The diabetes susceptibility gene Clec16a regulates mitophagy.

Clec16a has been identified as a disease susceptibility gene for type 1 diabetes, multiple sclerosis, and adrenal dysfunction, but its function is unknown. Here we report that Clec16a is a membrane-associated endosomal protein that interacts with E3 ubiquitin ligase Nrdp1. Loss of Clec16a leads to an increase in the Nrdp1 target Parkin, a master regulator of mitophagy. Islets from mice with pancreas-specific deletion of Clec16a have abnormal mitochondria with reduced oxygen consumption and ATP concentration, both of which are required for normal ß cell function. Indeed, pancreatic Clec16a is required for normal glucose-stimulated insulin release. Moreover, patients harboring a diabetogenic SNP in the Clec16a gene have reduced islet Clec16a expression and reduced insulin secretion. Thus, Clec16a controls ß cell function and prevents diabetes by controlling mitophagy. This pathway could be targeted for prevention and control of diabetes and may extend to the pathogenesis of other Clec16a- and Parkin-associated diseases.

Targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

ATF5 regulates ß-cell survival during stress.

The stress response and cell survival are necessary for normal pancreatic ß-cell function, glucose homeostasis, and prevention of diabetes. The homeodomain transcription factor and human diabetes gene pancreas/duodenum homeobox protein 1 (Pdx1) regulates ß-cell survival and endoplasmic reticulum stress susceptibility, in part through direct regulation of activating transcription factor 4 (Atf4). Here we show that Atf5, a close but less-studied relative of Atf4, is also a target of Pdx1 and is critical for ß-cell survival under stress conditions. Pdx1 deficiency led to decreased Atf5 transcript, and primary islet ChIP-sequencing localized PDX1 to the Atf5 promoter, implicating Atf5 as a PDX1 target. Atf5 expression was stress inducible and enriched in ß cells. Importantly, Atf5 deficiency decreased survival under stress conditions. Loss-of-function and chromatin occupancy experiments positioned Atf5 downstream of and parallel to Atf4 in the regulation of eIF4E-binding protein 1 (4ebp1), a mammalian target of rapamycin (mTOR) pathway component that inhibits protein translation. Accordingly, Atf5 deficiency attenuated stress suppression of global translation, likely enhancing the susceptibility of ß cells to stress-induced apoptosis. Thus, we identify ATF5 as a member of the transcriptional network governing pancreatic ß-cell survival during stress.

BARD1 germline variants induce haploinsufficiency and DNA repair defects in neuroblastoma.

BACKGROUND: High-risk neuroblastoma is a complex genetic disease that is lethal in more than 50% of patients despite intense multimodal therapy. Through genome-wide association studies (GWAS) and next-generation sequencing, we have identified common single nucleotide polymorphisms and rare, pathogenic or likely pathogenic germline loss-of-function variants in BARD1 enriched in neuroblastoma patients. The functional implications of these findings remain poorly understood. METHODS: We correlated BARD1 genotype with expression in normal tissues and neuroblastomas, along with the burden of DNA damage in tumors. To validate the functional consequences of germline pathogenic or likely pathogenic BARD1 variants, we used CRISPR-Cas9 to generate isogenic neuroblastoma (IMR-5) and control (RPE1) cellular models harboring heterozygous BARD1 loss-of-function variants (R112*, R150*, E287fs, and Q564*) and quantified genomic instability in these cells via next-generation sequencing and with functional assays measuring the efficiency of DNA repair. RESULTS: Both common and rare neuroblastoma-associated BARD1 germline variants were associated with lower levels of BARD1 mRNA and an increased burden of DNA damage. Using isogenic heterozygous BARD1 loss-of-function variant cellular models, we functionally validated this association with inefficient DNA repair. BARD1 loss-of-function variant isogenic cells exhibited reduced efficiency in repairing Cas9-induced DNA damage, ineffective RAD51 focus formation at DNA double-strand break sites, and enhanced sensitivity to cisplatin and poly (ADP-ribose) polymerase (PARP) inhibition both in vitro and in vivo. CONCLUSIONS: Taken together, we demonstrate that germline BARD1 variants disrupt DNA repair fidelity. This is a fundamental molecular mechanism contributing to neuroblastoma initiation that may have important therapeutic implications.

BARD1 germline variants induce haploinsufficiency and DNA repair defects in neuroblastoma.

Importance: High-risk neuroblastoma is a complex genetic disease that is lethal in 50% of patients despite intense multimodal therapy. Our genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) within the BARD1 gene showing the most significant enrichment in neuroblastoma patients, and also discovered pathogenic (P) or likely pathogenic (LP) rare germline loss-of-function variants in this gene. The functional implications of these findings remain poorly understood. Objective: To define the functional relevance of BARD1 germline variation in children with neuroblastoma. Design: We correlated BARD1 genotype with BARD1 expression in normal and tumor cells and the cellular burden of DNA damage in tumors. To validate the functional consequences of rare germline P-LP BARD1 variants, we generated isogenic cellular models harboring heterozygous BARD1 loss-of-function (LOF) variants and conducted multiple complementary assays to measure the efficiency of DNA repair. Setting: (N/A). Participants: (N/A). Interventions/Exposures: (N/A). Main Outcomes and Measures: BARD1 expression, efficiency of DNA repair, and genome-wide burden of DNA damage in neuroblastoma tumors and cellular models harboring disease-associated BARD1 germline variants. Results: Both common and rare neuroblastoma associated BARD1 germline variants were significantly associated with lower levels of BARD1 mRNA and an increased burden of DNA damage. Using neuroblastoma cellular models engineered to harbor disease-associated heterozygous BARD1 LOF variants, we functionally validated this association with inefficient DNA repair. These BARD1 LOF variant isogenic models exhibited reduced efficiency in repairing Cas9-induced DNA damage, ineffective RAD51 focus formation at DNA doublestrand break sites, and enhanced sensitivity to cisplatin and poly-ADP ribose polymerase (PARP) inhibition. Conclusions and Relevance: Considering that at least 1 in 10 children diagnosed with cancer carry a predicted pathogenic mutation in a cancer predisposition gene, it is critically important to understand their functional relevance. Here, we demonstrate that germline BARD1 variants disrupt DNA repair fidelity. This is a fundamental molecular mechanism contributing to neuroblastoma initiation that may have important therapeutic implications, and these findings may also extend to other cancers harboring germline variants in genes essential for DNA damage repair. Key Points: Question: How do neuroblastoma patient BRCA1-associated RING domain 1 ( BARD1 ) germline variants impact DNA repair? Findings: Neuroblastoma-associated germline BARD1 variants disrupt DNA repair fidelity. Common risk variants correlate with decreased BARD1 expression and increased DNA double-strand breaks in neuroblastoma tumors and rare heterozygous loss-of-function variants induce BARD1 haploinsufficiency, resulting in defective DNA repair and genomic instability in neuroblastoma cellular models. Meaning: Germline variation in BARD1 contributes to neuroblastoma pathogenesis via dysregulation of critical cellular DNA repair functions, with implications for neuroblastoma treatment, risk stratification, and cancer predisposition.

Pdx1 (MODY4) regulates pancreatic beta cell susceptibility to ER stress.

Type 2 diabetes mellitus (T2DM) results from pancreatic beta cell failure in the setting of insulin resistance. Heterozygous mutations in the gene encoding the beta cell transcription factor pancreatic duodenal homeobox 1 (Pdx1) are associated with both T2DM and maturity onset diabetes of the young (MODY4), and low levels of Pdx1 accompany beta cell dysfunction in experimental models of glucotoxicity and diabetes. Here, we find that Pdx1 is required for compensatory beta cell mass expansion in response to diet-induced insulin resistance through its roles in promoting beta cell survival and compensatory hypertrophy. Pdx1-deficient beta cells show evidence of endoplasmic reticulum (ER) stress both in the complex metabolic milieu of high-fat feeding as well as in the setting of acutely reduced Pdx1 expression in the Min6 mouse insulinoma cell line. Further, Pdx1 deficiency enhances beta cell susceptibility to ER stress-associated apoptosis. The results of high throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates a broad array of genes involved in diverse functions of the ER, including proper disulfide bond formation, protein folding, and the unfolded protein response. These findings suggest that Pdx1 deficiency leads to a failure of beta cell compensation for insulin resistance at least in part by impairing critical functions of the ER.

Preclinical Development of [211At]meta- astatobenzylguanidine ([211At]MABG) as an Alpha Particle Radiopharmaceutical Therapy for Neuroblastoma.

PURPOSE: [131I]meta-iodobenzylguanidine ([131I]MIBG) is a targeted radiotherapeutic administered systemically to deliver beta particle radiation in neuroblastoma. However, relapses in the bone marrow are common. [211At]meta-astatobenzylguanidine ([211At] MABG) is an alpha particle emitter with higher biological effectiveness and short path length which effectively sterilizes microscopic residual disease. Here we investigated the safety and antitumor activity [211At]MABG in preclinical models of neuroblastoma. EXPERIMENTAL DESIGN: We defined the maximum tolerated dose (MTD), biodistribution, and toxicity of [211At]MABG in immunodeficient mice in comparison with [131I]MIBG. We compared the antitumor efficacy of [211At]MABG with [131I]MIBG in three murine xenograft models. Finally, we explored the efficacy of [211At]MABG after tail vein xenografting designed to model disseminated neuroblastoma. RESULTS: The MTD of [211At]MABG was 66.7 MBq/kg (1.8 mCi/kg) in CB17SC scid-/- mice and 51.8 MBq/kg (1.4 mCi/kg) in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Biodistribution of [211At]MABG was similar to [131I]MIBG. Long-term toxicity studies on mice administered with doses up to 41.5 MBq/kg (1.12 mCi/kg) showed the radiotherapeutic to be well tolerated. Both 66.7 MBq/kg (1.8 mCi/kg) single dose and fractionated dosing 16.6 MBq/kg/fraction (0.45 mCi/kg) × 4 over 11 days induced marked tumor regression in two of the three models studied. Survival was significantly prolonged for mice treated with 12.9 MBq/kg/fraction (0.35 mCi/kg) × 4 doses over 11 days [211At]MABG in the disseminated disease (IMR-05NET/GFP/LUC) model (P = 0.003) suggesting eradication of microscopic disease. CONCLUSIONS: [211At]MABG has significant survival advantage in disseminated models of neuroblastoma. An alpha particle emitting radiopharmaceutical may be effective against microscopic disseminated disease, warranting clinical development.

Pre-clinical investigation of astatine-211-parthanatine for high-risk neuroblastoma.

Astatine-211-parthanatine ([211At]PTT) is an alpha-emitting radiopharmaceutical therapeutic that targets poly(adenosine-diphosphate-ribose) polymerase 1 (PARP1) in cancer cells. High-risk neuroblastomas exhibit among the highest PARP1 expression across solid tumors. In this study, we evaluated the efficacy of [211At]PTT using 11 patient-derived xenograft (PDX) mouse models of high-risk neuroblastoma, and assessed hematological and marrow toxicity in a CB57/BL6 healthy mouse model. We observed broad efficacy in PDX models treated with [211At]PTT at the maximum tolerated dose (MTD 36 MBq/kg/fraction x4) administered as a fractionated regimen. For the MTD, complete tumor response was observed in 81.8% (18 of 22) of tumors and the median event free survival was 72 days with 30% (6/20) of mice showing no measurable tumor >95 days. Reversible hematological and marrow toxicity was observed 72 hours post-treatment at the MTD, however full recovery was evident by 4 weeks post-therapy. These data support clinical development of [211At]PTT for high-risk neuroblastoma.

Calcineurin signaling regulates human islet {beta}-cell survival.

The calcium-regulated phosphatase calcineurin intersects with both calcium and cAMP-mediated signaling pathways in the pancreatic ß-cell. Pharmacologic calcineurin inhibition, necessary to prevent rejection in the setting of organ transplantation, is associated with post-transplant ß-cell failure. We sought to determine the effect of calcineurin inhibition on ß-cell replication and survival in rodents and in isolated human islets. Further, we assessed whether the GLP-1 receptor agonist and cAMP stimulus, exendin-4 (Ex-4), could rescue ß-cell replication and survival following calcineurin inhibition. Following treatment with the calcineurin inhibitor tacrolimus, human ß-cell apoptosis was significantly increased. Although we detected no human ß-cell replication, tacrolimus significantly decreased rodent ß-cell replication. Ex-4 nearly normalized both human ß-cell survival and rodent ß-cell replication when co-administered with tacrolimus. We found that tacrolimus decreased Akt phosphorylation, suggesting that calcineurin could regulate replication and survival via the PI3K/Akt pathway. We identify insulin receptor substrate-2 (Irs2), a known cAMP-responsive element-binding protein target and upstream regulator of the PI3K/Akt pathway, as a novel calcineurin target in ß-cells. Irs2 mRNA and protein are decreased by calcineurin inhibition in both rodent and human islets. The effect of calcineurin on Irs2 expression is mediated at least in part through the nuclear factor of activated T-cells (NFAT), as NFAT occupied the Irs2 promoter in a calcineurin-sensitive manner. Ex-4 restored Irs2 expression in tacrolimus-treated rodent and human islets nearly to baseline. These findings reveal calcineurin as a regulator of human ß-cell survival in part through regulation of Irs2, with implications for the pathogenesis and treatment of diabetes following organ transplantation.

The B7-H3-Targeting Antibody-Drug Conjugate m276-SL-PBD Is Potently Effective Against Pediatric Cancer Preclinical Solid Tumor Models.

PURPOSE: Patients with relapsed pediatric solid malignancies have few therapeutic options, and many of these patients die of their disease. B7-H3 is an immune checkpoint protein encoded by the CD276 gene that is overexpressed in many pediatric cancers. Here, we investigate the activity of the B7-H3-targeting antibody-drug conjugate (ADC) m276-SL-PBD in pediatric solid malignancy patient-derived (PDX) and cell line-derived xenograft (CDX) models. EXPERIMENTAL DESIGN: B7-H3 expression was quantified by RNA sequencing and by IHC on pediatric PDX microarrays. We tested the safety and efficacy of m276-SL-PBD in two stages. Randomized trials of m276-SL-PBD of 0.5 mg/kg on days 1, 8, and 15 compared with vehicle were performed in PDX or CDX models of Ewing sarcoma (N = 3), rhabdomyosarcoma (N = 4), Wilms tumors (N = 2), osteosarcoma (N = 5), and neuroblastoma (N = 12). We then performed a single mouse trial in 47 PDX or CDX models using a single 0.5 m/kg dose of m276-SL-PBD. RESULTS: The vast majority of PDX and CDX samples studied showed intense membranous B7-H3 expression (median H-score 177, SD 52). In the randomized trials, m276-SL-PBD showed a 92.3% response rate, with 61.5% of models showing a maintained complete response (MCR). These data were confirmed in the single mouse trial with an overall response rate of 91.5% and MCR rate of 64.4%. Treatment-related mortality rate was 5.5% with late weight loss observed in a subset of models dosed once a week for 3 weeks. CONCLUSIONS: m276-SL-PBD has significant antitumor activity across a broad panel of pediatric solid tumor PDX models.

Neonatal GLP1R activation limits adult adiposity by durably altering hypothalamic architecture.

OBJECTIVE: Adult obesity risk is influenced by alterations to fetal and neonatal environments. Modifying neonatal gut or neurohormone signaling pathways can have negative metabolic consequences in adulthood. Here we characterize the effect of neonatal activation of glucagon like peptide-1 (GLP-1) receptor (GLP1R) signaling on adult adiposity and metabolism. METHODS: Wild type C57BL/6 mice were injected with 1 nmol/kg Exendin-4 (Ex-4), a GLP1R agonist, for 6 consecutive days after birth. Growth, body composition, serum analysis, energy expenditure, food intake, and brain and fat pad histology and gene expression were assessed at multiple time points through 42 weeks. Similar analyses were conducted in a Glp1r conditional allele crossed with a Sim1Cre deleter strain to produce Sim1Cre;Glp1rloxP/loxP mice and control littermates. RESULTS: Neonatal administration of Ex-4 reduced adult body weight and fat mass, increased energy expenditure, and conferred protection from diet-induced obesity in female mice. This was associated with induction of brown adipose genes and increased noradrenergic fiber density in parametrial white adipose tissue (WAT). We further observed durable alterations in orexigenic and anorexigenic projections to the paraventricular hypothalamic nucleus (PVH). Genetic deletion of Glp1r in the PVH by Sim1-Cre abrogated the impact of neonatal Ex-4 on adult body weight, WAT browning, and hypothalamic architecture. CONCLUSION: These observations suggest that the acute activation of GLP1R in neonates durably alters hypothalamic architecture to limit adult weight gain and adiposity, identifying GLP1R as a therapeutic target for obesity prevention.

LIM domain-binding 1 maintains the terminally differentiated state of pancreatic ß cells.

The recognition of ß cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain ß cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic ß cells. Inducible ablation of LDB1 in mature ß cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted ß cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of ß cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative ß cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2) - factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of ß cells and is a component of active enhancers in both murine and human islets.

Tshz1 Regulates Pancreatic ß-Cell Maturation.

The homeodomain transcription factor Pdx1 controls pancreas organogenesis, specification of endocrine pancreas progenitors, and the postnatal growth and function of pancreatic ß-cells. Pdx1 expression in human-derived stem cells is used as a marker for induced pancreatic precursor cells. Unfortunately, the differentiation efficiency of human pancreatic progenitors into functional ß-cells is poor. In order to gain insight into the genes that Pdx1 regulates during differentiation, we performed Pdx1 chromatin immunoprecipitation followed by high-throughput sequencing of embryonic day (e) 13.5 and 15.5 mouse pancreata. From this, we identified the transcription factor Teashirt zinc finger 1 (Tshz1) as a direct Pdx1 target. Tshz1 is expressed in developing and adult insulin- and glucagon-positive cells. Endocrine cells are properly specified in Tshz1-null embryos, but critical regulators of ß-cell (Pdx1 and Nkx6.1) and α-cell (MafB and Arx) formation and function are downregulated. Adult Tshz1(+/-) mice display glucose intolerance due to defects in glucose-stimulated insulin secretion associated with reduced Pdx1 and Clec16a expression in Tshz1(+/-) islets. Lastly, we demonstrate that TSHZ1 levels are reduced in human islets of donors with type 2 diabetes. Thus, we position Tshz1 in the transcriptional network of maturing ß-cells and suggest that its dysregulation could contribute to the islet phenotype of human type 2 diabetes.

Diabetes Susceptibility Genes Pdx1 and Clec16a Function in a Pathway Regulating Mitophagy in ß-Cells.

Mitophagy is a critical regulator of mitochondrial quality control and is necessary for elimination of dysfunctional mitochondria to maintain cellular respiration. Here, we report that the homeodomain transcription factor Pdx1, a gene associated with both type 2 diabetes and monogenic diabetes of the young, regulates mitophagy in pancreatic ß-cells. Loss of Pdx1 leads to abnormal mitochondrial morphology and function as well as impaired mitochondrial turnover. High-throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates the expression of Clec16a, a type 1 diabetes gene and itself a key mediator of mitophagy through regulation of the E3 ubiquitin ligase Nrdp1. Indeed, expression of Clec16a and Nrdp1 are both reduced in Pdx1 haploinsufficient islets, and reduction of Pdx1 impairs fusion of autophagosomes containing mitochondria to lysosomes during mitophagy. Importantly, restoration of Clec16a expression after Pdx1 loss of function restores mitochondrial trafficking during mitophagy and improves mitochondrial respiration and glucose-stimulated insulin release. Thus, Pdx1 orchestrates nuclear control of mitochondrial function in part by controlling mitophagy through Clec16a. The novel Pdx1-Clec16a-Nrdp1 pathway we describe provides a genetic basis for the pathogenesis of mitochondrial dysfunction in multiple forms of diabetes that could be targeted for future therapies to improve ß-cell function.

The Polycomb protein, Bmi1, regulates insulin sensitivity.

OBJECTIVE: The Polycomb Repressive Complexes (PRC) 1 and 2 function to epigenetically repress target genes. The PRC1 component, Bmi1, plays a crucial role in maintenance of glucose homeostasis and beta cell mass through repression of the Ink4a/Arf locus. Here we have explored the role of Bmi1 in regulating glucose homeostasis in the adult animal, which had not been previously reported due to poor postnatal survival of Bmi1 (-/-) mice. METHODS: The metabolic phenotype of Bmi1 (+/-) mice was characterized, both in vivo and ex vivo. Glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps were performed. The insulin signaling pathway was assessed at the protein and transcript level. RESULTS: Here we report a negative correlation between Bmi1 levels and insulin sensitivity in two models of insulin resistance, aging and liver-specific insulin receptor deficiency. Further, heterozygous loss of Bmi1 results in increased insulin sensitivity in adult mice, with no impact on body weight or composition. Hyperinsulinemic-euglycemic clamp reveals increased suppression of hepatic glucose production and increased glucose disposal rate, indicating elevated glucose uptake to peripheral tissues, in Bmi1 (+/-) mice. Enhancement of insulin signaling, specifically an increase in Akt phosphorylation, in liver and, to a lesser extent, in muscle appears to contribute to this phenotype. CONCLUSIONS: Together, these data define a new role for Bmi1 in regulating insulin sensitivity via enhancement of Akt phosphorylation.

Pcif1 modulates Pdx1 protein stability and pancreatic ß cell function and survival in mice.

The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the ß cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus-interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse ß cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1(+/-) mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and ß cell function and normalized ß cell mass in Pdx1(+/-) mice by modulating ß cell survival. These findings indicate that in adult mouse ß cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of ß cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis.