Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression.

BACKGROUND: We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer. METHODS: The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO2, pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer. RESULTS: Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A2B-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon γ production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval = 3.35 to 41.13], n = 10, P < .001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity. CONCLUSIONS: Data identify A2B-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Brief report: STING expressed in tumor and non-tumor compartments has distinct roles in regulating anti-tumor immunity.

Type I interferon-mediated activation of immune cells can facilitate the generation of productive tumor antigen-specific T cell responses in solid tumors. The cGAS/STING DNA sensing pathway is a critical upstream mediator of type I interferon production and is an important regulator of anti-tumor immunity. Numerous STING pathway agonists are now being tested in clinical trials, but the effectiveness of this approach is not yet clear and a better understanding of the relative importance of this pathway in various tumor settings is needed. We have evaluated syngeneic tumor models with different baseline inflammatory states to determine the contributions of STING activity in both tumor and non-tumor cellular compartments to anti-tumor immune responses. We find that productive anti-tumor immune responses in the poorly immunogenic B16F10 model show a strong dependence on STING expression in non-tumor cells. In the immunogenic MC38 model, constitutive STING activation in tumor cells can partially bypass the requirement for STING-dependent activity from immune cells. Our findings reveal multiple, context-dependent roles for STING activity in the regulation of anti-tumor immunity and the response to immunotherapy. In preclinical models where STING is basally active, checkpoint inhibition is more likely to have a therapeutic effect and removal of STING signaling from either the tumor or the non-tumor compartment has a minimal effect. Removal of STING signaling in both, however, diminishes the efficacy derived from checkpoint therapy. Further work is needed to understand the heterogeneity of STING signaling in patients, both in tumor cells and the tumor microenvironment, and the best means of harnessing this pathway to generate anti-tumor immunity and improve therapeutic outcomes.

SMAD4 Suppresses Colitis-associated Carcinoma Through Inhibition of CCL20/CCR6-mediated Inflammation.

BACKGROUND & AIMS: We previously reported that colon epithelial cell silencing of Smad4 increased epithelial expression of inflammatory genes, including the chemokine c-c motif chemokine ligand 20 (CCL20), and increased susceptibility to colitis-associated cancer. Here, we examine the role of the chemokine/receptor pair CCL20/c-c motif chemokine receptor 6 (CCR6) in mediating colitis-associated colon carcinogenesis induced by SMAD4 loss. METHODS: In silico analysis of SMAD4, CCL20, and CCR6 messenger RNA expression was performed on published transcriptomic data from human ulcerative colitis (UC), and colon and rectal cancer samples. Immunohistochemistry for CCL20 and CCR6 was performed on human tissue microarrays comprising human UC-associated cancer specimens, Mice with conditional, epithelial-specific Smad4 loss with and without germline deletion of the Ccr6 gene were subjected to colitis and followed for up to 3 months. Tumors were quantified histologically, and immune cell populations were analyzed by flow cytometry and immunostaining. RESULTS: In human UC-associated cancers, loss of epithelial SMAD4 was associated with increased CCL20 expression and CCR6+ cells. SMAD4 loss in mouse colon epithelium led to enlarged gut-associated lymphoid tissues and recruitment of immune cells to the mouse colon epithelium and stroma, particularly T regulatory, Th17, and dendritic cells. Loss of CCR6 abrogated these immune responses and significantly reduced the incidence of colitis-associated tumors observed with loss of SMAD4 alone. CONCLUSIONS: Regulation of mucosal inflammation is central to SMAD4 tumor suppressor function in the colon. A key downstream node in this regulation is suppression of epithelial CCL20 signaling to CCR6 in immune cells. Loss of SMAD4 in the colon epithelium increases CCL20 expression and chemoattraction of CCR6+ immune cells, contributing to greater susceptibility to colon cancer.

Characterization of Immunopathology and Small Airway Remodeling in Constrictive Bronchiolitis.

Rationale: Constrictive bronchiolitis (ConB) is a relatively rare and understudied form of lung disease whose underlying immunopathology remains incompletely defined. Objectives: Our objectives were to quantify specific pathological features that differentiate ConB from other diseases that affect the small airways and to investigate the underlying immune and inflammatory phenotype present in ConB. Methods: We performed a comparative histomorphometric analysis of small airways in lung biopsy samples collected from 50 soldiers with postdeployment ConB, 8 patients with sporadic ConB, 55 patients with chronic obstructive pulmonary disease, and 25 nondiseased control subjects. We measured immune and inflammatory gene expression in lung tissue using the NanoString nCounter Immunology Panel from six control subjects, six soldiers with ConB, and six patients with sporadic ConB. Measurements and Main Results: Compared with control subjects, we found shared pathological changes in small airways from soldiers with postdeployment ConB and patients with sporadic ConB, including increased thickness of the smooth muscle layer, increased collagen deposition in the subepithelium, and lymphocyte infiltration. Using principal-component analysis, we showed that ConB pathology was clearly separable both from control lungs and from small airway disease associated with chronic obstructive pulmonary disease. NanoString gene expression analysis from lung tissue revealed T-cell activation in both groups of patients with ConB with upregulation of proinflammatory pathways, including cytokine-cytokine receptor interactions, NF-κB (nuclear factor-κB) signaling, TLR (Toll-like receptor) signaling, T-cell receptor signaling, and antigen processing and presentation. Conclusions: These findings indicate shared immunopathology among different forms of ConB and suggest that an ongoing T-helper cell type 1-type adaptive immune response underlies airway wall remodeling in ConB.

HLA-DR cancer cells expression correlates with T cell infiltration and is enriched in lung adenocarcinoma with indolent behavior.

Lung adenocarcinoma (ADC) is a heterogeneous group of tumors associated with different survival rates, even when detected at an early stage. Here, we aim to investigate whether CyTOF identifies cellular and molecular predictors of tumor behavior. We developed and validated a CyTOF panel of 34 antibodies in four ADC cell lines and PBMC. We tested our panel in a set of 10 ADCs, classified into long- (LPS) (n = 4) and short-predicted survival (SPS) (n = 6) based on radiomics features. We identified cellular subpopulations of epithelial cancer cells (ECC) and their microenvironment and validated our results by multiplex immunofluorescence (mIF) applied to a tissue microarray (TMA) of LPS and SPS ADCs. The antibody panel captured the phenotypical differences in ADC cell lines and PBMC. LPS ADCs had a higher proportion of immune cells. ECC clusters (ECCc) were identified and uncovered two ADC groups. ECCc with high HLA-DR expression were correlated with CD4+ and CD8+ T cells, with LPS samples being enriched for those clusters. We confirmed a positive correlation between HLA-DR expression on ECC and T cell number by mIF staining on TMA slides. Spatial analysis demonstrated shorter distances from T cells to the nearest ECC in LPS. Our results demonstrate a distinctive cellular profile of ECC and their microenvironment in ADC. We showed that HLA-DR expression in ECC is correlated with T cell infiltration, and that a set of ADCs with high abundance of HLA-DR+ ECCc and T cells is enriched in LPS samples. This suggests new insights into the role of antigen presenting tumor cells in tumorigenesis.

Adenosine/TGFß axis in regulation of mammary fibroblast functions.

Cancer associated fibroblasts (CAF) play a key role in cancer progression and metastasis. Diminished TGFß response on CAF correlates with poor outcome and recurrence in cancer patients. Mechanisms behind lost TGFß signaling on CAF are poorly understood, but, utilizing MMTV-PyMT mouse model, we have previously demonstrated that in tumor microenvironment myeloid cells, producing adenosine, contribute to downregulated TGFß signaling on CAFs. In the current work, we performed serial in vitro studies to investigate the role of adenosine/TGFß axis in mouse mammary fibroblast functions, i.e., proliferation, protein expression, migration, and contractility. We found that adenosine analog NECA diminished TGFß-induced CCL5 and MMP9 expression. Additionally, we discovered that NECA completely inhibited effect of TGFß to upregulate αSMA, key protein of cytoskeletal rearrangements, necessary for migration and contractility of fibroblasts. Our results show that TGFß increases contractility of mouse mammary fibroblasts and human fibroblast cell lines, and NECA attenuates theses effects. Using pharmacological approach and genetically modified animals, we determined that NECA effects on TGFß pathway occur via A2A/A2B adenosine receptor-AC-PKA dependent manner. Using isolated CD11b+ cells from tumor tissue of CD73-KO and CD39-KO animals in co-culture experiments with ATP and AMP, we confirmed that myeloid cells can affect functions of mammary fibroblasts through adenosine signaling. Our data suggest a novel mechanism of interaction between adenosine and TGFß signaling pathways that can impact phenotype of fibroblasts in a tumor microenvironment.

