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The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the hallmark genomic alteration of Ewing sarcoma, a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five-year survival rate. EWS-FLI1 is currently not clinically druggable, driving the need for more effective targeted therapies. Treatment with the H3K27 demethylase inhibitor, GSK-J4, leads to an increase in H3K27me and a decrease in H3K27ac, a significant event in Ewing sarcoma because H3K27ac associates strongly with EWS-FLI1 binding at enhancers and promoters and subsequent activity of EWS-FLI1 target genes. We were able to identify targets of EWS-FLI1 tumorigenesis directly inhibited by GSK-J4. GSK-J4 disruption of EWS-FLI1-driven transcription was toxic to Ewing sarcoma cells and slowed tumor growth in patient-derived xenografts (PDX) of Ewing sarcoma. Responses were markedly exacerbated by cotreatment with a disruptor of RNA polymerase II activity, the CDK7 inhibitor THZ1. This combination together suppressed EWS-FLI1 target genes and viability of ex vivo PDX Ewing sarcoma cells in a synergistic manner. In PDX models of Ewing Sarcoma, the combination shrank tumors. We present a new therapeutic strategy to treat Ewing sarcoma by decreasing H3K27ac at EWS-FLI1–driven transcripts, exacerbated by blocking phosphorylation of the C-terminal domain of RNA polymerase II to further hinder the EWS-FLI1–driven transcriptome.

Venetoclax is a small molecule inhibitor of the prosurvival protein BCL-2 that has gained market approval in BCL-2–dependent hematologic cancers including chronic lymphocytic leukemia and acute myeloid leukemia. Neuroblastoma is a heterogenous pediatric cancer with a 5-year survival rate of less than 50% for high-risk patients, which includes nearly all cases with amplified MYCN. We previously demonstrated that venetoclax is active in MYCN-amplified neuroblastoma but has limited single-agent activity in most models, presumably the result of other pro-survival BCL-2 family protein expression or insufficient prodeath protein mobilization. As the relative tolerability of venetoclax makes it amenable to combining with other therapies, we evaluated the sensitivity of MYCN-amplified neuroblastoma models to rational combinations of venetoclax with agents that have both mechanistic complementarity and active clinical programs. First, the MDM2 inhibitor NVP-CGM097 increases the prodeath BH3-only protein NOXA to sensitize p53-wild-type, MYCN-amplified neuroblastomas to venetoclax. Second, the MCL-1 inhibitor S63845 sensitizes MYCN-amplified neuroblastoma through neutralization of MCL-1, inducing synergistic cell killing when combined with venetoclax. Finally, the standard-of-care drug cocktail cyclophosphamide and topotecan reduces the apoptotic threshold of neuroblastoma, thus setting the stage for robust combination efficacy with venetoclax. In all cases, these rational combinations translated to in vivo tumor regressions in MYCN-amplified patient-derived xenograft models. Venetoclax is currently being evaluated in pediatric patients in the clinic, including neuroblastoma (NCT03236857). Although establishment of safety is still ongoing, the data disclosed herein indicate rational and clinically actionable combination strategies that could potentiate the activity of venetoclax in patients with amplified MYCN with neuroblastoma.

MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN-amplified neuroblastoma contributes to a large percent of pediatric cancer–related deaths. Therapy improvements for this subtype of cancer are a high priority. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1. Accumulating iron causes reactive oxygen species (ROS) production, and MYCN-amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. This dependence creates a marked vulnerability to targeting the system Xc-/glutathione (GSH) pathway with ferroptosis inducers. This reliance can be exploited through therapy with FDA-approved rheumatoid arthritis drugs sulfasalazine (SAS) and auranofin: in MYCN-amplified, patient-derived xenograft models, both therapies blocked growth and induced ferroptosis. SAS and auranofin activity was largely mitigated by the ferroptosis inhibitor ferrostatin-1, antioxidants like N-acetyl-L-cysteine, or by the iron scavenger deferoxamine (DFO). DFO reduced auranofin-induced ROS, further linking increased iron capture in MYCN-amplified neuroblastoma to a therapeutic vulnerability to ROS-inducing drugs. These data uncover an oncogene vulnerability to ferroptosis caused by increased iron accumulation and subsequent reliance on the system Xc-/GSH pathway. Significance: This study shows how MYCN increases intracellular iron levels and subsequent GSH pathway activity and demonstrates the antitumor activity of FDA-approved SAS and auranofin in patient-derived xenograft models of MYCN-amplified neuroblastoma.

Tumor cells undergo senescence in response to both conventional and targeted cancer therapies. The induction of senescence in response to cancer therapy can contribute to unfavorable patient outcomes, potentially including disease relapse. This possibiliy is supported by our findings that tumor cells induced into senescence by doxorubicin or etoposide can give rise to viable tumors in vivo. We further demonstrate sensitivity of these senescent tumor cells to the senolytic ABT‐263 (navitoclax), therefore providing a “two‐hit” approach to eliminate senescent tumor cells that persist after exposure to chemotherapy or radiation. The sequential combination of therapy‐induced senescence and ABT‐263 could shift the response to therapy toward apoptosis by interfering with the interaction between BCL‐XL and BAX. The administration of ABT‐263 after either etoposide or doxorubicin also resulted in marked, prolonged tumor suppression in tumor‐bearing animals. These findings support the premise that senolytic therapy following conventional cancer therapy may improve therapeutic outcomes and delay disease recurrence.

Triple negative breast cancer (TNBC) confers the poorest prognosis of the major subtypes of breast cancers, and often inflicts young, African-American and Hispanic women. Steadily improving survival in other subsets of breast cancers are attributed to the implementation of effective targeted therapies, which are non-chemotherapy drugs that target a specific gene in a cancer. For instance, hormone positive BCs are treated effectively with anti-hormone targeted therapy, and HER2 amplified BCs are treated effectively with anti-HER2 targeted therapy. The CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system, which enables specific gene editing to occur throughout the genome, allows for broad and unbiased discovery of new drug targets. We took advantage of this system to ask whether it may identify new druggable targets to treat TNBC and performed whole genome CRISPR/cas9 screening in two TNBC cell lines, BT549 and MDA-MB-468. To account for the heterogeneity found in TNBC, we further expanded the screen into seven other TNBC cell lines, by focusing on a select number of hits from the initial screen. This helped us identify a novel druggable target in TNBC, the ubiquitin activating enzyme 1 (UBA1). We took advantage of a recently reported small molecule inhibitor of UBA1, TAK-243, which is already in clinical trial evaluation in hematological cancers. Characterization of TAK-243 in cell culture models of TNBC and ex-vivo patient-derived xenografts (PDXs) demonstrated nanomolar IC50 toxicity, which was significantly lower than for tissue-derived normal cells. Mechanistically, TAK-243 therapy induced ER stress and the unfolded protein response in TNBC both in vitro and in vivo, leading to upregulation of activation transcription factor 4 (ATF4) and ATF6. Blocking ATF4 led to loss of NOXA and TAK-243 efficacy. TAK-243 induced tumor regressions in two PDX models of TNBC. Overall, these data demonstrate UBA1 is a novel drug target in TNBC. Citation Format: Sheeba Jacob, Tia H. Turner, Ann K. Yu, Colin M. Coon, Mohammad A. Alzubi, Charles T. Jakubik, Ynes Bouck, Mikhail G. Dozmorov, Sosipatros A. Boikos, Jennifer Koblinski, Joshua C. Harrell, Cyril H. Benes, Carlotta Costa, Anthony C. Faber. Genomic screening reveals UBA1 as a potent and druggable target in diverse models of triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-28.

Purpose: It was recently demonstrated that the EWSR1-FLI1 t(11;22)(q24;12) translocation contributes to the hypersensitivity of Ewing sarcoma to PARP inhibitors, prompting clinical evaluation of olaparib in a cohort of heavily pretreated Ewing sarcoma tumors. Unfortunately, olaparib activity was disappointing, suggesting an underappreciated resistance mechanism to PARP inhibition in patients with Ewing sarcoma. We sought to elucidate the resistance factors to PARP inhibitor therapy in Ewing sarcoma and identify a rational drug combination capable of rescuing PARP inhibitor activity. Experimental Design: We employed a pair of cell lines derived from the same patient with Ewing sarcoma prior to and following chemotherapy, a panel of Ewing sarcoma cell lines, and several patient-derived xenograft (PDX) and cell line xenograft models. Results: We found olaparib sensitivity was diminished following chemotherapy. The matched cell line pair revealed increased expression of the antiapoptotic protein BCL-2 in the chemotherapy-resistant cells, conferring apoptotic resistance to olaparib. Resistance to olaparib was maintained in this chemotherapy-resistant model in vivo, whereas the addition of the BCL-2/XL inhibitor navitoclax led to tumor growth inhibition. In 2 PDXs, olaparib and navitoclax were minimally effective as monotherapy, yet induced dramatic tumor growth inhibition when dosed in combination. We found that EWS-FLI1 increases BCL-2 expression; however, inhibition of BCL-2 alone by venetoclax is insufficient to sensitize Ewing sarcoma cells to olaparib, revealing a dual necessity for BCL-2 and BCL-XL in Ewing sarcoma survival. Conclusions: These data reveal BCL-2 and BCL-XL act together to drive olaparib resistance in Ewing sarcoma and reveal a novel, rational combination therapy that may be put forward for clinical trial testing.

Purpose: Effective targeted therapies are lacking for refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL). Suppression of the NOTCH pathway using gamma-secretase inhibitors (GSI) is toxic and clinically not effective. The goal of this study was to identify alternative therapeutic strategies for T-ALL. Experimental Design: We performed a comprehensive analysis of our high-throughput drug screen across hundreds of human cell lines including 15 T-ALL models. We validated and further studied the top hit, navitoclax (ABT-263). We used multiple human T-ALL cell lines as well as primary patient samples, and performed both in vitro experiments and in vivo studies on patient-derived xenograft models. Results: We found that T-ALL are hypersensitive to navitoclax, an inhibitor of BCL2 family of antiapoptotic proteins. Importantly, GSI-resistant T-ALL are also susceptible to navitoclax. Sensitivity to navitoclax is due to low levels of MCL-1 in T-ALL. We identify an unsuspected regulation of mTORC1 by the NOTCH pathway, resulting in increased MCL-1 upon GSI treatment. Finally, we show that pharmacologic inhibition of mTORC1 lowers MCL-1 levels and further sensitizes cells to navitoclax in vitro and leads to tumor regressions in vivo. Conclusions: Our results support the development of navitoclax, as single agent and in combination with mTOR inhibitors, as a new therapeutic strategy for T-ALL, including in the setting of GSI resistance.

MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN-amplified neuroblastoma contributes to a large percent of pediatric cancer-related deaths. Therapy improvements for this subtype of cancer are a high priority. Ferroptosis is an iron-dependent, oxidative form of cell death that is counteracted mainly by the production of Glutathione Peroxidase 4 (GPX4), a phospholipid hydroperoxidase that is produced through the glutathione pathway. The identification of cancers that may benefit from ferroptosis inducers are just emerging. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1 (TFR1). Accumulating iron causes reactive oxygen species (ROS) production, and MYCN-amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. By performing metabolomics, we demonstrate that the transsulfuration pathway is also activated by MYCN. The increased activation of both pathways leads to cysteine accumulation that results in inhibition of lipid peroxidation. Utilizing drugs that target the main components of the glutathione and transsulfuration pathway we sensitize the MYCN neuroblastomas to ferroptotic cell death. These data provide novel insights into how MYCN alters the transcriptome in neuroblastoma to confer growth and survival advantages and simultaneously sheds light on the mechanism of action of ferroptosis inducers with potential application in other types of cancer. Citation Format: Konstantinos V. Floros, Mia O. Johnson-Berro, Richard Kurupi, Carter K. Fairchild, Krista Dalton, Bin Hu, Madhavi Puchalapalli, Mikhail G. Dozmorov, Jennifer E. Koblinski, James A. Olzmann, Lauren A. Cowart, Anthony C. Faber. MYCN-amplified neuroblastoma is addicted to iron and vulnerable to ferroptosis [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 362.

Simple Summary Synovial sarcoma is a soft-tissue sarcoma that lacks effective systemic therapy and carries poor prognosis due to frequent late local recurrence and metastases. The cancer is known to be driven in part by increased expression of the pro-survival protein BCL-2. Surprisingly, synovial sarcoma proved resistant to BCL-2 inhibitors in pre-clinical trials. We identified increased activity of a second pro-survival protein, MCL-1, as responsible for this resistance. We showed that co-targeting both BCL-2 and MCL-1 proves to be an effective therapeutic approach both in cell culture and animal models of synovial sarcoma, supporting translation into clinical trials. Abstract Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.

Purpose: EGFR inhibitors (EGFRi) are effective against EGFR-mutant lung cancers. The efficacy of these drugs, however, is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M. We recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells [referred to as drug-tolerant cells (DTC)] prior to acquiring secondary mutations like T790M. Experimental Design: We have developed DTCs to EGFRi in EGFR-mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR-mutant lung cancer tumors in vivo. Results: We demonstrate surviving EGFR-mutant lung cancer cells upregulate the antiapoptotic protein MCL-1 in response to short-term EGFRi treatment. Mechanistically, DTCs undergo a protein biosynthesis enrichment resulting in increased mTORC1-mediated mRNA translation of MCL-1, revealing a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors. Moreover, MCL-1 is a key molecule governing the emergence of early EGFR-mutant DTCs to EGFRi, and we demonstrate it can be effectively cotargeted with clinically emerging MCL-1 inhibitors both in vitro and in vivo. Conclusions: Altogether, these data reveal that this novel therapeutic combination may delay the acquisition of secondary mutations, therefore prolonging therapy efficacy. Clin Cancer Res; 24(22); 5658–72. ©2018 AACR.

Despite the clinical success of anti-estrogen therapies, phosphatidylinositol 3-kinase inhibitors (PI3Ki), and mechanistic target of rapamycin complex I inhibitors (mTORC1i) for the treatment of patients with ER+ breast cancer, disease recurrence and progression are common. We found that a tumor transcriptional profile reflecting high stromal fibroblast content was associated with poor outcome in 3 cohorts of patients with ER+ breast cancer. We hypothesized that individual factors in the tumor microenvironment (TME) significantly contribute to drug resistance. To test this hypothesis, we screened 297 recombinant secreted proteins for ability to confer resistance to the anti-estrogen fulvestrant in MCF-7 and T47D ER+ breast cancer cells. Screen results were validated, and expansion screening included the anti-estrogen tamoxifen, the PI3Ki pictilisib, and the mTORC1i everolimus in 4 cell lines. To identify hits are most likely to be relevant to ER+ breast cancer, a bioinformatics filter was developed utilizing gene and protein expression in human tissues relevant to the TMEs of ER+ breast cancer. After filtering, the top screening hit was fibroblast growth factor 2 (FGF2), which confers resistance to anti-estrogens, PI3Ki, and mTORC1i, and is highly expressed in tissues and cell types associated with ER+ breast cancer. FGF2 did not rescue cells from the CDK4/6i palbociclib or the DNA-damaging agent doxorubicin, demonstrating pathway selectivity in the rescue phenotype. FGF2 rescued cells from anti-estrogen-, PI3Ki-, and mTORC1i-induced apoptosis and cell cycle arrest via activation of FGFR signaling through FRS2a, MEK1/2, ERK1/2, and downstream upregulation of cyclin D1 and degradation of Bim. FGF2-mediated anti-cancer effects were abrogated by co-treatment with the FGF2-neutralizing antibody GAL-F2, the pan-FGFR inhibitor PD-173074, the MEK inhibitor trametinib, or palbociclib. Cell cycle- and apoptosis-specific effects of FGF2 were abrogated by RNAi targeting cyclin D1 and Bim, respectively. We generated a transcriptional signature of FGF2 response by RNA-seq of fulvestrant-treated MCF-7 and T47D cells treated +/- FGF2. In 3 cohorts of patients with ER+ breast cancer, a signature of FGF2 signaling was significantly associated with poor prognosis and predictive of anti-estrogen resistance, including in a multivariate analysis including age, tumor grade, tumor stage, and FGFR amplification status. Finally, the therapeutic potential of targeting FGF2 was confirmed in 3 mouse models of ER+ breast cancer: 1) FGF2 rescue MCF-7 xenografts from fulvestrant; 2) GAL-F2 synergized with fulvestrant to suppress growth of 59-2-HI murine mammary adenocarcinomas that recruit FGF2-secreting stroma; 3) GAL-F2 synergized with fulvestrant to induce regression of HCI-003 patient-derived xenografts. Therapeutic effects coincided with increased tumor cell apoptosis and decreased proliferation, but not changes in tumor vasculature. These findings warrant consideration of FGF2 as a novel therapeutic target in ER+ breast cancer. Citation Format: Shee K, Hinds JW, Yang W, Hampsch RA, Patel K, Varn FS, Cheng C, Jenkins NP, Kettenbach AN, Demidenko E, Owens P, Lanari C, Faber AC, Golub TR, Straussman R, Miller TW. A microenvironment secretome screen reveals FGF2 as a mediator of resistance to anti-estrogens and PI3K/mTOR pathway inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-08.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract, thought to derive from neoplastic outgrowth of the interstitial cells of Cajal. Building on recent advances in recognition, classification and diagnosis, the past two decades have seen a changing paradigm with molecular diagnostics and targeted therapies.KITandPDGFRAmutations account for 85–90% of GIST carcinogenesis. However, the remaining 10–15% of GISTs, which until recently were calledKIT/PDGFRAwild-type GISTs, have been found to have one of the several mutations, including in theSDHA,B,C,D,BRAFandNF1genes. Though most of such GISTs are sporadic, a number of families with high incidence rates of GISTs and other associated clinical manifestations have been reported and found to harbor germline mutations inKIT,PDGFRA,SDHsubunits andNF1. The goal of this review is to describe the mutations, clinical manifestations and therapeutic implications of syndromic and inherited GISTs in light of recent studies of their clinicopathologic range and pathogenesis.

