Search

Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm characterized by activating mutations in the related receptor tyrosine kinases KIT and PDGFRA. GIST relies on expression of these unamplified receptor tyrosine kinase (RTK) genes through a large enhancer domain, resulting in high expression levels of the oncogene required for tumor growth. Although kinase inhibition is an effective therapy for many patients with GIST, disease progression from kinase-resistant mutations is common and no other effective classes of systemic therapy exist. In this study, we identify regulatory regions of the KIT enhancer essential for KIT gene expression and GIST cell viability. Given the dependence of GIST upon enhancer-driven expression of RTKs, we hypothesized that the enhancer domains could be therapeutically targeted by a BET bromodomain inhibitor (BBI). Treatment of GIST cells with BBIs led to cell-cycle arrest, apoptosis, and cell death, with unique sensitivity in GIST cells arising from attenuation of the KIT enhancer domain and reduced KIT gene expression. BBI treatment in KIT-dependent GIST cells produced genome-wide changes in the H3K27ac enhancer landscape and gene expression program, which was also seen with direct KIT inhibition using a tyrosine kinase inhibitor (TKI). Combination treatment with BBI and TKI led to superior cytotoxic effects in vitro and in vivo, with BBI preventing tumor growth in TKI-resistant xenografts. Resistance to select BBI in GIST was attributable to drug efflux pumps. These results define a therapeutic vulnerability and clinical strategy for targeting oncogenic kinase dependency in GIST. Significance: Expression and activity of mutant KIT is essential for driving the majority of GIST neoplasms, which can be therapeutically targeted using BET bromodomain inhibitors.

Gastrointestinal stromal tumor (GIST) is the most common GI sarcoma and is generally initiated by KIT or PDGFRA mutations which are compelling therapeutic targets for tyrosine kinase inhibitors (TKI). However, the emergence of secondary mutations results in clinical resistance to available TKIs. GIST progression is driven by genomic events which incrementally target the p16-CDK4/6-RB1 and p14-TP53-RB1 pathways to create CDK4/6 and CDK2 oncogenic co-dependency. Based on limited efficacy of single-agent CDK4/6-inhibitor (CDK4/6i) therapy in GIST, we evaluated strategies of co-targeting CDK2 and CDK4/6. Multiplexed protein imaging (via Immuno-SABER) was validated for the detection of cell cycle regulator aberrations in GIST clinical samples (N=18), 7 of which were TKI-resistant, and including 3 patients in whom multiple metastases were analyzed. The impact of various CDK perturbants using CDK2i (CDK2 inhibitor-II), CDK4/6i (palbociclib or abemaciclib), and CDK2/4/6i (PF-06873600) was determined through cell proliferation and protein detection assays in GIST cell lines and murine xenografts. Mechanisms of acquired CDK2i and CDK4/6i resistance were characterized in GIST cell lines after long-term exposure. Abnormal expression/biallelic inactivation of CDKN2A/p16, RB1, and TP53 were identified in 7 (39%), 2 (11%), and 2 (11%) of 18 GISTs, respectively. Identical aberrations of p16, RB1, and TP53 were present in all metastases from 3 patients. Since 5 of 7 RB1-intact advanced GISTs had co-dysregulation of the CDK2 and CDK4/6 pathways, we evaluated co-inhibition of CDK2 and CDK4/6 in vitro and in vivo which inhibited cell proliferation (P Citation Format: Inga-Marie Schaefer, Meijun Z. Lundberg, Matthew L. Hemming, Sinem K. Saka, Matthew P. Serrata, Isabel Goldaracena, Ninning Liu, Peng Yin, Joao A. Paulo, Steven Gygi, George D. Demetri, Ewa Sicinska, Adrian Mariño-Enríquez, Jason L. Hornick, Chandrajit P. Raut, Wen-Bin Ou, Jonathan A. Fletcher. Response and resistance to CDK2 and CDK4/6 inhibition in GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5648.

Introduction: Deconvolution of multi-nodal perturbations in cancer network architecture demands highly multiplexed profiling assays. We demonstrate the value of polyligand profiling of tumor systems states using libraries of single stranded oligodeoxynucleotides (ssODN) to distinguish between tumor tissue from breast cancer patients who did or did not derive benefit from treatment regimens containing trastuzumab. Methods: This study included cases from women with invasive breast cancer who received chemotherapy+ trastuzumab (C+T) or trastuzumab monotherapy with available retrospective data on the time to next treatment (TTNT). A library of 2x1012 unique ssODN was exposed to FFPE tissues from patients who benefited (B) or not (NB) from trastuzumab-based regimens in several rounds of positive and negative selection. Two enriched libraries were screened on independent set of 42 B and 19 NB cases using a modified IHC protocol for detection of bound ssODNs. Poly-Ligand Profiles (PLP) were scored by a blinded pathologist. Two libraries, EL-NB and EL-B, showed significant p-values between groups of responders and non-responders. A Cox-PH model was fitted using either tumors' HER2 status or PLP test results as the independent variable. Median survival time was calculated from the Kaplan-Meier estimate. A separate group of 63 cases with TTNT data from chemotherapy without trastuzumab was used as a control to distinguish prognostic from predictive performance. Results: The PLP scores of EL-NB and EL-B were assessed by receiver operating characteristic (ROC) curves and resulted in a combined AUC value of 0.81. EL-NB and EL-B were able to effectively classify B and NB patients with either HER2-negative/equivocal (AUC = 0.73) or HER2-positive cancers (AUC = 0.84). In contrast, HER2 status alone yielded an AUC value of 0.47. The combined PLP scores for the independent set of 63 patients treated with C excluding trastuzumab resulted in an AUC value of 0.53, indicating that the assay was predictive and not simply prognostic. Kaplan-Meier curves analysis shows that PLP+ cases have 429 days median TTNT, while PLP- cases have 129 days (HR = 0.38, log-rank p = 0.001). Analysis based on HER2 status showed no significant difference in TTNT between patients that were HER2+ (280 days) or HER2-negative/equivocal (336 days, HR = 1.27, log-rank p =0.45). Summary: Performance of the PLP assay in differentiating patients who did or did not benefit from trastuzumab therapy outperforms the standard IHC assay for HER2 status. These results represent a promising step towards the development of a CDx to identify the 50-70% of HER2+ patients who will not benefit from trastuzumab. In addition, PLP also has the potential to identify the HER2-negative/equivocal patients who may benefit from trastuzumab-containing regimens. Citation Format: Domenyuk V, Gatalica Z, Santhanam R, Wei X, Stark A, Kennedy P, Toussaint B, Levenberg S, Wang R, Xiao N, Greil R, Rinnerthaler G, Gampenrieder S, Heimberger AB, Berry DJ, Barker A, Demetri GD, Quackenbush J, Marshall JL, Poste G, Vacirca JL, Vidal GA, Schwartzberg LS, Halbert DD, Voss A, Miglarese MR, Famulok M, Mayer G, Spetzler D. Polyligand profiling differentiates cancer patients according to their benefit of treatment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-09.

