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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 pancreatic cancer (PDAC) model systems, we demonstrated the role of stromal fibroblasts in shaping PDAC single cell heterogeneity towards invasive (EMT) and proliferative (PRO) phenotypes. Using highcontent digital imaging of RNA in situ hybridization in 195 tumors, we observed these EMT and PRO subpopulations in 319,626 individual cancer cells. Interestingly, we found these EMT and PRO subpopulations form distinct tumor gland “units” associated with differences in stromal abundance and patient survival. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

CellMinerCDB provides a web-based resource and portal for integrating multiple forms of pharmacological and genomic analyses, unifying the richest cancer cell line datasets (the NCI-60, NCI-SCLC, Sanger/MGH GDSC, and Broad CCLE/CTRP). CellMinerCDB enables data queries for genomics and gene regulatory network analyses, and identifying pharmacogenomic determinants and drug signatures. It leverages overlaps of cell lines and drugs. A panel of 41 drugs was evaluated in parallel in the NCI-60 and GDSC, supporting reproducibility and further pathway analyses across databases. We demonstrate the value of CellMinerCDB for elucidating gene expression determinants, such as methylation and copy number variation; highlight difficulties in assessing mutation burden; the dominant role of SLFN11 for drug response; novel response determinants and genomic signatures for topoisomerase inhibitors and schweinfurthins; drug repositioning for bisacodyl and acetalax for triple negative breast cancer; and the discovery of LIX1L as a novel mesenchymal gene regulating cancer cellular migration and invasiveness.

Cancer's alternative means to an end To stay alive and proliferating, tumor cells must maintain their telomeres: the DNA sequences at the ends of chromosomes. The majority accomplish this by activating the enzyme telomerase. However, certain tumor types favor a different mechanism called alternative lengthening of telomeres (ALT), which involves DNA recombination. Flynn et al. delineated the molecular events that occur at the telomeres of ALT-proficient tumor cells by studying the function of a protein that is altered by mutation in these tumors. The analysis revealed a specific protein kinase that is essential for ALT, which could in principle be targeted to halt tumor growth. Science , this issue p. 273

// 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.

High-risk neuroblastoma is often distinguished by amplification of MYCN and loss of differentiation potential. We performed high-throughput drug screening of epigenetic-targeted therapies across a large and diverse tumor cell line panel and uncovered the hypersensitivity of neuroblastoma cells to GSK-J4, a small-molecule dual inhibitor of lysine 27 of histone 3 (H3K27) demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), and histone demethylase Jumonji D3 (JMJD3). Mechanistically, GSK-J4 induced neuroblastoma differentiation and endoplasmic reticulum (ER) stress, with accompanying up-regulation of p53 up-regulated modulator of apoptosis (PUMA) and induction of cell death. Retinoic acid (RA)–resistant neuroblastoma cells were sensitive to GSK-J4. In addition, GSK-J4 was effective at blocking the growth of chemorefractory and patient-derived xenograft models of high-risk neuroblastoma in vivo. Furthermore, GSK-J4 and RA combination increased differentiation and ER stress over GSK-J4 effects and limited the growth of neuroblastomas resistant to either drug alone. In MYCN-amplified neuroblastoma, PUMA induction by GSK-J4 sensitized tumors to the B cell lymphoma 2 (BCL-2) inhibitor venetoclax, demonstrating that epigenetic-targeted therapies and BCL-2 homology domain 3 mimetics can be rationally combined to treat this high-risk subset of neuroblastoma. Therefore, H3K27 demethylation inhibition is a promising therapeutic target to treat high-risk neuroblastoma, and H3K27 demethylation can be part of rational combination therapies to induce robust antineuroblastoma activity.

The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like APOBEC3A and APOBEC3B have emerged as key mutation drivers in cancer. Here, we show that APOBEC3A and APOBEC3B activities impose a unique type of replication stress by inducing abasic sites at replication forks. In contrast to cells under other types of replication stress, APOBEC3A-expressing cells were selectively sensitive to ATR inhibitors (ATRi), but not to a variety of DNA replication inhibitors and DNA-damaging drugs. In proliferating cells, APOBEC3A modestly elicited ATR but not ATM. ATR inhibition in APOBEC3A-expressing cells resulted in a surge of abasic sites at replication forks, revealing an ATR-mediated negative feedback loop during replication. The surge of abasic sites upon ATR inhibition associated with increased accumulation of single-stranded DNA, a substrate of APOBEC3A, triggering an APOBEC3A-driven feed-forward loop that ultimately drove cells into replication catastrophe. In a panel of cancer cell lines, ATRi selectively induced replication catastrophe in those harboring high APOBEC3A and/or APOBEC3B activities, showing that APOBEC3A and APOBEC3B activities conferred susceptibility to ATRi. Our results define an APOBEC-driven replication stress in cancer cells that may offer an opportunity for ATR-targeted therapy. Cancer Res; 77(17); 4567–78. ©2017 AACR.

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.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung tumors initially respond to small-molecule ALK inhibitors, but drug resistance often develops. Of tumors that develop resistance to highly potent second-generation ALK inhibitors, approximately half harbor resistance mutations in ALK, while the other half have other mechanisms underlying resistance. Members of the latter group often have activation of at least one of several different tyrosine kinases driving resistance. Such tumors are not expected to respond to lorlatinib-a third-generation inhibitor targeting ALK that is able to overcome all clinically identified resistant mutations in ALK-and further therapeutic options are limited. Herein, we deployed a shRNA screen of 1,000 genes in multiple ALK-inhibitor-resistant patient-derived cells (PDCs) to discover those that confer sensitivity to ALK inhibition. This approach identified SHP2, a nonreceptor protein tyrosine phosphatase, as a common targetable resistance node in multiple PDCs. SHP2 provides a parallel survival input downstream of multiple tyrosine kinases that promote resistance to ALK inhibitors. Treatment with SHP099, the recently discovered small-molecule inhibitor of SHP2, in combination with the ALK tyrosine kinase inhibitor (TKI) ceritinib halted the growth of resistant PDCs through preventing compensatory RAS and ERK1 and ERK2 (ERK1/2) reactivation. These findings suggest that combined ALK and SHP2 inhibition may be a promising therapeutic strategy for resistant cancers driven by several different ALK-independent mechanisms underlying resistance.

