.al Nat. Med, 2019). Consequently, DT2216 may represent an attractive strategy to target BCL-xL, reducing the apoptotic threshold and increasing responsiveness to chemotherapy selectively in tumor cells. In this study, we evaluated the pre-clinical efficacy of DT2216 in combination with ruxolitinib, 5-azacytidine (AZA), or MCL-1 inhibitor S63845 in JAK2-mut AML models. METHODS To validate the dependency of BCL2L1 (gene encoding BCL-xL) on JAK2-mut AML, we analyzed genome-wide CRISPR screen data, RNA-seq data from the DepMap portal and Beat AML 1.0 cohort. To assess the efficacy of DT2216-based treatments, including DT2216 alone, DT2216/AZA combination, DT2216/ruxolitinib combination, and DT2216/S63845 combination, we conducted the Cell Titer Glo (CTG) assay using JAK2-mut AML cell lines and primary leukemia samples. Since most JAK2-mut AML patients are resistant to ruxolitinib, we included ruxolitinib-resistant (ruxo-re) cell lines. The combination index (CI) values obtained at the ED 50, ED 75, and ED 90 were used to determine synergism (CI <1) between the agents, with lower values indicating stronger synergism. Clonogenic assays with three distinct JAK2-mut AML patient samples were performed using DT2216 and/or AZA to assess the anti-leukemia effects on hematopoietic stem and progenitor cells (HSPCs). Western blot was used to determine BCL-xL degradation and measure the expression of BCL-2 and MCL-1 after drug treatments. RESULTS The analysis of BCL2L1 gene expression (p<0.05, JAK2-mut AML cell lines versus other AML cell lines; p<0.01, post-MPN AML versus de novo AML) and BCL2L1 CRISPR gene effect (p<0.05, JAK2-mut AML cell lines versus other AML cell lines) confirmed the role of BCL-xL as a target in JAK2-mut AML. DT2216 as a single agent significantly reduced cell viability in JAK2-mut AML cell lines, with an average IC 50 of 1.61 μM in parental cell lines (range 0.74 - 2.35 μM) and an average IC 50 of 5.37 μM in ruxo-re cell lines (range 1.29 - 8.99 μM) at 72 hours. Notably, DT2216 also markedly reduced cell viability in primary samples (n=3), with an average IC 50 value of 1.21 μM (range 0.41 - 2.59 μM) at 24 hours. Moreover, the average CIs of DT2216/S68425 combination, DT2216/AZA and DT2216/ruxolitinib combination were 0.13 (range 0.07 - 0.17), 0.17 (range 0.61 - 0.22), 0.36 (range 0.14 - 0.54), respectively at 72 hours. Clonogenic assays in three primary JAK2-mut AML samples demonstrated a 69% (range 58.3 - 77.5%) reduction of colony count when treated with DT2216 alone (50 nM) (p<0.001, versus DMSO group) and 76% (range 70.8 - 79.8%) reduction when treated with the combination of DT2216 (50 nM) with AZA (100 nM) (p<0.0001, versus DMSO group) (Fig. 1A). Western blot analysis demonstrated efficient degradation of the BCL-xL protein, with average reductions of 78.5% (range: 70 - 90%), 82.75% (range: 68 - 96%) (Fig. 1B), and 86.75% (range: 83 - 97%) after 24 hours of treatment with DT2216 alone or in combination with AZA or ruxolitinib in both the parental and ruxo-re SET2 and HEL cell lines. CONCLUSIONS These findings highlight the promising efficacy of DT2216 in JAK2-mut AML, as evidenced by reduced cell viability, on-target degradation of BCL-xL, and synergistic anti-leukemia effects when combined with AZA, ruxolitinib or MCL-1 inhibitor. These results provide valuable insights into future therapeutic strategies for the treatment of JAK2-mut AML, particularly in the context of post-MPN AML.

Abstract 4792: Characterizing cellular senescence response to senolytic compounds using cell culture media analysis

Abstract Cellular senescence is an emerging hallmark of cancer. Senescent cells can lead to tumor development and chronic inflammation that stimulate age-related diseases. The secretion of senescent cells also generates an inflammatory environment that promotes tumor growth. Senolytics are pharmaceutical agents that selectively eliminate senescent cells through various mechanisms. Identifying the mechanism is crucial for the further development of senolytic drugs. ABT263 (also named navitoclax), a Bcl-2 and Bcl-xl dual inhibitor, is a well-known senolytic agent. Recently the first Bcl-2 and Bcl-xl dual-targeting proteolysis-targeting chimera (PROTAC), 753b, was developed, which has increased potency against senescent cells but minimized platelet toxicity. EF24 is another senolytic agent but the direct target is still unclear. Comprehensive analysis and comparison of the media environments from young cells, senescent cells, and senescent cells dosed with different senolytics can contribute toward solving senolytic mechanisms and make the drug discovery process more efficient and effective. WI38 cell culture media was used to grow young, senescence, and senolytic (753b or EF24)-dosed senescent cells for 72 hours. Cell culture media was collected at 10 different growth time points, subjected to protein crash using acetonitrile, and diluted with water prior to analysis. A 17-minute liquid chromatography triple quadrupole mass spectrometry method was developed for senescent cell and senolytic-added senescent cell culture media analysis. One hundred and forty-five cell culture related compounds including amino acids, nucleic acids, metabolites, sugars, and vitamins were simultaneously analyzed. A relative concentration trend graph was generated as the result for each of the cell growth related analytes. Thirteen compounds exhibited trends that distinguished at least one experimental group from the others in the 72 hours growth period. These compounds were asparagine, 5’-Methylthioadenosin, saccharopine, serotonin, alanyl-glutamine, urocanic acid, o-phosphoethanolamine, hypoxanthine, xanthosine, uracil, pyridoxal, nicotinic acid, and pyruvic acid. The differences observed in the cell environment reflect the differences of the activated pathways and mechanisms between the young, senescent, and senolytics-dosed senescent cells. This rapid and multicomponent cell culture analysis method enables scientists to analyze the environment as senescent cell and senolytic-dosed senescent cells grow. Comparing the chemical profile in the cell environments can be a potential strategy in understanding the mechanisms of the senolytic drugs. This information may also be valuable in predicting the toxicity and side effects of the new senolytic drugs. Citation Format: Evelyn Wang, Stephen Kurzyniec, Ethan Hain, Yoshiyuki Okamura, Yang Yang, Dongwen Lv. Characterizing cellular senescence response to senolytic compounds using cell culture media analysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4792.

Therapeutic potential of the novel Bcl-2/Bcl-XL dual inhibitor, APG1252, alone or in combination against non-small cell lung cancer.

Targeting the induction of apoptosis is a promising cancer therapeutic strategy with some clinical success. This study focused on evaluating the therapeutic efficacy of the novel Bcl-2/Bcl-XL dual inhibitor, APG1252-M1 (also named APG-1244; an in vivo active metabolite of APG1252 or pelcitoclax), as a single agent or in combination, against non-small cell lung cancer (NSCLC) cells. APG1252-M1 effectively decreased the survival of some NSCLC cell lines expressing low levels of Mcl-1 and induced apoptosis. Overexpression of ectopic Mcl-1 in the sensitive cells substantially compromised APG1252-M1's cell-killing effects, whereas inhibition of Mcl-1 greatly sensitized insensitive cell lines to APG1252-M1, indicating the critical role of Mcl-1 levels in impacting cell response to APG1252-M1. Moreover, APG1252-M1, when combined with the third generation epidermal growth factor receptor (EGFR) inhibitor, osimertinib, synergistically decreased the survival of EGFR-mutant NSCLC cell lines including those resistant to osimertinib with enhanced induction of apoptosis and abrogated emergence of acquired resistance to osimertinib. Importantly, the combination was effective in inhibiting the growth of osimertinib-resistant tumors in vivo. Collectively, these results demonstrate the efficacy of APG1252 alone or in combination against human NSCLC cells.

First-in-human study of pelcitoclax (APG-1252) in combination with paclitaxel in patients (pts) with relapsed/refractory small-cell lung cancer (R/R SCLC).

e20612 Background: Targeting BCL-2/BCL-xL proteins is considered an important approach for anticancer drug development. Investigational pelcitoclax (APG-1252) as a single agent is a novel BCL-2/BCL-xL dual inhibitor, which has demonstrated synergistic antitumor activity in combination with chemotherapy preclinically through targeting both BCL-xL and MCL-1 pathways. The phase I study of pelcitoclax suggested promising antitumor activity and a favorable safety profile. Methods: The primary aim of this study was to determine the safety and preliminary efficacy of pelcitoclax combined with paclitaxel in patients with R/R SCLC. Patients received pelcitoclax 160 mg or 240 mg IV infusion on Days 1, 8, and 15 plus paclitaxel 80 mg/m2 on Days 1 and 8 of a 21-day cycle. The primary endpoint of phase Ib was to characterize the safety and tolerability of the combination. The primary endpoint of phase 2 was the objective response rate (ORR) of pelcitoclax at the RP2D plus paclitaxel using RECIST v1.1. Results: On December 20, 2021, 28 pts (median age, 63 [range, 37-77] years) were enrolled. The median (range) duration of treatment was 84 (7-259) days. The RP2D of pelcitoclax was determined to be 240 mg. A total of 26 pts experienced any grade treatment-related adverse events (TRAEs), including anemia (32.1%); ALT or AST elevation (28.6% each); neutropenia (25%); fatigue, leukopenia, or thrombocytopenia (21.4% each); peripheral neuropathy, nausea, or alopecia (17.9% each); peripheral sensory neuropathy or decreased appetite (14.3% each); and lymphopenia, hyponatremia, or dizziness (10.7% each). A total of 15 pts had at least one ≥ grade 3-4 TRAE, and 11 (39.3%) had serious AEs. No dose-limiting toxicity was reported. A total of 5 of 20 evaluable pts (4 pts in the 240 mg cohort and 1 in the 160 mg cohort) reported PR (ORR 25%), with a median response duration of 83 days. The pharmacokinetics profile showed that pelcitoclax systemic exposure increased between 160 and 240 mg, with an average terminal half-life of 3.7 to 7.4 hours and without any drug-drug interaction. Conclusions: Pelcitoclax was well tolerated at doses of up to 240 mg/week. Treatment with pelcitoclax 240 mg and paclitaxel 80 mg/m2 demonstrated modest antitumor activity in pts with R/R SCLC. Further clinical exploration, preferably in selected patient populations with relevant biomarkers, is warranted to fully elucidate the clinical activity of pelcitoclax. Clinical trial information: NCT04210037.