More than 85% of GISTs are driven by activating mutations in KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor alpha (PDGFRA). Currently, the only approved treatments for GIST are KIT directed tyrosine kinase inhibitors (TKIs). However, treatment with approved TKIs eventually results in disease progression most often due to the development of secondary resistance mutations in KIT. In addition, these agents have limited activity in PDGFRA mutant GIST and KIT/PDGFRA wild type (WT) GIST as primary therapy. Therefore, it is essential to develop novel therapeutic strategies with different modes of action in advanced GIST. G protein-coupled receptor 20 (GPR20) is an orphan GPCR selectively and abundantly expressed in GIST. GPR20 expression is regulated by FOXF1 and ETV1, transcription factors that play critical roles in KIT-driven GIST initiation, proliferation, and survival. We hypothesize that GPR20 is a potential therapeutic target for ADC development for the treatment of GIST. In this study, 1) GPR20 and KIT protein expression was assessed by IHC staining on GIST samples from DFCI (n=144) and NCCHE (n=100) as well as on normal and malignant tissue microarrays obtained commercially, and 2) an anti-GPR20 ADC (DS-6157a) was generated to evaluate antitumor activity in GIST models and to assess safety. GPR20 was expressed in more than 88% of the GIST samples analyzed, with higher expression levels in samples that: (I) received multiple treatment lines compared to naïve/early treated samples, (II) expressed higher KIT levels, (III) were small intestinal GIST, and/or (IV) had no KIT mutation, including succinate dehydrogenase (SDH) deficient GIST and neurofibromatosis type 1 (NF1)-associated GIST. The interstitial cells of Cajal were the only normal cells positive for GPR20. Normal mast cells expressed KIT but not GPR20. DS-6157a is an ADC composed of a humanized anti-GPR20 antibody, a Gly-Gly-Phe-Gly tetra-peptide-based linker, and a DNA topoisomerase I (TOP1) inhibitor Dxd. DS-6157a exhibited GPR20 expression-dependent cell growth-inhibitory activity and induced tumor regression with dosing at 3 to 10 mg/kg in multiple GIST xenograft models. In addition, DS-6157a showed antitumor activity in a GIST patient-derived xenograft model that was resistant to imatinib, sunitinib, and regorafenib. In vitro, DS-6157a induced TOP1 inhibitor-associated markers of DNA damage (phosphorylation of Chk1) and apoptosis (cleaved PARP) in GPR20 expressing cells. In preclinical toxicology studies using rats and cynomolgus monkeys, the pharmacokinetics and safety profile of DS-6157a were favorable at up to 200 mg/kg and 30 mg/kg, respectively. These data support the clinical development of DS-6157a as a potential novel GIST therapy with activity in patients that are resistant, refractory, or intolerant to approved TKIs. Citation Format: Kenji Iida, Amr H. Abdelhamid, Akiko Kawano Nagatsuma, Tomoko Shibutani, Satoru Yasuda, Michiko Kitamura, Chiharu Hattori, Manabu Abe, Jun Hasegawa, Takuma Iguchi, Tsuyoshi Karibe, Takashi Nakada, Koichiro Inaki, Reiko Kamei, Yuki Abe, Jessica L. Andersen, Sandro Santagata, Matthew L. Hemming, Suzanne George, Toshihiko Doi, Atsushi Ochiai, George D. Demetri, Toshinori Agatsuma. Therapeutic targeting of GPR20, selectively expressed in gastrointestinal stromal tumor (GIST), with DS-6157a, an antibody-drug conjugate (ADC) [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 5181.

Introduction: GIST is the most common malignant mesenchymal neoplasm and features myogenic differentiation. Most GISTs depend upon oncogenic signaling through constitutively activated forms of KIT and exhibit remarkable responses to the first-line KIT inhibitor imatinib. Secondary resistance involves emergence of subclonal populations harboring heterogeneous KIT-secondary mutations that are not collectively inhibited by any approved therapy. We dissected KIT-downstream signaling nodes to identify mediators of the KIT oncogenic program, seeking targets of overarching relevance in polyclonal imatinib-resistant GIST. Methods: using clinically-representative human GIST cell models, we undertook pharmacological and functional screening to identify key targetable nodes within KIT-downstream PI3K/mTOR and RAS/MAPK pathways and to model therapeutic strategies in vitro and in vivo. Transcriptomic analysis (RNAseq) identified genes co-regulated by PI3K/mTOR and RAS/MAPK, and functional evaluations by lentiviral construct overexpression and gene knockdown elucidated roles in GIST biology. Results: PI3K/mTOR and MEK1/2 were the most essential KIT-dependent nodes irrespective of the type of KIT primary or secondary mutation. Single node ablation did not yield sustained antiproliferative and apoptotic effects, but concurrent intermittent inhibition was synergistic in vitro and in vivo, supporting the critical and complementary roles of these two pathways. Transcriptomic analyses underscored FBXO32 (a SCF E3 ubiquitin ligase and the main effector of muscular atrophy) as the most differentially expressed gene co-regulated by the PI3K/mTOR and RAS/MAPK pathways. FBXO32 was also the most differentially expressed gene after KIT inhibition in GIST cell lines, and enrichment in FBXO32 expression was found in post-imatinib GIST samples compared to baseline matched tumor tissue. We demonstrated that FBXO32 is transcriptionally upregulated by FOXO3a upon KIT or concurrent PI3K/mTOR and MEK1/2 blockage, paralleling human muscle regulation. Notoriously, FBXO32 proved to have a pro-survival role in GIST cells, enabling evasion of apoptosis. Microarray and in vitro and in vivo functional assays showed that FBXO32 mediates cell cycle exit and quiescence upon KIT or concurrent KIT-downstream pathways inhibition, thereby minimizing imatinib-induced apoptosis and facilitating GIST cell survival. Conclusions: PI3K/mTOR and MEK1/2 are critical mediators of KIT oncogenic signaling in GIST. FBXO32 emerges as a KIT-dependent E3 ubiquitin ligase transcriptionally repressed through KIT downstream pathways. However, FBXO32 induction upon therapeutic KIT signaling inhibition triggers quiescence and subsequent apoptosis evasion, thus emerging as a novel mechanism of adaptation to therapeutic inhibition of oncogenic KIT. Citation Format: Alfonso García Valverde, Jordi Rosell, Daniel Pilco Janeta, Sergi Sayols, Claudia Valverde, Anna Esteve, Marta Gut, Jordi Barretina, George D. Demetri, Jonathan A. Fletcher, Joan Carles, Joaquín Arribas, César Serrano. FBXO32 ubiquitin ligase mediates apoptosis evasion and adaptation to KIT inhibition in gastrointestinal stromal tumor - GIST [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 3767.

Background Gene fusions involving NTRK (neurotrophic receptor tyrosine kinase) genes result in the expression of chimeric TRK proteins with uncontrolled kinase activity, leading to oncogenic transformation and unrestrained cancer cell proliferation, which confers oncogenic potential across many histopathologies. Entrectinib is a central nervous system (CNS)-active, potent inhibitor of all TRK kinases, as well as ROS1. We present integrated efficacy and safety data for entrectinib in patients with locally advanced/metastatic NTRK fusion-positive (NTRK-FP) solid tumors enrolled in global (>150 sites, 15 countries) Phase I/II entrectinib trials (ALKA-372-001 [EudraCT 2012-000148-88], STARTRK-1 [NCT02097810], STARTRK-2 [NCT02568267]), focusing on a cohort of patients with NSCLC. Methods The analysis included patients with TRK inhibitor-naïve solid tumors (with or without baseline CNS disease) harboring an NTRK fusion identified via nucleic acid-based diagnostic platforms. Tumor assessments were performed at week 4 and every 8 weeks thereafter. Disease burden was assessed per blinded independent central review (BICR) by RECIST v1.1. Primary endpoints (per BICR): overall response rate (ORR) and duration of response (DOR). Key secondary endpoints: progression-free survival (PFS), overall survival (OS) and safety Results In the total efficacy-evaluable population (n=54 adult patients with advanced/metastatic NTRK-FP solid tumors, median age 57.5 years, 59.3% female, 22.2% baseline CNS disease, >19 histopathologies across 10 cancer types), ORR (BICR) was 57.4% (95% CI 43.2-70.8), with 4 complete responses (7.4%); responses were seen in all tumor types. Median DOR: 10.4 months (95% CI 7.1-NR); median PFS: 11.2 months (95% CI 8.0-14.9); median OS: 20.9 months (95% CI 14.9-NR). In the cohort of patients with NTRK-FP NSCLC (n=10), ORR (BICR) was 70.0% (7/10; 7/7 adenocarcinoma NSCLC, 0/3 squamous cell or unclassified/undifferentiated NSCLC). In NSCLC patients with investigator-assessed baseline CNS disease (n=6), 4/6 had an intracranial response (2 complete, 2 partial); 1 had stable disease, and 1 was not evaluable. In the safety population (68 patients with NTRK-FP solid tumors who received at least 1 dose of entrectinib) most treatment-related adverse events (AEs) were grade 1-2; grade 3: 32.4% of patients, grade 4: 2.9% of patients; no grade 5 treatment-related AEs. AEs resulted in dose reduction in 39.7% of patients and discontinuation in 4.4% of patients. Conclusion In this integrated analysis of global multicenter clinical trials, entrectinib was well tolerated and induced clinically meaningful, durable systemic and CNS responses in patients with NTRK-FP solid tumors, including those with NSCLC. Citation Format: Robert Doebele, Luis Paz-Ares, Anna F. Farago, Stephen V. Liu, Sant P. Chawla, Diego Tosi, Collin M. Blakely, John C. Krauss, Darren Sigal, Lyudmila Bazhenova, Tom John, Benjamin Besse, Jürgen Wolf, Takashi Seto, Edna Chow-Maneval, Chenglin Ye, Brian Simmons, George D. Demetri. Entrectinib in NTRK-fusion positive (NTRK-FP) non-small cell lung cancer (NSCLC): Integrated analysis of patients enrolled in three trials (STARTRK-2, STARTRK-1 and ALKA-372-001) [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 CT131.

