Faculty Bibliography

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.

No targeted treatments are currently approved for HER2 exon 20 insertion-mutant lung adenocarcinoma patients. Mobocertinib (TAK-788) is a potent irreversible tyrosine kinase inhibitor (TKI) designed to target human epidermal growth factor receptor 2 (HER2/ERBB2) exon 20 insertion mutations. However, the function of mobocertinib on HER2 exon 20 insertion-mutant lung cancer is still unclear. Here we conducted systematic characterization of preclinical models to understand the activity profile of mobocertinib against HER2 exon 20 insertions. In HER2 exon 20 insertion-mutant cell lines, the IC₅₀ of mobocertinib was higher than poziotinib and comparable with or slightly lower than afatinib, neratinib, and pyrotinib. Mobocertinib had the lowest HER2 exon 20 insertion IC₅₀/wild-type (WT) EGFR IC₅₀ ratio, indicating that mobocertinib displayed the best selectivity profile in these models. Also, mobocertinib showed strong inhibitory activity in HER2 exon 20^YVMA allograft and patient-derived xenograft models. In genetically engineered mouse models, HER2 exon 20^G776>VC lung tumors exhibited a sustained complete response to mobocertinib, whereas HER2 exon 20^YVMA tumors showed only partial and transient response. Combined treatment with a second antibody-drug conjugate (ADC) against HER2, ado-trastuzumab emtansine (T-DM1), synergized with mobocertinib in HER2 exon 20^YVMA tumors. In addition to the tumor cell autonomous effect, sustained tumor growth control derived from M1 macrophage infiltration and CD4⁺ T-cell activation. These findings support the ongoing clinical development of mobocertinib (NCT02716116) and provide a rationale for future clinical evaluation of T-DM1 combinational therapy in HER2 exon 20^YVMA insertion-mutant lung adenocarcinoma patients. SIGNIFICANCE: This study elucidates the potent inhibitory activity of mobocertinib against HER2 exon 20 insertion-mutant lung cancer and the synergic effect of combined mobocertinib and T-DM1, providing a strong rationale for clinical investigation.

SMAD4 is mutated in human lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generate a highly aggressive lung cancer mouse model bearing conditional Kras^G12D, p53^fl/fl LOF and Smad4^fl/fl LOF mutations (SPK), showing a much higher incidence of tumor metastases than the Kras^G12D, p53^fl/fl (PK) mice. Molecularly, PAK3 is identified as a downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice is achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3'UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components is observed in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.

Most EGFR exon 20 insertion (EGFRex20ins) driver mutations in non-small cell lung cancer (NSCLC) are insensitive to approved EGFR tyrosine kinase inhibitors (TKI). To address the limitations of existing therapies targeting EGFR-mutated NSCLC, mobocertinib (TAK-788), a novel irreversible EGFR TKI, was specifically designed to potently inhibit oncogenic variants containing activating EGFRex20ins mutations with selectivity over wild-type EGFR. The in vitro and in vivo activity of mobocertinib was evaluated in engineered and patient-derived models harboring diverse EGFRex20ins mutations. Mobocertinib inhibited viability of various EGFRex20ins-driven cell lines more potently than approved EGFR TKIs and demonstrated in vivo antitumor efficacy in patient-derived xenografts and murine orthotopic models. These findings support the ongoing clinical development of mobocertinib for the treatment of EGFRex20ins-mutated NSCLC. SIGNIFICANCE: No oral EGFR-targeted therapies are approved for EGFR exon 20 insertion (EGFRex20ins) mutation-driven NSCLC. Mobocertinib is a novel small-molecule EGFR inhibitor specifically designed to target EGFRex20ins mutants. Preclinical data reported here support the clinical development of mobocertinib in patients with NSCLC with EGFR exon 20 insertion mutations.See related commentary by Pacheco, p. 1617.This article is highlighted in the In This Issue feature, p. 1601.

Esophageal squamous cell carcinomas (ESCCs) harbor recurrent chromosome 3q amplifications that target the transcription factor SOX2. Beyond its role as an oncogene in ESCC, SOX2 acts in development of the squamous esophagus and maintenance of adult esophageal precursor cells. To compare Sox2 activity in normal and malignant tissue, we developed engineered murine esophageal organoids spanning normal esophagus to Sox2-induced squamous cell carcinoma and mapped Sox2 binding and the epigenetic and transcriptional landscape with evolution from normal to cancer. While oncogenic Sox2 largely maintains actions observed in normal tissue, Sox2 overexpression with p53 and p16 inactivation promotes chromatin remodeling and evolution of the Sox2 cistrome. With Klf5, oncogenic Sox2 acquires new binding sites and enhances activity of oncogenes such as Stat3. Moreover, oncogenic Sox2 activates endogenous retroviruses, inducing expression of double-stranded RNA and dependence on the RNA editing enzyme ADAR1. These data reveal SOX2 functions in ESCC, defining targetable vulnerabilities.

