Induction Of Bim Research Articles

BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAFV600E colorectal cancer

Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.

Abstract B072: Loss of the tumor and metastasis suppressor RasGAP DAB2IP mediates therapeutic resistance in HER2+ breast cancer

Abstract Resistance to HER2 inhibitors remains a clinical challenge in HER2+ breast cancer. Therefore, there is an urgent need to 1) understand the mechanisms that underlie resistance to these current treatments and 2) develop improved, and more importantly, curative combination therapies. We previously showed that the RasGAP DAB2IP is a tumor and metastasis suppressor in breast cancer. Interestingly, we have now generated robust data demonstrating that the loss of DAB2IP also mediates therapeutic resistance in HER2+ breast cancer. First, we genetically ablated DAB2IP in multiple HER2+ breast cancer cell lines and performed manual counting experiments after 6 days of HER2 TKI treatment. In all cell lines, DAB2IP knockdown conferred resistance to HER2 inhibitors. Since HER2 inhibitors are known to induce both apoptosis and senescence, we next investigated how DAB2IP loss specifically affects these phenotypes. Interestingly, DAB2IP knockdown prevented TKI-induced caspase-3/7 activation, measured by Incucyte live cell imaging, and enabled the regrowth of cells in long-term 10-day treatment and drug washout experiments, monitored by Incucyte or crystal violet staining. Mechanistically, stable knockdown of DAB2IP reduced the suppression of the AKT and ERK pathways and the induction of the pro-apoptotic protein Bim upon HER2i treatment. In addition, cell cycle progression pathways were enriched in DAB2IP-deficient cells on lapatinib treatment and immunoblots revealed higher residual levels of pRb and low levels of p27 in these cells. Interestingly, the levels of these proteins were restored and regrowth in low density assays was inhibited with co-treatment with an NF-kB inhibitor. We next evaluated the relevance of DAB2IP loss in mediating HER2 inhibitor resistance in a SUM190 orthotopic xenograft model. Importantly, while control tumors regressed upon lapatinib treatment, DAB2IP-deficient tumors did not regress and grew with kinetics comparable to vehicle-treated tumors after few days on treatment. Further understanding the pathways deregulated by DAB2IP loss upon HER2i treatment will be essential for developing effective therapeutic strategies to combat resistance induced by DAB2IP loss. Citation Format: Naiara Perurena, Amy Schade, Natalie Pilla, Patrick Loi, Carrie Rodriguez, Alycia Gardner, Karen Cichowski. Loss of the tumor and metastasis suppressor RasGAP DAB2IP mediates therapeutic resistance in HER2+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B072.

Circumvention of Gefitinib Resistance by Repurposing Flunarizine via Histone Deacetylase Inhibition.

Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) for treating advanced non-small cell lung cancer (NSCLC). However, drug resistance seriously impedes the clinical efficacy of gefitinib. This study investigated the repositioning of the non-oncology drug capable of inhibiting histone deacetylases (HDACs) to overcome gefitinib resistance. A few drug candidates were identified using the in silico repurposing tool "DRUGSURV" and tested for HDAC inhibition. Flunarizine, originally indicated for migraine prophylaxis and vertigo treatment, was selected for detailed investigation in NSCLC cell lines harboring a range of different gefitinib resistance mechanisms (EGFR T790M, KRAS G12S, MET amplification, or PTEN loss). The circumvention of gefitinib resistance by flunarizine was further demonstrated in an EGFR TKI (erlotinib)-refractory patient-derived tumor xenograft (PDX) model in vivo. The acetylation level of cellular histone protein was increased by flunarizine in a concentration- and time-dependent manner. Among the NSCLC cell lines evaluated, the extent of gefitinib resistance circumvention by flunarizine was found to be the most pronounced in EGFR T790M-bearing H1975 cells. The gefitinib-flunarizine combination was shown to induce the apoptotic protein Bim but reduce the antiapoptotic protein Bcl-2, which apparently circumvented gefitinib resistance. The induction of Bim by flunarizine was accompanied by an increase in the histone acetylation and E2F1 interaction with the BIM gene promoter. Flunarizine was also found to upregulate E-cadherin but downregulate the vimentin expression, which subsequently inhibited cancer cell migration and invasion. Importantly, flunarizine was also shown to significantly potentiate the tumor growth suppressive effect of gefitinib in EGFR TKI-refractory PDX in vivo. The findings advocate for the translational application of flunarizine to circumvent gefitinib resistance in the clinic.

Abstract P1-13-06: Loss of emerging tumor and metastasis suppressor RasGAPs mediates therapeutic resistance in HER2+ breast cancer

