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Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development. Significance: LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.

Many of the current approaches to personalized medicine rely on sequencing DNA to identify actionable mutations. However, growing evidence suggests that a multi-omic approach to more broadly assess biology is needed to improve patient outcomes. We have implemented a workflow for tissue acquisition, multi-omic clinical testing, and correlated computational biology analysis, within the Serial Measurements of Molecular and Architectural Responses to Therapy (SMMART) Program (Mitri et al, J Transl Med 2018). We report biopsy metrics, CLIA analytics utilized, the operation of a multi-omics tumor board, and clinical outcomes in metastatic breast cancer patients. A detailed clinical history of each patient was obtained, including demographics, tumor type, treatment and response to prior therapies, imaging, and blood tumor biomarkers. A comprehensive set of clinical assays was performed on newly obtained tumor biopsies, including immunohistochemistry (ER, PR, HER2, AR, BCL-2, and PD-L1), a targeted next-generation sequencing panel covering 225 genes (GeneTrails® Comprehensive Solid Tumor Panel), whole exome sequencing (Tempus xE), whole transcriptomic sequencing (Illumina TruSeq RNA exome), and a multiplex protein analysis of 22 key cancer proteins and phosphoproteins on the Nanostring platform (NanoString Vantage 3D™ Solid Tumor Panel). The integrated clinical and analytical information was made available to the multidisciplinary SMMART Clinical Tumor Board that provided treatment recommendations; the final treatment plan was at the discretion of the treating physician. Between 1/1/2017-1/1/2020, 53 breast cancer patients were consented. Seven screen-failed due to a lack of sites amenable to biopsy and 8 were actively co-consented to other clinical trials. The remaining 38 patients are included in this preliminary report. A total of 63 biopsies were collected from lymph node, liver, bone, soft tissue, lung, skin, breast, and brain. Serial biopsies (≥2) were obtained for 15 patients. Analytics were generated in 93.7% of biopsies. Tumor boards were held for 15 patients (17 total sessions). The experience and information gathered thus far have yielded the following unique cases: (1) single patient analysis of omics, imaging, and response over 42 months, (2) identification of an ERBB3 mutation with downstream pathway activation that responded to HER2-targeted therapy, and (3) clinically significant variation in hormonal and HER2 receptor status over time. We will provide an analysis across the 38 patients of treatment outcomes, analytics information content, biological changes observed through serial biopsies, and tumor board interventions. We demonstrate the feasibility of implementing a deep, real-time analytics platform for metastatic breast cancer patients that can provide new insight into therapeutic opportunities. The observed clinical responses support further use and investigation of this approach. Citation Format: Ben L. Kong, Brett E. Johnson, Jamie M. Keck, Souraya Mitri, Patrick Leyshock, Jayne M. Stommel, Kiara Siex, Marlana Klinger, Christina L. Zheng, Rochelle Williams-Belizaire, Shannon McWeeney, Jeremy Goecks, Annette Kolodzie, Alexander R. Guimaraes, George V. Thomas, Christopher L. Corless, Zahi I. Mitri, Joe W. Gray, Gordon B. Mills, Raymond C. Bergan. SMMART Program: A multi-omics tumor board with a focus on breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB010.

Chronic myeloid leukemia (CML) is defined by the presence of the BCR-ABL1 fusion protein, which results in constitutively active ABL1 tyrosine kinase activity. Although most chronic phase CML patients can be successfully treated with ABL1 tyrosine kinase inhibitors (TKIs), such as imatinib, up to one third of CML patients require alternative treatment. While the most common causes of TKI resistance in CML are BCR-ABL1 kinase domain mutations, many patients demonstrate BCR-ABL1 kinase-independent resistance through other poorly understood secondary molecular changes that mediate cell survival despite effective BCR-ABL1 kinase inhibition. Previous reports have described additional chromosomal rearrangements in CML patients at the time of disease transformation to blast crisis (Nucifora and Rowley, Blood 1995; Branford et al., Blood 2018), and we hypothesized that secondary fusion proteins may contribute to BCR-ABL1 kinase-independent resistance in CML. To explore this, we performed paired-end RNA sequencing on a cohort of 91 unique patients comprising three groups: BCR-ABL1 kinase-independent resistance (n=42), BCR-ABL1 kinase-dependent resistance (n=26), and newly diagnosed disease (n=23). Fusions were called using the STAR and TopHat methods, and in-frame fusion transcripts called by both methods were analyzed. We identified 11 secondary fusions which were recurrently observed among patients with BCR-ABL1 kinase-independent resistance, including both novel fusions and previously identified fusion proteins such as RUNX1-MECOM and CBFB-MYH11. Fusion breakpoint sequences were amplified via PCR in primary patient specimens at the time of resistance and confirmed by Sanger sequencing for 6 of the identified fusions: RUNX1-MECOM, CBFB-MYH11, KDM7A-MKRN1, TPM4-ACTB, TRDV2-TRAC, and ZNF292-PNRC1. To further evaluate the contribution of these fusion constructs to TKI resistance, we retrovirally co-expressed them with BCR-ABL1 in murine Ba/F3 cells and screened the cells against a panel of approved ABL1 TKIs in vitro using methanethiosulfonate (MTS)-based assays. Intriguingly, we confirmed that expression of KDM7A-MKRN1 was associated with varying degrees of decreased sensitivity to tested ABL1 TKIs, most pronouncedly for imatinib. Evaluation of drug sensitivity for additional fusions is underway and will be presented. Our findings suggest that secondary fusions, some of which are cytogenetically cryptic, beyond BCR-ABL1 are present in a subset of patients with BCR-ABL1 kinase-independent resistance and demonstrate decreased sensitivity to TKI treatment in vitro. Further characterization of the molecular mechanisms associated with these fusions in the context of BCR-ABL1 open opportunities for identifying new combination treatment strategies to overcome this type of resistance and improve outcomes for these patients. Citation Format: Evan J. Barnes, Christopher A. Eide, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, Cristina E. Tognon, Brian J. Druker. Secondary fusions as a mechanism of BCR-ABL1 kinase-independent resistance in chronic myeloid leukemia [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 38.

Background: Inflammation plays a crucial role in normal and malignant hematopoiesis. Patients with acute myeloid leukemia (AML) exhibit aberrant interleukin-1β (IL-1β) signaling which results in AML progression. Previously, we have shown that IL-1β promotes the expansion of leukemic progenitors while suppresses the growth of healthy cells. To understand this paradoxical effect, we applied transcriptome analysis of IL-1β-stimulated AML versus healthy progenitors and revealed differentially expressed genes involved in inflammation, cell cycle, and chromosome organization. We identified that ASF1B (anti-silencing function1B) is one of the highly expressed genes between AML and healthy progenitors upon IL-1β stimulation. ASF1B is a histone chaperone and delivers H3-H4 histone dimers onto DNA during replication and DNA damage response. ASF1B is overexpressed in various solid tumors and associated with poor prognosis. However, the functional roles of ASF1B in hematopoiesis and inflammation-driven leukemia have not been established. Methods and Results: We observed higher ASF1B expression in AML progenitors from various AML genetic subtypes (e.g. FLT3-ITD, NPM1, and MLL-ENL) compared to healthy cells at baseline levels and upon IL-1β stimulation. ASF1B upregulation is abolished upon treatment with a p38MAPK inhibitor, suggesting that ASF1B is downstream of IL-1β/p38 signaling. To determine whether ASF1B mediates IL-1β-driven leukemic growth, we overexpressed ASF1B with MLL-ENL oncogene in a murine bone marrow transplantation model. We found that IL-1β treatment accelerated AML progression compared to the vehicle-treated group (median survival = 64 vs. 85 days, P Conclusion: We demonstrate that the ASF1B pathway is activated in response to microenvironmental cues such as IL-1β in primary AML cells. In vivo evidence using murine AML and genetic models suggests that ASF1B contributes to IL-1β-driven AML progression. Therefore, we provide rationales for using ASF1B as a surrogate marker for AML progression and a safer therapeutic target to inhibit inflammation-driven growth in various AML genetic subtypes. Citation Format: Hsin-Yun Lin, Mona Mohammadhosseini, Marina Villamor, John McClatchy, Sophia Jeng, Andrew Adey, Shannon McWeeney, Travis Stracker, Anupriya Agarwal. Disruption of a histone chaperone pathway delays inflammation-driven AML progression [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 531.

In acute myeloid leukemia (AML), activating mutations in FLT3 are the most common genetic abnormality. Multiple FLT3 inhibitors have been developed, including the FDA-approved inhibitor gilteritinib. However, AML patients only respond to gilteritinib for approximately 6 months due to the emergence of drug resistance. While gilteritinib eliminates blasts in peripheral circulation, residual blasts in the bone marrow microenvironment are protected by cytokines and growth factors. Persistence of these residual cells represents early resistance to treatment. How these cells adapt to survive in the marrow microenvironment remains unclear, but over time resistant subclones resume growth and lead to relapsed disease. At relapse, many patients have intrinsic resistance mutations, what we term late resistance. In this study, we used a stepwise model that charts the temporal evolution of early to late gilteritinib resistance. To recapitulate early resistance, we cultured the FLT3-ITD+ AML cell lines, MOLM-14 and MV4;11, with exogenous microenvironmental ligands that allow cells to become resistant to gilteritinib in a ligand-dependent manner. After 7 weeks, all cultures with ligand resumed growth (early resistance), whereas cells without ligand never resumed growth. Following ligand withdrawal, the cells become transiently sensitive to gilteritinib but resumed growth after 2 months (late resistance). We comprehensively analyzed early and late resistance by integrating whole exome sequencing, CRISPR/Cas9 screening, proteomics, metabolomics, and small-molecule inhibitor screening. Early resistance is characterized by slowly dividing cells and metabolic reprogramming, particularly with respect to lipid metabolism. Early resistant cultures also became uniquely dependent on Aurora kinase B (AURKB) for survival. We then validated these findings in primary AML cells from patients (N=11) treated with gilteritinib and found that early resistant cells demonstrated reduced cell cycle and alterations in lipid metabolism. Gene expression analysis of sequential stromal cell samples from AML patients (N=13) pre- and post gilteritinib treatment showed an increase in lipid metabolism following gilteritinib treatment, indicating that the microenvironment is also dynamic and in crosstalk with neighboring AML cells. Primary early resistant AML cells also became dependent on AURKB signaling, and were exquisitely sensitive to the combination of AURKB inhibitors and gilteritinib. In contrast, late resistance is driven by an expansion of pre-existing NRAS mutant subclones, consistent with the resistance profile of AML patients on gilteritinib. Metabolic reprogramming continued to evolve in late resistance with further dependence upon lipid metabolism. Our study provides mechanistic understanding of how the marrow microenvironment contributes to extrinsic early resistance, which then leads to late intrinsic resistance. We also define a unique vulnerability to AURKB inhibitors in early resistance that may thwart the expansion of late resistant NRAS subclones. Citation Format: Sunil K. Joshi, Tamilla Nechiporuk, Daniel Bottomly, Paul Piehowski, Julie A. Reisz, Janét Pittsenbarger, Andy Kaempf, Sara J.C. Gosline, Yi-Ting Wang, Tao Liu, Cristina E. Tognon, Angelo D’Alessandro, Jeffrey W. Tyner, Shannon K. McWeeney, Karin D. Rodland, Brian J. Druker, Elie Traer. The AML microenvironment catalyzes a step-wise evolution to gilteritinib resistance [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT022.

