Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. Significance: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection. This article is highlighted in the In This Issue feature, p. 1861

We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8+ T cells specifically recognizing the model antigen in a H-2b–restricted manner. The assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell–mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8+ cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell–mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.

A.B., A.A. are inventors on a pending patent related on manipulating, culturing, and evaluating tumor of organotypic spheroids. to screen drug combinations C.P.P. has received honoraria from Bio-Rad and AstraZeneca Korea, is a co-founder of Xsphera Biosciences, and is on the scientific advisory board of DropWorks and Xsphera Biosciences. G.R.O has received consulting fees from AstraZeneca, DropWorks, LOXO, Jannsen, Inivata, GRAIL, Takeda and honoraria from Foundation Medicine and Guardant Health. K.K.W. has equity shares in G1 Therapeutics and is a consultant for G1, Janssen and Array Pharmaceuticals. I.D., and A.S.L. are employees and shareholders of Puma Biotechnology, Inc.

CPP and DAB are inventors on a pending patent related on manipulating, culturing, and evaluating tumor spheroids. DAB is a consultant for N of One and Tango Therapeutics, has received honoraria from Loxo Oncology, Merck, Esai/H3 Biomedicine, and Madalon Consulting, research grants from BMS, Novartis, Lilly, and Gilead Sciences, and is co-founder and on scientific advisory board of Xsphera Biosciences Inc. CPP has received honoraria from Bio-Rad and AstraZeneca, is on scientific advisory board of DropWorks, and is co-founder and on scientific advisory board of Xsphera Biosciences. RWJ has financial interest in XSphera Biosciences. MG receives research funding from Bristol-Myers Squibb and Merck. MP-O, WDH, SG and PSH are employees and shareholders of Novartis.

Succinate is known to act as an inflammatory signal in classically activated macrophages through stabilization of HIF-1α leading to IL-1β production. Relevant to this, hypoxia is known to drive succinate accumulation and release into the extracellular milieu. The metabolic alterations associated with succinate release during inflammation and under hypoxia are poorly understood. Data are presented showing that Mycoplasma arginini infection of VM-M3 cancer cells enhances the Warburg effect associated with succinate production in mitochondria and eventual release into the extracellular milieu. We investigated how succinate production and release was related to the changes of other soluble metabolites, including itaconate and 2-HG. Furthermore, we found that hypoxia alone could induce succinate release from the VM-M3 cells and that this could occur in the absence of glucose-driven lactate production. Our results elucidate metabolic pathways responsible for succinate accumulation and release in cancer cells, thus identifying potential targets involved in both inflammation and hypoxia. This article is part of a Special Issue entitled 20th European Bioenergetics Conference, edited by László Zimányi and László Tretter.