Abstract Resistance to HER2 inhibitors remains a clinical challenge in HER2+ breast cancer. Therefore, there is an urgent need to 1) understand the mechanisms that underlie resistance to these current treatments and 2) develop improved, and more importantly, curative combination therapies. We recently discovered that two emerging tumor suppressor RasGAPs, DAB2IP and RASAL2, cooperatively drive metastatic breast cancer when lost or inactivated. Interestingly, we have now generated robust data demonstrating that the loss of these RasGAPs also induces resistance to HER2 inhibitors in breast cancer. First, we genetically ablated both RASAL2 and DAB2IP in multiple HER2+ breast cancer cell lines (SKBR3, EFM192A, SUM190, BT474) and performed manual counting experiments after 6 days of TKI (lapatinib, tucatinib) treatment. In all cell lines, RASAL2/DAB2IP knockdown conferred resistance to HER2 inhibitors. Moreover, loss of these RasGAPs prevented TKI-induced caspase-3/7 activation, measured by Incucyte live cell imaging, and enabled the regrowth of cells in long-term 10-day treatment and drug washout experiments, monitored by Incucyte or crystal violet staining. Next, we sought to investigate the individual contribution of each RasGAP to these phenotypes. Surprisingly, we found that RASAL2 and DAB2IP functioned quite differently in this context. Specifically, while RASAL2 loss prevented apoptosis, DAB2IP loss prevented irreversible cell cycle arrest (measured by functional long-term experiments and EdU staining assays). Mechanistically, RASAL2 loss uniquely impaired BIM induction at both mRNA and protein levels, which is required for lapatinib-induced cell death of HER2+ cancer cells. By contrast, cell cycle progression pathways were uniquely enriched in DAB2IP-deficient cells on lapatinib treatment and immunoblots revealed higher residual levels of pRb and low levels of p27 in these cells. These data suggest that RASAL2 and DAB2IP (loss) mediate resistance to HER2 inhibitors by differentially deregulating unique pathways/phenotypes. We next evaluated the relevance of these findings in a SUM190 orthotopic xenograft model. Importantly, while control tumors (expressing both RASAL2 and DAB2IP) regressed upon lapatinib treatment, DAB2IP- and RASAL2-deficient tumors did not regress and grew with kinetics comparable to vehicle-treated tumors after few days on treatment. These data suggest that the unique phenotypes/pathways induced by both RASAL2 and DAB2IP are important mediators of resistance to HER2 inhibitors. Further understanding the contribution of these pathways to anti-HER2 resistance and determining how RASAL2 and DAB2IP differentially function will be essential for developing effective therapeutic strategies to bypass each type of resistance. Citation Format: Naiara Perurena, Natalie Pilla, Amy Schade, Marina Watanabe, Patrick Loi, Carrie L. Rodriguez, Alycia M. Gardner, Karen Cichowski. Loss of emerging tumor and metastasis suppressor RasGAPs mediates therapeutic resistance in HER2+ breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-06.

Subtype-selective induction of apoptosis in translocation-related sarcoma cells induced by PUMA and BIM upon treatment with pan-PI3K inhibitors

Translocation-related sarcomas (TRSs) harbor an oncogenic fusion gene generated by chromosome translocation and account for approximately one-third of all sarcomas; however, effective targeted therapies have yet to be established. We previously reported that a pan-phosphatidylinositol 3-kinase (PI3K) inhibitor, ZSTK474, was effective for the treatment of sarcomas in a phase I clinical trial. We also demonstrated the efficacy of ZSTK474 in a preclinical model, particularly in cell lines from synovial sarcoma (SS), Ewing’s sarcoma (ES) and alveolar rhabdomyosarcoma (ARMS), all of which harbor chromosomal translocations. ZSTK474 selectively induced apoptosis in all these sarcoma cell lines, although the precise mechanism underlying the induction of apoptosis remained unclear. In the present study, we aimed to determine the antitumor effect of PI3K inhibitors, particularly with regards to the induction of apoptosis, against various TRS subtypes using cell lines and patient-derived cells (PDCs). All of the cell lines derived from SS (six), ES (two) and ARMS (one) underwent apoptosis accompanied by the cleavage of poly-(ADP-ribose) polymerase (PARP) and the loss of mitochondrial membrane potential. We also observed apoptotic progression in PDCs from SS, ES and clear cell sarcoma (CCS). Transcriptional analyses revealed that PI3K inhibitors triggered the induction of PUMA and BIM and the knockdown of these genes by RNA interference efficiently suppressed apoptosis, suggesting their functional involvement in the progression of apoptosis. In contrast, TRS-derived cell lines/PDCs from alveolar soft part sarcoma (ASPS), CIC-DUX4 sarcoma and dermatofibrosarcoma protuberans failed to undergo apoptosis nor induce PUMA and BIM expression, as well as cell lines derived from non-TRSs and carcinomas. Thus, we conclude that PI3K inhibitors induce apoptosis in selective TRSs such as ES and SS via the induction of PUMA and BIM and the subsequent loss of mitochondrial membrane potential. This represents proof of concept for PI3K-targeted therapy, particularly such TRS patients.

BCL2L11 Induction Mediates Sensitivity to Src and MEK1/2 Inhibition in Thyroid Cancer.

Patients with advanced thyroid cancer, including advanced papillary thyroid cancer and anaplastic thyroid cancer (ATC), have low survival rates because of the lack of efficient therapies available that can combat their aggressiveness. A total of 90% of thyroid cancers have identifiable driver mutations, which often are components of the MAPK pathway, including BRAF, RAS, and RET-fusions. In addition, Src is a non-receptor tyrosine kinase that is overexpressed and activated in thyroid cancer, which we and others have shown is a clinically relevant target. We have previously demonstrated that combined inhibition of Src with dasatinib and the MAPK pathway with trametinib synergistically inhibits growth and induces apoptosis in BRAF- and RAS-mutant thyroid cancer cells. Herein, we identified the pro-apoptotic protein BCL2L11 (BIM) as being a key mediator of sensitivity in response to combined dasatinib and trametinib treatment. Specifically, cells that are sensitive to combined dasatinib and trametinib treatment have inhibition of FAK/Src, MEK/ERK, and AKT, resulting in the dramatic upregulation of BIM, while cells that are resistant lack inhibition of AKT and have a dampened induction of BIM. Inhibition of AKT directly sensitizes resistant cells to combined dasatinib and trametinib but will not be clinically feasible. Importantly, targeting BCL-XL with the BH3-mimeitc ABT-263 is sufficient to overcome lack of BIM induction and sensitize resistant cells to combined dasatinib and trametinib treatment. This study provides evidence that combined Src and MEK1/2 inhibition is a promising therapeutic option for patients with advanced thyroid cancer and identifies BIM induction as a potential biomarker of response.

Abstract 2668: Targeting of ERK and HDAC in the treatment of uveal melanoma

Abstract Uveal melanoma is one of the most common intraocular malignancies of the adult eye. GNAQ/GNA11 genetic alterations are present in 85% of cases resulting in activation of the MAPK pathway. Single agent approaches targeting the MAPK pathway and histone deacetylases (HDACs) have shown very modest activity in uveal melanoma. We hypothesize that a combination strategy may be an effective, novel therapeutic approach in uveal melanoma. We examined the activity of ASTX029, a novel dual-mechanism ERK inhibitor, which inhibits both the catalytic activity of ERK and its phosphorylation by MEK, and belinostat, a pan-HDAC inhibitor, in one GNAQQ209L- (92.1) and GNAQQ209P- (OMM1.3) and two GNA11Q209L-mutant (MP41, OMM1) uveal melanoma cell lines. The uveal melanoma cell lines were sensitive to ASTX029 with IC50 values between 50 to 100nM and to belinostat with IC50 values between 100 to 250nM. Combination index analysis of ASTX029 and belinostat by the Chou-Talalay method indicated synergism, with CI values ranging from 0.34 to 0.97. We explored other drug candidates for combinatorial approaches including the BCL-2 inhibitor navitoclax, but no combination effect was observed. After 48h of combination treatment with ASTX029 and belinostat, we examined the biochemical effects by western blot which demonstrated a sustained decrease in levels of p-ERK with the combination treatment. Western blot analysis confirmed reduction of downstream markers of MAPK pathway activity including cell cycle markers p-RB (780), cyclin D1 and c-myc with combination treatment. Apoptosis was evident from an increase in the subG1 population by flow cytometry analysis, which correlated with BIM induction and PARP cleavage on western blot. We also observed increased surface expression of immunological markers HLA-A, B, C (MHC Class 1), PD-L1, gp100 and MART-1. These results support that a combination strategy targeting ERK and HDAC in uveal melanoma should be investigated further. Citation Format: Elgilda Musi, Kim-Hien Dao, Chris Hindley, Gary K. Schwartz. Targeting of ERK and HDAC in the treatment of uveal melanoma [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 2668.

Abstract 4024: Combining selumetinib with BH3 mimetics enhances activity in MAPK-activated acute myeloid leukemia

Abstract Mitogen-activated protein kinase (MAPK) pathway alterations comprise some of the most frequent mutations in newly diagnosed acute myeloid leukemia (AML). Moreover, MAPK pathway alterations are also emerging as potential mechanisms of resistance to targeted therapy in AML including FLT3 inhibitors and venetoclax. In an ex vivo pharmacologic analysis of primary AML samples, sensitivity to the MEK inhibitor selumetinib (ARRY-142886) was enriched in patient samples resistant to venetoclax. Clinical activity of MAPK pathway inhibitors such as selumetinib has been explored in AML but monotherapy responses were modest. Another MEK inhibitor, cobimetinib, is currently being tested in combination with venetoclax in AML. We sought to understand whether combining selumetinib with BH3 mimetics such as venetoclax would improve efficacy in AML models. We evaluated combination activity of selumetinib plus venetoclax or the MCL1 inhibitor AZD5991 in a panel of AML cell lines. Cells were exposed to a 6x6 matrix of both agents for 72hrs and then viability assessed using CellTiter-Glo. Combination benefit was assessed using highest single agent (HSA) analysis. Selumetinib+AZD5991 demonstrated strong combination benefit (HSA score >0.1, Emax >0.5) in 4/19 AML cell lines. Selumetinib+venetoclax showed strong combination activity in 6/19 lines. Three lines showed benefit with both combinations. Many of these cell lines harbor MAPK pathway mutations including OCI-AML5 (SOS1N233Y, NF1K1385R), ML-2 (KRASA146T), HL-60 (NRASQ61L), and Nomo-1 (KRASG13D). Selumetinib treatment also led to robust BIM induction in vitro. Nomo-1 xenografts were evaluated for in vivo sensitivity to venetoclax (100 mg/kg qd PO), 5-azacytidine (1mg/kg BID q8h 3days on/4days off IP), AZD5991 (30mpk bid q2h qw IV), selumetinib (10 mg/kg bid q8h PO), as well as combinations of venetoclax+5-aza, AZD5991+selumetinib, and venetoclax+selumetinib. Venetoclax and venetoclax+5-aza treatment were ineffective. Selumetinib monotherapy led to 63% tumor growth inhibition (TGI) but tumors eventually grew out through treatment. The combination of selumetinib+venetoclax slightly improved efficacy (81% TGI) but markedly delayed tumor outgrowth (selumetinib monotherapy arm reached mean tumor volume >1000 mm3 on day 17, selumetinib+venetoclax reached average of ~966 mm3 on day 28). Reducing venetoclax dose (30mg/kg qd) or frequency (100mg/kg 3days on/4days off) maintained most of the combination efficacy. Selumetinb+AZD5991 also strongly improved efficacy compared to either monotherapy (88% TGI, mean tumor volume did not exceed ~400mm3 by day 28). Together these data suggest potential for combining MEK inhibitors with BH3 mimetics in AML. Work is ongoing to further understand how scheduling impacts combination efficacy, evaluate the triple combination of selumetinib+BH3 mimetic+azacytidine, and assess efficacy in disseminated models. Citation Format: Courtney L. Andersen, Azadeh Cheraghchi-Bashi, Patricia Jaaks, Elizabeth A. Coker, Kathleen Burke, Justin Cidado, Paul Smith, Corinne Reimer, Raoul Tibes, Mathew Garnett, Jerome Mettetal. Combining selumetinib with BH3 mimetics enhances activity in MAPK-activated acute myeloid leukemia [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 4024.

A chemoproteoinformatics approach demonstrates that aspirin increases sensitivity to MEK inhibition by directly binding to RPS5.