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it. Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance. Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes. Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

Targeted therapies have revolutionized treatment of several different types of cancers. However, in almost an invariable fashion, cancers eventually regrow in the presence of the targeted therapy, a phenomenon referred to as acquired resistance. In this issue of Cancer Research, Finn and colleagues demonstrate that modeling acquired resistance to MET tyrosine kinase inhibition in a MET-amplified gastric cancer cell line by a single, high exposure of the targeted therapy reveals clinically relevant acquired resistant mechanisms, which may be more faithful and comprehensive than the ones revealed through traditional ramp-up approaches. See related article by Finn et al., p. 79

Ever since Lynch et al . identified that some non-small cell lung cancers (NSCLCs) responded to the EGFR inhibitors gefitinib and erlotinib, and this could be correlated to activating mutations in EGFR, one of the great success stories in the era of targeted therapies was born.

MYCN-amplified neuroblastoma is responsible for ~10% of all cancer-related deaths in the pediatric population. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. The understanding that both compensatory pathways and intrinsic redundancy in cell systems exists implies that the use of combination therapies for effective and durable responses is necessary. Additionally, the most effective targeted therapies exploit an “Achilles’ heels” and are tailored to the genetics of the cancer under study. We performed an unbiased screen on select metabolic targeted therapy combinations and correlated sensitivity with over 20 subsets of cancer. We found that MYCN-amplified neuroblastoma are hypersensitive to the combination of AZD3965, an inhibitor of the lactate transporter MCT1, and phenformin, an inhibitor of complex I of the mitochondrion. Our data demonstrate MCT4 is highly correlated with resistance to the combination of AZD3965 and phenformin through the screen, is heavily methylated, and lowly expressed in MYCN-amplified neuroblastoma. Low MCT4 combines with high expression of the MCT2 and MCT1 chaperone CD147 in MYCN-amplified neuroblastoma, altogether conferring sensitivity to AZD3965 and phenformin combination. Metabolomic analysis showed that the result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum (ER) stress, and apoptosis, which was seen through cell viability assays and protein analysis. In mouse models of MYCN-amplified neuroblastoma, the combination was tolerable at concentrations where it shrank tumors and did not increase white blood cell toxicity compared to single-drugs. Therefore, we demonstrate a metabolic combination screen can identify vulnerabilities in subsets of cancer and put forth a metabolic combination therapy tailored for MYCN-amplified neuroblastoma that demonstrates efficacy and tolerability in vivo. Citation Format: Krista M. Powell, Timonthy L. Lochmann, Konstantinos V. Floros, Marissa L. Calbert, Richard Kurupi, Giovanna T. Stein, Joseph McClanaghan, Ellen Murchie, Regina K. Egan, Patricia Greninger, Mikhail Dozmorov, Sivapriya Ramamoorthy, Madhavi Puchalapalli, Bin Hu, Lisa Shock, Jennifer Koblisnski, John Glod, Sosipatros A. Boikos, Cyril H. Benes, Anthony C. Faber. Catastrophic ATP loss underlines a metabolic combination therapy tailored for MYCN-amplified neuroblastoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-024.

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression., Graphical Abstract, Highlights • Amplified MYCN is synthetic lethal with the BCL-2 inhibitor ABT-199 in neuroblastoma • MYCN upregulates the MCL-1 inhibitor, NOXA • MYCN-amplified neuroblastomas are further sensitized to ABT-199 with MLN8237 • ABT-199 with MLN8237 induce tumor regressions in MYCN-amplified neuroblastoma mice, Ham et al. show that MYCN-amplified neuroblastomas are sensitive to treatment with the BCL-2 inhibitor ABT-199 due to MYCN-driven increase of NOXA. Combination treatment with the Aurora Kinase A inhibitor MLN8237 and ABT-199 is synergistic in xenograft models of this tumor type, in part via reducing MCL-1.

e23561 Background: While Synovial Sarcoma (SS) only accounts for about 10% of soft tissue sarcomas (STS), or ~10,000 cases/year, ~4,000 of these will be fatal. Little benefit has been seen with newer combination chemotherapies and immune checkpoint inhibitors. SS is a transcription factor-driven cancer characterized by an t(X;18) chromosomal translocation resulting in the SS18-SSX fusion gene. SS is also known to overexpress the anti-apoptotic protein BCL-2, which has been used as a diagnostic marker for SS. Venetoclax, the FDA approved BH3 mimetic which specifically inhibits BCL-2, has been used to treat BCL-2-driven hematological cancers. Yet, preclinical studies of venetoclax, currently in clinical use for BCL-2 related hematologic malignancies, have failed in SS, raising the possibility of other BCL-2 family members playing a role in resistance. Methods: Publicly available gene expression databases of SS were queried for expression of BCL-2 family member expression, including the endogenous MCL-1 inhibitor, NOXA. Six SS cell lines, including ASKA, HS-SY-II, Yamato, SW982, SYO.1, and an ex vivo line from a SS patient-derived xenograft (PDX) were tested in vitro with combinations of venetoclax and the MCL-1 inhibitor S63845, using cell viability assays. In vivo testing employed both a patient-derived xenograft (PDX) model of SS and a cell line derived (SYO.1) xenograft model. Results: Queries of published gene expression data for SS demonstrated that SS expresses low levels of the endogenous MCL-1 inhibitor, NOXA, nominating a mechanism for intrinsic venetoclax resistance in these tumors, and suggesting rational combination of venetoclax and the MCL-1 inhibitor, S63845. In vitro, SS all cell lines possessing the SS18-SSX fusion gene demonstrated synergistic sensitivity to combined S63845 and venetoclax, despite generally insensitivity to either drug as monotherapy. Importantly, in an SS PDX, we demonstrated S63845 and venetoclax synergize to induce tumor regression. In the SYO.1 xenograft model, combination therapy significantly reduced tumor growth compared to no treatment or single agent groups. Conclusions: These findings provide the preclinical rationale for translation of the rational combination of BCL-2 and MCL-1 inhibitors into clinical trials for SS.

