ALBERT

Description: Recent studies, including studies from our group have shown that somatic mutations in DNA methyltransferase type 3A (DNMT3A) are frequently associated with systemic mastocytosis (SM), myelodysplastic syndrome and AML. Importantly, the presence of this mutation is associated with poor prognosis and overall patient survival. We have shown that approximately 12% of patients with SM possess DNMT3A mutations, which are commonly co-expressed with an activating mutation of KIT (KITD816V in humans and KITD814V in mice). However, it is unclear how DNMT3A mutations contribute to myeloid lineage derived mast cell growth and differentiation either alone or in conjunction with KITD816V. Further, the mechanisms by which loss of DNMT3A impairs various mast cell functions either alone or in cooperation with KITD816V are poorly understood. Utilizing mice conditionally deficient in Dnmt3a in the hematopoietic compartment, we show that loss of Dnmt3a in vivo results in increased numbers of mast cells relative to controls. In vitro, loss of Dnmt3a results in accelerated and enhanced differentiation of mast cells from its bone marrow (BM) precursors. Previous studies have shown that the level of expression of the transcription factor, Microthalamia (MITF) (high MITF expression drives mast cell commitment) and CEBPα (high C/EBPα drives basophil commitment) in Basophil-Mast cell common progenitor (BMPs) drives the fate of these cells towards either basophilic lineage or mast cell lineage. To assess why loss of Dnmt3a results in increased mast cell lineage commitment, we performed RNAseq analysis and found elevated expression of MITF and a profound repression in the expression of C/EBPα in BM precursors. Importantly, restoring the expression of Dnmt3a in Dnmt3a deficient BM cells, completely restored enhanced differentiation, along with the expression of MITF and C/EBPα. To our surprise, no appreciable differences in the expression of Gata-1, Gata-2 or PU.1 were noted in the absence of Dnmt3a relative to controls. These results demonstrate that early loss of C/EBPα in Dnmt3a null cells contributes to enhanced maturation of mast cells from their precursors. We next assessed the growth and survival potential of Dnmt3a mast cells and found a significant increase in SCF-mediated growth of these cells compared to controls. Biochemical analysis revealed greater PI3Kinase activation in Dnmt3a null cells, and these findings were supported by genome wide transcriptome analysis involving Ingenuity pathway analysis (IPA) and Gene set enrichment analysis (GSEA). Importantly, mast cells derived from Dnmt3a null BM lacking the expression of p85α regulatory subunit of PI3Kinase (Dnmt3a-/-:p85α-/-) or pharmacologic inhibition of PI3Kinase completely corrected SCF induced hyper proliferation in these cells to near normal levels. The increase in PI3Kinase activity in Dnmt3a null cells was associated with the loss of PTEN expression. To understand how loss of Dnmt3a and expression of an oncogenic form of KIT cooperate to drive MPN, we generated mice in which the expression of oncogenic KIT was temporally and spatially regulated in a cell type and tissue specific manner using the Mx-Cre system along with the expression of Dnmt3a. Consistent with earlier studies, we observed that KitD814V/+ mutation alone only resulted in the development of lymphoid neoplasms. In contrast, Dnmt3a-/- :KitD814V/+ mice manifested robust signs of MPN development including splenomegaly with increased numbers of neutrophils and monocytes in the peripheral blood associated with a significant reduction in lymphoid cells including B cells. Importantly, we noted a 5-fold increase in the fraction of LSKs and GMPs in the BM of Dnmt3a-/- :KitD814V/+ mice compared to controls and consequently an increase in the presence of mature Gr-1 and Mac-1 positive myeloid cells in the BM, spleen and in the PB of these mice relative to controls. The observed MPN was more aggressive in Dnmt3a-/- :KitD814V/+ mice compared to Dnmt3a+/- :KitD814V/+ mice and of stem cell origin as transplantation of BM cells from these mice recapitulated the development of MPN in recipient mice, similar to that observed in the original animals. The impact of PI3Kinase inhibition on mitigating oncogenic KIT and loss of Dnmt3a induced MPN and overall survival will be discussed. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3407 Poster Board III-295 Background: Diffuse Large B Cell (DLBCL) is the most common histological subtype of lymphoma diagnosed in the United States. Following failure with standard Rituximab- CHOP chemotherapy, salvage therapy followed by autologous stem cell transplant is the standard treatment for those who are transplant eligible. Previous data suggest that early lymphocyte recovery is associated with superior survival after autologous stem cell transplantation (ASCT) in lymphoma. However, there are no consistent data on whether prior rituximab therapy affects lymphocyte recovery early post- transplantation. In this study we present our transplant outcome experience regarding early lymphocyte recovery in patients exposed to prior rituximab therapy. Methods: One hundred fifteen patients undergoing autologous stem cell transplant for relapsed DLBCL at a single institution between January 2000 and December 2008 were included in our analysis after obtaining IRB approval. Descriptive statistics, Wilcoxon signed rank sum test and Kaplan-Meier analysis were performed as required using SPSS software. Results: Median age of patients undergoing ASCT was 50 years (range: 24-69 years). Nine patients who underwent ASCT for transformed DLBCL and 7 patients with T-cell rich B cell lymphoma were also included in the final analysis. Six patients had stage I disease, 29 patients with stage II, 33 patients with stage III and 36 patients were diagnosed with stage IV. Thirteen patients had bulky retroperitoneal adenopathy at the time of original diagnosis. Seventy one (66.3%) patients received one salvage therapy prior to transplant. Thirty six (33.7%) patients received two or more salvage therapies prior to transplant. Mobilization data was available on 89 patients. Of those, 82 patients were mobilized with chemotherapy and 5 patients underwent mobilization with GCSF alone. High dose cytclophosphamide (7200 mg/sq.m), etoposide (2000 mg/sq.m) and BCNU (400 mg/sq.m) [CBV] was the conditioning regimen in 88 (77.2%) patients. Sixty eight (59.6%) patients received pretransplant rituximab therapy. Sixty one (61.6%) patients were in radiographic complete response (CR) following salvage therapy at the time of transplant. Sixty nine (78.4%) patients continued to be in CR at day 100 evaluation. Nineteen (21.6%) patients had persistent disease requiring further therapy. The median survival of all patients was 38.7 months (range: 3 months- 186 months). At the time of the analysis, 69 (60.5%) patients were still alive. Prior rituximab therapy did not affect lymphocyte recovery on day 14 (p=0.95) or day 28 (p=0.27). Lymphocyte recovery on day 14 and day 28 (continuous and categorical variables) had no impact on transplant outcome. Other factors such as age, disease stage at presentation, presence of bulky retroperitoneal nodal disease, number of regimens, mobilization procedure, type of conditioning regimen, pre-transplant radiation therapy, pre-transplant disease status (CR vs. PR in chemo sensitive disease) had no impact on survival. Conclusions: our data demonstrate that prior rituximab therapy has no impact on lymphocyte recovery on day 14 or day 28. In this group of patients, lymphocyte recovery did not impact on autologous stem cell transplant outcome. The survival benefit with early lymphocyte recovery as mentioned in prior reports may be lost with longer follow up. Prior rituximab therapy may mitigate the effect of the number of treatment regimens or disease status (CR vs. PR) on transplant outcome. Disclosures: No relevant conflicts of interest to declare.