Anti-cancer small molecule ONC201 upregulates the integrated stress response (ISR) and acts as a dual inactivator of Akt/ERK, leading to TRAIL gene activation. ONC201 is under investigation in multiple clinical trials to treat patients with cancer. Given the unique imipridone core chemical structure of ONC201, we synthesized a series of analogs to identify additional compounds with distinct therapeutic properties. Several imipridones with a broad range of in vitro potencies were identified in an exploration of chemical derivatives. Based on in vitro potency in human cancer cell lines and lack of toxicity to normal human fibroblasts, imipridones ONC206 and ONC212 were prioritized for further study. Both analogs inhibited colony formation, and induced apoptosis and downstream signaling that involves the integrated stress response and Akt/ERK, similar to ONC201. Compared to ONC201, ONC206 demonstrated improved inhibition of cell migration while ONC212 exhibited rapid kinetics of activity. ONC212 was further tested in >1000 human cancer cell lines in vitro and evaluated for safety and anti-tumor efficacy in vivo. ONC212 exhibited broad-spectrum efficacy at nanomolar concentrations across solid tumors and hematological malignancies. Skin cancer emerged as a tumor type with improved efficacy relative to ONC201. Orally administered ONC212 displayed potent anti-tumor effects in vivo, a broad therapeutic window and a favorable PK profile. ONC212 was efficacious in vivo in BRAF V600E melanoma models that are less sensitive to ONC201. Based on these findings, ONC212 warrants further development as a drug candidate. It is clear that therapeutic utility extends beyond ONC201 to include additional imipridones.

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.

Summary: KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3′ kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention.

Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cells, but their efficacy in BRCA-deficient patients is limited by drug resistance. Here, we used derived cell lines and cells from patients to investigate how to overcome PARPi resistance. We found that the functions of BRCA1 in homologous recombination (HR) and replication fork protection are sequentially bypassed during the acquisition of PARPi resistance. Despite the lack of BRCA1, PARPi-resistant cells regain RAD51 loading to DNA double-stranded breaks (DSBs) and stalled replication forks, enabling two distinct mechanisms of PARPi resistance. Compared with BRCA1-proficient cells, PARPi-resistant BRCA1-deficient cells are increasingly dependent on ATR for survival. ATR inhibitors (ATRis) disrupt BRCA1-independent RAD51 loading to DSBs and stalled forks in PARPi-resistant BRCA1-deficient cells, overcoming both resistance mechanisms. In tumor cells derived from patients, ATRis also overcome the bypass of BRCA1/2 in fork protection. Thus, ATR inhibition is a unique strategy to overcome the PARPi resistance of BRCA-deficient cancers.

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.

Loss-of-function mutations in p16INK4A (CDKN2A) occur in approximately 80% of sporadic pancreatic ductal adenocarcinoma (PDAC), contributing to its early progression. Although this loss activates the cell-cycle–dependent kinases CDK4/6, which have been considered as drug targets for many years, p16INK4A-deficient PDAC cells are inherently resistant to CDK4/6 inhibitors. This study searched for targeted therapies that might synergize with CDK4/6 inhibition in this setting. We report that the IGF1R/IR inhibitor BMS-754807 cooperated with the CDK4/6 inhibitor PD-0332991 to strongly block proliferation of p16INK4A-deficient PDAC cells in vitro and in vivo. Sensitivity to this drug combination correlated with reduced activity of the master cell growth regulator mTORC1. Accordingly, replacing the IGF1R/IR inhibitor with the rapalog inhibitor temsirolimus broadened the sensitivity of PDAC cells to CDK4/6 inhibition. Our results establish targeted therapy combinations with robust cytostatic activity in p16INK4A-deficient PDAC cells and possible implications for improving treatment of a broad spectrum of human cancers characterized by p16INK4A loss. Cancer Res; 74(14); 3947–58. ©2014 AACR.

Staying one step ahead of tumors Cancer treatments require continual adjustment. A drug that works initially will lose its potency as the tumor acquires new mutations that allow it to bypass the drug's lethal effects. To stay ahead of the tumor, oncologists need a noninvasive way to collect tumor cells from patients over the course of their treatment. Analyzing the mutations in these samples may help them choose the right drugs as the tumors change. In a small study of breast cancer patients, Yu et al. show that rare tumor cells circulating in the blood can be captured in viable form and used for this purpose. Science , this issue p. 216

ONC201 is the first selective antagonist of dopamine receptor D2 (DRD2) and D3 (DRD3) for clinical oncology that has exhibited preliminary clinical activity in high grade gliomas. We investigated DRD2 dysregulation in glioma and its role in ONC201 efficacy. Investigating CRISPR screens across a spectrum of cancer revealed that glioma cell lines had the highest DRD2 gene essentiality scores, indicating that glioma is a tumor type with the most vulnerability to DRD2 antagonism. Investigation of TCGA revealed that DRD2 is highly expressed in glioblastoma relative to other dopamine receptor family members and is associated with a relatively poor clinical prognosis. Tissue microarray analysis confirmed DRD2 overexpression in glioblastoma relative to normal brain. A linear correlation between DRD2 mRNA and ONC201 GI50 was observed among NCI60 glioblastoma cell lines. Similarly, we found a significant concordance between a cell line’s sensitivity to ONC201 within the Genomics of Drug Sensitivity in Cancer (GDSC) panel and its DRD2 gene essentiality score. Next, we ranked the relative contribution of each dopamine receptor to ONC201 efficacy using a bioinformatics approach based on a generalized linear model. We found that the strongest negative contributor was DRD2 – where a negative contribution denotes a decreased IC50 value as expression increases. Interestingly, DRD5, a D1-like dopamine receptor that counteracts DRD2 signaling, was measured as having the highest positive score – indicating that low expression of DRD5 was correlated with ONC201 efficacy. DRD5 expression was significantly inversely correlated with ONC201 potency in the NCI60 and GDSC datasets. Furthermore, a missense DRD5 mutation was identified in tumor cells with acquired resistance to ONC201. Resistance could be recapitulated with overexpression of the mutant or wild-type DRD5 gene. In conclusion, DRD2 dysregulation and DRD5 expression predict preclinical ONC201 glioma sensitivity that may be used to identify additional settings for clinical evaluation.

Background T cell redirection strategies, such as CAR-T and bispecific antibodies (bsAb), are rapidly changing the way in which we approach and treat cancer. While CAR-T and bsAb have shown impressive clinical efficacy in a limited number of cancers, both strategies are ultimately limited by on-target toxicity that currently restricts application to B-cell lineage tumors as the number of genuinely tumor-specific surface antigens is extremely limited. BsAb also suffer from off-target toxicity relating their ability to directly active T-cells severely restricting the therapeutic window. We sought to solve these inherent problems with the current generation bsAb by re-designing the molecule to alter the mechanism of T-cell activation. By splitting the T-cell engaging VHVL antibody paratope between two separate molecules we created two molecules that formed an active T-cell engaging unit through protein domain complementation following proteolytic activation. Each antibody could target independent surface antigens vastly increasing targeting permutations. Thus, these two antibodies functioned as an "antibody circuit" permitting Boolean type logic to precisely control T-cell activation in multi-dimensional targeting space. We selected AML as model cancer to develop T-cell Engaging Antibody Circuits (TEACs) due to the highly characterized surface antigen landscape and the clear challenges and limitations of single-antigen targeting approaches. Results We first screened 10 AML cell lines for candidate surface antigens based upon prior studies of surface antigen display (Perna F et al, 2016) and identified CD33, CD123, CD49d, CD70, CD71, CD38, CLEC12A, Flt3, CD24, CD244, TIM3 and CCR1 as promising targets. We developed a secondary TEACs screening assay where the two TEAC molecules contained either a FITC or biotin binding domain and paired these to commercial FITC or biotin conjugated antibodies targeting the antigens above. We screened 72 TEAC pairs against the 10 cell lines which identified optimal antigen target combinations which included CD33xCD123, CD33xCLEC12A, CD33xCD49d and CD33xCD24. Using a FRET-based fluorescent peptide assay to identify peptide linkers susceptible to proteases we found MMP2 to be highly expressed in AML samples and thus designed all our TEACs with this cleavage site. We next generated IgG4 format TEACs targeting CD33, CD123, CLEC12A and CD24 that included the MMP2 cleavage activation site and tested these as TEAC pairs in vitro. This screen identified the CD123xCD33 as the most active TEAC pair which was active in 9/10 cells lines. To assess potential safety concerns, we tested TEACs and CD123 and CD33 BiTEs individually and as pairs on PBMCs and on plate-immobilized molecules. These data demonstrated that BiTEs were extremely active against healthy monocytes and also activate T-cells non-specifically once plate-immobilized. In contrast CD123xCD33 IgG TEACs pairs did not activate T-cells when plate-immobilized and did not target healthy monocytes.Finally, we examined the activity of both CD33 BiTEs and CD123xCD33 TEACs on primary patient AML samples. We conducted FRET based assays which confirmed high activity of MMP2 cleavage site on all primary AML samples. When we examined T-cell activation, CD123xCD33 TEACs were active in all CD123+ CD33+ AML samples evaluated with an EC50 of 30ug/ml. Conclusion These data suggest T-cell engaging antibody circuits is a new approach that could be safely applied toward AML. TEAC agents do not directly activate T-cells and CD123xCD33 TEAC pairs do not activate PBMC or monocytes. However, CD123xCD33 TEACs show strong activity against AML cell lines and primary CD123+CD33+ AML cells. Disclosures Millar: Revitope Oncology: Equity Ownership. Minshull:Atum Biotechnology: Employment, Equity Ownership. Narayan:Takeda: Other: Employment (spouse); Genentech: Other: Equity ownership (spouse); Merck: Other: Equity ownership (spouse). Graubert:Biogen: Other: Spouse Employee; Calico Life Sciences: Other: Research Support; Janssen Pharmaceuticals: Other: Research Support. Cobbold:Gritstone Oncology: Equity Ownership; Revitope Oncology: Consultancy; Revitope Oncology: Equity Ownership.

