KMT2D deficiency drives lung squamous cell carcinoma and hypersensitivity to RTK-RAS inhibition.

Lung squamous cell carcinoma (LUSC) represents a major subtype of lung cancer with limited treatment options. KMT2D is one of the most frequently mutated genes in LUSC (>20%), and yet its role in LUSC oncogenesis remains unknown. Here, we identify KMT2D as a key regulator of LUSC tumorigenesis wherein Kmt2d deletion transforms lung basal cell organoids to LUSC. Kmt2d loss increases activation of receptor tyrosine kinases (RTKs), EGFR and ERBB2, partly through reprogramming the chromatin landscape to repress the expression of protein tyrosine phosphatases. These events provoke a robust elevation in the oncogenic RTK-RAS signaling. Combining SHP2 inhibitor SHP099 and pan-ERBB inhibitor afatinib inhibits lung tumor growth in Kmt2d-deficient LUSC murine models and in patient-derived xenografts (PDXs) harboring KMT2D mutations. Our study identifies KMT2D as a pivotal epigenetic modulator for LUSC oncogenesis and suggests that KMT2D loss renders LUSC therapeutically vulnerable to RTK-RAS inhibition.

Nemvaleukin alfa, a novel engineered IL-2 fusion protein, drives antitumor immunity and inhibits tumor growth in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is a deadly disease with a 5-year survival of less than 7%. The addition of immunotherapy to chemotherapy was recently approved as first-line treatment; however, the improved clinical benefit is modest, highlighting an urgent need for new treatment strategies. Nemvaleukin alfa, a novel engineered interleukin-2 fusion protein currently in phase I-III studies, is designed to selectively expand cytotoxic natural killer (NK) cells and CD8+ T cells. Here, using a novel SCLC murine model, we investigated the effects of a mouse version of nemvaleukin (mNemvaleukin) on tumor growth and antitumor immunity. METHODS: A novel Rb1 -/- p53 -/- p130 -/- SCLC model that mimics human disease was generated. After confirming tumor burden by MRI, mice were randomized into four treatment groups: vehicle, mNemvaleukin alone, chemotherapy (cisplatin+etoposide) alone, or the combination of mNemvaleukin and chemotherapy. Tumor growth was measured by MRI and survival was recorded. Tumor-infiltrating lymphocytes and peripheral blood immune cells were analyzed by flow cytometry. Cytokine and chemokine secretion were quantified and transcriptomic analysis was performed to characterize the immune gene signatures. RESULTS: mNemvaleukin significantly inhibited SCLC tumor growth, which was further enhanced by the addition of chemotherapy. Combining mNemvaleukin with chemotherapy provided the most significant survival benefit. Profiling of tumor-infiltrating lymphocytes revealed mNemvaleukin expanded the total number of tumor-infiltrating NK and CD8+ T cells. Furthermore, mNemvaleukin increased the frequencies of activated and proliferating NK and CD8+ T cells in tumors. Similar immune alterations were observed in the peripheral blood of mNemvaleukin-treated mice. Of note, combining mNemvaleukin with chemotherapy had the strongest effects in activating effector and cytotoxic CD8+ T cells. mNemvaleukin alone, and in combination with chemotherapy, promoted proinflammatory cytokine and chemokine production, which was further confirmed by transcriptomic analysis. CONCLUSIONS: mNemvaleukin, a novel cytokine-based immunotherapy, significantly inhibited murine SCLC tumor growth and prolonged survival, which was further enhanced by the addition of chemotherapy. mNemvaleukin alone, and in combination with chemotherapy, drove a strong antitumor immune program elicited by cytotoxic immune cells. Our findings support the evaluation of nemvaleukin alone or in combination with chemotherapy in clinical trials for the treatment of SCLC.

DNA Methylation Profiling Identifies Subgroups of Lung Adenocarcinoma with Distinct Immune Cell Composition, DNA Methylation Age, and Clinical Outcome.

