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AbstractRapid and functional bone‐tendon (B‐T) healing remains a difficulty in clinical practice. Tissue engineering has emerged as a promising strategy to address this problem. However, the majority of tissue engineering scaffolds are loaded with stem cells to enhance the regenerability in B‐T healing, which is complicated and inconvenient for clinical application. Accordingly, developing a cell‐free scaffold with chemotactic function and chondrogenic inducibility may be an effective approach. In this study, a collagen affinity peptide derived from the A3 domain of von Willebrand factor (a hemostasis factor) was fused into the C‐terminal of a stromal cell‐derived factor‐1α (SDF‐1α) to synthesize a recombinant SDF‐1α capable of binding collagen and chemotactic activity. The recombinant SDF‐1α was then tethered on the collagen fibers of a book‐shaped acellular fibrocartilage scaffold (BAFS), thus fabricating a novel scaffold (C‐SDF‐1α/BAFS) with chemotactic function and chondrogenic inducibility. In vitro tests determined that this scaffold was noncytotoxic and biomimetic, could attract stem cells migrating to the scaffold using sustainably released C‐SDF‐1α, and inducedthe interacting stem cells down the chondrogenic lineage. In vivo, the C‐SDF‐1α/BAFS significantly enhanced the B‐T healing in a rabbit partial patellectomy model, as shown by the larger cartilaginous metaplasia region, better fibrocartilage regeneration, additional bone formation, and improved biomechanical properties. Therefore, the findings of the study demonstrate that the C‐SDF‐1α/BAFS could potentially be applied for B‐T healing.

INTRODUCTION The cerebral cortex underlies our complex cognitive capabilities. Variations in human cortical surface area and thickness are associated with neurological, psychological, and behavioral traits and can be measured in vivo by magnetic resonance imaging (MRI). Studies in model organisms have identified genes that influence cortical structure, but little is known about common genetic variants that affect human cortical structure. RATIONALE To identify genetic variants associated with human cortical structure at both global and regional levels, we conducted a genome-wide association meta-analysis of brain MRI data from 51,665 individuals across 60 cohorts. We analyzed the surface area and average thickness of the whole cortex and 34 cortical regions with known functional specializations. RESULTS We identified 369 nominally genome-wide significant loci ( P < 5 × 10 −8 ) associated with cortical structure in a discovery sample of 33,992 participants of European ancestry. Of the 360 loci for which replication data were available, 241 loci influencing surface area and 66 influencing thickness remained significant after replication, with 237 loci passing multiple testing correction ( P < 8.3 × 10 −10 ; 187 influencing surface area and 50 influencing thickness). Common genetic variants explained 34% (SE = 3%) of the variation in total surface area and 26% (SE = 2%) in average thickness; surface area and thickness showed a negative genetic correlation ( r G = −0.32, SE = 0.05, P = 6.5 × 10 −12 ), which suggests that genetic influences have opposing effects on surface area and thickness. Bioinformatic analyses showed that total surface area is influenced by genetic variants that alter gene regulatory activity in neural progenitor cells during fetal development. By contrast, average thickness is influenced by active regulatory elements in adult brain samples, which may reflect processes that occur after mid-fetal development, such as myelination, branching, or pruning. When considered together, these results support the radial unit hypothesis that different developmental mechanisms promote surface area expansion and increases in thickness. To identify specific genetic influences on individual cortical regions, we controlled for global measures (total surface area or average thickness) in the regional analyses. After multiple testing correction, we identified 175 loci that influence regional surface area and 46 that influence regional thickness. Loci that affect regional surface area cluster near genes involved in the Wnt signaling pathway, which is known to influence areal identity. We observed significant positive genetic correlations and evidence of bidirectional causation of total surface area with both general cognitive functioning and educational attainment. We found additional positive genetic correlations between total surface area and Parkinson’s disease but did not find evidence of causation. Negative genetic correlations were evident between total surface area and insomnia, attention deficit hyperactivity disorder, depressive symptoms, major depressive disorder, and neuroticism. CONCLUSION This large-scale collaborative work enhances our understanding of the genetic architecture of the human cerebral cortex and its regional patterning. The highly polygenic architecture of the cortex suggests that distinct genes are involved in the development of specific cortical areas. Moreover, we find evidence that brain structure is a key phenotype along the causal pathway that leads from genetic variation to differences in general cognitive function. Identifying genetic influences on human cortical structure. ( A ) Measurement of cortical surface area and thickness from MRI. ( B ) Genomic locations of common genetic variants that influence global and regional cortical structure. ( C ) Our results support the radial unit hypothesis that the expansion of cortical surface area is driven by proliferating neural progenitor cells. ( D ) Cortical surface area shows genetic correlation with psychiatric and cognitive traits. Error bars indicate SE. IMAGE CREDITS: (A) K. COURTNEY; (C) M. R. GLASS

Introduction: Emerging evidence supports that cytogenetic abnormalities (CAs) drive myelomagenesis and heterogeneity (e.g., clinical presentation, response to therapy, and prognosis) of multiple myeloma (MM). Diversity of CAs, including primary (IgH translocations and trisomies) and secondary CAs (copy number abnormalities) argues that MM is not a "single disease". Thus, identification of CAs at diagnosis is essential for risk stratification, guiding treatment, and prognostic estimation in daily practice. However, although frequency, configuration, and significance of CAs have been well documented in the Western countries, this information is lacking in the Asian population. To this end, a multi-center retrospective analysis was carried out to examine epidemiology and prognostic significance of CAs alone or in combination in a cohort of Chinese patients with newly-diagnosed MM (NDMM). Materials and Methods: A total of 1015 NDMM patients who had the baseline information of CAs detected by FISH at four institutes nationwide were included. According to the IMWG consensus updated in 2016, 1q gain, del(17p), t[4;14], and t[14;16] were defined as high-risk CAs (HRCAs), while another HRCA t[14;20] was not tested routinely in a majority of these patients. In addition, del(1p) and del(13q14) were considered as an adverse CA. According to the mSMART3.0 proposed by Mayo Clinic in 2018, double-hit (DHMM) and triple-hit MM (THMM) were defined as co-occurrence of 2 or >= 3 HRCAs, respectively. The Kaplan-Meier approach was used to estimate progression-free survival (PFS) and overall survival (OS). Results: In this cohort, the median age of 1015 patients was 61 years; 63.5% were male. The type of IgG, light chain, IgA, IgD, non/oligosecretory, IgM, or IgE accounted for 42.9%, 26.2%, 24.5%, 3.4%, 2.5%, 0.4%, and 0.1%, respectively. Del(13q) (46.4%) and 1q gain (46.1%; 3 copies = 73.8%, >= 4 copies = 26.2%) represented the most common CAs, followed by t(4;14) (14.0%), t(11;14) (11.8%), del(1p) (11.5%), del(17p) (9.9%; 20-50% cells = 35.6%, > 50% cells = 64.4%), and t(14;16) (5.1%). While none of these CAs was detected in 23.8% of cases, the frequency of patients who carried 1 - 5 CAs was 31.9%, 28.0%, 13.4%, 2.0%, and 0.9%, respectively. In the 1q+ cases, 36.4% patients carried second CA(s), including del(13q) (61.1%), t(4;14) (20.3%), del(1p) (14.8%), del(17p) (10.7%), t(11;14) (10.4%), and t(14;16) (8.1%). In the del(17p) cases, 57.5% patients had additional CA(s), including del(13q) (75.2%), 1q gain (49.5%), del(1p) (21.6%), t(4;14) (19.6%), t(14;16) (8.7%), and t(11;14) (2.2%). In the cases bearing IgH translocations, 26.0% patients also carried other CA(s), including del(13q) and 1q gain (61.9% for each), and del(17p) (9.3%). In the cases harboring HRCAs, the percentage of patients who carried 1 - 4 HRCAs was 70.2%, 24.8%, 3.7%, and 1.1%, respectively. Overall, 14.3% and 2.9% patients had DHMM or THMM, of whom 65.0%, 18.0%, 12.0%, and 5.0% had 2 - 5 HRCAs, respectively. While there was no significant difference in PFS between the cases carrying 1 and 2 CAs (P = 0.209), the patients who had 3 or more CAs displayed a sharp reduction in median PFS (P = 0.022 and P = 0.003 for 3 vs 1 or 2 CAs). Although multiple CAs was associated with shorter median OS, no statistical significance was observed for each comparison (P > 0.05). However, patients who carried >= 2 HRCAs had significantly shorter median PFS (12.1 months; P = 0.0004) and OS (29.3 months; P = 0.027) than those who had one single HRCA (32.2 and 65.6 months for median PFS and OS). Conclusion: In comparison with the Western countries, the incidence of secondary HRCAs (e.g., 1q gain and del(17p)) is relatively higher in Chinese patients at diagnosis, while the standard-risk CA such as t(11;14) is clearly less frequent. The proportion of Chinese patients who carry multiple CAs (up to 5) or HRCAs appear to be greater as well. In this context, patients carrying two or more HRCAs, so called DHMM or THMM, exhibit significantly worse outcome than those carrying only one HRCA. Together, this study builds up an up-to-date profile of CAs for Chinese patients, which might lay a foundation for revising the criteria for risk stratification and the guideline for treatment that is more feasible and practicable in China. It also provides the information about frequency and configuration of MM-driven CAs, which might be more relevant to the Asian population. Disclosures Kumar: Takeda: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

Abstract Abstract 64 Megakaryocytes are one of the few cell types that undergo a modified form of the cell cycle termed endomitosis, in which cells skip the late stages of mitosis to become polyploid. Murine and human megakaryocytes commonly reach modal ploidy states of 32N and 16N, respectively, and can sometimes achieve DNA contents as high as 64N. Polyploidization is associated with upregulation of megakaryocyte lineage specific genes, proplatelet formation and expression of genes related to apoptosis. RNA expression array studies have shown that high ploidy states are strongly correlated with megakaryocyte differentiation and maturation. Importantly, the choice of a megakaryocyte to undergo polyploidization and differentiation is inextricably linked to exit from the proliferative cell cycle. Given that megakaryocytes in patients with essential thrombocythemia are hyperproliferative and that those in primary myelofibrosis fail to undergo normal differentiation or polyploidization, we hypothesized that small molecule inducers of polyploidization would drive these cells to exit the proliferative cell cycle and undergo terminal differentiation or death. In collaboration with the Broad Institute, we performed a high throughput screen and identified small molecules that induce polyploidization and proliferative arrest of malignant megakaryocytes, including those that express MPLW515L and JAK2 V617F. We have shown that these compounds, including the Rho kinase inhibitor dimethylfasudil (diMF), selectively increase polyploidization, expression of megakaryocyte cell surface markers, and apoptosis of murine and human megakaryocytic cell lines and primary cells. Furthermore, diMF blocked the growth of primary human AMKL blasts both in vitro and in vivo. With respect to MPNs, diMF showed potent activity against megakaryocytic cell lines and primary cells expressing either JAK2 V617F or MPL W515L alleles commonly associated with these disorders. diMF inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis of megakaryocytes transduced with JAK2 V617F or MPLW515L. diMF also significantly reduced megakaryocyte colony forming units (CFU-MK), and induced polyploidization and differentiation of bone marrow and fetal liver megakaryocytes from Gata1 mutant mice, which develop a PMF-like disease. Given that diMF induces growth arrest, polyploidization and apoptosis of cells that express activated mutants of JAK2 and MPL, we predicted that it, as well as other small molecule inducers of polyploidy, would be efficient at restraining aberrant megakaryocyte proliferation in both PMF and ET. To assay the effectiveness of diMF in these disorders, we treated peripheral blood mononuclear cells from patients with either PMF or ET with diMF and monitored growth and maturation of megakaryocytes. We discovered that diMF induced polyploidization and subsequent apoptosis of both types of MPN primary samples. diMF also reduced CFU-MK of these MPN patient samples. Next, we assessed the activity of diMF in a model of MPN in which congenic recipients of MPLW515L transduced Balb/C bone marrow cells develop a rapid MPN characterized by leukocytosis, thrombocytosis, bone marrow fibrosis, and death. diMF led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen of a mouse model of PMF. diMF also led to a significant reduction in the platelet count and a trend towards decreased white cell count, with no effect on hematocrit. Overall, diMF results were comparable to intermediate doses of INCB16562. These encouraging results strongly suggest that diMF induces a decline in megakaryocyte lineage, which leads to reduction in platelet count, and support pre-clinical development of diMF for megakaryocytic subtypes of MPNs. Of note, diMF did not inhibit the phosphorylation of Stat5 or Stat3, suggesting that it acts through a mechanism distinct from JAK2 inhibitors. Interestingly, combination of diMF with a selective JAK2 inhibitor greatly enhanced the efficacy of diMF to inhibit proliferation and induce apoptosis in MPLW515L transduced megakaryocytic cell line. These data support combining JAK inhibition and induction of megakaryocyte polyploidy as a new therapeutic strategy for MPNs. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3862 Somatic mutations in JAK2 and MPL are associated with the BCR-ABL negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). While oral JAK2 inhibitors improve peripheral blood counts and splenomegaly, these drugs show varying effects on JAK2 mutant allele burden and do not eliminate the malignant clone in humans or in animal models of MPN. Although much of the research to date has focused on JAK/STAT signaling, other pathways downstream of the class I cytokine receptors, including PI3K/AKT and ERK are also activated in MPNs. Our hypothesis is that persistent activation of these signaling pathways contributes to the progression of myeloproliferative neoplasms (MPNs). Multiple studies have shown that overexpression of activated JAK2 and MPL mutants (e.g. JAK2 V617F or MPL W515L) in primary murine bone marrow cells causes robust AKT, STAT3, and STAT5, JNK, ERK phosphorylation. To determine the extent to which these signaling pathways are involved in the disease, we cultured JAK2 V617F and MPL W515L expressing clones of the G1ME and 6133 megakaryocytic cell lines with a panel of small molecule kinase inhibitors. We discovered that inhibition of the PI3K/AKT and MAPK/JNK signaling pathways with triciribine and SP600125 respectively, potently suppressed growth of these cells by inducing G1 arrest and apoptosis. In contrast, inhibitors of Ras, ERK, Src family tyrosine kinase, and PKC failed to significantly inhibit proliferation of the JAK2 or MPL mutant expressing cells. Murine bone marrow cells transduced with MPL W515L show a dramatic expansion of megakaryocytes (CFU-MKs) and this expansion was abrogated in the presence of PI3K/AKT inhibitors suggesting a requirement for this pathway in aberrant megakaryocyte expansion. Since AKT inhibition showed the strongest effect, we assayed the activity of MK-2206, a potent and selective allosteric AKT inhibitor, on multiple models of MPNs. We discovered that MK-2206 induced proliferative arrest and apoptosis accompanied by suppression of PI3K/AKT signaling in G1ME and 6133 cells expressing MPLW515L. MK-2206 also potently inhibited liquid culture growth and colony formation of PMF patient CD34+ cells in vitro. Finally, in preliminary murine transplants experiments with MPLW515L expressing bone marrow progenitors, treatment with MK-2206 led to significant reductions in peripheral blood leukocytosis and extramedullary hematopoiesis. Together, these findings demonstrate that the PI3K/AKT axis represents a rational target for therapy in human myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

Abstract Both genome wide association studies (GWAS) of common variation and exome wide association studies (EXWAS) of rare variation have successfully identified disease susceptibility variants for a variety of diseases. One GWAS of inherited susceptibility to Acute Myeloid Leukemia (AML) has been conducted, but no EXWAS have been performed to measure risk of AML attributable to low-frequency constitutional genetic variation. We performed the first EXWAS of risk of AML as a nested case-control study in the DISCOVeRY-BMT (Determining the Influence of Susceptibility Conveying Variants Related to one-Year mortality after BMT) cohorts. The DISCOVeRY-BMT parent study examined transplant-related mortality in leukemia patients undergoing unrelated donor allogeneic BMT. To identify low frequency variants and genes contributing to increased susceptibility to AML we used genotype data from the Illumina HumanExome BeadChip typed in the DISCOVeRY-BMT cohorts; the HumanExome BeadChip contains 242,901 variants, which are mainly protein-coding variants. The optimal sequence kernel association test (SKAT-O) was used to analyze gene-level associations with risk of AML. These gene-based tests evaluate the cumulative effects of multiple single gene variants on risk of AML. Analyses were performed in all European American AML cases and two subtypes: 1) de novo AML, 2) de novo AML with normal cytogenetics. Models were adjusted for age at transplant and principal components to control for population stratification. For gene-based tests at least 2 variants with minor allele frequency (MAF) ≤ 5%, were required to be present in the gene. This yielded a total of 13,687 genes tested, and a Bonferroni corrected significance level of P<3.65 x 10-6. Association tests were performed in 1,189 AML cases reported to CIBMTR 2000-08 (Cohort 1) and 327 AML cases reported to CIBMTR from 2009-11 (Cohort 2). Controls in Cohorts 1 (n=1,986) and 2 (n= 515) were 10/10 HLA-matched unrelated donors who passed a comprehensive medical exam and deemed healthy. We used metaSKAT to combine Cohorts 1 and 2 and obtain p-values of association with AML. We present the results of gene-level tests significant in both cohorts. The likely pathogenicity of these variants was determined in silico using SIFT, PolyPhen and MutationTaster. Patient characteristics are in Table 1. DNMT3A, on chromosome 2, was associated in the gene-based test with risk of AML (Pmeta=1.70x10-9, Table 2). Three missense variants at MAF <1% comprise both overall AML and de novo AML gene-based association: exm177559 (Asn->Ser), exm177507 (Arg->His), and exm177543 (Arg->Trp). Normal cytogenetics de novo AML gene-based assocations consisted of only 2 of these variants: exm177559 and exm177507 (Table 2). While prevalence of exm177507 is <1% for all AML cases, in de novo AML with normal cytogenetics the MAF was higher at 3%. The other 2 variants had a MAF<1% irrespective of subtype. Somatically, DNMT3A is most frequently mutated in hematologic malignancies, with >30% of de novo AML cases with a normal karyotype and >10% of MDS patients having DNMT3A mutations. Although these are germline gene associations all three of the variants found have been reported somatically in hematologic malignancies. In 200 AML cases from The Cancer Genome Atlas (TCGA) p.R882H (represented as exm177507 on the exome chip) was a frequent somatic mutation (25%). Exm177543 (p.R635W) and exm177559 (p.N501S) are reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) as somatic mutations involved in hematopoietic and lymphoid tissue in both cell lines and humans. Exm177507 and exm177543 show evidence of pathogenicity in all three in silico tools, while exm177559 was reported as deleterious and disease causing by Sift and MutationTaster, respectively. Our results show that multiple potentially pathogenic missense germline variants in DNMT3A comprise the gene-based association with AML, specifically de novo AML with normal cytogenetics. Given the functional nature of these variants it is possible germline risk stratification could be informative in determining AML risk, and subsequently development of AML harboring DNMT3A mutations. Confirmation of these findings in additional cohorts could have implications for individualized risk screening, prediction and prognosis. Additional cytogenetic subgroup analyses, including treatment-related AML, are underway. Disclosures Hahn: Novartis: Equity Ownership; NIH: Research Funding. McCarthy:Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sucheston-Campbell:NIH/NCI: Research Funding.

Abstract We recently reported that the induction of polyploidization of malignant megakaryocytes shows great promise as a new therapy for acute leukemia. Polyploidization inducers such as dimethylfasudil (diMF) and MLN8237, both of which target Aurora A kinase (AURKA), induce proliferation arrest, polyploidization, expression of megakaryocyte differentiation markers and apoptosis of leukemic megakaryocytes in vitro and in vivo. Since megakaryocytes in primary myelofibrosis (PMF) show impaired polyploidization and maturation, and likely directly contribute to the disease, we predicted that polyploidization inducers would provide a new therapeutic strategy. To determine the effect of these compounds on the growth of MPN cells, we first treated the JAK2 V617F mutant megakaryocytic SET2 cell line with varying doses of MLN8237 and diMF. Both compounds effectively and dose dependently inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis. Furthermore, MLN8237 synergized with ruxolitinib to induce apoptosis of the SET2 cells and also potently induced growth arrest of JAK2 inhibitor persistent SET2 cells. We observed a similar polyploidization and differentiating activity of MLN8237 and diMF on megakaryocytes derived from primary human PMF progenitors. The ability of these agents to induce polyploidization was specific, as the non-megakaryocyte fractions of the cultures were not affected. Next, we assayed the activity of polyploidization inducers on progression of MPNs in two mouse models: JAK2V617F conditional knockin mice and mice engrafted with MPLW515L expressing bone marrow progenitors. Of note, spleens from both mouse models displayed a robust increase in both total and phosphorylated forms of AURKA relative to control animals, further suggesting that AURKA is a rational target in this disease. We first assayed the activities of MLN8237 and diMF in the MPLW515L bone marrow transplantation model. Recipient mice develop a rapid MPN characterized by leukocytosis, thrombocytosis and bone marrow fibrosis. Both MLN8237 and diMF reduced the disease burden, as evidenced by significant reductions in the liver and spleen weights, white cell counts and platelet counts. Both compounds also led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen. Moreover, plasma levels of TGF-β a known myelofibrogenic cytokine, were decreased by more than 3-fold by the drug treatment. Both diMF and MLN8237 led to selective polyploidization of megakaryocytes in the spleen as well as marked reductions in the levels of p-AURKA. Of note, neither agent affected the extent of phosphorylation of STAT3 or STAT5. Therefore, we tested whether the combined use of a JAK inhibitor and a polyploidy inducer would show enhanced activity in vivo. Indeed, the combination of MLN8237 and ruxolitinib led to greater reductions in tumor burden in the MPLW515L mouse model than either agent alone. Similar results were obtained using the JAK2V617F knock-in model. To further validate our conclusion that AURKA is a target in PMF, we infected Aurkafl/fl floxed bone marrow progenitors with MPLW515L and transplanted the cells to irradiated recipients. Excision of both alleles of Aurka by Cre mediated recombination completely resolved the disease, while heterozygous deletion of Aurka significantly reduced the disease burden, in a manner similar to treatment with MLN8237. Given that heterozygous deletion of Aurka does not alter normal hematopoiesis in mice, the fact that a 50% reduction in kinase expression was associated with a significant decrease in disease burden suggests that there is an effective therapeutic window in which AURKA inhibitors will be effective against MPN while sparing normal hematopoiesis. Although JAK inhibitors provide symptomatic relief, it is becoming clear that they are not curative. Thus, there is an urgent need to develop new agents to use in combination with JAK inhibitors. Our data reveal that inducing polyploidization and differentiation of dysplastic megakaryocytes in PMF ameliorates features of the disease both in vitro and in vivo. These results support the initiation of clinical studies that combine a JAK inhibitor with an AURKA inhibitor. Disclosures: Crispino: Sanofi: Research Funding.

Abstract #the authors contributed equally to this work While survival outcomes after HLA-matched unrelated donor (URD) blood and marrow transplant (BMT) have significantly improved over the last two decades, about 40% of patients die before 1-year post URD BMT. Previously we performed a genome-wide association study (GWAS) named DISCOVeRY-BMT (Determining the Influence of Susceptibility COnveying Variants Related to one-Year mortality after BMT) to investigate the contributions of common genetic variants on survival outcomes. To address the specific contributions of low frequency (≤ 1%) exonic variants on survival outcomes, we used the DISCOVeRY-BMT cohorts typed with the Illumina HumanExome BeadChip containing 242,901 variants, which are mainly protein-coding variants. We used a gene-based test (the optimal sequence kernel association test (SKAT-O)) to evaluate the cumulative effects of multiple genetic variants within a gene on overall survival (OS), transplant-related mortality (TRM), and disease-related mortality (DRM) 1 year after URD BMT. SKAT-O measures the overall significance of the gene and allows the variants to have different directions and magnitude of effects. We used a 2-stage study design: SKAT-O analysis was carried out in Cohort 1 consisting of 1,972 AML, ALL or MDS patients reported to the Center for International Blood and Marrow Transplant Research from 2000 to 2008 and their 2,006 10/10 HLA-matched URD. Genes with a suggestive association with each outcome (P < 1×10-3) entered the final meta-analysis including Cohort 1 and an independent cohort (Cohort 2), consisting of 503 recipients and 520 10/10 HLA-matched URD from 2009 to 2011. The analysis included recipients and URD of European descent due to the low frequency of other populations. All association models included age at BMT, diagnosis (AML, ALL, MDS), disease status at BMT, cell source (peripheral blood, marrow), year of BMT, and principal components from EIGENSTRAT to control for population stratification. Only coding variants (missense and nonsense) with minor allele frequency ≤ 1% were included in our analysis, which resulted in > 12,000 genes tested in our gene-based analysis in recipients and donors. Exome-wide significance was set at Pmeta< 4.10×10-6 after Bonferroni correction for the total number of genes tested. The likely pathogenicity of these variants was determined in silico using SIFT and PolyPhen. In recipients, we identified OR51D1, a member of the olfactory receptor gene family, as significantly associated with both OS and TRM. The OS association with ORD51D1 is driven by TRM as the same six missense variants contribute to both TRM and OS, but not to DRM. In donors, four (ALPP, EMID1, SLC44A5, LRP1), one (HHAT), and two (LYZL4, NT5E) genes were significantly associated with OS, TRM, and DRM, respectively (Table). ALPP encodes an alkaline phosphatase. SLC44A5 is a member of the solute carrier gene family. LRP1 (low density lipoprotein receptor-related protein 1) encodes an endocytic receptor involved in several cellular processes, including intracellular signaling, lipid homeostasis, and clearance of apoptotic cells. HHAT encodes an enzyme that acts within the hedgehog secretory pathway, which is involved in hematologic malignancy. LYZL4 is a member of a family of lysozyme-like genes, which have a protective role in host defense. The encoded protein of NT5E, CD270, is used as a determinant of lymphocyte differentiation. The likely pathogenicity of several of the functional variants in all significant genes identified was predicted to be both (possibly or probably) damaging and deleterious (Table), thus indicating that the amino acid substitution of the variant may not be well tolerated. One damaging variant in LRP1 has been identified as a somatic mutation in hematopoietic and lymphoid tissue. Our study is the first analysis of the low-frequency coding variant contribution to URD BMT survival outcomes. We found that the missense variants contributing to many of these significant gene associations shows evidence of pathogenicity and thus it is biologically plausible these variants are contributing to survival outcomes. Further confirmation of these findings, and studies of the functional consequences of protein-coding changes in these genes, may provide more individualized risk prediction and prognosis as well as alternative donor selection strategies. Table. Table. Disclosures McCarthy: Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hahn:Novartis: Equity Ownership; NIH: Research Funding. Sucheston-Campbell:NIH/NCI: Research Funding.

Abstract Over the past decade, there have been numerous published candidate gene association studies of non-HLA variants, genes and/or pathways in relation to BMT survival outcomes. Most of these studies investigated a small number of candidate single nucleotide polymorphisms (SNPs), chosen based on known gene function, in a limited number of cases without replication. Here we test the association of previously reported SNPs with survival outcomes (overall survival (OS), progression-free survival (PFS), death due to disease (DD) or transplant-related mortality (TRM)) after URD BMT using data from the DISCOVeRY-BMT (Determining the Influence of Susceptibility Conveying Variants Related to one-Year mortality after BMT) study which included 2,052 European descent recipient-unrelated donor pairs (Cohort 1) and 763 recipient-unrelated donor pairs (Cohort 1) reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) between 2000-2009 and 2009-2011, respectively. DISCOVeRY-BMT is a Genome Wide Association Study (GWAS) that includes over 9 million typed or imputed SNPs. This represents the largest study to date and is well powered to test both SNP and gene-based association hypotheses. We conducted an extensive literature review of PubMed using MESH terms related to transplant and hematologic disease which yielded 76 candidate gene association studies that investigated a total of 46 genes with any survival outcome. Of the 46 genes, 11 genes (CTLA4, CYP2B6, CYP2C19, GSTM1, GSTT1, IL23R, IL6, MTHFR, NOD2/CARD15, TNFSRF1B, and VDR) were studied in at least 2 publications. A total of 16 SNPs in 8 of these genes reported a significant (P< 0.05) association with at least one survival outcome (OS, PFS, DD or TRM). We tested these 16 SNPs for an association with the survival outcome originally reported in each article (Table 1). All association models included age at BMT, diagnosis (AML, ALL, MDS), disease status at BMT, cell source (blood, bone marrow) and year of BMT. P-values from Cohorts 1 and 2 were combined using a fixed-effects model in METAL software. Since the candidate gene studies have a gene-based hypothesis, we also performed gene-level testing using the VErsatile Gene-Based Association Study (VEGAS2) software to measure the aggregate effect of all available typed and imputed SNPs from our GWAS data in these 8 genes. The candidate gene association studies included heterogeneous patient populations of autologous and/or allogeneic (related or unrelated donors) BMT for a variety of hematologic diseases. Most candidate gene studies performed genotyping on both donors and recipients, and SNPs were tested either independently for donor or recipient genomes, or considering the total number of variant alleles the recipient-donor pair had at a given locus (0-4) (Table 1). The 16 SNPs from the previous candidate gene association studies were not significantly associated at a nominal P<0.05 to either the survival outcome reported in the original published paper, or to any survival outcome (Table 1). Likewise, the VEGAS2 gene-based analysis did not reveal any statistically significant associations for any of the 8 genes at P<0.05. Our study also corroborated prior reports of SNPs and genes that were not associated with survival outcomes. Candidate gene association studies use a priori biological knowledge to select genes that are likely associated with outcome and subsequently SNPs within the gene thought likely to affect function based on knowledge at the time of the investigation. Our findings suggest that neither the genetic variation tested in these candidate genes, nor other individual SNPs in the gene independently or collectively are not associated with survival outcomes in recipients of URD BMT. Our failure to replicate the prior candidate gene studies may be due to these SNPs being dependent on the underlying disease (ie. AML, ALL, etc), the donor relation (related, unrelated, autologous), or that there is no true association of these SNPs with the outcomes studied. #These authors contributed equally Table 1. Table 1. Disclosures McCarthy: Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hahn:Novartis: Equity Ownership; NIH: Research Funding. Sucheston-Campbell:NIH/NCI: Research Funding.

Abstract DNA methylation at cytosine position 5 (5mC) is a genetic modification that regulates gene expression and is critical for cancer initiation and development. The methylcytosine dioxygenase TET2 participates in active DNA demethylation by converting 5mC to 5-hydroxymethylcytosine (5hmC) and its subsequent intermediates thereby regulating gene expression. TET2 gene is frequently mutated in hematological disorders, including 15-25% of myelodysplastic syndrome (MDS) cases. Loss of TET2 function leads to DNA hypermethylation and subsequent dysregulated gene expression in hematopoietic stem cells, and has been considered as an initial step of myeloid malignant transformation including MDS and acute myeloid leukemia (AML). While enzymatic activity of TET2 is well studied, little is known about post-translational modifications (PTMs) that regulate its activity in hematopoietic cells. Lysine acetylation, one of the most crucial PTMs, occurs in a variety of proteins and modulates protein-protein or protein-nucleic acids interactions. Here, we first observed that ectopic expression of histone acetyltransferase EP300 or CREB binding protein (CBP) increased the endogenous TET2 acetylation in 293T cells. Mass spectrometry analysis of TET2 identified several conserved acetylated lysine residues located on the exterior surface of its catalytic domain (CD), which is predicted to be essential for the interaction of TET2 with DNA. Modeling of TET2/DNA interaction using x-ray crystal structure (PDB code: 4NM6) indicates acetylated TET2 interferes with DNA binding thereby impairing the catalytic activity of the enzyme. Like other acetylated proteins, TET2 modification is reversible, since ectopic expression of the protein deacetylase Sirtuin1 (SIRT1) significantly decreases acetylation of TET2. A domain mapping analysis revealed that SIRT1 preferentially interacts with TET2 CD, further supporting the regulatory role of SIRT1 on TET2. To explore the role of SIRT1 mediated deacetylaion of TET2 in more physiological condition, SIRT1 knockdown (KD) MDS-L cells as well as SIRT1 knockout (KO) murine hematopoietic c-kit+ cells were used. In MDS-L cells derived from a MDS patient, SIRT1 KD promoted hyperacetylation of endogenous TET2 which is associated with 50% decrease in 5hmC levels compared to control (p=0.03, n=3). Similarly, SIRT1 KO murine c-Kit+ cells showed TET2 hyperacetylation and 62% decrease in 5hmC compared to wildtype (WT) counterpart (p=0.009, n=5). Phenotypically, the transforming ability of SIRT1 shRNA transduced MDS-L cells was evaluated in vitro through serial replating assay. In contrast to first plating which showed no difference between two groups, the number of colonies derived from SIRT1 KD cells was 2.39 fold higher than that from the control shRNA transduced group after two rounds of replating (p=0.0002, n=3). Importantly, we tested the in-vivo effect of SIRT1 KD on MDS-L cells engrafted in immunodeficient NSGS mice. SIRT1 KD significantly enhanced MDS-L cell engraftment compared to control shRNA (50±5% of human CD45+ cells in SIRT1 KD group vs. 19±4% human CD45+ cells in control group, p=0.003, n=6). Interestingly, increased transformation capacity of SIRT1 KD MDS-L cells was associated with decreased expression of TET2 regulated tumor suppressor genes. For example, expression levels of Mtss1 and Dusp6 were significantly decreased in SIRT1 KD MDS-L cells compared to control shRNA counterpart (Mtss1, 49±5% decrease compared to control, p=0.005; Dusp6, 25±2% decrease compared to control, p=0.01). Similar results were observed in SIRT1 KO cells. The validation of physical binding of TET2 to its targets is ongoing. Moreover, the effect of an SIRT1 allosteric activator-SRT1720 in MDS-L cells is similar to that of Vitamin C, a known TET2 activator. Specifically, SRT1720 significantly increased Mtss1 expression in MDSL cells (p=0.0005, n=3), indicating that SIRT1 agonist may lead to activation of TET2 downstream tumor suppressor genes. In summary, our data demonstrate that TET2 activity can be functionally modified by acetylation and may be enhanced in MDS patients that do not harbor loss-of-function mutations through the SIRT1 mediated deacetylation. These results support further exploration of molecular mechanisms inducing TET2 acetylation, and evaluation of SIRT1 activation as a potential therapeutic approach in MDS. Disclosures No relevant conflicts of interest to declare.