3576 Background: Studies of immune checkpoint blockade therapy (ICT) outcomes have been largely performed in melanoma and lung cancer patients, both of which are enriched for White patients. For example, a National Cancer Database study found that 97% of first-line ICT treatments in melanoma have been administered to White recipients (Patel, ASCO-SITC 2020). Given expanding indication in tumor types affecting more diverse populations, we sought to study whether minority populations might be projected to have differing checkpoint blockade response rates. Methods: Ethnicity information and RNAseq expression profiles and primary site information were obtained for 7087 patients from TCGA. Ethnicity was tested for association with RNA expression of targetable checkpoint genes ( PD1, PDL1, PDL2, CTLA4, IDO1, LAG3, TIM3, TIGIT, OX40, VISTA, and GITR) in 5 tumor histology types by Wilcoxon methods with Benjamini-Hochberg correction for multiple hypothesis testing. A dataset of > 2700 cases was obtained from NantHealth, with paired whole exome/RNAseq data. Ethnicity for 579 patients was assigned using allele-fraction from ~250 single nucleotide polymorphic sites found exclusively in 6 populations within the 1000 Genomes project. Ethnicity/checkpoint associations found in TCGA were tested within this dataset. Results: Within the TCGA cohort, ethnicity was not a factor in differential expression of checkpoint molecules in lung cancer. Within melanomas, in Asian patients PDL1, CTLA4, and IDO1 were expressed at lower levels than in White patients (each p = 0.04). These associations did not remain significant after correction for multiple hypothesis testing. Breast cancers in Black patients had significantly higher PD1, CTLA4, IDO1, LAG3, GITR, and OX40 expression compared to White patients, all remaining significant after correction (adj. p 3.7e-5 to 6.4e-3). Among White patients, colon cancers showed higher expression of PDL1/2, IDO1, LAG3, TIM3, and GITR (p 0.04 to 0.0017). IDO1 was significantly higher in White patients even after correction (adj. p = 0.03), and lower in Black patients (adj. p = 0.03). Conclusions: Ethnicity may represent a significant factor for efficacy checkpoint blockade therapies. White breast cancer patients might be anticipated to exhibit reduced sensitivity to PD1/CTLA4 blockade, while Black colon cancer patients may exhibit reduced sensitivity to IDO1 therapies such as epacadostat. A biomarker-driven approach to patient selection may ameliorate ethnic disparities in ICT outcomes.

The discovery of activated KIT mutations in gastrointestinal (GI) stromal tumors (GISTs) in 1998 triggered a sea change in our understanding of these tumors and has ushered in a new paradigm for the use of molecular genetic diagnostics to guide targeted therapies. KIT and PDGFRA mutations account for 85–90% of GISTs; subsequent genetic studies have led to the identification of mutation/epimutation of additional genes, including the succinate dehydrogenase (SDH) subunit A, B, C, and D genes. This review focuses on integrating findings from clinicopathologic, genetic, and epigenetic studies, which classify GISTs into two distinct clusters: an SDH-competent group and an SDH-deficient group. This development is important since it revolutionizes our current management of affected patients and their relatives, fundamentally, based on the GIST genotype.

Purpose: Small-cell lung cancer (SCLC) is an often-fatal neuroendocrine carcinoma usually presenting as extensive disease, carrying a 3% 5-year survival. Despite notable advances in SCLC genomics, new therapies remain elusive, largely due to a lack of druggable targets. Experimental Design: We used a high-throughput drug screen to identify a venetoclax-sensitive SCLC subpopulation and validated the findings with multiple patient-derived xenografts of SCLC. Results: Our drug screen consisting of a very large collection of cell lines demonstrated that venetoclax, an FDA-approved BCL-2 inhibitor, was found to be active in a substantial fraction of SCLC cell lines. Venetoclax induced BIM-dependent apoptosis in vitro and blocked tumor growth and induced tumor regressions in mice bearing high BCL-2–expressing SCLC tumors in vivo. BCL-2 expression was a predictive biomarker for sensitivity in SCLC cell lines and was highly expressed in a subset of SCLC cell lines and tumors, suggesting that a substantial fraction of patients with SCLC could benefit from venetoclax. Mechanistically, we uncover a novel role for gene methylation that helped discriminate high BCL-2–expressing SCLCs. Conclusions: Altogether, our findings identify venetoclax as a promising new therapy for high BCL-2–expressing SCLCs. Clin Cancer Res; 24(2); 360–9. ©2017 AACR.

がん進展制御研究所, Mutations in Kirsten rat-sarcoma (KRAS) are well appreciated to be major drivers of human cancers through dysregulation of multiple growth and survival pathways. Similar to many other non-kinase oncogenes and tumor suppressors, efforts to directly target KRAS pharmaceutically have not yet materialized. As a result, there is broad interest in an alternative approach to develop therapies that induce synthetic lethality in cancers with mutant KRAS, therefore exposing the particular vulnerabilities of these cancers. Fueling these efforts is our increased understanding into the biology driving KRAS mutant cancers, in particular the important pathways that mutant KRAS governs to promote survival. In this mini-review, we summarize the latest approaches to treat KRAS mutant cancers and the rationale behind them. © 2014 The Authors.

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers. Significance: Effective targeted therapies directed against colorectal cancer with activating mutations in KRAS remain elusive. We have leveraged drug-screen data from a large panel of human colorectal cancers to uncover an effective, rational targeted therapy strategy that has preferential activity in colorectal cancers with KRAS or BRAF mutations. This combination may be developed for clinical testing. Cancer Discov; 4(1); 42–52. ©2013 AACR. See related commentary by Russo et al., p. 19 This article is highlighted in the In This Issue feature, p. 1

The EWS-FLI1 translocation is the hallmark genomic alteration in the Ewing Family of Tumors (EWFT) a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five year survival rate. EWS-FLI is currently not pharmaceutically druggable, driving the need for more effective targeted therapies. It is well known that the EWS-FLI1 translocation induces a variety of epigenetic changes which impact tumorigenesis and disease progression. Using high throughput drug screening we have identified a novel epigenetic susceptibility in EWFT to the H3K27 demethylase inhibitor GSK-J4 (GlaxoSmithKline). Subsequent treatment with GSK-J4 leads to a decrease in H3K27 acetylation and ultimately silencing of EWS-FLI1 gene targets. We sought to fully characterize the hypersensitivity of EWFT to GSK-J4 and to elucidate the mechanisms of histone modifications caused by the EWS-FLI1 translocation in EWFT. We are examining key shifts in histone modifications at FLI1 target gene enhancer elements in the presence of GSK-J4 as a means of directly regulating FLI1 tumorigenesis. Additionally, we seek to determine the efficacy of GSK-J4 in combination with a CDK7 inhibitor (THZ1), which have shown synergy in vitro, through the utilization of patient derived xenograft models (PDX) and the identification of biomarkers of sensitivity to rationalize the use of this combination for clinical trials. Citation Format: Daniel A. Heisey, Timothy Lochmann, Marissa Calbert, Maninderjit Ghotra, Yuki Kato Maves, Sosipatros Boikos, Cyril Benes, C. Patrick Reynolds, Anthony Faber. Pharmaceutical means of targeting the fusion oncogene EWS-FLI1 in the Ewing family of tumors [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 4329.

1524 Background: Succinate Dehydrogenases and Fumarate Hydratase (SDH/FH) deficient tumors are characterized by succinate/fumarate accumulation and resultant pesudohypoxia that drives malignant transformation. This state of pseudohypoxia leads to dysregulation of PD-1 receptor-ligand signaling. In this study, we explored tumor mutation burden (TMB), gene expression of PD-L1, and expression of other immune checkpoint- associated genes in a diverse cohort of human tumors harboring SDH A, B, C, D and FH mutations. Methods: Retrospective analysis was performed on whole exome sequencing (WES; ~150x coverage) and whole transcriptomic RNAseq (~200x106 reads per tumor) data from NantHealth to identify tumors harboring SDHx and/or FH mutations. WES was performed on tumor tissue and matched normal for each patient to assess TMB. TMB was measured by counting all somatic-specific non-synonymous exonic mutations, with > 200 mutations qualified as TMB-high. Immune checkpoint therapy-related gene expression was evaluated for PDL1, CTLA4 , IDO, LAG3, FOXP3, PDL2, TIGIT, TIM3 and OX40 . Results: Among tumor samples from 3377 patients analyzed, 42 patients were found to harbor potentially-pathogenic & pathogenic mutations in the SDHA, B, C, D and FH genes. The most common tumor types with SDH/FH mutations were lung (n = 7), breast (n = 6), and colon cancer (n = 6). Our analysis revealed that TMB was positively correlated with the presence of SDH/FH mutations (p < 0.001). High PD-L1 expression was also significantly correlated with the presence of SDH/FH mutation (p < 0.05), while CTLA4, IDO, LAG3, FOXP3, and OX40 expression was significantly higher in SDH/FH mutated samples (p < 0.05). Conclusions: We report for the first time an association between increased TMB and increased PD-L1 expression in a variety of SDH/FH mutated tumors. These key parameters, imply that a higher TMB may drive the evolutionary pressure to select clones with a PDL1 high phenotype. This observation supports a potential therapeutic role for inhibition of PD-1/PD-L1 pathway in these tumors.

