KSR2 mutations are associated with obesity, insulin resistance, and impaired cellular fuel oxidation.

Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEKERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes.

Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

LX2761, a Sodium/Glucose Cotransporter 1 Inhibitor Restricted to the Intestine, Improves Glycemic Control in Mice.

LX2761 is a potent sodium/glucose cotransporter 1 inhibitor restricted to the intestinal lumen after oral administration. Studies presented here evaluated the effect of orally administered LX2761 on glycemic control in preclinical models. In healthy mice and rats treated with LX2761, blood glucose excursions were lower and plasma total glucagon-like peptide-1 (GLP-1) levels higher after an oral glucose challenge; these decreased glucose excursions persisted even when the glucose challenge occurred 15 hours after LX2761 dosing in ad lib-fed mice. Further, treating mice with LX2761 and the dipeptidyl-peptidase 4 inhibitor sitagliptin synergistically increased active GLP-1 levels, suggesting increased LX2761-mediated release of GLP-1 into the portal circulation. LX2761 also lowered postprandial glucose, fasting glucose, and hemoglobin A1C, and increased plasma total GLP-1, during long-term treatment of mice with either early- or late-onset streptozotocin-diabetes; in the late-onset cohort, LX2761 treatment improved survival. Mice and rats treated with LX2761 occasionally had diarrhea; this dose-dependent side effect decreased in severity and frequency over time, and LX2761 doses were identified that decreased postprandial glucose excursions without causing diarrhea. Further, the frequency of LX2761-associated diarrhea was greatly decreased in mice either by gradual dose escalation or by pretreatment with resistant starch 4, which is slowly digested to glucose in the colon, a process that primes the colon for glucose metabolism by selecting for glucose-fermenting bacterial species. These data suggest that clinical trials are warranted to determine if LX2761 doses and dosing strategies exist that provide improved glycemic control combined with adequate gastrointestinal tolerability in people living with diabetes.

Targeting neddylation and sumoylation in chemoresistant triple negative breast cancer.

Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.

Improved glycemic control in mice lacking Sglt1 and Sglt2.

Sodium-glucose cotransporter 2 (SGLT2) is the major, and SGLT1 the minor, transporter responsible for renal glucose reabsorption. Increasing urinary glucose excretion (UGE) by selectively inhibiting SGLT2 improves glycemic control in diabetic patients. We generated Sglt1 and Sglt2 knockout (KO) mice, Sglt1/Sglt2 double-KO (DKO) mice, and wild-type (WT) littermates to study their relative glycemic control and to determine contributions of SGLT1 and SGLT2 to UGE. Relative to WTs, Sglt2 KOs had improved oral glucose tolerance and were resistant to streptozotocin-induced diabetes. Sglt1 KOs fed glucose-free high-fat diet (G-free HFD) had improved oral glucose tolerance accompanied by delayed intestinal glucose absorption and increased circulating glucagon-like peptide-1 (GLP-1), but had normal intraperitoneal glucose tolerance. On G-free HFD, Sglt2 KOs had 30%, Sglt1 KOs 2%, and WTs <1% of the UGE of DKOs. Consistent with their increased UGE, DKOs had lower fasting blood glucose and improved intraperitoneal glucose tolerance than Sglt2 KOs. In conclusion, 1) Sglt2 is the major renal glucose transporter, but Sglt1 reabsorbs 70% of filtered glucose if Sglt2 is absent; 2) mice lacking Sglt2 display improved glucose tolerance despite UGE that is 30% of maximum; 3) Sglt1 KO mice respond to oral glucose with increased circulating GLP-1; and 4) DKO mice have improved glycemic control over mice lacking Sglt2 alone. These data suggest that, in patients with type 2 diabetes, combining pharmacological SGLT2 inhibition with complete renal and/or partial intestinal SGLT1 inhibition may improve glycemic control over that achieved by SGLT2 inhibition alone.

MicroCT analyses of mouse femoral neck architecture.

Hip fractures at the femoral neck are a major cause of morbidity and mortality, but aside from biomechanical strength testing, little is known about femoral neck architecture in mice. Procedures were optimized to analyze high-resolution (6 µm voxel size) microCT scans of the mouse femoral neck to provide bone mass and architectural information. Similar to histomorphometric observations in rats, the boundary between cortical and trabecular bone is difficult to identify in the mouse femoral mid-neck and these compartments were not analyzed separately. Analyses included total area, mineralized bone area, and bone volume fraction (BV/TV). Femoral neck architecture varies in C57BL/6J, 129/SvEv and BALB/c mouse strains. Bone cross sectional area and BV/TV were low in Lrp5 but elevated in Sost gene knockout mice. Sfrp4 gene knockout resulted in high total area, normal bone area, low BV/TV and, as indicated by BS/BV values, greater trabecularization. Femoral neck BV/TV declined with age and ovariectomy, but increased with teriparatide treatment. These findings demonstrate that the architecture of the mouse femoral neck mimics phenotypes and treatment effects observed at other skeletal sites and is a relevant bone site for translational studies examining osteoporosis therapies.

Single-cell evaluation reveals shifts in the tumor-immune niches that shape and maintain aggressive lesions in the breast.

There is an unmet clinical need for stratification of breast lesions as indolent or aggressive to tailor treatment. Here, single-cell transcriptomics and multiparametric imaging applied to a mouse model of breast cancer reveals that the aggressive tumor niche is characterized by an expanded basal-like population, specialization of tumor subpopulations, and mixed-lineage tumor cells potentially serving as a transition state between luminal and basal phenotypes. Despite vast tumor cell-intrinsic differences, aggressive and indolent tumor cells are functionally indistinguishable once isolated from their local niche, suggesting a role for non-tumor collaborators in determining aggressiveness. Aggressive lesions harbor fewer total but more suppressed-like T cells, and elevated tumor-promoting neutrophils and IL-17 signaling, disruption of which increase tumor latency and reduce the number of aggressive lesions. Our study provides insight into tumor-immune features distinguishing indolent from aggressive lesions, identifies heterogeneous populations comprising these lesions, and supports a role for IL-17 signaling in aggressive progression.

Reprogramming of H3K9bhb at regulatory elements is a key feature of fasting in the small intestine.

ß-hydroxybutyrate (ß-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine ß-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5+ stem cell-enriched epithelial spheroids treated with ß-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of ß-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting.

Fasting Reduces Intestinal Radiotoxicity, Enabling Dose-Escalated Radiation Therapy for Pancreatic Cancer.

PURPOSE: Chemotherapy combined with radiation therapy is the most commonly used approach for treating locally advanced pancreatic cancer. The use of curative doses of radiation in this disease setting is constrained because of the close proximity of the head of the pancreas to the duodenum. The purpose of this study was to determine whether fasting protects the duodenum from high-dose radiation, thereby enabling dose escalation for efficient killing of pancreatic tumor cells. METHODS AND MATERIALS: C57BL/6J mice were either fed or fasted for 24 hours and then exposed to total abdominal radiation at 11.5 Gy. Food intake, body weight, overall health, and survival were monitored. Small intestines were harvested at various time points after radiation, and villi length, crypt depth, and number of crypts per millimeter of intestine were determined. Immunohistochemistry was performed to assess apoptosis and double-strand DNA breaks, and microcolony assays were performed to determine intestinal stem cell regeneration capacity. A syngeneic KPC model of pancreatic cancer was used to determine the effects of fasting on the radiation responses of both pancreatic cancer and host intestinal tissues. RESULTS: We demonstrated that a 24-hour fast in mice improved intestinal stem cell regeneration, as revealed by microcolony assay, and improved host survival of lethal doses of total abdominal irradiation compared with fed controls. Fasting also improved survival of mice with orthotopic pancreatic tumors subjected to lethal abdominal radiation compared with controls with free access to food. Furthermore, fasting did not affect tumor cell killing by radiation therapy and enhanced γ-H2AX staining after radiation therapy, suggesting an additional mild radiosensitizing effect. CONCLUSIONS: These results establish proof of concept for fasting as a dose-escalation strategy, enabling ablative radiation in the treatment of unresectable pancreatic cancer.

NOTUM inhibition increases endocortical bone formation and bone strength.

The disability, mortality and costs caused by non-vertebral osteoporotic fractures are enormous. Existing osteoporosis therapies are highly effective at reducing vertebral but not non-vertebral fractures. Cortical bone is a major determinant of non-vertebral bone strength. To identify novel osteoporosis drug targets, we phenotyped cortical bone of 3 366 viable mouse strains with global knockouts of druggable genes. Cortical bone thickness was substantially elevated in Notum -/- mice. NOTUM is a secreted WNT lipase and we observed high NOTUM expression in cortical bone and osteoblasts but not osteoclasts. Three orally active small molecules and a neutralizing antibody inhibiting NOTUM lipase activity were developed. They increased cortical bone thickness and strength at multiple skeletal sites in both gonadal intact and ovariectomized rodents by stimulating endocortical bone formation. Thus, inhibition of NOTUM activity is a potential novel anabolic therapy for strengthening cortical bone and preventing non-vertebral fractures.

