Search

Mice with a targeted mutation in the pro-opiomelanocortin (Pomc) gene (Pomctm1/tm1 mice) are unable to synthesize desacetyl-α-MSH and α-MSH and they develop obesity when fed chow diet. In this study, we hypothesized that a chronic high-fat (HF) diet exacerbates Pomctm1/tm1 mouse obesity. Male and female Pomcwt/wt and Pomctm1/tm1 mice were fed low-fat (LF) (10 kcal percent fat) or HF (45 kcal percent fat) diets from weaning for 23 weeks. We show that Pomctm1/tm1 mouse obesity is sexually dimorphic and exacerbated by an HF diet. Male Pomctm1/tm1 mice develop obesity because they are hyperphagic compared with Pomcwt/wt mice when fed an LF or HF diet. Female Pomctm1/tm1 mice develop obesity when feeding on an LF or HF diet because they exhibit signs of reduced energy expenditure (no change in feed efficiency; body weight gained exceeding energy intake) compared with Pomcwt/wt mice. A chronic HF diet exacerbates male Pomctm1/tm1 and Pomcwt/wt mouse obesity, and the increased energy intake fully accounts for increased weight gain. In contrast, female Pomcwt/wt mice are protected from chronic HF diet–induced obesity because they reduce the amount of HF diet eaten, and they appear to increase their energy expenditure (no change in feed efficiency but energy intake exceeding body weight gained). A chronic HF diet exacerbates female Pomctm1/tm1 mouse obesity due to impaired ability to reduce the amount of HF diet eaten and apparent impaired HF diet–induced adaptive thermogenesis. Our data show that desacetyl-α-MSH and α-MSH are required for sexually dimorphic HF diet–induced C57BL/6J obesity. In conclusion, desacetyl-α-MSH and α-MSH play salutary roles in sexually dimorphic melanocortin obesity and sexually dimorphic HF diet–induced C57BL/6J obesity.

Uveitis is a complex ocular inflammatory disease often accompanied by bacterial or viral infections (infectious uveitis) or underlying autoimmune diseases (non-infectious uveitis). Treatment of the underlying infection along with corticosteroid-mediated suppression of acute inflammation usually resolves infectious uveitis. However, to develop more effective therapies for non-infectious uveitis and to better address acute inflammation in infectious disease, an improved understanding of the underlying inflammatory pathways is needed. In this review, we discuss the disease aetiology, preclinical in vitro and in vivo uveitis models, the role of inflammatory pathways, as well as current and future therapies. In particular, we highlight the involvement of the inflammasome in the development of non-infectious uveitis and how it could be a future target for effective treatment of the disease.

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1β and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1β, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line–derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models. Biomarker analysis used RNA sequencing, whole-exome sequencing, and whole-genome CRISPR knockout screens. Five advanced/metastatic HNSCC patients received evofosfamide monotherapy (480 mg/m2 qw × 3 each month) in a phase 2 study. Evofosfamide was potent and highly selective for hypoxic HNSCC cells. Proliferative rate was a predominant evofosfamide sensitivity determinant and a proliferation metagene correlated with activity in CDX models. Evofosfamide showed efficacy as monotherapy and with radiotherapy in PDX models, augmented CTLA-4 blockade in syngeneic tumors, and reduced hypoxia in nodes disseminated from an orthotopic model. Of 5 advanced HNSCC patients treated with evofosfamide, 2 showed partial responses while 3 had stable disease. In conclusion, evofosfamide shows promising efficacy in aggressive HPV-negative HNSCC, with predictive biomarkers in development to support further clinical evaluation in this indication.

Clinical islet transplantation using human pancreasdonors in patients with type 1 diabetes mellitus isan established cellular therapy, primarily, for thosepatients who suffer from severe hypoglycemicunawareness [1]. However, the low availability ofpancreata that are suitable for islet manufacturingand the variability in isolated human islet qualityhinders this treatment from becoming broadlyapplicable to meet the medical need. NeonatalKitzmann JP, Law L, Shome A, Muzina M, Elliott RB, Mueller KR,Schuurman H-J, Papas KK. Real-time assessment of encapsulatedneonatal porcine islets prior to clinical xenotransplantation.Xenotransplantation 2012; 19: 333–336. 2012 John Wiley & Sons A/S.Abstract: Background: Porcine islet transplantation is emerging as anattractive option for the treatment of patients with type 1 diabetes, withthe possibility of providing islets of higher and more consistent qualityand in larger volumes than available from human pancreata. The use ofencapsulated neonatal porcine islets (ENPI) is appealing because it canaddress islet supply limitations while reducing the need for anti-rejectiontherapy. Pre-transplant characterization of ENPI viability and potencyis an essential component of the production process. We applied thevalidated assay for oxygen consumption rate normalized for DNAcontent (OCR/DNA) to characterize ENPI viability.Methods: ENPI of low viscosity and high m alginate were preparedaccording to standard methods and characterized at various culture timepoints up to 5 weeks.Results: The OCR/DNA (nmol/minAEmgDNA ± SEM) of ENPI(235 ± 10, n = 9) was comparable to that of free NPI (255 ± 14,n = 13). After encapsulation, NPI OCR/DNA was sustained over aculture period of up to 5 weeks. The average OCR/DNA of ENPIcultured longer than 9 days was higher than that of freshly encapsulatedNPI.Conclusion: This is the first characterization of ENPI by a validated andmore sensitive method for product viability. The NPI encapsulationprocess does not compromise viability as measured by OCR/DNA, andENPI can be cultured for up to 5 weeks with maintenance of viability.ENPI meet or exceed current adult porcine islet product release criteria(established at the University of Minnesota) for preclinical xenotrans-plantation in terms of OCR/DNA.

Tumor hypoxia is prevalent in head and neck squamous cell carcinoma (HNSCC), where it limits radiotherapy outcomes. Hypoxia-activated prodrugs (HAPs) have been developed to target hypoxic regions of tumors. These agents undergo oxygen-sensitive reductive activation, thereby delivering cytotoxic species within hypoxic cells. This study investigated the efficacy and sensitivity determinants of the clinical-stage HAP evofosfamide (TH-302) using molecularly-characterized models of HNSCC. We deployed a collection of 27 HPV-negative HNSCC cell lines derived from lesions of varying TNM stages and primary, nodal or recurrent sites. The collection was characterized for gene expression by RNA-seq, from which somatic variants were also called. Their transcriptomic features were investigated in the context of pan-cancer TCGA data by hierarchical clustering. The potency and hypoxic selectivity of 3 HAPs - evofosfamide, PR-104A and SN30000 - were assessed by antiproliferative assay in 22 lines and compared to bromo-isophosphoramide mustard (Br-IPM), cisplatin and 5-FU. The antitumor activity of evofosfamide (50 mg/kg qdx5 for 2-3 cycles with or without a single 10 Gy dose of radiation on day 5 of cycle 1) was evaluated in HNSCC xenografts in addition to a PDX isolated from an SCC of the glottic larynx. The hypoxic fraction at baseline and after 5 days of treatment was quantified by pimonidazole staining. Genetic modifiers of sensitivity to evofosfamide and its cytotoxic metabolite Br-IPM were explored through whole-genome CRISPR-Cas9 screens using the GeCKO v2 library. High-throughput screens with a custom shRNA pool were performed in one HNSCC and two pancreatic ductal adenocarcinoma cell lines to identify reductases responsible for the activation of evofosfamide in hypoxic cells. Evofosfamide was more potent and more selective for hypoxic HNSCC cells in vitro than PR-104A or SN30000. Cell line sensitivity to evofosfamide was correlated with Br-IPM and cisplatin but not with PR-104A, SN30000 or 5-FU, indicating distinct sensitivity determinants. Evidence of antitumor activity with evofosfamide was observed in vivo. CRISPR screens identified potential evofosfamide sensitivity genes that were reproducibly enriched following drug exposure. Reductase-focused RNA interference screens defined a cluster of sensitivity genes that mapped to mitochondrial electron transport, whereas shRNA’s targeted against presumed activating enzymes such as POR were not enriched. Concentration-dependent oxidation of cytochrome a and decreased respiration was observed in cells exposed to evofosfamide, suggesting reduction by mitochondrial complexes. This study provides a rationale for the clinical evaluation of evofosfamide with radiotherapy in genetically defined subsets of HNSCC patients. Citation Format: Francis W. Hunter, Avik Shome, Dan Li, Way W. Wong, Peter Tsai, Nooriyah Poonawala, Purvi M. Kakadiya, Troy M. Ketelä, Maria K. Kondratyev, Courtney R. Lynch, Tet-Woo Lee, Khanh B. Tran, Jules B. Devaux, Rachel Zussman, Cho R. Hong, Dennis Kee, Andrew M. Macann, Anthony J. Hickey, Stefan K. Bohlander, Cristin G. Print, William R. Wilson, Bradly G. Wouters, Stephen M. Jamieson. Preclinical efficacy and sensitivity determinants of evofosfamide in molecularly defined models of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 169. doi:10.1158/1538-7445.AM2017-169