Mcl-1 levels critically impact the sensitivities of human colorectal cancer cells to APG-1252-M1, a novel Bcl-2/Bcl-XL dual inhibitor that induces Bax-dependent apoptosis

New treatment options, such as targeted therapies, are urgently needed for the treatment of colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide. The current study focuses on demonstrating the therapeutic efficacies of APG-1252-M1 (an active form of the prodrug, APG-1252 or pelcitoclax), a highly potent Bcl-2/Bcl-XL dual inhibitor in clinical trials, against CRC and understanding the underlying mechanisms. APG-1252-M1 effectively decreased the survival of CRC cell lines, particularly those expressing relatively low levels of Mcl-1, with the induction of apoptosis. High levels of Mcl-1 were significantly correlated with decreased sensitivity of CRC cell lines to APG-1252-M1. When combined with an Mcl-1 inhibitor, APG-1252-M1 synergistically decreased the survival and induced apoptosis of APG-1252-M1-insensitive cell lines with high levels of Mcl-1. This combination further decreased the survival and enhanced apoptosis even in sensitive cell lines with relatively low levels of Mcl-1, whereas enforced expression of ectopic Mcl-1 in these cells abrogated APG-1252-M1’s effects on decreasing cell survival and inducing apoptosis, which could be reversed by Mcl-1 inhibition. APG-1252-M1 rapidly induced cytochrome C and Smac release from mitochondria with caspase-3 and PARP cleavage. Deficiency of Bax in CRC cells abolished APG-1252-M1’s ability to induce apoptosis, indicating that APG-1252-M1 induces Bax-dependent apoptosis. The current study thus demonstrates the potential of APG-1252-M1 as a monotherapy in the treatment of CRC, particularly those with low Mcl-1 expression, or in combination with an Mcl-1 inhibitor, warranting further evaluation in vivo and in the clinic.

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

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

Abstract 977: Mcl-1 levels critically determines the sensitivities of human colon cancer cells to APG1244, a novel Bcl-2 and Bcl-XLinhibitor, that induces Bax- and caspase-8- dependent apoptosis

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide due to its poor prognosis. Most of the patients receive surgical resection when diagnosed as CRC, whereas the recurrence rate is very high. Therefore, additional treatment options, such as targeted therapy, are still urgently needed. The novel BH3 mimetic, APG1244 (a prodrug for APG1252), is a highly potent Bcl-2 and Bcl-XL dual inhibitor and being tested in clinical trials as a potential cancer therapeutic agent. The current study focuses on demonstrating the therapeutic efficacies of APG1244 against colorectal cancer and understanding the underlying mechanisms. A panel of CRC cell lines with different levels of Bcl-2, Bcl-XL and Mcl-1 possessed different sensitivities to APG1244. APG1244 effectively decreased the survival of CRC cell lines, particularly those expressing relative low levels of Mcl-1, with induction of apoptosis. High levels of Mcl-1 in these cell lines were significantly correlated to decreased sensitivity to APG1244. APG1244, when combined with a Mcl-1 inhibitor, synergistically decreased the survival and induced apoptosis of APG1244-insensitive cell lines with high levels of Mcl-1. This combination could further decreased the survival and enhanced apoptosis even in the cell lines with relatively low levels of Mcl-1, whereas enforced expression of ectopic Mcl-1 in these cells abrogated APG1244's effects on decreasing cell survival and inducing apoptosis, which can be reversed by Mcl-1 inhibition. In our study, it appears that APG1244 quickly activated caspase-8 and caspase-3 and induced PARP cleavage with induction of cytochrome C and Smac release from mitochondria. Deficiency of Bax, Smac, Bim or PUMA, but not FADD, in CRC cells protected (BAX-KO) or partially protected cells from APG1244-induced apoptosis, indicating that APG1244 induces Bax-dependent apoptosis. Furthermore, blockage of caspase-8 with a caspase-8 specific inhibitor abolished cleavage of caspase-3 and PARP including attenuation of cytochrome C and Smac release induced by APG1244, suggesting a caspase-8-dependent mechanism. The current study thus demonstrates the potential of APG1244 as a monotherapy in the treatment of CRC, particularly those with low Mcl-1 expression, or in combination with a Mcl-1 inhibitor for CRC treatment, warranting further evaluation in vivo and in the clinic. Citation Format: Weilong Yao, Shi-Yong Sun. Mcl-1 levels critically determines the sensitivities of human colon cancer cells to APG1244, a novel Bcl-2 and Bcl-XLinhibitor, that induces Bax- and caspase-8- dependent apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 977.

Abstract 3075: Anti-leukemic activity of BCL-2/BCL-XL dual inhibitor - AZD0466 in T-acute lymphoblastic leukemia preclinical models

Abstract Upregulation of BCL-2 family proteins, such as BCL-2 and BCL-XL, leads to imbalanced ratio between pro-death and pro-survival proteins that favors leukemic survival, tumorigenesis, and is an important driver of chemoresistance. B- and T-ALL JAK-STAT signaling pathway is positively correlated with increased BCL-2 family function. Importantly, this pathway plays an important role in the switch between BCL-2 and BCL-XL dependencies in normal developing T-cells. However, recent studies have shown that co-expression of BCL-2 and BCL-XL is linked with higher therapeutic resistance in different types of ALL. In this study, we tested BCL-2 and BCL-XL dependencies among different subtypes of ALL and the potential to revert these using AZD0466, a novel drug-dendrimer conjugate, of a BCL-2/ BCL-XL inhibitor AZD4320 and an FDA-approved BCL-2 inhibitor, ABT-199 (Venetoclax). The analysis of protein expression patterns in ALL cell lines revealed increased levels of BCL-2 in B-ALL cell lines, and of BCL-XL in Ph-like B-ALL and T-ALL cell lines. The analysis of cell viability assay revealed most B-ALL cell lines show sensitivity to both ABT-199 and AZD4320, except NALM6 which lacks the expression of the executioner Bax protein. Ph-like B-ALL and T-ALL cell lines on the other hand are most sensitive to the BCL-2/BCL-XL dual inhibitor, AZD4320. Additionally, Ph-like B-ALL and T-ALL PDX cells responded greater to dual inhibitor. The analysis of BH3 profiling of ALL cell lines demonstrated BCL-2 and BCL-XL codependence in Ph-like B-ALL and T-ALL cell lines while major BCL-2 dependence was seen in B-ALL cell lines. Our pre-clinical studies in T-ALL PDX models showed moderate response to ABT-199 and significantly prolonged survival and decreased leukemic burden mice treated with dual BCL-2/BCL-XL inhibitor AZD0466 (30mg/kg/weekly/IV). Importantly, AZD0466 treated cohorts did not show any significant change in the body weight and platelet counts when compared to control animals. Comparison of pharmacological response to dual inhibitor AZD0466 treatments in B-ALL PDX models demonstrated lower therapeutic efficacy than observed in T-ALL PDX, while in the same T-ALL PDX ABT-199 (100mg/kg/daily/oral) showed reduced efficacy. Further studies including BH3 profiling to measure the dynamic response of mitochondria and determine prior BH3 domain dependencies are in progress and will be reported. Our findings suggest that the novel dual BCL-2/BCL-XL inhibitor AZD0466 outperforms single BCL-2 inhibition by ABT-199 in T-ALL and Ph-like B-ALL. These findings will facilitate translation of AZD0466 dual BCL-2/BCL-XL inhibitor into ALL clinical trials, alone or in combination with standard chemotherapy and monoclonal antibodies. Citation Format: Sankaranarayanan Kannan, Sanaz Ghotbaldini, Julia E. Wells, Qi Zhang, Srividya Balachander, Justin Cidado, Marina Konopleva. Anti-leukemic activity of BCL-2/BCL-XL dual inhibitor - AZD0466 in T-acute lymphoblastic leukemia preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3075.

Abstract 6223: Combination of BCL-2/BCL-xL dual inhibitor APG-1252 and chemotherapeutics overcomes resistance to osimertinib in EGFR mutant NSCLC in preclinical models

Abstract Osimertinib (AZD9291) is the first-line treatment for EGFR-mutated NSCLC; however, the majority of patients inevitably develop resistance due to de novo genomic abnormalities, such as C797S mutation, EGFR exon 20 insertion, MET amplification and other unknown mechanisms. Hence, effective therapies to overcome acquired resistance are urgently needed. Inhibition of BCL-2/BCL-xL has been reported to enhance apoptosis in EGFR-TKI resistant cells with low sensitivity to EGFR inhibition. In this study, we evaluated whether the combination of a dual BCL-2/BCL-xL inhibitor APG-1252 and chemotherapeutics could overcome osimertinib resistance in preclinical xenograft models. First, EGFR C797S mutation was introduced to human NCI-H1975 NSCLC cells to construct an NCI-H1975-C797S resistant cell line using CRISPR technology. NCI-H1975-C797S derived-xenografts were treated with APG-1252, cisplatin/docetaxel or their combinations. Combination therapy with APG-1252 and cisplatin or docetaxel exhibited synergistic antitumor activity. APG-1252 plus docetaxel combination achieved 100% tumor partial regression (PR). Similar results were demonstrated in a patient-derived xenograft (PDX) tumor model derived from an osimertinib-resistant NSCLC patient harboring 19del-T790M-C797S mutations. Furthermore, the combinations also exhibited enhanced antitumor activity in an osimertinib-resistant PDX model that the resistant mechanism remained unknown. In summary, our results suggest that the combination treatment with APG-1252 and chemotherapeutics can overcome acquired resistance to osimertinib and the combination deserves further clinical evaluations. Citation Format: Ran Tao, Guangfeng Wang, Douglas D. Fang, Guoqin Zhai, Yuanbao Li, Jing Lv, Miaoyi Wu, Yangfeng Ge, Feifei Zhang, Danyi Wen, Dajun Yang, Yifan Zhai. Combination of BCL-2/BCL-xL dual inhibitor APG-1252 and chemotherapeutics overcomes resistance to osimertinib in EGFR mutant NSCLC in preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6223.

