Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Conversion from Roux-En-Y Gastric Bypass to Single Anastomosis Duodenoileal Bypass (SADI-S) for Weight Regain.

BACKGROUND: Revisional bariatric surgery presents a challenge for bariatric surgeons. This procedure can be considered for patients with inadequate weight loss or weight regain after an initial satisfactory response following bariatric surgery. However, the surgical management of weight regain following RYGB remains controversial. We present a case of successful weight gain management after a single anastomosis duodenoileal bypass with sleeve gastrectomy (SADIS) as a revisional procedure for patients with weight regain after RYGB. METHODS: A 23-year-old female with a body mass index (BMI) of 52 kg/m2 and no comorbidities underwent RYGB. Postoperatively, she reached an excess weight loss of 75% of her initial body weight, with a BMI of 32 kg/m2. Eight years after her RYGB, she started regaining weight, reaching a BMI of 47 kg/m2. The surgical team decided to perform a revisional surgery, a conversion of RYGB to SADIS. RESULTS: There were no intraoperative complications. An upper gastrointestinal series was obtained on the third postoperative day which resulted normal and oral feedings were resumed. The patient was then discharged on fifth postoperative day. There were no complications within the first 30 postoperative days. CONCLUSIONS: We attach a video that illustrates the management and technique used to deal with the weight regain after primary bariatric surgery RYGB. We consider that in patients with super morbid obesity refractory to RYGB, conversion to SADIS is an excellent alternative due to its safety and feasibility.

Obesity and the risk of Multiple Sclerosis. The role of Leptin.

OBJECTIVE: To investigate the effects of leptin on different T-cell populations, in order to gain more insight into the link between leptin and obesity. METHODS: Three hundred and nine RRMS patients and 322 controls participated in a cross-sectional survey, to confirm whether excess weight/obesity in adolescence or early adulthood increased the risk of MS. Serum leptin levels were determined by ELISA. MBP83-102 , and MOG63-87 peptide-specific T cells lines were expanded from peripheral blood mononuclear cells. Leptin receptor expression was measured by RT-PCR and flow cytometry. Bcl-2, p-STAT3, pERK1/2, and p27kip1 expression were assayed using ELISA, and apoptosis induction was determined by Annexin V detection. Cytokines were assessed by ELISPOT and ELISA, and regulatory T cells (Tregs) by flow cytometry. RESULTS: Logistic regression analysis, showed excess weight at age 15, and obesity at 20 years of age increased MS risk (OR = 2.16, P = 0.01 and OR = 3.9, P = 0.01). Leptin levels correlated with BMI in both groups. The addition of Leptin increased autoreactive T-cell proliferation, reduced apoptosis induction, and promoted proinflammatory cytokine secretion. Obese patients produced more proinflammatory cytokines compared to overweight/normal/underweight subjects. Inverse correlation was found between leptin levels and circulating Treg cells (r = -0.97, P < 0.0001). Leptin inhibited Treg proliferation. Effects of leptin on CD4+ CD25- effector T cells were mediated by increased STAT3 and ERK1/2 phosphorylation, and down modulation of the cell cycle inhibitor P27kip1 . In contrast, leptin effects on Tregs resulted from decreased phosphorylation of ERK1/2 and upregulation of p27kip1 . INTERPRETATION: Leptin promotes autoreactive T-cell proliferation and proinflammatory cytokine secretion, but inhibits Treg-cell proliferation.

Evaluation of growth production factors as predictors of the incidence and severity of white striping and woody breast in broiler chickens.

White striping (WS) and woody breast (WB) have been previously associated with older and heavier birds. However, there is limited information supporting the association between these 2 muscle conditions and growth parameters. The objectives of this study were 1) to investigate the relationship between WS and WB using different growth production factors and 2) to propose a predictive model that uses growth production factors to investigate the incidence and severity of WS and WB. A combined database of 4,332 broilers pooled from 7 research experiments conducted from 2016 to 2017 at Texas A&M University was used in this study. Parameters such as sex, age (4 wk, 6 wk, and 8 wk), strain (standard A vs. high-breast-yield [B and C]), live weight categories (500 g increments), and breast weight categories (250 g increments) were included in the model. Results showed that WS was 12% more likely to be present in non-WB fillets. The association between WS and WB suggests a moderate relationship between the ranks of both outcome variables (ρ = 0.57, P < 0.0001). Variables such as age, live weight, and sex were not as important as breast weight and strain in the severity prediction of WS and WB. Butterfly fillets above 750 g and with high-breast-yielding strains were more likely associated with higher severity of WS and WB scores. No post hoc variable selection was performed. Both models show good discrimination. The WS model produced an uncorrected area under the curve (AUC) of 0.739, with a bootstrap corrected estimate of 0.736. The WB model produced an uncorrected AUC of 0.753 and a bootstrap corrected estimate of 0.752. Therefore, the growth production factors analyzed in this study indicated that there is a moderate relationship between WS and WB myopathies and were jointly predictive of the severity of WS and WB. Potentially other factors not included in this study may play a major role in the relationship of these 2 myopathies. More research should be done to investigate this possibility.

PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models.

Immune checkpoint inhibitors (CPIs) are associated with a number of immune-related adverse events and low response rates. We provide preclinical evidence for use of a retroviral replicating vector (RRV) selective to cancer cells, to deliver CPI agents that may circumvent such issues and increase efficacy. An RRV, RRV-scFv-PDL1, encoding a secreted single chain variable fragment targeting PD-L1 can effectively compete with PD-1 for PD-L1 occupancy. Cell binding assays showed trans-binding activity on 100% of cells in culture when infection was limited to 5% RRV-scFv-PDL1 infected tumor cells. Further, the ability of scFv PD-L1 to rescue PD-1/PD-L1 mediated immune suppression was demonstrated in a co-culture system consisting of human-derived immune cells and further demonstrated in several syngeneic mouse models including an intracranial tumor model. These tumor models showed that tumors infected with RRV-scFv-PD-L1 conferred robust and durable immune-mediated anti-tumor activity comparable or superior to systemically administered anti-PD-1 or anti PD-L1 monoclonal antibodies. Importantly, the nominal level of scFv-PD-L1 detected in serum is ∼50-150 fold less than reported for systemically administered therapeutic antibodies targeting immune checkpoints. These results support the concept that RRV-scFv-PDL1 CPI strategy may provide an improved safety and efficacy profile compared to systemic monoclonal antibodies of currently approved therapies.

A Retroviral Replicating Vector Encoding Cytosine Deaminase and 5-FC Induces Immune Memory in Metastatic Colorectal Cancer Models.

Treatment of tumors with Toca 511, a gamma retroviral replicating vector encoding cytosine deaminase, followed by 5-fluorocytosine (5-FC) kills tumors by local production of 5-fluorouracil (5-FU). In brain tumor models, this treatment induces systemic anti-tumor immune responses and long-term immune-mediated survival. Phase 1 Toca 511 and Toca FC (extended-release 5-FC) clinical trials in patients with recurrent high-grade glioma show durable complete responses and promising survival data compared to historic controls. The work described herein served to expand on our earlier findings in two models of metastatic colorectal carcinoma (mCRC). Intravenous (i.v.) delivery of Toca 511 resulted in substantial tumor-selective uptake of vector into metastatic lesions. Subsequent treatment with 5-FC resulted in tumor shrinkage, improved survival, and immune memory against future rechallenge with the same CT26 CRC cell line. Similar results were seen in a brain metastasis model of mCRC. Of note, 5-FC treatment resulted in a significant decrease in myeloid-derived suppressor cells (MDSCs) in mCRC tumors in both the liver and brain. These results support the development of Toca 511 and Toca FC as a novel immunotherapeutic approach for patients with mCRC. A phase 1 study of i.v. Toca 511 and Toca FC in solid tumors, including mCRC, is currently underway (NCT02576665).

Melatonin Contributes to the Seasonality of Multiple Sclerosis Relapses.

Seasonal changes in disease activity have been observed in multiple sclerosis, an autoimmune disorder that affects the CNS. These epidemiological observations suggest that environmental factors influence the disease course. Here, we report that melatonin levels, whose production is modulated by seasonal variations in night length, negatively correlate with multiple sclerosis activity in humans. Treatment with melatonin ameliorates disease in an experimental model of multiple sclerosis and directly interferes with the differentiation of human and mouse T cells. Melatonin induces the expression of the repressor transcription factor Nfil3, blocking the differentiation of pathogenic Th17 cells and boosts the generation of protective Tr1 cells via Erk1/2 and the transactivation of the IL-10 promoter by ROR-α. These results suggest that melatonin is another example of how environmental-driven cues can impact T cell differentiation and have implications for autoimmune disorders such as multiple sclerosis.