MEK inhibitors are among the most successful molecularly targeted agents used as cancer therapeutics. However, to treat cancer more efficiently, resistance to MEK inhibitor-induced cell death must be overcome. Although previous genetic approaches based on comprehensive gene expression analysis or RNAi libraries led to the discovery of factors involved in intrinsic resistance to MEK inhibitors, a feasible combined treatment with the MEK inhibitor has not yet been developed. Here, we show that a chemoproteoinformatics approach identifies ligands overcoming the resistance to cell death induced by MEK inhibition as well as the target molecule conferring this resistance. First, we used natural products, perillyl alcohol and sesaminol, which induced cell death in combination with the MEK inhibitor trametinib, as chemical probes, and identified ribosomal protein S5 (RPS5) as their common target protein. Consistently, trametinib induced cell death in RPS5-depleted cancer cells via upregulation of the apoptotic proteins BIM and PUMA. Using molecular docking and molecular dynamics (MD) simulations, we then screened FDA- and EMA-approved drugs for RPS5-binding ligands and found that acetylsalicylic acid (ASA, also known as aspirin) directly bound to RPS5, resulting in upregulation of BIM and PUMA and induction of cell death in combination with trametinib. Our chemoproteoinformatics approach demonstrates that RPS5 confers resistance to MEK inhibitor-induced cell death, and that aspirin could be repurposed to sensitize cells to MEK inhibition by binding to RPS5.

The Mitochondrial Disruptor Devimistat (CPI-613) Synergizes with Genotoxic Anticancer Drugs in Colorectal Cancer Therapy in a Bim-Dependent Manner.

Colorectal cancer is one of the most frequent tumor entities, with an increasing incidence and mortality in younger adults in Europe and the United States. Five-year survival rates for advanced colorectal cancer are still low, highlighting the need for novel targets in colorectal cancer therapy. Here, we investigated the therapeutic potential of the compound devimistat (CPI-613) that targets altered mitochondrial cancer cell metabolism and its synergism with the antineoplastic drugs 5-fluorouracil (5-FU) and irinotecan (IT) in colorectal cancer. Devimistat exerted a comparable cytotoxicity in a panel of established colorectal cancer cell lines and patient-derived short-term cultures independent of their genetic and epigenetic status, whereas human colonic epithelial cells were more resistant, indicating tumor selectivity. These findings were corroborated in intestinal organoid and tumoroid models. Mechanistically, devimistat disrupted mitochondrial membrane potential and severely impaired mitochondrial respiration, resulting in colorectal cancer cell death induction independent of p53. Combination treatment of devimistat with 5-FU or IT demonstrated synergistic cell killing in colorectal cancer cells as shown by Combenefit modeling and Chou-Talalay analysis. Increased cell death induction was revealed as a major mechanism involving downregulation of antiapoptotic genes and accumulation of proapoptotic Bim, which was confirmed by its genetic knockdown. In human colorectal cancer xenograft mouse models, devimistat showed antitumor activity and synergized with IT, resulting in prolonged survival and enhanced therapeutic efficacy. In human tumor xenografts, devimistat prevented IT-triggered p53 stabilization and caused synergistic Bim induction. Taken together, our study revealed devimistat as a promising candidate in colorectal cancer therapy by synergizing with established antineoplastic drugs in vitro and in vivo.

Abstract P078: Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer

Abstract Oncogenic KRAS mutation is common in non-small cell lung cancer (NSCLC) and portends poor outcome. Direct KRASG12C inhibitors have clinical activity; however, intrinsic and acquired resistance to these drugs limits their utility. Aurora A Kinase (AURKA), a mitotic cell cycle regulator, has been considered as a key druggable KRAS effector and mediates adaptive resistance to direct KRASG12C inhibitors. We found that AURKA expression correlated with poor outcome of lung cancer patients in caBIG, GEO and TCGA, and its expression was greater in KRAS-mutated NSCLC cells resistant to KRASG12C inhibitor sotorasib (AMG510) compared to normal human tracheobronchial epithelial (NHTBE), lung fibroblast, and KRAS wild-type cells. We have previously demonstrated synergistic antitumor effects for combination AURKA and WEE1 inhibition. Here, we explored a novel combination of AURKA and KRASG12C inhibition in KRAS(G12C)-mutated NSCLC cells intrinsically resistant to the KRASG12C inhibitor, along with a combination of AURKA and WEE1 inhibition in mutant KRAS(G12C) lung cancer cells with acquired resistance to the inhibitor. To overcome resistance to the KRASG12C inhibitor sotorasib, we tested combination treatment with VIC-1911, a newly synthesized selective AURKA inhibitor, with sotorasib in sotorasib-resistant human lung cancer cells. Cooperative screening, Loewe plotting, and clonogenic survival assays were performed to determine synergistic antitumor effects. In addition, we established KRAS(G12C)-mutated human lung cancer cell lines with acquired resistance to sotorasib through the means of escalated incremental dosing or sorting non-quiescent cells after drug exposure. Synergy was further confirmed with cell cycle distribution, phenotypic mitotic catastrophe on confocal microscopy, and induction of apoptosis. Interestingly, cells intrinsically resistant to sotorasib showed profound synergistic suppressive effect on cancer cell survival with addition of VIC-1911 compared to sotorasib-sensitive cells (Loewe synergy scores: NCI-H1792=16.03; HCC44=14.37; NCI-H358=1.48). Further, the combination of AURKA and WEE1 inhibition synergistically induced greater cell death in NCI-H358 lung carcinoma cells harboring acquired resistance to sotorasib, with dramatic induction of Bim. Moreover, the combination of AURKA and WEE1 inhibition resulted in a synergistic tumor control in KRAS/TP53-mutated lung cancer xenograft models. Our findings suggest AURKA activation leads to both intrinsic and acquired resistance to sotorasib in KRAS(G12C)-mutated NSCLC; therefore, addition of AURKA inhibition to sotorasib may be a promising therapeutic approach in NSCLC with intrinsic resistance to direct KRASG12C inhibitors, while the combination of AURKA and WEE1 inhibition merits exploration in acquired resistance to these agents. Citation Format: Jong Woo Lee, Sundong Kim, Cindy Yang, Barbara Burtness. Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P078.

Succinate Ubiquinone Reductase Activity Predicts Multiple Myeloma Sensitivity to Venetoclax

Multiple myeloma (MM) is a plasma cell malignancy estimated to account for 12,590 deaths and 30,280 new diagnoses in the US in 2017. Despite the use of next generation immunomodulatory drugs (IMiDs), proteasome inhibitors and newer targeted therapies, the development of chemo-resistant MM is common, with approximately 20% of patients succumbing to aggressive treatment-refractory disease within a short period of diagnosis. Resistance is primarily associated with evasion of apoptosis connected to the inability to release sufficient pro-apoptotic BCL-2 proteins above a threshold required to elicit apoptosis. Thus, finding alternative strategies to shift a resistant cell closer to the apoptotic threshold could potentially circumvent resistance.Metabolites contribute to the development of resistance to apoptosis by regulating pro and anti-apoptotic BCL-2 family members. We previously reported that depriving cells of glutamine enhanced binding of BIM to BCL-2 thereby sensitizing myeloma cell lines and patient samples to the BH3 mimetic venetoclax (ABT-199), a highly selective, potent BCL-2 antagonist. ABT-199 is however effective only in 40% of MM patients exhibiting the (11;14) translocation, warranting investigation of strategies to increase the applicability of ABT-199. We were interested to explore if there were metabolic differences in ABT-199 sensitive t(11;14) and resistant cells that could inform us of a) predictors of sensitivity; and b) metabolic targets that could be inhibited to sensitize resistant cells to ABT-199.Given that, tumor cells differentially rely on glycolysis and mitochondrial oxidative phosphorylation we first evaluated oxygen consumption rates (OCR) in ABT-199 sensitive and resistant MM lines. All ABT-199 sensitive lines exhibited lower OCR in contrast to the resistant lines. Metabolite profiling and isotope tracing flux analyses of glutamine deprived myeloma cell lines revealed specific reduction of TCA cycle metabolites including succinate. TCA cycle intermediates are linked to mitochondrial respiration via electron transport chain (ETC) complexes (I-V). Among the five ETC complexes, only Complex II/succinate dehydrogenase (SDH) is directly connected to the TCA cycle and ETC. SDH facilitates the oxidation of succinate to fumarate in the TCA cycle through its subunit SDHA, and transfers the released electrons to ubiquinone via its SDHB, C and D subunits supporting the succinate ubiquinone reductase (SQR) activity of Complex II. Therefore, we sought to further interrogate succinate dehydrogenase in facilitating ABT-199 sensitization. We evaluated SDH ubiquinone reductase activity in ABT-199 sensitive and resistant myeloma lines and demonstrated that all ABT-199 sensitive cell lines had significantly lower SQR activity. Furthermore, inhibition of the SDH ubiquinone reductase with the Qp site inhibitor thenoyltrifluoroacetone (TTFA) sensitized resistant MM cells to ABT-199. Inhibition of Complex I with piericidin or the SDHA subunit with 3-NPA were not as effective in sensitizing MM to ABT-199. Protein expression and co-immunoprecipitation analyses demonstrated an induction of BIM and enhanced BIM-BCL-2 binding upon SDH inhibition with TTFA. TTFA treated cells exhibited a reduction of FOXO3a phosphorylation and concomitant nuclear localization upon SDH inhibition suggestive of FOXO3a playing a role in BIM induction. TTFA + ABT-199 co-treatment induced apoptosis in ABT-199 resistant myeloma patient cells without impacting viability of the associated normal populations.Our study thus identifies a tumor selective approach of metabolically-driven synthetic lethality that relies on inhibition of the mitochondrial enzyme SDH and use of the BCL-2 antagonist, ABT-199 for myeloma therapy. In addition, our results importantly suggest SDH activity predicts ABT-199 sensitivity and can be interrogated in MM patients as a predictive marker of ABT-199 sensitivity particularly within t(11;14) patients currently being enrolled for ABT-199 treatment. DisclosuresNooka:Amgen, Novartis, Spectrum, Adaptive: Consultancy. Boise:Abbvie: Consultancy; Eli Lilly and Company: Research Funding.

Hypoxia Induces Resistance to EGFR Inhibitors in Lung Cancer Cells via Upregulation of FGFR1 and the MAPK Pathway.

Development of resistance remains the key obstacle to the clinical efficacy of EGFR tyrosine kinase inhibitors (TKI). Hypoxia is a key microenvironmental stress in solid tumors associated with acquired resistance to conventional therapy. Consistent with our previous studies, we show here that long-term, moderate hypoxia promotes resistance to the EGFR TKI osimertinib (AZD9291) in the non-small cell lung cancer (NSCLC) cell line H1975, which harbors two EGFR mutations including T790M. Hypoxia-induced resistance was associated with development of epithelial-mesenchymal transition (EMT) coordinated by increased expression of ZEB-1, an EMT activator. Hypoxia induced increased fibroblast growth factor receptor 1 (FGFR1) expression in NSCLC cell lines H1975, HCC827, and YLR086, and knockdown of FGFR1 attenuated hypoxia-induced EGFR TKI resistance in each line. Upregulated expression of FGFR1 by hypoxia was mediated through the MAPK pathway and attenuated induction of the proapoptotic factor BIM. Consistent with this, inhibition of FGFR1 function by the selective small-molecule inhibitor BGJ398 enhanced EGFR TKI sensitivity and promoted upregulation of BIM levels. Furthermore, inhibition of MEK activity by trametinib showed similar effects. In tumor xenografts in mice, treatment with either BGJ398 or trametinib enhanced response to AZD9291 and improved survival. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased expression of FGFR1. The combination of EGFR TKI and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLC. SIGNIFICANCE: Hypoxia-induced resistance to EGFR TKI is driven by overexpression of FGFR1 to sustain ERK signaling, where a subsequent combination of EGFR TKI with FGFR1 inhibitors or MEK inhibitors reverses this resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4655/F1.large.jpg.

Mechanism of Zinc Excitotoxicity: A Focus on AMPK.

