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The menin-MLL1 interaction is critical for development of acute leukemias driven by MLL1 rearrangements (MLLr) or mutations in the Nucleophosmin 1 gene (NPM1c). Inhibition of the menin-MLL1 interaction by SNDX-5613 (revumenib) has demonstrated robust clinical responses in the current AUGMENT clinical trial (NCT04065399). During the trial, some responders relapsed during treatment due to acquired resistance in MEN1. Somatic MEN1 mutations were found at residues M327, G331 or T349 which diminished SDNX-5613 binding affinity and mediated therapeutic resistance. The presence of acquired resistance validates MEN1 as a therapeutic target in MLLr and NPM1c AML patients. Here we characterize the effects of these mutations on the activity of 6 menin inhibitor chemotypes currently in clinical trials (NCT04065399/Syndax, NCT04067336/Kura, NCT04811560/JNJ, NCT04988555/Sumitomo, NCT04752163/Daiichi, NCT05153330/Biomea). In vitro activity and binding modes for these compounds were evaluated in wild-type (WT) and mutant menins using (i) competition binding assays, (ii) cell-based proliferation assays and (iii) X-ray co-crystallography. For binding, His6-tagged MEN1 mutant proteins (G331R, M327I, M327V, T349M) were expressed and purified. Binding affinities were measured in competition binding format. The menin-MLL interaction was monitored by HTRF using Terbium labeled anti-His6 antibody and FITC labeled MLL peptide (4-43). Acquired mutations affected binding affinities (Ki) to varying degrees. Notably, M327I/V mutations reduced binding for all menin-MLL inhibitors ranging from ~30-300, indicating a class effect for this mutation. An irreversible Biomea chemotype did not inhibit menin-MLL binding in our assays. The decreased binding affinity to M327I was reflected in cell-based proliferation assays. Menin mutations were introduced into MV4;11 cells using CRISPR-Cas9 in conjunction with a homology directed repair template to edit the endogenous MEN1 coding sequence. Clonal lines were established harboring homozygous (homo) and heterozygous (het) M327I mutations. The M327I (het) MV4;11 cells experienced 15-50-fold shifts in IC50 vs WT cells, consistent with the reduced binding affinities. The molecular basis for sensitivity to M327 acquired resistance was examined by X-ray co-crystallography of inhibitors bound to M327I and WT menin. Both KO-539 and SNDX-5613 show notable changes in binding in M327I vs WT menin. The isoleucine creates a steric clash, displacing their position in the pocket as previously noted. The Janssen chemotype shows a novel binding mode. Although it has 30-fold lower affinity for M327I, it shows little change in its bound position to M327I menin. Given the clinical validation of menin inhibition in AML, the design of next generation compounds that block MLL1 binding while avoiding acquired MEN1 mutations may be a strategy to overcome acquired resistance to first generation menin inhibitors. Citation Format: Florian Perner, Sheng F. Cai, Daniela V. Wenge, Jeonghyeon Kim, Jevon Cutler, Radosław P. Nowak, Joel Cassel, Shivendra Singh, Shipra Bijpuria, William H. Miller, Eytan M. Stein, Ross L. Levine, Eric S. Fischer, Gerard M. McGeehan, Scott A. Armstrong. Characterization of acquired resistance mutations to menin inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3457.

Acute myeloid leukemia (AML) represents a set of heterogeneous myeloid malignancies hallmarked by mutations in epigenetic modifiers, transcription factors, and kinases that can cause epigenetic reshaping. It is unclear to what extent AML mutations drive chromatin 3D structure alteration and contribute to myeloid transformation. We first performed Hi-C and whole-genome sequencing in 25 AML patient samples and seven healthy donor samples, and identified recurrent alterations of A/B compartments, TADs, and chromatin loops that are unique to different subtypes. To investigate how altered chromatin organization contributes to transcriptional misregulation, we performed RNA-Seq, ATAC-Seq and CUT&ag for CTCF, H3K27ac, and H3K27me3 in the same AML samples. We identified extensive and recurrent AML-specific promoter-enhancer and promoter-repressor loops. We performed both CRISPR deletion and interference experiments and validated two repressor loops that downregulated cancer related genes IKZF2 and RTTN. Furthermore, by using our recently developed algorithm, we identified structural variation-induced enhancer-hijacking and repressor-hijacking events in AML samples. We further demonstrated the role of hijacked enhancers in AML cell growth by CRISPR screening, and the role of hijacked repressors by CRISPR de-repression. We performed whole-genome bisulfite sequencing in 20 AML and normal samples, and showed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Finally, by treating the AML cells with the DNA hypomethylating agent and performing triple knockdown of DNMT1/3A/3B, we demonstrated the impact of altered DNA methylation on gene expression and 3D genome organization. Overall this study provides an invaluable resource for leukemia studies and also highlighted the role of repressor-loops and hijacked cis-elements in gene regulation and human diseases. Citation Format: Jie Xu, Fan Song, Baozhen Zhang, Huijue Lyu, Mikoto Kobayashi, Ziyu Zhao, Ye Hou, Xiaotao Wang, Yu Luan, Bei Jia, Lena Stasiak, Qixuan Wang, Qi Jin, Qiushi Jin, Yihao Fu, Ross C. Hardison, Sinisa Dovat, Leonidas C. Platanias, Yue Yang, Tomoko Yamada, Aaron D. Viny, Ross L. Levine, David F. Claxton, James R. Broach, Hong Zheng, Feng Yue. Subtype-specific and structure variation induced 3D genome alteration in acute myeloid leukemia [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 2953.

OncoKB, Memorial Sloan Kettering Cancer Center’s (MSK) precision oncology knowledge base (www.oncokb.org), is a comprehensive database that annotates the oncogenic effects and clinical actionability of somatic alterations in cancer. OncoKB supports variant interpretation by the cBioPortal for Cancer Genomics and is used to annotate >12,000 MSK patient sequencing reports annually. Since its introduction in 2016, OncoKB has expanded to include 5685 alterations in 682 genes, and in October 2021, it became the first somatic knowledge base to be partially recognized by the FDA. The scope of the OncoKB FDA recognition includes clinically actionable variants that map to an FDA level of evidence, the processes of variant curation, and policies regarding database oversight, personnel training and transparency of data sources and operations. This recognition credentials OncoKB as providing accurate, reliable and clinically meaningful information to the medical and scientific communities. The OncoKB Therapeutic (Tx) Levels of Evidence categorize variants based on their tumor type-specific predictive value of sensitivity or resistance to matched standard care or investigational targeted therapies. To date, OncoKB includes 43 Level 1 genes (included in the FDA drug label), 23 Level 2 genes (included in professional guidelines), 25 Level 3A genes (predictive of drug response in well-powered clinical studies), 23 Level 4 genes (predictive of drug response based on compelling biological evidence), and 11 R1 or R2 resistance genes. Initially focused on solid tumors, OncoKB was expanded to include hematologic disease annotation in 2019 and introduced Diagnostic (Dx) and Prognostic (Px) levels of evidence. All three level of evidence systems (Tx, Dx and Px) are consistent with the guidelines for evidence-based categorization of somatic variants published as a joint consensus recommendation by AMP/ASCO/CAP. OncoKB is governed by a Clinical Genomics Annotation Committee, composed of MSK physicians and scientists who ensure that the information captured is accurate and current, and an external advisory board composed of leaders in the clinical oncology and genomics communities who oversee OncoKB updates and progress. OncoKB curation rules and processes are transparent and documented in the OncoKB Curation Standard Operating Procedure, which is publicly available via the website. User feedback to OncoKB content is encouraged via the website and through cBioPortal. Queries or suggestions by OncoKB users are addressed by the OncoKB team within 72 hours. OncoKB offers licenses for academic, commercial and hospital use, with which users can programmatically access the web API. Future work includes coverage of additional cancer-associated genes, annotation of germline alterations that are predictive of drug response and/or associated with increased heritable cancer risk and the development of a clinical trial matching system. Citation Format: Sarah P. Suehnholz, Moriah Nissan, Hongxin Zhang, Ritika Kundra, Calvin Lu, Benjamin Xu, Maria E. Arcila, Marc Ladanyi, Michael F. Berger, Ahmet Zehir, Aijaz Syed, Julia E. Rudolph, Ross L. Levine, Ahmet Dogan, Jianjiong Gao, David B. Solit, Nikolaus Schultz, Debyani Chakravarty. OncoKB, MSK’s precision oncology knowledge base [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 1189.