Purpose: BRAFV600E mutations are associated with poor clinical prognosis in colorectal cancer (CRC). Although selective BRAF inhibitors are effective for treatment of melanoma, comparable efforts in CRC have been disappointing. Here, we investigated potential mechanisms underlying this resistance to BRAF inhibitors in BRAFV600E CRC. Experimental Design: We examined phosphoinositide 3-kinase (PI3K)/mTOR signaling in BRAFV600E CRC cell lines after BRAF inhibition and cell viability and apoptosis after combined BRAF and PI3K/mTOR inhibition. We assessed the efficacy of in vivo combination treatment using a novel genetically engineered mouse model (GEMM) for BRAFV600E CRC. Results: Western blot analysis revealed sustained PI3K/mTOR signaling upon BRAF inhibition. Our BRAFV600E GEMM presented with sessile serrated adenomas/polyps, as seen in humans. Combination treatment in vivo resulted in induction of apoptosis and tumor regression. Conclusions: We have established a novel GEMM to interrogate BRAFV600E CRC biology and identify more efficacious treatment strategies. Combination BRAF and PI3K/mTOR inhibitor treatment should be explored in clinical trials. Clin Cancer Res; 19(10); 2688–98. ©2013 AACR.

KRAS is frequently mutated in lung cancer. Whereas MAPK is a well-known effector pathway of KRAS, blocking this pathway with clinically available MAPK inhibitors is relatively ineffective. Here, we report that epithelial-to-mesenchymal transition rewires the expression of receptor tyrosine kinases, leading to differential feedback activation of the MAPK pathway following MEK inhibition. In epithelial-like KRAS-mutant lung cancers, this feedback was attributed to ERBB3-mediated activation of MEK and AKT. In contrast, in mesenchymal-like KRAS-mutant lung cancers, FGFR1 was dominantly expressed but suppressed by the negative regulator Sprouty proteins; MEK inhibition led to repression of SPRY4 and subsequent FGFR1-mediated reactivation of MEK and AKT. Therapeutically, the combination of a MEK inhibitor (MEKi) and an FGFR inhibitor (FGFRi) induced cell death in vitro and tumor regressions in vivo. These data establish the rationale and a therapeutic approach to treat mesenchymal-like KRAS-mutant lung cancers effectively with clinically available FGFR1 and MAPK inhibitors. Significance: Adaptive resistance to MEKi is driven by receptor tyrosine kinases specific to the differentiation state of the KRAS-mutant non–small cell lung cancer (NSCLC). In mesenchymal-like KRAS-mutant NSCLC, FGFR1 is highly expressed, and MEK inhibition relieves feedback suppression of FGFR1, resulting in reactivation of ERK; suppression of ERK by MEKi/FGFRi combination results in tumor shrinkage. Cancer Discov; 6(7); 754–69. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 681

Purpose: Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor, often metastatic at presentation, for which current chemotherapeutic regimens are largely ineffective. As its pathogenesis is still unknown, we hypothesized that deregulation of signaling pathways commonly activated in cancer may contribute to MCC tumorigenesis and may provide insights into targeted therapy approaches for this malignancy. Experimental Design: We retrospectively profiled 60 primary MCC samples using a SNaPshot-based tumor genotyping assay to screen for common mutations in 13 cancer genes. Results: We identified mutations in 9 (15%) MCC primary tumors, including mutations in TP53 (3 of 60) and activating mutations in the PIK3CA gene (6 of 60). Sanger sequencing of the primary MCC tumors detected one additional PIK3CA mutation (R19K) that had not been previously described in cancer. Merkel cell polyoma virus (MCPyV) was detected in 38 (66%) MCC cases and patients with MCPyV-positive cancers showed a trend toward better survival. With one exception, the presence of MCPyV and activating mutations in PIK3CA appeared mutually exclusive. We observed that signaling through the PI3K/pAKT pathway was active in one MCPyV-positive and in all MCPyV-negative MCC cell lines, as evidenced by AKT phosphorylation. Importantly, the presence of a PIK3CA-activating mutation was associated with sensitivity to treatment with ZST474, a specific phosphoinositide 3-kinase (PI3K) inhibitor, and to NVP-BEZ235, a dual PI3K/mTOR inhibitor, targeted agents under active clinical development. Conclusions: PI3K pathway activation may drive tumorigenesis in a subset of MCC and screening these tumors for PIK3CA mutations could help identify patients who may respond to treatment with PI3K pathway inhibitors. Clin Cancer Res; 18(5); 1227–36. ©2012 AACR.

EGFR inhibitors (EGFRi) are effective at inducing transient tumor shrinkage in EGFR-mutant lung cancers. The efficacy of these drugs however is mitigated by the outgrowth of resistant cells: this is most often manifested by a secondary mutation in EGFR, T790M, which leads to reactivation of key intracellular signaling despite continued drug treatment. We recently demonstrated that T790M can occur both at low frequencies prior to initiation of EGFR inhibitor therapy, or alternatively arise de novo during treatment (Hata et al., Nat Med 2016). Since some cancers form T790M mutations de novo, one potential therapeutic strategy to thwart resistance is to identify the cells surviving initial therapy (referred to as persister cells or drug-tolerant cells [DTCs]) that eventually acquire the T790M mutation, and eliminate them prior to T790M acquisition. To this end, we hypothesized that some cells were refractory to EGFR inhibitor-induced apoptosis, surviving initial therapy and forming a reservoir of cells that could then eventually acquire T790M. We demonstrate that Western blots of lysates from EGFR-mutant lung cancers surviving initial therapy to the EGFR inhibitor gefitinib detect quick ( Citation Format: Kyung-A Song, Timothy L. Lochmann, Neha U. Patel, Jungoh Ham, Brad E. Windle, Hisashi Harada, Joel D. Leverson, Andrew J. Souers, Aaron N. Hata, Hiromichi Ebi, Anthony C. Faber. A protein synthesis switch underlies initial survival of EGFR-mutant lung cancer to EGFR inhibitors [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B31.

The ability of cancer cells to suppress apoptosis is critical for carcinogenesis. The BCL2 family proteins comprise the sentinel network that regulates the mitochondrial or intrinsic apoptotic response. Recent advances in our understanding of apoptotic signaling pathways have enabled methods to identify cancers that are “primed” to undergo apoptosis, and have revealed potential biomarkers that may predict which cancers will undergo apoptosis in response to specific therapies. Complementary efforts have focused on developing novel drugs that directly target antiapoptotic BCL2 family proteins. In this review, we summarize the current knowledge of the role of BCL2 family members in cancer development and response to therapy, focusing on targeted therapeutics, recent progress in the development of apoptotic biomarkers, and therapeutic strategies designed to overcome deficiencies in apoptosis. Significance: Apoptosis, long known to be important for response to conventional cytotoxic chemotherapy, has more recently been shown to be essential for the efficacy of targeted therapies. Approaches that increase the likelihood of a cancer to undergo apoptosis following therapy may help improve targeted treatment strategies. Cancer Discov; 5(5); 475–87. ©2015 AACR.

SummaryBYL719, which selectively inhibits the alpha isoform of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110a), is currently in clinical trials for the treatment of solid tumors, especially luminal breast cancers with PIK3CA mutations and/or HER2 amplification. This study reveals that, even among these sensitive cancers, the initial efficacy of p110α inhibition is mitigated by rapid re-accumulation of the PI3K product PIP3 produced by the p110β isoform. Importantly, the reactivation of PI3K mediated by p110β does not invariably restore AKT phosphorylation, demonstrating the limitations of using phospho-AKT as a surrogate to measure PI3K activation. Consistently, we show that the addition of the p110β inhibitor to BYL719 prevents the PIP3 rebound and induces greater antitumor efficacy in HER2-amplified and PIK3CA mutant cancers.

