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Summary: By sorting receptor tyrosine kinases into endolysosomes, the endosomal sorting complexes required for transport (ESCRTs) are thought to attenuate oncogenic signaling in tumor cells. Paradoxically, ESCRT members are upregulated in tumors. Here, we show that disruption of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a pivotal ESCRT component, inhibited tumor growth by promoting CD8+ T cell infiltration in melanoma and colon cancer mouse models. HRS ablation led to misfolded protein accumulation and triggered endoplasmic reticulum (ER) stress, resulting in the activation of the type I interferon pathway in an inositol-requiring enzyme-1α (IRE1α)/X-box binding protein 1 (XBP1)-dependent manner. HRS was upregulated in tumor cells with high tumor mutational burden (TMB). HRS expression associates with the response to PD-L1/PD-1 blockade therapy in melanoma patients with high TMB tumors. HRS ablation sensitized anti-PD-1 treatment in mouse melanoma models. Our study shows a mechanism by which tumor cells with high TMB evade immune surveillance and suggests HRS as a promising target to improve immunotherapy.

Objective We studied the safety and efficacy of hydroxychloroquine (HCQ) as pre-exposure prophylaxis for COVID-19 in healthcare workers (HCWs), using a meta-analysis of randomised controlled trials (RCTs).Data sources PubMed and EMBASE databases were searched to identify randomised trials studying HCQ.Study selection Ten RCTs were identified (n=5079 participants).Data extraction and synthesis The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were used in this systematic review and meta-analysis between HCQ and placebo using a Bayesian random-effects model. A pre-hoc statistical analysis plan was written.Main outcomes The primary efficacy outcome was PCR-confirmed SARS-CoV-2 infection and the primary safety outcome was incidence of adverse events. The secondary outcome included clinically suspected SARS-CoV-2 infection.Results Compared with placebo, HCWs randomised to HCQ had no significant difference in PCR-confirmed SARS-CoV-2 infection (OR 0.92, 95% credible interval (CI): 0.58, 1.37) or clinically suspected SARS-CoV-2 infection (OR 0.78, 95% CI: 0.57, 1.10), but significant difference in adverse events (OR 1.35, 95% CI: 1.03, 1.73).Conclusions and relevance Our meta-analysis of 10 RCTs investigating the safety and efficacy of HCQ as pre-exposure prophylaxis in HCWs found that compared with placebo, HCQ does not significantly reduce the risk of confirmed or clinically suspected SARS-CoV-2 infection, while HCQ significantly increases adverse events.PROSPERO registration number CRD42021285093.

Summary: Background: Only a minority of melanoma patients experience durable responses to immunotherapies due to inter- and intra-tumoral heterogeneity in melanoma. As a result, there is a pressing need for suitable preclinical models to investigate resistance mechanisms and enhance treatment efficacy. Methods: Here, we report two different methods for generating melanoma patient-derived organoids (MPDOs), one is embedded in collagen gel, and the other is inlaid in Matrigel. MPDOs in Matrigel are used for assessing the therapeutic effects of anti-PD-1 antibodies (αPD-1), autochthonous tumor infiltrating lymphocytes (TILs), and small molecule compounds. MPDOs in collagen gel are used for evaluating the chemotaxis and migratory capacity of TILs. Finding: The MPDOs in collagen gel and Matrigel have similar morphology and immune cell composition to their parental melanoma tissues. MPDOs show inter- and intra-tumoral heterogeneity and contain diverse immune cells such as CD4+, CD8+ T, Treg, CD14+ monocytic, CD15+, and CD11b+ myeloid cells. The tumor microenvironment (TME) in MPDOs is highly immunosuppressive, and the lymphoid and myeloid lineages express similar levels of PD-1, PD-L1, and CTLA-4 as their parental melanoma tissues. Anti-PD-1 antibodies (αPD-1) reinvigorate CD8+ T cells and induce melanoma cell death in the MPDOs. TILs expanded by IL-2 and αPD-1 show significantly lower expression of TIM-3, better migratory capacity and infiltration of autochthonous MPDOs, and more effective killing of melanoma cells than TILs expanded by IL-2 alone or IL-2 with αCD3. A small molecule screen discovers that Navitoclax increases the cytotoxicity of TIL therapy. Interpretation: MPDOs may be used to test immune checkpoint inhibitors and cellular and targeted therapies. Funding: This work was supported by the NIH grants CA114046, CA261608, CA258113, and the Tara Miller Melanoma Foundation.

