Anti-tumor activity of a novel LAIR1 antagonist in combination with anti-PD-1 to treat collagen-rich solid tumors.

We recently reported that resistance to PD-1-blockade in a refractory lung cancer-derived model involved increased collagen deposition and the collagen-binding inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and thus we hypothesized that LAIR1 and collagen cooperated to suppress therapeutic response. Here, we report LAIR1 is associated with tumor stroma and is highly expressed by intratumoral myeloid cells in both human tumors and mouse models of cancer. Stroma-associated myeloid cells exhibit a suppressive phenotype and correlate with LAIR1 expression in human cancer. NGM438, a novel humanized LAIR1 antagonist monoclonal antibody, elicits myeloid inflammation and allogeneic T cell responses by binding to LAIR1 and blocking collagen engagement. Further, a mouse-reactive NGM438 surrogate antibody sensitized refractory KP mouse lung tumors to anti-PD-1 therapy and resulted in increased intratumoral CD8+ T cell content and inflammatory gene expression. These data place LAIR1 at the intersection of stroma and suppressive myeloid cells and support the notion that blockade of the LAIR1/collagen axis can potentially address resistance to checkpoint inhibitor therapy in the clinic.

Autotaxin suppresses cytotoxic T cells via LPAR5 to promote anti-PD-1 resistance in non-small cell lung cancer.

Non-small cell lung cancers that harbor concurrent KRAS and TP53 (KP) mutations are immunologically warm tumors with partial responsiveness to anti-PD-(L)1 blockade; however, most patients observe little or no durable clinical benefit. To identify novel tumor-driven resistance mechanisms, we developed a panel of KP murine lung cancer models with intrinsic resistance to anti-PD-1 and queried differential gene expression between these tumors and anti-PD-1-sensitive tumors. We found that the enzyme autotaxin (ATX), and the metabolite it produces, lysophosphatidic acid (LPA), were significantly upregulated in resistant tumors and that ATX directly modulated antitumor immunity, with its expression negatively correlating with total and effector tumor-infiltrating CD8+ T cells. Pharmacological inhibition of ATX, or the downstream receptor LPAR5, in combination with anti-PD-1 was sufficient to restore the antitumor immune response and efficaciously control lung tumor growth in multiple KP tumor models. Additionally, ATX was significantly correlated with inflammatory gene signatures, including a CD8+ cytolytic score in multiple lung adenocarcinoma patient data sets, suggesting that an activated tumor-immune microenvironment upregulates ATX and thus provides an opportunity for cotargeting to prevent acquired resistance to anti-PD-1 treatment. These data reveal the ATX/LPA axis as an immunosuppressive pathway that diminishes the immune checkpoint blockade response in lung cancer.

Targeting immunosuppressive Ly6C+ classical monocytes reverses anti-PD-1/CTLA-4 immunotherapy resistance.

Introduction: Despite significant clinical advancement with the use of immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) there are still a major subset of patients that develop adaptive/acquired resistance. Understanding resistance mechanisms to ICB is critical to developing new therapeutic strategies and improving patient survival. The dynamic nature of the tumor microenvironment and the mutational load driving tumor immunogenicity limit the efficacy to ICB. Recent studies indicate that myeloid cells are drivers of ICB resistance. In this study we sought to understand which immune cells were contributing to resistance and if we could modify them in a way to improve response to ICB therapy. Results: Our results show that combination anti-PD-1/CTLA-4 produces an initial antitumor effect with evidence of an activated immune response. Upon extended treatment with anti-PD-1/CTLA-4 acquired resistance developed with an increase of the immunosuppressive populations, including T-regulatory cells, neutrophils and monocytes. Addition of anti-Ly6C blocking antibody to anti-PD-1/CTLA-4 was capable of completely reversing treatment resistance and restoring CD8 T cell activity in multiple KP lung cancer models and in the autochthonous lung cancer KrasLSL-G12D/p53fl/fl model. We found that there were higher classical Ly6C+ monocytes in anti-PD-1/CTLA-4 combination resistant tumors. B7 blockade illustrated the importance of dendritic cells for treatment efficacy of anti-Ly6C/PD-1/CTLA-4. We further determined that classical Ly6C+ monocytes in anti-PD-1/CTLA-4 resistant tumors are trafficked into the tumor via IFN-γ and the CCL2-CCR2 axis. Mechanistically we found that classical monocytes from ICB resistant tumors were unable to differentiate into antigen presenting cells and instead differentiated into immunosuppressive M2 macrophages or myeloid-derived suppressor cells (MDSC). Classical Ly6C+ monocytes from ICB resistant tumors had a decrease in both Flt3 and PU.1 expression that prevented differentiation into dendritic cells/macrophages. Conclusions: Therapeutically we found that addition of anti-Ly6C to the combination of anti-PD-1/CTLA-4 was capable of complete tumor eradication. Classical Ly6C+ monocytes differentiate into immunosuppressive cells, while blockade of classical monocytes drives dendritic cell differentiation/maturation to reinvigorate the anti-tumor T cell response. These findings support that immunotherapy resistance is associated with infiltrating monocytes and that controlling the differentiation process of monocytes can enhance the therapeutic potential of ICB.