Description: Multiple Myeloma (MM) remains an incurable malignancy, despite the advent of several new therapeutic agents, including immunomodulatory drugs (IMiDs, e.g., Lenalidomide (Len)) and proteasome inhibitors (PIs, e.g., Bortezomib (Btz)). Accordingly, there is an urgent need to identify new targetable vulnerabilities for MM patients. We developed an ex vivo 384-well platform that allows one to define drug sensitivities of primary patient CD138+ MM cells in the context of a reconstructed tumor microenvironment (TME), including allogeneic bone marrow stromal cells, extracellular matrix and MM patient serum. Using this platform and activity-based proteomic profiling (ABPP), we identified shared signaling pathways induced by the interactions of MM with stromal cells and integrated these data with screens performed using a bank of protein kinase inhibitors (PKI) and current anti-MM therapeutics. These analyses revealed that the serine/threonine kinases casein kinase-1δ (CK1δ) and CK1ε as high priority targets for MM. Indeed, a highly selective and potent dual inhibitor of CK1δ/CK1ε coined SR-3029 is the most potent PKI versus MM. Further, our studies revealed SR-3029 has potent activity in 138/153 primary patient MM specimens tested thus far, including quad and penta-refractory MM. Analysis of RNAseq data of over 600 Moffitt Cancer Center (MCC) MM patients revealed that patients with high expression of CK1ε had worse survival outcomes while no survival difference was seen with CK1δ expression. Importantly, using the established 5TGM1/Kal-Ridge (C57B6/KaLwRijHsd) syngeneic mouse model of multiple myeloma, we show that tumors derived from 5TGM1 MM cells, which rapidly die following exposure to SR-3029 ex vivo, are also sensitive to CK1δ/CK1ε inhibition in vivo, where SR-3029 treatment reduced tumor burden and significantly improved survival. Similar results were observed using NSG immune compromised animals inoculated with human MM1.S multiple myeloma cells (both flank and tail vein models), where SR-3029 treated animals had reduced tumor burden and extended survival. Analysis of RNAseq on patients' samples (on stroma) treated ex vivo with SR-3029 revealed CK1δ/CK1ε inhibition suppressed multiple metabolic pathways (oxidative phosphorylation, glycolysis, xenobiotic metabolism). Interestingly, analyses of MCC MM patient RNAseq data revealed upregulation of the genes identified in these metabolic pathways as patients progress from pre-treatment to relapse, and that patient MM samples that were resistant to CK1δ/CK1ε inhibition had an upregulation of some of these metabolic genes. Functional studies are being performed to define the mechanism(s) by which CK1δ/CK1ε inhibition disables MM metabolism. Collectively, these findings establish CK1ε and/or CK1δ as attractive targets for anti-myeloma therapy that are required to sustain MM metabolism. Disclosures Dai: M2Gen: Employment. Shain:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Description: Problem: Multiple myeloma (MM) is a treatable yet incurable hematologic cancer that lacks predictive biomarkers. Approach: Here we apply a systems biology approach to determine patient-specific mechanisms, as well as signatures of drug resistance in MM. To achieve this goal, we have combined ex vivo drug sensitivity data from 307 MM fresh primary samples tested with 162 drugs and combinations, with paired molecular data (RNAseq and mutational profiling) from a larger overlapping cohort of 606 MM samples from Moffitt's Multiple Myeloma Working Group (MMWG) repository in collaboration with M2Gen/Oncology Research Information Exchange Network (ORIEN). With the purpose of decoupling biological function from intracellular control mechanisms, we have re-constructed a MM-specific transcriptional regulatory network composed of clusters of co-expressing genes. We demonstrate how this gene cluster network regulates biology, and how different biological functions (e.g. Proteasome, Ribosome, Oxidative Phosphorylation) share common regulatory circuits. We have used gene set enrichment analysis (GSEA) to identify gene clusters with transcriptional profiles, and investigated mutations associated with drug resistance. Results: As a preliminary validation of this approach, we have confirmed established mechanisms of resistance (MOR) to targeted therapies, as well as proposed novel MOR to clinically relevant and experimental drugs in MM, as well as putative synergistic drug combinations. In addition, we have identified a list of low frequency mutations (