Abstract Background Gamma delta (γδ) T lymphocytes are promising candidate for adoptive T cell therapy, however, their treatment efficacy is not satisfactory. Vδ2 T cells are unique to primates and few suitable models are available to assay their anti‐tumour function. Methods We tested human γδ T cell activation, tumour infiltration, and tumour‐killing in four three‐dimensional (3D) models, including unicellular, bicellular and multicellular melanoma spheroids, and patient‐derived melanoma organoids. We studied the effects of checkpoint inhibitors on γδ T cells and performed a small molecule screen using these platforms. Results γδ T cells rapidly responded to melanoma cells and infiltrated melanoma spheroids better than αβ T cells in PBMCs. Cancer‐associated fibroblasts (CAFs) in bicellular spheroids, stroma cells in multicellular melanoma spheroids and inhibitory immune cells in organoids significantly inhibited immune cell infiltrates including γδ T cells and lessened their cytotoxicity to tumour cells. Tumour‐infiltrating γδ T cells showed exhausted immunophenotypes with high checkpoints expression (CTLA‐4, PD‐1 and PD‐L1). Immune checkpoint inhibitors increased γδ T cell infiltration of 3D models and killing of melanoma cells in all four 3D models. Our small molecule screen assay and subsequent mechanistic studies demonstrated that epigenetic modifiers enhanced the chemotaxis and cytotoxicity of γδ T cells through upregulating MICA/B, inhibiting HDAC6/7 pathway and downregulating the levels of PD‐L1 and PD‐L2 in CAFs and tumour cells. These compounds increased CXCR4 and CD107a expression, IFN‐γ production and decreased PD‐1 expression of γδ T cells. Conclusions Tumour‐infiltrating γδ T cells show exhausted immunophenotypes and limited anti‐tumour capacity in melanoma 3D models. Checkpoint inhibitors and epigenetic modifiers enhance anti‐tumour functions of γδ T cells. These four 3D models provided valuable preclinical platforms to test γδ T cell functions for immunotherapy.

Background Gamma-delta (γδ) T lymphocytes are primed to potently respond to pathogens and transformed cells by recognizing a broad range of antigens. However, adoptive immunotherapy with γδT cells has exhibited mixed treatment responses. Better understanding of γδT cell biology and stratifying healthy donors for allogeneic adoptive therapy is clinically needed to fully realize the therapeutic potential of γδT cells.Methods We examine 98 blood samples from healthy donors and measure their expansion capacity after zoledronate stimulation, and test the migration and cytotoxic effector function of expanded γδT cells in 2D culture, 3D tumor spheroid and patient-derived melanoma organoid assays.Results We find that γδT cell expansion capacity is independent of expansion methods, gender, age and HLA type. Basal γδT cell levels in Peripheral blood mononuclear cell (PBMC) correlate well with their expansion, migration and cytotoxic effector capacity in vitro. Circulating γδT cells with lower expression of PD-1, CTLA-4, Eomes, T-bet and CD69, or higher IFN-γ production expand better. γδT cells with central memory and effector memory phenotypes are significantly more abundant in good expanders. A cut-off level of 0.82% γδT cells in PBMC stratifies good versus poor γδT cell expansion with a sensitivity of 97.78%, specificity of 90.48% and area under the curve of 0.968 in a healthy individual. Donors with higher Vδ2 Index Score in PBMC have greater anti-tumor functions including migratory function and cytotoxicity.Conclusions Our results demonstrate that the interindividual γδT cell functions correlate with their circulating levels in healthy donors. Examination of circulating γδT cell level may be used to select healthy donors to participate in γδT-based immunotherapies.

