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While red blood cells (RBCs) and granulocytes have been more studied, platelets and reticulocytes are not commonly used in paroxysmal nocturnal hemoglobinuria (PNH) flow-cytometry and less is known about susceptibility to complement-mediated destruction and effects of anti-complement therapy on these populations. We performed flow-cytometry of RBCs and granulocytes in 90 PNH patients and of platelets and reticulocytes in a subgroup (N = 36), to unveil perturbations of these populations during PNH disease course before and after anti-complement treatment. We found that platelets and reticulocytes were less sensitive to complement-mediated lysis than RBCs but not as resistant as granulocytes, as shown by mean sensitive fraction (difference in a given PNH population vs. PNH granulocyte clone size). In treated patients, reticulocytes, platelets, RBCs (with differences between type II and III) and granulocytes significantly increased post-treatment, confirming the role of PNH hematopoiesis within the context of anti-complement therapy. Moreover, we found that PNH platelet clone size reflects PNH granulocyte clone size. Finally, we established correlations between sensitive fraction of PNH cell-types and thrombosis. In sum, we applied a flow-cytometry panel for investigation of PNH peripheral blood populations' perturbations before and after eculizumab treatment to explore complement-sensitivity and kinetics of these cells during the disease course.

The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5' long terminal repeat (LTR) but not on the 3' LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses.

TET2 mutations (TET2MT) occur around 40% of myeloid neoplasms (MN) and often constitute founder hits, as concluded from patient's clonal architecture and subclinical TET2MT clones in healthy individuals at risk for MN. The mechanisms of TET2MT in leukemogenesis involve at least two potential mechanisms: i) TET2 mutations could lead to stem cell expansion and altered differentiation and/or ii) they may convey a mutator phenotype with progression due to a higher rate of subsequent genetic hits. Both mechanisms are compatible with the weak driver function of TET2MT. Here, we studied whether TET2MT predispose to additional oncogenic mutations through faulty DNA repair,i.e., whether they produce a clonally acquired mutator phenotype. TET2MT lead to the inhibition of passive demethylation. Alternatively, they impair demethylation via excision repair of 5fC/5caC. Moreover, global 5mC accumulation may increase background C>T mutation rates via 5mC deamination linking TET2 to base excision repair machinery. We investigated this hypothesis because of the finding of increased mutability found in Tet2KO and Tet2kdin the mouse as determined by the numbers of coexisting subclonal hits in Tet2MTdisease (Pan F et.al., Nature Comm, 2017). Using WES analyses (n=435), we identified 95 cases with TET2MT stringently selected for high VAF and most damaging effects as confirmed by 2D-UPLC-MS/MS assay showing low levels of the TET2-dependent DNA oxidation products. To test whether these TET2MT indeed predisposed to additional mutations, we enumerated somatic SNV vs. TET2WT cases. Sequencing of MSH2, MSH3, MSH6, MLH1, CHEK2, and BRCA2 excluded the potential effects of alteration in these genes on our results. Similarly, the MMR gene expression were not significantly different among 20 TET2MT MDS, 71 TET2WT MDS pts, and 17 controls, indicating MMR downregulation was not the culprit. Consistent with our theory, TET2MT pts had a 1.5-fold increase in median WES SNV (p50% had a 2.1-fold increase in median WES SNV (p=.03), consistent with a gene-dose effect. HeLa TET2kd cells also had a 24-fold increase in spontaneous mutations, reversed with TET2 cDNA knock-in. In mice, 1.4-fold increased mutagenicity at Tet2-dependent active demethylation sites was found, in a manner also suggestive of a defective MMR. TET2MT cases (n=5) were shown to be microsatellite-stable at 5 TET2-independent poly-dA microsatellite loci, suggesting that hypermutagenicity was not driven by global MMR dysfunction and may occur only at CpG-containing microsatellites. MOLM-13 TET2kd increased PARP inhibitor sensitivity by almost 3-fold, suggesting that TET2kd renders cells vulnerable to DNA damage. In murine cells, by overexpressing Flag/V5-tagged TET2 in MEL cells and subjecting them to protein affinity purification, we identified MSH6 as a novel TET2 binding partner. Using HPRT mutability assay, we measured mutational frequency in the HPRT1 gene in control, TET2kd, MSH6kd,and TET2kd/MSH6kd HeLa cells. Moreover, HPRT1 mutational frequency in TET2kd HeLa cells increased 24-fold compared to parental controls while MSH6kd HeLa cells had a further 10-fold increase in HPRT1, likely reflecting the presence of TET2-independent mechanisms for MSH6-mediated MMR. Intriguingly, the dual TET2kd/MSH6kd HeLa cells had equivalent MSI and mutational frequencies to the MSH6kd HeLa cells, supporting MSH6-dependence for the mutator phenotype induced by TET2 loss. TET2:MSH6 interactions were validated in co-immunofluorescence experiments in MEL and MOLM-13 cells, and TET2kd altered MSH6 nuclear co-localization. TET2 immunoprecipitation from human CD34+ cells demonstrated abundant MSH2 binding to TET2, although we were unable to co-precipitate MSH6 in the same experiment. In sum, we uncovered novel connections among TET2, MMR proteins, epigenetic modifications, and genomic instability. Given that MSH2/6 is known to preferentially bind 5hmC, TET2 may target MSH2/6 to TET2-dependent DNA loci. Genomic instability due to TET2 dysfunction may allow therapeutic targeting of DNA repair proteins in the subset of pts with TET2MT-driven MN. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

IL-10 is a pleiotropic cytokine expressed during malaria, a disease characterized by short-lived, parasite-specific Ab responses. The role of IL-10 in regulating B cell responses during malaria is not known. In this study we report that IL-10 is essential for anti-Plasmodium humoral immunity. We identify that germinal center (GC) B cell reactions, isotype-switched Ab responses, parasite control, and host survival require B cell-intrinsic IL-10 signaling. IL-10 also indirectly supports humoral immunity by suppressing excessive IFN-γ, which induces T-bet expression in B cells. Genetic ablation of either IFN-γ signaling or T-bet expression in B cells substantially enhanced GC B cell responses and anti-Plasmodium Ab production. Together, our data show that B cell-intrinsic IL-10 enhances whereas B cell-intrinsic IFN-γ and T-bet suppress GC B cell responses and anti-Plasmodium humoral immunity. These data identify critical immunoregulatory circuits in B cells that may be targeted to promote long-lived humoral immunity and resistance to malaria.

Myelodysplastic syndromes (MDS) are unique among cancers because of the frequent occurrence of somatic mutations impacting spliceosome machinery. At least 65% of MDS patients harbor a mutation in one of several splicing factors including U2AF1, SF3B1 and SRSF2. Whole exome sequencing of MDS bone marrow uncovered somatic frameshift mutations in LUC7L2, the mammalian ortholog of a yeast splicing factor. LUC7L2 is located in the most commonly deleted region of chromosome 7. Deletions and frameshifts lead to haploinsufficient expression and therefore it can be approximated that a combined 14% of MDS patients have low expression of LUC7L2. Restoring expression of LUC7L2 in del(7q)-iPSCs partially rescues the differentiation of iPSCs into CD45+ myeloid progenitors. Although perhaps partly due to associated losses of other genes on chromosome 7, low expression of LUC7L2 correlates with a poorer patient prognosis, so its haploinsufficiency may play an important role in bone marrow failure. While U2AF1, SF3B1, and SRSF2 are well-characterized splicing factors, the function of LUC7L2 in pre-mRNA splicing is unexamined and its role in the MDS pathogenesis is undefined. We hypothesize that low expression of LUC7L2 results in the aberrant splicing of oncogenes and tumor suppressor gene transcripts thus reducing expression or altering function and contributing to the pathogenesis of MDS. We have characterized LUC7L2 as an alternative splicing regulatory protein that plays a repressive role in the regulation of alternative RNA splicing. We generated HEK-293 cells overexpressing V5-tagged LUC7L2 for immunoprecipitation-mass spectrometry, to ascertain protein interactions with LUC7L2. LUC7L2 co-immunoprecipitated with splicing regulators which are involved in splice site recognition. We performed cross-linking-IP-high-throughput-sequencing (CLIP-seq) to identify LUC7L2 binding sites on RNA. We identified 301 LUC7L2 RNA-binding sites as well as binding sites on U1 and U2 which is common for splicing regulatory proteins. Metagene analysis of these binding sites showed that LUC7L2 bound near splice sites in exonic sequences. We knocked down LUC7L2 expression in HEK293 and K562 cells to phenocopy the frameshifts and deletions observed in MDS patients. We used a PCR-based assay to measure the splicing efficiency of introns near LUC7L2-binding sites. Knockdown of LUC7L2 increased the splicing efficiency of 8/13 selected introns; this suggests that LUC7L2 represses selective splice site usage. We also performed RNA-seq to characterize global mis-splicing events. Analysis of RNA transcripts revealed a multitude of splicing changes, including enhanced exclusion of alternative introns. Knockdown LUC7L2 cells exhibited-altered expression of other splicing factors; this could have further contributed to the vast number of splicing changes observed. To identify specific splicing changes that could contribute to the pathogenesis of MDS, we compared the splicing profiles of LUC7L2-knockdown in K562 cells with RNA-seq data from K562 cells expressing U2AF1S34F, SRSF2P95H or SF3B1K700E. This analysis yielded several exon-skipping splicing patterns in cancer-relevant transcripts, such as oncogene PRC1, splicing factor PTBP1 and MRPL33. Additionally, we noticed commonly mis-spliced transcripts among the four datasets in which the missplicing events occurred in the functional domain, potentially conferring a functional change. Surprisingly, we observed missplicing of U2AF1 in LUC7L2-knockdown, SRSF2P95H, and SF3B1K700E K562 cells, which altered the length of the RNA-recognition UHM domain by inclusion of a mutually exclusive exon or retention of an intron. In this way, low expression of LUC7L2, or point mutants U2AF1S34F, SRSF2P95H, and SF3B1K700E,could alter U2AF1 function as a distal convergence point. In summary, we identified a novel splicing factor implicated in the pathogenesis of MDS. We characterized LUC7L2 as a splicing repressor and discovered many splicing changes caused by low expression of LUC7L2. Several genes were also mis-spliced in U2AF1S34F, SRSF2P95H and SF3B1K700E K562 cells targeting these for further study. Commonly mis-spliced targets such as U2AF1 may indicate that some of the novel therapeutics may have spliceosome mutation agnostic effects. If this applies to the LUC7L2 mutations, then they may also be effective in del7/del7q cases. Disclosures Carraway: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; FibroGen: Consultancy; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Saunthararajah:Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.

The homologous DNA repair pathway genes BRCA1 and BRCA2 are classically associated with increased susceptibility to hereditary breast and ovarian cancer due to increased vulnerability to double stranded DNA breaks (mutator phenotype). In addition to their role in breast/ovarian cancer, defects in these genes may predispose to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). For example, in large populations of breast cancer patients, those with inherited defects in homologous repair (HR) showed a propensity to therapy-related MDS and AML; however, a relationship between BRCA gene variants and spontaneous myeloid neoplasms has yet to be elucidated. Moreover, analyses are complicated by the large number of clinically uncharacterized single nucleotide variants (SNV) in BRCA gene loci. We thus evaluated the relationship between germline BRCA variants (GLVs) and the risk of adult MDS or AML. We applied next generation sequencing to a large cohort of patients (N =463) presenting with MDS (402) and AML (61). In these analyses, all mutations with a variant allele frequency of less than 30% were considered somatic. We then identified alterations known to be linked with breast/ovarian cancer development by linkage analyses. These mutations (n=2), along with missense mutations with an allele frequency 10, were considered Tier-1 mutations (n=37). Tier 2 mutations were defined as missense mutations with a population frequency >.01% or mutations in patients with incomplete clinical or sequencing data. After excluding Tier-2 variants, 39 of 88 GLVs were further analyzed. BRCA Tier-1 GLV were significantly over-represented in the MDS/AML cohort compared to the general population, estimated via publically available exome aggregation databases (ExAC, 8% vs 0.1% p BRCA GLVs (Tier-1) are over-represented in MDS/AML patients as compared to the general population, and linked with a higher rate of somatic point mutations but not common MDS/AML-associated cytogenetic abnormalities. This suggests the possibility that defects in HDR may facilitate the evolution of apparently spontaneous MDS/AML that is actually germline predisposed. As with breast and ovarian cancers, another possibility is that such variants could have implications in treatment selection, especially in the context of the evolution of PARP inhibition as a therapeutic modality in MDS/AML. Disclosures Saunthararajah: Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Carraway:Jazz: Speakers Bureau; FibroGen: Consultancy; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Agios: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees. Nazha:MEI: Consultancy. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Loss-of-function TET2 mutations (TET2mt) strongly associate with myeloid neoplasia. TET2 is an α-ketoglutarate (αKG) and Fe2+-dependent DNA-dioxygenase that hydroxylates 5-methylcytosine-DNA (5mC), producing 5-hydroxymethylcytosine-DNA (5hmC). Ascorbic acid (AA) activates TET2 and other dioxygenases via several direct and indirect mechanisms, including recharging Fe3+ to Fe2+. Recent studies, chiefly in murine models, indicated that increased AA availability may alleviate the decreased of dioxygenase activity in cells affected by hypomorphic mutations via up-modulation of the remaining functional activity, particularly in heterozygous TET2mt, but it may also act via increased compensatory activity of other TET enzymes. Beyond these observations, the mechanisms of AA action in TET2mt bone marrow cells are not well understood. Yet to be identified are factors that modulate the effects of AA. We performed in silico binding prediction validated by surface plasmon resonance assay coupled with confirmatory in vitro and in vivo assays. In cell free system we demonstrated the AA increases the production of 5mC-DNA oxidation products (TDOP, 5hmC/5fmC/5caC) in a mixture of recombinant TET2wt:TET2S1898F 1:1 mimicking heterozygous TET2mt neoplasia. Using cell-free system with recombinant TET2 protein and the aforementioned biophysicochemical analyses, we further demonstrated that AA binds to the TET2 catalytic domain adjacent to Fe2+/aKG binding site and recycle Fe2+ back into the catalysis. In vitro cultures demonstrated that AA increases TDOP and slows proliferation in Tet2+/- and Tet2-/- mouse bone marrow progenitor cells. A similar but less pronounced effect was also observed in shRNA TET2KD MOLM13 and K562 cells in which AA restored the original proliferation rate of these cells which was increased by TET2KD. Finally, using cultures of healthy human marrows (retrovirally transduced with TET2 or scrambled shRNA), we showed that AA decreased the proliferation rate of these cells similar to ectopic overexpression of TET2. When primary MDS bone marrow samples with various TET2mt (n=4) were studied, the effects of AA were less consistent. Consistent with the observation, in SIG-M5 cell line characterized by natural TET2 mutations, AA only modestly increased 5hmC 2-fold. While, in high TET2 expression CMK cell line, AA increased 5hmC 6-fold. In vivo, high-dose oral AA increased TDOP, decreased spleen size and myeloproliferation, and prolonged survival in sublethally-irradiated Tet2+/-Gulo-/- and Tet2+/- mice. However the AA treatment has only a modest effect on the overall survival of these mice. Recent reports indicated that TET2 activity is down regulated by HDAC1/2 by deacetylation at the N-terminus (Zhang et al., Molecular Cell 65, 323-335, 2017) and up regulated by Sirtuin 1 (Class III HDAC) via lysine deacetylation in the catalytic domain (Sun et al., abstract, Blood 2016 128:1053). In search of factors that modulate the effects of TET, experiments were performed to determine if acetylation also modulates the activity of TET in leukemia cells. Inhibition of class I and II but not class III HDACs by trichostatin A (TSA) increased the 5hmC level 1.5-fold. However, when combined with AA, TSA amplifies the 5hmC level nearly 4-fold. Acetylation of lysine in the catalytic site inactivates TET via structural perturbations. Therefore, activation of class III HDACs should activate TET2 and inhibition should inhibit it. Indeed, consistent with this presumption, in leukemia cells, we observed that activation of sirtuin, a class III HDAC, by small molecule SRT1720, significantly increased 5hmC while the inhibitors 3-TYP and sirtnol reduced 5hmC. Context-dependent TET2 acetylation/deacetylation significantly affects its AA-mediated activation, and thus MDS/MPN clones with dysregulation of histone acetyltransferases and/or histone deacetylases may be less responsive to AA alone. In sum, we demonstrate the mechanism and context of AA-mediated TET activation which may guide novel treatment strategies for TET2-deficient neoplasia. Our results show that TET2 activity can be modulated by various mechanisms including acetylation/deacetylation interactions with the effects of AA. Disclosures Nazha: MEI: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Different CSF3R mutations (CSF3RMT) result in aberrant G-CSF signaling pathways and are linked to a wide range of myeloid disorders. Loss-of-function mutations in its extracellular domain cause severe congenital neutropenia (SCN). Activating mutations in the juxtamembrane region have been associated with a variety of myeloid malignancies. Truncating mutations in the cytoplasmic domain are associated with SCN cases that progress to MDS or AML. In this study, we evaluate the extent to which different CSF3RMT associate with disease onset, progression to leukemia and neutrophil counts in patients (pts) diagnosed with myeloid malignancies. We identified CSF3RMT cases in a cohort of 1400 pts [median age 71 years (yrs)]. We analyzed somatic and germline mutational patterns, and cross-sectional correlation with other gene mutations in CSF3RMT. A stringent algorithm based on conserved amino acid residues and alterations of protein features was used to predict the pathogenic significance of CSF3RMT. We identified 44 CSF3RMT: 33 germline (CSF3RGL) and 11 somatic (CSF3RS) variants. Most CSF3RGL were found in pts (median age 63 yrs) with MDS or related conditions (87% of all mutant cases), conversely these mutations were present in 5% (n= 22/424) of MDS, 3% (n= 7/244) MDS/MPN and Overall combined allelic burden in pts cohort was 2% vs. 1.6% expected allelic burden in control populations for the same variants (P=.02). CSF3R S were found in 11 pts (median age 71 yrs) with AML or MDS related conditions (73% of all mutant cases), conversely these mutations were present in 1.4% (n= 6/424) of AML, Focusing on associations between CSF3RMT and mutations in the class III receptor tyrosine kinases CSF1R, FLT3, and KIT we identified only FLT3 to be co-mutated with CSF3RMT. All 3 pts (2 CSF3RGL and 1 CSF3RS) with such co-mutations evolved to AML. In sum, we found that CSF3RGL do not commonly co-occur with CSF3RS, suggesting that the neutropenia observed at the sampling time most likely is causative of undetected GL variants and/or is representative of a long unrecognized disease. Disclosures Nazha: MEI: Consultancy. Carraway:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau; FibroGen: Consultancy. Santini:Otsuka: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Discovery of many somatic lesions in leukemia enables development of targeted therapies. TET2 is one of the most frequently mutated genes in MDS/related disorders and is also present in a significant proportion of CHIP (clonal hematopoiesis of indeterminate potential) carriers. TET2 mutations (TET2mt) are mostly loss of function and occur in biallelic, heterozygous and hemi/homozygous configurations. TET2 encodes for Fe2+-dependent DNA dioxygenase that utilizes 2-ketoglutarate (αKG) for oxidation of 5-methylcytosine (mC) that results in demethylation either actively, by base excision repair of further oxidation products (fC or caC), or passively, via replication, due to DNA methyltransferase's inability to recognize 5-hydroxymethylcytosine (hmC). TET2mt are good targets for drug discovery because they often initiate the clonal evolution and are present in a large fraction of patients. However, except for ascorbic acid (AA) applied to augment TET2 activity and hypomethylating agents to which TET2mtmay be more susceptible, no specific therapies have been conceptualized for TET2mt disease. Synthetic lethality can be applied to genetic loci affected by loss of function mutations never occurring in homo/hemi/homozygous configuration. However, many tumor suppressor genes (TSG) are similar to TET2, where biallelic inactivation promotes oncogenicity and thus synthetic lethality would not be directly applicable. However, TET2 has TET1/TET3 homologs and a transient inhibition of TET enzymes could still yield a synthetic lethality (particularly in TET2mt). The idea for the proposed therapeutic strategy of TET inhibition was conceptualized based on in vivo observations of mutual exclusivity of TET2mt and IDH1/2 mutations which produces 2HG as a bone-fide natural TET inhibitor. Indeed, in our study of 485 TET2mt cases only 9 carried IDH1/2mt (mostly tiny subclones). Conversely, among 157 IDH1/2mtcases, there were only 11TET2mt (p=5.5x10-9) of which 4 were non-deleterious missense alterations or had a small clonal burden. To further support our hypothesis, we knocked in IDH1mt controlled by the doxycycline-inducible promoter into TET2mt cells. Induction of IDH1R132C or IDH1R132H (or IDH2mt), expression resulted in rapid cytotoxicity in TET2mt, and no growth perturbation was observed for TET2wt cells. Similar results have been observed in mice, where knockdown of TET3 in TET2 background shortened the life span. These observations led us to the idea of developing an aKG antagonist TET specific inhibitors (TETi). Using a structure-guided targeted discovery approach we designed, synthesized and characterized TETi, which demonstrated dose dependent inhibition of dioxygenase activity in a cell-free system. Using an iterative approach of design synthesis and activity, we selected the most potent 'hit', designated as TETi76, for further evaluation. Esterified TETi76 decreased 5hmC production and selectively induced cell death in TET2mt leukemia cell lines, SIGM5 (TET2-/-) and OCI-AML5 (TET2+/-), while a minimal effect was observed in K562 and CMK which are TET2+/+. The LD50 of TETi was 250-fold lower than 2HG in TET2mt cells. In TET2-/-engineered K562, TETi76 also showed cytotoxicity leaving a therapeutic window as compared with wild type K562. Normal bone marrows were resistant to TETi76 in clonogenic assay. In vitro mixing experiments in which Tet2mt/Tet2wt were subjected to methocult cultures at fixed ratios to mimic evolving Tet2mt clones, CD45.2 (either Tet2+/-or Tet2-/-) outcompeted CD45.1 marrow in a control setting, while treatment with TETi76 led to a gradual elimination of mutant marrow. This result was further recapitulated in vivo. In a competitive repopulation transplantation model using graft consist of a mixture of CD45 isotypes mismatched Tet2+/- or Tet2-/-and Tet2+/+marrow cells, TETi76 treatment selectively eliminated Tet2 deficient marrow. Our data have several important implications. It is likely that compensatory function from other TETs or remaining allele is needed for survival and elimination of dioxygenase appears to be lethal. This explains the exclusivity of TET2/IDH1/2 mutations. Dioxygenase inhibitors may have therapeutic applicability for selective elimination of TET2mt cells in MDS or potentially as a preventive measure in CHIP. TETi may represent a novel class of antileukemic agents. Disclosures Nazha: MEI: Consultancy. Maciejewski:Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

SF3B1 is a splicing factor gene whose mutations are pathognomonic of MDS with ring sideroblasts. Because of the ubiquitous importance of splicing, a major barrier in targeting cells with spliceosomal mutations is the discovery of agents decreasing the competitiveness of mutant cells while preserving the integrity of wild type cells. To date no specific therapies are FDA approved for SF3B1 mutant (SF3B1MT) MDS and few agents are in early clinical testing. We describe a novel targeted approach to drug development for SF3B1MT malignancies. Our investigative strategy started with a high throughput drug screen. We introduced K700E mutation into myeloid cells using CRISPR/Cas9. We then subjected K562+/K700E and matched-parental K562 cells to high throughput drug screen of a library of 3,000 mechanistically annotated, non-redundant, bioactive compounds. Top hits were validated by dose response testing (8 concentrations in half-log dilutions). Our interest focused on compounds with cytostatic activity towards K562+/K700E cells. Among these, a 4-pyridyl-2-anilinothiazole (PAT) showed preferential inhibition of growth in K562+/K700E cells with an IC50 of 3uM. Dose response showed that K562+/K700E cells were significantly sensitive to PAT with a growth inhibition of 20%, 32%, 51%, 65%, 95% at .3uM (P=.01), 1uM (P=.002), 3uM (P Using PAT as our lead, we employed a fragment based reiterative medicinal chemistry approach to synthesize selective compounds and improve therapeutic index. Libraries were constructed following Lipinski rules with ease of synthetic construction in mind. Pilot libraries were constructed via the classic Hantsch thiazole synthesis which involves condensation of α-halo ketones with substituted thioureas. This enterprise investigated SAR modifications of PAT by considering features such as regiochemistry and basicity of the nitrogen of the pyridine ring in the head region; replacement of the spacer 2,4-disubstituted thiazole ring with heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); alternatives for the aniline of the tail region e.g., sulfonamide, amide, and substituted amine linkers and substituted aromatic and aliphatic ring structures for the phenyl substituent. A major challenge in drug development of agents targeting spliceosomal mutations is the identification of key clusters of aberrantly spliced genes restored by drug treatment, e.g., identification of a pharmacodynamic endpoint that could be used to prove that that the drug reached its target. SF3B1 mutations induce aberrant 3′-ss selection by promoting use of alternative branch points. To identify biomarkers of splicing changes to screen our libraries, K562+/K700E and parental cells were treated with PAT (3uM) and RNA libraries were subjected to RNA Seq. The splicing pattern of parental cells was used as reference. We identified 328 cassette exons (±25% splicing inclusion difference, pFDR In sum, we described novel classes of compounds that inhibit the expansion of SF3B1MT cells by restoring the splicing defects intrinsically associated with SF3B1. Disclosures Carraway: Novartis: Speakers Bureau; Jazz: Speakers Bureau; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Subcellular compartmentalization of exoribonucleases (RNAses) is an important control mechanism in the temporal and spatial regulation of RNA processing and decay. Despite much progress towards understanding RNAse substrates and functions, we know little of how RNAses are transported and assembled into functional, subcellularly restricted complexes. To gain insight into this issue, we are studying the exosome-binding protein Dis3, a processive 3' to 5' exoribonuclease. Here, we examine the interactions and subcellular localization of the Drosophila melanogaster Dis3 (dDis3) protein. N-terminal domain mutants of dDis3 abolish associations with the 'core' exosome, yet only reduce binding to the 'nuclear' exosome-associated factor dRrp6. We show that nuclear localization of dDis3 requires a C-terminal classic nuclear localization signal (NLS). Consistent with this, dDis3 specifically co-precipitates the NLS-binding protein importin-alpha3. Surprisingly, dDis3 constructs that lack or mutate the C-terminal NLS retain importin-alpha3 binding, suggesting that the interaction is indirect. Finally, we find that endogenous dDis3 and dRrp6 exhibit coordinated nuclear enrichment or exclusion, suggesting that dDis3, Rrp6 and importin-alpha3 interact in a complex independent of the core. We propose that the movement and deposition of this complex is important for the subcellular compartmentalization and regulation of the exosome core.

CD4 T cell-dependent antibody responses are essential for limiting Plasmodium parasite replication and the severity of malaria; however, the factors that regulate humoral immunity during highly inflammatory, Th1-biased systemic infections are poorly understood. Using genetic and biochemical approaches, we show that Plasmodium infection-induced type I interferons limit T follicular helper accumulation and constrain anti-malarial humoral immunity. Mechanistically we show that CD4 T cell-intrinsic type I interferon signaling induces T-bet and Blimp-1 expression, thereby promoting T regulatory 1 responses. We further show that the secreted effector cytokines of T regulatory 1 cells, IL-10 and IFN-γ, collaborate to restrict T follicular helper accumulation, limit parasite-specific antibody responses, and diminish parasite control. This circuit of interferon-mediated Blimp-1 induction is also operational during chronic virus infection and can occur independently of IL-2 signaling. Thus, type I interferon-mediated induction of Blimp-1 and subsequent expansion of T regulatory 1 cells represent generalizable features of systemic, inflammatory Th1-biased viral and parasitic infections that are associated with suppression of humoral immunity., Author Summary Humoral immunity is essential for host resistance to pathogens that trigger highly inflammatory immune responses, including Plasmodium parasites, the causative agents of malaria. Long-lived, secreted antibody responses depend on a specialized subset of CD4 T cells called T follicular helper (Tfh) cells. However, anti-Plasmodium humoral immunity is often short-lived, non-sterilizing, and immunity rapidly wanes, leaving individuals susceptible to repeated bouts of malaria. Here we explored the relationship between inflammatory type I interferons, the regulation of pathogen-specific CD4 T cell responses, and humoral immunity using models of experimental malaria and systemic virus infection. We identified that type I interferons promote the formation and accumulation of pathogen-specific CD4 T regulatory 1 cells that co-express interferon-gamma and interleukin-10. Moreover, we show that the combined activity of interferon-gamma and interleukin-10 limits the magnitude of infection-induced Tfh responses, the secretion of parasite-specific secreted antibody, and parasite control. Our study provides new insight into the regulation of T regulatory 1 responses and humoral immunity during inflammatory immune reactions against systemic infections.