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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.

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.

Evolved resistance to tyrosine kinase inhibitor (TKI)-targeted therapies remains a major clinical challenge. In epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer (NSCLC), failure of EGFR TKIs can result from both genetic and epigenetic mechanisms of acquired drug resistance. Widespread reports of histologic and gene expression changes consistent with an epithelial-to-mesenchymal transition (EMT) have been associated with initially surviving drug-tolerant persister cells, which can seed bona fide genetic mechanisms of resistance to EGFR TKIs. While therapeutic approaches targeting fully resistant cells, such as those harboring an EGFRT790M mutation, have been developed, a clinical strategy for preventing the emergence of persister cells remains elusive. Using mesenchymal cell lines derived from biopsies of patients who progressed on EGFR TKI as surrogates for persister populations, we performed whole-genome CRISPR screening and identified fibroblast growth factor receptor 1 (FGFR1) as the top target promoting survival of mesenchymal EGFR mutant cancers. Although numerous previous reports of FGFR signaling contributing to EGFR TKI resistance in vitro exist, the data have not yet been sufficiently compelling to instigate a clinical trial testing this hypothesis, nor has the role of FGFR in promoting the survival of persister cells been elucidated. In this study, we find that combining EGFR and FGFR inhibitors inhibited the survival and expansion of EGFR mutant drug-tolerant cells over long time periods, preventing the development of fully resistant cancers in multiple vitro models and in vivo. These results suggest that dual EGFR and FGFR blockade may be a promising clinical strategy for both preventing and overcoming EMT-associated acquired drug resistance and provide motivation for the clinical study of combined EGFR and FGFR inhibition in EGFR-mutated NSCLCs.

Imipridones constitute a novel class of anti-tumor agents. Here, we report that a second-generation imipridone, ONC212, possesses highly increased anti-tumor activity compared to the first-generation compound ONC201. In vitro studies using human acute myeloid leukemia (AML) cell lines, primary AML, and normal bone marrow (BM) samples demonstrate that ONC212 exerts prominent apoptogenic effects in AML, but not in normal BM cells, suggesting potential clinical utility. Imipridones putatively engage G protein-coupled receptors (GPCRs) and/or trigger an integrated stress response in hematopoietic tumor cells. Comprehensive GPCR screening identified ONC212 as activator of an orphan GPCR GPR132 and Gαq signaling, which functions as a tumor suppressor. Heterozygous knock-out of GPR132 decreased the anti-leukemic effects of ONC212. ONC212 induced apoptogenic effects through the induction of an integrated stress response, and reduced MCL-1 expression, a known resistance factor for BCL-2 inhibition by ABT-199. Oral administration of ONC212 inhibited AML growth in vivo and improved overall survival in xenografted mice. Moreover, ONC212 abrogated the engraftment capacity of patient-derived AML cells in an NSG PDX model, suggesting potential eradication of AML initiating cells, and was highly synergistic in combination with ABT-199. Collectively, our results suggest ONC212 as a novel therapeutic agent for AML.

Many gene fusions are reported in tumours and for most their role remains unknown. As fusions are used for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their function in cancer. To systematically investigate the role of fusions in tumour cell fitness, we utilized RNA-sequencing data from 1011 human cancer cell lines to functionally link 8354 fusion events with genomic data, sensitivity to >350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness effects. Established clinically-relevant fusions were identified. Overall, detection of functional fusions was rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumour fitness. Therapeutically actionable fusions involving RAF1, BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have clinical implications., Gene fusions are observed in many cancers but their link to tumour fitness is largely unknown. Here, transcriptomic analysis combined with pharmacological and CRISPR-Cas9 screening of cancer cell lines was used to evaluate the functional linkage between fusions and tumour fitness.

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.

SummaryTargeted therapeutics have advanced cancer treatment, but single agent activity remains limited by de novo and acquired resistance. Combining targeted drugs is broadly seen as a way to improve treatment outcome, motivating the ongoing search for efficacious combinations. To identify synergistic targeted therapy combinations and study the impact of tumor heterogeneity on combination outcome, we systematically tested over 5,000 two drug combinations at multiple doses across a collection of 81 non-small cancer cell lines. Both known and novel synergistic combinations were identified. Strikingly, very few combinations yield synergy across the majority of cell line models. Importantly, synergism mainly arises due to sensitization of single agent resistant models, rather than further sensitize already sensitive cell lines, frequently via dual targeting of a single or two highly interconnected pathways. This drug combinations resource, the largest of its kind should help delineate new synergistic regimens by facilitating the understanding of drug synergism in cancer.

