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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.

Identifying robust, patient-specific, and predictive biomarkers presents a major obstacle in precision oncology. To optimize patient-specific therapeutic strategies, here we couple pathway knowledge with large-scale drug sensitivity, RNAi, and CRISPR-Cas9 screening data from 460 cell lines. Pathway activity levels are found to be strong predictive biomarkers for the essentiality of 15 proteins, including the essentiality of MAD2L1 in breast cancer patients with high BRCA-pathway activity. We also find strong predictive biomarkers for the sensitivity to 31 compounds, including BCL2 and microtubule inhibitors (MTIs). Lastly, we show that Bcl-xL inhibition can modulate the activity of a predictive biomarker pathway and re-sensitize lung cancer cells and tumors to MTI therapy. Overall, our results support the use of pathways in helping to achieve the goal of precision medicine by uncovering dozens of predictive biomarkers., Predicting an individual's response to therapy is an important goal for precision medicine. Here, the authors use an algorithm that takes into account the interaction type and directionality of signalling pathways in protein–protein interactions and find that their pathway analysis can predict essential genes, which may be a target for therapy.

Purpose: Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed. Experimental Design: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n = 101) or plasma (n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance. Results: MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib (P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy. Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET.

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.

Recent clinical data have linked KRAS/TP53 co-mutation (mut) to resistance to radiotherapy (RT), but supporting laboratory in vivo evidence is lacking. In addition, the ability of different radiation doses, with/without epidermal growth factor receptor (EGFR)-directed treatment, to achieve local tumour control as a function of KRAS status is unknown. Here, we assessed clonogenic radiation survival of a panel of annotated lung cancer cell lines. KRASmut/TP53mut was associated with the highest radioresistance in non-isogenic and isogenic comparisons. To validate these findings, isogenic TP53mut NCI-H1703 models, KRASmut or wild-type (wt), were grown as heterotopic xenografts in nude mice. A clinical RT schedule of 30 fractions over 6 weeks was employed. The dose that controlled 50% of tumours (TCD(50)) was calculated. The TCD(50) for KRASwt/TP53mut xenografts was 43.1 Gy whereas KRASmut/TP53mut tumours required a 1.9-fold higher TCD(50) of 81.4 Gy. The EGFR inhibitor erlotinib radiosensitized KRASmut but not KRASwt cells and xenografts. The TCD(50) associated with adding erlotinib to RT was 58.8 Gy for KRASmut, i.e., a ~1.4-fold dose enhancement. However, the EGFR antibody cetuximab did not have a radiosensitizing effect. In conclusion, we demonstrate for the first time that KRASmut in a TP53mut background confers radioresistance when studying a clinical RT schedule and local control rather than tumour growth delay. Despite the known unresponsiveness of KRASmut tumours to EGFR inhibitors, erlotinib radiosensitized KRASmut tumours. Our data highlight KRAS/TP53 co-mutation as a candidate biomarker of radioresistance that can be at least partially reversed by dose escalation or the addition of a targeted agent.

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.

MYCN-amplified neuroblastoma is a lethal subset of pediatric cancer. 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' heel" 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 is hypersensitive to the combination of an inhibitor of the lactate transporter MCT1, AZD3965, and complex I of the mitochondrion, phenformin. Our data demonstrate that MCT4 is highly correlated with resistance to the combination in the screen 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 the AZD3965 and phenformin combination. The result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum stress, and cell death. 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 that 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.

Acquired drug resistance to even the most effective anti-cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1‒6, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. The extent to which therapy actively drives tumor evolution by promoting mutagenic processes7 or simply provides the selective pressure necessary for the outgrowth of drug-resistant clones8 remains an open question. Here, we report that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Induction of A3A facilitated the formation of double-strand DNA breaks (DSBs) in cycling drug-treated cells, and fully resistant clones that evolved from drug-tolerant intermediates exhibited an elevated burden of chromosomal aberrations such as copy number alterations and structural variations. Preventing therapy-induced A3A mutagenesis either by gene deletion or RNAi-mediated suppression delayed the emergence of drug resistance. Finally, we observed accumulation of A3A mutations in lung cancer patients who developed drug resistance after treatment with sequential targeted therapies. These data suggest that induction of A3A mutagenesis in response to targeted therapy treatment may facilitate the development of acquired resistance in non-small-cell lung cancer. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to prevent or delay acquired resistance to lung cancer targeted therapy.

The development of molecular targeted drugs with radiation and chemotherapy is critically important for improving the outcomes of patients with hard-to-treat, potentially curable cancers. However, too many preclinical studies have not translated into successful radiation oncology trials. Major contributing factors to this insufficiency include poor reproducibility of preclinical data, inadequate preclinical modeling of intertumoral genomic heterogeneity that influences treatment sensitivity in the clinic, and a reliance on tumor growth delay instead of local control (TCD50) endpoints. There exists an urgent need to overcome these barriers to facilitate successful clinical translation of targeted radiosensitizers. To this end, we have used 3-dimensional (3D) cell culture assays to better model tumor behavior in vivo. Examples of successful prediction of in vivo effects with these 3D assays include radiosensitization of head and neck cancers by inhibiting epidermal growth factor receptor or focal adhesion kinase signaling, and radioresistance associated with oncogenic mutation of KRAS. To address the issue of tumor heterogeneity, we leveraged institutional resources that allow high-throughput 3D screening of radiation combinations with small-molecule inhibitors across genomically characterized cell lines from lung, head and neck, and pancreatic cancers. This high-throughput screen is expected to uncover genomic biomarkers that will inform the successful clinical translation of targeted agents from the National Cancer Institute Cancer Therapy Evaluation Program portfolio and other sources. Screening "hits" need to be subjected to refinement studies that include clonogenic assays, addition of disease-specific chemotherapeutics, target/biomarker validation, and integration of patient-derived tumor models. The chemoradiosensitizing activities of the most promising drugs should be confirmed in TCD50 assays in xenograft models with or without relevant biomarker and using clinically relevant radiation fractionation. We predict that appropriately validated and biomarker-directed targeted therapies will have a higher likelihood than past efforts of being successfully incorporated into the standard management of hard-to-treat tumors.

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.

While synthetic lethality (SL) holds promise in developing effective cancer therapies, SL candidates found via experimental screens often have limited translational value. Here we present a data-driven approach, ISLE (identification of clinically relevant synthetic lethality), that mines TCGA cohort to identify the most likely clinically relevant SL interactions (cSLi) from a given candidate set of lab-screened SLi. We first validate ISLE via a benchmark of large-scale drug response screens and by predicting drug efficacy in mouse xenograft models. We then experimentally test a select set of predicted cSLi via new screening experiments, validating their predicted context-specific sensitivity in hypoxic vs normoxic conditions and demonstrating cSLi’s utility in predicting synergistic drug combinations. We show that cSLi can successfully predict patients’ drug treatment response and provide patient stratification signatures. ISLE thus complements existing actionable mutation-based methods for precision cancer therapy, offering an opportunity to expand its scope to the whole genome., Synthetic lethality (SL) offers a new precision oncology approach, which is based on targeting cancer-specific vulnerabilities across the whole genome, going beyond cancer drivers. The authors develop an approach termed ISLE to identify clinically relevant SL interactions and use them for patient stratification and novel target identification.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-β signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Introduction Tumor immunotherapy have broadened therapeutic options for tumor treatment. The role of immune function in juvenile recurrent respiratory papillomatosis (JRRP) has not been investigated. Applying immunoblockade inhibitors as a novel disease treatment is unclear. Our study, for the first time, evaluates immune infiltration and immuno-suppressive molecule expression in JRRP. Our study provides insights in possibly treating this disease with tumor immunotherapies. We aimed to determine expression of programmed death-ligand 1 (PD-L1), a cancer escape protein, and presence of CD8+ T cell infiltration in tumor microenvironment. Material and methods Seven patients with JRRP (mean age: 7.43; age range 3–17) in this study routinely have their tumors surgical debulked at Massachusetts Eye and Ear Infirmary. Following surgery, samples were de-identified and sent to pathology where they were stained and analyzed. Results Six out of seven patients expressed PD-L1 on tumor cells to various extents. Three patients showed concurrent PD-L1 expression on tumor cells and abundant CD8+ tumor infiltrating lymphocytes as well as PD-L1+ stromal lymphocytes, while PD-L1 expression on tumor cells were not associated with CD8+ tumor infiltrating T cells nor PD-L1+ stromal lymphocytes in the other three patients. HPV 6/11 and p16 was detected in all the patients. There appeared to be no correlation between either PD-L1 expression and CD8+ infiltration and clinical severity as measured by both the number of surgeries per year or Derkay score. Conclusions Despite a small cohort, the expression of p16 and HPV 6/11 in all of the patients confirms the tissues were HPV tumor cells. PD-L1 expression was detected in the vast majority of tumor samples, while inflammatory cell compartments showed a higher degree of variation. Expression of PD-L1 on tumor cells but not inflammatory cells raises the possibility of a tumor cell intrinsic manner of PD-L1 expression. In contrast, a group of patients showed PD-L1 positivity in both tumor and inflammatory cells along with abundant CD8+ tumor infiltrating lymphocytes, suggesting adoptive immune resistance in these tumors and potential benefits from tumor immunotherapy.

ONC206 is a DRD2 antagonist and ClpP agonist that is a chemical derivative of ONC201, which is in Phase II clinical trials for H3 K27M-mutant glioma. We have previously reported that dopamine receptor expression correlates with ONC201 and ONC206 efficacy. Here, we evaluated additional predictive biomarkers for both agents in the GDSC panel using RNA-seq expression data. ClpP emerged as a strong predictor of efficacy for ONC206 (IC50: p = 1.83E-4, AUC: 9.92E-13). ClpP activation enhances degradation of its substrates, including electron transport chain members responsible for oxidative phosphorylation (OXPHOS). Imipridones activate the integrated stress response involving ATF4 and cause proteasomal degradation of c-myc, which also reduces OXPHOS. Accordingly, high ATF4 (IC50: p = 4.92E-5, AUC: p=2.84E-9) and elevated c-myc correlated with ONC206 efficacy (IC50: p = 6.42E-6, AUC: 3.31E-12). EGFR expression is inversely correlate with DRD2 expression in glioma and its activation is associated with glycolysis. Low EGFR expression correlated with increased ONC206 efficacy (IC50: p = 4.32E-7, AUC: p = 6.44E-20). Loss of HIF1 shunts cellular metabolism toward OXPHOS. Low HIF1 expression correlated with increased anticancer efficacy for ONC206 (IC50: p = 1.96E-3, AUC: p = 1.56E-11). Expression of each of the five markers was significantly different in cell lines that achieved an IC90 with ONC206 (~10%) versus those that did not. A similar analysis for ONC201 revealed that ClpP, MYC and EGFR are more predictive of efficacy relative to ATF4 and HIF1. Combinatorial biomarker analyses revealed MYC/EGFR as the most significant predictor of IC50 for both agents. ClpP/MYC and ClpP/HIF1 were the most significant predictors of AUC for ONC201 and ONC206, respectively. Ongoing studies are further investigating tumor type enrichment of biomarkers. Prediction of innate imipridone sensitivity using biomarkers identified in this study may guide patient and tumor type selection in clinical trials.

ONC201 is the first clinical bitopic antagonist of DRD2, an oncogenic receptor in brain and neuroendocrine tumors. ONC206, a derivative of ONC201 that shares the imipridone core structure, is also a bitopic DRD2 antagonist that exhibits enhanced non-competitive effects, high specificity, nanomolar potency, and disruption of DRD2 homodimers. Broad nanomolar efficacy of ONC206 (GI50 < 78-889nM, CellTitre-Glo, 72h) was observed in >1,000 GDSC cancer cell lines. Maximal ONC206 sensitivity was observed in pheochromocytoma, high-grade gliomas, neuroblastoma, medulloblastoma, sarcoma and cholangiocarcinoma cell lines exhibiting a DRD2+/DRD5- RNA expression signature. An exposure time of 48h at nanomolar concentrations was sufficient for maximal inhibition of tumor cell viability. ONC206 reduced the viability of normal human fibroblasts at higher doses (GI50 > 5µM), suggesting a wide therapeutic window. Antitumor efficacy without body weight loss was observed with 50 mg/kg weekly oral ONC206 in a dopamine-secreting HuCCT1 cholangiocarcinoma subcutaneous xenograft model. Biodistribution studies in Sprague-Dawley rats revealed a ~12 µM plasma Cmax with a systemic terminal half-life of ~6 hours upon a single oral dose of 50 mg/kg. Additionally, 5–10 fold higher ONC206 concentrations were observed in adrenal gland, bile duct, brain and bone marrow relative to plasma. Nanomolar concentrations were also observed in the CSF above DRD2 antagonism thresholds, unlike ONC201. GLP toxicology studies with weekly oral ONC206 in Sprague-Dawley rats and beagle dogs revealed no dose-limiting toxicities. Mild and reversible decreased body weight and/or body weight gain with no effects on food consumption were observed at the highest evaluated dose in both species. The highest non-severely toxic dose (HNSTD) was ≥ 16.7 mg/kg in dogs and ≥ 50 mg/kg in rats that exceeds efficacious doses in preclinical models. Using standard allometric scaling, a 90 mg starting dose of ONC206 was selected for the first-in-human clinical trial in biomarker-defined adult recurrent CNS tumors.