A requirement for p120-catenin in the metastasis of invasive ductal breast cancer.

We report here the effects of targeted p120-catenin (encoded by CTNND1; hereafter denoted p120) knockout (KO) in a PyMT mouse model of invasive ductal (mammary) cancer (IDC). Mosaic p120 ablation had little effect on primary tumor growth but caused significant pro-metastatic alterations in the tumor microenvironment, ultimately leading to a marked increase in the number and size of pulmonary metastases. Surprisingly, although early effects of p120-ablation included decreased cell-cell adhesion and increased invasiveness, cells lacking p120 were almost entirely unable to colonized distant metastatic sites in vivo The relevance of this observation to human IDC was established by analysis of a large clinical dataset of 1126 IDCs. As reported by others, p120 downregulation in primary IDC predicted worse overall survival. However, as in the mice, distant metastases were almost invariably p120 positive, even in matched cases where the primary tumors were p120 negative. Collectively, our results demonstrate a strong positive role for p120 (and presumably E-cadherin) during metastatic colonization of distant sites. On the other hand, downregulation of p120 in the primary tumor enhanced metastatic dissemination indirectly via pro-metastatic conditioning of the tumor microenvironment.

Loss of Myeloid BMPR1a Alters Differentiation and Reduces Mouse Prostate Cancer Growth.

The Bone Morphogenetic Protein (BMP) pathway is a member of the TGFß signaling family and has complex roles in cancer. BMP signaling is rarely mutated and can be frequently overexpressed in many human cancers. The dichotomous role of BMPs as both tumor promoters and suppressors appears to be largely context based in both the cancer cell and the surrounding microenvironment. Myeloid cells including macrophages and neutrophils have been shown to be tumor promoting when stimulated from BMPs. We found that conditional deletion of BMPR1a in myeloid cells (LysMCre) restricts tumor progression in a syngeneic mouse prostate cancer model. Specific changes occurred in myeloid cells both in tumor bearing mice and tumor naïve mice throughout multiple tissues. We profiled myeloid subsets in the bone marrow, spleen and primary tumor and found myeloid BMPR1a loss altered the differentiation and lineage capability of distinct populations by histologic, flow cytometry and high dimensional mass cytometry analysis. We further confirmed the requirement for BMP signaling with pharmacologic inhibition of THP-1 and Raw264.7 activated into M2 macrophages with the BMP inhibitor DMH1. M2 polarized primary bone marrow derived cells from LysMCre BMPR1a knockout mice indicated a distinct requirement for BMP signaling in myeloid cells during M2 activation. These results indicate a unique necessity for BMP signaling in myeloid cells during tumor progression.

Myeloid Cell-Derived TGFß Signaling Regulates ECM Deposition in Mammary Carcinoma via Adenosine-Dependent Mechanisms.

TGFß plays a crucial role in the tumor microenvironment by regulating cell-cell and cell-stroma interactions. We previously demonstrated that TGFß signaling on myeloid cells regulates expression of CD73, a key enzyme for production of adenosine, a protumorigenic metabolite implicated in regulation of tumor cell behaviors, immune response, and angiogenesis. Here, using an MMTV-PyMT mouse mammary tumor model, we discovered that deletion of TGFß signaling on myeloid cells (PyMT/TGFßRIILysM) affects extracellular matrix (ECM) formation in tumor tissue, specifically increasing collagen and decreasing fibronectin deposition. These changes were associated with mitigated tumor growth and reduced metastases. Reduced TGFß signaling on fibroblasts was associated with their proximity to CD73+ myeloid cells in tumor tissue. Consistent with these findings, adenosine significantly downregulated TGFß signaling on fibroblasts, an effect regulated by A2A and A2B adenosine receptors. METABRIC dataset analysis revealed that patients with triple-negative breast cancer and basal type harbored a similar signature of adenosine and ECM profiles; high expression of A2B adenosine receptors correlated with decreased expression of Col1 and was associated with poor outcome. Taken together, our studies reveal a new role for TGFß signaling on myeloid cells in tumorigenesis. This discovered cross-talk between TGFß/CD73 on myeloid cells and TGFß signaling on fibroblasts can contribute to ECM remodeling and protumorigenic actions of cancer-associated fibroblasts. SIGNIFICANCE: TGFß signaling on fibroblasts is decreased in breast cancer, correlates with poor prognosis, and appears to be driven by adenosine that accelerates tumor progression and metastasis via ECM remodeling.