Genomic sequencing of tumors is a routine part of cancer patient care. To address the need for a comprehensive resource that annotates the oncogenic effects and clinical actionability of somatic alterations in cancer, we developed OncoKB, a precision oncology knowledgebase. Since its introduction in 2016, OncoKB has grown to include annotations for >500 alterations in 682 genes. This includes 42 Level 1 genes (included in the FDA drug label), 12 Level 2 genes (included in the NCCN guidelines), and 29 Level 3A genes (predictive of drug response in well-powered clinical studies). We evaluated changes in the clinical actionability landscape and evolution of the OncoKB annotation rules and processes by comparing the AACR Project GENIE cohort (v8.1) annotated with the OncoKB version from February 2018 to that from November 2020. Even within a short window of time, this comparison reveals a significant shift of the proportion of samples that harbor a standard care alteration (Level 1 and 2), increasing from ~9% in 2018 to ~24% in 2020, and a Level 3A alteration, decreasing from ~11% to ~5%. This shift is partially attributable to the FDA approval of a PI3K inhibitor in PIK3CA-mutant ER+/HER2- breast cancer, approval of RAF inhibitors in BRAF V600E mutant anaplastic thyroid cancer and colorectal cancer, approval of NTRK-inhibitors in NTRK fusion-positive solid tumors, FGFR-inhibitor approval in FGFR2 fusion-positive bladder cancer and cholangiocarcinoma, RET-inhibitor approval in RET fusion-positive thyroid cancer and non-small cell lung cancer and expansion of indications for PARP inhibitors to include prostate cancer. The tumor agnostic approval of the checkpoint blockade inhibitor, pembrolizumab, in TMB-H solid tumors additionally gave rise to a ~4% increase in Level 1 samples. Analysis of the AACR Project GENIE cohort also revealed significant changes to the OncoKB process, including those reflected in the updated OncoKB Levels of Evidence v2.0. The refined levels system deprioritized the significance of standard care biomarkers when present in indications outside of the FDA-approved/NCCN listed indication. This change was based on clinical data demonstrating that patients with investigational predictive biomarkers for a specific tumor type based on compelling clinical evidence from phase 3 trials (currently Level 3A) are more likely to experience clinical benefit compared to patients with predictive biomarkers that are considered standard care in a different tumor type (previously Level 2B, currently Level 3B), and is consistent with guidelines published by ASCO/AMP/CAP and ESMO. Knowledgebases such as OncoKB have emerged as key informational resources that the clinical oncology and scientific communities have used to rapidly connect sequencing results to clinical actionability. Their utility depends on their ability to stay abreast of clinical data and seamlessly adapt their rules and processes to the evolving field of precision medicine. Citation Format: Sarah P. Suehnholz, Ritika Kundra, Hongxin Zhang, Shaleigh Smith, Moriah Nissan, Yifu Yao, Lindsay LaFave, Kinisha Gala, Linde Miles, Maria E. Arcila, Marc Ladanyi, Michael F. Berger, Ahmet Zehir, Aijaz Syed, Julia Rudolph, Paul Sabbatini, Ross Levine, Ahmet Dogan, Jianjiong Gao, David Solit, Nikolaus Schultz, Debyani Chakravarty. Evolution of OncoKB, a precision oncology knowledgebase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 99.

Introduction Acute myeloid leukemia (AML) is a disease characterized by the proliferation of undifferentiated myeloid blasts, promoting bone marrow failure, infectious complications, and death. Progress in developing treatments for AML has been slow and clinical outcomes outside of bone marrow transplant are poor. A limitation to progress is the availability of reproducible murine models of AML. As such, there is a critical need to develop new models to quickly screen new therapeutics and treatment combinations. Here we have optimized the engraftment of a spontaneous Idh2R140Q/Flt3ITD murine AML (developed by Shih et al 2017) for rapid in vivo screening of compounds and combination strategies. Methods Spleen derived AML cells from a spontaneous Idh2R140Q/Flt3ITD murine AML were engrafted i.v. into NCG mice. Disease progression was monitored by flow cytometry and LC-MS/MS measurement of plasma D-2-hydroxyglutarate (D2HG), the product of mutant IDH2. At 1 week post-engraftment animals were enrolled onto treatment arms, which included enasidenib (IDH2), gilteritinib (Flt3), a dihydroorotate dehydrogenase (DHODH) inhibitor, and combination arms. In follow-up studies, non-tumor bearing C57Bl/6 mice were treated with various doses of treatment combinations and blood counts, serum liver chemistry, and weight loss were used to evaluate toxicity. Results The time from engraftment to terminal disease can be precisely adjusted to experimental needs, enabling an aggressive 21-day disease course for rapid screening or a more protracted 1.5 month course that limits the initial number of cells required and allows for the testing of slower-acting differentiating agents. We show that enasidenib, gilteritinib, and a DHODH inhibitor all slow disease progression and prolong survival, and that DHODH inhibition may have a greater effect on survival than IDH2 inhibition. We also show that DHODH inhibition lowers plasma D2HG. Importantly, we identify that combining DHODH and IDH2 inhibition is untenable and results in lethal GI toxicity. This was validated in non-tumor bearing mice, where even low doses of DHODH/IDH2 inhibitors in combination resulted in cytopenias and rapid weight loss. Discussion Developing combination treatment regimens is likely to be key to achieving long-term remission for many AML patients; however, efforts to develop these combinations are often stymied by in vitro results that fail to translate to the in vivo setting, particularly unexpected toxicities that are not recognized in vitro. We show that our AML engraftment model can be used to identify and develop pre-clinical compounds and allow us to quickly identify and abandon treatment combinations that are toxic in vivo. Our hope is that this work enables the rapid identification and development of the best novel therapeutics and treatment combinations, such that valuable time and resources can be utilized to their greatest translational effect. Citation Format: Mariah L. Johnson, Ola A. Elgamal, Bonnie Harrington, Allison Mustonen, Amy Lehman, Chad Bennett, Kyle R. Dzwigalski, Kasey L. Hill, Mitch A. Phelps, Jean Truxall, Thomas Goodwin, Alan H. Shih, Ross L. Levine, Erin Hertlein, John C. Byrd. A primary mouse model of AML as a new way to screen novel therapeutics and treatment combinations [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 6118.

Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMT that catalyzes the formation of symmetrical dimethyl arginine (SDMA) on protein substrates and plays important roles in various cellular pathways including tumorigenesis. PRMT5 can methylate histones H3 and H4, thereby leading to repression of tumor suppressor genes at specific loci. PRMT5 also regulates gene expression on a global level by methylating and altering the function of transcription factors such as p53, E2F-1, NF-κB and others. Additionally, PRMT5 has been shown to interact with members of the spliceosome complex and regulate pre-mRNA processing of key target genes. In this study, we describe the in vitro and in vivo activity of PRT543, a novel, potent and selective inhibitor of PRMT5, in hematological and solid tumor models. In a scintillation proximity based radiometric assay, PRT543 inhibited the methyltransferase activity of the PRMT5/MEP50 complex with an IC50 of 10.8 nM. It was also highly selective against a panel of 36 other methyltransferases. PRT543 robustly inhibited cell proliferation in a panel of >50 cancer cell lines, representing both solid tumors and cancers of hematological origin, in a concentration-dependent manner with IC50 values ranging from 10 - 1000 nM. This anti-proliferative activity correlated with a reduction of symmetrically dimethylated SmD3 protein, a known PRMT5 substrate, demonstrating effective inhibition of PRMT5 enzymatic activity in cells. PRT543 demonstrated good oral bioavailability and favorable pharmacokinetic properties. Oral administration of this compound led to significant tumor growth inhibition in several subcutaneous mouse xenograft models including mantle cell lymphoma (Granta-519, Z-138), acute myeloid leukemia (MV4-11, SET2, HEL), small cell lung cancer (NCI-H1048) and bladder cancer (5637) at well-tolerated doses. This in vivo activity was accompanied by a corresponding decrease in symmetrically dimethylated SmD3 in tumor tissue collected from the treated animals. PRT543 was also tested in combination with other approved targeted therapies both in vitro and in vivo. Combining PRT543 with the BCL2 inhibitor, venetoclax, revealed a potent synergistic interaction in models of leukemia and lymphoma. Further ex vivo testing in genetically-defined primary patient tumor samples is ongoing. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Neha Bhagwat, Yang Zhang, Hong Lin, Min Wang, Dave Rominger, Tom Emm, Divya Chugani-Mahtani, Dimitrios Angelis, Rupa Shetty, Raul Leal, William Gowen-MacDonald, Alexander Grego, Juan Luengo, Taghi Manshouri, Friederike Pastore, Ross L. Levine, Srdan Verstovsek, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. Preclinical characterization of PRT543, a potent and selective inhibitor of protein arginine methyltransferase 5 (PRMT5), with broad antitumor activity in in vitro and in vivo models [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 2915.