Abstract Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS‐mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS‐mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS‐mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co‐targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment‐resistant tumor types.

Summary: Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups. : Krepler et al. have established a collection of melanoma patient-derived xenografts (PDX). Melanoma is a very heterogeneous cancer, and this large collection includes even rare subtypes and genetic aberrations in sufficient numbers. Multiple PDX from therapy-resistant patients are characterized and tested in pre-clinical trials for second line therapies. Keywords: melanoma, patient-derived xenografts, targeted therapy, immune checkpoint blockade, melanoma brain metastasis, in vivo models, BRAF inhibitor resistance, ERK inhibitor, MDM2 inhibitor, PI3K beta inhibitor

Summary: Tumor-sequencing studies have revealed the widespread genetic diversity of melanoma. Sequencing of 108 genes previously implicated in melanomagenesis was performed on 462 patient-derived xenografts (PDXs), cell lines, and tumors to identify mutational and copy number aberrations. Samples came from 371 unique individuals: 263 were naive to treatment, and 108 were previously treated with targeted therapy (34), immunotherapy (54), or both (20). Models of all previously reported major melanoma subtypes (BRAF, NRAS, NF1, KIT, and WT/WT/WT) were identified. Multiple minor melanoma subtypes were also recapitulated, including melanomas with multiple activating mutations in the MAPK-signaling pathway and chromatin-remodeling gene mutations. These well-characterized melanoma PDXs and cell lines can be used not only as reagents for a large array of biological studies but also as pre-clinical models to facilitate drug development. : Garman et al. have characterized melanoma PDXs and cell lines described in Krepler et al. (see the related paper in this issue of Cell Reports), identifying major and minor subtypes, some of which were previously not well defined, targeted and immunotherapy resistance, and tumor heterogeneity, creating a set of reagents for future drug discovery and biological studies. Keywords: melanoma, patient-derived xenografts, massively parallel sequencing, cell lines

Purpose: Dabrafenib (GSK2118436) is a potent inhibitor of mutated BRAF kinase. Our multicenter, single-arm, phase II study assessed the safety and clinical activity of dabrafenib in BRAF(V600E/K) mutation-positive metastatic melanoma (mut(+) MM)., Patients and Methods: Histologically confirmed patients with stage IV BRAF(V600E/K) mut(+) MM received oral dabrafenib 150 mg twice daily until disease progression, death, or unacceptable adverse events (AEs). The primary end point was investigator-assessed overall response rate in BRAF(V600E) mut(+) MM patients. Secondary end points included progression-free survival (PFS) and overall survival (OS). Exploratory objectives included the comparison of BRAF mutation status between tumor-specific circulating cell-free DNA (cfDNA) and tumor tissue, and the evaluation of cfDNA as a predictor of clinical outcome., Results: Seventy-six patients with BRAF(V600E) and 16 patients with BRAF(V600K) mut(+) MM were enrolled onto the study. In the BRAF(V600E) group, 45 patients (59%) had a confirmed response (95% CI, 48.2 to 70.3), including five patients (7%) with complete responses. Two patients (13%) with BRAF(V600K) mut(+) MM had a confirmed partial response (95% CI, 0 to 28.7). In the BRAF(V600E) and BRAF(V600K) groups, median PFS was 6.3 months and 4.5 months, and median OS was 13.1 months and 12.9 months, respectively. The most common AEs were arthralgia (33%), hyperkeratosis (27%), and pyrexia (24%). Overall, 25 patients (27%) experienced a serious AE and nine patients (10%) had squamous cell carcinoma. Baseline cfDNA levels predicted response rate and PFS in BRAF(V600E) mut(+) MM patients., Conclusion: Dabrafenib was well tolerated and clinically active in patients with BRAF(V600E/K) mut(+) MM. cfDNA may be a useful prognostic and response marker in future studies.