Summary Two-dimensional (2D) culture of tumor cells fails to recapitulate some important aspects of cellular organization seen in in vivo experiments. In addition, cell cultures traditionally use non-physiological concentration of nutrients. Here, we describe a protocol for a facile three-dimensional (3D) culture format for cancer cells. This 3D platform helps overcome the 2D culture limitations. In addition, it allows for longitudinal modeling of responses to cancer therapeutics. For complete details on the use and execution of this protocol, please refer to Lhuissier et al. (2017), Lehmann et al. (2016), Liu et al. (2016), and Duval et al. (2011)., Graphical Abstract, Highlights • A detailed protocol on hydrogel-based 3D culture of patient-derived tumor cell lines • No binding sites for cells in hydrogel polymers allowing for pure interaction of cells • Longitudinal 3D proliferation assays and drug-response assessments • Quick and easy recovery of 3D-cultured cells for downstream experiments, Two-dimensional (2D) culture of tumor cells fails to recapitulate some important aspects of cellular organization seen in in vivo experiments. In addition, cell cultures traditionally use non-physiological concentration of nutrients. Here, we describe a protocol for a facile three-dimensional (3D) culture format for cancer cells. This 3D platform helps overcome the 2D culture limitations. In addition, it allows for longitudinal modeling of responses to cancer therapeutics.

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: KRAS-mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy. Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C–activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient-derived xenograft models. Results: Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K–AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad-sensitizing effect of PI3K-AKT–mTOR pathway inhibitors. The G12Ci+PI3Ki combination was effective in vitro and in vivo on models resistant to single-agent ARS1620 including patient-derived xenografts models. Conclusions: Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.

PURPOSE: Triple-negative breast cancers (TNBC) are often resistant to treatment with ionizing radiation (IR). We sought to investigate whether pharmacologic inhibition of Chk1 kinase, which is commonly overexpressed in TNBC, preferentially sensitizes TNBC cells to IR. METHODS: Ten breast cancer cell lines were screened with small molecule inhibitors against Chk1 and other kinases. Chk1 inhibition was also tested in isogenic KRAS mutant or wild-type cancer cells. Cellular radiosensitization was measured by short-term and clonogenic survival assays and by staining for the DNA double-strand break (DSB) marker γ-H2AX. Radiosensitization was also assessed in breast cancer biopsies using an ex vivo assay. Aurora B kinase-dependent mitosis-like chromatin condensation, a marker of radioresistance, was detected using a specific antibody against co-localized phosphorylation of serine 10 and trimethylation of lysine 9 on histone 3 (H3K9me3/S10p). Expression of CHEK1 and associated genes was evaluated in TNBC and lung adenocarcinoma. RESULTS: Inhibition of Chk1 kinase preferentially radiosensitized TNBC cells in vitro and in patient biopsies. Interestingly, TNBC cells displayed lower numbers of IR-induced DSBs than non-TNBC cells, correlating with their observed radioresistance. We found that Chk1 suppressed IR-induced DSBs in these cells, which was dependent on H3K9me3/S10p – a chromatin mark previously found to indicate radioresistance in KRAS mutant cancers. Accordingly, the effects of Chk1 inhibition in TNBC were reproduced in KRAS mutant but not wild-type cells. We also observed co-expression of genes in this Chk1 chromatin pathway in TNBC and KRAS mutant lung cancers. CONCLUSIONS: Chk1 promotes an unexpected, common phenotype of chromatin-dependent DSB suppression in radioresistant TNBC and KRAS mutant cancer cells, providing a direction for future investigations into overcoming the treatment resistance of TNBC.

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.

BH3 mimetic drugs, which inhibit prosurvival BCL2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL1 inhibitors, we demonstrate that concurrent MEK + MCL1 inhibition induces apoptosis and tumor regression in KRAS-mutant non–small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL1 or BCL-xL was determined by the differential binding of proapoptotic BCL2 proteins to MCL1 or BCL-xL, respectively. The efficacy of dual MEK + MCL1 blockade was augmented by prior transient exposure to BCL-xL inhibitors, which promotes the binding of proapoptotic BCL2 proteins to MCL1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL2 family proteins for KRAS-mutant NSCLC. Significance: Defining the molecular basis for MCL1 versus BCL-xL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-xL and MCL1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS-mutant NSCLCs treated with MEK inhibitors. See related commentary by Leber et al., p. 1511. This article is highlighted in the In This Issue feature, p. 1494

Microtubule inhibitors as chemotherapeutic drugs are widely used for cancer treatment. However, the development of multidrug resistance (MDR) in cancer is a major challenge for microtubule inhibitors in their clinical implementation. From a high-throughput drug screen using cells transformed by oncogenic RAS, we identify a lead heteroaryl amide compound that blocks cell proliferation. Analysis of the structure-activity relationship indicated that this series of scaffolds (exemplified by MP-HJ-1b) represents a potent inhibitor of tumor cell growth. MP-HJ-1b showed activities against a panel of more than 1,000 human cancer cell lines with a wide variety of tissue origins. This compound depolymerized microtubules and affected spindle formation. It also induced the spike-like conformation of microtubules in vitro and in vivo, which is different from typical microtubule modulators. Structural analysis revealed that this series of compounds bound the colchicine pocket at the intra-dimer interface, although mostly not overlapping with colchicine binding. MP-HJ-1b displayed favorable pharmacological properties for overcoming tumor MDR, both in vitro and in vivo. Taken together, our data reveal a novel scaffold represented by MP-HJ-1b that can be developed as a cancer therapeutic against tumors with MDR. Significance: Paclitaxel is a widely used chemotherapeutic drug in patients with multiple types of cancer. However, resistance to paclitaxel is a challenge. This study describes a novel class of microtubule inhibitors with the ability to circumvent multidrug resistance across multiple tumor cell lines. Cancer Res; 78(20); 5949–57. ©2018 AACR.

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.

ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies. ONC201 demonstrated (GI50 1-8 µM) dose- and time-dependent efficacy in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), Hodgkin's lymphoma (nodular sclerosis) and multiple myeloma (MM) cell lines including cells resistant to standard of care (dexamethasone in MM) and primary samples. ONC201 induced caspase-dependent apoptosis that involved activation of the integrated stress response (ATF4/CHOP) pathway, inhibition of Akt phosphorylation, Foxo3a activation, downregulation of cyclin D1, IAP and Bcl-2 family members. ONC201 synergistically reduced cell viability in combination with cytarabine and 5-azacytidine in AML cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model induced tumor growth inhibition that was superior to either agent alone. ONC201 synergistically combined with bortezomib in MM, MCL and ALCL cells and with ixazomib or dexamethasone in MM cells. ONC201 combined with bortezomib in a Burkitt's lymphoma xenograft model reduced tumor cell density and improved CHOP induction compared to either agent alone. These results serve as a rationale for ONC201 single-agent trials in relapsed/refractory acute leukemia, non-Hodgkin's lymphoma, MM and combination trial with dexamethasone in MM, provide pharmacodynamic biomarkers and identify further synergistic combinatorial regimens that can be explored in the clinic.

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.

Transcriptional dysregulation induced by aberrant transcription factors (TF) is a key feature of cancer, but its global influence on drug sensitivity has not been examined. Here, we infer the transcriptional activity of 127 TFs through analysis of RNA-seq gene expression data newly generated for 448 cancer cell lines, combined with publicly available datasets to survey a total of 1,056 cancer cell lines and 9,250 primary tumors. Predicted TF activities are supported by their agreement with independent shRNA essentiality profiles and homozygous gene deletions, and recapitulate mutant-specific mechanisms of transcriptional dysregulation in cancer. By analyzing cell line responses to 265 compounds, we uncovered numerous TFs whose activity interacts with anticancer drugs. Importantly, combining existing pharmacogenomic markers with TF activities often improves the stratification of cell lines in response to drug treatment. Our results, which can be queried freely at dorothea.opentargets.io, offer a broad foundation for discovering opportunities to refine personalized cancer therapies. Significance: Systematic analysis of transcriptional dysregulation in cancer cell lines and patient tumor specimens offers a publicly searchable foundation to discover new opportunities to refine personalized cancer therapies. Cancer Res; 78(3); 769–80. ©2017 AACR.

Cells lacking the tumor suppressor gene LKB1/STK11 alter their metabolism to match the demands of accelerated growth, leaving them highly vulnerable to stress. However, targeted therapy for LKB1-deficient cancers has yet to be reported. In both Kras/p53/Lkb1 cell lines and a genetically engineered mouse model of Kras/p53/Lkb1–induced lung cancer, much higher rates of DNA damage occur, resulting in increased dependence on Chk1 checkpoint function. Here we demonstrate that short-term treatment with the Chk1 inhibitor AZD7762 reduces metabolism in pembrolizumab tumors, synergizing with the DNA-damaging drug gemcitabine to reduce tumor size in these models. Our results offer preclinical proof of concept for use of a Chk1 inhibitor to safely enhance the efficacy of gemcitabine, particularly in aggressive KRAS-driven LKB1-deficient lung adenocarcinomas. Cancer Res; 77(18); 5068–76. ©2017 AACR.

The formation of protein complexes and the co-regulation of the cellular concentrations of proteins are essential mechanisms for cellular signaling and for maintaining homeostasis. Here we use isobaric-labeling multiplexed proteomics to analyze protein co-regulation and show that this allows the identification of protein-protein associations with high accuracy. We apply this 'interactome mapping by high-throughput quantitative proteome analysis' (IMAHP) method to a panel of 41 breast cancer cell lines and show that deviations of the observed protein co-regulations in specific cell lines from the consensus network affects cellular fitness. Furthermore, these aberrant interactions serve as biomarkers that predict the drug sensitivity of cell lines in screens across 195 drugs. We expect that IMAHP can be broadly used to gain insight into how changing landscapes of protein-protein associations affect the phenotype of biological systems.

Lung cancers with activating KRAS mutations are characterized by treatment resistance and poor prognosis. In particular, the basis for their resistance to radiation therapy is poorly understood. Here, we describe a radiation resistance phenotype conferred by a stem-like subpopulation characterized by mitosis-like condensed chromatin (MLCC), high CD133 expression, invasive potential, and tumor-initiating properties. Mechanistic investigations defined a pathway involving osteopontin and the EGFR in promoting this phenotype. Osteopontin/EGFR–dependent MLCC protected cells against radiation-induced DNA double-strand breaks and repressed putative negative regulators of stem-like properties, such as CRMP1 and BIM. The MLCC-positive phenotype defined a subset of KRAS-mutated lung cancers that were enriched for co-occurring genomic alterations in TP53 and CDKN2A. Our results illuminate the basis for the radiation resistance of KRAS-mutated lung cancers, with possible implications for prognostic and therapeutic strategies. Cancer Res; 77(8); 2018–28. ©2017 AACR.

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.

BackgroundKRAS mutations are present in up to 30% of lung adenocarcinoma cases and are associated with poor survival. No effective targeted therapy against KRAS is currently available, and novel strategies to counteract oncogenic KRAS signaling are needed.ResultsWe used targeted proteomics to monitor abundance and site-specific phosphorylation in a network of over 150 upstream and downstream effectors of KRAS signaling in H358 cells (KRAS G12C). We compared patterns of protein regulation following sustained signaling blockade in the RAS/ERK module at two different levels, KRAS and MEK. Network-based analysis demonstrated complex non-linear patterns of regulation with wide-spread crosstalk among diverse subnetworks. Among 85 most regulated proteins in the network, only 12 proteins showed concordant regulation in response to signaling blockade at both KRAS and MEK levels, while the remainder were either specifically regulated in response to KRAS knockdown or MEK inhibition or showed orthogonal regulation in both conditions. Dephosphorylation of DNA methyltransferase 1 (DNMT1) at S714 was identified among the changes unique to KRAS knockdown, and here we elucidate the role of this phosphorylation in KRAS-dependent transcriptional silencing of tumor suppressor genes.ConclusionsNetwork-based analysis of the Ras signaling has shown complex non-linear patterns of regulation with wide-spread crosstalk among diverse subnetworks. Our work illustrates a targeted proteomics approach to functional interrogation of complex signaling networks focused on identification of readily testable hypotheses. These methods are widely applicable to diverse questions in tumor biology and other signaling paradigms.

