SUMMARY Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance.

systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens. Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts..

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy.

Glioblastoma is a universally lethal cancer with a median survival of approximately 15 months1. Despite substantial efforts to define druggable targets, there are no therapeutic options that meaningfully extend glioblastoma patient lifespan. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology2–4 for use in orthotopic patient-derived xenograft (PDX) models, creating a high-throughput negative selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators necessary for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, supporting targeting the transcription elongation machinery as a therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of ‘cancer dependencies’ not identified by previous in vitro approaches, which could supply untapped opportunities for therapeutic intervention.

While important strides have been made in cancer therapy by targeting certain oncogenes, KRAS, the most common among them, remains refractory to this approach. In recent years, a deeper understanding of the critical importance of inflammation in promoting KRAS-driven oncogenesis has emerged, and applies across the different contexts of lung, pancreatic, and colorectal tumorigenesis. Here we review why these tissue types are particularly prone to developing KRAS mutations, and how inflammation conspires with KRAS signaling to fuel carcinogenesis. We discuss multiple lines of evidence that have established NF-κB, STAT3, and certain cytokines as key transducers of these signals, and data to suggest that targeting these pathways has significant clinical potential. Furthermore, recent work has begun to uncover how inflammatory signaling interacts with other KRAS regulated survival pathways such as autophagy and MAPK signaling, and that co-targeting these multiple nodes may be required to achieve real benefit. In addition, the impact of KRAS associated inflammatory signaling on the greater tumor microenvironment has also become apparent, and taking advantage of this inflammation by incorporating approaches that harness T cell anti-tumor responses represents another promising therapeutic strategy. Finally, we highlight the likelihood that the genomic complexity of KRAS mutant tumors will ultimately require tailored application of these therapeutic approaches, and that targeting inflammation early in the course of tumor development could have the greatest impact on eradicating this deadly disease.

Molecularly targeted therapy and immunotherapy have dramatically changed the landscape of available treatment options for patients with advanced cancer. Improved understanding of the molecular and genomic features of cancers over the last decade has led to the development of successful targeted therapies and the field of precision cancer medicine. As a result of these advances, patients whose tumors harbor select molecular alterations are eligible for treatment with targeted therapies active against the unique molecular aberration. Concurrently, advances in tumor immunology have led to the development of immunomodulatory antibodies targeting T cell coinhibitory receptors CTLA-4 and PD-1 (programmed death-1) that have shown activity in several cancer histologies, reinvigorating antitumor immune responses in a subset of patients. These immunomodulatory antibodies offer the promise of durable disease control. However, discrete genomic determinants of response to cancer immunotherapy, unlike molecularly targeted therapies, have remained elusive, and robust biomarkers are lacking. Recent advances in tumor profiling have begun to identify novel genomic features that may influence response and resistance to cancer immunotherapy, including tumor mutational burden (e.g., microsatellite instability), copy-number alterations, and specific somatic alterations that influence immune recognition and response. Further investigation into the molecular and genomic features of response and resistance to cancer immunotherapy will be needed. We review the recent advances in understanding the molecular and genomic determinants of response to cancer immunotherapy, with an emphasis on immune checkpoint inhibitors.

Primary leptomeningeal melanoma (PLM) is a rare type of cancer that represents a major clinical and molecular diagnostic challenge. A definitive diagnosis requires consistent magnetic resonance imaging findings and cerebrospinal fluid (CSF) cytology. Due to the small number of malignant cells in the CSF, routine testing for mutations in the gene is difficult, which prevents the stratification of these patients to potentially beneficial therapies. We herein present the case of a 62-year old man with CSF cytology indicating PLM, where mutation testing, from cell-free (cf) tumor DNA isolated from the CSF and plasma was implemented to guide clinical decision making. Testing for mutation from the CSF and plasma was technically feasible, yielded concordant results, and guided the treatment for this patient. This case suggests that mutation testing of cfDNA isolated from the CSF is technically feasible and may guide therapy in cases where a tissue diagnosis is not possible for PLM and other malignancies with defined oncogenic driver mutations.

Immune checkpoint inhibition (ICI)-based approaches have transformed the treatment landscape of numerous solid tumors. Glioblastoma (GBM) is an aggressive and almost universally fatal disease which is in need of novel treatment options, and combinations of immune checkpoint inhibitors, including dual agent therapy, are starting to be explored in refractory GBM. Growing adoption of ICI-based approaches in solid tumors has been met with improved understanding of immune-related adverse events (IRAEs), including primary hematologic adverse events. Although management guidelines for multiple hematologic IRAEs have been established, the emergence of hemophagocytic lymphohistiocytosis (HLH) secondary to ICI therapy has only rarely been described, and its pathogenesis and optimal management are incompletely understood. We present the case of a 74-year-old male with a history of refractory GBM treated with PD-1 and indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition who experienced acute liver injury, followed by progressive fevers, altered mental status, and cytopenias. Serum studies and examination of spleen and bone marrow pathology were consistent with HLH, which was refractory to steroids and ultimately resulted in his rapid clinical decline. Here, we review prior cases of HLH secondary to ICI therapy across solid tumors, and explore potential mechanisms contributing to the rapid onset and refractory nature of our