PURPOSE: Lung adenocarcinoma (LUAD) is a clinically heterogeneous disease, which is highlighted by the unpredictable recurrence in low-stage tumors and highly variable responses observed in patients treated with immunotherapies, which cannot be explained by mutational profiles. DNA methylation-based classification and understanding of microenviromental heterogeneity may allow stratification into clinically relevant molecular subtypes of LUADs. EXPERIMENTAL DESIGN: We characterize the genome-wide DNA methylation landscape of 88 resected LUAD tumors. Exome sequencing focusing on a panel of cancer-related genes was used to genotype these adenocarcinoma samples. Bioinformatic and statistical tools, the immune cell composition, DNA methylation age (DNAm age), and DNA methylation clustering were used to identify clinically relevant subgroups. RESULTS: Deconvolution of DNA methylation data identified immunologically hot and cold subsets of LUADs. In addition, concurrent factors were analyzed that could affect the immune microenvironment, such as smoking history, ethnicity, or presence of KRAS or TP53 mutations. When the DNAm age was calculated, a lower DNAm age was correlated with the presence of a set of oncogenic drivers, poor overall survival, and specific immune cell populations. Unsupervised DNA methylation clustering identified six molecular subgroups of LUAD tumors with distinct clinical and microenvironmental characteristics. CONCLUSIONS: Our results demonstrate that DNA methylation signatures can stratify LUAD into clinically relevant subtypes, and thus such classification of LUAD at the time of resection may lead to better methods in predicting tumor recurrence and therapy responses.

Loss of TSC1/TSC2 sensitizes immune checkpoint blockade in non-small cell lung cancer.

Tuberous sclerosis complex subunit 1 (TSC1) and 2 (TSC2) are frequently mutated in non-small cell lung cancer (NSCLC), however, their effects on antitumor immunity remained unexplored. A CRISPR screening in murine KrasG12D/Trp53-/- (KP) model identified Tsc1 and Tsc2 as potent regulators of programmed cell death ligand 1 (Pd-l1) expression in vitro and sensitivity to anti-programmed cell death receptor 1 (PD-1) treatment in vivo. TSC1 or TSC2 knockout (KO) promoted the transcriptional and membrane expression of PD-L1 in cell lines. TSC2-deficient tumors manifested an inflamed microenvironment in patient samples and The Cancer Genome Atlas dataset. In syngeneic murine models, KP-Tsc2-KO tumors showed notable response to anti-PD-1 antibody treatment, but Tsc2-wild-type tumors did not. Patients with TSC1/TSC2-mutant NSCLC receiving immune checkpoint blockade (ICB) had increased durable clinical benefit and survival. Collectively, TSC1/TSC2 loss defines a distinct subtype of NSCLC characterized as inflamed tumor microenvironment and superior sensitivity to ICB.

Targeting the Atf7ip-Setdb1 Complex Augments Antitumor Immunity by Boosting Tumor Immunogenicity.

Substantial progress has been made in understanding how tumors escape immune surveillance. However, few measures to counteract tumor immune evasion have been developed. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR-Cas9 suppressor screen for genes encoding epigenetic modifiers. We found that disruption of the genes encoding either of the chromatin modifiers activating transcription factor 7-interacting protein (Atf7ip) or its interacting partner SET domain bifurcated histone lysine methyltransferase 1 (Setdb1) in tumor cells restored tumor antigen expression. This resulted in augmented tumor immunogenicity concomitant with elevated endogenous retroviral (ERV) antigens and mRNA intron retention. ERV disinhibition was associated with a robust type I interferon response and increased T-cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1. ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T-cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy.

Recent advances in preclinical models for lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) represents a major subtype of non-small cell lung cancer with limited treatment options. Previous studies have elucidated the complex genetic landscape of LUSC and revealed multiple altered genes and pathways. However, in stark contrast to lung adenocarcinoma, few targetable driver mutations have been established so far and targeted therapies for LUSC remain unsuccessful. Immunotherapy has revolutionized LUSC treatment and is currently approved as the new standard of care. To gain a better understanding of the LUSC biology, improved modeling systems are urgently needed. Preclinical models, particularly those mimicking human disease with an intact tumor immune microenvironment, are an invaluable tool to study cancer development and evaluate new therapeutic targets. Here, we discuss recent advances in LUSC preclinical models, with a focus on genetically engineered mouse models (GEMMs) and organoids, in the context of evolving precision medicine and immunotherapy.