Dopamine receptor D2 (DRD2) is a G protein-coupled receptor that is overexpressed and critical for survival in several cancers. ONC201, an imipridone small molecule, is a DRD2/3 antagonist in Phase II advanced cancer clinical trials with a compelling safety and efficacy profile. We evaluated the binding target, anti-tumor activity, biodistribution and safety of ONC206, a chemical derivative of ONC201 with the same imipridone core structure. GPCR profiling with β-Arrestin recruitment revealed that ONC206 selectively antagonizes dopamine receptors DRD2 and DRD3. 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. While 6 mutated residues were also critical for ONC201-mediated antagonism, the impact and magnitude of different mutants varied between the two compounds and one of the allosteric residues was unique to ONC206. In vitro profiling of ONC206 in >1000 GDSC cancer cell lines demonstrated broad nanomolar efficacy (GI50 5µM), suggesting a wide therapeutic window. Robust inhibition of tumor growth without body weight loss was observed in HuCCT1 cholangiocarcinoma and MHH-ES-1 Ewing’s sarcoma subcutaneous xenografts with 50-100 mg/kg oral ONC206 weekly or every 2 weeks. Biodistribution studies in Sprague-Dawley rats revealed a ~12 µM plasma Cmax with a 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 concentrations. GLP toxicology studies with weekly oral ONC206 in Sprague-Dawley rats and beagle dogs at doses above or equivalent to efficacious doses revealed no dose-limiting toxicities. In both species, observations at the highest dose were mild and reversible. The no observed adverse event level (NOAEL) was ≥ 16.7 mg/kg in dogs and ≥ 50 mg/kg in rats, which both correspond to a human dose of approximately 500 mg assuming standard allometric scaling. These results provide rationale for a 50 mg starting ONC206 dose in dose escalation clinical trials in patients with DRD2-dysregulated tumors. Citation Format: Varun V. Prabhu, Abed Rahman Kawakibi, Neel Madhukar, Mathew J. Garnett, Ultan McDermott, Cyril H. Benes, Lakshmi Anantharaman, Neil Charter, Sean Deacon, Alexander VanEngelenburg, Joseph B. Rucker, Benjamin J. Doranz, Jessica Rusert, Robert Wechsler-Reya, Olivier Elemento, Martin Stogniew, Wolfgang Oster, Sharon DeMorrow, R. Benjamin Free, David R. Sibley, Joshua E. Allen. IND-enabling characterization of DRD2/3 imipridone antagonist ONC206 for oncology [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 3877.

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.

G protein-coupled receptors (GPCRs) represent the most widely exploited superfamily of drug targets for FDA-approved therapies for many diseases, however, these receptors are underexploited for oncology. ONC201 is a selective antagonist of GPCRs dopamine receptor D2 (DRD2) and DRD3 that has been shown to induce tumor regressions with a benign safety profile in high grade glioma patients. ONC201 (benzyl-2-methylbenzyl-imipridone) is the founding member of the imipridone class of small molecules that share a unique tri-heterocyclic core chemical structure. Imipridones share several chemical and biological properties that are desirable drug-like characteristics: oral administration, wide therapeutic window, chemical stability and blood brain barrier penetrance. In this study, we profiled a series of imipridones for GPCR engagement and anti-cancer efficacy. Several imipridones were screened against a large panel of human GPCRs using a β-arrestin recruitment assay. The imipridones tested resulted in GPCR agonist/antagonist activity (threshold set at >20% activity) that was heterogenous, but exclusive among Class A GPCRs that represent the largest class. Minor chemical modifications to the ONC201 chemical structure caused large shifts in agonist versus antagonist activity and selectivity for GPCRs. Specifically, switching the ONC201 imipridone core from an angular to a linear isomer resulted in loss of DRD2 antagonist activity and impaired inhibition of cancer cell viability, indicating the imipridone core structure is critical for GPCR engagement and anti-cancer effects. The addition of electron withdrawing groups (e.g. di- or tri-halogen substitution) to the methyl benzyl ring improved potency for GPCR engagement and anti-cancer effects, but not for the benzyl ring. Loss of the benzyl ring impaired anti-cancer effects. Among all of the GPCR hits identified, maximal variance in imipridone GPCR engagement was identified for DRD2/DRD3 antagonism and GPR132 agonism that were prioritized considering their known biological relevance in oncology. ONC206 (benzyl-2,4-difluoromethylbenzyl-imipridone) emerged as the most selective and potent antagonist for D2-like dopamine receptors that are overexpressed and critical for survival in several cancers. ONC212 (benzyl-4-trifluoromethylbenzyl-imipridone) was the most selective and potent agonist for tumor suppressor GPR132. Both compounds were tested in the GDSC panel of >1000 cancer cell lines and demonstrated broad spectrum nanomolar inhibition of cancer cell viability and a wide therapeutic window. GPCR target expression correlated with anti-cancer efficacy in the GDSC panel for both compounds, providing potential biomarkers of response. Thus, chemical derivatization of ONC201 has generated a class of novel GPCR-targeting agents with promising preclinical efficacy and safety profiles in oncology. Citation Format: Varun V. Prabhu, Abed Rahman Kawakibi, Neel S. Madhukar, Lakshmi Anantharaman, Sean Deacon, Neil S. Charter, Mathew J. Garnett, Ultan McDermott, Cyril H. Benes, Wolfgang Oster, Olivier Elemento, Martin Stogniew, Joshua E. Allen. Defining structure activity relationships for GPCR engagement and anti-cancer efficacy of imipridone small molecules [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 2749.

MCL1, an anti-apoptotic member of the BCL-2 family of proteins, is a key regulator of cancer cell survival and a known resistance factor to anti-cancer drugs, making it a highly desirable target for therapeutic intervention. Recently several MCL1 inhibitors have entered Phase I clinical development. Data derived from large cancer cell line panels suggest, that cell lines of hematopoietic origin are more broadly sensitive to MCL1 inhibition, than cell lines derived from solid tumor types. In particular, for the therapy of solid tumor patients with an MCL1 inhibitor, a patient selection biomarker would be highly desirable. Published in vitro data using various MCL1 inhibitors, siRNA or CRISPR/Cas9 technology show that tumor cell lines with low BCL-XL gene expression are mostly sensitive to MCL1 inhibition, down-regulation or inactivation. Here we show that by adding the gene expression data of additional six genes (including the MCL1 binding partner BAK1) to BCL-XL, a supervised learning predictor was applied and could reach a performance of almost 80% correctly classified solid tumor cell lines. This new predictor has been applied to either tumor samples, adjacent normal tissues or normal tissue samples from TCGA and GTEx. Briefly, most normal tissue samples are categorized as being sensitive. Moreover, solid tumor samples in contrast to solid tumor cell lines are predicted to be broadly sensitive to MCL1 inhibition with a predicted anti-tumor effect rate of 60% to 95%. In summary, our work describes the translational challenges using cell line-derived predictors on ex vivo tumor samples. Citation Format: Andreas Wernitznig, Dorothea Rudolph, Matthias Samwer, Norbert Schweifer, Francesca Trapani, Tobias Wunberg, Heribert Arnhof, Teakyu Lee, John L. Sensintaffar, Edward T. Olejniczak, Cyril H. Benes, Stephen W. Fesik, Norbert Kraut. Predicting MCL1 inhibitor sensitivity in large cell line panels using a gene expression signature [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 3987.