KRASG12C inhibitors, including MRTX849, are promising treatment options for KRAS-mutant non-small cell lung cancer (NSCLC). PD-1 inhibitors are approved in NSCLC; however, strategies to enhance checkpoint inhibitor therapy (CIT) are needed. KRASG12C mutations are smoking-associated transversion mutations associated with high tumor mutation burden (TMB), PD-L1 positivity and an immunosuppressive tumor microenvironment. To evaluate the potential of MRTX849 to augment CIT, its impact on immune signaling and response to CIT was evaluated. In human tumor xenograft models, MRTX849 increased MHC class I protein expression and decreased RNA and/or plasma protein levels of immunosuppressive factors. In a KRASG12C-mutant CT26 syngeneic mouse model, MRTX849 decreased intratumoral myeloid-derived suppressor cells (MDSCs) and increased M1-polarized macrophages, dendritic cells, CD4+ and CD8+ T cells. Similar results were observed in lung KrasG12C-mutant syngeneic and a genetically engineered mouse (GEM) model. In the CT26 KrasG12C model, MRTX849 demonstrated marked tumor regression when tumors were established in immune-competent BALB/c mice; however, the effect was diminished when tumors were grown in T-cell deficient nu/nu mice. Tumors progressed following anti-PD-1 or MRTX849 single agent treatment in immune-competent mice; however, combination treatment demonstrated durable, complete responses (CRs). Tumors did not re-establish in the same mice that exhibited durable CRs when re-challenged with tumor cell inoculum, demonstrating these mice developed adaptive anti-tumor immunity. In a GEM model, treatment with MRTX849 plus anti-PD-1 led to increased progression-free survival compared to either single agent alone. These data demonstrate KRAS inhibition reverses an immunosuppressive tumor microenvironment and sensitizes tumors to CIT through multiple mechanisms.

INTRODUCTION/BACKGROUND:Lung adenocarcinomas harboring EGFR mutations do not respond to immune checkpoint blockade therapy and their EGFR wildtype counterpart. The mechanisms underlying this lack of clinical response have been investigated but remain incompletely understood. METHODS:We analyzed three cohorts of resected lung adenocarcinomas (Profiling of Resistance Patterns of Oncogenic Signaling Pathways in Evaluation of Cancer of Thorax, Immune Genomic Profiling of NSCLC, and The Cancer Genome Atlas) and compared tumor immune microenvironment of EGFR-mutant tumors to EGFR wildtype tumors, to identify actionable regulators to target and potentially enhance the treatment response. RESULTS:EGFR-mutant NSCLC exhibited low programmed death-ligand 1, low tumor mutational burden, decreased number of cytotoxic T cells, and low T cell receptor clonality, consistent with an immune-inert phenotype, though T cell expansion ex vivo was preserved. In an analysis of 75 immune checkpoint genes, the top up-regulated genes in the EGFR-mutant tumors (NT5E and ADORA1) belonged to the CD73/adenosine pathway. Single-cell analysis revealed that the tumor cell population expressed CD73, both in the treatment-naive and resistant tumors. Using coculture systems with EGFR-mutant NSCLC cells, T regulatory cell proportion was decreased with CD73 knockdown. In an immune-competent mouse model of EGFR-mutant lung cancer, the CD73/adenosine pathway was markedly up-regulated and CD73 blockade significantly inhibited tumor growth. CONCLUSIONS:Our work revealed that EGFR-mutant NSCLC has an immune-inert phenotype. We identified the CD73/adenosine pathway as a potential therapeutic target for EGFR-mutant NSCLC.

Neoantigens are critical targets of antitumor T-cell responses. The ATLAS bioassay was developed to identify neoantigens empirically by expressing each unique patient-specific tumor mutation individually in Escherichia coli, pulsing autologous dendritic cells in an ordered array, and testing the patient's T cells for recognition in an overnight assay. Profiling of T cells from patients with lung cancer revealed both stimulatory and inhibitory responses to individual neoantigens. In the murine B16F10 melanoma model, therapeutic immunization with ATLAS-identified stimulatory neoantigens protected animals, whereas immunization with peptides associated with inhibitory ATLAS responses resulted in accelerated tumor growth and abolished efficacy of an otherwise protective vaccine. A planned interim analysis of a clinical study testing a poly-ICLC adjuvanted personalized vaccine containing ATLAS-identified stimulatory neoantigens showed that it is well tolerated. In an adjuvant setting, immunized patients generated both CD4⁺ and CD8⁺ T-cell responses, with immune responses to 99% of the vaccinated peptide antigens. SIGNIFICANCE: Predicting neoantigens in silico has progressed, but empirical testing shows that T-cell responses are more nuanced than straightforward MHC antigen recognition. The ATLAS bioassay screens tumor mutations to uncover preexisting, patient-relevant neoantigen T-cell responses and reveals a new class of putatively deleterious responses that could affect cancer immunotherapy design.This article is highlighted in the In This Issue feature, p. 521.

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.