Background: Acute myeloid leukemia (AML) is a heterogeneous disease with a dismal 5 year survival rate below 30%. Current AML therapies have provided little improvement in achieving complete remission. This is attributed to the development of drug resistance and extrinsic factors from the microenvironment that promote AML progression. Previously, our lab demonstrated that proinflammatory cytokines from the bone marrow microenvironment such as IL-1, promote AML progression. This emphasizes the rationale to design and implement targeted therapies to block extrinsic pathways coupled with intrinsic pathways conferring drug resistance in AML. Methods: To identify factors that promote drug sensitivity and resistance in AML, we used a cohort of 350 AML patients with various genetic subtypes. We quantified levels of 41 growth factors in plasma samples using a multiplex luminex assay. The same cohort of AML samples were analyzed for drug response to 130 small molecule inhibitors in in vitro functional drug sensitivity assay, for RNA-seq gene expression, and whole exome sequencing. The data was integrated to identify differential pathways and markers for drug response. To model drug response in vitro we created a series of drug resistant AML cell lines by culturing these cells in specific drugs long-term. Results: Pathway analysis integrating drug sensitivity, transcriptome, and cytokine expression data identified distinct differentially regulated pathways for various inhibitors. We found that protein synthesis pathways were significantly enriched in sorafenib (FLT-3 inhibitor) resistant samples whereas cytokine and immune pathways (such as TLR and MCP1 signaling) were correlated with trametinib (MEK inhibitor) and venetoclax (Bcl-2 inhibitor) resistance. Specifically, venetoclax resistance correlated with augmented levels of cytokines such as IL-1 and IP-10 and growth factors such as PDGF. Further, trametinib resistance in AML samples is correlated with the upregulation of CCL11, MCP1, and TGFα but a downregulation of CCL22 levels. Accordingly, using AML cell line models, we observed an increase in MCP1 and TGFα at the transcript and secreted protein levels in trametinib resistant cell lines. We demonstrated that MCP1 stimulation activates survival pathways by activating pERK and pJNK signaling, thus reducing apoptosis in AML cells. Targeting MCP1 in combination with trametinib reverses these effects, thus offering a novel therapeutic approach to overcome drug resistance. Conclusion and perspectives: Our data suggest that distinct extrinsic pathways may regulate the response to specific targeted therapy, thus providing a strong basis to design new treatment regimens. Combination treatment incorporating extrinsic pathways would aid in sensitizing AML cells to therapy. Our study offers an integrated approach and a resource to identify such pathways for a large number of tested drugs, and narrows down functionally important pathways associated with drug response. Citation Format: Rucha V. Modak, Alisa Damnernsawad, Ted Laderas, Guanming Wu, Jeffery W. Tyner, Karin D. Rodland, Shannon K. McWeeney, Anupriya Agarwal. An integrated approach reveals novel extrinsic pathways and cytokine signatures associated with drug response in acute myeloid leukemia [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 5942.

Hematologic malignancies represent the fifth most common type of cancer and fourth most common cause of cancer-related deaths. These malignancies are characterized by a clonal expansion of hematopoietic progenitor cells that proliferate in the bone marrow, peripheral blood, lymph nodes and other organs. Genetic analyses of these diseases have led to the identification of specific genetic lesions that drive leukemogenesis, which has subsequently led to the development of targeted drugs. Acute myeloid leukemia (AML) is a fast progressing blood malignancy with impaired differentiation and proliferation of myeloid precursors. It is one of the most common leukemias in adults. AML is known for its molecular and biological heterogeneity, and a variety of genetic lesions have been implicated in the disease. FMS-like tyrosine kinase 3 (FLT3) mutations including FLT3 internal tandem duplication (ITD) or point mutations in the tyrosine kinase domain (TKD) are found in around 30% of AML patients. Sorafenib, a multi-kinase inhibitor that targets FLT3, RAF, VEGFR, FGFR, KIT and RET, is approved for use in hepatocarcinoma, renal cell carcinoma, and thyroid carcinoma treatments. Addition of sorafenib to standard treatment prolonged AML patient survival with or without FMS- like tyrosine kinase 3 (FLT3) mutations, although relapse caused by drug-resistance has limited its usefulness. Understanding the mechanism of resistance to targeted drugs, therefore, is necessary to improve treatment regimens for AML patients. We aim to elucidate resistance mechanisms to sorafenib in AML cells using genome-wide CRISPR screening. Two genome-scale human CRISPR knockout (KO) libraries were used to identify genes whose loss-of-function contribute to lower sensitivity to sorafenib in the MOLM13 AML cells. We verified 10 genes from the top hits showing consistency between the two CRISPR libraries, one of which was Tuberous Sclerosis 1 (TSC1). To validate these findings, TSC1 KO cells were generated using lentiCRISPRv2 system and single sgRNA sequence. Drug sensitivity assays confirmed increase in sorafenib resistance of TSC1 KO cells compared to parental cells or cells harboring non-targeting sgRNA. In addition to the sorafenib resistant phenotype, TSC1 KO cells were resistant to a panel of FLT3 inhibitors, quizartinib, crenolanib, gilteritinib, and UNC2025A. Moreover, RNA sequencing results from 271 AML patient peripheral blood or bone marrow samples revealed that lower RNA expression of Tuberous Sclerosis 1 (TSC1) correlated with lower sensitivity to sorafenib. TSC2 was also observed as a hit gene in both CRISPR libraries and lower TSC2 RNA levels also correlated with lower sensitivity to sorafenib, emphasizing TSC1/2 as an important pathway in sorafenib resistance. Citation Format: Alisa Damnernsawad, Tamilla Nechiporuk, Steve E. Kurtz, Wesley R. Horton, Olga Nikolova, Shannon K. McWeeney, Jeffrey W. Tyner. Genome-wide CRISPR screening identifies TSC1 as a regulator of sorafenib resistance in acute myeloid leukemia [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 4763.

