p53 modulates kinase inhibitor resistance and lineage plasticity in NF1-related MPNSTs.

Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.

Assessing Student Burnout, Treatment Acquisition, and Barriers to Care to Prompt Changes in a Student Mental Healthcare Program.

OBJECTIVE: Medical students demonstrate disproportionately higher levels of burnout and depression than their non-medical age-matched peers. Few studies have been conducted about rates of treatment acquisition and the barriers to care among students with mental health concerns. This study further characterizes rates of burnout, obstacles to treatment, and program preference for medical students at The University of Michigan. METHODS: In June 2020, a 31-question survey eliciting information regarding student burnout, well-being, barriers to care, and improvements to overcome such barriers was sent to 588 current and recently graduated medical students at The University of Michigan. Participation was anonymous and voluntary, with optional response to each question. RESULTS: Ultimately, 312 (53%) students responded. Pre-clinical and core clinical students were significantly more burned out than clinical elective students, with pre-clinical students' odds ratio (OR) of 2.45 and core clinical students' OR of 2.48. Most participants (81%) reported concerns regarding their emotional well-being. Two-thirds (66%) indicated a new or previously diagnosed mental health concern, with 37% of these students never having sought treatment. Commonly reported barriers to care and suggested improvement to mental health services are outlined. Commonly reported barriers to care were financial concerns, time constraints, and stigma-related fear of career-ending consequences. CONCLUSIONS: This study showed stratification of the high levels of burnout among medical students. Student-driven feedback and survey results can help prompt medical schools to develop more robust mental healthcare models and drive much-needed structural changes.

Pubertal Suppression and Surgical Management of a Patient With 5-Alpha Reductase Deficiency.

Five-alpha reductase type 2 deficiency (5αRD2) is a rare cause of atypical genitalia in newborns. There are no definitive guidelines regarding management of children with this disorder. While many children are raised as female given the under-virilized appearance of their external genitalia at birth, these patients are historically counseled to undergo male puberty, resulting in a change in gender identity from female to male in more than half of post-pubertal patients. Here we report the first case of a patient with 5αRD2who identified as female from a very early age, strongly desired gender-affirming surgery, and elected to initiate puberty-blocking therapy prior to the onset of male puberty.

The effect of preoperative tamsulosin on ureteroscopic access in school-aged children.

INTRODUCTION: Flexible ureteroscopes are not tailored for pediatric ureteral size and may not pass at first attempt into the pediatric ureter. Administration of tamsulosin preoperatively in adults has been shown to facilitate ureteral access sheath placement. Several studies have shown tamsulosin to be safe and effective when utilized for medical expulsive therapy in pediatric patients, but its utility for preoperative ureteral dilation has not been studied to date. OBJECTIVE: We hypothesized that preoperative tamsulosin reduces failed ureteroscopic access in children. MATERIALS AND METHODS: We conducted a retrospective review of patients aged 0-18 years undergoing flexible ureteroscopy (URS) from 2014 to 2019 at a single institution. Patients were divided into those taking 0.4 mg of tamsulosin daily for at least 1 week prior to surgery and those not taking tamsulosin. The primary outcome was failure to pass a 7.95 Fr flexible ureteroscope on initial attempt, requiring stent placement and staged management. Patients undergoing URS or stent placement within the prior year, requiring semi-rigid URS, or with genitourinary anomalies were excluded. RESULTS AND DISCUSSION: A total of 49 patients met inclusion criteria. The tamsulosin group (n = 13) and non-tamsulosin group (n = 36) were similar with respect to demographic data. The percentage of prepubertal patients was 53% vs 33% (p = 0.19). The tamsulosin group had lower failed URS (38% vs 61%, p = 0.20). When stratified by age, both prepubertal and postpubertal patients in the tamsulosin group had lower failed URS (43% vs 67%, p = 0.67 and 33% vs 58%, p = 0.38). These clinically significant results suggest initial passage of a flexible ureteroscope is more successful in pediatric patients on preoperative tamsulosin. This study was largely limited by its low power and flaws inherent to its retrospective design. CONCLUSION: Our results have implications for prescribing tamsulosin preoperatively to reduce multiple procedures and anesthetics in children. Adequately powered prospective trials are warranted to confirm preoperative tamsulosin reduces failed first-attempt flexible URS in children.

Spillover Effect of Opioid Reduction Interventions From Adult to Pediatric Surgery.