ZEB1 Is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis.

Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to posttranslational modifications (PTM). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non-small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (∼225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, whereas the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition. IMPLICATIONS: The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in patients with NSCLC.

ZEB1 is regulated by K811 acetylation to promote stability, NuRD complex interactions, EMT, and NSCLC metastasis.

Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to post-translational modifications (PTMs). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non-small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (&tilde225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, while the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition. Implications: The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in NSCLC patients.

Impad1 and Syt11 work in an epistatic pathway that regulates EMT-mediated vesicular trafficking to drive lung cancer invasion and metastasis.

Lung cancer is a highly aggressive and metastatic disease responsible for approximately 25% of all cancer-related deaths in the United States. Using high-throughput in vitro and in vivo screens, we have previously established Impad1 as a driver of lung cancer invasion and metastasis. Here we elucidate that Impad1 is a direct target of the epithelial microRNAs (miRNAs) miR-200 and miR∼96 and is de-repressed during epithelial-to-mesenchymal transition (EMT); thus, we establish a mode of regulation of the protein. Impad1 modulates Golgi apparatus morphology and vesicular trafficking through its interaction with a trafficking protein, Syt11. These changes in Golgi apparatus dynamics alter the extracellular matrix and the tumor microenvironment (TME) to promote invasion and metastasis. Inhibiting Impad1 or Syt11 disrupts the cancer cell secretome, regulates the TME, and reverses the invasive or metastatic phenotype. This work identifies Impad1 as a regulator of EMT and secretome-mediated changes during lung cancer progression.

The microRNA-183/96/182 cluster inhibits lung cancer progression and metastasis by inducing an interleukin-2-mediated antitumor CD8+ cytotoxic T-cell response.

One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8+ cytotoxic T lymphocytes (CD8+ CTLs) in m96cl-expressing tumors, and m96cl-mediated suppression of tumor growth and metastasis was CD8+ CTL-dependent. Using coculture assays with naïve immune cells, we show that m96cl expression drives paracrine stimulation of CD8+ CTL proliferation and function. Using tumor microenvironment-associated gene expression profiling, we identified that m96cl elevates the interleukin-2 (IL2) signaling pathway and results in increased IL2-mediated paracrine stimulation of CD8+ CTLs. Furthermore, we identified that the m96cl modulates the expression of IL2 in cancer cells by regulating the expression of transcriptional repressors Foxf2 and Zeb1, and thereby alters the levels of secreted IL2 in the tumor microenvironment. Last, we show that in vivo depletion of IL2 abrogates m96cl-mediated activation of CD8+ CTLs and results in loss of metastatic suppression. Therefore, we have identified a novel mechanistic role of the m96cl in the suppression of lung cancer growth and metastasis by inducing an IL2-mediated systemic CD8+ CTL immune response.

MEK inhibition invigorates chemoimmunotherapy by tumor mitophagy-induced CXCL10 expression.

A recent study by Limagne et al.1 in Cancer Cell demonstrates that addition of MEK inhibitor to standard-of-care platinum/pemetrexed promotes mitophagy-dependent CXCL10 expression via optineurin and TLR9. Tumor cell secretion of CXCL10 produces T cell recruitment and enhances immunotherapy efficacy.

Targeting CDK4 overcomes EMT-mediated tumor heterogeneity and therapeutic resistance in KRAS-mutant lung cancer.