Abstract 74: Co-targeting BCL-xL and HER2 high expression to overcome apoptosis blockade in gastric cancer

Abstract Background: Gastric cancer is one of the most prevalent cancers in the East Asia population. The five-year survival rate is approximately 20% globally. The analysis of gene expression data suggests that anti-apoptotic protein BCL-xL may be the oncogenic driver in gastric cancer as its expression levels are much higher than BCL-2 (Pan Cancer Atlas). In this study, we investigated if a clinical stage BCL-2/BCL-xL dual inhibitor APG-1252 would elicit therapeutic activity and associated mechanisms using a panel of gastric cancer patient-derived xenograft (PDX) models. Additionally, we explored if the combination with HER2 inhibition could enhance the antitumor activity in HER2+ gastric cancer models. Methods and Experiments: Eighteen PDX models of gastric cancer that bore high levels of BCL-xL expression (BCL-xLhigh) based on the gene expression profiles were selected for a mouse trial (n=2) with a dosage regimen of APG-1252 (100 mg/kg, BIW, IV) for three weeks. In a combination study (n=5), APG-1252 (50 mg/kg, BIW, IV) and lapatinib (100 mg/kg, QD, PO) were concurrently administered for three weeks. An advanced ELISA (MSD) and Western blot analysis were performed to detect BCL-xL complex and the protein level, respectively, in tumor samples. Results: APG-1252 treatment resulted in tumor growth inhibition (TGI greater than 50%) in four PDXs (20%), including three exhibiting TGI greater than 80%. PD study confirmed that BCL-xL protein levels were high, and bound with pro-apoptotic proteins PUMA (major binding partner) and BIM in the xenograft tumors of these models. Generally, in comparison with hematologic cancer cells which exhibit dominant BCL-2 complexes (i.e., Toledo), the levels of BCL-xL complexes were consistently higher than those of BCL-2 complexes in the gastric cancer PDXs, implicating a crucial role of BCL-xL in the solid tumors. After treatment with APG-1252, BCL-xL:PUMA, were disrupted and, interestingly, such a decrease in BCL-xL complex signal correlated with TGI. Additionally, the baseline levels of BCL-xL complexes also correlated with TGI. Furthermore, a consequent increase in MCL-1 complexes after APG-1252 treatment indicated that such a compensatory mechanism among anti-apoptotic proteins may confer the drug resistance. Finally, in mouse tumor models derived from HER2+ gastric cancer cell line NCI-N87 or patient-derived xenografts (PDXs), combined treatment with AGP-1252 and HER2 inhibitor lapatinib synergistically inhibited tumor growth, suggesting that lapatinib may be able to increase mitochondrial priming, thus sensitizing cancer cells to APG-1252. Conclusion: Our results demonstrate the on-target antitumor activity of APG-1252, the potential of BCL-xLhigh as a predictive biomarker, and the resistance mechanism conferred by MCL-1. Furthermore, the data provide a scientific rationale for the combined therapy with BCL-xL and HER2 inhibitors to achieve better clinical outcomes in a subset of HER2+ gastric cancers. Citation Format: Jing Deng, Xiaojing Huang, Guoqin Zhai, Kaixiang Zhang, Jiaxing Gu, Tingting Mao, Yan Yin, Ran Tao, Douglas D. Fang, Dajun Yang, Yifan Zhai. Co-targeting BCL-xL and HER2 high expression to overcome apoptosis blockade in gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 74.

Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity

Small molecules that selectively kill senescent cells (SCs), termed senolytics, have the potential to prevent and treat various age-related diseases and extend healthspan. The use of Bcl-xl inhibitors as senolytics is largely limited by their on-target and dose-limiting platelet toxicity. Here, we report the use of proteolysis-targeting chimera (PROTAC) technology to reduce the platelet toxicity of navitoclax (also known as ABT263), a Bcl-2 and Bcl-xl dual inhibitor, by converting it into PZ15227 (PZ), a Bcl-xl PROTAC, which targets Bcl-xl to the cereblon (CRBN) E3 ligase for degradation. Compared to ABT263, PZ is less toxic to platelets, but equally or slightly more potent against SCs because CRBN is poorly expressed in platelets. PZ effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia. With further improvement, Bcl-xl PROTACs have the potential to become safer and more potent senolytic agents than Bcl-xl inhibitors.

A novel Bcl-2 inhibitor, BM-1197, induces apoptosis in malignant lymphoma cells through the endogenous apoptotic pathway

BackgroundBcl-2 family members play an important role in the development of malignant lymphoma and can induce drug resistance in anticancer treatment. The development of small molecules targeting Bcl-2 family proteins may be a new strategy for the treatment of malignant lymphoma. In this study, we investigate the antitumor effect and cellular mechanism of a novel Bcl-2/Bcl-xL dual inhibitor, BM-1197, in DCBCL and Burkitt lymphoma cells.MethodsThe CCK-8 assay was used to detect cell viability. Apoptosis was determined by Hoechst 33258 staining and flow cytometry. The activity of caspase-3/caspase-9 was determined using a caspase-3/caspase-9 activity kit. Western blotting analysis was performed to evaluate the changes in protein expression. Functional analysis was performed via immunoprecipitation and siRNA interference. Human malignant lymphoma xenograft models in nude mice were established for in vivo efficacy detection.ResultsWe find that BM-1197 exerts potent growth-inhibitory activity against lymphoma cells that harbor high expression of Bcl-2 and Bcl-xL in vitro and has a synergistic effect with chemotherapeutic drugs. Mechanistically, we see that the intrinsic apoptosis pathway is activated upon BM-1197 treatment. BM-1197 affects the protein interactions of Bak/Bcl-xl, Bim/Bcl-2, Bim/Bcl-xl, and PUMA/Bcl-2 and induces conformational changes in the Bax protein, which result in the activation of Bax and release of cytochrome c, activate caspase − 9, − 3, and − 7 and finally induce cell apoptosis. Furthermore, our data demonstrate that BM-1197 exhibits strong anti-tumor effects against established human malignant lymphoma xenograft models.ConclusionsOur study demonstrated BM-1197 exerts potent antitumor effects both in vitro and in vivo and provides promising preclinical data for the further development of BM-1197 in malignant lymphoma.

A Novel BCL-2/BCL-Xl Dual Inhibitor Overcomes Ibrutinib-Resistance Conferred By the Upregulation of Integrin Pathway and BCL-Xl

Introduction Ibrutinib, a first-in-class oral covalent inhibitor of Bruton's tyrosine kinase (BTK), has been FDA-approved for the treatment of mantle cell lymphoma, chronic lymphocytic leukemia (CLL) and Waldenström's macroglobulinemia. In CLL, 80% overall response rate (OR) as a monotherapy or 96% OR in the combination with BCL-2 selective inhibitor venetoclax demonstrates a promising regimen for CLL patients. However, ibrutinib only yields to a modest (38%) OR in follicular lymphoma (FL) trial, especially doing poorly in patients with NF-kB constitutive activation via CARD11 mutation. To better understand ibrutinib resistance mechanism, we established a resistance model using sensitive follicular lymphoma cell line DOHH2, and characterized the gene expression profile and signaling pathway alteration, and tested potential approach to overcome the resistance. Methods 1. DOHH2-IR (ibrutinib resistant) cells were established by exposing to the increasing concentrations of the drug over 3 months and confirmed by cell viability assays. 2. RNA-seq was performed to reveal the transcriptome alteration between DOHH2 and DOHH2-IR cells. 3. Cell sensitivity to BCL-2/BCL-xL inhibitors was assessed by CTG assays. Results Parental DOHH2 and resistant DOHH2-IR cells were first confirmed ibrutinib sensitivity/resistance by CTG assay, which showed an IC50 of 0.074 uM for DOHH2, but 160 fold greater for DOHH2-IR at 12.19 uM. To decipher the mechanisms underlying the induced ibrutinib resistance, whole-transcriptome RNA-seq was performed on parental and resistant cells. Unsupervised hierarchical clustering showed significant alterations in gene expression signatures. Among them, 4,925 genes were significantly up-regulated and 3,727 genes were down-regulated. Kyoto Encyclopedia of Genes and Genome (KEGG) analysis showed that the components of BCR signaling pathway had been significantly downregulated in the resistant cells, so were those in pro-survival NF-kB and mTOR pathways, anti-apoptotic BCL-2 and BFL-1 genes, and TP53 pathway; in contrast, BCL-xL, MAP kinase and extracellular matrix (ECM)-receptor interaction genes, especially those integrin family genes, were increased (Figure 1). The upregulation of BCL-xL gene expression prompted us to investigate if inhibiting BCL-xL would resume cell sensitivity to killing. We thus treated cells with APG-1252-M1 (ester hydrolyzed active metabolite of APG-1252 for in vitro study), a novel BCL-2/BCL-xL dual inhibitor developed by Ascentage. In comparison, we also treated cells with BCL-2 selective inhibitor APG-2575, which is also a proprietary compound by Ascentage, and its reference compound ABT-199. APG-2575 and ABT-199 partially restored cell death in the resistant cells, with IC50s of 0.65 uM and 1.19 uM, respectively, which were greater than parental cells with IC50s of approximate 0.05 uM. However, the dual inhibitor APG-1252-M1 effectively killed DOHH2-IR cells with an IC50 of 0.017 uM, and cell death was potently elicited within 24 hours (Figure 2). This finding is consistent with RNA-seq data, that BCL-2 is downregulated, while BCL-xL is induced during ibrutinib resistant selection. Conclusions In an ibrutinib-resistant DOHH2 model, we show that reprogram of BTK and apoptosis pathway is associated with ibrutinib resistance. Instead of mutating BTK or its downstream PLCg2 gene in CLL, resistant cells have selected to bypass the pathway; and unexpectedly, NF-kB pathway is also downregulated in DOHH2-IR, which is contrast to previously report that it is one of resistant mechanisms for ibrutinib. Evading apoptosis is one of the key mechanisms in which cancer cells can survive or become resistant to therapies. Integrin family proteins or ECM - receptor interactions also play important roles in drug resistance. Further studies are needed to understand how the components of integrin pathway cross-talk with BCL-2 family proteins. However, the potent killing activity conferred by APG-1252 confirms that BCL-xL upregulation is key in this FL ibrutinib resistance model. Collectively, the results suggest that the combination of ibrutinib and BCL-2/BCL-xL dual inhibitor may produce deeper and longer duration response in follicular lymphoma patients. Disclosures Deng: Ascentage Pharma Group: Employment. Mao:Ascentage Pharma Group: Employment. Huang:Ascentage Pharma Group: Employment. Rui:Ascentage Pharma Group: Employment. Wang:Ascentage Pharma Group: Employment. Fang:Ascentage Pharma Group: Employment. Yang:Ascentage Pharma Group: Employment. Zhai:Ascentage Pharma Group: Employment.