Recent studies among healthy individuals show evidence of somatic mutations in leukemia-associated genes, referred to as clonal hematopoiesis (CH). To determine the relationship between CH and oncologic therapy we collected sequential blood samples from 525 cancer patients (median sampling interval time = 23 months, range: 6-53 months) of whom 61% received cytotoxic therapy or external beam radiation therapy and 39% received either targeted/immunotherapy or were untreated. Samples were sequenced using deep targeted capture-based platforms. To determine whether CH mutational features were associated with tMN risk, we performed Cox proportional hazards regression on 9,549 cancer patients exposed to oncologic therapy of whom 75 cases developed tMN (median time to transformation=26 months). To further compare the genetic and clonal relationships between tMN and the proceeding CH, we analyzed 35 cases for which paired samples were available. We compared the growth rate of the variant allele fraction (VAF) of CH clones across treatment modalities and in untreated patients. A significant increase in the growth rate of CH mutations was seen in DDR genes among those receiving cytotoxic (p=0.03) or radiation therapy (p=0.02) during the follow-up period compared to patients who did not receive therapy. Similar growth rates among treated and untreated patients were seen for non-DDR CH genes such as DNMT3A. Increasing cumulative exposure to cytotoxic therapy (p=0.01) and external beam radiation therapy (2x10-8) resulted in higher growth rates for DDR CH mutations. Among 34 subjects with at least two CH mutations in which one mutation was in a DDR gene and one in a non-DDR gene, we studied competing clonal dynamics for multiple gene mutations within the same patient. The risk of tMN was positively associated with CH in a known myeloid neoplasm driver mutation (HR=6.9, p Citation Format: Kelly L. Bolton, Ryan N. Ptashkin, Teng Gao, Lior Braunstein, Sean M. Devlin, Minal Patel, Antonin Berthon, Aijazuddin Syed, Mariko Yabe, Catherine Coombs, Nicole M. Caltabellotta, Mike Walsh, Ken Offit, Zsofia Stadler, Choonsik Lee, Paul Pharoah, Konrad H. Stopsack, Barbara Spitzer, Simon Mantha, James Fagin, Laura Boucai, Christopher J. Gibson, Benjamin Ebert, Andrew L. Young, Todd Druley, Koichi Takahashi, Nancy Gillis, Markus Ball, Eric Padron, David Hyman, Jose Baselga, Larry Norton, Stuart Gardos, Virginia Klimek, Howard Scher, Dean Bajorin, Eder Paraiso, Ryma Benayed, Maria Arcilla, Marc Ladanyi, David Solit, Michael Berger, Martin Tallman, Montserrat Garcia-Closas, Nilanjan Chatterjee, Luis Diaz, Ross Levine, Lindsay Morton, Ahmet Zehir, Elli Papaemmanuil. Oncologic therapy shapes the fitness landscape of clonal hematopoiesis [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 5703.

Background: In the last few decades, theoretical models of cancer growth and progression have long focused on the aberrations of cancer cells alone, such as the abnormal mitotic and invasive characteristics of cancer cells. More recent research across multiple solid tumors suggests a critical interplay between solid tumors and immune regulating cells. Mounting evidence suggests that the immune system can tip the scales of cancer progression, eliciting either an anti-tumor or pro-tumor immune response depending upon varying stimulating and inhibitory factors. Here, we are the first to demonstrate novel mutations including leukemogenic mutations among tumor infiltrating lymphocytes in breast cancer patients. Methods: We obtained 17 primary breast cancer samples from patients who presented for either a lumpectomy or mastectomy as part of an IRB approved biospecimen protocol. Of the 17 patient samples, 13 had triple negative breast cancer, 2 had ER+, HER2+ disease, and 2 had ER+, HER2- disease. In the 17 samples, we used fluorescent activated cell sorting to separate CD45-positive hematopoietic cells from CD45-negative epithelial cells. We then performed exome sequencing of tumor-infiltrating hematopoietic cells to investigate for the presence of pathogenic mutations in tumor-associated leukocytes. In this first step, we identified candidate mutations in known cancer genes, including BCOR, NOTCH2, TET2, NF1, EZH2, and JAK1. As a validation step, we then performed capture-based sequencing of tumor-infiltrating leukocytes in 20 breast cancer samples matched to each patient’s germline DNA sample (buccal swab). In 10 of the 20 patients, we identified and validated somatic mutations. Of note, 6 of these patients harbored mutations known to be associated with leukemia, including DNTM3A, TET2, and BCOR. Most of these mutations were present in at least 5-20% of reads. This suggests that these mutations were present in enriched subclones and were not rare alleles occurring in a minority of hematopoietic stem cells. Lastly, we performed 454 deep sequencing analysis of microdissected tumor DNA samples and confirmed the absence of these mutations in breast cancer cells. Conclusion: Our data demonstrate somatic mutations in tumor infiltrating leukocytes in breast tumors which were not identified in matched germline or tumor DNA samples. Notably, some of these mutations have been implicated in the pathogenesis of lymphoid and myeloid malignancies. This observation suggests a unique relationship between cancer cells and mutant infiltrating leukocytes. We are now investigating the functional interaction between cancer cells and hematopoietic cells. Our findings reframe our understanding of carcinogenesis and offer novel opportunities for cancer detection and treatment. Citation Format: Elizabeth A Comen, Maria Kleppe, Hannah Wen, Britta Weigelt, Lennart Bastian, Brian Blum, Franck T Rapaport, Matt Keller, Nicolas Socci, Agnes Viale, Daoqi You, Robert Benezra, Edi Brogi, Jorge Reis-Filho, Michael Berger, Ross Levine, Larry Norton. Somatic leukemogenic mutations associated with infiltrating white blood cells in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-4.

Mutations in ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, are the most common somatic alteration of the SWI/SNF complex across all cancers including estrogen receptor positive (ER)+ breast cancer. We have recently reported that ARID1A inactivating mutations are present at a high frequency in advanced endocrine resistant ER+ breast cancer. However, the mechanisms whereby disruption of ARID1A influences breast cancer progression and/or endocrine therapy resistance are unknown. In order to elucidate mechanisms of resistance to endocrine therapy, we performed an epigenome CRISPR/CAS9 knockout screen that identified ARID1A as the top candidate whose loss determines resistance to the ER degrader, fulvestrant. ARID1A knockout cells were found to be less responsive to endocrine therapy compared to intact ARID1A cells in vitro and in vivo. These observations led us to undertake a comprehensive chromatin-based mechanistic characterization of ARID1A loss in ER+ breast cancer and its role in endocrine therapy response. ARID1A disruption in ER+ breast cancer cells led to widespread changes in chromatin accessibility converging on the master transcription factors (TFs) that regulate gene expression programs critical for luminal (ER+) lineage identity. Global transcriptome profiling of ARID1A knockout cell lines and patient samples harboring ARID1A inactivating mutations revealed an enrichment for basal-like (ER-) gene expression signatures. The state of increased cellular plasticity of luminal cells that acquire a basal-like phenotype upon ARID1A inactivation is enabled by loss of ARID1A-dependent SWI/SNF complex targeting to genomic sites of the major luminal-lineage determining transcription factors including ER, FOXA1, and GATA3. Thus, through widespread chromatin reprograming and functional regulation of mater luminal TFs, tumor cells alter lineage fidelity and become less responsive to luminal-specific anti-ER therapy. We also show that ARID1A regulates genome-wide ER-chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in the determination of breast luminal cell identity and endocrine therapeutic response in breast cancer. Citation Format: Eneda Toska, Guotai Xu, Sagar Chhangawala, Emiliano Cocco, Pedram Razavi, Jordan Otto, Yanyan Cai, Carmen Chan, Drew R. D' Avino, Clayton Collings, Ross L. Levine, Maurizo Scaltriti, Jorge S. Reis-Filho, Cigall Kadoch, Christina Leslie, Jose Baselga. ARID1A is a critical regulator of luminal identity and therapeutic response in estrogen receptor-positive breast cancer [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 949.

Genome profiling represents a critical pillar for clinical, translational, and basic research studies. Hospitals, core facilities, and research enterprises invest significant resources to generate genomic data sets. Yet, data management and analysis is frequently manual, which demands significant operator time and often results in siloed resources rendering them as single-use assets. Centralization of the genomic capital in a framework that enables automated processing, metadata integration and continuous interrogation maximizes return for investment and serves as the critical catalyst for research innovation, clinical translation and reproducible research. We developed Isabl, a plug-and-play infrastructure for scalable bioinformatics operations. Isabl provides solutions for databasing, assets management, tracking, automated and reproducible data processing. Dynamic reporting and meta-analysis across data assets is enabled. Isabl is built on four main components. First, an individual-centric and extensible relational database with tracking support for samples (temporal, spatial, aliquot), experimental data (assays, platforms, sequencing runs), cohorts (clinical trials, research projects) and versioned bioinformatics applications (assembly aware, tools, results). Second, the database is exposed through a fully featured RESTful API that enables horizontal integration with information systems such as sequencing cores LIMS, variant visualization platforms like cBioPortal, and where applicable, clinical and biospecimen institutional databases. Third, a Software Development Kit (SDK) built for Next Generation Sequencing assets management. The SDK enables automated execution of data import and language-agnostic bioinformatics applications (alignment, variant calling, post-processing) with support for cohort and individual level reporting features. Furthermore, the SDK facilitates dynamic retrieval of results using vertical and horizontal queries (individual and cohort level, respectively). Lastly, Isabl comes with a Single Page Web Application that fosters user interaction with multidisciplinary teams (i.e. researchers, project coordinators, engineers, clinicians) facilitating tracking of analyses, results visualization, and dynamic query processing. Isabl is currently supporting the Memorial Sloan Kettering Genome Pediatrics Precision Medicine Initiative, a prototype platform that delivers integrated, real-time automated reporting of clinical targeted gene re-sequencing, research whole genome and transcriptome profiling data; as well as linked data from pre-clinical models (i.e. PDX) and single cells studies. As an open-source tool, Isabl democratizes access to a purpose built, automated, scalable and fully integrable bioinformatics architecture. Isabl will be available at https://github.com/isabl-io. Citation Format: Juan S. Medina-Martínez, Juan E. Arango-Ossa, Gunes Gundem, Max F. Levine, Minal Patel, Noushin R. Farnoud, Venkata D. Yellapantula, Gao Teng, Joseph G. Mccarter, Elsa Bernard, Franck Rapaport, Dominik Glodzik, Ross L. Levine, Andrew Kung, Elli Papaemmanuil. A plug-and-play infrastructure for scalable bioinformatics operations [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 5105.

Background: As the repertoire of molecular targeted therapies for hematologic malignancies continues to expand, so too does the opportunity for molecular profiling to inform treatment decisions. While mutations in certain genes, such as JAK2, MPL, MYD88 and BRAF have diagnostic utility, others such as FLT3, NPM1, IDH1, IDH2, DNMT3A, KIT and CEBPA have prognostic value. Here, we present the development and clinical experience of MSK-IMPACT Heme (Integrated Mutation Profiling of Actionable Cancer Targets for Hematologic malignancies), a comprehensive molecular profiling platform, utilizing hybridization capture and high coverage next generation sequencing of paired tumor and normal tissues. Methods: We designed custom DNA probes corresponding to all exons of 400 key oncogenes and tumor suppressor genes implicated in hematologic malignancies, including all genes that are targetable by approved and experimental therapies being investigated in clinical trials at our institution. The accuracy, precision, and sensitivity of MSK-IMPACT Heme was assessed on a validation set of 113 unique tumor samples with known SNVs and indels previously confirmed by orthogonal methods. We implemented a custom analysis pipeline to integrate the analysis of any number of normal samples with a given tumor and provide a reliable assessment of somatic alterations, even in post-transplant chimeric patients. The selection of matched nail, saliva, and/or blood tissue was determined at the time of test initiation as indicated by patient diagnosis and transplant history. The ability to detect somatic copy number alterations was demonstrated with samples previously characterized by SNP array platforms. Results: We sequenced 821 tumor samples, from 759 patients that represented over 50 tumor types to a mean depth of 758X. 429 patients were male (56.5%) and 20 cases were post allogeneic stem cell transplantation. The most common tumor types sequenced were Follicular lymphoma (11.9%), DLBCL (11.3%), and AML (11.0%). We identified 4,935 mutations from 732 samples. The most commonly altered genes were TP53, KMT2D, and CREBBP. Implementation of the MSK-IMPACT Heme workflow enabled the characterization of complex tumor specimens, including sorted cells and tumor samples from post-transplant chimeric patients. The joint utilization of matched patient and donor normal tissues enabled differentiation between somatic alterations and both host and donor derived common polymorphisms. Conclusions: The MSK-IMPACT Heme assay provides molecular profiling of hematologic malignancies with high accuracy and sensitivity. Paired analysis of tumors and patient and/or donor matched normal tissue samples enables the unambiguous detection of somatic alterations and the ability apply these data towards tumor classification, risk assessment, prognosis, disease monitoring, and treatment optimization. Citation Format: Ryan N. Ptashkin, Ryma Benayed, John Ziegler, Anoop Balakrishnan Rema, Justyna Sadowska, Iwona Kiecka, Caleb Ho, JinJuan Yao, Christine Moung, Kseniya Petrova-Drus, Khedoudja Nafa, Connie Batlevi, Martin Tallman, Ross Levine, Sergio Giralt, Anas Younes, Marc Ladanyi, Mike Berger, Ahmet Zehir, Maria E. Arcila. MSK-IMPACT Heme: Validation and clinical experience of a comprehensive molecular profiling platform for hematologic malignancies [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 3409.

Solid tumor patients often suffer from cytopenias and are at risk for therapy-related myeloid neoplasms (tMN). Somatic mutations in leukemia-associated genes can occur in normal healthy individuals, referred to as clonal hematopoiesis (CH). CH is associated with cytopenias, risk of leukemia and cardiovascular disease. We and others have shown that CH is frequent in cancer patients. Characterization of the relationship between exposure to specific oncologic regimens and CH and how these relate to cytopenias and tMN risk would inform treatment decisions and tMN prevention strategies. To determine the relationship between CH and oncologic therapy we interrogated CH in a cohort of 9045 solid tumor patients. Subjects were sequenced using a targeted panel of cancer-associated mutations used to screen tumor samples against a blood control sample. Mutation detection was performed on blood-derived sequencing data using the matched tumor as a comparator and accounted for background sequencing error rates. CH was identified in 23% of patients. In multivariate regression analyses adjusted by age, CH was more often found in current smokers (OR=1.20, 95%CI=1.07-1.35, p CH is frequent in solid tumor patients and can be reliably detected when a matched tumor normal targeted gene sequencing approach is performed. Beyond age, CH is strongly associated with race, smoking and importantly prior exposure to oncologic therapy with evidence of specific treatment effects. Screening of CH in cancer cohorts is critical to the development of future clinical guidelines and risk-adapted prevention strategies for tMN. Note: This abstract was not presented at the meeting. Citation Format: Kelly Bolton, Ryan Ptashkin, Lior Braunstein, Teng Gao, Sean M. Devlin, Daniel Kelly, Catherine Coombs, Minal Patel, Matahi Moarii, Elsa Bernard, Antonin Berthon, Laura Boucai, Dominik Glodzik, Axel Martin, Zsofia Stadler, Michael Walsh, Diana Mandelker, Akshar Patel, Jessica Schulman, Gunes Gundem, Aijazuddin Syed, Maria Arcila, David B. Solit, Mark E. Robson, Marc Ladanyi, Choonsik Lee, John Philip, Dean Bajorin, Montserrat Garcia-Closas, Stuart Gardos, David Hyman, Martin Tallman, Mariko Yabe, Kenneth Offit, Howard Scher, Virginia Klimek, Luis Diaz, Nilanjan Chatterjee, Michael F. Berger, Lindsay Morton, Ross Levine, Ahmet Zehir, Elli Papaemmanuil. Oncologic therapy for solid tumors alters the risk of clonal hematopoiesis [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-304.

Therapeutic advances for the treatment of acute myeloid leukemia (AML) have been limited in part due to the heterogeneity and complexity of the disease and a poor understanding of its underlying biology. The leukemia stem cell (LSC) arguably resists current therapy resulting in relapses for most initially treatment sensitive patients. AML with myelodysplastic syndrome related changes (AML-MRC) highlights this challenge, representing a very poor outlook subset. The present study sought to understand the underlying sphingolipid biology in and AML, and to evaluate the efficacy of nanoliposomal ceramide (Lip-C6). Sphingolipids play essential roles in cell survival and proliferation, as well as stress and death. Lip-C6, which delivers a short-chain analog of the pro-apoptotic sphingolipid ceramide, has been in development as an anticancer therapeutic. The efficacy of Lip-C6 therapy was evaluated in both in vitro and in vivo models using primary AML cells and AML cell lines. Evaluation and characterization of the effect of treatment with Lip-C6 was done through lipidomic, short term assays such as apoptosis, autophagy and colony formation assays. Efficacy of Lip-C6 and vinblastine was tested in patient derived xenograft models and mouse - human cell line xenograft MV-411. NOD SCID gamma (NSG) mice were injected with luciferase/YFP labeled cells and monitored by bioluminescence imaging (BLI) for the leukemia progression and efficacy. Sphingolipid metabolism was observed to be elevated in patient samples with De Novo AML but not those with AML-MRC. Apoptosis induced by Lip-C6 in CD34+ve/CD38-ve “LSCs” was robust in AML-MRC, but limited in De Novo AMLs. Similarly, AML colonies forming cells were more sensitive to Lip-C6 in AML- MRC than in De Novo cases. It was hypothesized that elevated sphingolipid metabolism and the upregulation of pro-survival pathways such as autophagy contributed to Lip-C6 resistance in De Novo AML. Vinblastine, when combined with Lip-C6, focused sphingolipid metabolism towards pro-apoptotic metabolites and blocked autophagy. In-vivo combination of Lip-C6 and vinblastine uniquely yielded long term control of leukemia progression without systemic toxicity, translating in to prolonged overall leukemia free survival compared to single agents. Altogether, this study shows fundamental biological differences in sphingolipid metabolism between De Novo AML and AML-MRC. The combination of Vinblastine and Lip-C6 targets the LSC and yields apparent cure of lethal human xenograft AML. Citation Format: Charyguly Annageldiyev, Arati Sharma, Brian M. Barth, Todd E. Fox, Tye Deering, Viola Devine, Nicole R. Keasey, Stephan T. Stern, Megan M. Young, Hong-Gang Wan Wang, Jason Liao, Junjia Zhu, Aaron D. Viny, Ross L. Levine, Thomas P. Loughran, Mark Kester, David F. Claxton. Sphingolipid metabolism determines the efficacy of nanoliposomal ceramide in acute myeloid leukemia [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 5832.

The oncogenetic events that transform chronic myeloproliferative neoplasms (MPN) to acute myeloid leukemias (AML) are not well characterized. We investigated the role of several genes implicated in leukemic transformation by mutational analysis of 63 patients with AML secondary to a preexisting MPN (sAML). Frequent mutations were identified in TET2 (26.3%), ASXL1 (19.3%), IDH1 (9.5%), and JAK2 (36.8%) mutations in sAML, and all possible mutational combinations of these genes were also observed. Analysis of 14 patients for which paired samples from MPN and sAML were available showed that TET2 mutations were frequently acquired at leukemic transformation [6 of 14 (43%)]. In contrast, ASXL1 mutations were almost always detected in both the MPN and AML clones from individual patients. One case was also observed where TET2 and ASXL1 mutations were found before the patient acquired a JAK2 mutation or developed clinical evidence of MPN. We conclude that mutations in TET2, ASXL1, and IDH1 are common in sAML derived from a preexisting MPN. Although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2, but not ASXL1, are commonly acquired at the time of leukemic transformation. Our findings argue that the mutational order of events in MPN and sAML varies in different patients, and that TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation. Given the presence of sAML that have no preexisting JAK2/TET2/ASXL1/IDH1 mutations, our work indicates the existence of other mutations yet to be identified that are necessary for leukemic transformation. Cancer Res; 70(2); 447–52

Myelofibrosis (MF) is a common feature of the Philadelphia-Chromosome negative (Ph-) myeloproliferative neoplasms (MPN), a group that includes Primary Myelofibrosis (PMF), Essential Thrombocytosis (ET) and Polycythemia Vera (PV). Current treatment with Jak2 inhibitors reduces splenomegaly, but does not eliminate malignant clones nor improve bone marrow (BM) or spleen fibrosis, underlining the need for alternative therapies. One promising target is TGF-β1, a cytokine crucial for the development of myelofibrosis that is elevated in PMF patient plasma, progenitors and megakaryocytes (MK), and is known to promote fibrosis through stimulation of fibroblasts and their progenitors, mesenchymal stroma cells (MSC). Dysregulation of TGF-β1 signaling has been described in a number of mouse models of myelofibrosis exhibiting aberrant megakaryopoiesis, one of the hallmarks of MPNs. Preliminary data from 93 MPN patients showed overexpression of TGF-ß in PMF samples (p=0.001), with increased activation of the canonical target SMAD2 (p=0.005). We tested the efficacy of the orally available TGF-β1 signaling inhibitor Galunisertib (LY2157299) in counteracting fibrosis in two MF mouse models of common MPN oncogenes Jak2 and Mpn. Mice in the first model express the constitutively active human Jak2V617F mutant under the control of the ubiquitous hematopoietic promoter vav1 and show a PV-like phenotype at 6 weeks old, progressing to fibrosis of bone marrow (BM) and spleen around 25-30 weeks. Thirty week old mice were treated with Galunisertib for 4 weeks and showed a significant decrease in fibrosis compared to control mice. Similar results were observed in 50 week old mice treated with a murine antibody against TGFβRII (IMC-TR1,LY3022859) for 4 weeks.Our second model relies on transplantation of mice with BM cells transduced with MPLW515L, a mutant mpl variant that is active in the absence of TPO. These mice rapidly develop MF, myeloproliferation and splenomegaly. Flow cytometry showed an expansion of immature and mature megakaryocytes with overexpression of TGF-β1 in MPLW515L cells. Treatment from day 12 to day 26 after transplantation reduced both white blood cell counts (a measure of myeloproliferation) and fibrosis as measured by reticulin staining. In vitro, Galunisertib counteracted the pro-fibrotic effects of TGF-ß1 on both murine and human MSC by antagonizing its stimulatory effects on collagen I and III production. Analysis of MSCs from Galunisertib-treated mice also showed reduced collagen I and III production compared to controls. In conclusion, we offer further evidence that aberrant megakaryopoiesis drives TGF-β1 overproduction in MPN by increasing megakaryocyte numbers and overexpressing TGF-β1 in mutant MKs. Galunisertib counteracts TGF-ß1 induced fibrosis in two MPN mouse-models and is a promising candidate for symptomatic treatment of myelofibrosis. Citation Format: Matthias Bartenstein, Lanzhu Yue, Wanke Zhao, Wanting Tina Ho, Cem Murdun, Adam W. Mailloux, Ling Zhang, Anjali Budhakoti, Kith Pradhan, Franck Rapaport, Huaquan Wang, Zonghong Shao, Ulrich Steidl, Ross L. Levine, Zhizhuang Joe Zhao, Amit K. Verma, Pearlie K. Epling-Burnette. TGF-ß signaling inhibition counteracts myelofibrosis in MPN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 954. doi:10.1158/1538-7445.AM2017-954

Background: Recent genomic studies have identified somatic mutations in leukemia genes in asymptomatic individuals without hematologic malignancy. A subset of patients (pts) with clonal hematopoiesis (CH) subsequently develops hematologic malignancies. However, the incidence of CH in pts with solid tumors has not been extensively studied. We sought to assess for CH and its associated clinical impact in pts with solid tumors who were profiled using paired tumor/blood sequencing. Methods: This study included pts who were consented on protocol NCT01775072. All had tumor and blood genomic profiling using the MSK-IMPACT hybridization capture-based next-generation sequencing assay, encompassing all protein-coding exons of 410 cancer-associated genes. In matched blood, we investigated for hotspot mutations from COSMIC database (v71) and non-hotspot mutations in leukemia-associated genes and genes reported in prior CH studies. Mutations were scored as present if variant allele frequency (VAF) in blood was greater than 2% and at least twice the VAF seen in tumor. For cases where at least one mutation exceeded these thresholds, we reduced the VAF threshold in blood to 1% to detect subsequent events. Mutations with VAF >35% in both blood and tumor were excluded as likely germline events. Results: We analyzed 2,146 pts, including 239 pts (11%) with CH. Most commonly identified mutations were in DNMT3A, TET2, and TP53, seen in 167, 52, and 18 pts, respectively. The mean age at time of testing was 62.6 years in pts with CH and 55.4 years in pts without CH (p< 0.001). When comparing baseline blood parameters, there were no statistically significant differences except for higher MCV in CH vs. non-CH pts (91 vs. 90, p = 0.047). 51% of pts with CH had previous radiation therapy compared to 45% of pts without clonal mutations in their blood (p = 0.057). There was no statistically significant difference in the proportion of pts who had received previous chemotherapy (71% of pts in each group). On prospective follow up of pts with CH, no pts have progressed to develop overt hematologic malignancy, with limited follow up (median 13.1 months). Overall survival (OS) was estimated for pts without CH (n = 1907; 1-yr OS: 0.70), CH with 1 mutation (n = 184; 1-yr OS: 0.64), and CH with >1 mutation (n = 55; 1-yr OS: 0.59). The univariate hazard ratios were 1.16 (p = 0.269) and 1.63 (p = 0.015) for 1 mutation and >1 mutation, respectively, when compared to pts without CH. After adjusting for age and sex, a marginal OS association persisted for >1 mutation compared to no CH (HR: 1.46, p = 0.060) Conclusions: CH is common among solid tumor pts without known hematologic malignancy, occurring in 11% of pts, and appears to be associated with decreased OS. Analyses will be replicated in a larger cohort with longer follow up to determine the clinical impact of these mutations in cancer pts. Citation Format: Catherine Coombs, Ahmet Zehir, Sean Devlin, Sumit Middha, Donavan Cheng, Ashwin Kishtagari, David Hyman, David Solit, Mark Robson, Jose Baselga, Maria Arcila, Martin Tallman, Ross Levine, Michael Berger. Clonal hematopoiesis identified by matched-normal blood sequencing of solid tumor patients without hematologic malignancy is common and is associated with decreased overall survival. [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 2640.