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion–positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion–positive ICC.Significance:ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.This article is highlighted in the In This Issue feature, p. 983

Introduction Non-small-cell lung cancer (NSCLC) is among the most common cancers, accounting for 85% of all lung cancer cases. Drug combinations are known to more effective than single drugs and can delay drug resistance. NSCLC has a poor prognosis and there are no approved targeted cancer drug combinations. Here we have conducted the first large-scale screen of drug combinations in NSCLC cell lines to identify new candidate synergistic combinations and map the landscape of synthetic lethal and synergistic pathways underlying them. Results We conducted the largest of its kind in vitro drug combination screening of 81 NSCLC cell-lines for 21 x 242 cancer drug combinations at 5 different doses. Our collection of approved and investigational drugs included mainly targeted drugs as well as some chemotherapy drugs. For each drug combination, we computed the synergy score using the Bliss model. Our key findings are as follows: 1. We identify drug combinations (like CHEK and WEE1 inhibitors) that are highly synergistic in many NSCLC cell lines. 2. Combinations of targeted therapies are overall more synergistic than chemotherapeutic drug combinations. 3. Synergistic combinations occur more within pathways. 4. We see that most drug combinations do not make cell lines more sensitive than the most sensitive cell line to the corresponding single agents across all the cell lines tested. 5. We identify a new class of drug combinations - called super-sensitizers - whose combined effects are larger than the effects observed for the individual drugs composing across all of the cohort cell-lines. 6. Combinations of multi-target drugs tend to be more synergistic than single-target drugs. 7. We identify and characterize a set of context-specific drug combinations that are differentially synergistic or differentially effective (i.e. viability after treatment) between wild-type vs mutant cell lines of various cancer drivers (KRAS, TP53, STK11, PIK3CA, EGFR). 8. Studying potential mechanistic underpinnings of drug synergism in NSCLC, we find that: (a) synergistic combinations target genes that have more proximal physical interactions, (b) some synergistic combinations target synthetic lethal combinations, (c) a subset of synergistic combinations have further support by NSCLC patients survival analysis. Conclusions We performed the first large in vitro drug combination screen in NSCLC. We identify top drug combinations that are highly synergistic and effective in many cell lines and characterize key biological properties and mechanisms underlying them. The screen results are provided in a user-friendly public data resource that can help facilitate the development of targeted drug combinations in lung cancer therapy. Citation Format: Nishanth Ulhas Nair, Adam Friedman, Patricia Greninger, Avinash D. Sahu, Ellen Murchie, Giovanna Stein Crowther, Joe McClanaghan, Joo Sang Lee, Daniel A. Haber, Jeffrey Engelman, Eytan Ruppin, Cyril Benes. A large cellular screen charting the landscape of synergistic drug combinations in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1053.

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER+/HER2- primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2+ and HER2- subpopulations: HER2+ circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2- circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2- circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2+ and HER2- circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2- phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.

Small-molecule inhibitors of the bromodomain and extraterminal (BET) family of proteins are being tested in clinical trials for a variety of cancers, but patient selection strategies remain limited. This challenge is partly attributed to the heterogeneous responses elicited by BET inhibition (BETi), including cellular differentiation, senescence, and death. In this study, we performed phenotypic and gene-expression analyses of treatment-naive and engineered tolerant cell lines representing human melanoma and leukemia to elucidate the dominant features defining response to BETi. We found that de novo and acquired tolerance to BETi is driven by the robustness of the apoptotic response, and that genetic or pharmacologic manipulation of the apoptotic signaling network can modify the phenotypic response to BETi. We further reveal that the expression signatures of the apoptotic genes BCL2, BCL2L1, and BAD significantly predict response to BETi. Taken together, our findings highlight the apoptotic program as a determinant of response to BETi, and provide a molecular basis for patient stratification and combination therapy development. Cancer Res; 76(6); 1313–9. ©2016 AACR.

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.

ONC201 is the first bitopic antagonist of dopamine receptor D2 (DRD2) and allosteric mitochondrial protease ClpP agonist, that is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201, is also a bitopic DRD2 antagonist that exhibits enhanced non-competitive effects, nanomolar potency, and disruption of DRD2 homodimers. In these studies, a FITC-casein degradation assay revealed that ONC206 also acts as an agonist of human ClpP and has a 3-fold improved potency. GEPIA database analysis showed ClpP mRNA was overexpressed in glioblastoma cells relative to normal cells. Broad nanomolar efficacy of ONC206 (GI50 < 78-889nM, 72h) was observed in >1,000 GDSC cancer cell lines with the highest sensitivity in cell lines exhibiting high ClpP and/or DRD2+/DRD5- RNA expression signatures. Among solid tumors, nervous system related cell lines were particularly sensitive. ONC206 reduced the viability of normal human fibroblasts at higher doses (GI50 > 5µM), suggesting a wide therapeutic window. Antitumor efficacy without body weight loss was observed with 50 mg/kg weekly oral ONC206 in a subcutaneous xenograft model of DRD2-overexpressing, dopamine-secreting tumor cells. Oral ONC206 at 50mg/kg exhibited a ~12 µM plasma Cmax and ~6 hours terminal half-life in Sprague-Dawley rats. Additionally, 5–10 fold higher ONC206 concentrations were observed in adrenal gland, bile duct, brain and bone marrow relative to plasma. GLP toxicology studies with weekly oral ONC206 in Sprague-Dawley rats and beagle dogs revealed no dose-limiting toxicities. Mild and reversible body weight changes were observed at the highest evaluated dose in both species. The no-observed-adverse-effect level was ≥ 16.7 mg/kg in dogs and ≥ 50 mg/kg in rats that exceed efficacious doses. A 50 mg starting dose of ONC206 was selected for the first-in-human open label, dose escalation, and food effect Phase I study in biomarker-enriched adult recurrent primary CNS tumors.

The combination of CDK4/6 inhibitors with anti-estrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Since PTEN loss also causes resistance to PI3Kα-inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment sequencing strategies. Citation Format: Carlotta Costa, Ye Wang, Amy Ly, Yasuyuki Hosono, Ellen Murchie, Charlotte S. Walmsley, Tiffany Huynh, Christopher Healy, Rachel Peterson, Shogo Yanase, Charles T. Jakubik, Laura E. Henderson, Leah J. Damon, Daria Timonina, Ioannis Sanidas, Christopher J. Pinto, Mari Mino-Kenudson, James Stone, Nicholas J. Dyson, Leif W. Ellisen, Aditya Bardia, Hiromichi Ebi, Cyril H. Benes, Jeffrey A. Engelman, Dejan Juric. PTEN loss mediates clinical cross-resistance to CDK4/6 and PI3Ká inhibitors in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1903.

The combination of CDK4/6 inhibitors with antiestrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells, ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because PTEN loss also causes resistance to PI3Kα inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment-sequencing strategies. Significance: Our analysis of serial biopsies uncovered RB and PTEN loss as mechanisms of acquired resistance to CDK4/6 inhibitors, utilized as first-line treatment for ER-positive advanced breast cancer. Importantly, these findings have near-term clinical relevance because PTEN loss also limits the efficacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting. This article is highlighted in the In This Issue feature, p. 1

The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges associated with inhibition of MCL1 protein–protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. Significance: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers. See related commentary by Leber et al., p. 1511. This article is highlighted in the In This Issue feature, p. 1494

Most patients with advanced cancer eventually acquire resistance to targeted therapies, spurring extensive efforts to identify molecular events mediating therapy resistance. Many of these events involvesynthetic rescue (SR) interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (therescuer). Here we perform a genome-wide prediction of SR rescuer genes by analyzing tumor transcriptomics and survival data of 10,000 TCGA cancer patients. Predicted SR interactions are validated in new experimental screens. We show that SR interactions can successfully predict cancer patients’ response and emerging resistance. Inhibiting predicted rescuer genes sensitizes resistant cancer cells to therapies synergistically, providing initial leads for developing combinatorial approaches to overcome resistance proactively. Finally, we show that the SR analysis of melanoma patients successfully identifies known mediators of resistance to immunotherapy and predicts novel rescuers.