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.

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

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

Cancer stem cells (CSCs) correlate with recurrence, metastasis and poor survival in clinical studies. Encouraging results from clinical trials of CSC inhibitors have further validated CSCs as therapeutic targets. ONC201 is a first-in-class small molecule imipridone in Phase I/II clinical trials for advanced cancer. We have previously shown that ONC201 targets self-renewing, chemotherapy-resistant colorectal CSCs via Akt/ERK inhibition and DR5/TRAIL induction. In this study, we demonstrate that the anti-CSC effects of ONC201 involve early changes in stem cell-related gene expression prior to tumor cell death induction. A targeted network analysis of gene expression profiles in colorectal cancer cells revealed that ONC201 downregulates stem cell pathways such as Wnt signaling and modulates genes (ID1, ID2, ID3 and ALDH7A1) known to regulate self-renewal in colorectal, prostate cancer and glioblastoma. ONC201-mediated changes in CSC-related gene expression were validated at the RNA and protein level for each tumor type. Accordingly, we observed inhibition of self-renewal and CSC markers in prostate cancer cell lines and patient-derived glioblastoma cells upon ONC201 treatment. Interestingly, ONC201-mediated CSC depletion does not occur in colorectal cancer cells with acquired resistance to ONC201. Finally, we observed that basal expression of CSC-related genes (ID1, CD44, HES7 and TCF3) significantly correlate with ONC201 efficacy in >1000 cancer cell lines and combining the expression of multiple genes leads to a stronger overall prediction. These proof-of-concept studies provide a rationale for testing CSC expression at the RNA and protein level as a predictive and pharmacodynamic biomarker of ONC201 response in ongoing clinical studies.

Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054–67. ©2017 AACR.

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.

Sorafenib is a RAF inhibitor approved for several cancers, including hepatocellular carcinoma (HCC). Inhibition of RAF kinases can induce a dose-dependent “paradoxical” upregulation of the downstream mitogen-activated protein kinase (MAPK) pathway in cancer cells. It is unknown whether “paradoxical” ERK activation occurs after sorafenib therapy in HCC, and if so, if it impacts the therapeutic efficacy. Here, we demonstrate that RAF inhibition by sorafenib rapidly leads to RAF dimerization and ERK activation in HCCs, which contributes to treatment evasion. The transactivation of RAF dimers and ERK signaling promotes HCC cell survival, prevents apoptosis via downregulation of BIM and achieves immunosuppression by MAPK/NF-kB-dependent activation of PD-L1 gene expression. To overcome treatment evasion and reduce systemic effects, we developed CXCR4-targeted nanoparticles to co-deliver sorafenib with the MEK inhibitor AZD6244 in HCC. Using this approach, we preferentially and efficiently inactivated RAF/ERK, upregulated BIM and down-regulated PD-L1 expression in HCC, and facilitated intra-tumoral infiltration of cytotoxic CD8+ T cells. These effects resulted in a profound delay in tumor growth. Thus, this nano-delivery strategy to selectively target tumors and prevent the paradoxical ERK activation could increase the feasibility of dual RAF/MEK inhibition to overcome sorafenib treatment escape in HCC.

RAS oncogenic mutations are common in human cancers, but RAS proteins have been difficult to target. We sought to identify pharmacological agents to block RAS oncogenic signaling by a distinct mechanism. Because the biological activity of RAS proteins relies upon lipid modifications and RAS regulates lipid metabolisms in cancer cells, we screened a bioactive lipid library using a RAS-specific cell viability assay. We report the discovery of a new class of inhibitors for RAS transformation. Compounds in the class represented by endocannabinoid N-arachidonoyl dopamine (NADA) can induce cell oncosis, independent of its ability to engage cannabinoid receptors. Further analyses show that NADA is more active in inhibiting the NRAS transformation and signaling than that of KRAS4B. Mechanistically, NADA blocks the plasma membrane translocation of NRAS, but not that of KRAS4B. In addition, NADA inhibits plasma membrane translocation and neoplastic transformation of oncogenic KRAS4A. Interestingly, NADA also redistributes the cytoplasmic NRAS to the Golgi apparatus in a palmitoylation-dependent manner. The results indicate that NADA inhibits NRAS and KRAS4A plasma membrane translocation by targeting a novel molecular process. The new findings would help to develop novel targeted therapies for a broad range of human cancers. Mol Cancer Ther; 16(1); 57–67. ©2016 AACR.

Hedgehog (HH) pathway Smoothened (Smo) inhibitors are active against Gorlin syndrome-associated basal cell carcinoma (BCC) and medulloblastoma where Patched (Ptch) mutations occur. We interrogated 705 epithelial cancer cell lines for growth response to the Smo inhibitor cyclopamine and for expressed HH pathway-regulated species in a linked genetic database. Ptch and Smo mutations that respectively conferred Smo inhibitor response or resistance were undetected. Previous studies revealed HH pathway activation in lung cancers. Therefore, findings were validated using lung cancer cell lines, transgenic and transplantable murine lung cancer models, and human normal-malignant lung tissue arrays in addition to testing other Smo inhibitors. Cyclopamine sensitivity most significantly correlated with high cyclin E (P=0.000009) and low insulin-like growth factor binding protein 6 (IGFBP6) (P=0.000004) levels. Gli family members were associated with response. Cyclopamine resistance occurred with high GILZ (P=0.002) expression. Newer Smo inhibitors exhibited a pattern of sensitivity similar to cyclopamine. Gain of cyclin E or loss of IGFBP6 in lung cancer cells significantly increased Smo inhibitor response. Cyclin E-driven transgenic lung cancers expressed a gene profile implicating HH pathway activation. Cyclopamine treatment significantly reduced proliferation of murine and human lung cancers. Smo inhibition reduced lung cancer formation in a syngeneic mouse model. In human normal-malignant lung tissue arrays cyclin E, IGFBP6, Gli1 and GILZ were each differentially expressed. Together, these findings indicate that Smo inhibitors should be considered in cancers beyond those with activating HH pathway mutations. This includes tumors that express genes indicating basal HH pathway activation.

Genetic approaches have shown that the p110β isoform of class Ia phosphatidylinositol-3-kinase (PI3K) is essential for the growth of PTEN-null tumors. Thus, it is desirable to develop p110β-specific inhibitors for cancer therapy. Using a panel of PI3K isoform-specific cellular assays, we screened a collection of compounds possessing activities against kinases in the PI3K superfamily and identified a potent and selective p110β inhibitor: KIN-193. We show that KIN-193 is efficacious specifically in blocking AKT signaling and tumor growth that are dependent on p110β activation or PTEN loss. Broad profiling across a panel of 422 human tumor cell lines shows that the PTEN mutation status of cancer cells strongly correlates with their response to KIN-193. Together, our data provide the first pharmacologic evidence that PTEN-deficient tumors are dependent on p110β in animals and suggest that KIN-193 can be pursued as a drug to treat tumors that are dependent on p110β while sparing other PI3K isoforms. Significance: We report the first functional characterization of a p110β-selective inhibitor, KIN-193, that is efficacious as an antitumor agent in mice. We show that this class of inhibitor holds great promise as a pharmacologic agent that could be used to address the potential therapeutic benefit of treating p110β-dependent PTEN-deficient human tumors. Cancer Discov; 2(5); 425–33. ©2012 AACR. Read the Commentary on this article by Shepherd and Denny, p. 393. This article is highlighted in the In This Issue feature, p. 377.

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

Therapies inhibiting receptor tyrosine kinases (RTKs) are effective against some human cancers when they lead to simultaneous downregulation of PI3K/AKT and MEK/ERK signaling. However, mutant KRAS has the capacity to directly activate ERK and PI3K signaling, and this is thought to underlie the resistance of KRAS mutant cancers to RTK inhibitors. Here, we have elucidated the molecular regulation of both the PI3K/AKT and MEK/ERK signaling pathways in KRAS mutant colorectal cancer cells and identified combination therapies that lead to robust cancer cell apoptosis. KRAS knockdown using shRNA suppressed ERK signaling in all of the human KRAS mutant colorectal cancer cell lines examined. However, no decrease, and actually a modest increase, in AKT phosphorylation was often seen. By performing PI3K immunoprecipitations, we determined that RTKs, often IGF-IR, regulated PI3K signaling in the KRAS mutant cell lines. This conclusion was also supported by the observation that specific RTK inhibition led to marked suppression of PI3K signaling and biochemical assessment of patient specimens. Interestingly, combination of RTK and MEK inhibitors led to concomitant inhibition of PI3K and MEK signaling, marked growth suppression, and robust apoptosis of human KRAS mutant colorectal cancer cell lines in vitro and upon xenografting in mice. These findings provide a framework for utilizing RTK inhibitors in the treatment of KRAS mutant colorectal cancers.

The mechanisms by which inhibition of the epidermal growth factor receptor (EGFR) sensitizes non–small cell lung cancer (NSCLC) cells to ionizing radiation remain poorly understood. We set out to characterize the radiosensitizing effects of the tyrosine kinase inhibitor erlotinib and the monoclonal antibody cetuximab in NSCLC cells that contain wild-type p53. Unexpectedly, EGFR inhibition led to pronounced cellular senescence but not apoptosis of irradiated cells, both in vitro and in vivo. Senescence was completely dependent on wild-type p53 and associated with a reduction in cell number as well as impaired clonogenic radiation survival. Study of ten additional NSCLC cell lines revealed that senescence is a prominent mechanism of radiosensitization in 45% of cell lines and occurs not only in cells with wild-type p53 but also in cells with mutant p53, where it is associated with an induction of p16. Interestingly, senescence and radiosensitization were linked to an increase in residual radiation-induced DNA double-strand breaks irrespective of p53/p16 status. This effect of EGFR inhibition was at least partially mediated by disruption of the MEK–ERK pathway. Thus, our data indicate a common mechanism of radiosensitization by erlotinib or cetuximab across diverse genetic backgrounds. Our findings also suggest that assays that are able to capture the initial proliferative delay that is associated with senescence should be useful for screening large cell line panels to identify genomic biomarkers of EGFR inhibitor–mediated radiosensitization. Cancer Res; 71(19); 6261–9. ©2011 AACR.

Multiple SRC-family kinases (SFKs) are commonly activated in carcinoma and appear to have a role in metastasis through incompletely understood mechanisms. Recent studies have shown that CDCP1 (CUB (complement C1r/C1s, Uegf, Bmp1) Domain-Containing Protein-1) is a transmembrane protein and an SRC substrate potentially involved in metastasis. Here we show that increased SFK and CDCP1 tyrosine phosphorylation is, surprisingly, associated with a decrease in FAK phosphorylation. This appears to be true in human tumors as shown by our correlation analysis of a mass spectrometric data set of affinity-purified phosphotyrosine peptides obtained from normal and cancer lung tissue samples. Induction of tyrosine phosphorylation of CDCP1 in cell culture, including by a mAb that binds to its extracellular domain, promoted changes in SFK and FAK tyrosine phosphorylation, as well as in PKC(TM), a protein known to associate with CDCP1, and these changes are accompanied by increases in adhesion and motility. Thus, signaling events that accompany the CDCP1 tyrosine phosphorylation observed in cell lines and human lung tumors may explain how the CDCP1/SFK complex regulates motility and adhesion.