Acute myeloid leukemia (AML) is a predominantly fatal hematopoietic malignancy with high inter-patient and intra-patient genetic and epigenetic heterogeneity. The prognosis of relapsed AML remains dismal, yet the epigenetic basis of relapse is still unclear. Here we investigated whether and how the epigenome evolution impacts AML progression with biological and clinical relevance. Methods: We obtained clinical annotation and AML specimens from 138 patients with paired diagnosis and relapsed samples. We used normal bone marrow (NBM) as epigenetic/transcriptomic controls and patients’ matched germline DNA as genetic controls. We then performed DNA methylation sequencing (ERRBS), RNA-seq, and Exome-seq. For one patient with 5 serial time points, we performed whole genome sequencing (WGS), ERRBS, and single cell RNA-seq. We measured the epigenetic allele burden using a compositional entropy-based approach (Methclone) and methylation heterogeneity using epipolymorphism. Results: We found that diagnosis stage epigenetic allele burden (ΔS < -90) was linked to an inferior clinical outcome (p = 0.0064, log-rank test of relapse-free survival). The higher significance in promoter regions implies the functional impact of epigenetic dynamics. Promoter epiallele shift was associated with more differential expression events (p = 3.8 × 10−6, Wilcoxon signed-rank test) and promoter epiallele diversity is significantly associated with single cell resolution transcriptional heterogeneity (p < 2.2 × 10−16, ANOVA test). The global methylation heterogeneity is decreased from diagnosis to relapse, indicating a selective impact of chemotherapy on epigenetic variability (p = 0.0056, paired Wilcoxon test). We investigated epigenetic allele burden progression from diagnosis to relapse by classifying patients into three clusters using K-means clustering: those with 1) decreased, 2) stable, or 3) increased abundance of epiallele burden. No association was seen between epigenetic clusters and patterns of genetic evolution, and the genetic abundance is higher in Cluster 3 than Cluster 1 (p = 0.048, Wilcoxon test), indicating divergent paths of genetic and epigenetic evolution. We next examined differential expression in the epigenetic cluster samples at diagnosis compared to NBM. Cluster 1 specific genes were enriched for cell cycle processes, while Cluster 3 genes were enriched for immune responses (p < 0.001, gene ontology hypergeometric tests). Integrating WGS and ERRBS data showed that epiallele burden is more dynamic than somatic mutations; a significant increase in epiallele burden preceded a major increase of somatic mutational abundance. Summary: Our results indicate that epigenetic dynamics may provide leukemia cells greater evolutionary fitness via transcriptional adaptation and is associated with clinical outcome. This provides an alternative mechanism of AML resilience during progression and a potential predictor of relapse. Citation Format: Sheng Li, Francine E. Garrett-Bakelman, Stephen S. Chung, Todd Hricik, Franck Rapaport, Jay Patel, Richard Dillon, Priyanka Vijay, Anna L. Brown, Alexander E. Perl, Joy Connon, Mathijs A. Sanders, Peter J.M. Valk, Lars Bullinger, Selina Luger, Michael W. Becker, Ian D. Lewis, Luen Bik To, Richard J. D’Andrea, David Grimwade, Ruud Delwel, Bob Löwenberg, Hartmut Döhner, Konstanze Döhner, Monica L. Guzman, Duane C. Hassane, Gail J. Roboz, Martin Carroll, Christopher Y. Park, Donna S. Neuberg, Ross L. Levine, Ari M. Melnick, Christopher E. Mason. Epigenome evolution in relapsed acute myeloid leukemia. [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 LB-073.

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non–small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal–regulated kinase (ERK)-1/2 in human 293T cells and to growth factor–independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma. [Cancer Res 2008;68(14):5524–8]

Background: Accurate characterization of chromosomal aberrations (cytogenetics) in hematologic malignancies is fundamental for proper disease diagnosis, prognosis and may inform treatment decisions. Current karyotyping and fluorescent in situ hybridization (FISH) clinical assays have important sensitivity and sample limitations and cannot detect many novel chromosomal abnormalities in cancer. We have developed a NGS-based targeted DNA and RNA sequencing assay (FoundationOne Heme)and analysis pipeline compatible with typical clinical specimens that can detect all types of variants including large chromosomal abnormalities at base-pair level. High accuracy for the detection of recurrent cytogenetic events was validated across 100 CLL and DLBCL specimens with known abnormalities Method: Adaptor-ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 405 blood cancer-related genes by DNA-seq, and 265 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 498x for DNA and ∼7M on-target unique pairs for RNA, to enable highly accurate detection of base substitutions, indels, copy number alterations and gene rearrangements. Cytogenetics calls were determined using a computational method which compares germline SNP allele frequencies to model expectations, taking into account tumor purity, ploidy, and local copy number. Result: Assay performance demonstrated highly accurate detection of known cytogenetic events, including large deletions, trisomy and translocations, in 61 CLL and 39 DLBCL specimens previously characterized with CLIA-certified karyotyping and FISH assays. In the CLL cohort, 40 of 41 (98%) known positives and 172 of 172 (100%) known negatives were successfully characterized, including deletion of chr11q, chr13q, chr17p and chr12 trisomy. No false positive was reported in any of the cytogenetic variants. In the DLBCL cohort, 35 of 39 (90%) known translocations involving IGH-MYC, IGH-BCL2 and IGH-BCL6 were detected. Accurate detection of these rearrangements is particularly difficult as they are not typical gene fusions and the genomic breakpoints are highly variable, distributed across upstream, genic and downstream of the targeted genes. In addition, deletion in chr17p including TP53 was detected from 37 of 165 DLBCL patients with large length distribution ranging from 500 kb to 79 Mb. Conclusion: Accurate detection of cytogenetic events at higher resolution than traditional cytogenetics and FISH techniques is possible using an optimized NGS-based assay and tailored algorithms. An improved understanding of the molecular details of chromosomal abnormalities, integrated with comprehensive profiling of other cancer relevant genomic alterations, may help the upfront characterization of these alterations and impact diagnosis, prognosis and therapeutic intervention. Citation Format: Jie He, Jeffrey Gardner, James X. Sun, Omar Abdel-Wahab, Andrew M. Intlekofer, Michelle K. Nahas, Jo-Anne Vergilio, Jeffery S. Ross, Roman Yelenskey, Ross Levine, Geoff Otto, Doron Lipson, Vincent Miller, Philip Stephens. Next-generation sequencing enables new approach to molecular cytogenetics. [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 4927. doi:10.1158/1538-7445.AM2015-4927

Despite significant advances in cancer research and treatment, therapeutic resistance remains a major obstacle for achieving stable remission in cancer patients. Acute myeloid leukemia (AML) is no exception, and most AML patients develop resistance to chemotherapy/targeted therapies, which results in disease relapse and progression. Recurrent mutations in the DNA methyltransferase 3A (DNMT3A) gene have been identified in 20-30% of AML cases and are predictive of unfavorable prognosis in patients treated with standard anti-leukemic regimens. In addition, DNMT3A-mutant AMLs appear to be relatively refractory to anthracycline family chemotherapeutics, such as daunorubicin. Half of all DNMT3A mutations affect amino acid residue R882, and recent work has shown that these mutants display decreased enzymatic activity and aberrant binding properties. In addition, previous studies have shown that wild-type DNMT3A functions as a pro-apoptotic switch in response to genotoxic stress induced by another anthracycline doxorubicin. We propose that mutant DNMT3A protects cells from apoptosis in response to DNA damage by altering molecular machinery involved in DNA-damage sensing, response and/or repair, through DNA methylation-dependent or independent mechanisms. Specifically, our data show that mutant DNMT3A affects recruitment of DNA repair proteins to chromatin, including aberrant distribution of homologous recombination marker RAD51. We are currently investigating molecular changes in DNA damage response in DNMT3A-mutant cells in vitro and ex vivo, and leukemogenic potential of the mutant Dnmt3a allele in vivo, whether alone or in combination with other cooperating oncogenes. Citation Format: Olga A. Guryanova, Kaitlyn Shank, Luisa Luciani, Evangelia Loizou, Matthew D. Keller, Abby R. Weinstein, Omar Abdel-Wahab, Siddhartha Mukherjee, Stephen S. Nimer, Ross L. Levine. Leukemia-associated DNMT3A R882 mutations and their role in anthracycline-induced DNA damage response and therapeutic resistance. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 44. doi:10.1158/1538-7445.CANSUSC14-44