A Novel Bcl-2 Inhibitor, BM-1197, Induces Apoptosis in Malignant Lymphoma Cells Through the Endogenous Apoptotic Pathway

Background: Bcl-2 family members play an important role in the development of malignant lymphoma and can induce drug resistance in anticancer treatment. The development of small molecules targeting Bcl-2 family protein can be new strategy for malignant lymphoma treatment. In this study, we investigate the antitumor effect and the cellular mechanism of a novel Bcl-2/Bcl-xL dual inhibitor BM-1197 in DCBCL and Burkitt lymphoma cells. Methods: CCK-8 assay was used to detect cell viability. Apoptosis was determined by Hoechst 33258 staining and flow cytometry. The activity of caspase-3/caspase-9 was determined using the caspase-3/ caspase-9 activity kit. Western blotting analysis was performed to evaluate the change of protein expression. The functional analysis was evalueated via immunoprecipitation and siRNA interference. Human malignant lymphoma xenograft models in nude mice were established for in vivo efficacy detection. Results: We find that BM-1197 exerts potent growth-inhibitory activity against lymphoma cells which harbor Bcl-2 and Bcl-xL high expression in vitro and has synergistic effect with chemotherapeutic drugs. Mechanistically, we see that the intrinsic apoptosis pathway is activated upon BM-1197 treatment. BM-1197 affects the protein interaction of Bak/Bcl-xl, Bim/Bcl-2, Bim/Bcl-xl, PUMA/Bcl-2 and induced conformational change in the Bax protein.which results in activation of Bax and release cytochrome c, and activated caspase -9, -3, -7 and finally induce cell apoptosis. Furthermore, our data demonstrates that BM-1197 exhibits strong anti-tumor effects against established human malignant lymphoma xenograft models. Conclusions: Our study demonstrated BM-1197 exerts potent antitumor effects both in vitro and in vivo, and provides promising preclinical data for further development of BM-1197 in malignant lymphoma. Funding Statement: This work is supported by National Natural Scientific Research Fund of China (Project Number: 81301904), Medical Scientific Technology and Research Fund of Guangdong (Project Number: A2016023), Special Project by Guangdong Science and Technology Department (2018YJ021), and National Natural Scientific Research Fund of China (Project Number: 81101671) Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal experiments were performed under the guidance of Sun Yat-Sen University Committee for Use and Care of Laboratory Animals and approved by the animal experimentation ethics committee.

Selective targeting of antiapoptotic BCL-2 proteins in cancer.

Circumvention of apoptotic machinery is one of the distinctive properties of carcinogenesis. Extensively established key effectors of such apoptotic bypass mechanisms, the antiapoptotic BCL-2 (apoptosis regulator BCL-2) proteins, determine the response of cancer cells to chemotherapeutics. Within this background, research and development of antiapoptotic BCL-2 inhibitors were considered to have a tremendous amount of potential toward the discovery of novel pharmacological modulators in cancer. In this review, milestone achievements in the development of selective antiapoptotic BCL-2 proteins inhibitors for BCL-2, BCL-XL (BCL-2-like protein 1), and MCL-1 (induced myeloid leukemia cell differentiation protein MCL-1) were summarized and their future implications were discussed. In the first section, the design and development of BCL-2/BCL-XL dual inhibitor navitoclax, as well as the recent advances and clinical experience with selective BCL-2 inhibitor venetoclax, were synopsized. Preclinical data from selective BCL-XL inhibitors, which are currently undergoing extensive testing as a single agent or in combination with other therapeutic agents, were further summarized. In the second section, MCL-1 inhibitors developed as potential anticancer agents were reviewed regarding their specificity toward MCL-1. Explicitly, studies leading to the identification of MCL-1, nonselective and selective targeting of MCL-1, and recently initiated clinical trials were compiled in chronological order. Based on these concepts, future directions were further discussed for increasing selectivity in the design of prosurvival BCL-2 member inhibitors.

Abstract 2530: Bcl-2 and Bcl-xL dual inhibitor, ABT-737, circumvents JNK mediated upregulation of anti-apoptotic molecules in cisplatin treated non-small cell lung cancer models

Abstract Introduction; Non-small cell lung cancer (NSCLC), which does not have druggable driver oncogene, is treated with cytotoxic chemotherapy, but clinical outcome is suboptimal. Cisplatin is still one of the most commonly used drugs for the treatment of NSCLC, and, however, there are limitations of the therapeutic effect because of the multiple resistance mechanisms. In particular, elevated level of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1) triggered by cisplatin-induced DNA damage response correlate with the drug resistance. In this study, we investigated synergic effects of combined treatment with cisplatin and dual inhibitor of Bcl-2/Bcl-xL, ABT-737, in NSCLC models. Materials and Methods; H460 and A549 NSCLC cells, and LSL K-Ras G12D mouse model were used. Protein expression was evaluated by immunoblotting and cytotoxicity was measured by FACS followed by annexin-V and PI staining. LSL Kras G12D mouse were inhaled 5 × 107 PFU AdCre virus at 8 weeks after birth and, in 24 weeks after inhalation, were taken microCT, randomized into 4 groups, and then treated with ABT-737 (50mg/kg, i.p., daily) and/or cisplatin (5 mg/kg, i.p., weekly) for two weeks. Results; There was a dose dependent phosphorylation of JNK, which is most prominent between 6 to 24hr after cisplatin treatment. Expression of anti-apoptotic molecules, Bcl-2, Bcl-w, Bcl-xL, and Mcl-1 were increased by cisplatin treatment in a dose and time dependent manner. JNK specific activator, anisomycin, increased mRNA level of anti-apoptotic molecules. ABT-737 displaced BCL-xL to the cytoplasm and aggregated Bak. Furthermore, ABT-737 released cytochrome C from mitochondria to the cytoplasm. ABT-737 itself showed cytotoxic effects and combination of ABT-737 potentiates cytotoxic effects of cisplatin with strong synergism. Combination of ABT-737 with cisplatin treatment induced significant tumor regression in 2 weeks in LSL K-ras G12D mouse model when compared those treated with single agents or vehicle. Conclusion; Combination of ABT-737 with cisplatin showed synergistic cytotoxic effects and anti-tumor activities in NSCLC preclinical models. These findings suggest that clinical trials using combination with ABT-737 and cisplatin may be beneficial in advanced NSCLC patients. Citation Format: Eun Young KIM, Arum Kim, Yoon Soo Chang. Bcl-2 and Bcl-xL dual inhibitor, ABT-737, circumvents JNK mediated upregulation of anti-apoptotic molecules in cisplatin treated non-small cell lung cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2530. doi:10.1158/1538-7445.AM2015-2530

Abstract PR01: Adaptive resistance of patient-derived ovarian cancer cells to PI3K/mTOR inhibition

Abstract Relapse due to drug resistance remains a major challenge for successful cancer treatment despite advances in chemotherapy efficacy and the development of targeted therapies. We have previously reported a therapy-induced adaptive response that is associated with treatment resistance and are investigating whether this adaptive response program could be targeted to increase treatment efficacy (Muranen et al, Cancer Cell 2012). The PI3K pathway is amplified in high-grade serous ovarian tumor and represents an attractive target in this cancer subtype. We found that both in vitro and in vivo exposure of patient-derived ovarian cancer cells to the PI3K/mTOR dual inhibitor GNE-493 increases the levels of the anti-apoptotic Bcl-2 protein, and multiple tyrosine kinase receptors, such as EGFR and HER2. Concurrent treatment of these cancer cells with GNE-493 and ABT737 (a Bcl-2 and Bcl-xl dual inhibitor) causes a reduction in cell number and increased cell death. Ongoing studies are underway to comprehensively characterize the adaptive response signature of a panel of patient-derived ovarian cancer cells and study whether targeting multiple different upregulated proteins (e.g. EGFR) may increase PI3K/mTOR treatment efficacy. Therapy resistance has also been associated with exposure of tumor cells to pro-survival factors secreted by supporting cells in the tumor microenvironment (Straussman et al, Nature 2012 and Wilson et al, Nature 2012). We found that the growth-factors EGF, inflammatory cytokine IL-6 and ascites fluid from ovarian cancer patients induces up-regulation of Bcl-2 as well as IGFR, which masked the adaptive response to PI3K/mTOR inhibitors. We are performing ongoing studies using orthotopic patient-derived ovarian cancer xenografts to investigate the patterns of residual disease in vivo with the ultimate goal to identify whether Bcl-2 targeted drugs will enhance the efficacy of PI3K/mTOR inhibition and whether there is differential sensitivity at distinct anatomical sites (pancreas, diaphragm, peritoneal wall). Our studies provide a framework to identify adaptive response programs and tumor microenvironment factors in patient-derived ovarian cancer cells that limit treatment efficacy of PI3K/mTOR pathway inhibition and to develop therapies to abrogate this resistance. Citation Format: Ioannis Zervantonakis, Hsing-Yu Chen, Taru Muranen, Joyce Liu, Ronny Drapkin, Ursula Matulonis, Joan Brugge. Adaptive resistance of patient-derived ovarian cancer cells to PI3K/mTOR inhibition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR01.