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK–positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 μM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations.

Dopamine receptor D2 (DRD2) is a G protein-coupled receptor that is overexpressed in several cancers and its antagonism has been shown to cause anti-cancer effects. ONC201, an imipridone small molecule, is a DRD2/3 antagonist in Phase II advanced cancer clinical trials. We evaluated the binding target and anti-tumor activity of ONC206, a chemical derivative of ONC201. Experimental GPCR profiling with β-Arrestin recruitment assays revealed that ONC206 selectively antagonizes dopamine receptors DRD2 and DRD3. ONC206 exhibited a Ki of ~320nM for DRD2 with complete specificity across human GPCRs at doses that resulted in complete inhibition. Schild analyses of ONC206 in cAMP and β-Arrestin recruitment assays revealed hallmarks of non-competitive DRD2 antagonism, unlike antipsychotics that act as competitive and often non-selective antagonists. Shotgun mutagenesis across DRD2 identified 7 residues within and outside of the orthosteric binding site that are critical for ONC206-mediated antagonism. While 6 mutated residues were also critical for ONC201-mediated antagonism, the impact and magnitude of different mutants varied between the two compounds and one of the mutated residues was unique to ONC206 in an allosteric location. In vitro profiling of ONC206 in >1000 GDSC cancer cell lines revealed broad nanomolar efficacy (GI50 5µM), suggesting a wide therapeutic window. Robust inhibition of tumor growth was observed with weekly oral ONC206 (50mg/kg) in subcutaneous xenografts of cholangiocarcinoma, a dopamine-secreting bile duct tumor with DRD2 overexpression. Thus, imipridone ONC206 acts as a selective non-competitive DRD2/3 antagonist at nanomolar concentrations with efficacy in tumors with dopamine receptor pathway dysregulation.

Since microRNAs (miRNAs, miRs) have been implicated in oncogenesis, many of them have been identified as therapeutic targets. Previously we have demonstrated that miRNA-10b acts as a master regulator of the viability of metastatic tumor cells and represents a target for therapeutic intervention. We designed and synthesized an inhibitor of miR-10b, termed MN-anti-miR10b. We showed that treatment with MN-anti-miR10b led to durable regression/elimination of established metastases in murine models of metastatic breast cancer. Since miRNA-10b has been associated with various metastatic and non-metastatic cancers, in the present study, we investigated the effect of MN-anti-miR10b in a panel of over 600 cell lines derived from a variety of human malignancies. We observed an effect on the viability of multiple cell lines within each cancer type and a mostly dichotomous response with cell lines either strongly responsive to MN-anti-miR10b or not at all even at maximum dose tested, suggesting a very high specificity of the effect. Genomic modeling of the drug response showed enrichment of genes associated with the proto-oncogene, c-Jun.

Endometrial cancer is the most common gynecologic malignancy in the developed world. These cancers harbor the highest rates of PI3K pathway alterations reported, with PTEN being the most frequently altered gene. Differently from other cancer types, in endometrial cancer a specific hotspot mutation in the lipid phosphatase domain of PTEN is present at residue 130. This hotspot mutation significantly co-occurs with other mutations in the PI3K pathway, such as PIK3CA and PIK3R1. Interestingly, in the TCGA data we also found a positive correlation between this mutation and the expression level of the estrogen receptor alpha. Although it has been reported that the PTEN-R130 mutant loses its lipid phosphatase activity in vitro, little is known regarding the other unconventional functions of PTEN, such as its protein phosphatase activity. We performed unbiased phospho-proteomics experiments in order to explore the impact of this specific mutation in multiple endometrial cancer cell models. Citation Format: Ye Wang, Charles Jakubik, Rachel Peterson, Cyril Benes, Wilhelm Haas, Jeffrey Engelman, Carlotta Costa. Characterization of a hotspot PTEN mutation in endometrial cancer [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 5502.

D2-like dopamine receptors (DRD2/3/4) are G protein-coupled receptors (GPCRs) that are overexpressed in glioblastoma (GBM) and their antagonism induces tumor cell apoptosis. We describe the first selective DRD2/3 antagonist for neuro-oncology using computational, receptor pharmacology, biochemical and clinical studies. Consistent with an in-silico prediction and in contrast to antipsychotics that target several dopamine receptors and other GPCRs, β-arrestin recruitment and cAMP assays determined that ONC201 is a selective DRD2/3 antagonist. Schild analyses and radioligand competition assays revealed competitive and non-competitive DRD2 antagonism with a potency (2-3 µM) that is consistent with anticancer activity and driven by an unusually slow association rate. Proof-of-concept studies show that selective DRD2 inhibition induces superior anti-cancer efficacy relative to pan-targeting of the dopamine receptor family. In accordance with superior selectivity, ONC201 also exhibited a wider therapeutic window compared to antipsychotics. Shotgun mutagenesis across 350 amino acids of DRD2 identified 8 residues that are critical for ONC201-mediated DRD2 antagonism. Consistent with competitive inhibition, several mutated residues were within the orthosteric binding site. However, distal residues were identified that were not involved in DRD2 antagonism by antipsychotics and may explain the selectivity and non-competitive antagonism of ONC201. In vitro and in vivo studies have previously demonstrated single agent ONC201 efficacy in GBM models (Allen et al 2013). Analyses of The Cancer Genome Atlas and tissue microarrays revealed high DRD2 expression relative to other dopamine receptors, correlation with poor prognosis and high DRD2 expression in primary rather than secondary GBM. A linear correlation between DRD2 mRNA and ONC201 GI50 was observed among GBM 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 dataset (P Citation Format: Varun Vijay Prabhu, Neel Madhukar, C. Leah B. Kline, Rohinton Tarapore, Wafik S. El-Deiry, Joseph Rucker, Benjamin Doranz, Faye Doherty, Alexander VanEngelenburg, Jessica Durrant, Cyril Benes, Sean Deacon, Neil Charter, R. Benjamin Free, Wolfgang Oster, David Sibley, Isabel Arrillaga, Olivier Elemento, Joshua E. Allen. Selective targeting of dopamine receptor dysregulation in high grade gliomas with imipridone ONC201 [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 3857.

Microtubule inhibitors, as chemotherapeutic drugs, are widely used for cancer treatment in clinic. However, the development of multidrug resistance (MDR) in cancer is a major challenge for microtubule inhibitors in its clinical implementation. From a high throughput drug screening using cells transformed by oncogenic RAS, we identified a small molecule AR8-1006 that blocks cell proliferation. The analysis of structure-activity relationship indicated that this serial of scaffold represents a potent inhibitor of tumor cell growth. AR8-1006 shows activities against a larger panel of more than 1000 human cancer cell lines with a wide variety of tissue origins. Further analysis showed that this compound depolymerizes microtubules and affects formation of the spindle. Interestingly, it induces the spike-like conformation of microtubules in vitro and in vivo, which is different from typical microtubule modulators. Structural analysis revealed that this serial of compounds binds to the colchicine pocket at the intra-dimer interface, though mostly not overlapping with colchicine binding. More importantly, AR8-1006 displays favorable pharmacological properties on overcoming tumor MDR both in vitro and in vivo. Taken together, our data reveal a novel scaffold represented by AR8-1006 that could be developed as cancer therapeutics, especially for tumor multidrug resistance. Citation Format: Nannan Ning, Chengyong Wu, Min Wu, Yamei Yu, Changjun Zhu, Cyril Benes, Jianming Zhang, Xianming Deng, Qiang Chen, Ruibao Ren. Discovering a novel microtubule inhibitor overcoming tumor multidrug resistance [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 3926.

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.

Dopamine receptor D2 (DRD2) is a G protein-coupled receptor (GPCR) overexpressed in many cancers and its antagonism causes anti-tumor effects. ONC201, founding member of the imipridone class of small molecules, is a DRD2 antagonist in Phase II advanced cancer clinical trials. We evaluated the binding target and anti-tumor activity of ONC206, an ONC201 analog. An orphan small molecule target prediction algorithm revealed that ONC206, like ONC201, antagonizes DRD2. Experimental GPCR profiling using the PathHunter® β-Arrestin assay, confirmed that ONC206 selectively antagonizes D2-like (DRD2/3) but not D1-like (DRD 1/5) dopamine receptors. In this assay, ONC206 possesses a ~10-fold increased potency for DRD2 compared to ONC201 with a Ki of ~320nM with selectivity that was superior to approved antipsychotics. Shotgun mutagenesis across 350 amino acids of DRD2 identified 7 residues critical for ONC206-mediated antagonism of DRD2-induced calcium flux. Consistent with competitive inhibition, mutated residues were within the orthosteric binding site. While 6 mutated residues were also critical for ONC201 activity, one of the mutated residues was unique to ONC206, suggesting differentiated receptor pharmacology. TCGA analysis and immunohistochemistry of patient-derived tissue microarrays revealed DRD2 was overexpressed in neuroblastoma, sarcoma and pheochromocytoma specimens relative to normal tissues. In vitro profiling of ONC206 in the Genomic of Drug Sensitivity in Cancer collection of cell lines revealed broad nanomolar efficacy across most tumor types (GI50 5µM). Efficacy evaluation in the MHH-ES-1 Ewing's sarcoma xenograft model demonstrated that ONC206 (100 mg/kg PO every 10 days) causes significant tumor growth inhibition that was comparable to methotrexate (400 mg/kg, IP) while being better tolerated. Thus, imipridone ONC206 acts as a selective antagonist of DRD2/3 at nanomolar concentrations and may address tumor types where the properties of ONC201 do not permit for complete therapeutic engagement in vivo. Citation Format: Varun Vijay Prabhu, Neel Madhukar, Rohinton Tarapore, Mathew Garnett, Ultan McDermott, Cyril Benes, Neil Charter, Sean Deacon, Alexander VanEngelenburg, Joseph Rucker, Benjamin Doranz, Olivier Elemento, Wolfgang Oster, Martin Stogniew, Joshua Allen. Receptor pharmacology and anti-cancer activity of selective DRD2/3 antagonist imipridone ONC206 [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 4874.

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.

The ATR-Chk1 pathway is critical for DNA damage responses and cell cycle progression. Chk1 inhibition is more deleterious to cycling cells than ATR inhibition, raising questions about ATR and Chk1 functions in the absence of extrinsic replication stress. Here, we show that a key role of ATR in S phase is to coordinate RRM2 accumulation and origin firing. ATR inhibitor (ATRi) induces massive ssDNA accumulation and replication catastrophe in a fraction of early S-phase cells. In other S-phase cells, however, ATRi induces moderate ssDNA and triggers a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. The backup pathway creates a threshold such that ATRi selectively kills cells under high replication stress, whereas Chk1 inhibitor induces cell death at a lower threshold. The levels of ATRi-induced ssDNA correlate with ATRi sensitivity in a panel of cell lines, suggesting that ATRi-induced ssDNA could be predictive of ATRi sensitivity in cancer cells.