Phenotypic differences between primary patient tumors and models of tumors that are amenable to chemical screening represents a key barrier to drug discovery and drug repurposing. High-throughput technologies that can accurately identify active drugs using primary patient tumors are therefore valuable additions to the drug development toolbox and may have direct clinical utility as predictive biomarkers. Here we report a method called high-throughput dynamic BH3 profiling (HT-DBP), a robust microscopy-based assay with single-cell resolution that enables chemical screens of hundreds of candidate drugs on freshy isolated tumor cells to identify chemical inducers of mitochondrial apoptotic signaling. HT-DBP requires only 24 hours of ex vivo culture which enables the direct study of fresh primary tumor cells and minimizes adaptive changes that occur with prolonged ex vivo culture. Using 1650 compounds, we performed HT-DBP on freshly isolated cells from the MMTV-PyMT genetically engineered mouse model of breast cancer to identify drugs that sensitize these tumors for apoptosis. Selected compounds identified by HT-DBP induced regressions in the MMTV-PyMT genetically engineered mouse model of breast cancer in vivo, and in a patient derived xenograft model of breast cancer. To evaluate how chemical vulnerabilities evolve in cell culture, we performed HT-DBP on a cancer cell line from MMTV-PyMT tumors. We observe different patterns of chemical vulnerabilities between freshly isolated tumor cells and in the derived cell lines, which is consistent with the mixed track record of cancer cell line chemical screens. We demonstrate that HT-DBP can be used to generate personalized apoptotic chemical vulnerabilities (which we refer to as pharmacotypes) for a set of colon cancer PDX models. We find that a PDX model derived from a primary site tumor and a metastatic lesion from the same patient have different pharmacotypes. Using annotations of small molecule targets, we can identify proteins and signaling pathways that represent apoptotic vulnerabilities in colon PDX models. Finally, we apply HT-DBP to primary human thyroid tumors and sarcomas to identify potential active therapies. In sum, HT-DBP can efficiently predict therapeutic sensitivity upon short-term ex vivo drug exposure and may empower functional precision medicine approaches in the clinic. Citation Format: Patrick D. Bhola, Eman Ahmed, Jennifer Guerriero, Ewa Sicinska, Emily Su, Jing Ni, Elizaveta Lavrova, Otari Chipashvili, Timothy Hagan, Kimmie Ng, Andrew Aguirre, Jochen Lorch, Suzanne George, George Demetri, Jean Zhao, Anthony Letai. High-throughput dynamic BH3 profiling identifies active cancer therapies in solid 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 4478.

The ALK kinase inhibitor crizotinib (PF-02341066) is clinically effective in patients with ALK-translocated cancers, but its efficacy will ultimately be limited by acquired drug resistance. Here we report the identification of a secondary mutation in ALK, F1174L, as one cause of crizotinib resistance in a patient with an inflammatory myofibroblastic tumor (IMT) harboring a RANBP2-ALK translocation who progressed while on crizotinib therapy. When present in cis with an ALK translocation, this mutation (also detected in neuroblastomas) causes an increase in ALK phosphorylation, cell growth, and downstream signaling. Furthermore, the F1174L mutation inhibits crizotinib-mediated downregulation of ALK signaling and blocks apoptosis in RANBP2-ALK Ba/F3 cells. A chemically distinct ALK inhibitor, TAE684, and the HSP90 inhibitor 17-AAG are both effective in models harboring the F1174L ALK mutation. Our findings highlight the importance of studying drug resistance mechanisms in order to develop effective clinical treatments for patients with ALK-translocated cancers. Cancer Res; 70(24); 10038–43. ©2010 AACR.

Metabolic lesions with profound effects on epigenetic regulation are widely implicated in cancer, yet the mechanistic links between this epigenetic dysregulation and tumorigenesis remain unclear. Succinate dehydrogenase (SDH) deficiency, responsible for a subset of gastrointestinal stromal tumors (GISTs), causes accumulation of the metabolite succinate and DNA hypermethylation. We identified convergent mechanisms involving altered chromosomal conformation and pseudo-hypoxia that mediate the tumorigenic effects of SDH deficiency in GIST. To investigate epigenetic alterations in this disease, we created epigenetic maps of 14 clinical GIST specimens; including KIT and PDGFRA mutant, and SDH-deficient tumors. We characterized the landscapes of enhancers, genetic regulatory elements which can drive gene expression, through histone H3 lysine 27 acetylation chromatin immunoprecipitation sequencing (ChIP-seq). We characterized both the DNA methylation and CTCF occupancy of insulators, elements which help control chromatin conformation and restrict enhancer-gene interactions, through hybrid selection bisulfite sequencing and CTCF ChIP-seq, respectively. Analyzing these data, we uncovered thousands of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. One of the strongest disrupted insulators protected the receptor tyrosine kinase and known driver of GIST, c-KIT, from a nearby superenhancer. Chromatin conformation studies confirmed an SDH-deficient-specific interaction of this superenhancer with the KIT gene. CRISPR-mediated excision of the insulator in an SDH-intact GIST model resulted in enhancer interaction and KIT upregulation. Immunohistochemical studies confirm strong expression of c-KIT in SDH-deficient GIST clinical samples. SDH deficiency has also been reported to cause pseudohypoxia in tumors. We confirmed that the enhancer landscape of SDH-deficient tumors had a signature of pseudohypoxia. Additionally, following pseudohypoxia induction in a SDH-intact GIST model, the c-KIT ligand Stem Cell Factor (SCF/KITLG) was upregulated 12-fold. While activating KIT mutations drive the majority (~75%) of GIST tumors and are mutually exclusive with SDH deficiency, we show that a primary consequence of SDH loss is in fact induction of KIT signaling. Our findings demonstrate how metabolic lesions can provide alternate epigenetic mechanisms to activate classic tumorigenic pathways in the absence of canonical genetic mutations. Citation Format: William A. Flavahan, Yotam Drier, Sarah E. Johnstone, Daniel R. Tarjan, Esmat Hegazi, Ewa T. Sicinska, Matthew L. Hemming, Chandrajit P. Raut, Jason L. Hornick, George D. Demetri, Bradley E. Bernstein. Insulator dysfunction and epigenetic oncogene activation in SDH-deficient gastrointestinal stromal tumor [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 2996.

Background: Leucine rich repeat containing 15 (LRRC15) is a TGFβ-regulated structural protein that is highly expressed on cancer-associated fibroblasts (CAFs) in the stromal microenvironment of many solid tumors, as well as directly on cancer cells of mesenchymal origin. Soft tissue sarcomas (STS) and bone sarcomas (BS) represent a diverse family of mesenchymal malignancies that can develop at any anatomic site and that comprise more than 70 histopathologic subtypes. After screening LRRC15 expression across a variety of sarcoma histologies, we evaluated the antitumor activity of ABBV-085, an MMAE (monomethyl auristatin E) containing antibody-drug conjugate directed against LRRC15 (mouse, cyno, human), in patient-derived xenograft (PDX) models of selected sarcomas with varying levels of LRRC15 expression. Methods: LRRC15 expression/intensity in sarcoma histologies was performed by immunohistochemical (IHC) staining (Leica Bond RX, Bond Polymer Refine Kit) with a human LRRC15 specific mouse IgG2b antibody. Based on LRRC15 expression levels, STS and BS tumor fragments (PDXs) were implanted into NSG mice. Mice with growing tumors were then selected to evaluate the in vivo efficacy of ABBV-085 monotherapy (6 mg/kg). Results: LRRC15 expression was evaluated in 340 human sarcoma tumors representing a variety of STS and BS histologic subtypes. LRRC15 IHC expression was determined by scoring the percentage of positive cells, together with the intensity of staining (score of 0 for negative, 1 for weak, 2 for moderate, and 3 for strong staining), for the cancer cells and stroma, respectively. A stringent cut-off for strong LRRC15 positivity was defined as ≥2+ intensity in ≥50% of the cancer or stromal area. Strong LRRC15 expression was observed in several sarcoma subsets: 67% (14/21) of osteosarcoma (OS) tumor samples, 64% (23/36) of undifferentiated pleomorphic sarcoma (UPS), 18% (8/44) of leiomyosarcomas (LMS) and 17% (6/35) of liposarcomas (LPS). Significant antitumor activity including regressions and cures was induced by ABBV-085 in LRRC15-positive STS and BM PDX models, when compared with isotype-control treated mice. The ABBV-085-induced efficacy in osteosarcoma PDX models was superior to current standard-of-care therapies when dosed maximally in mice. In addition, ABBV-085 demonstrated efficacy in different LRRC15 positive STS subtypes including UPS, LMS and LPS. ABBV-085 was well tolerated with minimal to no body weight loss observed. Conclusions: LRRC15 is highly expressed in the majority of human osteosarcomas and undifferentiated pleomorphic sarcomas, as well as in a range of other STS and BS subtypes. ABBV-085 demonstrates promising preclinical antitumor efficacy in LRRC15-positive PDX models of STS and BS. ABBV-085 is currently being investigated in an ongoing phase 1 study in soft tissue sarcomas (including undifferentiated pleomorphic sarcoma) and osteosarcoma. Citation Format: Eytan Ben-Ami, Ying Huang, Prafulla C. Gokhale, Benjamin Eschle, Lisa Durkin, Jonathan Hickson, Mien Sho, Susan Morgan-Lappe, Kurt Gish, Dominic W. Lai, Randy R. Robinson, Diane Hollenbaugh, Eric D. Hsi, Debra T. Chao, George D. Demetri, James W. Purcell. LRRC15 is a novel antigen in sarcoma and the therapeutic target of the antibody-drug conjugate (ADC) ABBV-085 [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 953.

Introduction: Genotype-directed therapies are revolutionizing care for gastrointestinal stromal tumors (GIST). Somatic mutations in KIT exons (ex) 11 and 9 induce constitutive kinase activity and account for ~95% of the primary oncogenic events in KIT-driven GIST; KIT mutations correlate with prognosis and predict clinical activity of tyrosine kinase inhibitors (TKI), such as imatinib. After a median of 24 months on imatinib, 85% of patients (pts) develop resistance and progress multifocally, mostly due to secondary KIT mutations in ex 13 and/or ex 17. We describe here a rapid, clinical grade droplet digital PCR (ddPCR) assay to detect and quantify KIT mutations associated with TKI resistance in cfDNA from plasma of metastatic (met) GIST pts. Unlike allele-specific ddPCR that requires splitting input DNA into many reactions, we designed pan-ex 17 KIT assay to capture the polyclonal mutational landscape of KIT in a single reaction. Methods: Tumor and plasma samples and clinical data were collected from pts with met GIST. ddPCR assays were developed for the detection in cfDNA of all common secondary KIT mutations associated with imatinib resistance (V654A, T670I, A829P and multiple ex 17 mutations). The pan-ex 17 mutation drop-off assay uses a FAM-labeled mutant (mut) probe that spans sequences encoding amino acids 819~824, and a VIC-labeled reference probe that is shared by both wild-type (wt) and mut alleles. Common ex 17 mutations were detected via cluster separation. V654A, T670I, A829P are detected with allele-specific assays that use a FAM-labeled mut probe and a VIC-labeled wt probe. To demonstrate analytical sensitivity/specificity of each assay, ddPCR cycling conditions were optimized to yield the maximum fluorescence signal with minimal noise signal. Results: The KIT ddPCR assays detect a mutation prevalence of 0.01 - 0.05%, with a sensitivity of 5 - 50 KIT mut copies in a background of 10,000 KIT wt copies, depending on the mutation assayed, with absolute specificity. Results were validated in a pilot observational cohort of pts who were treated with imatinib and progressed, for which matching plasma (1 ml) and tumor samples were analyzed to reveal identical mutations. In a separate cohort, ddPCR results were validated by sensitive plasma NGS. Conclusions: We have developed rapid, highly sensitive, and 100% specific quantitative assays to enable plasma-based tumor genotyping for secondary KIT resistance mutations, which has been technically optimized for translation into clinical practice. Analysis of cfDNA by ddPCR can successfully detect KIT secondary mutations in met GIST pts. In an ongoing study, serial monitoring of KIT mutations in cfDNA from pts with advanced GIST should allow for early detection of secondary mutations and optimization of genotype-targeted therapeutic management. Citation Format: Adrian Marino-Enriquez, Suzanne George, Julianna Supplee, Grace Heavey, Pasi A. Janne, Chandrajit Raut, Jonathan Fletcher, George D. Demetri, Cloud P. Paweletz, Yanan Kuang. Development of a rapid clinical grade assay to detect and monitor secondary KIT mutations in circulating free DNA (cfDNA) for personalization of targeted therapy for gastrointestinal stromal tumors (GIST) [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 4578.

Angiosarcoma (AS) is an exceedingly rare soft tissue sarcoma, with an incidence of 300 cases/yr and a 5-year disease-specific survival of 30%. The low incidence has impeded large-scale research efforts that may lead to improved clinical outcomes. To address this, we launched a nationwide clinical-genomics study in order to empower patients to accelerate research by sharing their normal and tumor samples and clinical information remotely. Patients can access the study through an online portal (ASCproject.org). Enrolled patients are mailed saliva and blood draw kits. The study team obtains medical records and stored FFPE tumor samples. All received FFPE samples are examined by an expert pathologist to confirm a diagnosis of angiosarcoma. In order to validate that our processes would enable the generation of a robust dataset from tissues acquired from multiple institutions, we sought to characterize previously described genes known to be altered in angiosarcoma (e.g., TP53, NF1, KDR, BRCA2, MET, ARID1A, POT1, BRCA1, ASXL1, KDM6A, BRAF, SETD2, PTPRB, NRAS). A total of 251 patients have enrolled since the project launched in March of 2017. Primary locations of AS are primary breast 59 (25%), breast with prior radiation 45 (19%), head/face/neck/scalp 52 (22%), bone/limb 26 (11%), abdomen 5 (2%), heart 5 (2%), lung 2 (1%), liver 1 (1%), lymph 1 (0.4%), multiple locations 25 (11%), and other locations 12 (5%); 107 (52%) reported being disease free at the time of enrollment. To date, we have received 129 saliva kits, 106 medical records, 19 blood samples, and 36 tissue samples. Whole-exome sequencing (WES) was performed on 21 FFPE/saliva matched pairs with a goal mean target coverage of 150x for tumors. Ultra-low pass whole-genome sequencing (0.1x) was performed on cell free DNA (cfDNA) from plasma in order to determine tumor fraction. Of 10 cfDNA samples sequenced, 4 samples met criteria to perform WES. Additionally, transcriptome sequencing was performed on 9 FFPE samples. Sequence data processing and analysis has been completed on the first 10 samples and is in progress for the subsequent samples. Alterations were detected in genes previously described to be affected in angiosarcoma. Recurrent mutations in TP53 were detected in 50% (5/10) of analyzed samples, comprising 3 missense mutations, 1 frameshift deletion, and 1 frameshift insertion. Alterations were seen in at least one sample in all other genes selected for this initial analysis. This initiative demonstrates the feasibility of studying tissues from geographically dispersed patients and serves as proof of concept that patient-driven genomics efforts can democratize research for exceedingly rare cancers. Enrollment is still in progress, and additional samples will be sequenced and analyzed at scale. The data generated from these studies will be deposited into the public domain in six-month intervals. Citation Format: Michael Dunphy, Esha Jain, Elana Anastasio, Mary McGillicuddy, Rachel Stoddard, Beena Thomas, Sara Balch, Kristin Anderka, Katie Larkin, Niall Lennon, Yen-Lin Chen, Andrew Zimmer, Esme O. Baker, Simone Maiwald, Jen Hendrey Lapan, Jason Hornick, Chandrajit Raut, George Demetri, Eric Lander, Todd Golub, Nikhil Wagle, Corrie Painter. The Angiosarcoma Project: Generating the genomic landscape of an exceedingly rare cancer through a nationwide patient-driven initiative [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 5384.