Identification of unsuspected Wolfram syndrome cases through clinical assessment and WFS1 gene screening in type 1 diabetes mellitus patients.

OBJECTIVE: Wolfram syndrome (WS) is a severe autosomal recessive pleiotropic disease primarily characterized by the association of juvenile-onset diabetes mellitus and optic atrophy. Earlier reports have shown that a proportion of WS cases may remain unrecognized due to misdiagnosis as type 1 diabetes mellitus (T1DM). The objectives of this work were to estimate the prevalence of patients fulfilling clinical criteria for WS in a cohort of subjects diagnosed as T1DM and to identify causal WFS1 gene mutations in those individuals meeting clinical criteria for the disease. METHODS: A cohort of 131 unrelated Mexican T1DM patients was collected, including 77 females and 54 males. Additional clinical anomalies suggesting WS were identified through review of medical files, detailed physical examination and/or specialized tests. WFS1 gene analysis was performed using exon-by-exon PCR amplification and direct Sanger sequencing on genomic DNA from patients reaching WS clinical criteria. RESULTS: Clinical criteria for a WS diagnosis were reached in 6 probands, corresponding to a 4.58% frequency of the disease. WFS1 mutations were identified in 4 out of 5 (80%) individuals fulfilling WS clinical criteria, including two homozygous, one compound heterozygous, and one patient with a single allele mutation. No WFS1 mutations were identified in the remaining subject. CONCLUSIONS: In our cohort, approximately 6% of cases diagnosed as T1DM were in fact patients with Wolfram syndrome. WFS1 mutations were identified in 4 out of 5 individuals (80%) fulfilling clinical criteria for WS. Clinical and genetic analyses of large cohorts of T1DM patients from different ethnic origins would help to better estimate the occurrence of WS and will lead to a better management of such patients.

Low familial risks for multiple sclerosis in Buenos Aires, Argentina.

BACKGROUND: Multiple sclerosis is a complex disease in which genetic susceptibility plays a role and familial occurrence has long been recognized. To date, no studies of familial occurrence have been conducted in Argentina, a country with low to intermediate prevalence. METHODS: As part of a cross-sectional study on multiple sclerosis in Buenos Aires, immediate and extended pedigree details were collected on 219 patients. Crude and age-adjusted recurrence risks for patient relatives were also assessed. RESULTS: Details on age or age of death and disease status were obtained for 4227 relatives. Ten percent of patients reported at least one relative with MS diagnosis, the highest risk (1.54%) was observed in daughters of patients who presented 92-times greater risk than the general population. Sibling recurrence risk ratio was similar to that reported in other locations. CONCLUSIONS: Relatives of patients with MS living in Argentina are at greater risk of developing the disease, although not as high as the risk reported for other geographic regions.

Autoimmune disease prevalence in a multiple sclerosis cohort in Argentina.

Background. Comorbid autoimmune diseases in MS patients have been studied extensively with controversial results. Moreover, no such data exists for Latin-American MS patients. Methods. We conducted a case-control study aimed to establish the prevalence of autoimmune disorders in a cohort of Argentinean MS patients. Results. There were no significant differences in autoimmune disease prevalence in MS patients with respect to controls. The presence of one or more autoimmune disorders did not increase risk of MS (OR 0.85, 95% CI 0.6-1.3). Discussion. Our results indicate absence of increased comorbid autoimmune disease prevalence in MS patients, as well as of increased risk of MS in patients suffering from other autoimmune disorders.

Sex-specific environmental influences affecting MS development.

Vitamin D status, smoking, and Epstein-Barr virus infection (EBV) may all contribute to explain differences in disease prevalence and incidence of Multiple Sclerosis (MS). MS affects women more often than men, and recent cross-sectional study assessments provide evidence of increased female to male prevalence in relapsing remitting MS patients, suggesting that sex hormones may exert an active role in disease pathogenesis. Studies in both humans and animal disease models demonstrate a functional synergy for the immunomodulatory effects of Vitamin D3 and 17-ß estradiol. Both smoking and EBV infection clearly increase MS risk, and smoking history has also been associated with poorer disease prognosis. However, neither factor can explain the recent trend indicating greater female prevalence. Therefore, large population-based case-control studies from well defined geographic areas with homogeneous populations should be performed, in order to define environmental factor effects, and sex hormone influences, to better understand prevalence and incidence gender differences observed.