Acute Myeloid Leukemia (AML) remains a highly fatal disease due to the development of drug resistance. Mutational activation of kinases is a frequent event in leukemia, driving malignant cell growth. The most commonly mutated kinase in AML is the FMS-like tyrosine kinase 3 (FLT3) receptor. Internal tandem duplication (ITD) events in the juxtamembrane domain of FLT3 have been reported in ~ 20% of patients and lead to constitutive activation of FLT3. Previous work from our laboratory has shown that fibroblast growth factor 2 (FGF2) secreted by mesenchymal stromal cells protects FLT3-ITD AML cells from quizartinib, a FLT3 inhibitor (Cancer Research, 2016). FGF2 binds the FGFR1 receptor and activates MAPK signaling, enabling leukemia cells to become drug resistant. Over months of treatment, FGF2-protected leukemia cells acquired FLT3 resistance mutations. This two-step mechanism of resistance in vitro mirrors what happens in patients treated with quizartinib. Newer FLT3 inhibitors, such as gilteritinib, have been developed that have activity against both ITD and kinase domain mutations that impart resistance to quizartinib. We used a similar approach and treated the FLT3-ITD+ AML cell line, MOLM-14, with 100 nM of gilteritinib in media alone (n=4), or supplemented with 10 ng/ml FGF2 (n=4) or FLT3 ligand (FL, n=4). After 7 weeks, all cultures supplemented with FGF2 or FL eventually resumed growth. In contrast, no MOLM-14 cells cultured in gilteritinib alone resumed growth even after 16 weeks, suggesting the importance of extrinsic factors in facilitating early drug resistance. Selective pressure was then put on the ligand-dependent cultures by removal of FGF2 and FL. This transiently restored sensitivity to gilteritinib, however within a month the cultures resumed exponential growth. To further explore the mechanism of resistance, we performed immunoblots of kinase signaling pathways on MOLM-14 resistant cultures pre- and post-withdrawal of ligands. We saw robust activation of FLT3 receptor in resistant cultures with FL. FGF2 activated the MAPK pathway, circumventing inhibition by gilteritinib. After removal of extrinsic FGF2 and FL, there was re-activation of MAPK signaling but FLT3 itself remained inhibited. Whole Exome Sequencing of resistant cells identified NRAS mutations as the source of MAPK signaling after ligand withdrawal. Two of the four FGF2-supplemented cultures also developed novel FLT3 point mutations, although there was no clear re-activation of FLT3 signaling in these cultures. Our in vitro results suggest that extrinsic signals from the microenvironment can re-activate MAPK pathways enough to keep cells alive but continued selective pressure eventually leads to direct activation of MAPK through RAS mutations. As such, there is a need to consider rational inhibitor combinations early in disease therapy to prevent the emergence of resistance. Citation Format: Sunil K. Joshi, Stephen Christy, Renata Scopim Ribeiro, Shannon McWeeney, Jeffrey W. Tyner, Cristina E. Tognon, Brian J. Druker, Elie Traer. Extrinsic and intrinsic activation of RAS/MAPK signaling enables resistance to FLT3 inhibitor, gilteritinib, in acute myeloid leukemia [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 926.

Therapeutics targeted to specific proteins known to drive or modulate progression of acute myeloid leukemia (AML) have shown limited effectiveness as resistance frequently develops during their clinical use. Similar to many cancers, a stalled process of programmed cell death in apoptosis-primed cells is one of the prevalent mechanisms of drug resistance in AML. The intrinsic mitochondrial apoptotic pathway is controlled by a rheostat of anti-apoptotic and pro-apoptotic proteins and perturbing the balance toward pro-apoptotic responses remains a focus of many small molecular therapeutics. Venetoclax, an inhibitor of anti-apoptotic gene BCL2, approved for treatment of a certain subtypes of chronic lymphocytic leukemia (CLL), shows limited success as a monoagent in AML clinical trials due to an inherent resistance in approximately 37% of all patients (IC50 > 5μM). To identify mechanisms underlying intrinsic and acquired resistance to the BCL2 inhibitor, venetoclax, we used a genome-wide CRISPR/Cas9 screen to identify genes whose inactivation results in loss of venetoclax sensitivity in MOLM13 cells, derived from an AML patient that harbored mutation in FLT3-ITD, one of the major genetic landscapes of AML. Interrogation with two independently derived genome-wide libraries yielded identical top candidates TP53, BAX and PMAIP1. We also observed other gene targets that implicate the mitochondrial intrinsic, TP53-controlled pro-apoptotic pathway in the acquisition of venetoclax resistance. Correlatively, in a large AML cohort (Beat AML dataset), patients with loss of function mutations in TP53, or either low expression of TP53 or BAX exhibited statistically significant lower levels of responses to venetoclax ex vivo. Resultant venetoclax resistant cells with inactivation of TP53 or BAX had significantly reduced capacity for apoptosis when treated with venetoclax and exhibited elevated pro-survival signaling exemplified by increased levels of MAPK and AKT. In addition, loss of TP53 led to changes in the ratio of anti-apoptotic proteins BCL2, BCL-xL and MCL1 implicating TP53 as a major transcriptional regulator of the apoptotic rheostat in MOLM13 cells. Evaluation of the TP53 and BAX knockout cells for sensitivities to a panel of small molecule inhibitors revealed loss of sensitivities to a wide range of additional inhibitors including FLT3, AKT, multi-kinase inhibitors as well as a surprising gain of sensitivity to NTRK inhibitors relying on alternative pathways of cell death. Our results provide an independent confirmation of TP53 and its apoptotic network involvement to the venetoclax response in AML cells and suggest strategies to overcome resistance. Citation Format: Tamilla Nechiporuk, Stephen E. Kurtz, Olga Nikolova, Amanda d'Almeida, Kevin Watanabe-Smith, Mara Rosenberg, Brian Druker, Jeffrey W. Tyner, Shannon K. McWeeney. A genome-wide CRISPR screen on AML cells reveals the TP53 apoptotic network is a primary mediator of resistance to BCL2 inhibition [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 325.

CG-806 is a pan-FLT3 / pan-BTK inhibitor that is more potent (IC50 = 0.08 µM, n=265, p < 0.001) than other FLT3 inhibitors including midostaurin, sorafenib, sunitinib, dovitinib, quizartinib, crenolanib and gilteritinib against acute myeloid leukemia (AML) primary patient samples containing wild-type or mutated FLT3. CG-806 has significant activity against AML cell lines with or without FLT3 internal tandem duplication (ITD) / tyrosine kinase domain (TKD) mutations and in mouse AML xenograft models. Oral CG-806 has a desirable safety profile in the pre-IND studies of rodent and dog 28-day GLP toxicology, rodent respiratory and central nervous system safety, and the bacterial reverse mutation assay. The current study explored the relationship between genetic abnormalities in bone marrow and peripheral mononuclear cells isolated from AML patients and sensitivity to CG-806 using an ex vivo cytotoxicity assay. To correlate CG-806 sensitivity with clinical status, gene abnormalities and expression levels, whole exome sequencing (n=118) and RNA sequencing (n=111) were performed. CG-806 was equally potent against cells from patients in the adverse, intermediate and favorable risk groups (2017 ELN risk stratification), and cells from patients with relapsed or transformed AML (WHO classification) were as sensitive as those from patients with de novo AML. CG-806 had equivalent potency in cases of TP53 WT and TP53 mutations, whereas cases with TP53 mutations were resistant (FDR-corrected p Citation Format: Hongying Zhang, Andrea Local, Jeffrey W. Tyner, Stephen E. Kurtz, Beth Wilmot, Shannon Mcweeney, Brian J. Druker, Stephen B. Howell, William G. Rice. CG-806, a pan-FLT3 / pan-BTK inhibitor, demonstrates superior potency against cells from IDH-1 mutant and other non-favorable risk groups of AML patients [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 1323.

Anti-apoptotic BCL-2 family proteins contribute to AML pathogenesis and chemotherapy resistance. Venetoclax (VEN) monotherapy has limited activity in relapsed/refractory AML with a 19% overall response rate. PI3K pathway is constitutively active in 50-80% of AML pts and is tightly linked to the apoptotic pathway. Using clinical, genomic and ex vivo drug sensitivity (IC50) data from 802 AML pts from the multi-institutional Beat AML 1.0 collaboration, we tested the hypothesis that aberrant activation of the PI3K/FLT3 pathway is a mechanism of VEN resistance, with potential utility for identifying pt subsets likely to respond to novel drug combinations. IC50 values were determined by ex vivo drug sensitivity assay, and RNAseq analysis was performed on Illumina HiSeq 2500. To identify pts with activated PI3K signaling, we derived a novel 24-gene expression signature from genes enriched among PIK3CA-mutant tumors, and calculated a PI3K signaling score by applying singular value decomposition to the expression value matrix. Drug response biomarkers were evaluated using a student's t-test without adjustments for multiple comparisons. 45.2% (161/356) of AML pt samples were sensitive to VEN (median IC50, 1.64μM), 70% (190/272) to taselisib (tas), a selective PI3Kα and δ but β-sparing inhibitor (median IC50, 0.06μM) and 95.8% (115/120) to VEN-tas combination (median IC50, 0.02μM). Response to PI3K inhibition varied; 23% (150/659) of AML samples were sensitive to PI3Kδ inhibitor idelalisib (idela; median IC50, 7.23μM) and 63% (337/533) to pan-PI3K inhibitor pictilisib (pic; median IC50, 0.45μM; p=0.03 vs tas). 92% (47/51) of VEN-resistant AML samples tested were sensitive to VEN-tas. VEN sensitivity correlated with high BCL-2 expression, and was greatest among pts with both high BCL-2 and low MCL-1 expression or low PI3K signature. Low expression of PIK3CD (but not PIK3CA/B/G) and the PI3K signature were associated with VEN sensitivity. High BCL-2 expression was associated with resistance to pic, idela and FLT3 inhibitor quizartinib (quiz), with a weaker trend for tas that did not reach statistical significance. Activities of tas, pic and quiz were significantly greater among AML samples with FLT3-ITD mutations, a previously reported VEN resistant subgroup. Expression of activated PI3K signature was higher among AML samples sensitive to pic and quiz. Among 30 AML samples refractory to tas, 18 (60%) were sensitive to VEN and 28 (93%) to VEN-tas. In summary, we identified novel AML subsets characterized by either high gene expression of an activated PI3K gene signature that are refractory to BCL-2 inhibitors or by high BCL-2 expression associated with resistance to PI3K/FLT3 inhibitors. 95.8% of AML pt samples were sensitive to combined inhibition of PI3K and BCL-2. Dual inhibition of PI3K and BCL-2 apoptotic pathways may be a promising combination and should be evaluated in clinical trials. Citation Format: Teiko Sumiyoshi, Chris Bolen, Jeff Tyner, Steve Kurtz, Uma Borate, Shannon McWeeney, Beth Wilmot, Dan Bottomly, Erik Segerdell, Monique Dail, Timothy R. Wilson, Wan-Jen Hong, Jeffrey M. Venstrom. Novel and established acute myeloid leukemia (AML) subsets correlate with ex vivo sensitivity to BCL-2 and PI3K inhibition [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 3599.

Background: Medical castration that interferes with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, clinical progression is universal, and tumors with AR-independent resistance mechanisms appear to be increasing in frequency. Consequently, there is an urgent need to develop new treatments targeting molecular pathways enriched in lethal prostate cancer. Lysine specific demethylase 1 (LSD1) is a histone demethylase and important regulator of gene expression. Here we describe a distinct role of LSD1 as a driver of proliferation and survival of castration-resistant prostate cancer (CRPC) cells independently of its demethylase function and of the AR. Methods: We used loss of function studies to determine the importance of LSD1 for survival of prostate cancer cells. To identify transcriptional networks that contribute to cell survival, we suppressed LSD1 with RNAi and performed gene expression profiling. To determine the importance of LSD1's demethylase function in regulation of target genes, we measured LSD1's canonical histone substrates (H3K4me2 and H3K9me2) using genome-wide chromatin immunoprecipitation-sequencing and measured the effect of ectopically expressed wild type or catalytically deficient LSD1 on cell survival and target gene expression. We used mass spectrometry (MS) to identify important LSD1 protein complex members. Finally, we used multiple biochemical and cellular assays and an in vivo study to confirm the mechanism of action of a small molecule inhibitor that targets critical, non-canonical functions of LSD1 in CRPC cells. Results: Cell viability assays demonstrated that LSD1 is important for proliferation and survival of CRPC cells independently of the AR. Gene expression profiles demonstrated that LSD1 activates androgen-independent genes that are enriched in lethal human tumors. Importantly, our global epigenomic studies and biochemical experiments demonstrated that LSD1's demethylase function was dispensable for these effects, while our MS studies identified cooperating LSD1 binding proteins. Finally, we identified a reversible small molecule inhibitor of LSD1 that blocked critical, demethylase-independent functions in vitro and in vivo. Conclusions: Our results demonstrate that LSD1 promotes survival of prostate cancer cells, including those that are castration-resistant, independently of its demethylase function and of the AR. Importantly, this effect is explained in part by activation of a lethal prostate cancer gene network in collaboration with specific binding proteins. Finally, that a reversible small molecule LSD1 inhibitor blocks important demethylase-independent functions and suppresses CRPC cell viability demonstrates the potential of LSD1 inhibition in this disease. Citation Format: Archana Sehrawat, Dae-Hwan Kim, Lina Gao, Yuliang Wang, Armand Bankhead, Shannon McWeeney, Carly J. King, Jacob Schwartzman, Junior Tayou, Joshua Urrutia, William H. Bisson, Daniel J. Coleman, Sunil K. Joshi, David A. Sampson, Sheila Weinmann, Bhaskar VS Kallakury, Deborah L. Berry, Reina Haque, Stephen K. Van Den Eeden, Sunil Sharma, Jared Bearss, Tomasz M. Beer, George V. Thomas, Laura M. Heiser, Joshi J. Alumkal. LSD1 activates a lethal prostate cancer gene network independently of its demethylase function [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 LB-240.