SMARCB1/BAF47/INI1 is a core subunit of the mammalian SWI/SNF (BAF) family of ATP-dependent chromatin remodeling complexes, which remodel nucleosome architecture to achieve coordinated regulation of gene expression. Genetic loss of SMARCB1 has been identified in several cancer types, including malignant rhabdoid tumor (MRT, 98%) and epithelioid sarcoma (EpS, 90%), strongly implicating this event as the oncogenic driver in these malignancies. However, the precise mechanism underpinning the tumor suppressive function of BAF47 to date remains unclear. In order to elucidate the underlying mechanism and to identify direct genetic targets of aberrant BAF complexes in this context, we comprehensively evaluated the effects of BAF47 reintroduction in BAF47-deficient sarcomas with respect to complex subunit and associated protein factor composition and stability, global chromatin structure, and gene regulation. Reintroduced BAF47 stably integrated into BAF complexes, and remarkably, stabilized a highly specific set of BAF subunits, resulting in an increased complex molecular weight and stoichiometric nuclear abundance. These biochemical changes inducing the formation of wild-type complexes in MRT and EpS cell settings were directly linked to reproducible changes in BAF complex localization genome-wide, particularly, in the targeting to H3K4me3-marked promoter regions of direct target genes to establish DNA accessibility. Importantly, changes in BAF47-dependent BAF complex targeting between oncogenic and induced wild-type conditions were reproducibly associated with differential chromatin architecture and gene expression signatures hallmark to both MRT and EpS, and uniformly resulted in proliferative senescence of MRT and EpS cell lines in culture. These studies highlight, for the first time, the full spectrum of structural and functional contributions of the BAF47 subunit, implicating its role as a keystone in heteromorphic BAF complex assembly; BAF47 is required for the stable integration of several BAF subunits and novel interacting factors, which we determine govern specific genome-wide targeting mechanisms and chromatin-templated activities. These results reveal the mechanisms underlying the oncogenesis of BAF47-deficient sarcomas and point toward novel therapeutic strategies for this group of human sarcomas. Citation Format: Robert Nakayama, Robert T. Williams, Seth H. Cassel, Mingxiang Teng, Rafael A. Irizarry, George D. Demetri, Cigall Kadoch. Genome-wide mistargeting of oncogenic SWI/SNF(BAF) complexes in SMARCB1(BAF47)-deficient sarcomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2658.

Sarcomas represent a heterogeneous set of malignant tumors originating from mesenchymal cells. Liposarcomas (LPS) and Leiomyosarcomas (LMS) are two of the most common histotypes, accounting for approximately 25% and 30% of all soft tissue sarcomas, respectively. The most frequent form of LPS is represented by a spectrum of disease including well-differentiated LPS (WDLPS), a non-metastasizing but locally recurrent disease with adipocytic differentiation, and de-differentiated LPS (DDLPS), an aggressive, frequently metastasizing high grade sarcoma which can arise in conjunction with elements of WDLPS. Besides the known oncogenic KIT or PDGFRA driver mutations in GIST, somatic alterations in RB1, deletions in PTEN, MDM2 amplifications in LPS, and TP53 mutations in LMS are the most commonly reported genomic aberrancies identified in sarcomas. Both WDLPS and DDLPS share amplification of 12q13-15, a genomic region containing the MDM2 and CDK4 loci, yet little is understood about the other genetic processes that yield such different phenotypes. In LMS, recurrent mutations in TP53 have been identified, but a detailed analysis of primary and metastatic tumors has not previously been described. To evaluate genetic events in these transitions, we performed whole exome sequencing (WES) on trios comprising normal tissue, WDLPS, and DDLPS obtained from individual patients (n = 19), as well as on trios of normal tissue, primary and metastatic LMS tumors obtained from separate individual patients (n = 8). Consistent with prior reports of the molecular characterization of LPS, we observed arm-level amplifications in Chr 12 in all LPS samples, and Chr 17 amplification in many LMS cases. There were no shared somatic alterations across paired WDLPS and DLPS pairs from individual patients aside from the common Chr 12 amplification (MDM2 & CDK4). A higher mutation rate was observed in LMS, and there were many clonal somatic alterations shared between the primary and metastatic lesions of individuals. This study demonstrates the heterogeneity of evolution between different subtypes of sarcomas using whole exome sequencing of clinical specimens. This finding has implications for better understanding of the molecular drivers of these tumors and opportunities to identify subtype-specific therapeutic targets. Citation Format: Ali Amin-Mansour, Stefano Sioletic, Scott L. Carter, Levi A. Garraway, George D. Demetri, Suzanne George, Eliezer M. Van Allen, Andrew J. Wagner. Differential evolution of tumor heterogeneity in leiomyosarcomas and liposarcomas suggested by genomic profiling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4819. doi:10.1158/1538-7445.AM2015-4819

KIT expression is a key diagnostic feature of gastrointestinal stromal tumors (GISTs), and virtually all of the GISTs express oncogenic forms of the KIT or PDGFRA receptor tyrosine kinase proteins, which serve as therapeutic targets of imatinib mesylate (Gleevec; Novartis, Basel, Switzerland). However, KIT expression can be low in PDGFRA-mutant GISTs, increasing the likelihood of misdiagnosis as other types of sarcoma. We report that the signaling intermediate protein kinase C θ (PKCθ) is a diagnostic marker in GISTs, including those that lack KIT expression and/or contain PDGFRA mutations. PKCθ is strongly activated in most GISTs and hence may serve, along with KIT/PDGFRA, as a novel therapeutic target.

Chromosomal Region Maintenance Protein 1/Exportin 1 (CRM1/XPO1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of Tumor Suppressor Proteins (TSPs) and renders cancer cells susceptible to apoptosis. Selinexor is orally bioavailable and represents a novel class of small molecule compounds with potent activity against a wide variety of cancers. Selinexor is currently in Phase 1 clinical studies in hematological and solid cancer patients (Clinicaltrials.gov NCT01607892 and NCT01607905). We tested the activity of Selinexor in a soft tissue sarcoma – ASPS that is resistant to traditional chemotherapy and irradiation treatment. Here we report in vitro activity of Selinexor against ASPS and in vivo efficacy results in xenograft models. Methods We used MTT, FACS, qPCR, immunofluorescence, immunostaining and immunoblots to measure the in vitro and in vivo effects of KPT-330 on the ASPS-KY cell line and in xenograft models. Results The IC50 of the ASPS-KY cell line treated with Selinexor for 72 hours was 10 μM. This concentration induced nuclear accumulation of p53, p21, IκB and FOXO3A within 4 hours of treatment, and by 24 hours cells stopped DNA synthesis and were arrested at G1 phase of the cell cycle. By 72 hours, 25% of the cells died. Prior to cell death, the drug reduced the survival protein BCL2 as well as other pro-proliferative proteins such as CDK4, Cyclin D and E2F. In addition, Selinexor induced dephosphorylation of pRb activating its tumor suppressor activity and also induced Caspase 3/7 and PARP cleavage. To assess the in vivo activity of Selinexor in a xenograft model of ASPS, we treated mice with 10 or 20 mg/kg of KPT-330 using a 3 times weekly oral dosing schedule. Following a week of treatment, tumors showed accumulation of TSPs as well as significant reduction of the proliferation marker Ki-67. Following treatment with Selinexor for 40 days, tumor growth was inhibited by 70% at 10 mg/kg and by 80% at 20 mg/kg compared with vehicle treated animals. Analysis of immunoblots from these tumors showed induction of p21, with corresponding reduction of the pro-survival and proliferation markers c-Jun, c-Met, survivin, ERK and HSP70. Histological analysis revealed apoptosis and large areas of fibrosis in treated tumors. These results indicated that Selinexor not only inhibited tumor growth, but also induced ASPS cell death in vivo. Conclusions Our results demonstrated that Selinexor is effective against ASPS in vivo as a single agent and further support the development of SINE-based therapies for alveolar soft part sarcoma that has currently no cure. We will perform further studies to test Selinexor in drug combination studies to identify synergism with other therapies. Citation Format: Marsha L. Crochiere, Trinayan Kashyap, Boris Klebanov, William Senapedis, Diego del Alamo, Sharon Tamir, Erkan Baloglu, Dilara McCauley, Robert Carlson, Michael Kauffman, Sharon Shacham, George Demetri, Andrew Wagner, Ewa Sicinska, Prafulla Gokhale, Nancy Kohl, Amy Saur, Yosef Landesman. Selinexor (KPT-330), a novel selective inhibitor of nuclear export (SINE), shows single agent efficacy against alveolar soft part sarcoma (ASPS) in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3810. doi:10.1158/1538-7445.AM2014-3810