Background: Next-generation sequencing (NGS) is rapidly becoming an indispensable cancer diagnostic, as it can detect most genomic alterations in a single assay from limited tissue. We developed a novel, NGS-based assay designed to provide targeted assessment of the genomic landscape of hematologic malignancies from archived FFPE, blood and bone marrow aspirate samples, sequencing both DNA and RNA to improve sensitivity for driver fusion events which are common in these tumors. Methods: The high accuracy of the assay for detection of substitutions, indels and CNAs was previously demonstrated by extensive validation studies achieving 95-99% across all alteration types with high specificity (PPV>99%) [Frampton et al, Nat Biotech, 2013]. To validate assay performance in detecting gene fusions we created reference samples by mixing 21 cell-lines with previously characterized fusions in 39 combinations representing 167 fusion events in 10-50% tumor cell fractions. In addition, we confirmed accuracy in 76 clinical hematologic FFPE and bone-marrow samples profiled for 212 substitutions, indels and fusions in 11 genes by Sanger sequencing, PCR, fragment sizing and FISH. DNA and RNA were extracted from all samples; adaptor ligated sequencing libraries were captured by solution hybridization using custom baits targeting 405 cancer related genes by DNA-seq, and 265 frequently rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine), averaging 467x for DNA and 6M unique pairs for RNA. Results: On reference samples, sensitivity for detection of fusions events reached >99% for tumor cell fractions of 20-50%, and 97% for tumor fraction of 10%, all with high specificity (PPV>95%). Robust performance translated to the clinical samples: we observed a concordance rate of 98.6% relative to prior calls, with only 3/212 differing calls (2+, 1-) by NGS. 129 additional known oncogenic alterations in 57 different genes were detected in these samples, for a total of 3.1 alterations per sample. Analysis of 290 additional leukemia, lymphoma and myeloma patient samples revealed known and novel gene fusions in 15% of cases, more than half of which were identifiable only by RNA-seq. Conclusions: We describe the analytic validation of a sensitive, high throughput assay to detect somatic alterations in hundreds of genes known to be deregulated in hematologic malignancies, which can be used to identify a spectrum of somatic alterations from blood, bone marrow and paraffin embedded patient samples. We demonstrate that targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations, including gene fusions, with high accuracy. This approach offers the opportunity to streamline the characterization of genomic alterations in hematologic malignancies and to expand targeted treatment options for patients with liquid tumors. Citation Format: Doron Lipson, Michelle K. Nahas, Geoff A. Otto, Jie He, Kai Wang, Kristina M. Knapp, Kristina W. Brennan, Amy L. Donahue, Lauren E. Young, Geneva Young, Alex Fichtenholtz, Jeffrey S. Ross, Roman Yelensky, Philip J. Stephens, Vincent A. Miller, Ross Levine. Development and validation of a clinical next generation sequencing-based assay for hematologic malignancies. [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 3570. doi:10.1158/1538-7445.AM2014-3570

Background: Cancer metabolism represents an emerging field of novel cancer target discovery. Somatic point mutations in the metabolic enzymes isocitrate dehydrogenase 1/2 (IDH1/2) confer a novel gain-of-function in cancer cells, which results in the accumulation and secretion of the onco-metabolite, R-2-hydroxyglutarate (2-HG). High levels of 2-HG have been shown to inhibit α᠄KG dependent dioxygenases including histone and DNA demethylases, which regulate the epigenetic state of cells and result in altered cellular differentiation. IDH2 mutations have been identified in a spectrum of solid tumors and hematologic malignancies including chondrosarcoma, glioblastoma, acute myeloid leukemia (AML), and myelodyplastic syndromes (MDS). AG-221 is the first IDH mutant inhibitor in clinical trials; it is an oral, potent, reversible, and selective inhibitor of the mutated IDH2 protein. In a primary human AML xenograft model, AG-221 treatment reduced 2-HG levels and demonstrated a dose dependent survival benefit. Early results of the ongoing first in human phase I study of AG-221 in patients with advanced IDH2 mutant positive hematologic malignancies are reported here. Study Methods: This phase I study of oral AG-221 is designed to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD) including assessment of 2-HG levels, and clinical activity in patients with advanced hematologic malignancies. AG-221 is administered orally 2 times per day (BID) in continuous 28-day cycles. Sequential cohorts of up to 5 PK-evaluable patients are enrolled at higher dose levels, followed by multiple planned expansion cohorts. The eligible patient population includes those with relapsed or refractory AML, myelodysplastic syndromes (MDS,) and elderly untreated AML that harbor an IDH2 mutation. Blood and bone marrow is collected at multiple time points for determination of the PK and PD effects of AG-221. Response assessments via bone marrow examination are performed on Days 15, 29, 57, and every 56 days (2 cycles) thereafter. Study Status and Results: The study was activated in September 2013. As of February 26th 2014, a total of 19 patients have been enrolled; 18 with AML and 1 with MDS. All patients were IDH2-mutant positive by local testing. AG-221 doses administered included 30 mg BID (n=7), 50 mg BID (n=5), 75 mg BID (n=5), and 100 mg QD (n=2). Two patients were added to the 30 mg BID cohort to replace PK-unevaluable patients. Fourteen of 19 patients remain on study drug treatment. Therapy has been well tolerated; with no dose-limiting toxicities reported. The maximum tolerated dose has not been reached. Possible drug-related severe adverse events were reported in two patients: grade 2 hyperleukocytosis and grade 3 confusion. In the first cohort there were three deaths due to sepsis within 30 days of study drug termination. One of these was attributed as possibly related to study drug treatment. Preliminary analysis of PK at 30 and 50 mg doses demonstrated excellent oral AG-221 exposure and a mean plasma half-life of greater than 40 hours. Evaluation of the PD response demonstrated sustained reduction in 2-HG plasma levels of up to 97% following AG-221 dosing in cohort 1 and 2. Ten AML patients are evaluable for efficacy at this time: (N=5 at 30 mg BID, N=5 at 50 mg BID), 5 men and 5 women, with a median age (range) of 62.5 years (53-74). Eight patients had an R140Q mutation and two had a R172K mutation. The median number of prior regimens was 2 (1-4) including one patient who had relapsed after an allogeneic bone marrow transplant. Currently, 6 of 10 patients have had objective responses, including 2 complete remissions defined by the International Working Group Criteria (1 at 30mg BID and 1 at 50mg BID). The four other responses are ongoing and will be updated. Marked differentiation of myeloblasts into mature forms, consistent with preclinical models, was associated with responses. Only one patient experienced disease progression. One patient with a CR was removed from study to undergo allogeneic BMT. Five of the 6 responding patients remain on AG-221. Dose escalation continues along with exploration of a once daily dosing regimen. Expansion cohorts are being planned. Conclusions: AG-221, a novel, oral, potent IDH2 mutant inhibitor is well tolerated and shows promising initial clinical and pharmacodynamic activity in patients with relapsed and refractory IDH2 mutant hematologic malignancies, even in the lowest dose cohort. These data provide early validation of mutant IDH2 as a therapeutic target in hematologic malignancies. Additional safety and efficacy data from the ongoing study will be presented. Clinical Trial Information: NCT01915498 Citation Format: Eytan Stein, Martin Tallman, Daniel A. Pollyea, Ian W. Flinn, Amir T. Fathi, Richard M. Stone, Ross L. Levine, Samuel Agresta, David Schenkein, Hua Yang, Bin Fan, Kate Yen, Stephane De Botton. Clinical safety and activity in a phase I trial of AG-221, a first in class, potent inhibitor of the IDH2-mutant protein, in patients with IDH2 mutant positive advanced hematologic malignancies. [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 CT103. doi:10.1158/1538-7445.AM2014-CT103

Diffuse Large B-cell Lymphomas (DLBCLs) are a heterogeneous group of diseases initiating from germinal center (GC) B cells. GC B cells are uniquely specialized to tolerate rapid proliferation, and physiological genomic instability in order to generate a diverse set of clones of cells encoding high affinity antibodies. This accelerated micro-evolutionary process requires the concerted action of transcription and epigenetic factors that collectively reprogram B cells to overcome many of their normal checkpoint restraints and tolerate the various stresses associated with affinity maturation. The GC phenotype poses a significant risk of malignant transformation to B cells, which must accordingly balance suppression of checkpoints with suppression of pro-oncogenic genes. Epigenetic regulatory complexes play a critical role in lymphomagenesis. EZH2, which epigenetically silences genes through histone 3 lysine 27 methylation is upregulated in normal and malignant GC B cells. EZH2 is often affected by gain of function mutations in lymphomas that alter its enzymatic specificity. EZH2 mediates GC formation by transiently suppressing checkpoint genes and terminal differentiation genes through formation of bivalent chromatin domains. EZH2 somatic mutations induce germinal center hyperplasia and malignant transformation, and cooperate with other oncogenes such as BCL2. EZH2 specific inhibitors can suppress the growth of GC derived lymphoma cells in vitro and in vivo, and are currently being evaluated in early phase clinical trials. DNA methyltransferase 1 (DNMT1) is required for B cells to form GCs, and GC B cells display cytosine methylation redistribution as compared to resting or naïve B cells. DLBCLs in turn exhibit prominent and heterogeneous disruption of cytosine methylation distribution, with specific and distinct DNA methylation profiles occurring in different lymphoma subtypes. Epigenetic heterogeneity is associated with unfavorable outcome in B-cell lymphomas, suggesting that epigenetic diversity may provide a survival advantage to lymphoma cell populations. DNA methyltransferase inhibitors can reprogram lymphoma cells to develop a form of incomplete senescence that sensitizes lymphoma cells to more readily respond to chemotherapy. DNA methyltransferase inhibitors can be safely combined with standard lymphoma therapies for the up-front treatment of patients with high-risk lymphomas. Citation Format: Wendy Beguelin, Matt Teater, Relja Popovic, Yanwen Jiang, Leandro Cerchietti, Rita Shaknovich, Randy Gascoyne, Ross Levine, Omar Abdel-Wahab, Jonathan Licht, Olivier Elemento, Ari M. Melnick. How deregulation of histone methyltransferases drive malignant transformation of B-cells. [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 SY09-03. doi:10.1158/1538-7445.AM2014-SY09-03