Lack of sustained response to therapeutic agents in patients with KRAS-mutant lung cancer poses a major challenge and arises partly due to intratumor heterogeneity that defines phenotypically distinct tumor subpopulations. To attain better therapeutic outcomes, it is important to understand the differential therapeutic sensitivities of tumor cell subsets. Epithelial-mesenchymal transition is a biological phenomenon that can alter the state of cells along a phenotypic spectrum and cause transcriptional rewiring to produce distinct tumor cell subpopulations. We utilized functional shRNA screens, in in vitro and in vivo models, to identify and validate an increased dependence of mesenchymal tumor cells on cyclin-dependent kinase 4 (CDK4) for survival, as well as a mechanism of resistance to MEK inhibitors. High zinc finger E-box binding homeobox 1 levels in mesenchymal tumor cells repressed p21, leading to perturbed CDK4 pathway activity. Increased dependence on CDK4 rendered mesenchymal cancer cells particularly vulnerable to selective CDK4 inhibitors. Coadministration of CDK4 and MEK inhibitors in heterogeneous tumors effectively targeted different tumor subpopulations, subverting the resistance to either single-agent treatment.

p53 loss activates prometastatic secretory vesicle biogenesis in the Golgi.

Cancer cells exhibit hyperactive secretory states that maintain cancer cell viability and remodel the tumor microenvironment. However, the oncogenic signals that heighten secretion remain unclear. Here, we show that p53 loss activates prometastatic secretory vesicle biogenesis in the Golgi. p53 loss up-regulates the expression of a Golgi scaffolding protein, progestin and adipoQ receptor 11 (PAQR11), which recruits an adenosine diphosphate ribosylation factor 1-containing protein complex that loads cargos into secretory vesicles. PAQR11-dependent secretion of a protease, PLAU, prevents anoikis and initiates autocrine activation of a PLAU receptor/signal transducer and activator of transcription-3-dependent pathway that up-regulates PAQR11 expression, thereby completing a feedforward loop that amplifies prometastatic effector protein secretion. Pharmacologic inhibition of PLAU receptor impairs the growth and metastasis of p53-deficient cancers. Blockade of PAQR11-dependent secretion inhibits immunosuppressive processes in the tumor microenvironment. Thus, Golgi reprogramming by p53 loss is a key driver of hypersecretion in cancer.

Th17 cells contribute to combination MEK inhibitor and anti-PD-L1 therapy resistance in KRAS/p53 mutant lung cancers.

Understanding resistance mechanisms to targeted therapies and immune checkpoint blockade in mutant KRAS lung cancers is critical to developing novel combination therapies and improving patient survival. Here, we show that MEK inhibition enhanced PD-L1 expression while PD-L1 blockade upregulated MAPK signaling in mutant KRAS lung tumors. Combined MEK inhibition with anti-PD-L1 synergistically reduced lung tumor growth and metastasis, but tumors eventually developed resistance to sustained combinatorial therapy. Multi-platform profiling revealed that resistant lung tumors have increased infiltration of Th17 cells, which secrete IL-17 and IL-22 cytokines to promote lung cancer cell invasiveness and MEK inhibitor resistance. Antibody depletion of IL-17A in combination with MEK inhibition and PD-L1 blockade markedly reduced therapy-resistance in vivo. Clinically, increased expression of Th17-associated genes in patients treated with PD-1 blockade predicted poorer overall survival and response in melanoma and predicated poorer response to anti-PD1 in NSCLC patients. Here we show a triple combinatorial therapeutic strategy to overcome resistance to combined MEK inhibitor and PD-L1 blockade.

Contextual cues from cancer cells govern cancer-associated fibroblast heterogeneity.

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.

Dual Inhibition of MEK and AXL Targets Tumor Cell Heterogeneity and Prevents Resistant Outgrowth Mediated by the Epithelial-to-Mesenchymal Transition in NSCLC.

The epithelial-to-mesenchymal transition (EMT) is a dynamic epigenetic reprogramming event that occurs in a subset of tumor cells and is an initiating step toward invasion and distant metastasis. The process is reversible and gives plasticity to cancer cells to survive under variable conditions, with the acquisition of cancer stem cell-like characteristics and features such as drug resistance. Therefore, understanding survival dependencies of cells along the phenotypic spectrum of EMT will provide better strategies to target the spatial and temporal heterogeneity of tumors and prevent their ability to bypass single-inhibitor treatment strategies. To address this, we integrated the data from a selective drug screen in epithelial and mesenchymal KRAS/p53 (KP)-mutant lung tumor cells with separate datasets including reverse-phase protein array and an in vivo shRNA dropout screen. These orthogonal approaches identified AXL and MEK as potential mesenchymal and epithelial cell survival dependencies, respectively. To capture the dynamicity of EMT, incorporation of a dual fluorescence EMT sensor system into murine KP lung cancer models enabled real-time analysis of the epigenetic state of tumor cells and assessment of the efficacy of single agent or combination treatment with AXL and MEK inhibitors. Both two- and three-dimensional culture systems and in vivo models revealed that this combination treatment strategy of MEK plus AXL inhibition synergistically killed lung cancer cells by specifically targeting each phenotypic subpopulation. In conclusion, these results indicate that cotargeting the specific vulnerabilities of EMT subpopulations can prevent EMT-mediated drug resistance, effectively controlling tumor cell growth and metastasis. SIGNIFICANCE: This study shows that a novel combination of MEK and AXL inhibitors effectively bypasses EMT-mediated drug resistance in KRAS/p53-mutant non-small cell lung cancer by targeting EMT subpopulations, thereby preventing tumor cell survival.

AXL Inhibition Induces DNA Damage and Replication Stress in Non-Small Cell Lung Cancer Cells and Promotes Sensitivity to ATR Inhibitors.

AXL, a TAM (TYRO3, AXL, and MERTK) family receptor tyrosine kinase, is increasingly being recognized as a key determinant of resistance to targeted therapies, as well as chemotherapy and radiation in non-small cell lung cancer (NSCLC) and other cancers. We further show here that high levels of AXL and epithelial-to-mesenchymal transition were frequently expressed in subsets of both treatment-naïve and treatment-relapsed NSCLC. Previously, we and others have demonstrated a role for AXL in mediating DNA damage response (DDR), as well as resistance to inhibition of WEE1, a replication stress response kinase. Here, we show that BGB324 (bemcentinib), a selective small-molecule AXL inhibitor, caused DNA damage and induced replication stress, indicated by ATR/CHK1 phosphorylation, more significantly in TP53-deficient NSCLC cell lines. Similar effects were also observed in large-cell neuroendocrine carcinoma (LCNEC) cell lines. High AXL protein levels were also associated with resistance to ATR inhibition. Combined inhibition of AXL and ATR significantly decreased cell proliferation of NSCLC and LCNEC cell lines. Mechanistically, combined inhibition of AXL and ATR significantly increased RPA32 hyperphosphorylation and DNA double-strand breaks and induced markers of mitotic catastrophe. Notably, NSCLC cell lines with low levels of SLFN11, a known predictive biomarker for platinum and PARP inhibitor sensitivity, were more sensitive to AXL/ATR cotargeting. These findings demonstrate a novel and unexpected role for AXL in replication stress tolerance, with potential therapeutic implications. IMPLICATIONS: These findings demonstrate that the combination of AXL and ATR inhibitors could be a promising therapeutic combination for NSCLC, LCNEC, and other cancers.

A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma.

Therapeutic strategies designed to target TP53-deficient cancer cells remain elusive. Here, we showed that TP53 loss initiated a pharmacologically actionable secretory process that drove lung adenocarcinoma (LUAD) progression. Molecular, biochemical, and cell biological studies showed that TP53 loss increased the expression of Golgi reassembly and stacking protein 55 kDa (G55), a Golgi stacking protein that maintains Golgi organelle integrity and is part of a GOLGIN45 (G45)-myosin IIA-containing protein complex that activates secretory vesicle biogenesis in the Golgi. TP53 loss activated G55-dependent secretion by relieving G55 and myosin IIA from miR-34a-dependent silencing. G55-dependent secreted proteins enhanced the proliferative and invasive activities of TP53-deficient LUAD cells and promoted angiogenesis and CD8+ T cell exhaustion in the tumor microenvironment. A small molecule that blocks G55-G45 interactions impaired secretion and reduced TP53-deficient LUAD growth and metastasis. These results identified a targetable secretory vulnerability in TP53-deficient LUAD cells.

PI4KIIIß is a therapeutic target in chromosome 1q-amplified lung adenocarcinoma.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.

Ntrk1 Promotes Resistance to PD-1 Checkpoint Blockade in Mesenchymal Kras/p53 Mutant Lung Cancer.

