Notch mediated patterning and cell fate allocation of pancreatic progenitor cells

A perturbation calculation is shown to give a satisfactory analytical description of the dc voltage characteristics of a coupled pair of superconducting weak links. In particular it predicts locking intervals over which the individual voltages of the junctions will either be equal or integer multiples of each other. Numerical simulations corroborate the perturbation approach and, as well, reveal the phenomenon of phase slippage between the junctions

A case of mistaken identity: When lupus masquerades as primary myelofibrosis

Introduction: Autoimmune myelofibrosis is an uncommon hematologic disease characterized by anemia, bone marrow myelofibrosis, and an autoimmune feature. Myelofibrosis is often associated with other conditions, including infections, nutritional/endocrine dysfunction, toxin/drug exposure, and connective tissue diseases, including scleroderma and systemic lupus erythematosus. Absence of clonal markers ( JAK2 ) and heterogeneity of the symptoms often complicate the diagnosis. Case presentation: Here, we present two cases of systemic lupus erythematosus–induced autoimmune myelofibrosis. The first case is of a 36-year-old African American female with diagnosis of systemic lupus erythematosus at the age of 12 years. The second patient is a 44-year-old African American male with family history of systemic lupus erythematosus who developed anemia and constitutional symptoms later on. Both patients showed hypercellularity and fibrotic changes of the bone marrow. Moreover, mutational analysis showed that both patients were wild type for JAK2 (V617F and exon 12) and MPL (exon 10). Conclusions: These two cases illustrate that anemic patients with fibrotic changes in the bone marrow without other clinicopathologic features associated with primary myelofibrosis in the presence of clinical manifestations and history of an autoimmune disease should suggest an autoimmune myelofibrosis. These cases demonstrate that a good clinical history combined with molecular technologies and pathomorphologic criteria are helpful in distinguishing between primary myelofibrosis and a nonclonal myelofibrosis from an associated condition

A case of mistaken identity: When lupus masquerades as primary myelofibrosis

Introduction: Autoimmune myelofibrosis is an uncommon hematologic disease characterized by anemia, bone marrow myelofibrosis, and an autoimmune feature. Myelofibrosis is often associated with other conditions, including infections, nutritional/endocrine dysfunction, toxin/drug exposure, and connective tissue diseases, including scleroderma and systemic lupus erythematosus. Absence of clonal markers ( JAK2 ) and heterogeneity of the symptoms often complicate the diagnosis. Case presentation: Here, we present two cases of systemic lupus erythematosus–induced autoimmune myelofibrosis. The first case is of a 36-year-old African American female with diagnosis of systemic lupus erythematosus at the age of 12 years. The second patient is a 44-year-old African American male with family history of systemic lupus erythematosus who developed anemia and constitutional symptoms later on. Both patients showed hypercellularity and fibrotic changes of the bone marrow. Moreover, mutational analysis showed that both patients were wild type for JAK2 (V617F and exon 12) and MPL (exon 10). Conclusions: These two cases illustrate that anemic patients with fibrotic changes in the bone marrow without other clinicopathologic features associated with primary myelofibrosis in the presence of clinical manifestations and history of an autoimmune disease should suggest an autoimmune myelofibrosis. These cases demonstrate that a good clinical history combined with molecular technologies and pathomorphologic criteria are helpful in distinguishing between primary myelofibrosis and a nonclonal myelofibrosis from an associated condition

Spliceosome Gene Mutations Are Frequently Found In JAK2 Negative Myelofibrosis and Associated With Worse Clinical Outcomes

Abstract Myelofibrosis (MF), a Philadelphia chromosome negative myeloproliferative neoplasm (MPN) requires clonal markers for accurate diagnosis according to the 2008 World Health Organization classification scheme. However, molecular mutations in Janus Kinase 2 (JAK2) are found in only 50% of MF patients (pts). Various somatic mutations identified in other myeloid cancers have also been found in MF with pre-existing JAK2 V617F mutations. We previously reported in 57 pts diagnosed with Triple Negative MF (TN-MF: JAK2 exon12/14 and wild type (WT) MPL) the absence of mutations in genes commonly identified in other myeloid malignancies, specifically TET2, DNMT3A, CBL, IDH1/2, SH3B2 (LNK), N/KRAS and EZH2. Only somatic mutations in ASXL1 were identified in a small cohort of TN-MF (16%). Mutations in components of the RNA splicing machinery have been recently identified in varying frequencies in different myeloid malignancies. We performed RNA-sequencing on 2 samples (JAK2 mutant (MUT) and 1 JAK2 WT) and found that 2 spliceosome genes (U2AF1 and SF3B1) had higher mRNA expression levels in the JAK2 WT (MFC=0.76 and 0.82, respectively) compared to JAK2 MUT patient. We hypothesized that somatic mutations in RNA splicing factor genes occur in TN-MF pts and may be helpful in the biological characterization of this group of pts. Therefore, we isolated DNA from bone marrow (BM) or peripheral blood mononuclear cells from a cohort of MF pts (N=132; JAK2 MUT:75 and JAK2 WT:57) and performed Sanger sequencing for SF3B1 (exons 13-16), U2AF1 (exons 2, 6 and 7), and SRSF2 (exons 1 and 2). Baseline characteristics of pts and clinical data including hematologic parameters, BM results, and presence of splenomegaly by palpation were collected. Pts were stratified based on the Dynamic International Prognostic Scoring System-Plus risk as high=40, Int-2=51, Int-1=11 and low-risk=5 pts. The mean duration of follow-up was 16.5 months in JAK2 MUT and 12.8 months in JAK2 WT. Spliceosome mutations were found in 35/132 (27%) (SRSF2=17%, U2AF1=8% and SF3B1=2%) of pts. Interestingly, the frequency of spliceosome mutations in JAK2 WT was higher (31%; SRSF2=26%, U2AF1= 5%, SF3B1=0) compared to JAK2 MUT (18%; SRSF2=10%, U2AF1=6%, SF3B1=2%) pts. Of note, the 2 pts with SF3B1 mutations (K700E) had ring sideroblast in the BM (occasional and 50%). A large number of WT JAK2 patients (50%) had concomitant SRSF2 and ASXL1 mutations. Both SRSF2 and ASXL1 have been associated with poor prognosis in MF. Indeed, in our JAK2 WT cohort, pts who harbored SRSF2 mutations had a higher mean percentage of BM blasts compared to the ones that were SRSF2 WT (5.6% vs 1.8%; P=.006). Moreover, JAK2 WT pts carrying spliceosome mutations had more severe anemia as shown by lower hemoglobin (8.98 g/dL vs 10.5) and higher leukocyte counts (27.5 x109/L vs 20.5) compared to WT cases. Furthermore, we noticed that they also had higher frequency of RBC (Red Blood Cells) transfusions compared to WT (60% vs 40%) cases. Spleen examination by palpation below the left sub-costal margin showed that JAK2 WT cases carrying spliceosome mutations had larger splenomegaly compared to WT cases [10.1 cm (range 4-26), vs 8.7cm (range 2-20); P=0.05]. In conclusion, molecular alterations in the spliceosome machinery are frequently found in MF pts who are WT for JAK2/MPL and are associated with higher BM blast percentage, more severe anemia, higher leukocyte counts, transfusion dependence and more prominent splenomegaly. The observation of appreciable frequency of spliceosome mutations in JAK2 WT MF pts opens the possibility of using spliceosome inhibitors in the management of this disease group. Disclosures: No relevant conflicts of interest to declare

Patients with SF3B1 Mutation Have Good Prognosis Even in the Presence of Other Poor Prognostic MDS Features and Have Better Outcomes During Treatment with Low Intensity Chemotherapy

Abstract Abstract 3831 The discovery of mutations in components of the RNA splicing machinery has underlined the importance of this pathogenetic mechanism in MDS biology. We and others reported that mutations in SF3B1 are frequent in refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T). We recently reported good survival outcomes in a large cohort of SF3B1 mutant patients similar to the findings of Papaemmanuil et al, NEJM< 2011. Here, we aimed to evaluate the correlation between SF3B1 mutations in association with other poor prognostic clinical and pathologic factors in MDS. We focused on a large cohort of patients with several myeloid malignancies (n=340). Patients were grouped in MDS (n=176), MDS/MPN (n=88) and secondary AML (sAML) from a previous MDS (n=76). Diseases were classified according to the 2008 WHO classification. Survival outcomes (overall survival [OS], progression free survival [PFS] and event free survival [EFS]) were defined according to MDS IWG criteria. We studied a total of 340 patients with myeloid malignancies. median age, 69 years old (range 19–92), sex (Male, n=222; females, n=118). We performed direct sequencing for SF3B1 (exon 13–16) on a large cohort of MDS, MDS/MPN and sAML patients and found 11.8% SF3B1 mutants. The outcomes of SF3B1 mutant vs WT patients were compared based on the presence of poor clinicopathologic factors associated with MDS like RBC transfusion dependence, presence of SNP-A lesions, presence of acquired somatic uniparental disomy (AS-UPD), Age ≥ 60 years, presence of reticulin fibrosis in the bone marrow. We also assessed the effects of therapies in the prognostic effect of SF3B1 mutations. We previously reported that the presence of new SNP-A lesions in myeloid malignancies including MDS and AML are associated with poor prognostic outcomes. However, the good prognostic effects of SF3B1 mutation is still apparent even in the patients with new SNP-A abnormalities (OS: 40 vs 16 mos, p=.003; PFS: 40 vs 10 mos, p=.003, 40 vs 10 mos, p=.0007. However, when analysis is limited to acquired somatic uniparental disomy defects which we recently reported as the worst lesion among SNP-A abnormalities, the good prognostic effect of SF3B1 is lost (OS: 19 vs 9 mos, p=.29, PFS: 18 vs 7 mos, p=.20, EFS: 19 vs 8 mos, p=.17). Age is an important predictor of outcomes in MDS with higher age associated with worse outcomes. SF3B1 remained predictive of good outcomes in patients ≥ 60 years of age (OS: 40 vs 16, p=.002; PFS: 40 vs 11 mos, p=.003; EFS: 40 vs 10 mos, p=.0005). Persistent RBC transfusions are also associated with inferior survival in MDS, yet SF3B1 mutant patients continued to have good outcomes (OS 34 vs 13, p=.002; PFS: 27 vs 8 mos, p=.004; EFS: 14 vs 9 mos, p=.001). Reticulin fibrosis in the bone marrow is a characteristic feature of myeloproliferative neoplasms but their presence in MDS is associated with unfavorable results, SF3B1 mutant retain their good outcomes even in the presence of reticulin fibrosis in the BM (OS: 40 vs 15 mos, p=.09; PFS: 47 vs 12 mos, p=.04; EFS: 75 vs 13 mos, p=.008). Patients screened for SF3B1 were also further stratified according to treatments received. No patients with SF3B1 mutations underwent allogeneic hematopoietic stem cell transplantation and high intensity chemotherapy specifically induction chemotherapy or high dose cytarabine. However based on treatment with LIC, SF3B1 mutant patients have better survival outcomes compared to WT patients (OS: 61 vs 17 mos, p=.004; PFS: not reached [NR] vs 7 mos; p=.009; EFS: 61 vs 10 mos, p=.001). These observed survival outcomes remain significant when patients were stratified according to disease subtypes. MDS and MDS/MPN patients treated with LIC have better outcomes if they have SF3B1 mutation (OS: 61 vs 23 mos, p=.005; PFS: NR vs 16 mos, p=.01; EFS: 61 vs 16 mos, p=.001). A high number of TET2/DNMT3A mutations are found in SF3B1 mutants. TET2/DNMT3A mutations have been previously association with improved response to hypomethylating agents which is one of the possible reasons why SF3B1 mutants treated with LIC did better than their WT counterpart. In conclusion, SF3B1 retains its favorable prognostic effect even in the face of poor prognostic factors such as RBC transfusion dependence, presence of SNP-A lesions, Age ≥ 60 years, presence of reticulin fibrosis in the bone marrow except in the presence of AS-UPD. SF3B1 mutants treated with LIC also have better outcomes. Disclosures: No relevant conflicts of interest to declare

BCL-2 Family Of Genes Is a Key Regulator In The Pathogenesis Of SF3B1 Mutant and Wild Type MDS With Ring Sideroblasts and Represents a Novel Drug Target In This Disease

Abstract Myelodysplastic syndromes (MDS) are a group of neoplastic diseases characterized by impairment in hematopoietic cell differentiation which leads to disease related consequences like cytopenias and cellular dysplasia. Mutations in splicing factor 3b subunit 1 (SF3B1) are the most frequently identified genetic abnormalities in refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). However, the mechanisms whereby cells acquire clonal dominance and evolution, cytopenias and impairment in differentiation remain unknown. To understand the role of SF3B1 mutations in the pathogenesis of RARS/-T, we analyzed the transcriptome of SF3B1 mutant (MUT) and wild type (WT) RARS/-T patients (pts). RNA was isolated from bone marrow of healthy subjects (n=3), SF3B1 MUT (n=3) and WT (n=3) RARS/-T, and other MDS (n=5). cDNA was made from RNA (1.5-3ug) and fragmented for library preparation. RNA-sequencing was performed on 20 million sequence reads on an Illumina HiSeq2000 and aligned to the human genome 19. RNA-splicing patterns were analyzed by a published bioinformatics algorithm at three levels (exon usage, gene expression, and pathway analysis) (Visconte V; Blood. 2012). Differential exon usage identified 271 and 71 genes with at least 1 exon alternatively spliced in SF3B1 MUT, WT, and healthy subjects (P<.05). SF3B1 preferentially affected the 3’-end regions. The chromosomes (chrs) with the highest number of genes displaying differential exon usage were chr1:30, chr17:25, chr7:22, and chrs19: 20, chr11:17, chrs:6 and 2:15 following by chrs:8, 5, and 16: 11. No significant differences in mRNA levels of genes relevant to MDS (U2AF1, TET2, DNMT3A, ASXL1, EZH2, TP53, RUNX1, ETV6, CBL, IDH1/2, and N/KRAS) were found leading us to think that SF3B1 affects alternative targets. Using GSEA/ MiSigDB v3.0 analysis we found a specific molecular signature: out of 27 mitochondrial gene sets, one gene set differed between SF3B1 MUT and WT RARS pts (P=.01). This gene set contains 11 genes involved in mitochondrial membrane/organization/biogenesis network. BCL-2 family genes were highly expressed: BCL-2 (mean: 22.8 vs 68.1 vs 15.2), BNIP3 (mean: 50.1 vs 55.3 vs 31.9) and BCL2-L1 (mean: 3221 vs 2905 vs 595) in SF3B1 MUT vs WT pts and vs healthy subjects. Changes in BCL2 mRNA levels were also confirmed by western blotting in MUT (n=2) vs WT (n=2). In myeloid cell lineage, the relationship between BCL-2 protein levels is inversely related to the maturation stage with myeloblasts and promyelocytes having higher BCL-2 while more differentiated cells like metamyelocytes and polymorphonuclear cells have less to no BCL-2 expression. Contrary to prevailing knowledge and consistent with what would be expected in explaining MDS persistence by development of clonal advantage, in our hands RARS/-T pts were not characterized by increased cleaved PARP and cellular stress as evidenced by no difference in reactive oxygen species between SF3B1 MUT and WT (93.83% ± 7.94 vs 83.28% ± 13.82; n=3 vs 4) further supported by the overexpression of an antiapoptotic gene BCL-2. We previously presented the biological rationale of the different clinical outcomes between SF3B1 MUT and WT (Visconte V; Blood (ASH Annual Meeting Abstracts), Nov 2012;120: 922). SF3B1 WT carries more high risk chromosomal defects and higher g-H2AX levels indicative of greater DNA damage. Higher g-H2AX and genomic instability are associated with BCL-2 over-expressing lymphoid malignancies (Laulier C; Cancer Res. 2011) which may explain the higher number of chromosomal defects and propensity for leukemia transformation in WT pts. SF3B1 WT tends to respond less to hypomethylating therapy (HMT) vs MUT (n=2 vs 2). Cells presenting an immature hematopoietic phenotype have lower response rate to HMT in-vitro. Indeed, differences in erythroid maturation were found in SF3B1 MUT vs WT RARS. SF3B1 MUT showed higher mRNA levels of GATA-1, a marker of hematopoietic maturation (FC: 4.0), and a down-regulation (FC: 0.48) of GATA-2, an early hematopoietic marker vs WT. In conclusion, BCL-2 is a key regulator in the pathogenesis of RARS/-T and may provide clonal advantage. Differences in BCL-2 expression can explain different survival outcomes in SF3B1 MUT vs WT. BCL-2 may be a novel pathway in the pathogenesis of RARS/-T opening the possibility that a BCL2- antagonist either with/without a spliceosome inhibitor will be an ideal therapeutic approach in RARS/-T. Disclosures: No relevant conflicts of interest to declare

The Molecular and Cytokine Profile of Triple-Negative (JAK2 V617F, JAK2 exon 12, MPL negative) Myelofibrosis, a Myeloproliferative Neoplasm with Distinct Clinico-Pathologic Characteristics

Abstract Abstract 3805 Myelofibrosis (MF) is a clonal Philadelphia chromosome negative myeloid neoplasm characterized by cytopenias with or without proliferative hematologic changes, extramedullary hematopoiesis and an increased propensity for transformation to acute myeloid leukemia. Identification of somatic mutations in Janus kinase 2 (JAK2) has revolutionized our understanding of the pathogenesis of some MF patients. However, there is a wide range of clinical heterogeneity in MF patients and somatic mutations of JAK2 (exon 14 or exon12) and MPL are only found in 50–60%/1–2% and 5–10% of MF patients respectively. Therefore, a large proportion of MF patients remain molecularly unaccounted after sequencing for these commonly analyzed mutations. We hypothesized that MF patients who are lack of JAK2 and MPL mutations, collectively called “triple-negative MF,” have a distinct clinicopathologic profile compared to their mutant counterparts. Our study included 76 MF patients (primary MF=48, post-essential thrombocythemic-MF=21, post-polycythemic-MF=7), with amedian age of 65 years (25–80) and predominantly intermediate 1 or 2 risk by the D-IPSS grouping (low=8, Int-1=20, Int-2=42, high=6). We compared hematologic and clinical variables between JAK2 mutant (N=40) and triple-negative MF (N=36) and found that triple-negative MF patients had a higher WBC count (14.4±23.6. vs 11±14.9) and higher platelet counts (365±434.1 vs 252±220.5) but similar hemoglobin levels, LDH levels and D-IPSS plus risk profiles. No difference in overall survival was noted (25.5 months in triple-negative vs 24 months in JAK2 positive). Newly discovered molecular mutations in myeloid malignancies have provided insight into the biology of MDS and some MPN cases. We performed direct sequencing on 9 genes including TET2 (Exon 3), DNMT3A (Exon 18/19, 20, 21, 22 and 23), IDH1/2 (Exon4), LNK (exon2–8), EZH2 (Exon 18/19, N/KRAS (Exon 1–2), CBL (Exon 8–9) and did not find any mutation in the triple-negative cohort. Recently, L3MBTL1 has been reported to be important in the pathogenesis of polycythemia vera, but sequencing of this gene in MF patients did not yield any mutations. Next generation sequencing technologies have been instrumental in the discovery of new genes important in disease pathogenesis in myeloid neoplasias. We performed whole exome sequencing in the triple-negative cases and found somatic mutations in the following genes, ODZ1 (cellular signal transducer), ZNFE391 (transcriptional regulator), DENND3 (protein-coding), ARHGEF11 (modulator of cellular process through G protein) and PIK3CD (Phosphorylate Inositol Lipids regulator involving in Immune response) in one patient and subsequently confirmed as somatic by direct sequencing. This is the first time, which these genetic mutations were reported in hematologic malignancies specifically in MF. However, these mutations did not recur when screening, a limited cohort of patients (N=20). For better understanding of this disease subtype, we compared the cytokine profile in these two groups based on the fact that, cytokines have been crucial in the pathogenesis of MF, and we found marked elevation in the levels of IL-1B (1.63 vs 0.42), IL-8 (2.89 vs 0.56), IL-17A (2.56 vs 1.12) and IFN-δ (2.07 vs 0.37) in the JAK2 positive patients compared to the triple-negative cases. Comparison of both groups showed similar responses to JAK inhibitors by spleen size shrinkage and constitutional symptoms. In conclusion, despite the discovery of JAK2 and MPL mutations, a subset of MF patients remains molecularly uncharacterized. We found that triple negative MF patients had higher leukocyte and platelet counts, but a similar prognosis. Moreover, we identified novel somatic mutations in 1 triple-negative MF patient, which has not been previously described interestingly, despite lower cytokine levels; responses to JAK-inhibitors were still noted. Whole exome sequencing and other immunogenetic studies may reveal novel genes which may elucidate the pathogenesis of these MF cases. Disclosures: No relevant conflicts of interest to declare

Triple Negative (JAK2 exon 12 /14 and MPL wild type) Myelofibrosis Have Higher Expression Of CDC25A and Greater Sensitivity To CDC25A Inhibition Compared To JAK2 Mutant Cases

Abstract Pharmacologic therapies that target the JAK-STAT pathway are clinically used to alleviate splenomegaly and disease-related constitutional symptoms in MF. However, it is clear that some patients develop intolerance or resistant to this therapy. Furthermore, there are MF related complications especially cytopenias that are not alleviated by these therapies. Therefore, alternative and complementarytherapies are warranted in the management of MF. We hypothesized that other pathways downstream of the JAK-STAT signaling pathway can play a role in the pathophysiology of MF. We used whole exome (WES) and RNA sequencing technologies to interrogate new molecular markers and pathways which can serve as novel targets for this disease. In 4 MF patients [JAK2 mutant (MUT) =2, and wild type (WT) =2], WES was performed using the Illumina platform. All of the variants were filtered based on PHRED score (>=30) with coverage was set at 30X. Analysis of data in JAK2/MPL WT patients demonstrated the presence of 263 candidate genes. After clarifying the status of tumor nucleotide variants in each gene compared to germline (CD3+) fraction, 7 genes (RBL1, ADSS, ZNF717,MUC4, TUBB4Q and CDC25A) were selected for further somatic confirmation by direct sequencing. Among these genes, only alteration in CDC25A, a regulator of cyclinE/cdk2 (cyclin-dependent kinase-2) and cyclinA/cdk2 kinase, was confirmed to be somatic. This genetic change was previously reported as somatic by WES in lung cancer although not confirmed by direct sequencing (Bartkova et al, Nature, 2005, Apr 14; 434 (7035):864-70). Based on these observations and since CDC25A acts as a downstream effector of JAK-STAT signaling, we hypothesized that, CDC25A phosphatase, may be a driver in MF pathogenesis. The transcriptome of two patients, one MUT and one WT for JAK2 was then analyzed. RNA was isolated from bone marrow (BM) cells of healthy individuals (HI) (N=3). cDNA was made from 1.5-3 ug of RNA and fragmented for library preparation. RNA-sequencing was performed on 20 million sequence reads. Paired-end 90 base pair reads were generated on an Illumina HiSeq2000 sequencer and aligned to the human genome 19. RNA-splicing patterns were analyzed by a bioinformatics algorithm and gene expression analysis was carried out using GSEA (Visconte V; Blood. 2012). By using FDR80%) while JAK2 MUT samples had only a few positive megakaryocytes (<20%). To test the feasibility of targeting this pathway in patients with MF and to assess for differential response between JAK2 MUT and WT cases, a potent cell permeable 7-substituted quinolinedione derivedCDC25 phosphatase inhibitor (NSC663284) was tested in JAK2 MUT (N=2) vs WT (N=1). Cell proliferation was determined by Trypan Blue and MTT assay after cell exposure to different concentrations of the inhibitor [3, 5, 7, 10 and 30uM] in 24 hours observation. NSC663284 induced higher dose-dependent cell growth inhibition in JAK2 WT compared to MUT cases (% of viable cells in WT vs MUT using previously mentioned concentrations, 3 uM= 98% vs 86%, 5 uM= 93% VS 77%, 7 uM= 88% vs 65%, 10 uM= 71% vs 43% and 30 uM= 25% vs 61%; p=0.01).In conclusion, CDC25A is more highly expressed in patients with wild type JAK2 compared to the mutant counterpart and primary cells from WT JAK2 patients demonstrate higher sensitivity to CDC25A inhibition, warranting further clinical testing of this therapeutic strategy. Disclosures: No relevant conflicts of interest to declare

Spliceosome Mutations Are Frequent In Patients With Myelodysplastic Syndromes Who Failed Hypomethylating Therapy: Possible Implications Of Spliceosome Inhibitors As Alternative Treatments

Abstract Myelodysplastic syndromes (MDS) are a heterogeneous group of blood cancers characterized by bone marrow (BM) failure, peripheral blood cytopenias, dysplasia, chromosomal abnormalities and an increased risk for transformation to acute myeloid leukemia (AML). Patients (pts) with higher risk disease are primarily treated with pharmacologic treatments like hypomethylating therapy (HMT) (5-azacytidine and decitabine). 5-azacytidine (AZA) and decitabine (DAC) can result in overall response rates of 36% with a median duration of response of 15 months and 17-21% with a median duration of response of 10 months, respectively. Pts refractory to HMT have poor outcomes with a median overall survival of ∼4 months. Spliceosome gene mutations are frequently found in certain subtypes of MDS specifically SF3B1 (∼28%), U2AF1 (6-12%) and SRSF2 (6-12%). The prognostic value of spliceosome mutations in different MDS subtypes has been largely investigated while the impact of these mutations on treatment response is still unknown. We aim to investigate the frequency of three commonly mutated spliceosome genes (SF3B1, U2AF1, and SRSF2) in pts who failed HMT in order to define mutational frequency and evaluate the feasibility of targeted therapy with next generation spliceosome inhibitors. We screened a cohort of 120 pts (MDS, 70; MDS/MPN, 33; MDS/sAML, 17; median age: 69; male/female: 85/35) that underwent HMT (AZA: 58; DAC: 21; AZA/DAC: 7; AZA/REV: 25; DAC/REV: 4; AZA/DAC/REV: 5). Forty-eight percent of pts failed HMT therapy as refractory or relapse. We performed Sanger sequencing on BM/peripheral blood DNA for known pathways involved in MDS pathogenesis including methylation (TET2, DNMT3A, IDH1/2), histone (ASXL1, UTX, EZH2), signaling (CBL, N/KRAS), transcription (RUNX1, TP53, JAK2), and RNA splicing (SF3B1, U2AF1, SRSF2). Data analysis was available for 90 pts. We detected a total of 131 mutations in different pathways. In total, spliceosome mutations were observed in 28/90 (31%) of pts. When we analyzed the presence of the mutations in relation to the rate of response, we found that pts who failed HMT have frequent spliceosome mutations: 17/58 (29%). We have reported that molecular mutations in TET2 and DNMT3A can predict response to treatment to HMT (Traina F, Blood (ASH Annual Meeting Abstracts), Nov 2011; 118: 461). Indeed, the frequency of mutations in methylation genes was lower in the group of pts who failed HMT (11/58; 18.9%) compared to pts who achieved hematological response (11/32; 34%). Spliceosome inhibitors have been proposed for targetted therapy in MDS. The presence of spliceosome mutations in pts who failed HMT can open a new era of investigation leading to the possibility of using spliceosome inhibitors in pts who fail conventional therapy. We performed RNA-sequencing analysis on BM cells of pts who failed HMT compared to pts who achieved hematological response (n=2 vs 2) in order to define any specific gene signature explaining the differences in response to HMT. We performed differential gene expression testing on 11,459 expressed genes. In total, 158 genes were differentially expressed at FDR < .2 in responders compared to not responders. We identified several interesting genes involved in tumorigenesis and epigenetic regulation such as YPEL3, and ST14, which were up-regulated in responders vs not responders (FC: 4 and 7.5; P<.00001). In sum, spliceosome mutations are frequent in pts who failed HMT and may affect downstream pathways most probably in the epigenetic network leading to differences in susceptibility to HMT and can open the possibility of treatment with spliceosome inhibitors. Disclosures: No relevant conflicts of interest to declare