Resistance to neoadjuvant chemotherapy in triple-negative breast cancer mediated by a reversible drug-tolerant state.

Eradicating triple-negative breast cancer (TNBC) resistant to neoadjuvant chemotherapy (NACT) is a critical unmet clinical need. In this study, patient-derived xenograft (PDX) models of treatment-naïve TNBC and serial biopsies from TNBC patients undergoing NACT were used to elucidate mechanisms of chemoresistance in the neoadjuvant setting. Barcode-mediated clonal tracking and genomic sequencing of PDX tumors revealed that residual tumors remaining after treatment with standard frontline chemotherapies, doxorubicin (Adriamycin) combined with cyclophosphamide (AC), maintained the subclonal architecture of untreated tumors, yet their transcriptomes, proteomes, and histologic features were distinct from those of untreated tumors. Once treatment was halted, residual tumors gave rise to AC-sensitive tumors with similar transcriptomes, proteomes, and histological features to those of untreated tumors. Together, these results demonstrated that tumors can adopt a reversible drug-tolerant state that does not involve clonal selection as an AC resistance mechanism. Serial biopsies obtained from patients with TNBC undergoing NACT revealed similar histologic changes and maintenance of stable subclonal architecture, demonstrating that AC-treated PDXs capture molecular features characteristic of human TNBC chemoresistance. Last, pharmacologic inhibition of oxidative phosphorylation using an inhibitor currently in phase 1 clinical development delayed residual tumor regrowth. Thus, AC resistance in treatment-naïve TNBC can be mediated by nonselective mechanisms that confer a reversible chemotherapy-tolerant state with targetable vulnerabilities.

6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase-2 Regulates TP53-Dependent Paclitaxel Sensitivity in Ovarian and Breast Cancers.

PURPOSE: Paclitaxel is an integral component of primary therapy for breast and epithelial ovarian cancers, but less than half of these cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases.Experimental Design: Twelve kinases that regulate metabolism were depleted in multiple ovarian and breast cancer cell lines to determine whether they regulate sensitivity to paclitaxel in Sulforhodamine B assays. The effects of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) depletion on cell metabolomics, extracellular acidification rate, nicotinamide adenine dinucleotide phosphate, reactive oxygen species (ROS), and apoptosis were studied in multiple ovarian and breast cancer cell lines. Four breast and ovarian human xenografts and a breast cancer patient-derived xenograft (PDX) were used to examine the knockdown effect of PFKFB2 on tumor cell growth in vivo. RESULTS: Knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP53). Silencing PFKFB2 significantly inhibited tumor growth and enhanced paclitaxel sensitivity in four xenografts derived from two ovarian and two breast cancer cell lines, and prolonged survival in a triple-negative breast cancer PDX. Transfection of siPFKFB2 increased the glycolysis rate, but decreased the flow of intermediates through the pentose-phosphate pathway in cancer cells with wtTP53, decreasing NADPH. ROS accumulated after PFKFB2 knockdown, which stimulated Jun N-terminal kinase and p53 phosphorylation, and induced apoptosis that depended upon upregulation of p21 and Puma. CONCLUSIONS: PFKFB2 is a novel target whose inhibition can enhance the effect of paclitaxel-based primary chemotherapy upon ovarian and breast cancers retaining wtTP53.

High-resolution clonal mapping of multi-organ metastasis in triple negative breast cancer.

Most triple negative breast cancers (TNBCs) are aggressively metastatic with a high degree of intra-tumoral heterogeneity (ITH), but how ITH contributes to metastasis is unclear. Here, clonal dynamics during metastasis were studied in vivo using two patient-derived xenograft (PDX) models established from the treatment-naive primary breast tumors of TNBC patients diagnosed with synchronous metastasis. Genomic sequencing and high-complexity barcode-mediated clonal tracking reveal robust alterations in clonal architecture between primary tumors and corresponding metastases. Polyclonal seeding and maintenance of heterogeneous populations of low-abundance subclones is observed in each metastasis. However, lung, liver, and brain metastases are enriched for an identical population of high-abundance subclones, demonstrating that primary tumor clones harbor properties enabling them to seed and thrive in multiple organ sites. Further, clones that dominate multi-organ metastases share a genomic lineage. Thus, intrinsic properties of rare primary tumor subclones enable the seeding and colonization of metastases in secondary organs in these models.

A functional genomic screen in vivo identifies CEACAM5 as a clinically relevant driver of breast cancer metastasis.

Tumor cells disseminate early in tumor development making metastasis-prevention strategies difficult. Identifying proteins that promote the outgrowth of disseminated tumor cells may provide opportunities for novel therapeutic strategies. Despite multiple studies demonstrating that the mesenchymal-to-epithelial transition (MET) is critical for metastatic colonization, key regulators that initiate this transition remain unknown. We serially passaged lung metastases from a primary triple negative breast cancer xenograft to the mammary fat pads of recipient mice to enrich for gene expression changes that drive metastasis. An unbiased transcriptomic signature of potential metastatic drivers was generated, and a high throughput gain-of-function screen was performed in vivo to validate candidates. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver. CEACAM5 overproduction enriched for an epithelial gene expression pattern and facilitated tumor outgrowth at metastatic sites. Tissues from patients with metastatic breast cancer confirmed elevated levels of CEACAM5 in lung metastases relative to breast tumors, and an inverse correlation between CEACAM5 and the mesenchymal marker vimentin was demonstrated. Thus, CEACAM5 facilitates tumor outgrowth at metastatic sites by promoting MET, warranting its investigation as a therapeutic target and biomarker of aggressiveness in breast cancer.

Sotagliflozin improves glycemic control in nonobese diabetes-prone mice with type 1 diabetes.

PURPOSE: Oral agents are needed that improve glycemic control without increasing hypoglycemic events in patients with type 1 diabetes (T1D). Sotagliflozin may meet this need, because this compound lowers blood glucose through the insulin-independent mechanisms of inhibiting kidney SGLT2 and intestinal SGLT1. We examined the effect of sotagliflozin on glycemic control and rate of hypoglycemia measurements in T1D mice maintained on a low daily insulin dose, and compared these results to those from mice maintained in better glycemic control with a higher daily insulin dose alone. MATERIALS AND METHODS: Nonobese diabetes-prone mice with cyclophosphamide-induced T1D were randomized to receive one of four daily treatments: 0.2 U insulin/vehicle, 0.05 U insulin/vehicle, 0.05 U insulin/2 mg/kg sotagliflozin or 0.05 U insulin/30 mg/kg sotagliflozin. Insulin was delivered subcutaneously by micro-osmotic pump; the day after pump implantation, mice received their first of 22 once-daily oral doses of sotagliflozin or vehicle. Glycemic control was monitored by measuring fed blood glucose and hemoglobin A1c levels. RESULTS: Blood glucose levels decreased rapidly and comparably in the 0.05 U insulin/sotagliflozin-treated groups and the 0.2 U insulin/vehicle group compared to the 0.05 U insulin/vehicle group, which had significantly higher levels than the other three groups from day 2 through day 23. A1c levels were also significantly higher in the 0.05 U insulin/vehicle group compared to the other three groups on day 23. Importantly, the 0.2 U insulin/vehicle group had, out of 100 blood glucose measurements, 13 that were <70 mg/dL compared to one of 290 for the other three groups combined. CONCLUSION: Sotagliflozin significantly improved glycemic control, without increasing the rate of hypoglycemia measurements, in diabetic mice maintained on a low insulin dose. This sotagliflozin-mediated improvement in glycemic control was comparable to that achieved by raising the insulin dose alone, but was not accompanied by the increased rate of hypoglycemia measurements observed with the higher insulin dose.

Profound obesity secondary to hyperphagia in mice lacking kinase suppressor of ras 2.

The kinase suppressor of ras 2 (KSR2) gene resides at human chromosome 12q24, a region linked to obesity and type 2 diabetes (T2D). While knocking out and phenotypically screening mouse orthologs of thousands of druggable human genes, we found KSR2 knockout (KSR2(-/-)) mice to be more obese and glucose intolerant than melanocortin 4 receptor(-/-) (MC4R(-/-)) mice. The obesity and T2D of KSR2(-/-) mice resulted from hyperphagia which was unresponsive to leptin and did not originate downstream of MC4R. The kinases AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are each linked to food intake regulation, but only mTOR had increased activity in KSR2(-/-) mouse brain, and the ability of rapamycin to inhibit food intake in KSR2(-/-) mice further implicated mTOR in this process. The metabolic phenotype of KSR2 heterozygous (KSR2(+/minus;)) and KSR2(-/-) mice suggests that human KSR2 variants may contribute to a similar phenotype linked to human chromosome 12q24.