// Avital Lev 1 , Amriti R. Lulla 1 , Jessica Wagner 1 , Marie D. Ralff 1 , Joshua B. Kiehl 1 , Yan Zhou 2 , Cyril H. Benes 3 , Varun V. Prabhu 4 , Wolfgang Oster 4 , Igor Astsaturov 5 , David T. Dicker 1 and Wafik S. El-Deiry 1 1 Department of Hematology/Oncology, Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, USA 2 Biostatistics Department, Fox Chase Cancer Center, Philadelphia, PA, USA 3 Massachusetts General Hospital, Boston, MA, USA 4 Oncoceutics, Inc., Philadelphia, PA, USA 5 Department of Hematology/Oncology, Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, USA Correspondence to: Wafik S. El-Deiry, email: // Keywords : pancreatic cancer, ONC201, ONC212, AG1024, IGF1-R Received : July 25, 2017 Accepted : August 17, 2017 Published : September 12, 2017 Abstract Pancreatic cancer is chemo-resistant and metastasizes early with an overall five-year survival of ~8.2%. First-in-class imipridone ONC201 is a small molecule in clinical trials with anti-cancer activity. ONC212, a fluorinated-ONC201 analogue, shows preclinical efficacy in melanoma and hepatocellular-cancer models. We investigated efficacy of ONC201 and ONC212 against pancreatic cancer cell lines ( N =16 including 9 PDX-cell lines). We demonstrate ONC212 efficacy in 4 in-vivo models including ONC201-resistant tumors. ONC212 is active in pancreatic cancer as single agent or in combination with 5-fluorouracil, irinotecan, oxaliplatin or RTK inhibitor crizotinib. Based on upregulation of pro-survival IGF1-R in some tumors, we found an active combination of ONC212 with inhibitor AG1024, including in vivo . We show a rationale for targeting pancreatic cancer using ONC212 combined with targeting the unfolded-protein response and ER chaperones such as GRP78/BIP. Our results lay the foundation to test imipridones, anti-cancer agents, in pancreatic cancer, that is refractory to most drugs.

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.

Summary Personalized cancer therapy is based on a patient’s tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient’s living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care., Graphical Abstract, Highlights • Successful culture of cancer cells from tumor biopsies • An immunofluorescence-based assay to quantify drug sensitivity in mixed cell cultures • NSCLC patients’ biopsy culture sensitivities reflect clinical response, Kodack et al. report on the development of cancer models from tumor biopsies and technologies toward a functional approach for personalized medicine. They describe the ability to reliably test drug response in patient-derived samples of mixed cell populations. In doing so, they show that patient biopsy cultures may predict patient clinical responses.

Juvenile Recurrent Respiratory Papillomatosis is a Human Papillomavirus (HPV) infection associated tumor that primarily affects children. Currently, there are no effective therapeutic reagents to treat this disease and patients often receive repeated surgeries at papilloma recurrence. Previous studies have shown promising results in treating HPV related tumors with immunological approaches. However, immune cell infiltration and immune response in JRRP has not yet to be investigated. Using immunochemistry staining as well as flow cytometry, we evaluated expression of program death ligand 1 (PD-L1) on tumor cells and presence of lymphocytes in these tumors. In our small cohort of samples (n=7), we detected PD-L1 positive tumor cells in all the samples with varying degree of signal strength and percentage of positivity. We also detected presence of immune cells including CD4 and CD8 T cells in these tumors. Our results suggest the possibility of managing this disease through modulation of immune cells. Citation Format: Tingyu Liu, Carissa Wentland, Max Greenberg, Brandon Sepe, Tiffany Huynh, David Kodack, Mari Mino-Kenudson, Cyril Benes, Christopher Hartnick, Jeffrey Engelman. Detection of PD-L1 and lymphocytes infiltration in Juvenile Recurrent Respiratory Papillomatosis (JRRP). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A22.

Targeted cancer therapies have produced substantial clinical responses, but most tumors develop resistance to these drugs. Here, we describe a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome resistance. We established cell culture models derived from biopsy samples of lung cancer patients whose disease had progressed while on treatment with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors and then subjected these cells to genetic analyses and a pharmacological screen. Multiple effective drug combinations were identified. For example, the combination of ALK and MAPK kinase (MEK) inhibitors was active in an ALK -positive resistant tumor that had developed a MAP2K1 activating mutation, and the combination of EGFR and fibroblast growth factor receptor (FGFR) inhibitors was active in an EGFR mutant resistant cancer with a mutation in FGFR3 . Combined ALK and SRC (pp60c-src) inhibition was effective in several ALK-driven patient-derived models, a result not predicted by genetic analysis alone. With further refinements, this strategy could help direct therapeutic choices for individual patients.

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.

Background: Most patients with advanced cancer eventually acquire resistance to targeted therapies, spurring extensive efforts to identify molecular events mediating therapy resistance. Here we demonstrate that many of these molecular events involve synthetic rescue (SR) genetic interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (the rescuer). Methods: Analyzing recently published large scale in vitro functional screens we identify 100,000s candidate SR interactions that show evidence of rescue events in cancer cell lines. We then analyze tumor transcriptomic, genomic, and genetic profiles together with survival and clinical characteristics of 10,000 TCGA cancer patients to identify the few thousand SR interactions that have strong evidence that they are mediators of emerging resistance in patient tumors. Results: We identify the first rescue network, composed of SR interactions common to many cancer types. The identified SRs successfully match the resistance mediators identified in recently published clinical studies. We perform multiple in vitro analyses in head and neck and lung cancer, showing that targeting predicted rescuer genes successfully re-sensitizes resistant cancer cells, providing specific leads for targeting resistance proactively. We further demonstrate that SR-based combination therapy can improve the progression free survival in mouse xenografts models. Notably, we show that SR interactions successfully predict cancer patients' response in the TCGA compendium, showing performance superior to existing machine learning based predictive models. Finally, we show that SR analysis of melanoma patients successfully identifies known mediators of resistance to checkpoint immunotherapy (reported previously in mice) and suggests new combination therapies that counteract the resistance. Conclusions: This work presents a new paradigm identifying and harnessing synthetic rescue interactions to counteract resistance to both targeted- and immuno-therapies. Future implementations of this approach will have two broad implications in the precision oncology era: first for determining the most effective treatment regimen based on the molecular characteristics of individual patient’s tumor; second for identifying conjunct drugs to counteract resistance to existing primary therapies, for both targeted and immune checkpoint therapies. Citation Format: Avinash Das, Joo Sang Lee, Gao Zhang, Zhiyong Wang, Arnaud Amzallag, Genevieve Boland, Sridhar Hannenhalli, Meenhard Herlyn, Cyril Benes, J. Silvio Gutkind, Keity Flaherty, Eytan Ruppin. Genome-wide prediction of synthetic rescue mediators of resistance to targeted and immunotherapy [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 LB-149.

ONC201 is the founding member of a novel class of anti-cancer compounds called imipridones that is currently in Phase II clinical trials in multiple advanced cancers. Since the discovery of ONC201 as a p53-independent inducer of TRAIL gene transcription, preclinical studies have determined that ONC201 has anti-proliferative and pro-apoptotic effects against a broad range of tumor cells but not normal cells. The mechanism of action of ONC201 involves engagement of PERK-independent activation of the integrated stress response, leading to tumor upregulation of DR5 and dual Akt/ERK inactivation, and consequent Foxo3a activation leading to upregulation of the death ligand TRAIL. ONC201 is orally active with infrequent dosing in animals models, causes sustained pharmacodynamic effects, and is not genotoxic. The first-in-human clinical trial of ONC201 in advanced aggressive refractory solid tumors confirmed that ONC201 is exceptionally well-tolerated and established the recommended phase II dose of 625 mg administered orally every three weeks defined by drug exposure comparable to efficacious levels in preclinical models. Clinical trials are evaluating the single agent efficacy of ONC201 in multiple solid tumors and hematological malignancies and exploring alternative dosing regimens. In addition, chemical analogs that have shown promise in other oncology indications are in pre-clinical development. In summary, the imipridone family that comprises ONC201 and its chemical analogs represent a new class of anti-cancer therapy with a unique mechanism of action being translated in ongoing clinical trials.

Imipridones are first-in-class anti-tumor compounds including ONC201, which has shown promising clinical activity. ONC212 was designed as a second-generation imipridone. We first confirmed the ONC212 effects in a collection of 1,088 human cancer cell lines available from the Genomic of Drug Sensitivity in Cancer Project; leukemia was identified as the most sensitive tumor type. In fact, ONC212 exerted prominent apoptogenic effects in acute myeloid leukemia (AML) cell lines and primary AML, but not normal bone marrow (BM) cells. We investigated the effects of ONC212 in vivo in an aggressive systemic AML xenograft model using OCI-AML3 cells. ONC212 markedly inhibited AML expansion and prolonged median survival (controls: 43 d, ONC212: 49 d; p = 0.0003). For in vivo functional assessment of ONC212's anti-tumor effects against leukemia stem and progenitor cells (LSPCs), we treated patient-derived xenograft (PDX) cells with ONC212 (250 nM, 36 hr) ex vivo, and then injected into recipient NSG mice. After one month, the human leukemic CD45+ cells in the peripheral blood, spleen, and BM were significantly decreased in the ONC212 treated group. The median survival was remarkably prolonged (controls: 36 d, ONC212: 82 d; p < 0.0001). These results indicate that ONC212 has anti-LSPC effects to reduce the engraftment potential. We previously demonstrated that the prototype it compound ONC201 induces apoptosis via an atypical integrated stress response (ISR; Ishizawa et al., Sci Signal, 2016). As expected, ONC212 induced the transcription factor ATF4, a key effector of ISR. Because BCL-2 is generally considered to be protective against ISR-mediated apoptosis, we hypothesized that the BCL-2 inhibitor ABT-199 could further sensitize AML cells to ONC212. Indeed, the in vitro combination of ONC212 plus ABT-199 synergistically induced apoptosis in AML cells. Furthermore, the combination showed highly significant synergistic anti-leukemia effects in vivo. The combinatorial treatment prolonged overall median survival (controls: 20 d, each agent: 21 d, the combination 30 d; p < 0.0001). Since the G-protein-coupled receptor (GPCR) dopamine receptor D2 is the putative target of ONC201, we hypothesized that ONC212 also targets GPCRs. The PathHunter β-arrestin screening discovered that ONC212 specifically activated the orphan GPCR GPR132. Consistently, the GPR132 mRNA expression was correlated with ONC212 sensitivity. On the other hand, GPR132 overexpression induced cell death in AML cells, which is consistent with previous reports implicating GPR132 as a tumor suppressor. Furthermore, ONC212 increased GPR132 mRNA expression. These results suggest that GPR132 could be a potential therapeutic target in AML. Taken together, ONC212 has potential as a novel agent for AML therapy. This study provides the first reported opportunity to therapeutically target GPR132 in oncology. Citation Format: Takenobu Nii, Jo Ishizawa, Varun V. Prabhu, Vivian Ruvolo, Neel Madhukar, Ran Zhao, Hong Mu, Lauren Heese, Kensuke Kojima, Mathew Garnett, Ultan McDermott, Cyril Benes, Neil Charter, Sean Deacon, Olivier Elemento, Joshua Allen, Wolfgang Oster, Martin Stogniew, Michael Andreeff. The novel imipridone ONC212 highly synergizes with the BCL-2 inhibitor ABT-199 in AML and activates orphan receptor GPR132 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4957.

Background: Tyrosine kinase inhibitors (TKI) have yielded promising responses in non-small-cell lung cancer (NSCLC) with EGFR mutations and ALK translocations. However, these and other targeted therapies are limited by intrinsic and acquired drug resistance. The previous study from our group investigated tumor autonomous resistance mechanisms by developing patient-derived cancer models (PDCs). In this study, we aimed to decipher the non-autonomous resistance mechanisms via tumor microenvironment by developing patient-derived fibroblast (PDF) models. Method: Cancer-associated fibroblast cell lines are established directly from individual EGFR mutant NSCLC biopsies. These cell lines, as representative of each patient's tumor microenvironment, are further subjected to functional analysis. An imaging-based high-throughput platform is developed to screen for non-autonomous resistance by co-culturing PDC and PDF models in vitro. In the parallel, two independent approaches are performed to further identify mechanisms underlying the non-autonomous resistance. These include a drug screen to determine the pathway maintaining the cancer cells' survival, and a secretomic analysis on the PDFs to identify the plausible cytokine(s) responsible for the resistance. Result: By co-culturing screening, non-autonomous resistance can be found in a wide spectrum of models. The subsequent drug screen reveals both a canonical HGF dependent and novel HGF independent mechanisms contributing to EGFR TKI resistance. Both of these can be explained by the PDF's variable cytokine secretion and can be overcome by specific therapeutic combinations. Moreover, the microenvironment-driven EGFR TKI resistance has also been validated in vivo. And the prevalence of the identified cytokine is further tested in clinical specimens. Conclusion: PDFs provide a new avenue to explore non-autonomous resistance for targeted therapy. Applying this approach, we identified both the canonical HGF dependent and novel HGF independent mechanisms that putatively conferring EGFR TKI resistance. Taking EGFR TKI therapy as a paradigm, these findings will be valuable to optimize targeted therapy and to inform the design of personalized pharmaceutical interventions. Citation Format: Haichuan Hu, Lecia Sequist, Zosia Piotrowska, David Kodack, Aaron Hata, Matt Niederst, Cyril Benes, Jeffrey Engelman. Decoding tumor microenvironment to enhance NSCLC targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4954.