The purpose of this study is the development of a novel combination therapy that targets HER2-amplified breast cancer. About one quarter of breast cancers harbor amplification of HER2. HER2 is a transmembrane receptor tyrosine kinase (RTK) belonging to the ERBB family of receptors (ERBB1-4). Upon hetero- and homo-dimerization, HER2 activates several key intracellular pathways, regulating many cellular functions including proliferation and survival. HER2 inhibitors (HER2i) (e.g. the receptor tyrosine kinase (RTK) inhibitor, lapatinib) are now part of standard care for treating HER2-amplified breast cancers. However, despite their anti-cancer benefit, these drugs have limited efficacy as monotherapies, which contrasts to other RTK inhibitors in other RTK-driven cancers. To better understand this apparent dichotomy, in the present study, we evaluated potential modifiers of HER2i therapy. Here, we found that the pro-apoptotic NOXA, a member of the B-cell CLL/lymphoma 2 (BCL2) family which acts mainly via inhibitory binding of the pro-survival MCL-1, was markedly down-regulated in breast cancers compared to other cancers, and this was largely attributed to the HER2-amplified subset. Experimentally, overexpressing NOXA or silencing MCL-1 dramatically sensitizes HER2 amplified breast cancer cell lines to lapatinib via apoptosis. Consistently, pharmaceutical inhibition of MCL-1 sensitizes HER2-amplified breast cancers to lapatinib in vitro and in vivo. Mechanistically, disruption of MCL-1:BIM complexes and MCL-1:BAK underlie dual HER2i/MCL-1i therapy. Therefore, deficient NOXA expression constitutes a bonafide apoptotic block in HER2 amplified breast cancers, contributes to mitigated HER2i responses, and presents a rational combination therapy that may improve HER2i responses. Citation Format: Konstantinos V. Floros, Kyung-A Song, Timothy L. Lochmann, Mark T. Hughes, Daniel A. Heisey, Hisashi Harada, Bin Hu, Jennifer Koblinski, Andrew J. Souers, Joel D. Leverson, Anthony C. Faber. Deficient NOXA in HER2-amplified breast cancer drives kinase inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3082. doi:10.1158/1538-7445.AM2017-3082

Introduction The BIM deletion polymorphism in intron 2 was found in a significant percent of the Asian population. Patients with epidermal growth factor receptor (EGFR) mutant lung cancers harboring this BIM polymorphism have shorter progression free survival and overall response rates to EGFR tyrosine kinase inhibitors. However, the association between the BIM deletion polymorphism and lung cancer risk is unknown. Methods The BIM deletion polymorphism was screened by polymerase chain reaction in 765 lung cancer cases and 942 healthy individuals. Results Carriers possessing one allele of the BIM polymorphism were observed in 13.0% of control cases and 12.8% of lung cancer cases, similar to incidence rates reported earlier in healthy individuals. Homozygote for the BIM polymorphism was observed in four of 942 healthy controls and three of 765 lung cancer cases. The frequency of the BIM deletion polymorphism in lung cancer patients was not related to age, sex, smoking history, or family history of lung cancer. The BIM deletion polymorphism was found in 30 of 212 patients with EGFR wild type lung cancers and 16 of 120 patients with EGFR mutant lung cancers. The frequency of the BIM polymorphism is similar between cancers with wild type EGFR and mutated EGFR (p = 0.78). Conclusion The BIM deletion polymorphism was not associated with lung cancer susceptibility. Furthermore, the BIM polymorphism is not associated with EGFR mutant lung cancer.

Although several groups have demonstrated that concomitant use of MEK and phosphoinositide 3-kinase (PI3K) inhibitors (MEKi/PI3Ki) can induce dramatic tumor regressions in mouse models of KRAS-mutant non–small cell lung cancer (NSCLC), ongoing clinical trials investigating this strategy have been underwhelming to date. While efficacy may be hampered by a narrow therapeutic index, the contribution of biologic heterogeneity in the response of KRAS-mutant NSCLCs to MEKi/PI3Ki has been largely unexplored. In this study, we find that most human KRAS-mutant NSCLC cell lines fail to undergo marked apoptosis in response to MEKi/PI3Ki, which is key for tumor responsiveness in vivo. This heterogeneity of apoptotic response occurs despite relatively uniform induction of growth arrest. Using a targeted short hairpin RNA screen of BCL-2 family members, we identify BIM, PUMA, and BCL-XL as key regulators of the apoptotic response induced by MEKi/PI3Ki, with decreased expression of BIM and PUMA relative to BCL-XL in cell lines with intrinsic resistance. In addition, by modeling adaptive resistance to MEKi/PI3Ki both in vitro and in vivo, we find that, upon the development of resistance, tumors have a diminished apoptotic response due to downregulation of BIM and PUMA. These results suggest that the inability to induce apoptosis may limit the effectiveness of MEKi/PI3Ki for KRAS-mutant NSCLCs by contributing to intrinsic and adaptive resistance to this therapy. Cancer Res; 74(11); 3146–56. ©2014 AACR.

PI3K inhibition in combination with other agents has not been studied in the context of PIK3CA wild-type, KRAS mutant cancer. In a screen of phospho-kinases, PI3K inhibition of KRAS mutant colorectal cancer cells activated the MAPK pathway. Combination PI3K/MEK inhibition with NVP-BKM120 and PD-0325901 induced tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer, which was mediated by inhibition of mTORC1, inhibition of MCL-1, and activation of BIM. These findings implicate mitochondrial-dependent apoptotic mechanisms as determinants for the efficacy of PI3K/MEK inhibition in the treatment of PIK3CA wild-type, KRAS mutant cancer. Keywords: PI3K; MEK; KRAS; Colorectal cancer; Mouse model of cancer

Effective targeted therapies are lacking for refractory and relapsed T-cell Acute Lymphoblastic Leukemia (T-ALL). Analysis of a large cell line screen revealed that NOTCH1 mutant T-ALL cells are sensitive to the Bcl-2/xL inhibitor ABT-263. We show that NOTCH1 inhibits the mTOR complex 1, resulting in low MCL-1 levels and sensitization to ABT-263. Further suppression of mTORC1 using small molecule inhibitors in combination with ABT-263 results in very high levels of apoptosis and tumor regressions in vivo. This suggests new therapeutic opportunities for NOTCH1 mutant T-ALL, including in the setting of gamma-secretase inhibitor (GSI) resistance. Citation Format: Anahita Dastur, Carlotta Costa, Anthony Faber, Cyril Benes. Sensitivity of NOTCH1 mutant T-ALL to ABT-263. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3846.

There is no effective molecularly targeted therapy for T-cell Acute Lymphoblastic Leukemias (T-ALL) and prognosis for relapsed or chemotherapeutic refractory cases remains poor. A large unbiased drug screen performed across cell lines from a broad spectrum of cancers revealed that NOTCH1 mutant T-ALL cells, including lines resistant to gamma-secretase inhibitors are very sensitive to the Bcl-2/xL inhibitor ABT-263. We found that NOTCH1 up regulates the mTOR suppressor REDD1, leading to suppression of the mTOR Complex 1. This in turn results in low MCL-1 levels and ABT-263 sensitivity. Inhibition of NOTCH1 activity in this context leads to robust and sustained up regulation of mTOR signaling which could limit the activity of gamma-secretase inhibitors. Analysis of a small set of patient samples support the findings that NOTCH1 mutant T-ALL have low MCL-1 levels. Most importantly, we show that suppression of mTORC1 by a small molecule inhibitor further sensitizes NOTCH1 mutant T-ALL to ABT-263 resulting in massive apoptosis and in vivo efficacy. SIGNIFICANCE: This study uncovers a previously unappreciated link between two major oncogenic pathways, the NOTCH and mTOR pathways, with NOTCH1 repressing mTORC1 through REDD1. Further, we show that this suppression results in low MCL-1 levels and that combining mTORC inhibition with BCL-2 inhibition is a promising therapeutic strategy for GSI-resistantNOTCH1-mutant T-ALL. Citation Format: Anahita Dastur, Anthony Faber, Carlotta Costa, Cyril Benes. Repression of mTORC1 activity in NOTCH1 mutant T-ALL results in sensitivity to the BCL-2 inhibitor ABT-263. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A77.

