ALBERT

Description: 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.

Description: 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

Description: 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

Description: 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.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is usually associated with reduced bone marrow (BM) capacity caused by acquired idiopathic aplastic anemia (AA). PIGA mutations lead to a partial or total deficiency of glycosylphosphatidyl-inositol (GPI) anchor proteins (AP). AA is characteristically accompanied by the presence of often tiny GPI-AP deficient clones, which in a significant proportion of patients (10-15%), irrespective of the initial success of immunosuppressive therapy, will evolve to produce manifest hemolytic PNH. Indeed in our cohort of BM failure patients (n=319), 41% of AA patients had a PNH clone present (0.02-20% of granulocytes) (AA/PNH), 14% of patients had primary PNH (primary PNH), and 8% had a history of PNH post AA (secondary PNH). To date, drug development for PNH has focused on designing supportive therapies to prevent transfusions due to hemolysis or thrombotic complications. In addition to the current FDA approved C5 inhibitor eculizumab, new, more convenient and effective complement blockers are under development. Apart from hematopoietic stem cell (HSC) transplantation, no direct strategies targeting basic pathophysiologic mechanisms of PNH have been ventured to prevent evolution of PNH clones and cure the disease. In early AA/PNH syndrome, the PIGA mutant HSCs are rare and unlikely contribute to significant blood cell production. While in later stages of manifest hemolytic PNH, hematopoiesis relies most frequently on mutant HSCs and thus elimination of these cells would result in AA. We hypothesized that if a selective inhibitor of GPI-AP-deficient [GPI-AP (-)] cells can be developed, it could be used primarily in AA/PNH patients with a small clone size. The hope would be to prevent both later expansion of GPI-AP d(-) cells and development of manifest PNH. To discover compounds acting selectively against GPI-AP (-) cells, we subjected wild type (WT) and GPI-AP (-) cell lines (K562, TF-1) to a high-throughput screen using a platform of 3000 bio-active molecules to identify hits and chemical compounds capable of selectively eliminating GPI-AP (-) cells. Our robotic screen yielded several top hits including GR -89696 fumarate, D-cycloserine and CGS-15943. Dose-response experiments confirmed CGS-15943 as a candidate growth inhibitor of GPI-AP (-) cells. CGS-15943 is an adenosine receptor antagonist and non-phosphodiesterase inhibitor which has previously been shown to inhibit cancer cell growth via PI3K/Akt pathway. Low range dose CGS-15943 (1uM) induced cell growth inhibition in K562 and TF-1 GPI-AP (-) cells by 4.7 fold and 3.2 fold, respectively. No cell growth arrest was observed in K562 WT and TF-1 WT cells, as the percentage of alive cells was 〉95% upon drug treatment. Mixed competition assays were conducted in vitro using equal ratios of K562 and TF-1 WT and GPI-AP (-) cells exposed to CGS-15943 (1uM). Six days after culture, flow cytometric analysis of CD59 surface expression revealed that CGS-15943 allowed for preferential survival of WT cells (84.7 % K562, 96.3% TF-1) vs. GPI-AP (-) cells (15.3% K562, 3.7% TF-1). CGS-15943 induced an increase in the % of AnnexinV+/PI- and AnnexinV+/PI+ in TF-1 GPI-AP (-) cells (12.04% and 44.82, respectively). Similar results were obtained in K562 GPI-AP (-) cells (15.84% and 21.08%). Mononuclear cells of a PNH patient were stimulated with CD3/28 beads in presence of CGS-15943. Flow cytometric analysis indicates a dose dependent growth inhibition effect on GPI-AP (-) lymphocytes after 3 days of culture. Previous reported observations from our group identified that the survival differences between GPI-AP (-) and WT cells largely depend on active PI3K signaling pathway. Our pilot investigation of CGS-15943 - indicates that CGS-15943 induces an decrease in the protein expression of the PI3K isoform - p110γ - exclusively in GPI-AP (-) cells possibly suggesting that CGS-15943 inhibits the catalytic subunit of- p110γ. In sum, we describe that the small molecule compound CGS-15943 selectively eliminates GPI-AP (-) cells in vitro, in both cell lines and in primary PNH cells most likely interfering with the PI3K/AKT survival pathway. Disclosures Maciejewski: Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Description: 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

Description: 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.