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Introduction. Approximately 40% of patients (pts) with newly diagnosed AML either fail to achieve complete remission with intensive induction therapy or experience disease recurrence after a short remission (CR1 6 months), the probability of response for PIF/ER pts is particularly poor (~12%) with median expected overall survival of ~3.5 month and no approved therapy for this specific population. We have recently shown that increased immune infiltration of the tumor microenvironment (TME) is associated with induction failure and poor prognosis; conversely, an infiltrated TME predisposes for immunotherapy response1. We provide an update of the first-in-human study of flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule currently in clinical development for PIF/ER AML pts. Methods. In this phase of the study, PIF is defined as being refractory to induction with: ≥1 high-intensity cytarabine-based chemotherapy (CTx) cycles, or ≥2 but ≤4 Bcl-2 inhibitor-based combinations, or gemtuzumab ozogamicin only. ER is defined as relapse following CR1 < 6 months. Pts who receive up to one prior salvage attempt are included. Pts whose AML recurred following HSCT are excluded. The recommended Phase 2 dose (RP2D) of FLZ is 500 ng/kg/day administered as a continuous infusion in 28-day cycles following a step-up ('priming') lead-in dose during Cycle 1 Week 1. Disease status is assessed by modified IWG criteria. Duration of response is measured from initial response to relapse or death. Results. As of July 1, 2020, 38 PIF/ER (as defined above) AML patients have been treated at the RP2D (median age 63yrs [range 28-81]; 31.6% [12] pts female). Most pts (63.2%, 24/38) were PIF and the large majority (94.7%, 36/38) had non-favorable risk by ELN 2017 criteria (25 pts adverse, 11 pts intermediate); 34.2% (13/38) had secondary AML. For ER pts, median duration of CR1 was 2.9 months (range: 0.7-4.0 months). Cytokine release syndrome (CRS) was the most frequently reported treatment related adverse event (TRAE), with all pts experiencing mild-to-moderate (grade ≤ 2) CRS. No grade ≥ 3 CRS events have been reported in this cohort. Most CRS events (51.5%) occurred in the first week of treatment during step-up dosing. The incidence of CRS progressively decreased during dosing at RP2D (34.8% in week 2, 4.5% in week 3, and 6.1% in week 4), allowing outpatient treatment in most cases. Neurologic AEs have been infrequent, with the most prominent event being grade 1 or grade 2 headache in 23.7% (9/38) treated at the RP2D. Two pts experienced grade 3 confusion of short duration (1-2 days) that was fully reversible. Over half (57.9%) of pts had evidence of antileukemic activity (reduction in blast count) with a median decrease of 92.7% in BM blasts (Fig. 1). The overall complete response rate (CRR, Conclusion: FLZ demonstrated encouraging activity in pts with PIF/ER AML, a population with poor prognosis and high unmet medical need, with 42.1% achieving CRR and over half of those receiving a stem cell transplant. Treatment is tolerable with a minimum 8 day inpatient treatment. The study is currently enrolling patients [NCT02152956] 1 Vadakekolathu J, Minden MD, Hood T, Church SE, Reeder S, Altmann H et al. Immune landscapes predict chemotherapy resistance and immunotherapy response in acute myeloid leukemia. Sci Trans Med 2020. Disclosures Aldoss: abbvie: Consultancy, Research Funding; agios: Honoraria; kite: Consultancy; autolus limited: Consultancy; JAZZ: Honoraria, Speakers Bureau; Amgen: Consultancy; Agios: Consultancy. Uy:Genentech: Consultancy; Agios: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Astellas Pharma: Honoraria. Emadi:Amgen: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Research Funding; Jazz Pharmaceuticals: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; KinaRx: Other: co-founder and scientific advisor; Servier: Membership on an entity's Board of Directors or advisory committees. Walter:Aptevo Therapeutics: Research Funding. Foster:Daiichi Sankyo: Consultancy; Bellicum Pharmaceuticals: Research Funding; Macrogenics: Consultancy, Research Funding. Arellano:Hanmi: Research Funding; Gilead Sciences, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cephalon Oncology: Research Funding. Wieduwilt:Amgen: Research Funding; Macrogeneics: Research Funding; Leadiant: Research Funding; Merck: Research Funding; Shire: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Michaelis:Jazz Pharmaceuticals: Research Funding. Stiff:Kite, a Gilead Company: Research Funding; Gamida Cell: Research Funding; Atara: Research Funding; Unum: Research Funding; Delta-Fly: Research Funding; Macrogenics: Research Funding; Amgen: Research Funding. Advani:Novartis: Consultancy, Other: advisory board; Pfizer: Honoraria, Research Funding; Takeda: Research Funding; OBI: Research Funding; Kite: Other: Advisory board/ honoraria; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Immunogen: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Wermke:MacroGenics: Honoraria. Erba:AbbVie, Daiichi Sankyo, Forma, ImmunoGen, Jazz Pharmaceuticals, MacroGenics, Novartis, PTC: Research Funding; Glycomimetics: Other: member of Scientific Steering Committee; Celgene: Other: chair of the Scientific Steering Committee; Covance (AbbVie): Other: chair of the Independent Review Committee; AbbVie, Agios, Celgene, Incyte, Jazz Pharmaceuticals, and Novartis: Speakers Bureau; AbbVie, Agios, Amgen, Astellas, Celgene, Daiichi Sankyo, Glycomimetics, ImmunoGen, Incyte, Jazz Pharmaceuticals, MacroGenics, Novartis, and Pfizer: Consultancy. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Ravandi:Abbvie: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Xencor: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Macrogenics: Research Funding; Celgene: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; Orsenix: Consultancy, Honoraria, Research Funding. Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Collins:IQVIA: Other: I have worked as a contractor for IQVIA in the past, within the past 24 months.; MacroGenics: Current equity holder in publicly-traded company, Other: I currently work as a contractor for MacroGenics. Guo:Macrogenics: Current Employment. Tran:MacroGenics: Current Employment. Kaminker:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Patel:MacroGenics: Current Employment. Bakkacha:MacroGenics: Current Employment. Jacobs:MacroGenics: Current Employment. Seiler:MacroGenics: Current Employment. Rutella:Kura Oncology: Research Funding; MacroGenics Inc.: Research Funding; NanoString Technologies Inc.: Research Funding. Bonvini:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Davidson-Moncada:Macrogenics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Introduction: CRS is a potentially life-threatening toxicity observed following T cell-redirecting therapies. CRS is associated with elevated cytokines, including IL6, IFNγ, TNFα, IL2 and GM-CSF. Glucocorticosteroids (GC) and the IL6 receptor blocking antibody tocilizumab (TCZ) can reduce CRS severity; however, CRS may still occur and limit the therapeutic window of novel immunotherapeutic agents. Disruption of cytokine signaling via Janus kinase (JAK) pathway interference may represent a complementary approach to blocking CRS. Ruxolitinib (RUX), an oral JAK1/2 inhibitor approved for the treatment of myelofibrosis and polycythemia vera, interferes with signaling of several cytokines, including IFNγ and IL6, via blockade of the JAK/STAT pathway. We hypothesized that RUX may reduce the frequency and severity of CRS in R/R AML patients (pts) undergoing treatment with flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule. Methods: Relapse/refractory (including primary induction failure, early relapse and late relapse) AML pts were included in this study. RUX pts were treated at a single site, Washington University, St. Louis, MO. RUX was dosed at 10 mg or 20mg BID days -1 through 14. Comparator (non-RUX) pts (n=23) were treated at other clinical sites. FLZ was administered at 500 ng/kg/day continuously in 28-day cycles following multi-step lead-in dosing in week 1 of cycle 1. CRS was graded per Lee criteria1. Results: As of July 1st, 2020, 10 R/R AML pts, median age 65 (range 40-82) years, have been enrolled and treated in the RUX cohort (6 at 10mg, 4 at 20 mg of RUX). All pts had non-favorable risk by ELN 2017 criteria (8 adverse and 2 intermediate); 1 (10.0%) pt had secondary AML; pt characteristics in the RUX and non-RUX cohorts were balanced, except for median baseline BM blasts which was higher in non-RUX pts: 15% (range 5-72) vs (40% (range 7-84), RUX and non-RUX pts respectively. Cytokine analysis showed statistically significant (p Conclusion: Prophylactic RUX produced a clear difference in cytokine profiles but no discernable improvement in clinical CRS or response rates in FLZ treated patients. A larger study may be required to determine the prophylactic role of RUX in CRS. References: 1. Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood 2014; 124(2): 188-195. doi: 10.1182/blood-2014-05-552729 Disclosures Uy: Pfizer: Consultancy; Agios: Consultancy; Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo: Consultancy; Astellas Pharma: Honoraria. Aldoss:abbvie: Consultancy, Research Funding; kite: Consultancy; agios: Honoraria; autolus limited: Consultancy; JAZZ: Honoraria, Speakers Bureau; Amgen: Consultancy; Agios: Consultancy. Arellano:Cephalon Oncology: Research Funding; Gilead Sciences, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Hanmi: Research Funding. Foster:Daiichi Sankyo: Consultancy; Bellicum Pharmaceuticals: Research Funding; Macrogenics: Consultancy, Research Funding. Ravandi:Celgene: Consultancy, Honoraria; Xencor: Consultancy, Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Orsenix: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding. Advani:Takeda: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding; Immunogen: Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Pfizer: Honoraria, Research Funding; Novartis: Consultancy, Other: advisory board; OBI: Research Funding. Wieduwilt:Macrogeneics: Research Funding; Amgen: Research Funding; Leadiant: Research Funding; Merck: Research Funding; Shire: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Emadi:Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Research Funding; Jazz Pharmaceuticals: Research Funding; KinaRx: Other: co-founder and scientific advisor; Servier: Membership on an entity's Board of Directors or advisory committees. Michaelis:Jazz Pharmaceuticals: Research Funding. Stiff:Macrogenics: Research Funding; Kite, a Gilead Company: Research Funding; Delta-Fly: Research Funding; Unum: Research Funding; Atara: Research Funding; Gamida Cell: Research Funding; Amgen: Research Funding. Wermke:MacroGenics: Honoraria. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Collins:MacroGenics: Current equity holder in publicly-traded company, Other: I currently work as a contractor for MacroGenics; IQVIA: Other: I have worked as a contractor for IQVIA in the past, within the past 24 months.. Guo:Macrogenics: Current Employment. Tran:MacroGenics: Current Employment. Kaminker:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Patel:MacroGenics: Current Employment. Bakkacha:MacroGenics: Current Employment. Jacobs:MacroGenics: Current Employment. Seiler:MacroGenics: Current Employment. Rutella:MacroGenics Inc.: Research Funding; Kura Oncology: Research Funding; NanoString Technologies Inc.: Research Funding. Walter:Aptevo Therapeutics: Research Funding. Bonvini:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Davidson-Moncada:Macrogenics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive, male-biased (>3:1 M:F) hematologic malignancy in which some patients have bone marrow involvement at diagnosis (50%) and most have tumor formation in the skin (~90%), often preceding marrow disease. The prognosis is poor (median survival of 12-24 months) and there is unmet need for biological insight. TET2, ASXL1, and RNA splicing genes (SRSF2, SF3B1, and ZRSR2) are recurrently mutated in BPDCN. The X chromosome gene ZRSR2 was the most frequently mutated spliceosome gene reported in a prior BPDCN cohort (7 of 24, 29.2%; Taylor, ASH 2013). Our goal was to define the functional consequences of ZRSR2 mutations in BPDCN. First, we confirmed the frequency of ZRSR2 mutations in a larger cohort from the US and Europe; we found ZRSR2 mutations in 24 of 93 (25.8%). Notably, ZRSR2 mutations were almost exclusively in males (23/73 males vs 1/20 females, P=0.019). Next, we compared the global mutation pattern to 30 predefined signatures from >7000 cancers in COSMIC. Analysis of all somatic single nucleotide variants in 11 tumor-normal pairs using whole exome sequencing (tumor was sorted BPDCN cells from marrow) revealed that BPDCN had an ultraviolet (UV)-induced mutation signature (score >0.25 in 6/11 or 55%; Figure 1A). For comparison, we detected the UV signature in melanoma but not in AML from The Cancer Genome Atlas. These data suggest that mutations acquired in the skin stage of BPDCN evolution are retained in subsequent leukemic disease. Next, we performed RNA-sequencing from sorted BPDCN and normal plasmacytoid dendritic cells (pDCs). Differentially expressed genes between BPDCN and pDCs (BCL2, MYB, IRF4, CEP70, IGLL1, GZMB) were similar to those that distinguish BPDCNs from pDCs by bulk and single cell RNA-sequencing. By gene set enrichment analysis (GSEA), BPDCNs were enriched for overexpression of MYC/E2F targets and PI3K/AKT/MTORC1 signaling pathway-associated genes. BPDCNs transcriptomes were also enriched for gene sets associated with RNA splicing machinery and RNA nonsense mediated decay (NMD). To link RNA splicing with functional consequences of ZRSR2 mutations, we generated ZRSR2-knockout BPDCN cells (CAL1) using CRISPR/Cas9. This models primary tumors because ZRSR2-mutant BPDCNs have complete loss of ZRSR2 protein. Activation marker (CD80) upregulation and type 1 interferon secretion after Toll-like receptor (TLR) stimulation with lipopolysaccharide (LPS) or R848 were reduced in ZRSR2-deficient cells. We found similar defective cytokine production in stimulated primary BPDCN cells compared to normal pDCs. After activation, normal pDCs undergo apoptosis in a negative feedback process. In contrast, ZRSR2-knockout, but not control cells, were protected from TLR activation-induced apoptosis. Reexpression of wild-type ZRSR2 in knockout cells restored activation-induced apoptosis (Figure 1B). These data suggested that ZRSR2-mutant BPDCNs have defects downstream of TLR stimulation. By RNA-sequencing, we found that IRF7 mRNA was mis-spliced in all ZRSR2- (2/2), SRSF2- (4/4), and SF3B1- (1/1) mutant BPDCNs compared to those with no mutated splicing gene (4/4). IRF7 (interferon regulatory factor 7) is a transcription factor activated by TLR signaling that is important for pDC activation and apoptosis. The IRF7 mRNA transcript contains a "weak intron" (intron 4) that is subject to intron retention, which leads to NMD and reduced IRF7 protein level in stimulated dendritic cells (Luke, Mol Cell 2019). IRF7 intron 4 was mis-spliced in ZRSR2-, SRSF2-, and SF3B1-mutant BPDCNs. ZRSR2-knockout CAL1 cells had severely impaired ability to upregulate IRF7 after LPS stimulation, which was partially rescued by reepxression of wild-type ZRSR2 (Figure 1C). Expression of constitutively activated IRF7 inhibited growth of both ZRSR2-knockout and control cells, confirming that the inability to activate IRF7 is important for the effect of ZRSR2 loss on TLR agonist-induced growth inhibition. In conclusion, male-biased ZRSR2 mutations are frequent in BPDCN and impair pDC activation and apoptosis, at least in part via TLR-IRF7. These data may explain why BPDCNs have an impaired activation state (Bierd, BCJ 2019). They also suggest that splicing factor mutations affect cell type-specific pathways to promote transformation, underscoring the importance of studying cancer genes in relevant contexts. Figure Disclosures Griffin: Moderna Therapeutics: Consultancy. Ghandi:Monte Rosa Therapeutics: Consultancy; Cambridge Data Science LLC: Current Employment, Current equity holder in private company. Seiler:Remix Therapeutics: Current Employment. Konopleva:Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Eli Lilly: Research Funding; Genentech: Consultancy, Research Funding; Agios: Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; AbbVie: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding; Calithera: Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Ablynx: Research Funding; Kisoji: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Pemmaraju:Cellectis: Research Funding; Daiichi Sankyo: Research Funding; DAVA Oncology: Honoraria; Plexxikon: Research Funding; Blueprint Medicines: Honoraria; Incyte Corporation: Honoraria; SagerStrong Foundation: Other: Grant Support; Celgene: Honoraria; Pacylex Pharmaceuticals: Consultancy; Affymetrix: Other: Grant Support, Research Funding; MustangBio: Honoraria; Roche Diagnostics: Honoraria; Novartis: Honoraria, Research Funding; LFB Biotechnologies: Honoraria; Stemline Therapeutics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Samus Therapeutics: Research Funding. Abdel-Wahab:H3 Biomedicine Inc.: Consultancy, Research Funding; Merck: Consultancy; Janssen: Consultancy; Envisagenics Inc.: Current equity holder in private company. Lane:Qiagen: Consultancy; Abbvie: Research Funding; Stemline Therapeutics: Research Funding.

Recurrent somatic mutations in core components and modulators of the cohesin ring - a multimeric protein complex that forms a ring structure around DNA and provides spatial genome organization - have been identified across multiple cancer types, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), where they are associated with poor overall survival. Cohesin proteins are involved in sister chromatid cohesion, chromatin organization into loops, transcriptional activation, and DNA damage repair. The mechanisms underlying clonal expansion of these driver mutations are unknown and no therapies have selective efficacy in cohesin-mutant cancers. We sought to determine the effects of mutations in the most frequently mutated cohesin subunit, STAG2, on cohesin complex composition using immunoprecipitation followed by quantitative mass spectrometry (IP-MS), genetic dependencies of STAG2-mutant cells by genome-wide CRISPR screening, and mutant cohesin association with chromatin using chromatin immunoprecipitation followed by sequencing (ChIP-Seq). Our goal was to understand how these mutations contribute to cellular transformation and to identify possible therapeutic targets. Applying IP-MS in AML cell lines engineered with different STAG2 mutations, we identified and validated a switch from STAG2- to its paralog STAG1-containing cohesin complexes. In addition, we observed changes in the interaction of the mutant cohesin complex with proteins involved in DNA repair and replication, including PARP1, and RNA-mediated interaction with RNA splicing machinery, including SF3B family members. We next hypothesized that these cohesin-dependent alterations could lead to shifts in genetic dependencies. Using genome-scale CRISPR-Cas9 screens, we identified preferential dependency of STAG2-mutant cells on STAG1, consistent with our proteomics studies. We also found a striking concordance between additional cellular processes highlighted by IP-MS experiments and observed increased dependency of STAG2-mutant cells on DNA damage repair and mRNA processing. Therefore, STAG2 mutations lead to changes in cohesin complex structure and alter interactions with proteins involved in DNA damage, replication, and RNA modification, which become genetic dependencies in this context. Prompted by this concordance, we evaluated DNA replication, DNA damage and splicing in cohesin-mutant cells. We observed a 4-fold increase in replication fork stalling in STAG2-mutant cells, which was associated with accumulation of double strand DNA breaks and activation of the ATR and ATM DNA damage checkpoints. STAG2-mutant cells demonstrated ~100-fold increased sensitivity to the PARP inhibitor talazoparib, which was consistent across models of other cohesin-mutant subunits. In addition, cohesin-mutant cells showed aberrant splicing and increased sensitivity to treatment with SF3B1 inhibitors E7107 and H3B-8800. In aggregate, genetic or pharmacologic perturbation of DNA damage repair or splicing created a synthetic vulnerability for cohesin-mutant cells in vitro and in vivo. Finally, we explored how STAG1-containing complexes alter cohesin-mediated genome compartmentalization in cohesin-mutant cells. Using ChIP-Seq, we observed that STAG2 loss leads to a global decrease in cohesin binding to chromatin, including at sites of insulated neighborhood boundaries, with subsequent gene expression changes. Loss of cohesin binding was associated with increased enhancer activity and super-enhancer expansion in STAG2-mutant cells. In addition, we identified changes in the co-localization of the mutant cohesin complex with super-enhancer enriched factors, DNA damage repair and splicing machinery. These findings are consistent with a model in which wild type and mutant cohesin complexes, defined by their unique composition and patterns of chromatin binding and architecture, have differential abilities to maintain chromatin organization as it relates to spatial organization of super-enhancers, coactivators and transcription factors, as well as DNA damage repair and splicing machinery. Perturbation of any of these components, which have been recently proposed to form phase-separated nuclear bodies, creates vulnerabilities that may be exploited therapeutically with existing drugs in patients with cohesin-mutated malignancies. Disclosures Abraham: Syros Pharmaceuticals: Equity Ownership. Seiler:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. D'Andrea:Intellia Therapeutics: Consultancy; Cedilla Therpeutics: Consultancy, Equity Ownership; EMD-Serono: Consultancy, Research Funding; Sierra: Consultancy, Research Funding; Ideaya: Consultancy, Equity Ownership; Lilly: Consultancy, Research Funding; Formation Biologics: Consultancy. Young:Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Many key oncogenic pathways converge to adapt tumor cell metabolism to requirements of cancer cells. Aberrant proliferation that is frequently associated with cancer cells is also linked to an adjustment of metabolism in order to fuel cell growth and division. Cancer cells prefer utilizing glycolysis for energy production and providing essential building blocks for a variety of macromolecules. Hexokinases (HKs) are rate-limiting enzymes that catalyze the first and irreversible step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. Four HK isoforms are expressed in mammalian cells, HK1, HK2, HK3 and HK4 (also known as glucokinase). HKs promote and sustain a concentration gradient that facilitates glucose entry, which ensures the initiation of glucose dependent pathways. In general, HKs have a cytoprotective role that was highlighted by enhanced sensitivity of cancer cells to drugs when HKs were inhibited. Previously we have reported that HK3 was transcriptionally regulated by PU.1 (SPI-1) in myeloid cells. Further, HK3 expression was significantly reduced in patient acute myeloid leukemia (AML) cells, particularly in acute promyelocytic leukemia (APL) cells expressing the PML-RARA oncofusion protein. We now report on the expression and regulatory function of HKs, particularly HK3, during myeloid differentiation and granulocyte associated cell death. First, we analyzed mRNA HK levels in human CD34+ hematopoietic progenitors cells differentiated towards granulocytes or macrophages by qPCR. Interestingly, while HK1 and HK2 levels remain stable during all stages of myeloid differentiation, HK3 mRNA levels significantly increased. The same pattern of HK mRNA expression was seen in NB4 APL and HL60 AML cell lines differentiated towards granulocytes and monocytes using all-trans retinoic acid (ATRA) and vitamin D3, respectively. To determine a specific role for HK1-3 function in myeloid cells, HK1-3 knockdowns (KD) and knockouts (KO) in NB4 and HL60 cell lines, using shRNA or gRNAs (Cas9/CRISPR technology), respectively, were generated. NB4 HK KD AML cells were tested for their differentiation upon ATRA treatment. Knockdown of HKs generally resulted in a decreased differentiation response of about 20% as assessed by the differentiation marker CD11b. We next determined energy metabolism of HK altered KD and KO cells, relative to parental cells, using a seahorse analyzer. While lowering HK1 or HK3 levels in NB4 and HL60 AML cells did not affect glycolytic capacity at steady state, HK2 inhibition significantly reduced steady state glycolytic capacity. In contrast, knocking down or knocking out HK3 resulted in a higher sensitivity to ATRA-induced cell death during differentiation, which was coupled with higher glycolytic capacity. Together, our findings suggest that HK2 has an important role in steady state metabolism of AML cells while HK3 appears to be a metabolic switch for cell survival during myeloid differentiation. Disclosures No relevant conflicts of interest to declare.

In iron deficiency anaemia patients, intravenous administration of either ferric carboxymaltose (FCM) or iron isomaltoside (IM) both allow high dosage iron substitution within a single outpatient visit. In contrast, other iron compounds require repetitive, low dosage infusions. Recently, FCM was reported to frequently induce acute, reversible hypophosphatemia. It remains enigmatic whether these hypophosphataemic effects of FCM are substance-specific, or whether they generally occur after high dosage iron substitution. A direct comparison of phosphorus regulation after high dosage iron substitution with either FCM or IM is clinically important, as hypophosphatemia after FCM has anecdotally been associated with osteomalacia and bone fractures. In the HOMe AFers 1 (HOMburg evaluations on application of Ferrum study 1) trial, we recruited normophosphatemic women with gynaecological bleeding and subsequent iron deficiency anaemia, in whom we assessed the longitudinal biochemical response over 28 days to a single intravenous injection of equivalent doses of randomly-assigned FCM and IM (1000 mg). The primary study hypothesis was that the incidence of hypophosphatemia - defined as plasma phosphorus < 2.0 mg/dl at least out of three post-infusion study time points (day 1, day 8, and week 5) - differs between FCM and IM. HOMe AFers 1 initially planned to recruit 60 women. An interim analysis was pre-specified and scheduled in July 2018, with the option to stop further patient recruitment if the incidence of hypophosphatemia differs significantly at this interim analysis. At the time point of the interim analysis, 26 patients have been recruited. One patient withdrew her acceptance to participate after day 1, leaving 25 patients for our per protocol interim analysis. Baseline plasma phosphorus did not differ significantly between FMC (3.3 ± 0.4 mg/dl) and IM (3.6 ± 0.6 mg/dl; p = 0.135). Analysis on the primary endpoint demonstrated that significantly more women developed hypophosphatemia < 2.0 mg/dl after FMC infusion (9 out of 12 patients) than after IM infusion (1 out of 13 patient) (p=0.001). Further, the minimum plasma phosphorus during any of the three post-infusion study time points was lower after FMC (1.8 ± 0.3 mg/dl) than after IM (2.7 ± 0.6 mg/dl; p < 0.001). As expected, ferritin and haemoglobin increased in both study groups after iron infusion. No severe adverse events occurred in either group. In conclusion, while both FCM and IM provide efficient iron substitution in iron deficiency anaemia, FCM induced a substantially higher incidence of hypophosphatemia. Disclosures Emrich: Pharmacosmos: Consultancy, Honoraria, Research Funding. Stilgenbauer:Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Brandenburg:Pharmacosmos: Consultancy, Honoraria, Research Funding; Vifor: Consultancy, Honoraria. Heine:Pharmacosmos: Consultancy, Honoraria, Research Funding.

In chronic lymphocytic leukemia (CLL), biologic risk factors such as immunoglobulin variable heavy chain gene (V(H)) mutation status, CD38 expression level, and genomic aberrations have recently been identified, but the relative prognostic impact of the individual parameters is unknown. In the current study, we analyzed V(H) mutation status by polymerase chain reaction and sequencing (n = 300), genomic aberrations by fluorescence in situ hybridization (+3q, 6q-, +8q, 11q-, +12q, 13q-, t(14q), 17p-) (n = 300), and CD38 expression by triple-color FACS (CD5, CD19, CD38) (n = 157) in a unicentric CLL cohort. The prognostic influence of V(H) mutation rate and CD38 expression level was tested by maximally selected log-rank statistics. A corrected P value (P(cor)) for a cutoff level allowing the best separation of 2 subgroups with different survival probabilities was identified at 97% V(H) homology (95% confidence interval [CI], 96%-98% homology, P(cor)

Genome-wide knockout screening employing CRISPR-Cas9 genome-editing is a powerful tool for functional genomics. However, identifying actionable targets for cancer therapy has been challenging due, in part, to the complex genetic background of cell lines used for screening. To overcome this obstacle, we generated two acute myeloid leukemia (AML) lines from mouse leukemia models based on activity of the leukemia oncogenes CALM/AF10 or MLL/AF9. Both lines exhibited a normal karyotype and intact Tp53 activity. Using these lines, we performed genome-wide CRISPR-Cas9 screening, followed by a second screen in vivo . We then selected genes meeting the following criteria: 1) they encoded a protein with an available inhibitor or 2) their germline mutation loss-of-function phenotype was relatively moderate based on the literature or the human exome-sequencing database. We excluded genes with a well-defined function in leukemogenesis as well as those encoding components of basal cellular machineries. Among genes significantly depleted in our primary screen was the mRNA decapping enzyme scavenger (Dcps), which encodes a mRNA 5' cap binding protein implicated in mRNA decay. Read counts for each Dcps-targeted single-guide RNA (sgRNA) significantly decreased in vitro (AML cell lines) and in vivo (mouse AML model). A negative selection CRISPR-Cas9 mutagenesis scan of all Dcps coding exons revealed that the C-terminal Dcps domain, namely aa 230-240, plays a critical role in AML survival. RG3039, an orally active quinazoline derivative, is a DCPS inhibitor that was originally developed to treat spinal muscular atrophy (SMA) and has been judged safe in a phase I trial in healthy volunteers (Van Meerbeke JP et al. Hum Mol Genet. 2013). We validated RG3039 binding to DCPS protein in AML cells via a cellular thermal shift assay (CETSA). We then assessed anti-leukemia effects of RG3039 by treating human AML lines with RG3039 and generating growth curves. That analysis showed that RG3039 had dose-dependent anti-proliferative effects. RG3039 treatment induced cell cycle arrest and apoptosis, revealed by EdU incorporation assay and Annexin V stain, respectively. Since DCPS protein was predominantly nuclear in human primary AML cells, we hypothesized that DCPS primarily functions in that compartment rather than in the cytoplasmic mRNA 3' end decay pathway. To search for nuclear proteins potentially interacting with DCPS, we undertook immunoprecipitation with an anti-DCPS antibody of lysates of AML cells followed by mass spectrometry analysis. Among highly significant interactors, we identified components of pre-mRNA processing machineries including spliceosomes. To assess whether DCPS inhibition would impair pre-mRNA splicing, we treated AML cells with RG3039 and performed RNA-sequencing to determine potential transcriptome-wide splicing changes. Alternative 5' splice site selection was most frequently observed in RG3039-treated cells, and most aberrant splicing events involved the first exon. Bioinformatic prediction analysis revealed that approximately 40% of the aberrantly-spliced genes were NMD (nonsense-mediated mRNA decay)-sensitive. Gene Set Enrichment Analysis identified gene signatures representing a type-I interferon response, reminiscent of that observed in RNAi-treated cells, in RG3039-treated AML cells. We next explored effects of DCPS deficiency on normal hematopoiesis in humans. To do so, we examined peripheral blood counts of three children harboring germline homozygous loss-of-function mutations as well as heterozygous relatives in a family reported previously (Ng et al. Hum Mol Genet 2015). They exhibited normal blood counts, indicating that DCPS is dispensable for steady-state hematopoiesis in humans. Finally, to investigate potential anti-leukemia effects of RG3039 in vivo, we tested RG3039 efficacy using patient-derived xenograft (PDX) AML models established from three human AML lines. RG3039 exhibited anti-leukemic activity, as evidenced by the lower leukemia burden in PB and BM of RG3039-treated mice. They survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of RG3039 monotherapy against AML in vivo. In summary, our findings shed a new light on a pre-mRNA metabolic pathway regulated by DCPS. They also identify DCPS as a novel target for AML therapy and suggest potential "repurposing" of RG3039 as an anti-leukemia drug. Disclosures Seiler: H3 Biomedicine, Inc.: Employment. Orkin: Epizyme Inc.: Consultancy; Bioverativ: Consultancy. Buonamici: H3 Biomedicine Inc.: Employment.

Mutations in RNA splicing factors confer an alteration of function and are common in patients with myelodysplastic syndrome (MDS, ~45%), chronic myelomonocytic leukemia (CMML, ~60%), and acute myeloid leukemia (AML) derived from these conditions. Recent data suggest that spliceosome-mutant cells are preferentially sensitive to genetic or pharmacologic splicing modulation compared with wildtype (WT) counterparts. Here, we describe the discovery of H3B-8800, a potent and orally bioavailable modulator of the SF3b complex, and demonstrate efficacy in models of spliceosome mutant myeloid malignancies including a novel xenograft system for CMML. H3B-8800 was identified through a medicinal chemistry approach aimed at identifying compounds with preferential lethality in spliceosome mutant cells. Using a scintillation proximity assay, we demonstrated that H3B-8800 potently binds to SF3b complexes containing either WT or mutant SF3B1 protein. Consistent with this, H3B-8800 showed dose-dependent modulation of splicing in in vitro biochemical splicing assays and cellular pharmacodynamic assays. Selectivity of H3B-8800 for the SF3b complex was confirmed through observing resistance in cells expressing SF3B1R1074H, an SF3B1 mutation previously shown to confer resistance to natural product splicing modulators. In the above biochemical and cellular assays, H3B-8800 affected splicing similarly regardless of spliceosome genotype. However, preferential inhibition of in vitro cell growth was observed in isogenic AML cells with endogenous knock-in of SF3B1K700E or SRSF2P95H mutations compared to WT counterparts. In animals xenografted with SF3B1K700E knock-in K562 cells, oral H3B-8800 treatment demonstrated dose-dependent splicing modulation and inhibited tumor growth, while no therapeutic impact was seen in WT controls. Similarly, anti-leukemic efficacy and improved survival were observed with H3B-8800 treatment in mice transplanted with Srsf2P95H/MLL-AF9 mouse AML cells, a result not seen in Srsf2 WT/MLL-AF9 counterpart leukemias. To understand the preferential effects on spliceosome mutant cells, RNA-seq analysis of isogenic K562 cells treated with H3B-8800 was performed. H3B-8800 induced intron retention and exon skipping, however these effects were not global and introns preferentially retained by H3B-8800 were shorter and more GC-rich compared to those unaffected by drug (Figure A). Interestingly, a substantial number of genes experiencing intron retention with H3B-8800 themselves encoded spliceosome components (Figure B). This suggests that the preferential effect of H3B-8800 on spliceosome mutant cells is due to the exquisite dependency of these cells on normal expression of spliceosome proteins. Next we aimed to understand the therapeutic potential of H3B-8800 in the context of CMML due to the high frequency of SRSF2 mutations and the need for improved outcome in this disorder. To this end, we developed a xenotransplantation model through direct intrafemoral injection of CD34+ cells from CMML patients into "NSGS" mice: a variant of NSG mice that express human IL3, SCF and GM-CSF. We specifically focused on CMML with 200,000 CD34+ cells achieved robust engraftment for all patients (n=7) with rapid lethality (median of 39 days). In vivo H3B-8800 administration substantially reduced leukemic burden in spliceosome-mutant but not spliceosome-WT CMML PDX (Figure C). Furthermore, 2.2-fold reductions in immunophenotypically-defined leukemia initiating cells were seen with H3B-8800 versus vehicle treatment in spliceosome-mutant CMML compared with no change in those mice engrafted with spliceosome-WT CMML. These data identify a novel therapeutic approach with selective lethality in myeloid cells bearing a spliceosome mutation. Despite the essential nature of splicing, CMML/AML cells without a spliceosome mutation were less sensitive to H3B-8800 compared with potent eradication of mutant counterparts. These data demonstrate the therapeutic potential of splicing modulation in spliceosome mutant cancers and H3B-8800 is currently undergoing clinical evaluation in patients with MDS, AML and CMML. Figure. Figure. Disclosures Buonamici: H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Caleb:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Karr:H3 Biomedicine: Employment. Liu:H3 Biomedicine: Employment. Meeske:H3 Biomedicine: Employment. Mizui:Eisai: Employment. Pazolli:H3 Biomedicine: Employment. Prajapati:H3 Biomedicine: Employment. Wang:Eisai: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment.

Mutations in genes encoding RNA splicing factors constitute the most common class of alterations in patients with myelodysplasticsyndromes (MDS). These occur predominantly as heterozygous point mutations at restricted residues in SF3B1, SRSF2, and U2AF1 in a mutually exclusive manner, suggesting that spliceosomal gene mutations confer gain-of-function with converging biological effects. However, recent studies suggest that mutations in each splicing factor result in distinct alterations in pre-mRNA splicing. It is therefore unclear if such mutual exclusivity is due to overlapping biological effects and/or synthetic lethal interactions. Furthermore, although cells bearing mutant splicing factors have been shown to require the wildtype allele for survival, whether these mutations can exist in a homozygous state is unknown. Here we addressed these questions by analyzing the effects of expressing SF3B1 and SRSF2 mutations simultaneously or in a homozygous state in vivo. Re-analysis of published sequencing data revealed that only 2% of MDS patients (86/4,032) have mutations in >1 splicing factor simultaneously (whether these mutations are present in the same cell or not is unclear). Of these 86 patients, co-mutations in SRSF2 and SF3B1 represent the most prevalent combination (n=23/86) with all SRSF2 mutations affecting the P95 residue while all co-existing SF3B1 mutations occurred outside of the most commonly mutated K700 residue. To understand the basis for exclusivity of SRSF2P95 and SF3B1K700 mutations, we generated mice for inducible heterozygous expression of Sf3b1K700E/+ and Srsf2P95H/+ mutations simultaneously (Mx1-cre Sf3b1K700E/+/Srsf2P95H/+). We next performed competitive bone marrow transplantation (BMT) assays where each mutation was induced, alone or together, following stable engraftment (Figure 1A). Simultaneous expression of Sf3b1K700E and Srsf2P95H mutations resulted in severe defects on the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPC), which were outcompeted by wildtype and single-mutant HSPCs (Figure 1B). In noncompetitive BMT assays, HSPCs co-expressing Sf3b1K700E and Srsf2P95H mutations had severe defects in multi-lineage reconstitution (Figure 1C). Analyses of hematopoietic organs 6 months post-BMT revealed a near complete absence of Sf3b1K700E/+/Srsf2P95H/+ double-mutant cells, which was distinct from expression of Sf3b1K700E or Srsf2P95H mutationalone. In addition, mice with conditional homozygous expression of the SRSF2P95H mutation (Mx1-cre Srsf2P95H/P95H) had severe defects in HSPC self-renewal as well as multi-lineage reconstitution, analogous to those seen with hemizygous Srsf2P95H expression (Mx1-cre Srsf2P95H/KO) (Figure 1D). As noted earlier, SF3B1 and SRSF2 mutations cause different effects on mRNA splicing. However, there has never been a direct comparison of the effects of each of these mutations in an isogenic context. To address this and to understand the mechanistic basis for exclusivity of these mutations, we performed RNA-seq on lineage- c-Kit+ cells from Mx1-cre Sf3b1K700E/+/Srsf2P95H/+ andcontrols 2 weeks after conditionally expressing each mutation alone or together. As evidence of the intolerability of combined SF3B1/SRSF2 mutations, mean allelic ratio of Sf3b1K700E and Srsf2P95H expressed in double-mutant mice was 20.7% and 33.5%, respectively, markedly lower than the near 50% expression seen in single-mutant controls. Despite this, principle component analysis of differentially spliced genes revealed distinct changes mediated by expression of Sf3b1K700E and Srsf2P95H mutations (Figure 1E). Moreover, previously described changes to alternative 3" splice site selection as well as cassette exon splicing were seen in cells bearing Sf3b1K700E and Srsf2P95H mutation, respectively, as well as in Sf3b1K700E/+/Srsf2P95H/+ double-mutantcells. These findings indicate thatspliceosomalgene mutations, despite imparting distinct alterations on gene expression and splicing, are not tolerated when co-expressed in the same cell, thus providing a basis for their strong mutual exclusivity in MDS. These data, combined with the fact that neitherhemizygousnor homozygous expression of splicing factor mutations is tolerated, further establishes the unique requirement ofspliceosomalmutant cells on the remaining function ofwildtypespliceosomecomponents. Figure 1. Figure 1. Disclosures Palacino: H3 Biomedicine Inc.: Employment. Seiler:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Smith:H3 Biomedicine Inc.: Employment.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive malignancy thought to result from transformation of plasmacytoid dendritic cells (pDCs). Clinical outcomes are poor and pathogenesis is unclear. To better understand BPDCN genomics and disease mechanisms, we performed whole exome- (12 BPDCNs), targeted DNA- (additional 12 BPDCNs), bulk whole transcriptome RNA- (12 BPDCNs and 6 BPDCN patient-derived xenografts [PDXs]), and single cell RNA-sequencing (scRNA-seq) compared to normal DCs. We observed RNA splicing factor mutations in 16/24 cases (7 ZRSR2, 6 SRSF2, 1 each SF3B1, U2AF1, SF3A2, SF3B4). Additional recurrent alterations were in genes known to be mutated in other blood cancers: TET2, ASXL1, TP53, GNB1, NRAS, IDH2, ETV6, DNMT3A, and RUNX1. From exome sequencing we also discovered recurrent mutations in CRIPAK (6/12 cases), NEFH (4/12), HNF1A (2/12), PAX3 (2/12), and SSC5D (2/12) that may be unique to BPDCN. ZRSR2 is notable among the recurrently mutated splicing factors in hematologic malignancies in that all mutations are loss-of-function (e.g., nonsense, frameshift). Of note, BPDCN is very male predominant, ZRSR2 is located on chrX and all mutations are in males. ZRSR2 plays a critical role in "minor" or U12-type intron splicing (only 0.3% of all introns). Thus, we hypothesized that mis-splicing, possibly of U12 genes, contributes to BPDCN pathogenesis. Using RNA-seq, we measured aberrant splicing in BPDCN. Intron retention was the most frequent abnormality in ZRSR2 mutant BPDCNs and PDXs compared to non-mutant cases. ZRSR2 mutant intron retention predominantly affected U12 introns (patients: 29.4% of retained introns, P Disclosures Seiler: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Lane:Stemline Therapeutics: Research Funding; N-of-1: Consultancy.

Introduction: Several targeted agents have been introduced for CLL, including the CD20-antibody obinutuzumab (GA101, G) and the Bcl-2 antagonist venetoclax (ABT-199, A). Both agents show exciting efficacy which comes at the cost of an increased risk of TLS and IRRs and mandate several safety precautions especially during the first treatment cycles and in patients (pts) with a higher tumor load and/or renal insufficiency. Based on the theoretical "sequential triple-T" concept [Hallek M., Blood 2013; 122(23): 3723-34] of a tailored and targeted treatment aiming for total eradication of minimal residual disease (MRD), the GCLLSG designed the CLL2-BAG trial combining G and A in an induction and maintenance treatment. To reduce the risk of TLS and IRRs, G and A were started sequentially during the induction phase and patients with high tumor burden received bendamustine (B) as a debulking step before the induction treatment. Methods: This prospective, open-label, multicenter phase-II trial investigates the safety and efficacy of a sequential treatment with B, G and A in an all-comer population of physically fit and unfit, treatment-naïve and relapsed/refractory CLL pts requiring treatment, irrespective of high-risk genetics. Pts with an absolute lymphocyte count (ALC) ≥ 25.000/µl and/or lymph nodes (LN) ≥ 5cm received 2 cycles of B as debulking treatment (70mg/m² d1&2 q28 days). In the induction G was administered 3 times in cycle 1 (days 1/2, 8 & 15) and every 4 weeks in cycles 2-6. Daily intake of A started in cycle 2 with a slow dose ramp-up over 5 weeks and several safety precautions including blood sampling, hydration, allopurinol and rasburicase (depending on patient's TLS risk category). In the maintenance phase, A was continued and G administered every 3 months until achievement of a MRD-negative complete response or for up to 24 months. The primary endpoint was the overall response rate (ORR) at the end of induction therapy; secondary endpoints include safety parameters, MRD evaluations and survival parameters. Results: Between May 2015 and January 2016, 66 pts were enrolled, among them 35 with treatment naïve and 31 with relapsed/refractory CLL (median number of prior therapies: 2, range: 1-8). Median age was 59 (28-77) years and the median CIRS score was 2 (0-14). 12 of 48 pts (25%) had an impaired renal function at baseline with a creatinine clearance of 30-70ml/min. 11 of 62 pts (18%) had a del(17p) and 48 of 64 (75%) had an unmutated IGHV status. 47 (71%) pts received B debulking and 19 (29%) pts immediately started with G due to a low tumor burden (6), contraindications for B [known hypersensitivity or refractoriness] (4) and/or physicians decision (13). Median ALC was 52.3 (0.6-423.5) at baseline and 0.9 (0.2-102.0) after first induction cycle. Risk categories for TLS at baseline were: low (LR: ALC 10cm): 17 (27%), 3 missing. After debulking and/or 1st cycle with G risk categories were re-assessed in 19 pts and were LR in 13 (68%) and IR in 6 (32%) pts. As of July 20th2016, 76 serious adverse events (SAEs) were reported in 36 of the 66 pts, among them 64 (84%) related to study drug. 57 SAEs (75%) were CTC°3-4 and 3 deaths (septicaemias in heavily pretreated pts). So far 13 (17%) SAEs occurred during B debulking, 62 (82%) in the induction and 1 (1%) in the maintenance phase. Most common SAEs were infections (26 in 15 pts; among them 12 CTC°3-5) and hematological disorders (18 in 11 pts; 10 CTC°3-4); especially pneumonias (8 in 3 pts), sepsis (3 in 3 pts; all CTC°5) and neutropenias with/without fever (5 and 7 each; in 7 pts). Six serious IRRs occurred in 6 pts (4 CTC°3-4), 5 occurred during the first infusion of G; 3 of the affected pts had not received prior B. Five SAEs were laboratory TLS, which all resolved quickly; 1 occurred during the debulking with B and 4 during the induction therapy (1 in induction cycle 1 with G, 2 in cycle 3 and 1 in cycle 4 with G and A), all in pts without prior B debulking. Conclusion: The administration of B as a debulking step, followed by G helps to effectively reduce the patient's tumor load providing the ability to prevent serious IRRs and TLS. With this concept and the established safety precautions no clinical TLS occurred so far. Aside from 3 septicaemias with fatal outcome in heavily pretreated pts, the toxicity profile of this regimen is acceptable. Disclosures Cramer: Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Novartis: Consultancy, Research Funding; Roche: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Gilead: Other: Travel, Accommodations, Expenses, Research Funding; GlaxoSmithKline: Research Funding; Astellas: Other: Travel, Accommodations, Expenses; Mundipharma: Other: Travel, Accommodations, Expenses. von Tresckow:Janssen-Cilag: Honoraria, Other: Travel grants, Research Funding; Celgene: Other: Travel grants; Hoffmann-LaRoche: Other: Travel grants, Research Funding. Bahlo:Roche: Honoraria, Other: Travel grants. Engelke:Hoffmann-LaRoche: Other: Travel grants. Langerbeins:Hoffmann-LaRoche: Honoraria, Other: Travel grants, Research Funding; Janssen-Cilag: Honoraria, Other: Travel grants, Research Funding; Mundipharma: Honoraria, Other: Travel grants, Research Funding. Fink:Mundipharma: Other: Travel grants; Celgene: Other: Travel grants, Research Funding; AbbVie: Other: Travel grants; Hoffmann-LaRoche: Other: Travel grants. Al-Sawaf:Gilead: Other: Travel grants. Fischer von Weikersthal:Roche: Consultancy, Other: Travel Grant; Novartis: Consultancy; Pfizer: Honoraria. Fischer:Hoffmann-LaRoche: Other: Travel grants. Wendtner:AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau. Stilgenbauer:Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding. Eichhorst:GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau. Hallek:F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau.

Single cytokine therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) has been shown to be effective in decreasing the respective periods of neutropenia and thrombocytopenia following radiation- or drug-induced marrow aplasia. The combined administration of IL-3 and GM-CSF in normal primates suggested that a sequential protocol of IL-3 followed by GM-CSF would be more effective than that of GM-CSF alone in producing neutrophils (PMN). We investigated the therapeutic efficacy of two combination protocols, the sequential and coadministration of recombinant human IL-3 and GM-CSF relative to respective single cytokine therapy, and delayed GM-CSF administration in sublethally irradiated rhesus monkeys. Monkeys irradiated with 450 cGy (mixed fission neutron:gamma radiation) received either IL-3, GM-CSF, human serum albumin (HSA), or IL-3 coadministered with GM-CSF for days 1 through 21 consecutively postexposure, or IL-3 or HSA for days 1 through 7 followed by GM-CSF for days 7 through 21. All INEAGE-SPECIFIC CYTOKINES, granulocyte-macL rophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) have demonstrated therapeutic efficacy in reducing the periods of neutropenia associated with radiation- and drug-induced marrow aplasia.’-3 Interleukin-3 (IL-3) has shown efficacy in stimulating the production of platelets (PLTs) in animals4 and some patients, but is less effective in generating neutrophils (PMNS).~.~ Previous studies in normal primates have indicated that the combination of GM-CSF and IL-3 promoted a synergistic rise in peripheral white blood cells (WBC) and PLT levels when IL-3 was administered before GM-CSF.7-9 Whereas the coadministration of GM-CSF and IL-3 resulted in diminished PMN production relative to GM-CSF alone (Farese et al, unpublished data). The sequential combination of IL-3 and GM-CSF is based on the concept that IL-3 will expand GM-CSF sensitive target cells for more efficient production of neutrophils in addition to generating an increase of PLT precursors. Whereas, the combined administration of IL-3 and GM-CSF, may result in downregulation ofGM

Single cytokine therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) has been shown to be effective in decreasing the respective periods of neutropenia and thrombocytopenia following radiation- or drug-induced marrow aplasia. The combined administration of IL-3 and GM-CSF in normal primates suggested that a sequential protocol of IL-3 followed by GM-CSF would be more effective than that of GM-CSF alone in producing neutrophils (PMN). We investigated the therapeutic efficacy of two combination protocols, the sequential and coadministration of recombinant human IL- 3 and GM-CSF relative to respective single cytokine therapy, and delayed GM-CSF administration in sublethally irradiated rhesus monkeys. Monkeys irradiated with 450 cGy (mixed fission neutron:gamma radiation) received either IL-3, GM-CSF, human serum albumin (HSA), or IL-3 coadministered with GM-CSF for days 1 through 21 consecutively postexposure, or IL-3 or HSA for days 1 through 7 followed by GM-CSF for days 7 through 21. All cytokines and HSA were injected subcutaneously at a total dose of 25 micrograms/kg/d, divided twice daily. Complete blood counts (CBC) and platelet (PLT) counts were monitored over 60 days postirradiation. The respiratory burst activity of the PMN was assessed flow cytometrically, by measuring hydrogen peroxide (H2O2) production. Coadministration of IL-3 and GM-CSF reduced the average 16-day period of neutropenia and antibiotic support in the control animals to 6 days (P = .006). Similarly, the average 10-day period of severe thrombocytopenia, which necessitated PLT transfusion in the control animals, was reduced to 3 days when IL-3 and GM-CSF were coadministered (P = .004). The sequential administration of IL-3 followed by GM-CSF had no greater effect on PMN production than GM-CSF alone and was less effective than IL-3 alone in reducing thrombocytopenia. PMN function was enhanced in all cytokine-treated animals.

Despite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epitopes for U- and M-CLL Igs differed significantly. M-CLL–derived peptides exhibited better amino acid motifs, were more similar to each other, aligned more easily, and formed tighter clusters than U-CLL–derived peptides. Mono-, oligo-, and polyreactivity of peptides correlated with structural changes within antigen-binding sites of selecting M-CLL mAbs. Although M-CLL–isolated peptides and certain U-CLL mAbs bound more effectively to the selecting mAb, others were not as specific, reacting with M-CLL and U-CLL mAbs; these data suggest that in vivo structurally diverse epitopes could bind smIgs of distinct CLL clones, thereby altering survival and growth. Finally, an M-CLL–derived peptide inhibited, in a dose-dependent manner, binding of its homologous mAb to human B lymphocytes; therefore peptides that inhibit or alter the consequences of antigen-smIg interactions may represent therapeutic modalities in CLL.

INTRODUCTION The Bcl-2 inhibitor venetoclax (GDC-0199/ABT-199) has yielded promising results in patients with relapsed/refractory chronic lymphocytic leukemia (CLL), both as monotherapy and in combination with rituximab. The CLL11 trial demonstrated that combining obinutuzumab, a type 2, glycoengineered anti-CD20-antibody, with chlorambucil resulted in improved overall survival compared to chlorambucil alone in patients with previously untreated CLL and coexisting medical conditions. Prior to opening the randomized phase of the current CLL14 trial, the successor trial to CLL11, a safety run-in phase of previously untreated patients with CLL and coexisting medical conditions was performed to assess the tolerability of obinutuzumab and venetoclax. Here, we report preliminary results of this safety run-in phase after completion of recruitment. METHODS The protocol specified to enroll 12 previously untreated patients with confirmed CLL and with coexisting medical conditions assessed by cumulative illness rating scale (CIRS) total score > 6 and/or estimated creatinine clearance (CrCl) < 70 mL/min requiring treatment (according to NCI/iwCLL criteria into the safety run-in phase of the CLL14 trial. All patients received 6 cycles of obinutuzumab and venetoclax followed by 6 additional cycles of venetoclax. Obinutuzumab was administered intravenously with 100 mg on day 1, 900 mg on day 2 (option to deliver 1000 mg on day 1), 1000 mg on day 8 and day 15 of cycle 1 and 1000 mg on day1 for cycles 2-6. A gradual weekly dose ramp-up of venetoclax with 20 mg, 50 mg, 100 mg, 200 mg up to 400 mg was administered starting at day 22 of cycle 1. Risk assessment for tumor lysis syndrome (TLS) based on absolute lymphocyte count and the largest diameter of measurable lymph nodes was performed before treatment in order to direct prophylactic measures. Study defined stopping criteria for all 12 patients included: one treatment-related death or one grade 4 adverse event related to a clinical TLS despite protocol-specified prophylaxis. Adverse events were graded per the NCI CTCAE v.4 criteria. RESULTS Between December 2014 and April 2015, 13 previously untreated patients from Australia, Canada, Germany, New Zealand, United States and Spain were enrolled; baseline patient characteristics are summarized in Figure 1. The median age was 75 years (range 59 - 88) and 54% of the patients were classified as Binet stage C; six patients were assessed medium risk and 7 patients high risk for TLS. At the timepoint of data cut-off, 12 of 13 patients have been on treatment for at least 4 weeks and completed the venetoclax dose ramp up. The median time on treatment with venetoclax is 64.5 days (range 34-153 days). One patient developed a grade 4 infusion related reaction (IRR) during the first dose of obinutuzumab and was therefore withdrawn from the trial according to the protocol requirements. All patients experienced at least 1 adverse event. Adverse events are summarized in Figure 2. Of note, 1 patient developed a self-limited grade 4 elevation of transaminases secondary to obinutuzumab. No clinical TLS was reported. Two patients developed laboratory TLS. One patient was classified as medium risk for TLS and developed hyperkalemia and hyperuricemia after the 100 mg infusion of obinutuzumab on day 1 of cycle 1. The second patient was classified as high risk for TLS (including a lymph node measuring 110 mm in diameter) and developed hypocalcemia, hyperphosphatemia and hyperuricemia after receiving 200 mg of venetoclax on day 15 of cycle 2. Neither event resulted in interruption or dose modification of study treatment. Rapid reduction in the peripheral lymphocyte count was observed in all 12 patients treated with the combination regimen. Initial response data is anticipated for all 12 patients in the next 6 months per the protocol schedule. CONCLUSIONS The treatment regimen developed for the experimental arm of the CLL14 trial comprising obinutuzumab monotherapy for one cycle followed by venetoclax and obinutuzumab in previously untreated, elderly patients with CLL and coexisting medical conditions appears tolerable. The majority of enrolled patients were older than 70 years of age and many of them had clinically meaningful comorbidities in addition to CLL. None of the protocol defined stopping criteria for the safety run-in phase of the study were met. The randomized phase of the CLL14 trial was opened in August 2015. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Fischer: Roche: Other: Travel Grants. Off Label Use: GAZYVA (obinutuzumab) is a CD20-directed cytolytic antibody and is indicated, in combination with chlorambucil, for the treatment of patients with previously untreated chronic lymphocytic leukemia (CLL). This abstract reports on obinutuzumab in combination with venetoclax for previously untreated CLL. Venetoclax is an investigational drug that is not yet approved in this indication. Fink:Roche: Honoraria, Other: travel grant. Bishop:Roche: Employment. Dixon:Roche: Employment, Equity Ownership. Choi:Gilead: Consultancy, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Other: Advisory Board, Research Funding. Weinkove:Avalia Immunotherapies: Consultancy; Janssen New Zealand: Consultancy; Roche New Zealand: Other: Travel support for conference. Robinson:Lundbeck: Other: Advisory boards; Novartis: Other: Advisory boards; CSL: Other: Advisory boards; Bering: Other: Advisory boards; Roche: Honoraria, Other: Advisory boards; Bayer: Other: Advisory boards; Pfizer: Other: Advisory boards; Celgene: Other: Advisory boards; Biogen Idec: Other: Advisory boards; Gilead: Other: Advisory boards; Janssen: Other: Advisory boards; Baxter: Other: Advisory boards. Dreyling:Roche: Honoraria, Research Funding. Opat:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Subsidised drug. Owen:Gilead: Honoraria; Lundbeck: Honoraria; Janssen: Honoraria; Roche: Honoraria. Tausch:Gilead: Other: Travel support. Ritgen:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Humerickhouse:AbbVie: Employment, Equity Ownership. Humphrey:Roche: Employment. Wenger:Genentech, Inc.: Employment. Goede:Bristol-Myers Squibb: Honoraria; Mundipharma: Honoraria; GSK: Honoraria; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding. Eichhorst:AbbVie: Consultancy; Roche: Consultancy, Research Funding, Speakers Bureau; MundiPharma: Consultancy, Research Funding, Speakers Bureau. Wendtner:AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Genentech, Inc.: Consultancy, Honoraria, Research Funding. Stilgenbauer:Roche: Honoraria, Research Funding. Kipps:Pharmacyclics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau. Hallek:Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Board; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding.

Heterozygous mutations in several core members of the spliceosome complex have been reported in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). In particular high frequency SF3B1 hotspot mutations, a component of the U2 complex involved in the interaction with the branch point (BP) and recognition of the 3' splice sites (ss) during splicing, have been identified in Refractory Anemia with Ringed Sideroblasts (RARS) a subtype of MDS. Using computational analyses of RNAseq from several cancer types including RARS, we identified that SF3B1 hotspot mutations induce aberrant 3'ss selection by recognizing a cryptic AG located between 15 to 24 nucleotides upstream of the canonical AG. Experimental confirmation of these motif features was performed using minigenes in SF3B1 mutant cells. Furthermore, we discovered that SF3B1 mutant utilized a different BP from that used by SF3B1 wild-type providing novel mechanistic insights into changes in function induced by the hotspot mutations. The induction of aberrant splicing can introduce premature termination codons thus targeting mRNA for degradation by Nonsense Mediated Decay (NMD). We predicted that close to 50% of the aberrantly spliced genes would be subject to NMD and showed (using isogenic Nalm-6 cells engineered by AAV homology to express SF3B1K700E or SF3B1K700K) that several of these genes were downregulated at the transcript and protein levels. These downregulated genes/proteins might be involved in the pathogenesis of SF3B1 mutant cancers. Interestingly, pathway analysis of genes differentially expressed or aberrantly spliced in SF3B1 mutant compared to wild-type in RARS samples identified cell differentiation and epigenetics as the primary misregulated pathways. To study the impact of SF3B1 mutations on differentiation, we used the TF-1 differentiation cell model where erythroid differentiation is induced by treatment with erythropoietin (EPO). EPO treatment, as expected, induced erythroid differentiation in TF-1 cells transduced with SF3B1WT, but a block in erythroid differentiation was observed in TF-1 cells transduced with SF3B1K700E (the most common mutation in MDS and chronic lymphocytic leukemia (CLL)). Intriguingly, SF3B1G742D, which is found mutated in CLL but not MDS, did not block differentiation in this myeloid differentiation model, implying that specific SF3B1 mutations and splicing aberrations have important context dependent effects. Pathway analysis comparing SF3B1K700E vs. SF3B1WT or SF3B1G742D identified several genes involved in heme biosynthesis or downstream of GATA1 to be downregulated (such as, AHSP, ALAS2, CCL5, CD36, EPOR, GP1BB, HBB, HBE1, HBG1, PRG2) in SF3B1K700E cells only. This is consistent with the role of SF3B1K700E in RARS. In our analyses, we also identified that ABCB7 is aberrantly spliced and that the aberrant transcript is subject to NMD, causing downregulation of the canonical transcript and protein. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and in heme production; moreover, partial loss of function mutation in ABCB7 has been identified in X-linked sideroblastic anemia and ataxia, demonstrating an iron overload phenotype in cells with defective ABCB7. Interestingly, when ABCB7 was knocked down in TF-1 cells we observed block in differentiation similar to that observed in SF3B1K700E cells suggesting a link between SF3B1 mutation and ABCB7 levels and impaired differentiation. Taken together, these data suggest that SF3B1 mutations induce aberrant splicing and as a consequence downregulation of several genes that contribute to the block in erythroid differentiation, one of the key biological defects observed in MDS. Disclosures Buonamici: H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Agrawal:H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Keaney:H3 Biomedicine: Employment. Kumar:H3 Biomedicine: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Matijevic:Eisai: Employment. Mizui:H3 Biomedicine: Employment. Myint:Eisai: Employment. Park:H3 Biomedicine: Employment. Pazolli:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Furman:Acerta Pharma BV: Research Funding; Gilead: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Ebert:Celgene: Consultancy; H3 Biomedicine: Consultancy; Genoptix: Consultancy, Patents & Royalties. Smith:H3 Biomedicine: Employment.

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by dysplastic hematopoiesis and peripheral blood cytopenias. Recently, somatic mutations affecting components of the spliceosomal machinery have been discovered in the majority of MDS patients. SF3B1 mutations are most frequent and strongly correlate with the presence of bone marrow ring sideroblasts and a favorable prognosis. SF3B1 mutations, including the K700E substitution which accounts for more than 50% of all mutations, are missense, heterozygous and cluster in a hotspot within the heat domain of the protein suggesting that they are gain-of-function variants. The molecular effects of SF3B1 mutations and the mechanisms through which they drive clonal expansion and dyserythropoiesis remain obscure. Therefore, to assess their molecular and phenotypic consequences, we generated a mouse model carrying a conditional floxed knock-in allele (Sf3b1flox-K700E/+) by homologous recombination of JM8 murine embryonic stem cells. To induce expression of Sf3b1 K700E in adult hematopoietic stem and progenitor cells, Sf3b1flox-K700E/+/Mx1-Cre+ were injected with pIpC from 4-8 weeks of age. Here we report the initial characterization of these animals. Monthly peripheral blood counts from mutants and wild-type (WT) littermates starting one month post-pIpC injection showed a reduction in hemoglobin levels (at 8 weeks WT=17g/dl mut=14.5g/dl, p Finally, as Sfb31 mutations are predicted to affect splicing of pre-mRNA and consequently alter the gene expression, we performed RNAseq analysis in unselected and Lin-ve bone-marrow cells from mutant and controls animals. Comparison between wt and mutant samples showed deregulated expression of genes implicated in human MDS (Mmp9, Puma, Bcl2l1). We then looked at the pattern of aberrant splicing promoted by Sf3b1flox-K700E, and found that mutant animals have an increased use of cryptic 3'' splice sites (ss) throughout their genome. We showed that the majority of these alternative 3' ss are novel and we characterized them as being located 15 to 24 nucleotides upstream from the canonical 3' ss and associated with sequence features including a shorter polypyrimidine tract and an enrichment of adenines -8 to -18 bases upstream of the cryptic 3' ss. Interestingly, similar features have been reported in human cancers with SF3B1 hotspot mutations. We predict that ~33% of the mRNAs affected by aberrant splicing will include an aberrant premature termination codon, promoting RNA degradation through nonsense-mediated decay. In conclusion, our conditional Sf3b1K700E knock-in mouse is a faithful molecular model of the consequences of these mutations in the mouse hematopoietic system. The mild phenotype we observe in comparison to SF3B1-mutant human MDS may be explained by the requirement for additional mutations to progress to overt MDS and is more reminiscent of SF3B1-associated clonal hemopoiesis, relatively common phenomenon in elderly humans without overt hematological abnormalities. Additionally, our initial characterization of novel splice sites preferentially recognised by the mutant Sf3b1 protein suggests that transcriptional consequences of the mutation may differ between species, dependant on the degree of conservation of the relevant intronic regions. Disclosures Seiler: H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Campbell:14M genomics: Other: Co-founder and consultant.

The mutation status and usage of specific VH genes such as V3-21 and V1-69 are potentially independent pathogenic and prognostic factors in chronic lymphocytic leukemia (CLL). To investigate the role of antigenic stimulation, we analyzed the expression of genes involved in B-cell receptor (BCR) signaling/activation, cell cycle, and apoptosis control in CLL using these specific VH genes compared to VH mutated (VH-MUT) and VH unmutated (VH-UM) CLL not using these VH genes. V3-21 cases showed characteristic expression differences compared to VH-MUT (up: ZAP70 [or ZAP-70]; down: CCND2, P27) and VH-UM (down: PI3K, CCND2, P27, CDK4, BAX) involving several BCR-related genes. Similarly, there was a marked difference between VH unmutated cases using the V1-69 gene and VH-UM (up: FOS; down: BLNK, SYK, CDK4, TP53). Therefore, usage of specific VH genes appears to have a strong influence on the gene expression pattern pointing to antigen recognition and ongoing BCR stimulation as a pathogenic factor in these CLL subgroups.

Introduction: T-cell prolymphocytic leukemia (T-PLL) is a very rare & aggressive disease. Therapeutic advances during recent years have been very limited. Even after initial response, the vast majority of T-PLL patients (pts) relapse quickly and the median overall survival remains below 2 years (ys). This also holds true for induction therapies with the anti-CD52 antibody Alemtuzumab (A). Furthermore, life-threatening viral infections are a severe problem in pts treated with this antibody. In a precursor trial (Hopfinger et al, Cancer 2013), we evaluated a consolidation therapy with A after polychemotherapy induction. In the T-PLL2 trial, we evaluated feasibility, safety and efficacy of the addition of A s.c. to an induction treatment with Fludarabine, Mitoxantrone & Cyclophosphamide (A-FMC), followed by an A s.c. maintenance therapy. Patients and Methods: 16 pts (12 untreated and 4 with pretreatments) with T-PLL were enrolled between 06/2010-09/2013. Pts received an induction treatment with A 10mg s.c. on day (d) 1-3 combined with 20mg/m2 Fludarabine i.v. d 1-3, 6mg/m2 Mitoxantrone i.v. d1 & 200mg/m2 Cyclophosphamide d1-3. After 2 cycles the A dose was increased to 30 mg s.c., if a stable disease (SD) or a partial remission (PR) was achieved. A-FMC treatment was administered every 28 days for up to 4 cycles, followed by a maintenance treatment with 30mg A s.c. starting 1 month (mo) after final staging. During the first 6 mo, A s.c. was administered monthly and in addition once in mo 10 and 13. For younger and fit pts, the option of allogeneic transplantation (tx) was explicitly recommended after induction therapy. Peripheral blood (PB) samples were taken at diagnosis & at the time of relapse/progression. Valganciclovir was recommended for prophylaxis. Results: 16 pts with a median age of 68 ys (range 32-78) and a median score on the cumulative illness rating scale of 3 (range 0-6) were enrolled. The diagnosis of T-PLL was established based on clinico-pathologic characteristics, with the lead finding of a monoclonal mature T-cell population in PB. All leukaemias (100%) were CD52 positive, 15/16 cases (93.75%) expressed TCL1 & the most frequent abnormalities by FISH/classical karyotyping were aberrations involving 14q32.1 in 14/14 cases (100%), gains of chromosome 8q in 10/14 cases (71%), & deletion 11q23 in 8/14 cases (57%). A median number of 4 courses (range 2-4) were administered for induction treatment. Six pts (37.5%) proceeded with maintenance treatment with a median of 4 (range 1-6) A applications. In total, 94 non-infectious CTC grade 3-4 adverse events (AE) & 28 episodes of CTC grade 1-4 infections were documented. Non-infectious grade 3/4 AEs were most frequently due to myelosuppression: neutropenia/leukopenia occurred in 14/16 pts (87.5%), anaemia in 7/16 pts (43.75%) & thrombocytopenia in 10/16 pts (62.5%). Two cases of cytomegalovirus (CMV) & 1 case of varicella zoster infection were documented. Most AEs could be successfully managed, however 2 (12.6%), possibly treatment related deaths occurred, both from fatal bleeding in thrombocytopenia. In all pts response data after induction treatment was available. The overall response rate was 68.75% (11/16pts) with complete remissions (CR) in 4 pts (25%), CR with insufficient bone marrow recovery in 1 pt (6.25%) & a PR in 6 pts (37.5%). Progressive disease (PD) was documented in 4 pts (25%), a SD in 1 pt (6.25%). The trial was terminated in May 2014. Until today, 11 deaths (68.75%) were reported & 1 pt (6.25%) was lost to follow-up. Prophylaxis with Valgancyclovir was administered in 12/16 pts (75%), 2 pts (12.5%) received prophylactic Valaciclovir & 2 pts (12.5%) received no viral prophylaxis at all. Seven pts (43.75%) received an allogeneic tx after the study treatment: 3 pts after the induction phase, 1 pt after 5 maintenance applications & 3 pts after bridging/salvage therapy. Tx outcomes were documented with 5 CRs & 2 PDs. Conclusion: A s.c. combined with FMC & A maintenance therapy is a relatively safe and feasible regimen in pts with T-PLL. However, safety seems to come at the cost of response quality. Therefore, the authors currently recommend using A i.v. as part of the initial therapy for T-PLL pts. Prophylaxis with Valganciclovir was effective in preventing CMV infection, one of the major threats to pts treated with A. Most importantly, the study emphasizes the need for new therapies and intensified clinical research for pts with T-PLL. Disclosures Off Label Use: Therapeutic off-label use of Alemtuzumab, Fludarabine, Cyclophophamide and Mitoxantron in T-PLL. Hopfinger:Genzyme: Research Funding. Weit:Beckman Coulter GmbH, Krefeld, Germany: Employment. Stilgenbauer:Genzyme : Consultancy, Honoraria, Research Funding. Eichhorst:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grant Other; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grant, Travel grant Other; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy. Hallek:Genzyme, Bayer: Research Funding.

Natural killer cell lymphoma (NKL) is a highly aggressive form of hematolymphoid malignancy that arises from natural killer cells, a critical component of the innate immune system. Early diagnosis of the tumor is critical however there are no biomarkers which are useful currently for the diagnosis and monitoring of patients with NKLs. We hypothesized that comprehensive analysis of differentially expressed proteins by NKL and normal NK cells would be a rational strategy for the discovery of NKL biomarkers and lead to better understanding of pathogenetic mechanisms of NKL. We used total cell lysates obtained from highly enriched peripheral blood NK cells (CD3-, CD56dim) and an Epstein-Barr virus negative, IL-2 dependent cell line (NK-92MI) for quantitative proteomic analysis. Isotopecoded affinity tags (ICATTM)-labeling followed by liquid chromatography-tandem mass spectrometry analyses (LC-MS/MS) identified 114 differentially expressed proteins in NKL compared to the normal NK cells. Out of these, 80 were upregulated 1.5-fold or greater and 34 were downregulated 1.5-fold or greater in NKL. Proteins within diverse cellular functional categories including cell signaling, transcriptional regulation, DNA metabolism, and immune response were differentially expression. Furthermore, known proteins relevant to the function of NK cells such as perforin, granzyme and defensins were downregulated in the NKL cell line. We selected eight proteins to validate the changes identified by ICAT-LC-MS/MS using Western blot analysis. HSP90, PCNA, alpha-enolase, GSTP1, galectin-1, and PARK7 were upregulated, and lactoferrin and annexin A1 were downregulated by western blot analysis demonstrating high concordance with the results of ICAT-LC-MS/MS analysis. We validated the functional significance of HSP90 as a potential therapeutic target in NKL by investigating the effect of inhibitors of HSP90 (geldanamycin) on the survival of an NKL cell line. Inhibition of HSP90 led to significant decrease in cell viability and apoptotic cell death as confirmed by western blotting demonstration of PARP-1 induction and detection of its cleavage products. This study represents the first global quantitative proteomic cataloguing of proteins expressed by NKL and normal NK cells, and reveals potential candidate proteins that may be useful for the diagnosis and function as therapeutic targets in patients with NKL.