Myeloproliferative neoplasms (MPNs) are clonal myeloid malignancies which are initiated by somatic mutations in hematopoietic stem and progenitor cells. Of the classic MPNs, the disease myelofibrosis (MF) is characterized by the shortest median survival as well as progressive anemia, splenomegaly, and constitutional symptoms. Somatic JAK2V617F mutations are observed in approximately 60% of MF patients; however, murine studies have shown that JAK2V617F mutations are not sufficient to induce MF in the absence of additional mutations. Notably, the most common mutations that co-occur with JAK2V617F in MF are in the polycomb gene Additional Sex Combs Like-1 (ASXL1). Importantly, ASXL1 mutations are the strongest predictor of poor overall survival in MF. Given the role of ASXL1 mutations in MF pathogenesis and outcome, we hypothesized that loss of ASXL1 and JAK2V617F would collaborate to modulate epigenetic regulation and facilitate hematopoietic disease transformation. Previous studies have shown that JAK2V617F can translocate to the nucleus and phosphorylate histone H3 on tyrosine 41 (H3Y41), allowing for regulation of specific target genes including JAK2 itself. We used RNA interference to knock down ASXL1 in two JAK2V617F-mutant leukemic cell lines, SET2 and UKE1, which revealed that global and locus-specific H3Y41 phosphorylation levels increased after ASXL1 silencing leading to increased expression of JAK2/H3Y41 target genes. To evaluate whether H3Y41 levels were changed in vivo as well, we crossed a Jak2V617F knock-in mouse to our Asxl1 hematopoietic conditional knockout allele. Heterozygous expression of Jak2V617F and heterozygous deletion of Asxl1 driven by Vav-Cre hastened myeloid expansion and decreased overall survival in primary mice consistent with in vivo cooperativity between Jak2V617F and Asxl1. Multiparameter flow cytometric staining revealed that Vav+ Jak2V617F/+ Asxl1f/+ mice have an expansion of immature erythrocytes in their bone marrow as compared to Vav+ Jak2V617F/+ mice, suggestive of impaired hematopoietic differentiation, which was more severe in Vav+ Jak2V617 VF/+ Asxl1 f/f mice. We then used the Mx1-Cre transgene to induce Jak2V617F and delete Asxl1 in the adult hematopoietic compartment. Mx1-Cre+ Jak2V617F/+ Asxl1f/f mice developed severe, progressive anemia, erythroid precursor expansion in the peripheral blood, bone marrow and spleen, and increased disease burden in primary mice and serially transplanted mice. Importantly, mice expressing Jak2V617F/+ and concomitant Asxl1 loss/haploinsufficency have increased Jak2 protein expression, and increased elevated expression of H3Y41 target genes consistent with coordinate dysregulation of chromatin state by JAK2V617F and ASXL1 loss. These findings suggest ASXL1 loss contributes to myeloid transformation in part through modulation of H3Y41 phosphorylation levels, leading to amplified JAK2 expression, increased JAK-STAT signaling, and disease progression. Citation Format: Lindsay M. LaFave, Young Rock Chung, Matt Keller, Maria Kleppe, Ann Mullally, Omar Abdel-Wahab, Ross L. Levine. Coordinate regulation of chromatin state by JAK2 and ASXL1 mutations in myeloid malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A09.

Background: Acute lymphoblastic leukemia (ALL) is the most common cancer occurring in children. Treatment strategies have been based on risk stratification comprising clinical and biologic features. Despite success in treating ALL, children continue to relapse, and recurrent ALL is difficult to cure with contemporary chemotherapy, suggesting a drug-resistant phenotype and an urgent need of a better understanding of the biology of ALL that will ultimately lead to the development of an alternative therapeutic approach targeting specific molecules associated with the pathogenesis of ALL. JAK3 is preferentially expressed in hematopoietic cells and mediates signals through a common gamma chain shared by receptors for various cytokines. Consistent with JAK3 function in the hematopoietic development, activating mutant alleles of JAK3 have recently been identified in several different types of blood cancer, and functional analyses of a subset of these alleles showed that the mutations can cause lethal hematopoietic malignancies in animal models. Given the central role of JAK3 in the regulation of hematopoiesis, it is likely that additional genetic alterations that activate JAK3 will be identified in other blood malignancies. Objective: We proposed that somatic mutations in JAK3 occur in a subset of pediatric patients with ALL, and that these mutations represent an important therapeutic target which can be leveraged to improve outcomes for ALL patients with JAK3 mutant. Results: A large set of DNA samples extracted from fresh bone marrows of ALL patients for mutations in JAK3 were analyzed. In 91 clinical specimens, a substitution of Val for Ile at amino acid position 722 was identified in 12, and an identical silent mutation was identified in 4. We found a significant increase in the phosphorylation levels of JAK3, Stat5, Erk1/2 and Akt in Ba/F3-JAK3V722I cells, compared with those in Ba/F3-JAK3WT cells. Furthermore, we found the expression of JAK3V722I transforms Ba/F3 cells to factor-independent growth, whereas JAK3WT-transduced cells retain the dependence on IL-3 for proliferation. Importantly, the transforming ability of JAK3V722I in the cells was abolished by treatment with the known JAK3 inhibitor CP-690550. Conclusion and Future Directions: These results suggest that IL-3-independent growth of Ba/F3-JAK3V722I cells resulted from persistent JAK3 activation and JAK3V722I-mediated transformation can be blocked by JAK3 inhibitors. In order to model the effects of JAK3V722I in vivo, we are employing a murine bone marrow transplantation assay to express MIG-JAK3WT and MIG-JAK3V722I constructs. Our preliminary studies reveal engraftment (>10% GFP positive) of both MIG-JAK3WT and MIG-JAK3V722I transduced cells 2 weeks after transplantation. 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 155. doi:1538-7445.AM2012-155

To maintain homeostasis, cells employ intricate molecular machineries comprised of thousands of proteins programmed to execute well-defined functions. Dysregulation of these pathways, through protein mis-expression or mutation, provides biological advantages that confer the malignant phenotype. At the molecular level, this requires cells to invest energy in maintaining the stability and function of these proteins, and for this reason cancer cells co-opt molecular chaperones, including Hsp90. Hsp90 is recognized to play important roles in maintaining the transformed phenotype - the chaperone and its associated co-chaperones assist in the correct folding of cellular proteins, collectively referred to as “client proteins,” many of which are effectors of signal transduction pathways controlling cell growth, differentiation, the DNA damage response, and cell survival. Tumor cell addiction to these proteins (i.e. through mutations, aberrant expression, improper cellular translocation, etc) thus makes them critically reliant on Hsp90. While Hsp90 is expressed in all cells and tissues, it was shown that tumors preferentially contain Hsp90 that is in a higher order multi-chaperone complex with high affinity for certain Hsp90 inhibitors, while normal tissues harbor a latent, uncomplexed Hsp90 that has low affinity for these inhibitors. We here extend this model and propose that Hsp90 forms biochemically distinct complexes in cancer cells. In this view, a major fraction of cancer cell Hsp90 retains “house keeping” chaperone functions similar to normal cells, whereas a functionally distinct Hsp90 pool enriched or expanded in cancer cells specifically interacts with oncogenic proteins required to maintain tumor cell survival. Perhaps this Hsp90 fraction represents a cell stress specific form of chaperone complex that is expanded and constitutively maintained in the tumor cell context. Our data suggest that it may execute functions necessary to maintain the malignant phenotype. One such role is to regulate the folding of mutated (i.e. mB-Raf) or chimeric proteins (i.e. Bcr-Abl). We here also present experimental evidence for an additional role; that is, to facilitate scaffolding and complex formation of molecules involved in aberrantly activated signaling complexes. 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 3029. doi:1538-7445.AM2012-3029