Cancers with specific genetic mutations are susceptible to selective kinase inhibitors. However, there is a wide spectrum of benefit among cancers harboring the same sensitizing genetic mutations. Herein, we measured apoptotic rates among cell lines sharing the same driver oncogene following treatment with the corresponding kinase inhibitor. There was a wide range of kinase inhibitor-induced apoptosis despite comparable inhibition of the target and associated downstream signaling pathways. Surprisingly, pretreatment RNA levels of the BH3-only pro-apoptotic BIM strongly predicted the capacity of EGFR, HER2, and PI3K inhibitors to induce apoptosis in EGFR-mutant, HER2-amplified, and PIK3CA-mutant cancers, respectively, but BIM levels did not predict responsiveness to standard chemotherapies. Furthermore, BIM RNA levels in EGFR-mutant lung cancer specimens predicted response and duration of clinical benefit from EGFR inhibitors. These findings suggest assessment of BIM levels in treatment-naïve tumor biopsies may indicate the degree of benefit from single-agent kinase inhibitors in multiple oncogene-addiction paradigms. Significance: In several oncogene-addiction paradigms, assessment of BIM RNA levels identifies those cancers that fail to have substantial apoptotic responses to kinase inhibitors. BIM RNA levels may be assessed in diagnostic cancer specimens to predict which patients will receive less benefit from single-agent kinase inhibitors. Cancer Discovery: 1(4); 352–65. ©2011 AACR. Read the Commentary on this article by Yoshida and Haura, p. 289 This article is highlighted in the In This Issue feature, p. 275

The development of acquired drug resistance limits the effectiveness of targeted therapies for lung cancer. In 50% of patients with EGFR mutant non-small cell lung cancer (NSCLC), acquired resistance to EGFR inhibitors can be attributed to the development of a secondary T790M mutation that prevents drug binding. Third generation irreversible EGFR inhibitors that effectively inhibit EGFR with T790M are currently under clinical development. Using patient-derived EGFR mutant NSCLC models of T790M+ acquired resistance, we observed that the presence of a mesenchymal phenotype was associated with a decreased response to subsequent irreversible EGFR inhibitor therapy. Epithelial-to-mesenchymal transformation (EMT) led to suppression of pro-apoptotic signaling and a blunted apoptotic response to EGFR inhibition. Combination BCL-XL and irreversible EGFR inhibitor therapy restored the apoptotic response and resulted in regressions of T790M+ resistant tumors that exhibited poor response to single agent irreversible EGFR inhibitors. This suggests that combining BCL-XL and irreversible EGFR inhibitors may be an effective treatment strategy for T790M+ resistant EGFR mutant lung cancers with suppressed apoptotic signaling due co-acquisition of a mesenchymal phenotype. Citation Format: Aaron N. Hata, Matthew J. Niederst, Hannah L. Archibald, Hillary Mulvey, Jungoh Ham, Maria Gomez-Caraballo, Anuj Kalsy, Anthony C. Faber, Jeffrey Engelman. Co-acquisition of T790M and EMT in resistant EGFR mutant non-small cell lung cancer can be overcome by combined irreversible EGFR and BCL-XL inhibition. [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 2845. doi:10.1158/1538-7445.AM2015-2845

BH3 mimetics like ABT-263 induce apoptosis in subsets of cancer. However, they have shown limited clinical efficacy as single agents, and rational combination strategies remain underexplored. Toward the development of a novel combined therapy, we examined efficacy of ABT-263 across 331 cancer cell lines. Cell lines with high expression levels of the pro-apoptotic gene BIM were more sensitive to ABT-263 activity. This was particularly evident in small cell lung cancer (SCLC), which tends to have higher BIM levels and is among the most sensitive to ABT-263. However, MCL-1 expression mitigated the impact of ABT-263-induced apoptosis by sequestering BIM released from BCL-2 and BCL-XL. Since MCL-1 expression is dependent on TORC1 activity in some cancers, we attempted to overcome the apoptotic block in SCLC by combining ABT-263 with the TORC1/2 inhibitor AZD8055. AZD8055 treatment alone downregulated MCL-1 expression; combination AZD8055 and ABT-263 induced marked apoptosis and growth arrest in vitro, as well as tumor regressions in multiple SCLC xenograft models. Strikingly, low-dose AZD8055 was sufficient to promote tumor regressions in the combination strategy. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve single-agent ABT-263 activity in SCLC. Citation Format: Anthony C. Faber, Carlotta Costa, Anahita Dastur, Cyril Benes, Jeffrey Engelman. Assessment of ABT-263 activity across a comprehensive cancer cell line collection leads to a novel, potent combination therapy for small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2933. doi:10.1158/1538-7445.AM2014-2933

The phosphatidylinositol 3-kinase (PI3K) family of lipid kinases plays a prominent role in the growth and survival of several types of cancer, including breast cancers where the PI3K pathway is altered in up to 70% of cases. Due to the technical challenges of measuring the PI3K product, PIP3 directly, the phosphorylation of the downstream effector AKT has historically been used to indirectly assess the activity of class I PI3K. While this approach has proven to be rather useful, it is based on the assumption that this readout is proportional to the levels of PIP3, which is likely to apply only to a limited range of activation levels and within specific contexts. Indeed, evaluation of the efficacy of PI3K inhibitors with such indirect measurements may approximate and oversimplify the complex transduction mechanism of PI3K. During time course experiments using the p110A selective inhibitor, BYL719, we observed that both PIP3 and p-AKT levels were similarly restored after 24 hours in HER2 amplified breast cancer cells, while the rebound of p-AKT was minimal and did not correlate with increased PIP3 levels in PIK3CA mutant breast cancer models. Both p110B inhibition and p110B depletion were effective in blocking the PIP3 rebound observed. Consistent with this, the combination of p110A and p110B inhibitors decreased cell growth and induced a better apoptotic response compared to BYL719 alone. We further demonstrated that only the combination of p110A and p110B inhibitors induced tumor regression. Importantly, PIP3 levels correlated with efficacy more reliably than p-AKT in a PIK3CA mutant model in vivo. These data suggest that measurement of PIP3 levels following treatment may serve as a more effective biomarker than p-AKT levels to predict response to PI3K inhibitors within this subset of breast cancers. Citation Format: Carlotta Costa, Hiromichi Ebi, Anthony C. Faber, Madhuri Nishtala, Youngchul Song, Cyril H. Benes, Jeffrey A. Engelman. Meausuring PIP3 reveals the unexpected role of p110B in luminal breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2619. doi:10.1158/1538-7445.AM2014-2619

Treatment of EGFR-mutant lung cancer with erlotinib results in dramatic tumor regression but it is invariably followed by drug resistance. In characterizing early transcriptional changes following drug treatment of mutant EGFR-addicted cells, we identified the stem cell transcriptional regulator SOX2 as being rapidly and specifically induced, both in vitro and in vivo. Suppression of SOX2 sensitizes cells to erlotinib-mediated apoptosis, whereas its ectopic expression reduces drug-induced cell death. We show that erlotinib relieves EGFR-dependent suppression of FOXO6, leading to SOX2 induction and repression of the pro-apoptotic BH3-only genes BIM and BMF. Together, these observations point to a physiological feedback mechanism that attenuates oncogene addiction-mediated cell death and may contribute to the acquisition of drug resistance. Citation Format: Stephen Michael Rothenberg, Kyle Concannon, Sarah Cullen, Alexa B. Turke, Anthony C. Faber, Jeffrey A. Engelman, Shyamala Maheswaran, Daniel A. Haber. Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6 dependent survival pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4773. doi:10.1158/1538-7445.AM2014-4773

The PI3K pathway is alterated in up to 70% of breast cancers. Although both isoform-specific and pan-PI3K inhibitors are available, it is still unclear which compounds are more effective among the different subtypes of breast cancer. We performed time course experiments using the selective p110α inhibitor, BYL-719, in PIK3CA mutant and HER2 amplified cell lines in order to test whether p110α inhibition alone was sufficient to abrogate PI3K-AKT signaling. We observed a rebound of PIP3, the PI3K product, 24 hours after treatment in all cell lines. Notably, both PIP3 and p-AKT levels increased after 24 hours in HER2 amplified cells, while the rebound of p-AKT in PIK3CA mutant was minimal and did not correlate with PIP3 levels. Both p110β inhibitor and siRNA againt p110β were effective in blocking the PIP3 rebound observed after 24 hours of treatment with BYL-719 alone. Consistent with this, the combination of p110α and p110β inhibitors reduce cell growth in long term viability assays and induced a greater apoptotic response in comparison to BYL-719 alone. Importantly, p110α and p110β inhibitors in combination induced tumor regression in vivo, while with single agent BYL-719 we observed only tumor stasis. Finally, we demonstrated, both in vitro and in vivo, that PIP3 levels correlate with inhibitor efficacy more accurately than p-AKT in PIK3CA model. Thus, PIP3 may serve as a more effective biomarker than p-AKT levels to predict response to PI3K inhibitors in PIK3CA mutant and HER2 amplified breast cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B186. Citation Format: Carlotta Costa, Hiromichi Ebi, Anthony C. Faber, Madhuri Nishtala, Young Chul Song, Jeffrey A. Engelman. The unexpected role of p110β in HER2 amplified and PIK3CA mutant breast cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B186.

Colorectal cancers (CRCs) harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that combines targeting of the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS mutant (MT) and BRAF MT but not wild-type (WT) CRC cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not wild-type CRCs, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS MT colorectal cancer xenograft and genetically-engineered mouse models of CRC, but not in the corresponding KRAS WT CRC models. These data suggest that the combination of BCL-2/XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C263. Citation Format: Erin M. Coffee, Anthony C. Faber, Carlotta Costa, Anahita Dastur, Hiromichi Ebi, Aaron N. Hata, Alan T. Yeo, Elena J, Youngchul Song, Ah Ting Tam, Jessica L. Boisvert, Randy J. Milano, Jatin Roper, David P. Kodack, Rakesh K. Jain, Ryan B. Corcoran, Miguel N. Rivera, Sridhar Ramaswamy, Kenneth E. Hung, Cyril H. Benes, Jeffrey A. Engelman. mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C263.

The clinical development of selective RAF and MEK inhibitors has transformed the treatment of the ∼50% of melanoma patients whose tumors harbor BRAF mutations. However, a substantial percentage of these patients fail to respond to therapy, and most responses are partial and short-lived. Although multiple mechanisms of resistance have been identified in BRAF-mutant melanoma, no clinically useful biomarkers have been established to predict which patients are most likely to demonstrate sensitivity or resistance to RAF or MEK inhibitors. We found that suppression of TORC1 activity in response to RAF or MEK inhibitors, as measured by decreased phosphorylation of ribosomal protein-S6 (P-S6), was a functional biomarker that effectively predicted sensitivity in BRAF-mutant melanoma cell lines in vitro and in mouse tumor xenografts. In sensitive melanomas, TORC1 and P-S6 were suppressed in response to RAF or MEK inhibitors, but in resistant melanomas, TORC1 activity was maintained, in some cases despite robust suppression of MAPK signaling by these inhibitors. In mouse models, suppression of TORC1 after MAPK inhibition was necessary for induction of apoptosis and tumor response in vivo. Notably, in paired biopsies obtained from patients with BRAF-mutant melanoma before treatment and after initiation of RAF inhibitor therapy, P-S6 suppression was associated with significantly improved progression-free survival [HR 0.19, 95% CI 0.01-0.84, p=0.03]. Finally, we found that changes in P-S6 in patients’ tumor cells could be readily monitored in real-time by multiplexed, quantitative immunofluorescence microscopy of serial fine-needle aspiration biopsies obtained from patients before and during the first 2 weeks of RAF inhibitor therapy. This approach provides a minimally-invasive means of rapidly monitoring the efficacy of treatment, before changes in tumor volume are apparent by traditional radiographic imaging. Together, these results establish suppression of P-S6 after initiation of RAF inhibitor therapy as a robust potential functional biomarker to guide the treatment of patients with BRAF-mutant melanoma, and present a powerful methodology for monitoring changes in potentially any signaling pathway in response to targeted therapies in patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C137. Citation Format: Ryan B. Corcoran, S. Michael Rothenberg, Aaron N. Hata, Anthony C. Faber, Adriano Piris, Rosalynn M. Nazarian, Ronald D. Brown, Jason T. Godfrey, Daniel Winokur, John Walsh, Mari Mino-Kenudson, Shyamala Maheswaran, Jeffrey Settleman, Jennifer A. Wargo, Keith T. Flaherty, Daniel A. Haber, Jeffrey A. Engelman. Rapid assessment of TORC1 suppression as a functional biomarker predicting responsiveness to RAF and MEK inhibitors in BRAF-mutant melanoma patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C137.

The phosphatidylinositol 3-kinase (PI3K) pathway is genetically activated in excess of 70% of breast cancers, often throughPIK3CA mutation and HER2 amplification. Previous studies with PI3K inhibitors have suggested enhanced sensitivity in this subset of breast cancers, however, the mechanism underlying this sensitivity is mainly unknown. In this study, we investigated the signaling consequences of PI3K inhibition in PIK3CA mutant and HER2 amplified breast cancers, and unexpectedly observed suppression of ERK signaling in these cancers. In contrast to PI3K inhibitors, AKT inhibitors failed to block MEK/ERK signaling and to induce sustained apoptosis, suggesting that a different PI(3,4,5)P3-dependent pathway regulated ERK. We determined that suppression of ERK was due to loss of active Rac1, which led to suppression of PAK/c-RAF/MEK/ERK signaling. Expression of a constitutively active form of Rac1 abrogated the capacity for PI3K inhibitors to inhibit ERK, induce apoptosis, and have anti-tumor activity in vivo. Further studies identified P-Rex1 as the PI(3,4,5)P3-dependent guanine exchange factor for Rac1 responsible for regulation of the Rac1/PAK/c-RAF/MEK/ERK pathway in these breast cancer cells. Furthermore, the expression level of P-Rex1 in these breast cancer cells predicted for capacity for PI3K inhibitors to suppress ERK signaling and correlated with sensitivity to PI3K inhibitors. Thus, PI3K inhibitors have enhanced sensitivity in PIK3CA mutant and HER2 amplified breast cancers where PI3K inhibition downregulates both the AKT and Rac1/ERK pathways. In addition, P-Rex1 may serve as a biomarker to predict response to single-agent PI3K inhibitors within this subset of breast cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A29. Citation Format: Hiromichi Ebi, Carlotta Costa, Anthony Faber, Dejan Juric, Patricia Della Pelle, Youngchul Song, Seiji Yano, Mari Mino-Kenudson, Cyril H. Benes, Jeffrey A. Engelman. PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF, P-Rex1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A29.

HER2 amplification is found in up to 20% of breast cancers and PIK3CA mutations occur in up to 30%. These cancers are susceptible to targeted therapies directed against oncogenic HER2 and PI3K. Emerging evidence from clinical trials has revealed a surprising heterogeneity in response to targeted therapies, for example within HER2 positive breast cancer patients, the efficacy of HER2 inhibitors has varied. We have previously shown in breast cancer cell lines and a small cohort of HER2 positive breast cancer patients that low levels of the pro-apoptotic BIM can confer intrinsic resistance to HER2 inhibitors, by repressing the apoptotic response following treatment. Here in, we investigated the ability of a BCL-2/Bcl-XL inhibitor to enhance the apoptotic response specifically in low BIM HER2 amplified and PIK3CA mutant breast cancers with intrinsic resistance to targeted therapies, by freeing up BIM from BCL-2:BIM and BCL-xL:BIM complexes. We found that in these cancers, the addition of BCL-2/BCL-xL inhibitors synergized with HER2 inhibitors and PI3K inhibitors to induce apoptosis, and to similar levels induced by targeted therapy alone in the high BIM expressing breast cancers. Biochemical analysis revealed that PI3K inhibition downregulated MCL-1 in PIK3CA mutants, which leads to a further increase in the amount of free BIM, further “maximizing” the otherwise low levels of cellular BIM in these cells. These results translated to tumor regressions in mouse xenografts treated with the combination, similar to what was seen with single-agent PI3K inhibitor in a high BIM expressing PIK3CA mutant breast cancer xenograft model. Thus, combining a BCL-2/BCL-xL inhibitor with the appropriate targeted therapy may overcome intrinsic resistance caused by low BIM expression in HER2 amplified and PIK3CA mutant breast cancer. Citation Format: Anthony C. Faber, Sadhna Vora, Jeffrey A. Engelman. Sensitizing low BIM expressing breast cancers to targeted therapies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A062.