Description: Trisomy 8 CD34 cells persist and even expand in patients with bone marrow failure despite a potent specific immune response against them(Sloand EM et al; Blood2005; 106(3):841). We previously demonstrated dramatic increases in c-myc, survivin, and CD1 in trisomy 8 CD34 cells by microarray analysis, realtime PCR, and immunoblot (Sloand et al; Blood2007; 109:2399) and postulated that upregulation of c-myc located on chromosome 8 was responsible for increases in CD1 and survivin, an anti-apoptotic protein. Knock-down of either survivin or c-myc resulted in selective trisomy 8 apoptosis and death. The styryl sulfones (Reddy, M et al Acta Chim Hungarica1984;115:269), are novel small molecule anticancer agents that inhibit cell cycle progression in cancer cells, and phase I studies for solid tumors have demonstrated little toxicity in humans (Donehower, R et al, J. Clin. Oncol. 24. 2006 abstract #13026). Initial studies in our laboratory demonstrated significant decreases in CD1 measured after trisomy 8 cells were co-cultured with increasing concentrations (10–30 nM). In this study, we examined the effect 01910.Na, a styryl sulfone, on trisomy 8 cell growth and survival. Short-term culture of mononuclear cells from four normal bone marrows with increasing concentrations of the drug (10–200nM) showed no adverse effect on hematopoietic colony formation (N=4). MDS bone marrow cells from ten trisomy 8 patients; 4 with RAEB and 6 with RA and two monosomy 7 patients with RAEB were studied. All patients with cytogenetics demonstrating trisomy 8 either had it as the sole karyotypic abnormality or with monosomy 7 in the same clone. Bone marrow was cultured with drug for 2 weeks at four different drug concentrations and fluorescent in situ hybridization performed by three blinded investigators counting 300 cells each. There was a substantial decline in the number and percent of aneuploid cells containing trisomy 8 (p=0.03; N=6) Fig 1. Other cells, including those with monosomy 7 as the sole abnormality and diploid cells were relatively unaffected. Continued treatment of the cells with a 100–250nM concentration for a 14 day period resulted in optimal killing of aneuploid cells when compared to one treatment at the beginning of the two week period. The number of blasts also decreased over the two week period when slides were prepared, stained with H&E, and read blindly by a pathologist; this effect was dose-dependent (N=3; p=0.02; Fig 2). Flow cytometric examination of a fourth patient confirmed these findings, demonstrating increased proportions of mature CD15 positive myeloid cells and decreased number of immature CD33 cells or blasts staining with CD34 concomitant with decreased numbers of trisomy 8 cells (Fig3). ON 01910.Na may prove a targeted therapy for patients with MDS and a trisomy 8 clone. A phase I/II trial is set to commence shortly. Figure Figure

Description: Chronic myelogenous leukemia is characterized by the Philadelphia chromosome, which at the molecular level results from the fusion of the bcr gene on chromosome 22 and the abl gene on chromosome 9. The bcr-abl fusion gene encodes a novel tyrosine kinase with transforming activity. In this study, we have synthesized a multi-unti ribozyme that targets bcr-abl mRNA. In vitro ribozyme cleavage reactions show increased cleavage efficiency of this multi-unit ribozyme compared with single or double ribozymes. The multiunit ribozyme was then transfected into murine myeloblasts transformed with the bcr-abl gene (32D cells). Ribozyme transfection was accomplished either by liposomes or using follic acid-polylysine as a carrier. Multi-unit ribozyme transfection reduced the level of bcr-abl mRNA 3 logs when transfected via folate receptor-mediated uptake into transformed 32D cells. These results suggest that a multi-unit ribozyme could be an effective therapeutic agent for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia.

Description: Multiple myeloma (MM) is an incurable plasma cell malignancy accounting for more than 10,000 deaths in the US each year. Hence the pursuit for novel therapeutic agents remains critically important. Myeloma pathogenesis is associated in part with aberrant cell cycle progression. Inhibition of cyclin dependent kinases CDK4/6 results in cell cycle arrest and sensitization to Bortezomib and other active agents in MM (Huang, Blood 2012). Here, we show that ARK5, a novel member of the human AMPKfamily, is overexpressed in 70% of MM and helps promote proliferation and cell cycle progression via G1/S phase activation in an mTOR dependent manner. We examined the role of ARK5 using loss of function studies by ARK5 siRNA transfection in MM1.S, NCI-H929 cells as well as treatment with ON 123300, a dual CDK4/ARK5 kinase inhibitor. ARK5 siRNA knockdown decreased MM cell viability and cell proliferation via G1/S arrest compared to control siRNA. ARK5 siRNA treatment significantly (~70%) induced apoptosis in MM cells as detected by Annexin V/PI staining. We observed that phosphorylation of Rb, a critical cell cycle protein was significantly reduced in ARK5 depleted cells. Moreover, mTOR pathway inhibition was confirmed by reduction of pS6K in ARK5depleted cells as compared to control siRNA treated cells. ON 123300 decreased viability in MM cell lines and patient cells but was not lethal to normal PBMCs. A single treatment of 50nM drug stratified MM cell lines into 2 groups, 5 resistant (MM.1R, KMS11, U266, RPMI-8226 and ARP1) and 4 sensitive cell lines (〉80% cell kill-MM.1S, EJM, JJN3, NCI-H929). ARK5 protein expression by western blot analysis was much higher in sensitive cell lines. ON 123300 triggered G0/G1 cell cycle arrest and induced apoptosis similar to the effect of ARK5 siRNA (80% vs 70%). ON 123300 treatment also reduced phosphorylation of pRb and pS6K downstream of mTOR pathway. These results confirm that cell inhibitory effects of ON 123300 in MM are mediated in a large part via inhibition of ARK5. Co-culture experiments with BMSCs showed that ON 123300 not only targets MM cells but also overcomes the cytoprotective effects of the MM-host BM microenvironment. 4/5 ARK5 positive primary samples with adverse cytogenetics including 1q amplification and CyclinD1 translocation were sensitive to ON 123300 (〉80% cytotoxicity) at 50nM. Further, IP injection of ON 123300 (100mg/kg) in tumor xenograft models (MM1.S, NCI-H929) showed that ON 123300 is well tolerated and significantly inhibits tumor growth in vivo(p

Description: Introduction Diffuse large B cell lymphoma (DLBCL), the most common lymphoma world-wide, is strikingly heterogeneous. This heterogeneity creates a daunting challenge for conducting well-powered studies connecting molecular features to clinical outcome. Not only is the association of genetic mutations with clinical outcome in DLBCL mostly unknown, the relative importance of other well-described features, such as MYC and BCL2 translocation/expression and cell of origin based subsets (ABC and GCB DLBCL), is difficult to interpret due to conflicting reports. We sought to comprehensively define the spectrum of genetic mutations and their association with clinical outcome in DLBCL. Our calculations indicated that 500 tumor-normal pairs would provide 95% power to define mutations occurring in at least 5% of patients, and that 800 cases would be required to define the clinical correlations with cross-validation. Methods We enrolled 1001 de novo DLBCL patients, with complete IPI and survival data, who were treated uniformly with standard rituximab and anthracycline containing regimens. All tumors were subjected to whole exome and transcriptome sequencing (RNAseq), as well as SNP arrays to confirm genetic alterations. ABC (38%) and GCB DLBCL (36%) subtypes were defined using microarrays and RNAseq in these patients to examine subgroup-based differences in mutations and outcome. MYC and BCL2 expression were quantified separately. Results Gene discovery analysis of somatic mutations and copy number alterations in exome sequencing data from 502 tumor-normal pairs of DLBCL identified 197 recurrently mutated genes, including 155 genes previously identified to be mutated in DLBCLs. In addition, our study uncovered 42 novel driver genes in DLBCL (e.g. BTK, SPEN, CD70). Exome sequencing results were validated by Sanger sequencing of 1120 variants with over 90% concordance. We also identified copy number alterations in these genes, with strong agreement (90%) of amplifications and/or deletions to those detected on Illumina high resolution SNP microarrays. These 197 genes were found to comprise 15 functionally related subnetworks, including those related to histone modification, NFkB, B cell receptor, PI3K and cell cycle (Figure 1). Within each subnetwork, the vast majority of the gene alterations occurred in a mutually exclusive (P

Description: Oncogenic activation of RAS via point mutations occurs in more than 30% of all human cancers, including hematopoietic malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Investigations to understand the critical biochemical and biological mechanisms of RAS function are at the forefront of cancer research. Studies have shown that RAS interacts with a large number of effector proteins by a highly conserved mechanism that involves the switch region of RAS and the RAS-binding domains (RBDs) of its effector proteins. Because these interactions play an essential role in oncogenic RAS function, inhibiting them constitutes an attractive and important therapeutic approach for myeloid neoplasias and other cancers. Rigosertib is a novel styryl benzyl sulfone, which is in a Phase III clinical trial (ONTIME) for MDS. Here, we delineate the way rigosertib interacts with the RBDs of several RAS effector proteins: RAF, the PI3K family of proteins and RalGDS. To identify residues in the B-RAF RBD that interact with rigosertib, we recorded a series of 15N-1H HSQC spectra of 15N-labeled B-RAF RBD with increasing concentration of rigosertib. Strikingly, the chemical shift perturbations caused by addition of rigosertib are localized to the very region of the B-RAF-RBD implicated in RAS binding, namely the beta1 and beta2 strands and alpha3 helix (Fig 1). Additionally, this cluster of residues with largest chemical shift perturbation contains many of the same residues involved in RAS binding, namely Ile156, Lys164, Arg166, Thr167, Val168, Ala184 and Met187. These key residues are conserved within RAF RBDs, suggesting that rigosertib would bind to similar regions of the A- and c-RAF RBDs. Next, we examined the binding of rigosertib and GTP-RAS to wild type and mutant forms of c-RAF RBD that harbor mutations in residues that mediate binding to rigosertib. Our studies show that all mutations that cause dissociation of GTP-RAS binding also inhibit rigosertib binding to these mutant proteins. Taken together, the chemical shift data and mutagenesis data provide powerful evidence that rigosertib binds the B-RAF RBD at the same location as the RAS switch I region. A consequence of inhibiting RAS binding to RAF appears to be a block in growth factor-induced activation of RAF kinase activity. We also show that a result of this block in RAS/RAF interactions is an inability of RAF proteins to form dimers and activate MEK and ERK. This block in the activation of MEK/ERK pathways can be seen in cells that express wild-type RAS and RAF proteins (HeLa), in cells that express a constitutively active form of oncogenic RAS (HeLa-N-RAS-G12D), and in cells that exhibit amplification of EGF receptors (A431). Rigosertib also inhibits the phosphorylation of c-RAF serine 338, which has been shown to be essential for the activation of its kinase activity and for its association with and activation of PLK-1. Our results showing rigosertib-mediated inhibition of the PLK-1/RAF interaction might help explain the ability of this compound to induce mitotic arrest of human tumor cells and the ability of rigosertib to reduce blast counts in MDS patients (Seetharam et al, Leuk Res 2012). We have also demonstrated the binding of rigosertib to the RBDs of the PI3K family of kinases and RalGDS, both of which constitute important effectors of RAS. A consequence of the interaction of rigosertib with the RBD domains of PI3Ks appears to be a block in growth factor-induced AKT activation. These studies suggest that the disruption of multiple RAS-driven signaling pathways by rigosertib is mediated via rigosertib’s binding to RBDs of RAS effector proteins, leading to their inactivation. Figure 1 Figure 1. Disclosures Reddy: Onconova Therapeutics Inc: Research Funding. Divakar:Onconova Therapeutics Inc: Research Funding. Vasquez-Del Carpio:Onconova Therapeutics Inc: Research Funding. Dutta:Onconova Therapeutics Inc: Research Funding. Baker:Onconova Therapeautics Inc: Consultancy. Reddy:Onconova Therapeutics Inc: Consultancy. Aggarwal:Onconova Therapeutics Inc: Research Funding.

Description: Follicular Lymphoma (FL) is the most common indolent lymphoma derived from light zone germinal center B cells and characterized by a t(14;18) translocation resulting in upregulation of BCL2 in over 80% of cases. This translocation alone is not sufficient for tumorogenesis, and must be combined with additional genetic mutations to transform B cells. FL is incurable and the disease course can be highly varied, with survival ranging from a few months to decades following diagnosis and treatment with standard chemoimmunotherapy. The heterogeneity of FL poses major challenges to identifying the association of genetic alterations and clinical outcome. Current WHO guidelines recommend establishing grade for each FL case with grade 3 thought to be more aggressive than 1 and 2. The genetic basis and clinical implications of grade in FL are unclear. Recent sequencing studies have identified many genes found to be recurrently mutated in FL including KMT2D and CREBBP. However, the degree to which genetic alterations cooperate with each other or contribute to clinical outcome is unclear. Based on the observed mutational rates in follicular lymphoma, we estimated 900 cases were needed to comprehensively delineate the genetic alterations that underlie histologic grade and clinical outcome. Accordingly, we enrolled a cohort of 1042 patients with newly diagnosed FL. All treated patients received rituximab-containing standard regimens. To go beyond the identification of gene-coding events, we developed a very large panel of 110 Mbp covering exonic (~40Mbp) and non-exonic regions (~70Mbp) of interest to enable a wide range of genomic analysis including mutation calling in both coding and non-coding regions, rearrangement detection, viral identification, and copy number analysis. In addition to the whole exome, we extended coverage to include introns, promoters, and untranslated regions of all known driver genes in cancer. We included the entirety of the immunoglobulin loci, T-cell receptor loci and CD3 loci to detect clonotypes and rearrangements. We also included lymphoma-relevant long non-coding RNAs, microRNAs, enhancers, and breakpoint-prone regions. For viral detection, we targeted the genomes of eight cancer-related viruses: Epstein-Barr virus, human papillomavirus, human immunodeficiency virus, hepatitis B, hepatitis C, Kaposi's sarcoma-associated herpesvirus, human T-lymphotropic virus, and Merkel cell polyomavirus. In addition, to enable high resolution identification of copy number variation (CNV) calls, the entire genome was tiled with probes spaced 10kb apart. DNA and RNA were extracted from all tumors and their paired normal samples, prepared into DNA and RNA sequencing libraries and subjected to sequencing on the Illumina platform to a targeted coverage of 150X. Somatic events were identified and further filtered to identify driver events in both coding and non-coding regions. FLs demonstrated a significant degree of genetic heterogeneity with over 100 genes mutated with a frequency of at least 2%. Nearly 100% of FL cases had a mutation in at least one chromatin-modifying gene. The most frequently mutated genes in follicular lymphoma were KMT2D, BCL2, IGLL5 and CREBBP. In addition, we identified frequent mutations in SPEN, BIRC6 and SETD2. To our knowledge, this is the first description of alterations in these genes in FL. Transcriptome analysis indicated a strong correlation between BIRC6 mutations and the previously described immune response 2 signature that is associated with a poor prognosis. We further performed unbiased clustering of genetic alterations in these FL cases. We identified a cluster that was specifically enriched in BCL6 and TP53 alterations and was strongly associated with grade 3 FLs which are predicted to have poorer outcomes with low intensity therapies. We further examined the genetic profiles of 1001 DLBCLs in comparison to this cohort of FLs. Our data indicate a continuum of highly overlapping genetic alterations with DLBCL displaying more complex patterns that included alterations in MYC, TP53 and CDKN2A (mainly copy number losses), indicating shared pathogenetic mechanisms underlying FL and DLBCL, particularly those germinal center B cell origin. Disclosures Koff: Burroughs Wellcome Fund: Research Funding; V Foundation: Research Funding; Lymphoma Research Foundation: Research Funding; American Association for Cancer Research: Research Funding. Leppä:Roche: Honoraria, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Gang:ROCHE: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Hsi:Abbvie: Research Funding; Eli Lilly: Research Funding; Cleveland Clinic&Abbvie Biotherapeutics Inc: Patents & Royalties: US8,603,477 B2; Jazz: Consultancy. Flowers:AbbVie: Consultancy, Research Funding; Denovo Biopharma: Consultancy; BeiGene: Consultancy, Research Funding; Burroughs Wellcome Fund: Research Funding; Eastern Cooperative Oncology Group: Research Funding; National Cancer Institute: Research Funding; V Foundation: Research Funding; Optimum Rx: Consultancy; Millenium/Takeda: Research Funding; TG Therapeutics: Research Funding; Gilead: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Karyopharm: Consultancy; AstraZeneca: Consultancy; Pharmacyclics/Janssen: Consultancy, Research Funding; Spectrum: Consultancy; Bayer: Consultancy; Acerta: Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding. Neff:Enzyvant: Consultancy; EUSA Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fedoriw:Alexion Pharmaceuticals: Other: Consultant and Speaker. Reddy:Genentech: Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; KITE Pharma: Consultancy; Abbvie: Consultancy. Mason:Sysmex: Honoraria. Behdad:Loxo-Bayer: Membership on an entity's Board of Directors or advisory committees; Thermo Fisher: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: Speaker. Burton:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dave:Data Driven Bioscience: Equity Ownership.