Invasive lobular carcinoma (ILC) is the second most frequently occurring histological breast cancer subtype after invasive ductal carcinoma (IDC), accounting for around 10% of all breast cancers. The molecular processes that drive the development of ILC are still largely unknown. We have performed a comprehensive genomic, transcriptomic and proteomic analysis of a large ILC patient cohort and present here an integrated molecular portrait of ILC. Mutations in CDH1 and in the PI3K pathway are the most frequent molecular alterations in ILC. We identified two main subtypes of ILCs: (i) an immune related subtype with mRNA up-regulation of PD-L1, PD-1 and CTLA-4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone related subtype, associated with Epithelial to Mesenchymal Transition (EMT) and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using the somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization may help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies.

Background: ONC201, an imipridone that is a selective antagonist of the G protein-coupled receptors dopamine receptor D2 (DRD2) and D3 (DRD3), has exhibited biologic activity and an exceptional safety profile in a phase II study in bevacizumab-naïve recurrent glioblastoma (Arrillaga et a.l, 2017). Single-agent ONC201 efficacy has been observed in preclinical glioblastoma models in addition to robust penetrance of the blood-brain barrier (Allen et al., 2013). DRD2 antagonism induces tumor cell apoptosis via the same signaling pathways affected by ONC201. In addition, DRD2 is expressed on NK and other immune cells and DRD2 antagonism can induce their activation. Methods: Cell viability assays were performed with ONC201 in >1000 Genomic of Drug Sensitivity in Cancer (GDSC) cell lines and NCI60. Immunohistochemistry staining of DRD2/DRD5 was performed in glioblastoma tissue microarrays and archival tumor tissues. Whole exome sequencing was performed in RKO cells with acquired resistance to ONC201. DRD5 wild-type and mutant constructs were generated for overexpression studies. ELISA was used to quantitate serum prolactin and immune effector (perforin) levels. Intratumoral drug concentrations were evaluated by LC-MS assays conducted on glioblastoma tissue resected from patients following the second dose of 625mg ONC201. Results: Evaluation of ONC201 in GDSC cell lines confirmed broad anticancer efficacy with high sensitivity (~1-3 µM) in human brain cancer. The Cancer Genome Atlas (TCGA) revealed that DRD2 is highly expressed in glioblastoma relative to other dopamine receptors and that genetic aberrations are rare. High expression of DRD2 occurred in primary, rather than secondary, glioblastoma and was associated with a poor prognosis. Immunohistochemistry of tissue microarrays revealed DRD2 overexpression in glioblastoma relative to normal brain. A linear correlation between DRD2 mRNA and ONC201 GI50 was observed among glioblastoma cell lines in the NCI60 panel. Interestingly, expression of DRD5, a D1-like dopamine receptor that counteracts DRD2 signaling, was significantly inversely correlated with ONC201 potency in the NCI60 and GDSC datasets (P 5 month had no detectable expression of DRD5, unlike those with PFS Citation Format: Varun Vijay Prabhu, Neel Madhukar, C. Leah B. Kline, Rohinton Tarapore, Wafik El-Deiry, Olivier Elemento, Faye Doherty, Alexander VanEngelenburg, Jessica Durrant, Andrew Zloza, Cyril Benes, Isabel Arrillaga, Wolfgang Oster, Joshua E. Allen. Targeting DRD2 dysregulation in recurrent glioblastoma with imipridone ONC201: predictive and pharmacodynamic clinical biomarker analyses [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A060.

ONC201, founding member of the imipridone class of compounds, is a highly selective small molecule G protein-coupled receptor (GPCR) antagonist that is in Phase I/II advanced cancer clinical trials. In this study, we evaluated GPCR engagement and antitumor activity of ONC212, an imipridone that is a chemical analogue of ONC201. Experimental GPCR profiling using the PathHunter® β-Arrestin assay, determined that ONC212 is a selective agonist of the orphan GPCR GPR132/G2A. Multidose validation confirmed nanomolar GPR132 agonist activity (EC50 ~400nM). GPR132 is a stress-inducible orphan GPCR that causes G2/M arrest. The tumor suppressive role of GPR132 has been demonstrated most notably in lymphoid leukemia. Gene expression analysis of samples in the Cancer Genome Atlas (TCGA) revealed GPR132 is expressed in a range of tumor types with highest expression in leukemia and lymphoma. The in vitro efficacy of ONC212 was assessed in the Genomic of Drug Sensitivity in Cancer (GDSC) collection of cell lines (>1,000 cell lines). Cell viability assays were performed at 72 hours post-treatment to generate dose responses curves at concentrations from 78nM up to 20μM. ONC212 was broadly efficacious across most solid tumors and hematological malignancies in the low nanomolar range. Ranking the sensitivity of cancer lines to ONC212 by tumor type revealed that leukemia and lymphoma are the most responsive tumor types based on completeness of response (area under the dose-response curve). Interestingly, we also observed that high expression of GPR132 significantly correlated with ONC212 efficacy in GDSC cell lines, suggesting the importance of GPR132 agonist activity for ONC212 efficacy. ONC212 was tested in 62 human leukemia cell lines that included acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) and hairy cell leukemia. ONC212 demonstrated broad spectrum anti-leukemic activity and was equally efficacious across all leukemia subtypes tested. Most cell lines (60/62) were responsive to ONC212 with GI50 ranging from ONC212 was also tested in 58 lymphoma cell lines comprised of anaplastic large cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma (CTCL), diffuse large B-cell lymphoma (DLBCL), Mantle cell lymphoma (MCL), Follicular Lymphoma and Hodgkin’s lymphoma. ONC212 reduced cell viability in most cell lines (56/58) and was equally efficacious regardless of subtypes with GI50 ranging from Thus, GPR132 agonist ONC212 possesses robust anti-cancer activity in hematological malignancies irrespective of leukemia and lymphoma subtype and provides further validation of the anti-cancer efficacy of the novel imipridone class of small molecules. Citation Format: Varun Vijay Prabhu, Neel Madhukar, Rohinton Tarapore, Mathew Garnett, Ultan McDermott, Cyril Benes, Neil Charter, Sean Deacon, Wolfgang Oster, Michael Andreeff, Olivier Elemento, Martin Stogniew, Joshua Allen. Potent anti-cancer effects of selective GPR132/G2A agonist imipridone ONC212 in leukemia and lymphoma [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 1155. doi:10.1158/1538-7445.AM2017-1155

