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SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5′ UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non–rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation.

Resistance to therapy is one of the most significant challenges in the treatment of acute myeloid leukemia (AML). While great efforts have uncovered genetic mechanisms of resistance to certain AML-directed therapies, to date, treatment resistance in AML has only partially explained by acquired genetic alterations. Here, we performed genome-wide CRISPR/Cas9 screens to identify drug-gene interactions that modulate therapeutic response to treatments commonly used in AML. Interestingly, our findings uncovered several genes that regulate pre-mRNA splicing whose loss strongly synergized with venetoclax, a BH3 mimetic that blocks the antiapoptotic protein BCL-2. To further delineate the role of RNA processing in response to AML treatments, we performed secondary CRISPR screens with a domain-focused gRNA library targeting 490 RNA processing factors in the presence of various AML drugs. Overall, these genetic screens identified a number of RNA splicing factors whose loss-of-function sensitized AML cells to BCL2 inhibition (Fig. A). Among the top gene candidates whose loss promoted venetoclax efficacy was the splicing factor RBM10 (Fig.B). Strikingly, loss of RBM10 exclusively synergized with venetoclax-based treatments across AML therapeutics, including in TP53 mutant lines (Fig.C-D). Moreover, RBM10 loss restored venetoclax sensitivity to AML cell line variants with acquired venetoclax resistance. Interestingly, while many RNA splicing factors are pan-essential, generation of an Rbm10 conditional knockout mouse revealed that Rbm10 is completely dispensable for steady-state normal hematopoiesis (Fig.E). Since RBM10 has not been studied previously in hematopoiesis, we mapped the impact of RBM10 on mRNA expression and splicing using RNA-seq and direct RNA binding partners genome-wide by eCLIP-Seq (Fig. F). RBM10 loss was strongly associated with downregulation of BCL2A1, an anti-apoptotic factor whose expression is correlated with venetoclax resistance in AML (Fig.G-H). This was dependent on RBM10's ability to bind RNA and expression of BCL2A1 cDNA fully rescued the growth-inhibitory effect of RBM10 KO-venetoclax treated AML cells. Overall, the above data support RBM10 as a synthetic lethal vulnerability in venetoclax therapy. Beyond RBM10, our genetic screens also identified several splicing factors belonging to the family of serine and arginine-rich (SR) proteins whose loss synergized with venetoclax treatment (Fig. I). SR proteins are essential for pre-mRNA splicing and are substrates for phosphorylation by conserved family of kinases, such as Cdc2-like kinases (CLKs) and (dual-specificity tyrosine-regulated kinases) DYRKs. We therefore utilized a series of selective pan-CLK/DYRK1A inhibitors, including SM09419 and SM08502, that potently suppress SR protein phosphorylation. Interestingly, BCL2 is one of the top genetic dependencies upon DYRK1A genetic suppression in prior work from the DepMap (Fig. J). Pharmacologic inhibition of CLK/DYRK1A exhibited high in vitro efficacy at nanomolar range across a diverse range of AML subtypes including cell lines with acquired venetoclax resistance (Fig.K). Consistent with this, combined SM09419 and venetoclax displayed synergistic anti-leukemic effects and venetoclax-sensitive AML cell lines (Fig.L). Taken together, these data support the notion of targeting CLK/DYRK1A in the context of BCL2 inhibition. In this study, we systematically defined gene interactions that mediate the response to a wide range of AML drugs. Recent studies have begun to show that dysfunctional RNA processing promotes AML development. However, the role of RNA processing in modulating drug responsiveness in AML is not well understood. Here, we have uncovered that synthetic lethal targeting of splicing factors, such as RBM10, increases sensitivity of AML cells to BCL2 inhibition. Therapeutically, pharmacologic inhibition of SR protein function via inhibiting CLK/DYRK1A-mediated phosphorylation of splicing factors is an effective strategy used in combination with venetoclax or to overcome venetoclax resistance. Overall, our findings underscore the central importance of RNA splicing in drug response and provides a therapeutic rationale for modulating RNA splicing to enhance current AML therapies. Figure 1 Figure 1. Disclosures McMillan: Prizer: Ended employment in the past 24 months. Bossard: Biosplice Therapeutics: Current Employment. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Mutations in RNA splicing factors are amongst the most common genetic alterations in myeloid malignancies. Mutations in the splicing factors SF3B1, SRSF2, and U2AF1 occur as heterozygous, missense mutations and have been shown to confer a change-of-function. In contrast, the X chromosome encoded ZRSR2 is enriched in nonsense/frameshift mutations in males, consistent with loss of function. To date however, we do not understand the basis for enrichment of ZRSR2 mutations in leukemia. Moreover, ZRSR2 is the only one of these factors that primarily functions in the minor spliceosome. While most introns are spliced by the major spliceosome, a small subset ( The rarity and conservation of minor introns offered a unique opportunity to investigate splicing factor mutations and identify potential tissue-specific roles of the minor spliceosome. Modeling loss-of-function mutations in ZRSR2 via a mouse model for induced deletion of Zrsr2 revealed strikingly enhanced self-renewal of Zrsr2-deficient male and female hematopoietic cells (Fig. A-C). This was in stark contrast to the effects of hotspot mutations in Sf3b1and Srsf2 and similar to those of Tet2 loss on increasing self-renewal and numbers of HSCs. Zrsr2 loss was also associated increased myeloid cells in the blood and long-term hematopoietic stem cells (HSCs) in the marrow (Fig. C). To understand the mechanistic basis by which ZRSR2 loss causes aberrant HSC self-renewal, we quantified transcriptome-wide splicing patterns in MDS patients. ZRSR2-mutant samples had widespread, dysfunctional recognition of minor introns- 48% of minor introns exhibiting significantly increased retention (Fig. D). We next systematically mimicked the effects of nonsense-mediated decay caused by minor intron retention in ZRSR2-mutants. Every gene containing a ZRSR2-regulated minor intron was targeted by 4 sgRNAs via a positive-enrichment CRISPR screen using pools of lentiviral sgRNAs in cytokine-dependent human and mouse hematopoietic cell lines. This identified several minor intron-containing genes whose downregulation conferred cytokine independence. Strikingly, just one gene was enriched in all lines (Fig. E): LZTR1, a cullin-3 adaptor for ubiquitin-mediated suppression of RAS-related GTPases which is subject to loss-of-function mutations in several cancers and the RASopathy Noonan Syndrome. Minor intron retention in LZTR1 correlated with reduced LZTR1 protein in MDS patients (Fig. F-G). Inducing mutations in either the protein-coding region of LZTR1 or its minor intron resulted in cytokine independence (Fig. H), reduced LZTR1, and dramatic accumulation of RIT1, a RAS GTPase substrate of LZTR1. In a Noonan Syndrome family wherein one child died of AML, the mother and all children carried an intronic mutation within LZTR1's minor intron (Fig. I-J). Fibroblasts from each family member revealed clear LZTR1 minor intron retention with impaired LZTR1 protein expression and RIT1 accumulation in subjects bearing the LZTR1 minor intron mutation (Fig. J). We next interrogated LZTR1 minor intron splicing across all cancers in the TCGA. While LZTR1's minor intron was efficiently excised in normal samples, a notable subset of tumors in almost all cancer types exhibited significantly increased retention within LZTR1's minor intron. These data indicate LZTR1 is frequently dysregulated via perturbed minor intron splicing - much more so than by protein-coding mutations alone. Here we uncover a heretofore unrecognized role of minor intron excision in regulating HSC self-renewal, a molecular link between ZRSR2 mutations and aberrant LZTR1 splicing and expression, and frequent LZTR1 minor intron retention in diverse cancers and cancer predisposition syndromes. Given frequent post-transcriptional disruption of LZTR1 in the absence of protein-coding mutations, our data additionally motivate study of other cancer-associated minor intron-containing genes which may be dysregulated via similar, and as-yet-undetected, aberrant splicing. Figure Disclosures Abdel-Wahab: Merck: Consultancy; Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy.

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous gain-of-function mutations in a core U2 spliceosome factor SF3B1 are present in ~25% of myelodysplastic syndrome (MDS) patients. Strikingly, SF3B1 mutations are present in ~80% of MDS with ring sideroblasts (MDS-RS) patients suggesting a causal connection between mutant SF3B1 and ring sideroblasts (RS), erythroid precursors with iron-laden mitochondria (Yoshida et al. Nature 2011, Papaemmanuil et al.NEJM 2011). However, the mechanism by which SF3B1 mutations cause RS formation remains poorly understood since existing models of SF3B1-mutant MDS do not recapitulate RS formation in vitro. We have established an induced pluripotent stem cell (iPSC) model of MDS-RS that recapitulates mutant SF3B1-mediated mis-splicing and in vitro ring sideroblast formation. We reprogrammed SF3B1-mutant and SF3B1-wild-type iPSCs from individual MDS-RS patients enabling internal normalization to the isogenic normal clone (Hsu et al. Blood 2019). We established expandable multipotential HPC lines by conditional expression of five transcription factors (5F-HPC), followed by an 18-day erythroid differentiation. We monitored RS formation during the erythroid differentiation of 5F-HPCs using Prussian blue histological staining. RS formation increased during terminal erythropoiesis and peaked at 25-40% on day 18 in SF3B1-mutant lines, whereas SF3B1-wild-type lines showed no detectable RS formation. The frequency of RS was similar in isogenic SF3B1-only and SF3B1/EZH2 co-mutant cells suggesting that the mutant SF3B1 is sufficient to drive RS formation. To identify mis-splicing events that contribute to RS formation, we performed RNA-sequencing and splicing analysis of SF3B1-mutant and SF3B1-wild-type iPSCs at three stages of erythroid differentiation: CD34+ progenitor, CD71+ early erythroblast, and CD71+Glycophorin A+ erythroblast. Global isoform usage was dramatically altered during erythropoiesis, but was more similar in stage-matched SF3B1-mutant and SF3B1-wild-type cells suggesting that mutant SF3B1 selectively mis-splices a subset of transcripts. We identified 2300 transcripts with >10% mis-splicing in SF3B1-mutant lines, and only 120 transcripts with more significant >40% mis-splicing. Of these, TMEM14C and PPOX, inner mitochondrial membrane components of the heme synthesis pathway were strongly mis-spliced throughout erythroid differentiation, consistent with previous studies (Conte et al.BJHaem 2015, Shiozawa et al. Nat Commun 2018). The transcript levels of PPOX but not TMEM14C were reduced in SF3B1-mutant lines as a result of mis-splicing. The expression of ABCB7, a mitochondrial iron sulfur cluster biogenesis component mutated in inherited X-linked sideroblastic anemia (Allikmets et al. Hum Mol Genet 1999), was also reduced in SF3B1-mutant cells as expected (Shiozawa et al. Nat Commun 2018, Dolatshad et al. Leukemia 2016). To investigate the role of these mis-splicing events in ring sideroblast formation, we performed lentiviral overexpression of TMEM14C, PPOX, and ABCB7, in SF3B1-mutant 5F-HPCs and quantified RS formation during late stages of erythroid differentiation. Overexpression of TMEM14C and ABCB7 in SF3B1-mutant cells partially but not completely rescued RS formation compared to luciferase control. These findings confirm the long-standing hypothesis that mis-splicing of mitochondrial iron metabolism genes causes RS formation. Furthermore, these findings suggest that RS formation in MDS is a multigenic event caused by coordinated but incomplete mis-splicing of several critical iron metabolism genes. Disclosures No relevant conflicts of interest to declare.

Glucocorticoids, such as dexamethasone and prednisone, are mainstays in the treatment of lymphoid malignancies and autoimmune diseases. To understand the mechanism of how glucocorticoids induce cell death, we integrated a comprehensive, genome-wide shRNA screen with differential gene expression analysis in B-cell acute lymphoblastic leukemia (B-ALL). In addition to activating pro-apoptosis genes and repressing anti-apoptosis genes, we found that repression of B-cell development genes contributes to dexamethasone-induced cell death in B-ALL. Similarly, elevated glucocorticoid levels brought on by stress in stroke victims causes lymphocytopenia by inducing of apoptosis in immature B-cells, but not hemopoietic stem cells or mature B-cells. Additionally, chronic stress has been associated with immune suppression and increased risk for autoimmune disease. Together these findings suggest that glucocorticoids play a normal role in B-cell development, and that elevated glucocorticoid levels in response to stress can alter that development. A control point in this mechanism may be MBNL1. MBNL1 is a sequence-specific RNA binding factor that alters mRNA splicing, stability, and localization. In this work, we show that glucocorticoid suppression of MBNL1 contributes to induction of cell death in B-ALL. Deletion of MBNL1 by CRISPR/Cas9 in the B-ALL cell line NALM-6 induces accumulation and depletion of mRNA levels for hundreds of genes. A large number of B-cell specification genes, including CD19, CD79A, SPI1, and LEF1, are repressed upon MBNL1 deletion, whereas the most highly activated gene is CD34, which is a marker for the less mature lymphoid progenitor cells. Depletion of MBNL1 also changed the splicing of B-cell development genes, including quantitative depletion of LEF1 exon 6. These data suggest that depletion of MBNL1 induces de-differentiation of B-cell precursors. Consistent with this model, an analysis of gene expression in differentiating B-cells revealed a rapid elevation of MBNL1 expression during B-cell specification (after the lymphoid precursor stage), and a concomitant repression of CELF2, an RNA-binding splicing factor that has been shown to oppose MBNL1. We therefore propose that glucocorticoids play a physiological role in B-cell development by restraining B-cell specification through repression of key genes, including MBNL1. Figure Disclosures Tasian: Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corportation: Research Funding.

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is widely appreciated to be a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous mutations in a core U2 spliceosome factor SF3B1 are strongly associated with MDS with ring sideroblasts (MDS-RS). MDS-RS is characterized by iron retention in mitochondria and ineffective erythropoiesis. How aberrant splicing induced by mutant SF3B1 causes ring sideroblasts and erythroid defects remains poorly understood. There are no genetically accurate models of MDS-RS that recapitulate ring sideroblast formation. Our lab has established an iPSC reprogramming approach to generate an in vitro model of MDS-RS. This method captures genetically distinct MDS subclones as well as normal cells in individual patients enabling internal normalization to the patient's isogenic normal clone. By differentiating iPSCs we generated inducibly immortalized MDS-RS CD34+ progenitor cell lines with robust expansion and erythroid differentiation potential enabling extensive analysis of SF3B1-mutant aberrant splicing throughout erythroid differentiation. We show that SF3B1-mutant lines form abundant ring sideroblasts compared to normal isogenic controls. We next performed RNA-sequencing and splicing analysis at different stages of erythroid differentiation. We show that global splicing patterns are maintained in SF3B1-mutant cells, while revealing distinct subsets of aberrantly spliced transcripts in CD34+ progenitors and erythroblast populations. This suggests that different aberrant splicing events contribute to erythroid progenitor expansion and inefficient erythropoiesis and iron dysregulation in MDS-RS. Taken together, this novel iPSC model of MDS-RS is a robust tool for studying the role of aberrant splicing in SF3B1-mutant erythroid progenitor expansion, inefficient erythropoiesis and ring sideroblast formation. Disclosures No relevant conflicts of interest to declare.

Mutations in the RNA splicing factor SF3B1 are common in MDS and other myeloid malignancies. SF3B1 mutations promote expression of mRNAs that use an aberrant, intron proximal 3' splice site (ss). Despite the consistency of this finding, linking aberrant splicing changes to disease pathogenesis has been a challenge. Here we identify aberrant splicing and downregulated expression of BRD9, a member of the recently described ATP-dependent non-canonical BAF (ncBAF) chromatin remodeling complex, across SF3B1 mutant leukemias. In so doing, we identify a novel role for altered ncBAF function in hematopoiesis and MDS. To systematically identify functionally important aberrant splicing events created by mutant SF3B1, we integrated differential splicing events in SF3B1 mutant versus wild-type MDS with a positive enrichment CRISPR screen mimicking splicing changes induced by mutant SF3B1 that promote NMD (non-sense mediated mRNA decay). We tested whether loss of any gene functionally inactivated by SF3B1 mutations promoted transformation of Ba/F3 and 32D cells. This identified a specific NMD-inducing aberrant splicing event in BRD9 which promoted cytokine independence (Fig. A) and exhibited striking aberrant splicing across CLL and MDS and across all mutational hotspots in SF3B1 (Fig. B). SF3B1 mutations cause exonization of a normally intronic sequence in BRD9, resulting in inclusion of a poison exon that interrupts BRD9's reading frame (Fig. C) and reduced BRD9 mRNA and protein expression through NMD (Fig. D). We confirmed that mutant SF3B1 suppressed full-length BRD9 levels without generating truncated BRD9 protein. Loss of BRD9 impaired ncBAF complex formation as indicated by abolished interaction between the ncBAF specific component GLTSCR1 and the ATPase subunit BRG1 upon chemical or spliceosomal BRD9 ablation (Fig. D). Given that prior work has linked mutant SF3B1 to use of aberrant 3' ss, we sought to understand the molecular basis for aberrant exon inclusion in BRD9 by mutant SF3B1. Lariat sequencing of SF3B1 mutant versus WT K562 cells and BRD9 minigene analyses identified use of a deep intronic branchpoint adenosine by mutant SF3B1 to promote BRD9 poison exon inclusion (Fig. E). The data above suggest a role for BRD9 downregulation in SF3B1 mutant leukemia. While prior work indicated that BRD9 is required in MLL-rearranged AML (Hohmman et al. Nature Chemical Biology 2016), the role of BRD9 in normal hematopoiesis or other subtypes of myeloid neoplasms has not been evaluated. Genetic downregulation of BRD9 in normal human hematopoietic progenitors from cord blood promoted myelopoiesis while impairing megakaryopoiesis. Interestingly and unexpectedly, BRD9 loss in CD34+ cells promoted terminal erythroid differentiation in vitro. To further evaluate BRD9's role in hematopoiesis in vivo, we also generated mice with inducible knockout of the bromodomain of BRD9 (required for BRD9 function) and generation of a frameshift transcript resulting in reduced Brd9 expression (Fig. F). Loss of Brd9 resulted in macrocytosis with bone marrow erythroid dysplasia in a dosage-dependent manner, along with impaired lymphopoiesis and myeloid skewing. Moreover, competitive transplantation of hematopoietic precursors from these mice revealed that ablation of Brd9 function impaired lymphoid reconstitution while promoting advantage of myeloid cells and hematopoietic precursors (Fig. G-I). In myeloid leukemia cells, introduction of SF3B1K700E or downregulation of BRD9 resulted in increased chromatin accessibility at promoters with a significant overlap in commonly upregulated genes. This finding suggests shared epigenetic effects of SF3B1K700E mutations and BRD9 loss (Fig. J). These data identify aberrant splicing of BRD9 across the spectrum of SF3B1 mutant cancers and identify a novel role for downregulation of ncBAF function in MDS pathogenesis. Consistent with human genetic data, genetic ablation of BRD9 function in mouse and human hematopoietic cells resulted in myeloid skewing and dyserythropoiesis. These data suggest that targeted correction of aberrant BRD9 splicing might serve as a novel therapeutic approach for SF3B1-mutant leukemias. Of note, treatment with drugs impairing the binding of mutant SF3B1 to RNA resulted in a dose-dependent rescue of aberrant BRD9 splicing in vitro (Fig. K) and in treatment of an SF3B1 mutant AML patient-derived xenograft in vivo. Figure Disclosures Kadoch: Foghorn Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Mutations in genes encoding RNA splicing factors constitute the most common class of genetic alterations in patients with myelodysplastic syndromes (MDS). These occur as heterozygous point mutations at specific amino acid residues in SF3B1, SRSF2, and U2AF1, and are almost always mutually exclusive with one another. Recent studies have identified that mutations in each of these genes results in activation of the innate immune signaling through altered splicing of mRNAs encoding key enzymes in this pathway. Now, through an unbiased genetic screen as well as focused genetic studies, we have identified that SF3B1-mutant MDS depends on aberrant immune signaling for cell survival. Recent work has identified that aberrant splicing of MAP3K7 (also known as TAK1; TGF-b Activating Kinase 1) is pervasive across SF3B1-mutant human and mouse cells and results in reduced MAP3K7 protein expression and increased NF-κB signaling. Consistent with this, Map3k7 haploinsufficiency in myeloid cells is known to cause myeloproliferation, while at the same time, complete loss of Map3k7 is intolerable for hematopoietic cells. We therefore hypothesized that partial inhibition of MAP3K7 might preferentially impact SF3B1-mutant cells. To test this hypothesis, we generated mice with inducible deletion of 1 or 2 copies of Map3k7 (Mx1-cre Map3k7fl/+,Mx1-cre Map3k7fl/fl) alone or in the presence of mutant Sf3b1K700E (Mx1-cre Map3k7fl/+Sf3b1K700E/+,Mx1-cre Map3k7fl/flSf3b1K700E/+), along with all controls (Mx1-cre Sf3b1+/+ Map3k7+/+ (Wildtype; WT) and Mx1-cre Sf3b1K700E/+ mice). We then performed bone marrow transplantation (BMT) to assess the effect of Map3k7 deletion on aberrant hematopoiesis driven by mutant SF3B1. Consistent with prior reports, heterozygous deletion of Map3k7did not affect repopulating potential in BMT assays compared to controls while homozygous deletion of Map3k7 resulted in complete failure of hematopoiesis (Figure A). Interestingly, however, in the presence of Sf3b1K700E mutation, deletion of a single copy of Map3k7 completely rescued the hematopoietic defects characteristic of mutant SF3B1 in both mature and immature cells (Figure B-C). These data suggest that inhibition of residual MAP3K7 function may preferentially target SF3B1-mutant MDS cells. In parallel to the above studies, we also performed a negative selection RNAi screen to uncover novel genetic dependencies in SF3B1-mutant myeloid neoplasms. We performed pooled lentiviral infection of shRNAs targeting ~2,200 genes encoding proteins which are drug targets ("The Druggable Genome") under the control of a doxycycline-inducible vector in isogenic K562 cells expressing the two most commonly occurring SF3B1 mutations, SF3B1K666N and SF3B1K700E, from the endogenous SF3B1 locus. Two individual clones per SF3B1-mutant line were used to improve the robustness of the screen. On Day 21 following shRNA activation, genes with ≥3 shRNAs depleted in SF3B1-mutant cells while remaining unchanged in parental K562 cells were selected. This identified 101 candidates that are potentially synthetic-lethal with SF3B1 mutation (Figure D). Interestingly, pathway analysis of these potential candidates revealed of genes involved in immune and inflammatory signaling as well as in metabolic processes (Figure E). Further target validation was performed using in vitro competitive growth assay in K562 cells, and another set of SF3B1 isogenic lymphoid leukemia cell lines (NALM-6) expressing the same mutations. This revealed consistent dependency of SF3B1-mutant cells on STAT1, an essential component of the interferon (IFN) signaling pathway (Figure F). Upon exposure to Type-I IFNs, SF3B1-mutant K562 cells showed increased transcriptional response in IFN-responsive genes containing interferon stimulated response elements (ISREs) compared with SF3B1 WT cells (Figure G). These data highlight that SF3B1-mutant cells are hyper-responsive to IFN signaling and require intact IFN-signaling responses for cell survival. Taken together, the above studies indicate that sustained IFN signaling as well as activated innate immune signaling downstream of TAK1 are required for the survival of SF3B1-mutant myeloid cells. These results therefore have important therapeutic implications as they suggest that pharmacologic inhibition of STAT1/Type I IFN activation and/or TAK1 may serve as important therapeutic agents for SF3B1-mutant MDS. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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.

Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. Mutations in the spliceosomal component SF3B1 (Splicing Factor 3B, subunit 1) are prevalent in MDS, but how this leads to aberrant hematopoiesis is unclear. We elucidated the in vivo consequences of sf3b1 misregulation on hematopoiesis and splicing using a zebrafish sf3b1 (sf3b1hi3394) loss-of-function mutant. Primitive erythropoiesis initiates normally in sf3b1 mutants, as evidenced by normal expression of scl (stem cell leukemia), gata1 and beta-globin, but maturation is defective as shown by decreased hemoglobinization and an increase in immature erythroblasts at 48 hours post fertilization (hpf). Similarly, primitive myelopoiesis initiates normally as early expression of scl and pu.1 are normal, but expression of the differentiation markers l-plastin and myeloperoxidase are diminished in mutants at 24 and 28 hpf. Hemogenic endothelial and HSPC markers runx1 and gata2b are also diminished, while the pan-endothelial marker kdrl (kinase insert domain receptor like) and aorta-specific markers notch1b and notch3 are normally expressed at 24 hpf, indicating hematopoietic induction is more sensitive to sf3b1 loss than vascular formation. RNA-seq analysis of sf3b1 mutants revealed large-scale splicing defects and alterations, particularly affecting introns with suboptimal splice sites. Comparison of RNA-seq data from zebrafish sf3b1 mutants and MDS patients harboring SF3B1 mutations revealed a significant overlap in misspliced genes and in the corresponding affected pathways, including many pathways implicated in MDS pathology such as TGFβ (Transforming Growth Factor-Beta) and immune signaling. Together, these results show conservation between zebrafish mutants and human cells with SF3B1 mutations. We next took a chemical genetic approach and discovered sf3b1 mutants were more sensitive than wild type siblings to treatment with E7107, a small molecule inhibitor of the spliceosome, indicating a potential therapeutic window for spliceosome modulators in MDS. Motivated by this finding, we performed a large-scale chemical modifier screen and identified many new lead compounds (6 out of 562 screened) that also show synthetic lethality in sf3b1 mutants. These small molecules are enriched in those with anti-inflammatory properties, suggesting aberrant immune signaling could underlie some of the defects in sf3b1 mutants. Together, our data demonstrate the molecular and cellular consequences of sf3b1 loss in vivo and establish sf3b1 zebrafish mutants as a novel model for discovery of therapeutics for MDS. Disclosures Smith: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment.

A major recent advance for the biology of Myelodysplastic Syndrome (MDS) was the discovery of recurrent mutations in genes encoding splicing factors (SFs). SRSF2 is a SF that binds to RNA sequences called exonic splicing enhancers (ESEs) to promote inclusion of exons containing these motifs. Mutations of SRSF2 are found in 20-30% of MDS patients, are associated with adverse prognosis and are almost always heterozygous missense mutations (P95 L/R/H). This strongly suggests a gain or alteration of function mechanism, corroborated by recent findings by us and others in a knockin mouse model and in K562 cells expressing mutant SRSF2, showing that the SRSF2 P95H mutation changes the normal RNA binding specificity of SRSF2 (Kim et al. Cancer Cell, 2015; Zhang et al. PNAS, 2015). While mis-splicing of hematopoietic regulators, such as EZH2, was proposed as a potential downstream mechanism of SF mutations, systematically identifying the key downstream mediators remains a challenge. No human hematopoietic cell lines harboring SF mutations in the context of a normal diploid genome exist, primary MDS cells afford limited experimental opportunities and mouse modeling is likely to miss disease relevant targets, as only a quarter of regulated alternative splicing events are conserved between human and mouse. Our lab has pioneered the modeling of MDS with human induced pluripotent stem cells (iPSCs). We previously derived iPSCs with the SRSF2 P95L mutation and a chr7q deletion (del7q), as well as normal iPSCs from residual normal hematopoietic cells from the same MDS patient (Kotini et al. Nat. Biotech, 2015). To develop a model of mutant SRSF2, we used CRISPR/Cas9 technology to generate a panel of isogenic iPSCs with or without the SRSF2 P95 mutation, isolated or with the cooperating del(7q), all in the same genetic background, by both introducing the SRSF2 P95L mutation in normal iPSCs and correcting it in MDS-iPSCs from the same patient. Both patient-derived and genetically engineered SRSF2 P95L-iPSCs showed decreased growth and increased cell death of hematopoietic progenitor cells (HPCs), decreased clonogenic capacity and features of morphologic dysplasia. This phenotype is consistent with the SRSF2 mutation being an early, potentially initiating, event, supported by its frequent presence in the dominant clone and in individuals with clonal hematopoiesis of indeterminate potential (CHIP) without overt MDS. Using a competitive cell growth assay, we found that the splicing inhibitor E7107, as well as small molecule inhibitors of kinases modulating splicing, preferentially inhibit the growth of SRSF2 mutant, but not of isogenic normal, iPSC-derived HPCs. To investigate the effects of mutant SRSF2 in mRNA splicing, we performed RNA sequencing of purified CD34+, CD34+/CD45+ HPCs and undifferentiated iPSCs from mutant and isogenic wild-type (WT) iPSCs. SRSF2 mutant cells exhibited genome-wide alterations in ESE preferences, recapitulating the altered RNA binding found in patient cells, with mutant SRSF2 preferentially recognizing a CCNG motif versus a GGNG motif, while WT SRSF2 binds to both with similar affinity. Genes found mis-spliced in the SRSF2 mutant cells included previously reported genes of potential disease relevance, like EZH2 and FYN. While the majority of differentially spliced genes overlapped among the 3 cell states, cell type-specific differences were also noted, highlighting the importance of performing these analyses in the appropriate cell type. Importantly, iPSC-derived HPCs recapitulated a higher percentage of the mis-spliced events observed in patient cells than either the knockin mouse or the K562 models, thus capturing disease-relevant splicing alterations more faithfully than other models. To identify critical direct targets of mutant SRSF2, we used a second round of CRISPR/Cas9 gene editing to introduce a 3xFLAG epitope tag at the carboxyl terminus of the endogenous SRSF2 locus in isogenic SRSF2mutant and WT iPSCs. CLIP-seq experiments in undifferentiated cells and HPCs derived from them are underway to identify genes differentially bound by the mutant vs the WT SRSF2. The model we describe here will be valuable for dissecting the pathogenesis of MDS with SF mutations, testing drugs and identifying new therapeutic targets for drug development. Disclosures No relevant conflicts of interest to declare.

Mutations in the spliceosomal genes SRSF2, U2AF1 and SF3B1 are commonly found in patients with myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations occur at highly restricted amino acid residues, are always heterozygous, and rarely co-occur with one another. These data suggest that splicing factor mutations confer an alteration of splicing function and/or that cells may only tolerate a certain degree of splicing modulation. Consistent with this, it has been recently shown that mice expressing the heterozygous Srsf2P95H mutation develop MDS-like features due to altered mRNA recognition. To begin to identify therapeutic vulnerabilities created by spliceosomal gene mutations, we first generated Mx1-Cre Srsf2P95H/KO mice to study the effects of wildtype Srsf2 deletion with concomitant activation of the Srsf2P95H mutant allele. In noncompetitive transplantation, hemizygous Mx1-Cre Srsf2P95H/KO mutant mice exhibited severe bone marrow failure due to complete loss of hematopoietic stem/progenitor cells (HSPCs), suggesting that Srsf2P95H cells depend on wildtype Srsf2 for survival (Fig. 1A). RNA-seq analyses revealed an increase in mis-splicing due to altered mRNA recognition in Srsf2P95H/KO hemizygous mutant HSPCs compared with heterozygous Srsf2P95H/+ mice, which resulted in strong repression of genes involved in hematopoietic self-renewal, most notably Runx1, Erg, and the entire HoxA cluster. Based on these observations, we hypothesized that spliceosomal-mutant leukemias might display greater sensitivity to pharmacologic modulation of splicing than their wildtype counterparts. To address this, we tested the efficacy of a known spliceosome modulator, E7107, in myeloid leukemias with or without splicing factor mutations in vivo. We created a murine leukemia model using MLL-AF9 overexpression on either Srsf2+/+ or Srsf2P95H/+ background, a mutational combination which represents ~10% of adult MLL-rearranged leukemias (LavallŽe et al., 2015). In vivo E7107 treatment resulted in decreased disease burden and ultimately survival benefit in Srsf2P95H/+ mice, whereas the same treatment regimen had no impact on overall survival of Srsf2+/+ mice (Fig. 1B). RNA-seq analysis of leukemic cells after 5 consecutive days ofE7107 treatment in vivo revealed massive global splicing inhibition in response to E7107 in both genotypes, most notably global cassette exon skipping and retention of both constitutive and alternative introns. E7107-induced splicing inhibition was more severe in Srsf2P95H/+ versus Srsf2+/+ mice, consistent with its differential effect on survival in these 2 genotypes. Interestingly, among the most differentially spliced genes in Srsf2P95H/+ mice in response to E7107 are genes involved in maintaining the leukemogenic programs in MLL-rearranged leukemias, including Dot1l, Meis1 and Lsd1 (Fig. 1C). To confirm that preferential sensitivity of spliceosomal-mutant myeloid leukemias to splicing modulation is directly translatable to a human context, we next generated patient-derived xenografts (PDX) from AML patients wildtype (n=2) or mutant (n=3) for splicing factors. 10 mice were successfully engrafted per patient via tail-vein injection in NSG mice and treated with vehicle or E7107 for 10 days once human bone marrow chimerism was >50%. In each PDX model, targeted genomic analysis of human leukemic cells confirmed faithful engraftment of the major leukemic clones as found in patients pre-engraftment and that spliceosomal mutations were present in the major leukemic clone engrafted in mice. All spliceosomal mutant AML PDXs showed significant reductions in human leukemic burden in response to E7107 while the response in splicing wildtype AML cells was less robust (Fig. 1D). Collectively, these data provide both genetic and pharmacologic evidence that splicing factor mutant leukemias are preferentially sensitive to splicing modulation in vivo compared with spliceosomal wildtype counterpart cells. This has important therapeutic implications for the wide range of patients with MDS and AML bearing these alterations. Figure 1. Figure 1. Disclosures Thomas: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Smith:H3 Biomedicine Inc.: Employment.

Mutations affecting the spliceosomal protein U2AF1 are among the most common mutations observed in patients with MDS and related disorders. However, it is unclear how these mutations affect the normal RNA splicing process, and how the resulting changes in splicing contribute to myeloid dysplasia. Here, we combined the strengths of data from primary AML patient samples with the controlled context of isogenic cell lines. We generated K562 erythroleukemic cell lines stably expressing each of the four common U2AF1 mutations (S34F, S34Y, Q157P, and Q157R). We compared expression of each of these mutant alleles with knock down of endogenous U2AF1 to compare the relative consequences of U2AF1 mutations versus loss of function. We first sought to identify changes in splicing driven by U2AF1 mutations that contribute to myeloid dysplasia. We compared the splicing of ~125,000 annotated alternative splicing events and ~160,000 constitutively spliced junctions between AML samples with or without mutations (TCGA cohort), as well as our isogenic K562 cell lines stably expressing either mutant (S34F, S34Y, Q157P, and Q157R) or wild-type (WT) alleles of U2AF1. Unsupervised cluster analysis revealed that S34F/Y versus Q157P/R samples clustered together in both the AML data and our cell lines, suggesting that U2AF1 mutations affecting different residues of the protein have different molecular consequences. Intersecting the AML and K562 data, we identified >300 splicing events that were consistently differentially spliced in association with S34 mutations, and a similar number for Q157 mutations. Many of these splicing events affected biological pathways that have been implicated in myeloid malignancies, including DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). For example, two exons of DNMT3B are differentially spliced in both AML samples and our K562 cells (Figure A), including an exon lying within the methyltransferase domain. We next identified mechanistic changes in the splicing process caused by U2AF1 mutations. U2AF1 binds the intron-exon boundary by sequence-specifically recognizing the AG dinucleotide and flanking sequence positions that define the 3' splice site. Comparing AML samples and K562 cells with and without U2AF1 mutations, we found that S34 and Q157 mutations give rise to specific and distinct alterations in 3' splice site preference. S34 mutations alter the consensus nucleotide immediately before the AG dinucleotide, while Q157 mutations alter the consensus nucleotide immediately after the AG (Figure B). We observed highly similar allele-specific alterations in 3' splice site preference in every AML patient with a U2AF1 mutation, as well as all K562 cell lines expressing a U2AF1 mutant allele. In contrast, knock down of endogenous U2AF1 caused no alterations in the consensus sequence at those positions, indicating that U2AF1 mutations do not cause loss of function at the level of RNA splicing. To confirm that the nucleotides immediately before and after the AG determine whether a splice site responds to U2AF1 mutations, we created minigenes of cassette exons within the ATR and EPB49 genes. We found that response to U2AF1 S34 and Q157 mutations requires the endogenous nucleotides immediately before and after the AG, as predicted by our genomics analysis, and that mutating these positions abolishes response to U2AF1 mutations. Finally, we recapitulated the RNA splicing process in vitro using nuclear extract from blood cells expressing either wild-type or mutant U2AF1 to show that identical changes in splice site preference occur in a controlled in vitro context (Figure C). Together, our data show that U2AF1 mutations cause allele-specific alterations in normal 3' splice site recognition in patients, in isogenic cell lines, and in vitro. These alterations in splice site preference give rise to mis-splicing that affects many genes previously implicated in myeloid malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Spliceosomal mutations account for the most frequent class of mutations in patients with MDS and CMML, yet the mechanism by which these mutations perform their driver function is not well understood. Moreover, no genetically engineered murine models for expression of spliceosomal gene mutations from their endogenous loci have been reported. Given the genetic heterogeneity of primary patient samples, we generated a model for conditional expression of the commonly occurring SRSF2P95H mutation from the endogenous murine locus of Srsf2. We compared expression of the Srsf2P95H mutation with genetic inactivation of 0, 1 or 2 copies of Srsf2 to understand the biological and mechanistic consequences of spliceosomal mutations relative to loss-of-function. Mx1-cre Srsf2P95H/wildtype mice exhibited significant morphologic dysplasia, leukopenia, macrocytic anemia, and preserved bone marrow (BM) cellularity as early as 2 weeks after mutation expression (Figure A). Moreover, Mx1-cre Srsf2P95H/wildtype mice exhibited an increase in hematopoietic stem/progenitor cells (HSPCs) with an increase in lineage-negative Sca1+ c-Kit+ cells (LSK cells) in S-phase and early apoptosis. In competitive transplantation, Srsf2P95H mice HPSCs were expanded in the BM at 16 weeks post-transplantation despite having a reduced contribution to peripheral blood chimerism. In contrast, mice with homozygous deletion of Srsf2 exhibited anemia and leukopenia due to BM aplasia with striking loss of HSPCs (Figure B). Collectively, these data show that Srsf2 is required for hematopoiesis, while mutations in Srsf2 provide a competitive advantage at the level of HSPCs but impair differentiation into mature circulating blood elements. Next, to identify transcriptional and post-transcriptional alterations caused by SRSF2 mutations, we performed deep RNA-seq on sorted HSPC populations from wildtype and Srsf2P95H mice, stable K562 cell lines ectopically expressing an empty vector or a single allele of SRSF2 (WT, P95H, P95L, P95R), as well as primary CMML and AML patient samples. We quantified global changes in splicing of ~125,000 alternative splicing events and ~160,000 constitutive splice junctions associated with SRSF2 mutations in these datasets. Intersection of differentially spliced genes in primary murine HSPC, CMML, and AML samples identified 75 genes that were differentially spliced in association with SRSF2 mutations in murine HSPCs and at least one primary patient cohort. Many of the genes that were differentially spliced in SRSF2 mutant cells participate in biological processes of known importance in myeloid malignancies. For example, a cassette exon of EZH2 that alters the reading frame likely to induce nonsense-mediated decay was promoted by SRSF2 mutations, while a frame-preserving cassette exon of BCOR was repressed by SRSF2 mutations. We next sought to determine how SRSF2 mutations alter SRSF2Õs normal role in RNA splicing. As SRSF2 recognizes exonic splicing enhancer (ESE) elements within the pre-mRNA to promote exon recognition, we hypothesized that SRSF2 mutations might alter its normal sequence-specific activity. To test this, we performed an ab initio motif identification screen to identify motifs that were enriched or depleted in cassette exons promoted versus repressed in primary Srsf2P95H cells relative to wildtype. This analysis identified CCAG and GGTG as the most enriched and depleted consensus motifs, respectively. A recent solution structure of SRSF2 in complex with RNA revealed that SRSF2 normally recognizes the motifs CCNG and GGNG equally well. Analysis of the spatial distribution of CCNG and GGNG motifs across genomic loci containing cassette exons that were promoted or repressed in association with SRSF2 mutations revealed that CCNG and GGNG were respectively enriched and depleted specifically over the cassette exons, and not over the flanking introns or exons, that were differentially spliced in association with SRSF2 mutations (Figure C). Together, our data indicate that mutant SRSF2 drives widespread changes in splicing due to alterations in its sequence-specific recognition of exonic splicing enhancers. The biological as well as molecular data here identify an effect of the SRSF2P95H mutation distinct from haploinsufficient or complete loss of SRSF2 and reveal that mutations in SRSF2 alter ESE preference to contribute to key aspects of MDS. Figure 1 Figure 1. Disclosures Feala: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment.

Abstract 2898 Background: STA-9090 is a potent, second-generation, small-molecule Hsp90 inhibitor, with a chemical structure unrelated to the first-generation, ansamycin family of Hsp90 inhibitors (e.g., 17-AAG or IPI-504). STA-9090 induces the loss of Hsp90 client proteins that are important in hematologic cancers, including BCR-ABL, c-KIT, FLT3, WT1, and JAK2. In preclinical studies, STA-9090 has shown potency up to 100 times greater than the first-generation Hsp90 inhibitors as well as activity against a wider range of kinases. In in vitro and in vivo models, STA-9090 has shown potent activity against a broad range of leukemias, lymphomas, and multiple myeloma. Methods: The primary objective of this Phase I multicenter study (NCT00858572) was to determine the maximum tolerated dose (MTD) of STA-9090 given twice weekly in patients with acute myeloid leukemia (AML), advanced myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), and myeloproliferative neoplasms (MPN). An adaptive statistical design methodology was used to determine dose escalation. Safety, pharmacokinetics (PK), pharmacodynamic (PD), and clinical activity were evaluated. Dosing for 4 consecutive weeks constituted one dosing cycle. Serial pharmacokinetic (PK) samples were collected pre-dose, 0.5, 1, 1.5, 2, 4, 6, 8, and 24 hours post-dose on study days 1 and 22, and pre-dose and 1 hour post at all other visits in cycle 1. Plasma samples were collected for HSP70 protein analysis. Safety assessments included the number and grade of adverse events (AEs), changes from baseline in laboratory parameters, and evaluation of electrocardiogram changes. Results: 22 patients (14 males, 8 females; median age 62 years, range 33–80; Eastern Cooperative Oncology Group [ECOG] status range [0-2]) received STA-9090 twice weekly. Patients with the following disease types were treated: AML (n=7), CML (n=8), MDS (n=3), and MPN (n=4). The median time from initial diagnosis to first treatment was 19 months; patients had received a median of 2 (range, 1–9) prior treatments and 27% were refractory to their most recent therapy. Dosing with STA-9090 was at 14 mg/m2 (4 patients), 22 mg/m2 (4 patients), 30 mg/m2 (3 patients), 70mg/m2 (3 patients), and 110 mg/m2 (8 patients). Patients received a median of 2 (range, 1–8) cycles of STA-9090. AEs reported in ≥25% of patients were diarrhea, nausea, fatigue, abdominal pain, anemia, increased alanine aminotransferase, arthralgia, increased aspartate aminotransferase, dyspnea, hypokalaemia, thrombocytopenia, and vomiting. The majority of AEs were mild to moderate in severity. There have been 3 patients with dose limiting toxicities: one at 14 mg/m2 (grade 3 hyponatremia and hyperbilirubinemia) and two at 110 mg/m2 (grade 3 prolonged QTc and transaminitis). At the time of abstract submission, the MTD has not been defined. STA-9090 exhibited linear PK with exposures (AUC and Cmax) increasing in proportion to dose. One hour following infusion termination, plasma concentrations declined by approximately 10 fold, and by 100 fold within approximately 8 to 10 hours. STA-9090 showed no drug accumulation with twice weekly dosing. STA-9090 was well distributed, exhibiting an apparent volume of distribution greater than total body water. HSP70 protein levels increased following STA-9090 administration and remained elevated prior to subsequent doses indicating a durable biological impact of the drug. Clinical responses include 1 patient with CML (dosed at 14 mg/m2) and 1 patient with AML (dosed at 110 mg/m2) who had best responses of hematological improvement lasting 2 and 3 months, respectively. In addition, 4 patients with myelofibrosis (1 patient dosed at 14 mg/m2 and 3 dosed at 70 mg/m2) had stable disease as best response; 1 of these responses lasted for 7 months and the patient went on to receive an allogenic stem cell transplant. Further response evaluation is ongoing. Conclusions: In patients with hematologic malignancies, STA-9090 is well tolerated up to dose levels of 110 mg/m2 given twice weekly. Induction of HSP70 protein levels provides evidence of in vivo biological activity. A favorable PK profile, preliminary signs of pharmacodynamic activity, and early clinical activity signals warrant continued evaluation of single-agent STA-9090 using a twice weekly dosing regimen. Disclosures: Off Label Use: Phase I clinical study. Bradley:Synta Pharmaceuticals Corp.,: Employment, Equity Ownership. Teofilovici:Synta Pharmaceuticals Corp.,: Employment, Equity Ownership.