Over the last 20 years, it has been shown that complex signaling cascades are involved in zinc excitotoxicity. Free zinc rapidly induces PKC activation, which causes reactive oxygen species (ROS) production at least in part through NADPH oxidase. It also promotes neuronal nitric oxide synthase, thereby increasing nitric oxide (NO) production. Extracellular signal-regulated kinase activation and Egr-1 transcription factor activity were quickly induced by zinc, too. These concurrent actions of kinases consequently produce oxygen free radical, ROS, and NO, which may cause severe DNA damage. Following the excessive activity of poly(ADP-ribose) polymerase-1 depletes NAD+/ATP in the cells. Zinc excitotoxicity exhibits distinct characteristics of apoptosis, too. Activation of caspase-3 is induced by liver kinase B1 (LKB1)-AMP-activated kinase (AMPK)-Bim cascade signaling and induction of p75NTR receptors and p75NTR-associated Death Executor. Thus, zinc excitotoxicity is a mechanism of neuronal cell death showing various cell death patterns. In addition to the above signaling cascades, individual intracellular organelles also play a crucial role in zinc excitotoxicity. Mitochondria and lysosomes function as zinc reservoirs, and as such, are capable of regulating zinc concentration in the cytoplasm. However, when loaded with too much zinc, they may undergo mitochondrial permeability transition pore (mPTP) opening, and lysosomal membrane permeabilization (LMP), both of which are well-established mechanisms of cell death. Since zinc excitotoxicity has been reported to be associated with acute brain injuries, including stroke, trauma, and epilepsy, we performed to find the novel AMPK inhibitors as therapeutic agents for these diseases. Since we thought acute brain injury has complicated neuronal death pathways, we tried to see the neuroprotection against zinc excitotoxicity, calcium-overload excitotoxicity, oxidative damage, and apoptosis. We found that two chemicals showed significant neuroprotection against all cellular neurotoxic models we tested. Finally, we observed the reduction of infarct volume in a rat model of brain injury after middle cerebral artery occlusion (MCAO). In this review, we introduced the AMPK-mediated cell death mechanism and novel strategy for the development of stroke therapeutics. The hope is that this understanding would provide a rationale for acute brain injury and eventually find new therapeutics.

Abstract 1821: Reversal of PD-L1-mediated chemoresistance by MCL-1/BCL-2 antagonism in colon cancer cells

Abstract Background: PD-L1 is an immune checkpoint protein that may display intrinsic, non-immune functions in human cancer cells. Previously, we discovered that PD-L1 is regulated by BRAFV600E via YAP and c-Jun, and whose deletion can confer chemoresistance due to a decrease in the BH3-only BIM protein in colorectal cancer (CRC) cells. Here, we hypothesize that release of pro-apoptotic BIM from its sequestration by MCL-1 or BCL-2 can overcomes drug resistance mediated by depletion of PD-L1. Materials and Methods: Parental and PD-L1 knockout human (RKO) and murine (MC38) CRC cells with or without reconstituted PD-L1 were utilized. RKO cells with knockdown of MCL-1 or BCL-2 by shRNA were generated. Cells were treated with the MCL-1 selective inhibitor AZD5591 or the BCL-2/BCL-xL inhibitor ABT-263 alone or combined with the MEK inhibitor, cobimetinib. Apoptosis was measured by immunoblotting of cleaved caspase-3 and by Annexin V labeling using FACS. Clonogenic cell survival assay was performed. Protein/protein interaction was analyzed by immunoprecipitation assay. Results: BIM induction by cobimetinib was insufficient to overcome chemoresistance due to loss of PD-L1. Using a potent and selective inhibitor of MCL-1 (AZD-5591), we demonstrate that concurrent antagonism of MCL-1 and MEK can overcome apoptosis resistance in both human and murine PD-L1 knockout CRC cells. Specifically, inhibition of MCL-1 by AZD-5591 or gene knockdown combined with cobimetinib suppresses clonogenic cell survival and enhances apoptosis in cells with PD-L1 knockout, wild type or those with reconstituted PD-L1. Mechanistically, the interaction of BIM with MCL-1 is increased in PD-L1 knockout (versus wild-type cells) in the presence of cobimetinib, and enhanced binding of BIM to MCL-1 is associated with BIM phosphorylation at Thr116. Targeting MCL-1 using AZD-5591 or MCL-1 shRNA releases BIM from its sequestration by MCL-1 and results in enhanced apoptotic signaling induced by cobimetinib to an equivalent extent in wild type and PD-L1 knockout cells with or without reconstitution of PD-L1. Similarly, targeting BCL-2 by ABT-263 or by BCL-2 shRNA releases BIM from its binding to BCL-2, and enhances apoptosis induction by cobimetinib independent of PD-L1 status. Enhanced binding of BIM to BCL-2 was also associated with BIM phosphorylation at Thr116. Conclusion: Chemoresistance mediated by loss of PD-L1 can be reversed by the use of BH3 mimetic drugs combined with MEK inhibition in CRC cells to exploit their apoptotic dependency on BIM expression. Citation Format: Lei Sun, Arpad Patai, Bo Qin, Frank A. Sinicrope. Reversal of PD-L1-mediated chemoresistance by MCL-1/BCL-2 antagonism in colon cancer cells [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 1821.

Functional miRNA Screening Identifies Wide-ranging Antitumor Properties of miR-3622b-5p and Reveals a New Therapeutic Combination Strategy in Ovarian Tumor Organoids.

Novel therapeutic strategies are urgently required for the clinical management of chemoresistant ovarian carcinoma, which is the most lethal of the gynecologic malignancies. miRNAs hold promise because they play a critical role in determining the cell phenotype by regulating several hundreds of targets, which could constitute vulnerabilities of cancer cells. A combination of gain-of-function miRNA screening and real-time continuous cell monitoring allows the identification of miRNAs with robust cytotoxic effects in chemoresistant ovarian cancer cells. Focusing on miR-3622b-5p, we show that it induces apoptosis in several ovarian cancer cell lines by both directly targeting Bcl-xL and EGFR-mediating BIM upregulation. miR-3622b-5p also sensitizes cells to cisplatin by inhibiting Bcl-xL in ovarian cancer cell lines escaping BIM induction. miR-3622b-5p also exerts antimigratory capacities by targeting both LIMK1 and NOTCH1. These wide-ranging antitumor properties of miR-3622b-5p in ovarian cancer cells are mimicked by the associations of pharmacologic inhibitors targeting these proteins. The combination of an EGFR inhibitor together with a BH3-mimetic molecule induced a large decrease in cell viability in a panel of ovarian cancer cell lines and several ovarian patient-derived tumor organoids, suggesting the value of pursuing such a combination therapy in ovarian carcinoma. Altogether, our work highlights the potential of phenotype-based miRNA screening approaches to identify lethal interactions which might lead to new drug combinations and clinically applicable strategies.

Blockade of HSP70 by VER-155008 synergistically enhances bortezomib-induced cytotoxicity in multiple myeloma

Proteasome inhibitor bortezomib is one of the most effective drugs currently available for the treatment of multiple myeloma (MM). However, the intrinsic and acquired resistance to bortezomib can limit its effectiveness. The activation of heat shock response has been characterized as a potential resistance mechanism protecting MM cells from bortezomib-induced cell death. In this study, in response to bortezomib therapy, we discovered that HSP70 is one of the most substantially upregulated heat shock proteins. In order to further explore approaches to sensitizing bortezomib-based treatment for MM, we investigated whether targeting HSP70 using a specific inhibitor VER-155008 combined with bortezomib could overcome the acquired resistance in MM. We found that HSP70 inhibitor VER-155008 alone significantly decreased MM cell viability. Moreover, the combination of VER-155008 and bortezomib synergistically induced MM cell apoptosis markedly in vitro. Notably, the combined treatment was found to increase the cleavage of PARP, an early marker of chemotherapy-induced apoptosis. Importantly, the reduction of anti-apoptotic Bcl-2 family member Bcl-2, Bcl-xL, and Mcl-1 and the induction of pro-apoptotic Bcl-2 family member BH3-only protein NOXA and Bim were confirmed to be tightly associated with the synergism. Finally, the ER stress marker CHOP (CCAAT-enhancer binding protein homologous protein), which can cause transcriptional activation of genes involved in cell apoptosis, was markedly induced by both VER-155008 and bortezomib. Taken together, our finding of a strong synergistic interaction between VER-155008 and bortezomib may support for combination therapy in MM patients in the future.

Abstract A123: Reversal of glucocorticoid resistance in pediatric acute lymphoblastic leukemia is dependent on restoring BIM expression

Abstract Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Glucocorticoids (e.g. dexamethasone, prednisolone) form a critical component of chemotherapy regimens for pediatric ALL and initial resistance to glucocorticoid therapy is predictive of poor outcome. Acquired resistance to glucocorticoids is common at relapse and is disparate compared with other chemotherapeutic drugs used in the treatment of pediatric ALL. Glucocorticoids induce apoptosis in lymphoid cells which is mediated through the glucocorticoid receptor (GR). Resistance to glucocorticoids in pediatric ALL is associated with upregulation of the oncogene C-MYC and failure to induce the proapoptotic gene BIM. The purpose of this study was to identify small molecules that are able to reverse glucocorticoid resistance in pediatric ALL patient-derived xenografts (PDXs). Methods: A 40,000 compound high-throughput screening (HTS) campaign was carried out to identify compounds that potentiated the cytotoxicity of dexamethasone against a pediatric ALL PDX derived from a patient with aggressive and fatal relapse. Cytotoxicity assays were carried out by alamarBlue assay. Gene expression analysis was carried out using Illumina microarrays. mRNA and protein analysis was carried out by RT-PCR and immunoblotting, respectively. Results: The HTS campaign identified a novel glucocorticoid sensitiser, 2-((4,5-dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phenylacetamide (GCS-3). The sensitizing effect was specific to glucocorticoids, with synergy observed when GCS-3 was combined with dexamethasone or prednisolone, but not with other chemotherapeutic drugs. Synergy was observed in a range of dexamethasone-resistant and -sensitive xenografts representative of B-ALL, T-ALL, early T-cell precursor ALL and Philadelphia chromosome positive ALL. GCS-3 required a functional GR to sensitise ALL xenografts to dexamethasone. Gene expression analysis showed that GCS-3 in the presence of dexamethasone upregulated a previously published gene expression signature induced by glucocorticoid treatment of B-ALL patients (Rhein et al. Leukemia, 2007, 21:897-905). Moreover, GCS-3 in combination with dexamethasone significantly downregulated C-MYC and upregulated BIM expression in a glucocorticoid-resistant ALL xenograft. Treatment with the GR antagonist, RU486, significantly abrogated the cytotoxic effects of the GCS-3/dexamethasone combination. Although RU486 increased C-MYC expression in the dexamethasone treated cells, there was no increase in C-MYC expression in the GCS-3/dexamethasone combination. RU486 suppressed the marked induction of BIM following treatment with the GCS-3/dexamethasone combination, indicating that BIM upregulation is critical for the cytotoxicity of the GCS-3/dexamethasone combination. The GCS-3/dexamethasone combination significantly increased binding of the GR to an intronic binding site at the BIM locus which is associated with BIM transcription and glucocorticoid sensitivity in pediatric ALL. Conclusion: This study describes the potential of the novel glucocorticoid sensitizing agent, GCS-3, as a biological tool to understand glucocorticoid action and resistance. Citation Format: Richard B Lock, Cara E Toscan, Duohui Jing, Chelsea Mayoh. Reversal of glucocorticoid resistance in pediatric acute lymphoblastic leukemia is dependent on restoring BIM expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A123. doi:10.1158/1535-7163.TARG-19-A123