Serial single-cell profiling analysis of metastatic TNBC during Nab-paclitaxel and pembrolizumab treatment.

PURPOSE: Immunotherapy has recently been shown to improve outcomes for advanced PD-L1-positive triple-negative breast cancer (TNBC) in the Impassion130 trial, leading to FDA approval of the first immune checkpoint inhibitor in combination with taxane chemotherapy. To further develop predictive biomarkers and improve therapeutic efficacy of the combination, interrogation of the tumor immune microenvironment before therapy as well as during each component of treatment is crucial. Here we use single-cell RNA sequencing (scRNA-seq) on tumor biopsies to assess immune cell changes from two patients with advanced TNBC treated in a prospective trial at predefined serial time points, before treatment, on taxane chemotherapy and on chemo-immunotherapy. METHODS: Both patients (one responder and one progressor) received the trial therapy, in cycle 1 nab-paclitaxel given as single agent, in cycle 2 nab-paclitaxel in combination with pembrolizumab. Tumor core biopsies were obtained at baseline, 3 weeks (after cycle 1, chemotherapy alone) and 6 weeks (after cycle 2, chemo-immunotherapy). Single-cell RNA sequencing (scRNA-seq) of both cancer cells and infiltrating immune cells isolated were performed from fresh tumor core biopsy specimens by 10 × chromium sequencing. RESULTS: ScRNA-seq analysis showed significant baseline heterogeneity of tumor-infiltrating immune cell populations between the two patients as well as modulation of the tumor microenvironment by chemotherapy and immunotherapy. In the responding patient there was a population of PD-1high-expressing T cells which significantly decreased after nab-paclitaxel plus pembrolizumab treatment as well as a presence of tissue-resident memory T cells (TRM). In contrast, tumors from the patient with rapid disease progression showed a prevalent and persistent myeloid compartment. CONCLUSIONS: Our study provides a deep cellular analysis of on-treatment changes during chemo-immunotherapy for advanced TNBC, demonstrating not only feasibility of single-cell analyses on serial tumor biopsies but also the heterogeneity of TNBC and differences in on-treatment changes in responder versus progressor.

Clinical Characteristics and Outcomes of COVID-19-Infected Cancer Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: Previous studies have indicated coronavirus disease 2019 (COVID-19) patients with cancer have a high fatality rate. METHODS: We conducted a systematic review of studies that reported fatalities in COVID-19 patients with cancer. A comprehensive meta-analysis that assessed the overall case fatality rate and associated risk factors was performed. Using individual patient data, univariate and multivariable logistic regression analyses were used to estimate odds ratios (OR) for each variable with outcomes. RESULTS: We included 15 studies with 3019 patients, of which 1628 were men; 41.0% were from the United Kingdom and Europe, followed by the United States and Canada (35.7%), and Asia (China, 23.3%). The overall case fatality rate of COVID-19 patients with cancer measured 22.4% (95% confidence interval [CI] = 17.3% to 28.0%). Univariate analysis revealed age (OR = 3.57, 95% CI = 1.80 to 7.06), male sex (OR = 2.10, 95% CI = 1.07 to 4.13), and comorbidity (OR = 2.00, 95% CI = 1.04 to 3.85) were associated with increased risk of severe events (defined as the individuals being admitted to the intensive care unit, or requiring invasive ventilation, or death). In multivariable analysis, only age greater than 65 years (OR = 3.16, 95% CI = 1.45 to 6.88) and being male (OR = 2.29, 95% CI = 1.07 to 4.87) were associated with increased risk of severe events. CONCLUSIONS: Our analysis demonstrated that COVID-19 patients with cancer have a higher fatality rate compared with that of COVID-19 patients without cancer. Age and sex appear to be risk factors associated with a poorer prognosis.

Investigation of a Mobile Health Texting Tool for Embedding Patient-Reported Data Into Diabetes Management (i-Matter): Development and Usability Study.

BACKGROUND: Patient-reported outcomes (PROs) are increasingly being used in the management of type 2 diabetes (T2D) to integrate data from patients' perspective into clinical care. To date, the majority of PRO tools have lacked patient and provider involvement in their development, thus failing to meet the unique needs of end users, and lack the technical infrastructure to be integrated into the clinic workflow. OBJECTIVE: This study aims to apply a systematic, user-centered design approach to develop i-Matter (investigating a mobile health [mHealth] texting tool for embedding patient-reported data into diabetes management), a theory-driven, mobile PRO system for patients with T2D and their primary care providers. METHODS: i-Matter combines text messaging with dynamic data visualizations that can be integrated into electronic health records (EHRs) and personalized patient reports. To build i-Matter, we conducted semistructured group and individual interviews with patients with T2D and providers, a design thinking workshop to refine initial ideas and design the prototype, and user testing sessions of prototypes using a rapid-cycle design (ie, design-test-modify-retest). RESULTS: Using an iterative user-centered process resulted in the identification of 6 PRO messages that were relevant to patients and providers: medication adherence, dietary behaviors, physical activity, sleep quality, quality of life, and healthy living goals. In user testing, patients recommended improvements to the wording and timing of the PRO text messages to increase clarity and response rates. Patients also recommended including motivational text messages to help sustain engagement with the program. The personalized report was regarded as a key tool for diabetes self-management by patients and providers because it aided in the identification of longitudinal patterns in the PRO data, which increased patient awareness of their need to adopt healthier behaviors. Patients recommended adding individualized tips to the journal on how they can improve their behaviors. Providers preferred having a separate tab built into the EHR that included the personalized report and highlighted key trends in patients' PRO data over the past 3 months. CONCLUSIONS: PRO tools that capture patients' well-being and the behavioral aspects of T2D management are important to patients and providers. A clinical trial will test the efficacy of i-Matter in 282 patients with uncontrolled T2D. TRIAL REGISTRATION: ClinicalTrials.gov NCT03652389; https://clinicaltrials.gov/ct2/show/NCT03652389.

Epigenetic CRISPR Screens Identify Npm1 as a Therapeutic Vulnerability in Non-Small Cell Lung Cancer.

Despite advancements in treatment options, the overall cure and survival rates for non-small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (Npm1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC. SIGNIFICANCE: Epigenome-wide CRISPR knockout screens identify NPM1 as a novel metabolic vulnerability and demonstrate that targeting NPM1 is a new therapeutic opportunity for patients with NSCLC.

In Vivo Epigenetic CRISPR Screen Identifies Asf1a as an Immunotherapeutic Target in Kras-Mutant Lung Adenocarcinoma.

Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS-mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a Kras G12D/Trp53 -/- LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identified Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a deficiency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.See related commentary by Menzel and Black, p. 179.This article is highlighted in the In This Issue feature, p. 161.

CDK7 Inhibition Potentiates Genome Instability Triggering Anti-tumor Immunity in Small Cell Lung Cancer.

Cyclin-dependent kinase 7 (CDK7) is a central regulator of the cell cycle and gene transcription. However, little is known about its impact on genomic instability and cancer immunity. Using a selective CDK7 inhibitor, YKL-5-124, we demonstrated that CDK7 inhibition predominately disrupts cell-cycle progression and induces DNA replication stress and genome instability in small cell lung cancer (SCLC) while simultaneously triggering immune-response signaling. These tumor-intrinsic events provoke a robust immune surveillance program elicited by T cells, which is further enhanced by the addition of immune-checkpoint blockade. Combining YKL-5-124 with anti-PD-1 offers significant survival benefit in multiple highly aggressive murine models of SCLC, providing a rationale for new combination regimens consisting of CDK7 inhibitors and immunotherapies.