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.

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.

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 IC 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.

The tumor microenvironment plays an important role in the processes of tumor growth, metastasis and drug resistance. We have utilized 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 cancer cells proliferation to different classes of drugs. 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 pre-formed 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 pre-formed spheres compared to forming spheroids 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

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.

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.

Malignant peripheral nerve sheath tumors (MPNSTs) are rare and deadly sarcomas with few therapeutic options. Particularly common in individuals with the tumor predisposition syndrome Neurofibromatosis Type 1 (NF1), MPNSTs are the leading cause of death for NF1 patients and are largely resistant to chemotherapy, with large burden of genomic alterations that make any early response to treatment quickly followed by the development of resistance. NF1 is the major negative regulator of RAS, therefore one of the hallmarks of MPNST is deregulated RAS signaling leading to activation of MEK/ERK. Currently, no targeted therapy has been approved for NF1-related MPNSTs, highlighting the need for a better understanding of the complex signaling in MPNSTs and how mechanisms of resistance arise. To investigate the molecular mechanisms behind NF1-related MPNST resistance, we are using both in vitro and in vivo assays including a drug matrix combination platform and an in vivo model of drug resistance. Using xenografts of NF1-related MPNST germline models, we use a drug holiday approach to promote resistance to targeted kinase inhibition, such as MET and MEK inhibitors. For example, treatment with the MEK inhibitor trametinib initially reduce tumor burden, however after a brief “drug holiday” where the mice received no treatment, tumors regrew and were treated with either a MET inhibitor or a combination of MET and MEK inhibitors. Analysis of these tumors indicate a number of altered pathways that may contribute to resistance, including an upregulation of AKT signaling. These results indicate the need for a closer look at the PI3K/AKT signaling and their role in the resistance in NF1-related MPNSTs. To target adaptive AKT activation upon MEK inhibition we utilized the upregulation of AKT with MEK inhibitor treatment in a matrix drug combination platform using human NF1-related MPNST cell lines. In human MPNST cells, we observed that AKT inhibitors alone or in combination with a MEK inhibitor are not effective in reducing human MPNST cell viability. This is likely due to the presence of complex genomic alterations in these lines and RTK crosstalk signaling that promotes kinome plasticity. Currently we are evaluating other pathways that have been found to drive MEK inhibitor resistance in other RAS-driven cancers. Additionally, we are applying the “drug holiday” approach to a panel of patient derived xenograft (PDX) MPNST models to identify the diversity of resistance mechanisms that are likely present in NF1-related MPNST patients. These studies will enhance our understanding of how genomic alterations in NF1-related MPNST patients promote resistance and identify potential novel therapeutic targets. Citation Format: Lauren McGee, Jamie Grit, Curt Essenburg, Carrie Graveel, Matt Steensma. Defining mechanisms of resistance in NF1-related malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-115.

Malignant peripheral nerve sheath tumors (MPNSTs) are a rare and deadly sarcoma with few therapeutic options that are the leading cause of death for patients with Neurofibromatosis Type 1 (NF1). MPNSTs are characterized by a large burden of genomic alterations, chemoresistance, and a 5-year survival rate of 25-50%. NF1 is the major negative regulator of RAS, therefore a hallmark of MPNSTs is deregulated RAS/MAPK signaling. To date, no targeted therapies have been approved for MPNST treatment, highlighting the need for an understanding of adaptive signaling mechanisms that drive resistance. The HIPPO pathway is a key regulator of organ growth and cellular differentiation and is a central driver of resistance to RAS pathway inhibition in other cancers. The key HIPPO effector, YAP1, interacts with RAS and AKT signaling pathways, creating alternate routes for resistance in multiple cancers. In our studies, we identified strong YAP activation in response to MEK and AKT inhibition in MPNST cell lines. Since YAP acts as a transcriptional co-activator through interaction with TEAD transcription factors, we proposed that targeting the Hippo pathway in MPNST will abrogate MAPK inhibitor resistance. To investigate tumor signaling responses and efficacy in vivo, we utilized 3 genomically distinct MPNST patient-derived xenograft (PDX) models. To assess drivers of MPNST resistance, we have developed a preclinical model of drug resistance that simulates clinical treatment schedules. Using a cross-over and a drug holiday design, we are able to evaluate patterns of response and resistance to resumed treatment. We observed distinctive responses to MEK and AKT inhibitors in each PDX line; however, the impact on MPNST growth was minimal with single agent treatment. Comparison of pathway activation between the “drug holiday” biopsies and the treatment endpoint, demonstrated distinct YAP and RAS (pERK) activation levels in resistant tumors. For example, strong YAP activation is observed in correlation with decreased pERK in resistant tumors. YAP activation was strongest at the invasive edge of viable tumor regions. Currently, we are evaluating the efficacy and signaling adaptations to investigate the underlying molecular mechanisms in these MPNST models. Overall, we have demonstrated the development of a clinically relevant model of MPNST resistance and a potential switch between RAS and YAP signaling that promotes resistance to MEK or AKT inhibition. Citation Format: Lauren McGee, Curt Essenburg, Lisa Turner, Angela Hirbe, Anwesha Dey, Jason Zbieg, Carrie Graveel, Matt Steensma. YAP signaling promotes resistance to MEK and AKT inhibition in NF1-related MPNSTs [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A018.