Rationale for Targeting BCL6 in MLL-Rearranged B-ALL

Background and significance: MLL-gene rearrangements occur in ~10% of B-cell acute lymphoblastic leukemia (B-ALL) and 70% of infant B-ALL and define a group of patients with dismal outcomes. MLL belongs to the family of histone methyltransferases and plays a critical role in maintaining hematopoietic stem cells (Jude et al. 2007). Analyzing gene expression data from 207 children with high-risk ALL, we found that BCL6 is a predictor of poor clinical outcome in B-ALL, particularly in cases with MLL rearrangements. Thus, while the mechanisms of drug-resistance in MLL-rearranged B-ALL are largely unknown, we studied here a potential role of BCL6 in promoting the aggressive and refractory phenotype in this subtype of B-ALL. Results: Immunohistochemical staining of bone marrow biopsies from 10 of 11 MLL-rearranged B-ALL samples studied revealed aberrant expression of BCL6, a transcription factor that promotes oncogenic B-cell transformation and drug-resistance in B-ALL (Duy et al. Nature 2011). Our genetic and ChIP-seq analyses showed that MLL-AF4 and MLL-ENL fusions directly bound to the BCL6 promoter and upregulated BCL6 expression. While oncogenic MLL-fusions strongly induced aberrant BCL6 expression in B-ALL cells, germline MLL was required to upregulate Bcl6 in response to physiological stimuli during normal B-cell development. Inducible expression of Bcl6 increased MLL mRNA levels, which was reversed by genetic deletion and pharmacological inhibition of Bcl6, suggesting a positive feedback loop between MLL and BCL6. Highlighting the central role of BCL6 in MLL-rearranged B-ALL, conditional deletion and pharmacological inhibition of BCL6 compromised leukemogenesis in transplant recipient mice and restored sensitivity to vincristine chemotherapy in MLL-rearranged B-ALL patient samples. Oncogenic MLL-fusions strongly induced transcriptional activation of the pro-apoptotic BH3-only molecule BIM, while BCL6 was required to curb MLL-induced expression of BIM. Notably, peptide (RI-BPI) and small-molecule (FX1) BCL6-inhibitors derepressed BIM and synergized with the BH3-mimetic ABT-199 in eradicating MLL-rearranged B-ALL cells. These findings uncover MLL-dependent transcriptional activation of BCL6 as a previously unrecognized requirement of malignant transformation by oncogenic MLL-fusions and identified BCL6 as a novel target for the treatment of MLL-rearranged B-ALL. Discussion: Pharmacological inhibition of BCL6 using a retro-inverso peptide inhibitor (RI-BPI) compromised MLL-rearranged B-ALL leukemia-initiation and subverted vincristine-resistance. A central mechanistic aspect of BCL6-function in MLL-rearranged B-ALL involves transcriptional repression of the pro-apoptotic BH3-only molecule Bim (BCL2L11). Profiling for BH3-only proteins in various B-ALL subtypes revealed that MLL-rearranged B-ALL is associated with increased expression of the pro-apoptotic protein BIM (Benito et al. 2015). ABT-199 (venetoclax) is a BH3-mimetic that disrupts the interaction between BCL2 and BIM, leading to BIM release and induction of apoptosis. Previous studies have found that MLL-rearranged leukemia cells are sensitive to the BCL2 inhibitor ABT-199. Besides BCL2, we here identified BCL6 as a central antagonist of pro-apoptotic BIM function in MLL-rearranged B-ALL cells. In genetic experiments, we showed that oncogenic MLL-fusions strongly activated BCL2L11 transcription reinforcing the notion that constitutively high Bim expression levels represents an important and selective vulnerability in MLL-rearranged B-ALL. Both BCL2 and BCL6 represent crucial antagonists of BIM in MLL-rearranged B-ALL, BCL2 mediating BIM-sequestration and BCL6 being required for transcriptional repression of BIM. In support of this scenario, peptide (RI-BPI) and small molecule (FX1) inhibitors of BCL6 strongly synergized with blockade of BCL2-mediated BIM sequestration (ABT-199) in killing MLL-rearranged B-ALL cells. Figure 1 Disclosures Armstrong: AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; Janssen: Research Funding; Novartis: Research Funding; Accent Therapeutics: Consultancy, Equity Ownership; Mana Therapeutics: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership. Melnick:Janssen: Research Funding; Constellation: Consultancy; Epizyme: Consultancy. Milne:OxStem Ltd.: Equity Ownership.

Abstract 698: Enhancing apoptosis to improve the therapy of EGFR-mutant lung cancer

Abstract EGFR-TKIs have shown clinical efficacy in EGFR-mutant lung cancer patients, but most of the cases eventually develop resistance to the drugs, including the third-generation EGFR-TKI. Here, we develop a cell death mechanism-based combination strategy to enhance the therapeutic effect of EGFR-TKIs as well as to overcome the acquired resistance to EGFT-TKI in EGFR-mutant lung cancer. Through high-throughput screening of a custom pathway inhibitor library (-1000 drugs), we discovered Aurora kinase inhibitors that synergized with a third-generation EGFR-TKI (AZD9291) to induce apoptosis in EGFR-mutant lung cancer cells. Aurora kinase inhibitors enhanced AZD9291-mediated induction of BIM and PUMA, two important sentinels interconnecting the EGFR signal transduction pathway and the mitochondrial apoptotic machinery BAX and BAK. Combined inhibition of Aurora kinases and EGFR prevented the emergence of EGFR-TKI resistant clones in clonogenic assays. Interestingly, AZD9291 resistant lung cancer cells exhibited greater sensitivity to Aurora kinase inhibitors compared with parental cells. In conclusion, we found that the combination of AZD9291 and Aurora kinase inhibitors synergistically induced BIM and PUMA-mediated apoptosis, which may render a promising strategy to improve the therapy of EGFR-mutant lung cancer. <!–EndFragment–> Citation Format: Kosuke Tanaka, Song Han, Yogesh T. Ganesan, James J. Hsieh, Emily H. Cheng. Enhancing apoptosis to improve the therapy of EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 698.