INTRODUCTION: Extensive tumor sequencing efforts have transformed the way in which cancer driver genes are identified. Appropriate statistical modeling is crucial for distinguishing true drivers from passenger events that accumulate during tumorigenesis but provide no fitness advantage to cancer cells. A central assumption used in discovering driver genes and specific driver mutations is that exact positional recurrence is unlikely by chance: Seeing exactly the same DNA base pair mutated recurrently across patients is taken as proof that the mutation must be under functional selection for contributing to tumor fitness. The assumption is that mutational processes, being essentially random, are unlikely to hit the exact same base pair over and over again. However, although functional selection is clearly a key cause of recurrent mutations in cancers, whether it is the only prominent cause is not known. RATIONALE: To distinguish driver mutations from passengers, it is critical to understand the landscape of background mutations in cancer genomes. Recent pan-cancer mutation analyses have revealed rules of mutation distribution at the smallest (one to three base pairs) and largest (megabase) scales. At the small scale, mutational processes such as those attributable to sunlight, cigarette smoke, or random DNA copying errors generate patterns known as mutational signatures at the trinucleotide level. At the opposite extreme, the cell’snucleus is organized into two large compartments known as A and B, each consisting of multi-megabase chromatin domains. Compartment A contains gene-rich, open, active, early-replicating euchromatin. Compartment B contains gene-poor, closed, inactive, and late-replicating heterochromatin. Mutation frequency is generally higher in compartment B. Cancer genomes have been studied in detail at these two opposite scales, but less attention has been paid so far to the intervening “mesoscale.” RESULTS: We investigated the influence of mesoscale genomic features on mutational recurrence. We found that mutagenesis by the cytidine deaminase APOBEC3A is uniquely sensitive to mesoscale features, specifically the ability of DNA to adopt particular “hairpin” (stem-loop) structures while transiently single-stranded. Identifying DNA loci that can form hairpins requires sequence analysis at the mesoscale (~30–base pair) level. Combining biochemistry and bioinformatics, we deduced the features of APOBEC3A’s optimal DNA substrates, revealing that cytosine bases presented in a short loop at the end of a strongly paired stem can be mutated up to 200 times as frequently as nonhairpin sites. Analyzing the most frequent APOBEC mutations in protein-coding regions of cancer genomes, we identified numerous recurrent mutations at optimal hairpins in genes unconnected to cancer. Conversely, we found that mutational hotspots at nonoptimal sites are enriched in known cancer driver genes. CONCLUSION: Our results indicate that there are multiple possible routes to mutational hotspots in cancer. Functional mutations in oncogenes or tumor suppressors can rise to prominence through positive selection. These driver hotspots are not restricted to the “favorite” sites of any particular mutagen. In contrast, DNA sites that happen to be perfect substrates for a mutagen can give rise to “passenger hotspot mutations” that owe their prevalence to substrate optimality, not to any effects on tumor fitness. In light of these findings, we recommend caution in interpreting the long lists of putative novel cancer driver hotspots being produced by high-throughput sequencing projects.

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 involve synthetic rescue (SR) interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (the rescuer). Here, we perform a genome‐wide in silico 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.

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.

Advanced, anaplastic lymphoma kinase (ALK)–positive lung cancer is currently treated with the first-generation ALK inhibitor crizotinib followed by more potent, second-generation ALK inhibitors (e.g., ceritinib and alectinib) upon progression. Second-generation inhibitors are generally effective even in the absence of crizotinib-resistant ALK mutations, likely reflecting incomplete inhibition of ALK by crizotinib in many cases. Herein, we analyzed 103 repeat biopsies from ALK-positive patients progressing on various ALK inhibitors. We find that each ALK inhibitor is associated with a distinct spectrum of ALK resistance mutations and that the frequency of one mutation, ALKG1202R, increases significantly after treatment with second-generation agents. To investigate strategies to overcome resistance to second-generation ALK inhibitors, we examine the activity of the third-generation ALK inhibitor lorlatinib in a series of ceritinib-resistant, patient-derived cell lines, and observe that the presence of ALK resistance mutations is highly predictive for sensitivity to lorlatinib, whereas those cell lines without ALK mutations are resistant.Significance: Secondary ALK mutations are a common resistance mechanism to second-generation ALK inhibitors and predict for sensitivity to the third-generation ALK inhibitor lorlatinib. These findings highlight the importance of repeat biopsies and genotyping following disease progression on targeted therapies, particularly second-generation ALK inhibitors. Cancer Discov; 6(10); 1118–33. ©2016 AACR.See related commentary by Qiao and Lovly, p. 1084.This article is highlighted in the In This Issue feature, p. 1069

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.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines, including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multikinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant “gatekeeper” mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness.Significance: IDH mutations define a distinct subtype of ICC, a malignancy that is largely refractory to current therapies. Our work demonstrates that IDHm ICC cells are hypersensitive to dasatinib and critically dependent on SRC activity for survival and proliferation, pointing to new therapeutic strategies against these cancers. Cancer Discov; 6(7); 727–39. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 681

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.

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.

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.

Summary Multidrug resistance (MDR) in cancer remains a major challenge for the success of chemotherapy. Natural products have been a rich source for the discovery of drugs against MDR cancers. Here, we applied high-throughput cytotoxicity screening of an in-house natural product library against MDR SGC7901/VCR cells and identified that the cyclodepsipeptide verucopeptin demonstrated notable antitumor potency. Cytological profiling combined with click chemistry-based proteomics revealed that ATP6V1G directly interacted with verucopeptin. ATP6V1G, a subunit of the vacuolar H+-ATPase (v-ATPase) that has not been previously targeted, was essential for SGC7901/VCR cell growth. Verucopeptin exhibited strong inhibition of both v-ATPase activity and mTORC1 signaling, leading to substantial pharmacological efficacy against SGC7901/VCR cell proliferation and tumor growth in vivo. Our results demonstrate that targeting v-ATPase via its V1G subunit constitutes a unique approach for modulating v-ATPase and mTORC1 signaling with great potential for the development of therapeutics against MDR cancers.

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.

ONC201 is the first clinical bitopic antagonist of dopamine receptor D2 (DRD2), that is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. 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. ONC206 exhibited a Ki of ~320nM for DRD2 with complete specificity across human GPCRs and complete DRD2 antagonism. Schild analyses of ONC206 in cAMP and β-Arrestin recruitment assays revealed hallmarks of non-competitive DRD2 antagonism, unlike antipsychotics but similar to ONC201. Shotgun mutagenesis across DRD2 identified 7 residues critical for ONC206-mediated antagonism at orthosteric and allosteric sites. Six residues were critical for ONC201 and ONC206, however the impact varied between the two compounds and one allosteric residue was exclusive to ONC206 located at the region that mediates the DRD2 homodimer interface. Gene expression profiling revealed ONC206 and ONC201 (upon 200nM treatment, 72 h) induce distinct signatures in U87 glioblastoma cells, further supporting distinct functional effects. Cell lines resistant to ONC201 and ONC206 are being generated to profile acquired-resistance mechanisms. Broad nanomolar efficacy of ONC206 (GI50 1,000 GDSC cancer cell lines with the highest sensitivity in cell lines exhibiting a DRD2+/DRD5- RNA expression signature. 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. 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. 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 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 clinical trial in biomarker-enriched adult recurrent CNS tumors. In summary, ONC206 is poised for clinical introduction as the next imipridone bitopic DRD2 antagonist for oncology that exhibits differentiated target engagement, signaling, and biodistribution profiles. Citation Format: Varun Vijay Prabhu, Sara Morrow, Caroline A. Cuoco, Abed R. Kawakibi, Jinkyu Jung, Neel Madhukar, Matthew J. Garnett, Ultan McDermott, Cyril H. Benes, Robert Wechsler-Reya, Lakshmi Anantharaman, Neil Charter, Joseph B. Rucker, Benjamin J. Doranz, Joel Basken, Olivier Elemento, R. Benjamin Free, David R. Sibley, Martin Stogniew, Wolfgang Oster, Mark R. Gilbert, Sharon DeMorrow. IND-enabling characterization of ONC206 as the next bitopic antagonist for oncology [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 5688.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

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