BACKGROUND: Procedure-specific prescribing guidelines and trainee education have reduced opioid overprescribing in adult surgical patients, but tailored interventions do not yet exist for children. It is unknown what effect these adult interventions have had on postoperative opioid prescribing in children at the same institution, where trainees rotate across both adult and pediatric services. MATERIALS AND METHODS: This retrospective study of patients (<18 y) undergoing pediatric surgery (PS), pediatric otolaryngology (ENT), or pediatric urology (URO) procedures at a single tertiary academic center assessed opioid doses per patient before (January 01, 2015 to September 30, 2016) and after (January 01, 2017 to March 31, 2018) opioid prescribing guidelines and trainee education were instituted for adult laparoscopic cholecystectomy. Patient demographics, postoperative opioid prescribing, opioid refills, and emergency department (ED) visits <21 d after surgery were compared using chi-squared analyses and t-tests. Interrupted time-series analyses (ITSA) assessed changes in the rate of opioid prescribing pre- and postintervention for each subspecialty. RESULTS: There were 3371 patients preintervention and 2439 patients postintervention. After the intervention, fewer patients were prescribed opioids (ENT: 97% versus 93%, P < 0.001; URO: 98% versus 94%, P < 0.001; PS: 61% versus 25%, P < 0.001) and fewer opioid doses were prescribed in each prescription (ENT: 63.8 ± 26.1 versus 50.8 ± 22.0 doses, P < 0.001; URO: 33.5 ± 23.4 versus 22.1 ± 11.3, P < 0.001; PS: 20.4 ± 12.8 versus 13.8 ± 11.4 doses, P < 0.001). There were no changes in opioid refill or ED visit rates postintervention. A decreasing rate in ENT prescribing was seen preintervention, with no significant change postintervention (-2.3 ± 1.1 versus -3.3 ± 0.7; P = 0.24). Whereas, the rate of decrease in PS and URO prescribing significantly slowed postintervention (PS: -2.0 ± 0.1 versus -0.9 ± 0.1, P < 0.001; URO: -4.2 ± 0.2 versus -2.3 ± 0.5, P = 0.005). CONCLUSIONS: Opioid prescribing rates are decreasing, but adult interventions did not achieve reductions in pediatric opioid prescribing at the same institution. There was no concomitant rise in postoperative ED visits or opioid refills as prescribing declined, indicating that the risks of reducing opioid prescriptions may be minimal. Development of evidence-based, procedure-specific prescribing guidelines that specifically address pediatric patients are needed to effectively minimize opioid overprescribing in this population.

High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer.

Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. Herein, we have optimized a quantitative high throughput screen (qHTS) assay to identify compounds that modulate exosome biogenesis and/or release by aggressive prostate cancer (PCa) CD63-GFP-expressing C4-2B cells. A total of 4,580 compounds were screened from the LOPAC library (a collection of 1,280 pharmacologically active compounds) and the NPC library (NCGC collection of 3,300 compounds approved for clinical use). Twenty-two compounds were found to be either potent activators or inhibitors of intracellular GFP signal in the CD63-GFP-expressing C4-2B cells. The activity of lead compounds in modulating the secretion of exosomes was validated by a tunable resistive pulse sensing (TRPS) system (qNano-IZON) and flow cytometry. The mechanism of action of the lead compounds in modulating exosome biogenesis and/or secretion were delineated by immunoblot analysis of protein markers of the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways. The lead compounds tipifarnib, neticonazole, climbazole, ketoconazole, and triademenol were validated as potent inhibitors and sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol as activators of exosome biogenesis and/or secretion in PC cells. Our findings implicate the potential utility of drug-repurposing as novel adjunct therapeutic strategies in advanced cancer.

Manumycin A suppresses exosome biogenesis and secretion via targeted inhibition of Ras/Raf/ERK1/2 signaling and hnRNP H1 in castration-resistant prostate cancer cells.

Emerging evidence links exosomes to cancer progression by the trafficking of oncogenic factors and neoplastic reprogramming of stem cells. This necessitates identification and integration of functionally validated exosome-targeting therapeutics into current cancer management regimens. We employed quantitative high throughput screen on two libraries to identify exosome-targeting drugs; a commercially available collection of 1280 pharmacologically active compounds and a collection of 3300 clinically approved compounds. Manumycin-A (MA), a natural microbial metabolite, was identified as an inhibitor of exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) C4-2B, but not the normal RWPE-1, cells. While no effect was observed on cell growth, MA attenuated ESCRT-0 proteins Hrs, ALIX and Rab27a and exosome biogenesis and secretion by CRPC cells. The MA inhibitory effect is primarily mediated via targeted inhibition of the Ras/Raf/ERK1/2 signaling. The Ras-dependent MA suppression of exosome biogenesis and secretion is partly mediated by ERK-dependent inhibition of the oncogenic splicing factor hnRNP H1. Our findings suggest that MA is a potential drug candidate to suppress exosome biogenesis and secretion by CRPC cells.

Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays.

Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.

A High-Throughput Screening Model of the Tumor Microenvironment for Ovarian Cancer Cell Growth.

The tumor microenvironment plays an important role in the processes of tumor growth, metastasis, and drug resistance. We have used a multilayered 3D primary cell culture model that reproduces the human ovarian cancer metastatic microenvironment to study the effect of the microenvironment on the pharmacological responses of different classes of drugs on cancer cell proliferation. A collection of oncology drugs was screened to identify compounds that inhibited the proliferation of ovarian cancer cells growing as monolayers or forming spheroids, on plastic and on a 3D microenvironment culture model of the omentum metastatic site, and also cells already in preformed spheroids. Target-based analysis of the pharmacological responses revealed that several classes of targets were more efficacious in cancer cells growing in the absence of the metastatic microenvironment, and other target classes were less efficacious in cancer cells in preformed spheres compared to forming spheroid cultures. These findings show that both the cellular context of the tumor microenvironment and cell adhesion mode have an essential role in cancer cell drug resistance. Therefore, it is important to perform screens for new drugs using model systems that more faithfully recapitulate the tissue composition at the site of tumor growth and metastasis.

Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models.

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 µm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.