The implementation of cancer immunotherapeutics for solid tumors including lung cancers has improved clinical outcomes in a small percentage of patients. However, the majority of patients show little to no response or acquire resistance during treatment with checkpoint inhibitors delivered as a monotherapy. Therefore, identifying resistance mechanisms and novel combination therapy approaches is imperative to improve responses to immune checkpoint inhibitors. To address this, we performed an in vivo shRNA dropout screen that focused on genes encoding for FDA-approved drug targets (FDAome). We implanted epithelial and mesenchymal Kras/p53 (KP) mutant murine lung cancer cells expressing the FDAome shRNA library into syngeneic mice treated with an anti-PD-1 antibody. Sequencing for the barcoded shRNAs revealed Ntrk1 was significantly depleted from mesenchymal tumors challenged with PD-1 blockade, suggesting it provides a survival advantage to tumor cells when under immune system pressure. Our data confirmed Ntrk1 transcript levels are upregulated in tumors treated with PD-1 inhibitors. Additionally, analysis of tumor-infiltrating T cell populations revealed that Ntrk1 can promote CD8+ T cell exhaustion. Lastly, we found that Ntrk1 regulates Jak/Stat signaling to promote expression of PD-L1 on tumor cells. Together, these data suggest that Ntrk1 activates Jak/Stat signaling to regulate expression of immunosuppressive molecules including PD-L1, promoting exhaustion within the tumor microenvironment.

A novel ex vivo tumor system identifies Src-mediated invasion and metastasis in mesenchymal tumor cells in non-small cell lung cancer.

Lung cancer is the foremost cause of cancer related deaths in the U.S. It is a heterogeneous disease composed of genetically and phenotypically distinct tumor cells surrounded by heterotypic cells and extracellular matrix dynamically interacting with the tumor cells. Research in lung cancer is often restricted to patient-derived tumor specimens, in vitro cell cultures and limited animal models, which fail to capture the cellular or microenvironment heterogeneity of the tumor. Therefore, our knowledge is primarily focused on cancer-cell autonomous aberrations. For a fundamental understanding of lung cancer progression and an exploration of therapeutic options, we focused our efforts to develop an Ex Vivo Tumor platform to culture tumors in 3D matrices, which retains tumor cell heterogeneity arising due to in vivo selection pressure and environmental influences and recapitulate responses of tumor cells to external manipulations. To establish this model, implanted syngeneic murine tumors from a mutant KRAS/p53 model were harvested to yield multicellular tumor aggregates followed by culture in 3D extracellular matrices. Using this system, we identified Src signaling as an important driver of invasion and metastasis in lung cancer and demonstrate that EVTs are a robust experimental tool bridging the gap between conventional in vitro and in vivo models.

Targeting DNA Damage Response Promotes Antitumor Immunity through STING-Mediated T-cell Activation in Small Cell Lung Cancer.

Despite recent advances in the use of immunotherapy, only a minority of patients with small cell lung cancer (SCLC) respond to immune checkpoint blockade (ICB). Here, we show that targeting the DNA damage response (DDR) proteins PARP and checkpoint kinase 1 (CHK1) significantly increased protein and surface expression of PD-L1. PARP or CHK1 inhibition remarkably potentiated the antitumor effect of PD-L1 blockade and augmented cytotoxic T-cell infiltration in multiple immunocompetent SCLC in vivo models. CD8+ T-cell depletion reversed the antitumor effect, demonstrating the role of CD8+ T cells in combined DDR-PD-L1 blockade in SCLC. We further demonstrate that DDR inhibition activated the STING/TBK1/IRF3 innate immune pathway, leading to increased levels of chemokines such as CXCL10 and CCL5 that induced activation and function of cytotoxic T lymphocytes. Knockdown of cGAS and STING successfully reversed the antitumor effect of combined inhibition of DDR and PD-L1. Our results define previously unrecognized innate immune pathway-mediated immunomodulatory functions of DDR proteins and provide a rationale for combining PARP/CHK1 inhibitors and immunotherapies in SCLC. SIGNIFICANCE: Our results define previously unrecognized immunomodulatory functions of DDR inhibitors and suggest that adding PARP or CHK1 inhibitors to ICB may enhance treatment efficacy in patients with SCLC. Furthermore, our study supports a role of innate immune STING pathway in DDR-mediated antitumor immunity in SCLC.See related commentary by Hiatt and MacPherson, p. 584.This article is highlighted in the In This Issue feature, p. 565.

CD38-Mediated Immunosuppression as a Mechanism of Tumor Cell Escape from PD-1/PD-L1 Blockade.

Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNß in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment.Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8+ T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156-75. ©2018 AACR.See related commentary by Mittal et al., p. 1066This article is highlighted in the In This Issue feature, p. 1047.