Significance: The identification of Synthetic Lethal interactions (SLi) has long been considered a foundation for the advancement of cancer treatment. The rapidly accumulating large-scale patient data now provides a golden opportunity to infer SLi directly from patient samples. Here we present a new data-driven approach termed ISLE for identifying SLi, which is then shown to be predictive of clinical outcomes of cancer treatment in an unsupervised manner, for the first time. Methods: ISLE consists of four inference steps, analyzing tumor, cell line and gene evolutionary data: It first identifies putative SL gene pairs whose co-inactivation is underrepresented in tumors, testifying that they are selected against. Second, it further prioritizes candidate SL pairs whose co-inactivation is associated with better prognosis in patients, testifying that they may hamper tumor progression. Finally, it eliminates false positive SLi using gene essentiality screens (testifying to causal SLi relations) and prioritizing SLi paired genes with similar evolutionary phylogenetic profiles. Results: We applied ISLE to analyze the TCGA tumor collection and generated the first clinically-derived pan-cancer SL-network, composed of SLi common across many cancer types. We validated that these SLi match the known, experimentally identified SLi (AUC=0.87), and show that the SL-network is predictive of patient survival in an independent breast cancer dataset (METABRIC). Based on the predicted SLi, we predicted drug response of single agents and drug combinations in a wide variety of in vitro, mouse xenograft and patient data, altogether encompassing >700 single drugs and >5,000 drug combinations in >1,000 cell lines, 375 xenograft models and >5,000 patient samples. Of note, these predictions were performed in an unsupervised manner, reducing the known risk of over-fitting the data commonly associated with supervised prediction methods. Our prediction is based on the notion that a drug is likely to be more effective in tumors where many of its targets’ SL-partners are inactive, and drug synergism may be mediated by underlying SLi between their targets. Most importantly, we demonstrate for the first time that an SL-network can successfully predict the treatment outcome in cancer patients in multiple large-scale patient datasets including the TCGA, where SLis successfully predict patients’ response for 75% of cancer drugs. Conclusions: ISLE is predictive of the patients’ response for the majority of current cancer drugs. Of paramount importance, the predictions of ISLE are based on SLi between (potentially) all genes in the cancer genome, thus prioritizing treatments for patients whose tumors do not bear specific actionable mutations in cancer driver genes, offering a novel approach to precision-based cancer therapy. The predictive performance of ISLE is likely to further improve with the expected rapid accumulation of additional patient data. Citation Format: Joo Sang Lee, Avinash Das, Livnat Jerby-Arnon, Seung Gu Park, Matthew Davidson, Dikla Atias, Arnaud Amzallag, Chani Stossel, Ella Buzhor, Welles Robinson, Kuoyuan Cheng, Joshua J. Waterfall, Paul S. Meltzer, Sridhar Hannenhalli, Cyril H. Benes, Talia Golan, Emma Shanks, Eytan Ruppin. Harnessing synthetic lethality to predict clinical outcomes of cancer treatment [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 543. doi:10.1158/1538-7445.AM2017-543

ONC201 is the lead small molecule of the imipridone class of anti-cancer compounds that is currently being evaluated in phase I/II advanced cancer clinical trials. ONC201 is a highly selective antagonist of the G protein-coupled receptor dopamine receptor D2 (DRD2) that has exhibited promising anti-cancer efficacy and an exceptional safety profile. In the current study, we evaluated the influence of the DRD2 pathway on the responsiveness of tumors to ONC201 in preclinical and clinical studies. In vitro and in vivo studies have previously demonstrated robust ONC201 efficacy in glioblastoma (Allen et al 2013) and lymphoma (Ishizawa et al 2016) models. ONC201 Phase I trials have also revealed evidence of clinical benefit in endometrial cancer (Stein et al 2016). In vitro efficacy profiling of ONC201 in the Genomic of Drug Sensitivity in Cancer (GDSC) collection of cell lines confirmed broad-spectrum anti-cancer efficacy with particularly high sensitivity in lymphoma, neuroblastoma, endometrial and brain cancer. DRD2 is overexpressed in many cancers and DRD2 antagonism kills cancer cells via the same signaling pathways that are altered in response to ONC201. Results from the Project Achilles screen indicate that anti-cancer effects of DRD2 knockdown in various tumor types correlated with overall ONC201 efficacy. In particular, we noted that lymphoma cells are highly sensitive to DRD2 knockdown- a tumor type where ONC201 performs well. Gene expression analysis of samples in the Cancer Genome Atlas (TCGA) revealed high DRD2 expression in ONC201-sensitive tumor types, such as lymphoma and glioblastoma, and that high expression of DRD2 in glioma was associated with a poor prognosis. High DRD2 expression was also observed in neuroendocrine prostate cancer relative to other prostate cancer subtypes. In immunohistochemistry analyses of patient-derived tumor tissue microarrays, DRD2 overexpression was particularly noted in endometrial cancer, neuroblastoma and pheochromocytoma relative to normal tissues. The anti-cancer activity of ONC201 in pheochromocytoma and neuroendocrine prostate cancer was confirmed in cell viability assays. In ONC201-treated patients, ELISA was used to quantitate serum prolactin levels, a clinical biomarker of DRD2 antagonism. A 2-fold mean induction of prolactin, was detected in the serum of ONC201-treated patients, in accordance with physiological DRD2 antagonism. Interestingly, expression of DRD5 (a D1-like dopamine receptor), which counteracts DRD2 signaling, was significantly negatively correlated with ONC201 in vitro potency in the NCI60 and GDSC dataset (P Citation Format: Neel Madhukar, Varun Vijay Prabhu, Etienne Dardenne, Faye Doherty, Alexander VanEngelenburg, Rohinton Tarapore, Mathew Garnett, Ultan McDermott, Cyril Benes, Wolfgang Oster, Wafik El-Deiry, Mark Stein, David Rickman, Joshua Allen, Olivier Elemento. The small molecule imipridone ONC201 is active in tumor types with dysregulation of the DRD2 pathway [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 2792. doi:10.1158/1538-7445.AM2017-2792

Background Preclinical studies have documented antitumor activity of PARP inhibition both in vitro and in vivo, against Ewing sarcoma cells. This study aimed to translate that observation into a clinical trial to assess the efficacy and tolerability of olaparib, a PARP inhibitor, in patients with advanced Ewing sarcoma (EWS) progressing after prior chemotherapy. Methods In this nonrandomized phase II trial, adult participants with radiographically measureable metastatic EWS received olaparib tablets, 400 mg orally twice daily, until disease progression or drug intolerance. Tumor measurements were determined by CT or MRI at 6 and 12 weeks after starting olaparib administration, and then every 8 weeks thereafter. Tumor response determinations were made according to RECIST 1.1, and adverse event determinations were made according to CTCAE, version 4.0. A total of 22 participants were planned to be enrolled using a conventional 2-step phase II study design. If no objective responses were observed after 12 participants had been followed for at least 3 months, further accrual would be stopped. Results 12 participants were enrolled, and all were evaluable. There were no objective responses (PR/CR), 4 SD (duration 10.9, 11.4, 11.9, and 17.9 wks), and 8 PD as best response. Of the SD, 2 had minor responses (−9% and −11.7% by RECIST 1.1). The median time to disease progression was 5.7 weeks. Further enrollment was therefore discontinued. No significant or unexpected toxicities were observed with olaparib, with only a single case each of grade 3 anemia and grade 3 thrombocytopenia observed. Conclusions This study is the first report of a prospective phase II trial to evaluate the safety and efficacy of a PARP inhibitor in patients with advanced Ewing sarcoma after failure of standard chemotherapy. Olaparib administration was safe and well tolerated when administered to this small heavily pre-treated cohort at the 400 mg BID dose, although the median duration of dosing was for only 5.7 weeks. No significant responses or durable disease control was seen, and the short average interval to disease progression underscores the aggressiveness of this disease. Other studies to combine cytotoxic chemotherapy with PARP inhibition in EWS are actively ongoing. Trial registration ClinicalTrials.gov Identifier: NCT01583543

Purpose: Improved therapeutic approaches are needed for the treatment of pancreatic ductal adenocarcinoma (PDAC). As dual MEK and PI3K inhibition is presently being used in clinical trials for patients with PDAC, we sought to test the efficacy of combined targeting of these pathways in PDAC using both in vitro drug screens and genetically engineered mouse models (GEMM). Experimental Design: We performed high-throughput screening of >500 human cancer cell lines (including 46 PDAC lines), for sensitivity to 50 clinically relevant compounds, including MEK and PI3K inhibitors. We tested the top hit in the screen, the MEK1/2 inhibitor, AZD6244, for efficacy alone or in combination with the PI3K inhibitors, BKM120 or GDC-0941, in a KrasG12D-driven GEMM that recapitulates the histopathogenesis of human PDAC. Results: In vitro screens revealed that PDAC cell lines are relatively resistant to single-agent therapies. The response profile to the MEK1/2 inhibitor, AZD6244, was an outlier, showing the highest selective efficacy in PDAC. Although MEK inhibition alone was mainly cytostatic, apoptosis was induced when combined with PI3K inhibitors (BKM120 or GDC-0941). When tested in a PDAC GEMM and compared with the single agents or vehicle controls, the combination delayed tumor formation in the setting of prevention and extended survival when used to treat advanced tumors, although no durable responses were observed. Conclusions: Our studies point to important contributions of MEK and PI3K signaling to PDAC pathogenesis and suggest that dual targeting of these pathways may provide benefit in some patients with PDAC. Clin Cancer Res; 21(2); 396–404. ©2014 AACR.

The wealth of genomic information available today to researcher and clinician has dramatically changed the way some diseases are defined. At the same time leveraging this information is a major challenge in part because previously established concepts and tools used for research and development need are largely inadequate. Using the example of cancer genomics this Translation focuses on systems biology approaches to inform the development of personalized medicine.

ONC201 is the founding member of the imipridone class of anti-cancer small molecules that possess a unique core chemical structure. ONC201 is currently being evaluated in several Phase I/II clinical trials for advanced cancers. In the current study, we evaluated the single agent and combinatorial efficacy of ONC201 in preclinical models of acute leukemia and multiple myeloma (MM). In acute leukemia, we evaluated ONC201 anti-cancer effects in acute myeloid leukemia (AML) (Kasumi-1, HL60) and acute lymphoblastic leukemia (ALL) (Reh, Jurkat and MOLT-4) cell lines. We observed a time- and dose-dependent decrease in cell viability for every cell line in the panel (EC50 1-5 µM). Vincristine-resistant cells HL60/VCR were also sensitive to single agent ONC201 with EC50 values on par with corresponding vincristine-sensitive parental cells. Dose- and time-dependent induction of apoptosis was noted in Western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201 for 48 hr. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously reported late-stage signaling effects of ONC201 in solid tumor cells (Allen et al, 2013). Sub-G1 analysis indicated that ONC201 induces apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. Western blot analysis revealed ONC201-mediated apoptosis involves PARP cleavage and caspase-9 activation in ALL cells. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells. We observed inhibition of Akt phosphorylation upon ONC201 treatment of ALL cells. Fresh AML patient cells were also found to be sensitive to ONC201 in cell viability and caspase 3/7 activity assays at 5µM. We observed that independent clones of cancer cells with acquired resistance to ONC201 were more sensitive to cytarabine compared to parental ONC201-sensitive cancer cells. In addition, ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine in AML cell lines. Determination of combination indices (CI) revealed synergy at several concentrations (CI 0.336-0.75 in CMK cells). Also, ONC201 combined additively with midostaurin in CMK cells and vincristine in HL60/VCR cells. Thus, ONC201 is a promising combinatorial partner for AML therapies based on these preclinical sensitization results. In accordance with ONC201-mediated activation of the integrated stress response that B cells are highly sensitive to (Kline et al and Ishizawa et al, 2016), MM was identified as one of the most ONC201-sensitive tumor types in the Genomics of Drug Sensitivity in Cancer collection of cell lines. Three human MM cell lines were used for validation (KMS18, MM.1S and RPMI-8226), which revealed a time- and dose-dependent decrease in cell viability (EC50 1-2.5 µM). Bortezomib-resistant cells MM.1S 33X were sensitive to ONC201 as a single agent with EC50 values comparable to bortezomib-sensitive parental cells. We observed an average of 10-fold induction of ONC201-mediated apoptosis using Sub-G1 analyses in MM cells at 5 µM, 48 hrs post-treatment. Rescue of ONC201-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, Western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Furthermore, ONC201 demonstrated synergistic reduction in cell viability at various concentrations in combination with either ixazomib or dexamethasone, which are used in the clinical treatment of MM, in RPMI8226 cells (CI 0.228-0.75). Also, ONC201 combined additively with bortezomib in RPMI8226 and MM.1S 33X cells. In summary, these preclinical studies support the ongoing ONC201 single agent trials in acute leukemias and MM. Our findings suggest that ONC201 may be an important therapeutic option for patients with hematological malignancies who have developed resistance to approved therapies. Additionally, our results point to specific standard-of-care therapies that may be combined with ONC201 to exert durable responses without adding to the burden of toxicity. Disclosures Prabhu: Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership.

ONC201, the founding member of the imipridone class of anti-cancer compounds, is a highly selective small molecule GPCR antagonist that is in Phase I/II advanced cancer clinical trials. In this study, we evaluated the anti-cancer effects of ONC212, an ONC201 analogue that possess the same unique core chemical structure shared by imipridones. The in vitro efficacy of ONC212 was assessed in the Genomics of Drug Sensitivity in Cancer collection of cell lines (>1,000 cell lines). Cell viability assays were performed to generate dose responses curves at concentrations from 78nM upto 20uM and at 72 hours post-treatment. ONC212 was broadly efficacious across most solid tumors and hematological malignancies in the low nanomolar range. Ranking the ONC212 sensitivity dataset revealed that leukemia is the most responsive tumor type based on completeness of response (area under the dose-response curve, AUC). ONC212 was tested in 65 leukemia cell lines in this study that is comprised of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) and hairy cell leukemia. ONC212 demonstrated broad spectrum anti-leukemic activity and was equally efficacious across all leukemia subtypes tested, in terms of AUC. Most cell lines (63/65) were responsive to ONC212 with GI50 ranging from Thus, ONC212 possesses robust anti-leukemic activity irrespective of subtype and provides further validation of the anti-cancer efficacy of the novel imipridone class of small molecules. Disclosures Prabhu: Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership. Stogniew:Oncoceutics Inc.: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership.