Genetic variation in SIRT1 affects obesity-related phenotypes in several populations. The purpose of this study was to determine whether variation in SIRT1 affects susceptibility to obesity or type 2 diabetes in Pima Indians, a population with very high prevalence and incidence rates of these diseases. Genotypic data from single nucleotide polymorphisms (SNPs) identified by sequencing regions of SIRT1 combined with SNPs in/near SIRT1 from a prior genome-wide association study determined that 4 tag SNPs (rs7895833, rs10509291, rs7896005, and rs4746720) could capture information across this gene and its adjacent 5′ region. The tag SNPs were genotyped in a population-based sample of 3501 Pima Indians (44% had diabetes, 58% female) for association with type 2 diabetes and BMI. Metabolic trait data and adipose biopsies were available on a subset of these subjects. Two tag SNPs, rs10509291 and rs7896005, were nominally associated with type 2 diabetes (P = 0.01, OR = 1.25 95%CI 1.05-1.48, and P = 0.02, OR = 1.17 95%CI 1.02-1.34, respectively; additive P values adjusted for age, sex, birth year, and family membership), but not BMI (adjusted P values 0.52 and 0.45, respectively). Among metabolically characterized subjects with normal glucose tolerance (N = 243), those carrying the diabetes risk allele (T) for rs10509291 and (G) for rs7896005 had a reduced acute insulin response (AIR) to an intravenous glucose bolus (adjusted P = 0.045 and 0.035, respectively). SIRT1 expression in adipose biopsies was negatively correlated with BMI (adjusted P = 0.00001). We conclude that variation in SIRT1 is nominally associated with reduced AIR and increased risk for type 2 diabetes. SIRT1 expression in adipose is correlated with BMI, but it remains unknown whether this is a cause or consequence of obesity.

ObjectiveA genome-wide association study (GWAS) was recently completed in 1120 Pima Indians to identify loci that influence BMI. Among the top 100 signals were three variants that mapped within the lysophosphatidylglycerol acyltransferase 1 (LPGAT1) gene. LPGAT1 belongs to a large family of acyltransferases, which are involved in a variety of biological processes including pathways that regulate energy homeostasis and body weight. Therefore LPGAT1 was analyzed as a candidate gene for obesity in Pima Indians.Design and MethodsVariants (n = 26) located within and adjacent to LPGAT1 including a novel 27bp deletion in the 5′-untranslated region identified by sequencing were genotyped in a population-based sample of 3,391 full-heritage Pima Indians living in the Gila River Indian Community. Replication of selected variants was assessed in a second sample of 3,327 mixed-heritage Native Americans from the same community.ResultsVariants with nominal associations with BMI in each of the two independent samples (tagged by rs112662024 and rs12058008) had associations of P = 1-4 × 10−5 in the combined sample (n = 6718). A haplotype that includes the novel 27bp deletion, which does not occur in Caucasians, showed the strongest association with BMI in the full-heritage Pima Indians. In vitro functional studies provided suggestive evidence that this 27bp deletion may affect transcriptional or posttranscriptional regulation. Analysis of LPGAT1 cDNA from human preadipocytes identified an additional exon whose sequence could potentially serve as a mitochondrial targeting peptide.ConclusionsLPGAT1 is a novel gene that influences BMI in Native Americans.

Objective: A genome-wide association study (GWAS) was recently completed in 1120 Pima Indians to identify loci that influence BMI. Among the top 100 signals were three variants that mapped within the lysophosphatidylglycerol acyltransferase 1 (LPGAT1) gene. LPGAT1 belongs to a large family of acyltransferases, which are involved in a variety of biological processes including pathways that regulate energy homeostasis and body weight. Therefore LPGAT1 was analyzed as a candidate gene for obesity in Pima Indians. Design and Methods: Variants (n ¼ 26) located within and adjacent to LPGAT1 including a novel 27bp deletion in the 5 0 -untranslated region identified by sequencing were genotyped in a population-based sample of 3,391 full-heritage Pima Indians living in the Gila River Indian Community. Replication of selected variants was assessed in a second sample of 3,327 mixed-heritage Native Americans from the same community. Results: Variants with nominal associations with BMI in each of the two independent samples (tagged by rs112662024 and rs12058008) had associations of P ¼ 1-4 Â 10 À5 in the combined sample (n ¼ 6718).A haplotype that includes the novel 27bp deletion, which does not occur in Caucasians, showed the strongest association with BMI in the full-heritage Pima Indians. In vitro functional studies provided suggestive evidence that this 27bp deletion may affect transcriptional or posttranscriptional regulation. Analysis of LPGAT1 cDNA from human preadipocytes identified an additional exon whose sequence could potentially serve as a mitochondrial targeting peptide. Conclusions: LPGAT1 is a novel gene that influences BMI in Native Americans.

Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm with five-year overall survival rates <30% on standard of care therapy. Relapse is common and thought to originate from AML stem cells that survive in the protective bone marrow (BM) microenvironment. Next generation sequencing (NGS) has revealed the somatic mutation spectrum of AML and the approval of midostaurin for FLT3 ITD- and enasidenib for IDH2 -mutated AML is evidence that the molecular knowledge is being translated clinically. To identify survival-critical AML genes irrespective of mutational status, we performed an shRNA screen on patient samples (N=12) using a barcoded lentiviral shRNA library. Transduced cells were cultured for 9 days on HS-5 stromal cells (to mimic the BM microenvironment) and subjected to NGS for barcode quantification. Sirtuin 5 (SIRT5), the only known enzyme with lysine desuccinylase, demalonylase, or deglutarylase activity, was amongst the top candidates. SIRT5 is implicated in regulating metabolic pathways, including energy metabolism. We first confirmed growth inhibition upon SIRT5 knockdown (KD) in cryopreserved AML cells from the original screen. Next, we transduced a panel of 25 AML cell lines with doxycycline (dox) - inducible shSIRT5 (dox-shSIRT5). SIRT5 KD strongly inhibited growth, reduced colony formation and increased apoptosis in 18/25 lines (SIRT5-dependent cell lines). Seven cell lines showed <20% colony reduction despite >80% reduction of SIRT5 protein and were considered SIRT5-independent. SIRT5 dependence was neither correlated with specific mutations nor basal SIRT5 expression. To control for off-target effects we tested three unique shSIRT5s targeting different sequences in three SIRT5-dependent and three SIRT5-independent cell lines, and confirmed the differential sensitivity for all lines and all constructs. We next transduced CD34+ cells from AML patients (n=15) or cord blood (CB, n=5) with dox-shSIRT5 and plated cells in colony assays. SIRT5 KD (~40% in AML and CB) reduced AML colonies by ~50%, with no effect on CB. Colony formation by SIRT5 null mouse bone marrow infected with FLT3-ITD or MLL-AF9 retrovirus was reduced by 50-60% compared to wild type controls. Metabolic profiling showed that SIRT5 KD induced a profound reduction of oxidative phosphorylation and glycolysis in SIRT5-dependent, but not SIRT5-independent cell lines. Mitochondrial reactive oxygen species (ROS) in SIRT5-dependent cell lines were strongly increased upon SIRT5 KD, and this increase preceded apoptosis. Ectopic expression of superoxide dismutase 2 (SOD2, mitochondrial) abrogated the increase in ROS and rescued cells from apoptosis. Metabolomics and RNAseq suggest that SIRT5-dependent cells exhibit profound metabolic disruption, including reduced TCA cycle activity, and recurrent transcriptional changes, while SIRT5 KD in SIRT5-independent cells is inconsequential. We next injected mice with CMK-1 cells (SIRT5-dependent) expressing dox-shSIRT5 and luciferase. Mice were randomized to dox-supplemented (dox-water) or control water 18 hours post injection, and monitored by luminescence imaging. Control mice showed abundant luminescence at week 3, and died before week 5, while mice receiving dox-water survived throughout the 13-week experiment without evidence of leukemia. When mice with established leukemia were switched to dox-water following week 3, luminescence rapidly decreased and the mice survived without evidence of leukemia until termination of the experiment (week 13). Dox-water had no effect on mice engrafted with OCI-AML3 cells (SIRT5-independent), all of whom died with extensive leukemic involvement despite downregulation of SIRT5 in leukemia cells. We are currently testing the requirement of SIRT5 in MLL-AF9-mediated AML using a retroviral BM transplant mouse model. Preliminary data suggest that absence of SIRT5 may prolong survival. Our data suggest that SIRT5 KD preferentially targets AML cells over normal cells. As SIRT5 null mice are viable with only minor metabolic abnormalities at steady state, these data implicate SIRT5 as a potential therapy target in AML and support the development of clinical SIRT5 inhibitors. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.