Many common human mesenchymal tumors, including gastrointestinal stromal tumor (GIST), rhabdomyosarcoma (RMS), and leiomyosarcoma (LMS), feature myogenic differentiation. Although presumptive initiating mutations have been identified in these cancers, the subsequent mechanisms of malignant progression are not known. Here we report that intragenic deletion of the dystrophin-encoding and muscular dystrophy-associated DMD gene is a frequent mechanism by which myogenic tumors progress to high-grade, lethal sarcomas. Genome-wide SNP screens demonstrated DMD intragenic deletions in 23 of 38 myogenic sarcomas (61%) compared to 0 of 58 non-myogenic sarcomas (p < 0.0001). Although DMD is an X-linked gene, somatic DMD deletions affected both sexes equally and DMD deletions in female patients involved the active X chromosome, resulting in functional DMD nullisomy. Dystrophin was expressed strongly in nonneoplastic and benign counterparts for GIST, RMS and LMS, and the DMD deletions in the malignant myogenic sarcomas clustered at the 5′ end of the gene, inactivating larger dystrophin isoforms, including 427kDa dystrophin, while preserving expression of an essential 71kDa isoform (Dp71) which is encoded by the DMD 3′ end. Dystrophin restoration inhibited myogenic sarcoma cell migration, invasion, anchorage independence, and invadopodia formation, and dystrophin inactivation was found in 96%, 100%, and 62% of metastatic GIST, embryonal RMS, and LMS, respectively. The genomic, clinicopathological and functional evidence validate dystrophin as a tumor suppressor and likely anti-metastatic factor, suggesting that therapies in development for muscular dystrophies may also have relevance in treatment of cancer. Citation Format: Yuexiang Wang, Adrian Marino-Enriquez, Richard Bennett, Meijun Zhu, Grant Eilers, Cristina Antonescu, Christopher Fletche, Chandrajit Raut, Matt van de Rijn, Louis Kunkel, George Demetri, Jonathan Fletcher. Dystrophin Is a tumor suppressor in human cancers with myogenic programs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1572. doi:10.1158/1538-7445.AM2014-1572

Intro/Objective: Several inhibitors of histone deacetylase have been shown to enhance chemotherapy induced apoptosis and reduce sarcoma tumor burden in preclinical models. We sought to determine the MTD, PK/PD, safety and toxicity of the histone deacetylase inhibitor (HDACi) abexinostat when administered with doxorubicin in patients with metastatic sarcomas. Methods: Participants were enrolled in a standard 3x3 dose escalation phase I study design. Abexinostat was administered on days 1-5 with 75 mg/m2 of doxorubicin administered on day 4 of every 21 day cycle until disease progression, drug intolerance, or a cumulative lifetime dose of 450 mg/m2 of doxorubicin was reached. GCSF support was provided at physician discretion on Arm A or provided to all participants in Arm B. 3-6 subjects were administered abexinostat at doses of 15, 30, 45, and 60 mg/m2 BID. All patients without progressive disease after receiving a cumulative lifetime dose of 450 mg/m2 of doxorubicin continued with abexinostat as a single agent until disease progression. Results: 22 participants (10 with prior doxorubicin therapy) were enrolled (6 in Arm A, 14 in Arm B), 20 were evaluable for DLT, and 17 were evaluable for radiologic response. In Arm A, participants were administered abexinostat at 30 or 15 mg/m2 BID. DLTs of grade 3 and 4 ANC were observed in two out of three participants dosed at 30 mg/m2 BID. Neither of these patients received GCSF prophylaxis. In Arm B, participants were administered abexinostat at 30, 45, or 60 mg/m2 BID, all with mandated GCSF support. Two DLTs were observed on the 60 mg/m2 BID dose (grade 3 infection and grade 4 thrombocytopenia). The pharmacokinetics of abexinostat was not affected by doxorubicin. HDAC activity, as measured by histone acetylation in PBMC, was maximally inhibited at 30mg/m2 BID. In the 17 participants evaluable for radiologic response there was 1 PR, 11 SD, and 5 PD as best response, with 7 participants completing 5 cycles or more. 3 of those participants remain in SD as their last disease status when this abstract was submitted. 4 participants who continued on monotherapy remained in SD for a median of 9.8 weeks after completing doxorubicin. The most common toxicities were fatigue, thrombocytopenia, and anemia (see Table 1). No study related deaths were observed. Conclusion: The MTD for abexinostat is 45 mg/m2 BID when administered on days 1-5 when doxorubicin is given at 75 mg/m2 on day 4 of a 3-week cycle and GCSF support is provided. Toxicities were manageable and tumor responses were seen. Additional studies are needed to further define the specific contributions of HDAC inhibition for patients receiving doxorubicin to treat metastatic sarcoma. Grade Type 1 2 3 4 Totals Fatigue 13 6 0 0 19 Platelets 10 3 1 2 16 Hemoglobin 11 4 1 0 16 Nausea 13 1 0 0 14 Neutrophils 4 1 4 3 12 Leukocytes 6 3 3 0 12 Diarrhea w/o prior colostomy 7 3 1 0 11 Hyperglycemia 7 1 1 0 9 QTc interval 7 2 0 0 9 Alopecia 1 5 1 0 7 Hypophosphatemia 3 4 0 0 7 Constipation 5 2 0 0 7 Extremity/limb pain 6 1 0 0 7 Anorexia 7 0 0 0 7 Lymphopenia 0 2 4 0 6 Pain: other 5 1 0 0 6 Hypomagnesemia 4 1 0 0 5 Hyponatremia 5 0 0 0 5 Taste disturbance 5 0 0 0 5 Citation Format: Edwin Choy, Sriram Balasubramanian, James Butrynski, Jeffrey Morgan, Mint Sirisawad, Chitra Mani, Suzanne George, Andrew Wagner, David Harmon, Yael Rubinstein, Zhenfeng Duan, Francis Hornicek, George Demetri. Phase I dose escalation study of abexinostat and doxorubicin in patients with metastatic sarcomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-167. doi:10.1158/1538-7445.AM2013-LB-167

Based on preclinical biology (1, 2), this translational trial aimed to assess the efficacy and tolerability of the PARP inhibitor, olaparib, in patients with advanced Ewing sarcoma progressing after prior chemotherapy. In this uncontrolled phase II pilot, adult participants with radiographically measureable disease received 400 mg of olaparib capsules, twice daily, until disease progression or drug intolerance. Tumor measurements were determined by CT or MRI, at 6 and 12 weeks after starting olaparib administration, and then every 8 weeks thereafter. Tumor response determinations were made according to RECIST 1.1, and adverse event determinations were made according to CTCAE, version 4.0. A total of 22 participants were planned to be enrolled using a conventional 2-step phase II study design. If no objective responses were observed after 12 participants had been followed for at least 3 months, further accrual would be stopped. 12 participants were enrolled, and all were evaluable. A single case each of grade 3 anemia and grade 3 thrombocytopenia was observed (see Table One). Otherwise, no significant or unexpected toxicities were observed while participants were receiving study drugs. There were 0 PR/CR, 4 SD (duration 10.9, 11.4, 11.9, and 18.4 wks), and 8 PD as best response. Of the SD, 2 had minor responses (-16.7% and -11.7% by RECIST 1.1). The median time to disease progression was 5.7 weeks. Further enrollment was therefore discontinued. This study is the first report of a prospective phase II trial to evaluate the safety and efficacy of a PARP inhibitor in patients with Ewing sarcoma. Olaparib tablets were safe and well tolerated when administered to this small cohort at the 400 mg BID dose, although the median duration of dosing was for only 5.7 weeks. However, no significant responses were seen, and the short average interval to disease progression underscores the aggressiveness of this disease. Preclinical modeling supports exquisite in vivo sensitivity of this pathway in combination with chemotherapy, and such a trial of olaparib with temozolomide will begin shortly at our Center. Toxicity Type # of Patients Grade 1 2 3 4 Anemia 1 0 0 0 0 Blood and lymphatic systems disorders: other 1 1 0 0 0 Fatigue 1 1 0 0 0 Fever 1 1 0 0 0 Pain 2 1 0 1 0 Rash maculo-papular 1 1 0 0 0 Nausea 1 1 1 0 0 Vomiting 1 1 0 0 0 Urinary tract infection 1 1 0 0 0 Platelet count decreased 2 1 1 0 Hypoglycemia 1 1 0 0 0 Metabolism and nutrition disorders: other 1 1 0 0 0 Arthritis 1 1 0 0 0 Musculoskeletal and connective tissue disorder: other 1 1 0 0 0 Dizziness 1 1 0 0 0 Nervous system disorders: other 1 1 1 0 0 Cough 3 3 0 0 0 Postnasal drip 1 1 0 0 0 Respiratory, thoracic and mediastinal disorders: other 1 1 0 0 0 Hypertension 1 1 0 0 0 Total 25 19 3 0 0 Citation Format: Edwin Choy, James Butrynski, David Harmon, Jeffrey Morgan, Suzanne George, Andrew Wagner, David D'Adamo, Greg Cote, Yael Rubinstein, Cyril Benes, Daniel Haber, Jose Baselga, George Demetri. Translation of preclinical predictive sensitivity of Ewing sarcoma to PARP inhibition: Phase II study of olaparib in adult patients with recurrent/metastatic Ewing sarcoma following failure of prior chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-174. doi:10.1158/1538-7445.AM2013-LB-174

Background: GRID is a phase III study for patients with advanced gastrointestinal stromal tumors (GIST) following failure of imatinib (I) and sunitinib (S) who were randomized to receive either the multikinase inhibitor regorafenib (R) or placebo (P). R demonstrated a highly significant improvement in progression-free survival compared with P (HR 0.27, p Methods: DNA was isolated from archival tumor tissue and analyzed for KIT mutations via Sanger sequencing. The expectation was that primary KIT mutations would be detectable in archival tissue but that secondary KIT mutations may be undetectable in tissues obtained before treatment with I or S. To overcome this potential limitation, plasma samples drawn at GRID study entry, post I and S failure, were used as a source of circulating DNA for evaluation of GIST oncogenic mutations (KIT, PDGFRA, BRAF) via BEAMing technology. Results: KIT mutations were detected in 83 of 138 (60%) plasma samples and 64 of 99 (65%) tumor tissue samples analyzed. Primary KIT exon 11 and 9 mutations were identified in approximately 42% and 18% of the tissue samples, respectively. The frequency of the canonical exon 9 mutations was similar for plasma and tissue samples, showing consistency between mutation-detection technologies. With limitations of tumor-based assays, a lower incidence of secondary KIT resistance mutations was detected in patient-matched archival tumor tissue compared with plasma samples: resistance mutations were detected in 12% of tissue samples vs 48% of plasma samples. Most (76%) secondary KIT mutations detected in plasma DNA were located in the KIT activation loop encoding structural alterations known to mediate resistance to I and S. Nearly half of the plasma samples in which secondary KIT mutations were identified harbored multiple secondary mutations, consistent with the results of previous studies on fresh tumor biopsies taken following resistance to both I and S. R was clinically active compared with P in all KIT mutational subgroups evaluated (HR 0.27 in patients with KIT exon 9 mutations; HR 0.25 in patients with secondary KIT mutations identified via plasma DNA). Conclusions: In GIST patients from the GRID trial, driver oncogenic mutations and secondary oncogenic mutations leading to I and S resistance are readily detectable via BEAMing of circulating DNA from plasma. BEAMing may provide a real-time assessment of tumor genotype in GIST and other cancers using blood-derived circulating DNA, that may be more comprehensive than tumor sampling. GIST patients with a wide spectrum of primary and secondary mutations in oncogenic kinases benefit from treatment with R. Citation Format: George D. Demetri, Michael Jeffers, Peter Reichardt, Yoon-Koo Kang, Jean-Yves Blay, Piotr Rutkowski, Hans Gelderblom, Peter Hohenberger, Michael Leahy, Margaret von Mehren, Heikki Joensuu, Giuseppe Badalamenti, Martin Blackstein, Axel Le Cesne, Patrick Schöffski, Robert G Maki, Sebastian Bauer, Binh Bui Nguyen, Jianming Xu, Toshirou Nishida, John Chung, Chetan D. Lathia, Christian Kappeler, Iris Kuss, Dirk Laurent, Paolo G Casali. Detection of oncogenic kinase mutations in circulating plasma DNA and correlation with clinical benefit in the phase III GRID study of regorafenib vs placebo in TKI-refractory metastatic GIST. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-295. doi:10.1158/1538-7445.AM2013-LB-295

Introduction: Activating protein 1 (AP-1) is a dimeric transcription factor comprising proteins whose common denominator is the possession of basic leucine zipper (bZIP) domains that are essential for dimerization and DNA binding. Among this family, the c-Jun protein is a transcription factor that is activated among the earliest responses to mitogenic signaling. It can promote cell division and differentiation but also can act as a potent inducer of apoptosis in other contexts. Recently, c-Jun was directly implicated as a proto-oncogene in a human cancer when it was found to be genomically amplified in ∼30% of dedifferentiated liposarcomas (DDLPS). It was hypothesized that c-Jun overexpression might directly impair adipogenesis, thereby mediating the transition from well differentiated liposarcomas (WDLPS) to DDLPS. However, recent work from our lab has suggested that c-Jun may regulate other cellular processes in DDLPS, including proliferation. We are currently elucidating the precise mechanisms by which c-Jun amplification drives tumorigenesis in DDLPS. Methods We used lentiviral-mediated RNA interference to inhibit c-Jun in two DDLPS cell lines, one with c-Jun amplification and overexpression (LP6) and one with normal c-Jun copy number (LPS141). We then analyzed changes in mRNA levels by Affymetrix Exon array and changes in miRNA levels via Nanostring. Ingenuity Pathways (IPA) was used to screen the results for biological trends. We validated changes in gene expression via qRT-PCR and immunoblotting. Results: Analysis of 754 microRNAs revealed that 85 (11%) miRNAs exhibited a >2 fold decrease, and 23 (3%) exhibited >2 fold increase after c-Jun knockdown in LP6 cells. mRNA analysis showed that 116 genes were downregulated and 12 genes were upregulated >2 fold after c-Jun knockdown in LP6 cells. In LPS141 cells, 341 genes were downregulated and 57 upregulated. We identified only 10 common differentially expressed genes between these 2 cell lines, suggesting that c-Jun may acquire additional functions when genomically amplified. Pathway analysis showed that c-Jun promotes cell proliferation in both cell lines. However, genes involved in “cellular movement and connective tissue development” were significantly downregulated by c-Jun knockdown only in LP6 cells. We have validated these gene expression changes and are testing the hypothesis that c-Jun amplification promotes cell migration in DDLPS. Discussion: Liposarcomas exhibit nearly universal genomic amplification of MDM2 and CDK4, two oncogenes which are readily targeted by small molecules. Transcription factors such as c-Jun are difficult to target directly by small molecule inhibitors. Our studies suggest that amplified c-Jun may regulate genes that stimulate both proliferation and cell migration in DDLPS. We expect that this work will provide a starting point for developing targeted therapies for c-Jun-amplified DDLPS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-270. doi:1538-7445.AM2012-LB-270

The tumor suppressor p53 is commonly inactivated in human malignancies. One mechanism is by genomic amplification and consequent overexpression of the ubiquitin E3 ligase MDM2, which targets p53 for proteosome-mediated degradation. The MDM2 gene is amplified in >95% of human liposarcomas, a disease for which the efficacy of systemic therapies is limited. Here, we compared the effects of two novel highly potent and selective inhibitors of the MDM2-p53 interaction, with the classic MDM2 inhibitor Nutlin-3 on the expression and activity of p53 and the resulting changes in human liposarcoma cell proliferation and survival. We determined MDM2 copy number, p53 mutational status, and relative expression levels of MDM2 and p53 proteins in 8 human liposarcoma cell lines and normal human preadipocytes/adipocytes. TP53 status was wild type in 7 of 8 liposarcoma cell lines, which also had genomic amplification of MDM2. MDM2 protein expression correlated with the degree of amplification and was highly expressed in comparison to normal preadipocytes/adipocytes. The two spiro-oxindole derivatives - Compounds A and B- (1) and Nutlin-3 were applied to liposarcoma cell lines with or without p53 siRNA and the biological consequences were determined by immunoblot, cell viability, cell cycle and apoptosis assays. Treatment with MDM2 inhibitors led to increased p53 protein levels and induction of its transcriptional targets MDM2 and p21. Cell viability was markedly decreased in a dose-dependent manner in liposarcoma cells with wild-type p53, with IC50 values of 0.40-4.23 μM, 0.37-1.88 μM and 2.72-9.83 μM for Compound A, Compound B, and Nutlin-3, respectively. These effects were markedly abrogated by siRNA-mediated p53 knockdown and in a cell line harboring a de novo p53 mutation. Compound A and Compound B also induced cell cycle arrest and apoptosis at approximately 5 fold lower concentrations than Nutlin-3. At low concentrations, G0/G1 cell arrest occurred, whereas higher concentrations resulted in G2/M arrest and induction of apoptosis. Cell line and tumor xenograft experiments are ongoing. These data demonstrate that Compounds A and B are highly potent and specific inhibitors of MDM2 with activity in genetically characterized liposarcoma cells. Further study and clinical development in this disease is warranted. 1. Wang S. et al., AACR, Orlando FL, 2011, Abstract LB-204 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2786. doi:1538-7445.AM2012-2786

Background: The anaplastic lymphoma kinase (ALK) inhibitor crizotinib is clinically effective in cancer patients whose tumors harbor ALK translocations. However, drug resistance will ultimately develop in most if not all patients. We studied drug resistance mechanisms using both in vitro models and analyses from tumors of patients that had developed crizotinib resistance. Methods and Results: We identified a secondary mutation in ALK, F1174L, as one cause of crizotinib resistance in a patient with an inflammatory myofibroblastic tumor (IMT) harbouring a RANBP2-ALK translocation who progressed while crizotinib therapy. When present in cis with an ALK translocation, this mutation (also detected in neuroblastomas) causes an increase in ALK phosphorylation, cell growth and downstream signaling. Furthermore, the F1174L mutation inhibits crizotinib mediated downregulation of ALK signaling and blocks apoptosis in RANBP2-ALK Ba/F3 cells. We also evaluated the impact of mutations in the ALK gatekeeper residue (L1196) and engineered all possible gatekeeper mutations resulting from a single nucleotide change into the background of EML4-ALK. Three of the mutations (L1196M, L1196Q and L1196V) in the background of EML4-ALK led to IL-3 independent growth. Furthermore the EML4-ALK L1196M mutant resulted in a greater baseline ALK phosphorylation, cell growth and downstream signaling compared to EML4-ALK alone. A chemically distinct ALK inhibitor, TAE684, or the HSP90 inhibitor 17-AAG are both effective in models harbouring the F1174L or the L1196M ALK mutations. Conclusions: We identify secondary mutations in ALK that confer crizotinib resistance. In addition, we identify therapeutic strategies, alternative ALK inhibitors or HSP90 inhibitors that may be clinically effective in patients that have developed crizotinib resistance. Understanding the mechanistic basis of drug resistance is necessary in order to develop effective clinical treatments for patients with ALK-translocated cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1541. doi:10.1158/1538-7445.AM2011-1541

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is standard of care for patients with metastatic GIST. However, most patients eventually develop clinical resistance to imatinib and other KIT/PDGFRA kinase inhibitors due to acquisition of secondary mutations in the KIT kinase domain, genomic amplification of KIT, or activation of alternate oncoproteins. Approximately 10% of KIT-dependent GIST metastases lose KIT expression at time of clinical progression during imatinib therapy. We performed whole transcriptome sequencing in KIT-expressing GIST vs. KIT-negative GIST, and thereby identified strong cyclin D1 expression only in KIT-negative GISTs. KIT-negative GISTs showed complete loss of the GIST biomarker, PKCθ, and restoration of PKCθ expression in these cells resulted in loss of Cyclin D1 expression. Lentivirus-mediated CCND1 (Cyclin D1) short hairpin RNA (shRNA) knockdown resulted in profound anti-proliferative and pro-apoptotic effects in KIT-negative GIST cell lines, and was associated with activation (dephosphorylation) of the tumor suppressor Rb and upregulation of the p27 CDK inhibitor. c-Jun expression levels paralleled the Cyclin D1 expression in the GIST lines, and c-Jun shRNA knockdown resulted in downregulation of cyclin D1 in the KIT-negative GIST lines, and was accompanied by profound anti-proliferative effects, including p27 upregulation, and pro-apoptotic effects as evidenced by PARP cleavage. By contrast, cyclin D1 and c-Jun silencing showed little anti-proliferative and pro-apoptotic effects in KIT-positive, KIT-dependent GIST lines. These findings show that cyclin D1 and c-Jun overexpression serve a crucial oncogenic role in KIT-independent GISTs, including those enabling clinical resistance to imatinib or sunitinib targeted kinase inhibitor therapies. Our findings also show that PKCθ downregulation and c-Jun upregulation coordinately enable cyclin D1 overexpression in KIT-independent GISTs. These novel findings highlight the relevance of c-Jun/cyclin D1 as novel therapeutic targets in GISTs that have lost KIT oncoprotein expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 952. doi:10.1158/1538-7445.AM2011-952

Sarcoma represents a class of tumors arising from mesenchymal tissue. New approaches that target sarcoma-specific molecular alterations hold a promise of more effective therapies. Currently available sarcoma models do not accurately resemble relevant properties of the human disease and very often are inadequate for predicting the efficacy of investigational drugs in preclinical settings. Thus, in order to test novel targeted therapies, it is essential to develop in vivo models of sarcomas. To this end, we have created a panel of well-characterized xenografts of primary human sarcomas that represent a spectrum of histotypes. Specifically, we transplanted intact human sarcomas under the skin of immunodeficient mice. We then compared the morphologic, phenotypic, and genetic characteristics of the tumor tissues before and after implantation in recipient mice. To date, we have implanted into Nu/Nu recipients tumor tissue fragments from 32 gastrointestinal stromal tumors (GIST) 10 dedifferentiated liposarcomas and 6 leiomyosarcomas. Successful tumor growth was detected in 86% cases of GIST, 44% of liposarcomas and 33% of leiomyosarcomas. Upon serial transplantation, 35% of GISTs, 44% of liposarcomas and 33% of leiomyosarcomas have grown beyond passage F3. Histological analysis of the primary tumors and corresponding xenografts revealed that morphology and immunophenotype (c-Kit status for GISTs, MDM2 for liposarcomas, Smooth Muscle Actin for leiomyosarcomas) was retained throughout all passages. Moreover, all major genetic abnormalities present in the primary tumors were retained in the xenografts, as revealed by genome-wide Single Nucleotide Polymorphism (SNP) array analyses. Our results show that this approach can be used to generate animal models that faithfully reproduce characteristics of human intact tumors. These models can be used for in vivo preclinical drug testing, diagnostic and prognostic molecular markers discovery, and for identification of molecular mechanisms of drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4197.

Clear Cell Sarcoma (CCS), a soft tissue malignancy of tendons and aponeuroses in children and young adults, is notoriously resistant to radiation and chemotherapy, and new approaches to treatment are needed. We recently demonstrated the involvement of the MET receptor tyrosine kinase (RTK) in regulation of the growth, migration, and invasion of CCS cells. However, pharmacologic agents targeting the HGF:MET axis could neither completely inhibit cell growth nor significantly induce apoptosis. To determine whether other RTKs might cooperate with MET in control of survival and proliferation of CCS cells, we used phospho-RTK arrays to assess the activation status of RTKs in three CCS cell lines (CCS292, DTC1, SU-CCS-1). We found that in addition to MET, ERBB3 was strongly activated in CCS. Western blot analysis confirmed constitutive activation of ERBB3 in CCS292 and DTC-1 cell lines and expression in all three lines. Since ERBB3 lacks intrinsic kinase activity, we searched for potential heterodimerization partners. Interestingly, we found that the activation of ERBB3 in different CCS cell lines relied on different partners: ERBB2 as a pivotal partner in CCS292, and MET as a pivotal partner in DTC-1. Moreover, we explored the possibility of a linkage between the CCS pathognomonic EWS-ATF1 fusion product and ERBB3 activation. We found that EWS-ATF1 knockdown resulted in both decreased expression of the ERBB3 ligand Neuregulin 1 and less ERBB3 phosphorylation in CCS292 cells, but had no effects on ERBB3 phosphorylation in DTC-1 cells. To explore the biological function of ERBB3, we applied siRNA or small molecule inhibitors of RTKs to CCS cell lines and determined the biological consequences by cell viability, cell cycle and apoptosis assays; these studies show that ERBB3 in conjunction with MET played an important regulatory role in the proliferation and survival of CCS cells. These data suggest that combined inhibition of the MET and EGFR signaling pathways may be a rationally designed strategy for treatment of patients with advanced CCS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 268.