The recently developed concept of cancer stem cells (CSC) sheds new light on various aspects of tumor growth and progression. Here, we present a mathematical model of malignancies to investigate how a hierarchical organized cancer cell population affects the fundamental properties of solid malignancies. We establish that tumors modeled in a CSC context more faithfully resemble human malignancies and show invasive behavior, whereas tumors without a CSC hierarchy do not. These findings are corroborated by in vitro studies. In addition, we provide evidence that the CSC model is accompanied by highly altered evolutionary dynamics compared with the ones predicted to exist in a stochastic, nonhierarchical tumor model. Our main findings indicate that the CSC model allows for significantly higher tumor heterogeneity, which may affect therapy resistance. Moreover, we show that therapy which fails to target the CSC population is not only unsuccessful in curing the patient, but also promotes malignant features in the recurring tumor. These include rapid expansion, increased invasion, and enhanced heterogeneity. Cancer Res; 70(1); 46–56

Background: Androgen deprivation therapy (ADT) or interference with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, progression is universal, and therapies following the emergence of castration resistance do not offer durable control of the disease. Lysine specific demethylase 1 (LSD1) is a histone demethylase and a key regulator of gene expression in cancer. Prior work demonstrates that LSD1 may act as a cofactor of the AR in androgen-dependent prostate cancer cells. In this report, we describe a distinct role of LSD1 as a driver of proliferation and survival of castration-resistant prostate cancer (CRPC) cells independently of the AR and independently of histone demethylation. Methods: We used gain and loss of function studies to determine the importance of LSD1 for survival of prostate cancer cells. To identify transcriptional networks that contribute to cell survival, we suppressed LSD1 with RNAi and measured gene expression changes with microarrays. To determine the importance of histone demethylation in regulation of these gene networks, we suppressed LSD1 and measured levels of LSD1 canonical histone substrates (H3K4me2 and H3K9me2) genome-wide with chromatin immunoprecipitation-sequencing. Results: Cell viability assays demonstrated that LSD1 is important for proliferation and survival of CRPC cells independently of the AR. Microarray studies demonstrated that LSD1 activates androgen-independent genes that comprise cell cycle and embryonic stem cell maintenance gene sets that are enriched in lethal human tumors. Importantly, our global epigenomic studies after LSD1 suppression demonstrated that LSD1 activates these gene sets independently of demethylation of its canonical histone substrates. Conclusions: Our results demonstrate that LSD1 promotes CRPC cell survival independently of the AR and suggest that LSD1 regulates key pathways in CRPC through demethylation of non-histone substrates or via a scaffold function―mechanisms we are currently investigating. In summary, LSD1 contributes to CRPC cell survival through non-canonical mechanisms and represents an attractive therapeutic target in lethal prostate cancer. Citation Format: Archana Sehrawat, Lina Gao, Junior Tayou, Armand Bankhead, Laura M. Heiser, Carly J. King, Yuliang Wang, Jacob Schwartzman, Joshua Urrutia, Daniel J. Coleman, Sheila Weinmann, Bhaskar V. Kallakury, Deborah L. Berry, Reina Haque, Stephen K. Van Den Eeden, Tomasz M. Beer, George V. Thomas, Shannon McWeeney, Joshi J. Alumkal. LSD1 promotes castration-resistant prostate cancer cell survival independently of the androgen receptor and of histone demethylation [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 2406. doi:10.1158/1538-7445.AM2017-2406

Background: HNSCC is the sixth most common cancer worldwide. EGFR is overexpressed in up to 90% of HNSCC and associated with poor outcome. An EGFR monoclonal antibody is the only approved molecular targeted therapy for HNSCC, however, resistance eventually occurs in all patients. Methods: Functional screens including a small-molecule kinase inhibitor panel and an RNAi screen panel were used to identify agents that synergized with EGFR inhibitors in reducing cell viability in HNSCC patient-derived tumor cells. Effective combination therapies were validated in scale-up experiments in 2D and 3D, and their true targets were evaluated using siRNAs to rule out off-target effects of the drugs. Cell viability, cell number and cell colony formation ability were tested upon siRNA treatment. qRT-PCR and immunofluorescent staining was used to test ALK expression in tumor cells before and after anti-EGFR treatment. Results: Two ALK inhibitors on the drug screen panel showed synergistic effects with EGFR inhibitors in 6/8 HNSCC patients’ tumor cells, despite ineffectiveness of single drug. Scale-up dose-response experiments confirmed patient cell sensitivity to 4 different ALK inhibitors in combination with the EGFR inhibitor Gefitinib. siRNA targeting ALK synergized with Gefitinib in reducing cell viability in the patient cells that responded to EGFR/ALK inhibitor combinations, indicating specificity to ALK. Scale-up RNAi experiments confirmed synergy between siRNAs targeting ALK and EGFR in reducing cell viability, cell number, and colony formation ability. Inhibition of EGFR by siRNA or Gefitinib each increased ALK expression at the mRNA level and protein level, suggesting induction of ALK as a novel mechanism of EGFR inhibitor resistance in HNSCC. Conclusion: We identified EGFR/ALK inhibitor combinations as a potential strategy for treating EGFR inhibitor resistant HNSCC. Citation Format: Xiaoming Ouyang, Ashley Barling, Aletha Lesch, Jeffrey Tyner, Sophia Jeng, Christina Zheng, Sara A. Courtneidge, Shannon McWeeney, Molly Kulesz-Martin. Co-targeting epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase overcomes EGFR inhibitor resistance in head and neck squamous cell carcinoma patient-derived models [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 1212. doi:10.1158/1538-7445.AM2017-1212

Mutations in the CSF3R extracellular domain have been reported so far only in neutropenia patients. These extracellular mutations are loss of function mutations, which interrupt ligand binding. Here we screened CSF3R extracellular domain variants in a variety of hematological malignances and described for the first time the identification of an activating CSF3R extracellular mutation W341C in a leukemia patient. Functional assays revealed that this mutation conferred Ba/F3 cell cytokine-independent growth and induced constitutive JAK-STAT activation. We further characterized that cysteine substitutions at other amino acid positions (342, 356 and 477) and other amino acid substitutes of W341 (A/G/K/R/S) did not transform cells, indicating that both the amino acid position and cysteine substitution are essential for the transforming capacity. In agreement, increased dimer formation of W341C was observed in the co-immunoprecipitation assay and non-reducing condition immunoblot, which could be abrogated in an immunoblot run under reducing conditions, highly suggesting that dimerization is mediated by the formation of intermolecular disulfide bonds. Surprisingly, W356C demonstrated loss of function properties, however, showed increased dimer formation similar to W341C, indicating that only the increased dimerization is not sufficient for transforming capacity. Computational modeling based on IL6ST showed opposite directions of W341 and W356, which may orientate the cytoplasmic domain towards or away from one another. Interestingly, a CSF3R cytoplasmic truncation mutation at amino acid W791 was found to be on the same allele as W341C in this patient. The W341C/W791X compound mutation transformed Ba/F3 with faster kinetics comparing to the W341C single mutation. Furthermore, the compound mutation, but not W341C alone demonstrated delayed receptor degradation, indicating enhanced oncogenic potential. Notably, the primary patient sample and the Ba/F3 cells transformed by W341C or the compound mutation were all sensitive to JAK inhibitors. The patient harboring these CSF3R mutations displayed myelodysplastic morphology with BCOR mutation at disease diagnosis, and showed good response to Azacytidine treatment. However her white blood count (mature neutrophils in particular) increased after 15-18 months’ treatment, which was concomitant with the acquisition and expansion of CSF3R W341C and W791X and disease progression. We further investigated the oncogenic potential of disrupting original cysteine pairs in the CSF3R extracellular domain. Increased dimers are observed in these mutations, whereas two functional consequences were observed: loss of function and constitutive activity. This, therefore, represents the first characterization of unpaired cysteines that mediate both loss and gain of function phenotypes. This paradigm may apply to other cytokine receptors. Citation Format: Haijiao Zhang, Sophie Means, Anna R. Schultz, Kevin Watanabe-Smith, Bruno C. Medeiros, Tim Kükenshöner, Oliver Hantschel, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, Jeffrey W. Tyner. Identification of unpaired cysteine-mediated gain and loss of function CSF3R extracellular mutations [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 532. doi:10.1158/1538-7445.AM2017-532

FMS-like tyrosine kinase 3 (FLT3) activating mutations are primary molecular targets for the treatment of acute myeloid leukemia (AML) due to high prevalence and unfavorable prognosis. Several type II FLT3 inhibitors have shown clinical benefits but acquisition of secondary FLT3 mutations was reported as a common mechanism of resistance. Previous studies showed that crenolanib, a type I FLT3 inhibitor, had clinical activity without acquisition of secondary FLT3 mutations. To identify the molecular mechanisms associated with crenolanib sensitivity and resistance, we performed exome sequencing on crenolanib treated patients with FLT3-mutant multiply relapsed or refractory AML. Baseline mutational analysis revealed distinct mutational profiles in patients with prior FLT3 inhibitor exposure, especially in the following pathways: NRAS, IDH1, WT1 and RUNX1. Further analysis of patients who had poor response to crenolanib showed mutations in other cell signaling genes such as NRAS and PTPN11. Variant allele frequency (VAF) analysis showed that these mutations sometimes occurred in subclones independent of the FLT3 mutation-bearing clone or sometimes were acquired by the FLT3 mutation-bearing clone. To characterize the influence of these mutations on crenolanib sensitivity, we transduced genes of interest into cell lines harboring FLT3 activating mutations and treated these cells with crenolanib at various concentrations. Significantly increased crenolanib IC50 and IC90 were observed in NRAS G12V MOLM14 and PTPN11 A72D/FLT3 D835 Ba/F3 cells relative to the respective control NRAS WT MOLM14 and PTPN11 WT/FLT3 D835 Ba/F3 cells. Notably, addition of trametinib restored crenolanib sensitivity and demonstrated synergistic cytotoxic effects on cells with FLT3 and NRAS or PTPN11 mutations. In addition, increases in TET2 nonsense/frameshift mutations were observed in patients who did not respond to crenolanib. VAF analysis demonstrated that TET2 mutations co-occurred with FLT3 mutations. We also observed that bone marrow cells from FLT3-ITD knock-in/TET2 knock-out mice are resistant to crenolanib at low concentrations, but remain sensitive to azacytidine at the same level as FLT3-ITD knock-in/TET2 WT cells. The remaining patients exhibited a diverse spectrum of secondary mutations associated with chromatin modifiers, cohesion, spliceosomes and transcription factors which mostly expanded during treatment, suggesting an elaborate genetic/epigenetic mechanism of resistance to crenolanib. Our data suggest that comprehensive sequencing should be carried out on patient samples prior to treatment to identify and pre-emptively target problematic clones. In addition, even with high VAF FLT3 mutations, although FLT3 inhibitor monotherapy provide some clinical benefit, combining agents targeting cooperative lesions will be imperative to eradicate both the dominant clone and resistant subclones and improve patient responses. Citation Format: Haijiao Zhang, Anna M. Schultz, Samantha Savage, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, Christopher Eide, Hoang Ho, Yee L. Lam, Richard Sweat, Jaime Faulkner, Evan Lind, Jeffrey W. Tyner. Diverse non-FLT3 molecular mechanisms of crenolanib resistance [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 3199. doi:10.1158/1538-7445.AM2017-3199

Purpose: Chronic neutrophilic leukemia (CNL), chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), and unclassified myeloproliferative neoplasms (MPN-U) are a group of heterogeneous disorders belonging to rare entities of myeloproliferative or myelodysplastic/myeloproliferative (MDS/MPN) syndromes. Due to lack of specific molecular markers and the limited understanding of pathogenesis, the treatment of these diseases remains empirical, resulting in poor outcomes. Recently, recurrent mutations in ASXL1, TET2, SRSF2 and cell signaling genes have been identified in these diseases. In this study we aim to analyze the co-occurrence patterns of these gene mutations, as well as the association of different mutations with disease subtypes and treatment outcomes. Methods and results: We performed whole exome sequence and RNA-Seq on primary patient samples. Consistent with previous studies, high frequencies of ASXL1, SRSF2, TET2, SETBP1 and signaling pathway mutations were observed; whereas mutations of MPL, CEBPa, IDH1/2, and TP53 were rare. Further variant allelic frequency analysis demonstrated that mutations of the chromatin modifiers, epigenetic markers and splicing factors are mostly present in the major clones indicating early acquisition of these mutations. In addition, ASXL1/2, splicing factor and signaling pathway mutations co-occur in around 45% of all patients with these mutations, indicating that they drive these diseases in a cooperative manner. RNA-seq analysis demonstrated two major gene expression clusters with high levels of either RAS or JAK-STAT signaling alterations respectively, indicating a potential need for distinct therapeutic targeting of specific subgroups of these diseases. In addition, we observed that 17% of patients have more than one signaling pathway mutation, possibly in distinct subclones, providing rationale for drug combination treatment. Furthermore, we observed that mutations related to RAS signaling pathway are prevalent in CMML; CSF3R and JAK2 mutations are enriched in CNL; whereas, RAS and JAK-STAT pathway double mutations are more frequent in aCML. Similar incidence of ASXL1, splicing factor and epidemics mutations are observed across all these diseases. Conclusions: CNL/aCML/MPN-U/CMML is a group of heterogeneous diseases associated with chromatin modifier, epigenetic, splicing factor and signaling pathway mutations in concomitant manner. We propose future studies of rational drug combinations with agents, targeting epigenetic and splicing factors, together with the appropriate signaling pathway inhibitors. Our study represents a large-scale comprehensive genomic analysis of these diseases, which reveals novel insight into patterns of mutation co-occurrence which could translate into new treatment paradigms for these difficult to treat hematologic malignancies. Citation Format: Haijiao Zhang, Beth Wilmot, Daniel Bottomly, Libbey White, Erik Segerdell, Shannon K. McWeeney, Vishesh Khanna, Angela Rofelty, Sophie Means, Brian Junio, Samantha Savage, Emily Stevens, Kim-Hien Dao, Julia E. Maxson, Jeffrey W. Tyner. Detailed genomic characterization of CNL/aCML/MPN-U/CMML reveals disease subgroups that may benefit from rationally-designed combination therapies [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 2452. doi:10.1158/1538-7445.AM2017-2452

Large scale sequencing efforts are currently being applied to numerous cancers and although significant numbers of mutations are being identified, functional validation to identify driver mutations is lagging. To accelerate progress in the identification of genetic drivers in hematological malignances, we are taking an integrated approach of deep sequencing coupled with functional screens using molecularly targeted drugs and siRNA screening of primary patient leukemia samples. The functional screens allow prioritization of mutations for validation. Using this approach, we identified a CSF3R mutation in a patient with chronic neutrophilic leukemia (CNL). Patients with CNL and the related disorder, atypical chronic myeloid leukemia (aCML), have neoplastic expansion of granulocytic cells with the diagnosis including exclusion of genetic drivers known to occur in other types of World Health Organization-defined myeloproliferative neoplasms (MPN) or myelodysplastic syndrome/MPN overlap disorders. After identifying a CSF3R mutation in our index case, we examined 21 additional cases of CNL and aCML and found that 59% of patients with CNL/aCML harbor CSF3R mutations. Two distinct regions of mutations within the CSF3R gene were identified. Expression of these classes of mutations in a growth factor-dependent myeloid cell line, BaF3, transformed these cells to growth factor independence. Interestingly, these two mutational classes produce preferential downstream kinase signaling (via TNK2/SRC or JAK kinases) and differential sensitivity to kinase inhibitors. Specifically, mutants signaling through TNK2/SRC are sensitive to SRC inhibitors such as dasatinib while mutants signaling thought JAK kinases were sensitive to JAK inhibitors such as ruxolitinib. One patient with CNL carrying a CSF3R mutation that signals through JAK kinases showed a dramatic and durable clinical improvement after treatment with the JAK1/2 kinase inhibitor, ruxolitinib. Mutations in the CSF3R define a large subset of patients with CNL/aCML and testing for these mutations will complement histopathologic diagnosis of CNL and aCML. Patients with CSF3R mutations may benefit from clinical administration of TNK2/SRC inhibitors (such as dasatinib) or JAK inhibitors (such as ruxolitinib). Our studies demonstrate that combining functional screens with genome sequencing can significantly accelerate target validation to define driving mutations that can be matched with therapies. Citation Format: Julia Maxson, Jason Gotlib, Daniel Pollyea, Angela Fleischman, Christopher Eide, Daniel Bottomly, Beth Wilmot, Shannon McWeeney, Cristina Tognon, J. Blake Pond, Robert Collins, Michael Deininger, Bill Chang, Marc Loriaux, Brian Druker, Jeffrey Tyner. Rapid identification of targetable CSF3R mutations that define neutrophilic leukemia by combining functional and genomic screens. [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 2282. doi:10.1158/1538-7445.AM2013-2282

Prostate cancer is the most common cancer in men in the United States and the second leading cause of cancer death. The androgen receptor (AR) is the central signaling pathway in prostate cancer. Because of this, androgen deprivation therapy (ADT), which involves reducing levels of androgen ligands or interfering with binding of ligands to AR, has been the major therapeutic focus for the past 70 years. However, in many patients there is no benefit from ADT, and all prostate cancers eventually progress. At progression on ADT, the AR is ubiquitously expressed. For this reason, we contend that AR-dependent mechanisms are critical for prostate cancer progression after ADT. Indeed, recent work demonstrates that the AR can function independently of ligands to promote prostate cancer cell survival. However, specific ligand-independent AR target genes that account for this effect were not well-characterized. We show here that c-Myc, which is a key mediator of ligand-independent prostate cancer progression, is a key ligand-independent AR target gene. Using microarray analysis, we found that c-Myc and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates c-Myc transcription in a ligand-independent manner, that AR and c-Myc suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-tumor activity of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer - AR and c-Myc - and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control. Citation Format: Lina Gao, Jacob Schwartzman, Angela Gibbs, Richard Kleinschmidt, Beth Wilmot, Daniel Bottomly, Ilsa Coleman, Peter Nelson, Shannon McWeeney, Joshi J. Alumkal. Androgen receptor promotes ligand-independent prostate cancer progression through c-Myc upregulation. [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 3131. doi:10.1158/1538-7445.AM2013-3131