Gas6/MerTK signaling is negatively regulated by NF-κB and supports lung carcinogenesis.

Growth arrest-specific 6 (Gas6) has been implicated in carcinogenesis through activation of its receptors, particularly MerTK. To investigate whether Gas6 plays a role in resistance to NF-κB inhibitors, which have not proven to be effective agents for lung cancer therapy, we studied lung cancer models induced by urethane injection or expression of mutant Kras (KrasG12D). We found that Gas6 is primarily produced by macrophages during tumorigenesis and that Gas6 is negatively regulated by NF-κB. Since Gas6 is a vitamin K dependent protein, we used low-dose warfarin to block Gas6 production and showed that this treatment inhibited tumorigenesis in both the urethane and KrasG12D models, most prominently in mice with targeted deletion of IKKß in myeloid cells (IKKßΔMye mice). In addition, MerTK deficient mice had reduced urethane-induced tumorigenesis. Inhibition of the Gas6-MerTK pathway in all these models reduced macrophages and neutrophils in the lungs of tumor-bearing mice. Analysis of mouse lung tumors revealed MerTK staining on tumor cells and in vitro studies showed that Gas6 increased proliferation of human lung cancer cell lines. To assess the therapeutic potential for combination treatment targeting NF-κB and Gas6-MerTK, we injected Lewis Lung Carcinoma cells subcutaneously and treated mice with Bay 11-70852 (NF-κB inhibitor) and/or Foretinib (MerTK inhibitor). While individual treatments were ineffective, combination therapy markedly reduced tumor growth, blocked tumor cell proliferation, reduced tumor-associated macrophages, and increased CD4+ T cells. Together, our studies unmask a role for Gas6-MerTK signaling in lung carcinogenesis and indicate that up-regulation of Gas6 production in macrophages could be a major mechanism of resistance to NF-κB inhibitors.

Bone marrow-derived fibroblasts are a functionally distinct stromal cell population in breast cancer.

Cancer-associated fibroblasts (CAFs) are highly prominent in breast tumors, but their functional heterogeneity and origin are still largely unresolved. We report that bone marrow (BM)-derived mesenchymal stromal cells (MSCs) are recruited to primary breast tumors and to lung metastases and differentiate to a distinct subpopulation of CAFs. We show that BM-derived CAFs are functionally important for tumor growth and enhance angiogenesis via up-regulation of Clusterin. Using newly generated transgenic mice and adoptive BM transplantations, we demonstrate that BM-derived fibroblasts are a substantial source of CAFs in the tumor microenvironment. Unlike resident CAFs, BM-derived CAFs do not express PDGFRα, and their recruitment resulted in a decrease in the percentage of PDGFRα-expressing CAFs. Strikingly, decrease in PDGFRα in breast cancer patients was associated with worse prognosis, suggesting that BM-derived CAFs may have deleterious effects on survival. Therefore, PDGFRα expression distinguishes two functionally unique CAF populations in breast tumors and metastases and may have important implications for patient stratification and precision therapeutics.

Loss of CXCR4 in Myeloid Cells Enhances Antitumor Immunity and Reduces Melanoma Growth through NK Cell and FASL Mechanisms.

The chemokine receptor, CXCR4, is involved in cancer growth, invasion, and metastasis. Several promising CXCR4 antagonists have been shown to halt tumor metastasis in preclinical studies, and clinical trials evaluating the effectiveness of these agents in patients with cancer are ongoing. However, the impact of targeting CXCR4 specifically on immune cells is not clear. Here, we demonstrate that genetic deletion of CXCR4 in myeloid cells (CXCR4MyeΔ/Δ) enhances the antitumor immune response, resulting in significantly reduced melanoma tumor growth. Moreover, CXCR4MyeΔ/Δ mice exhibited slowed tumor progression compared with CXCR4WT mice in an inducible melanocyte BrafV600E/Pten -/- mouse model. The percentage of Fas ligand (FasL)-expressing myeloid cells was reduced in CXCR4MyeΔ/Δ mice as compared with myeloid cells from CXCR4WT mice. In contrast, there was an increased percentage of natural killer (NK) cells expressing FasL in tumors growing in CXCR4MyeΔ/Δ mice. NK cells from CXCR4MyeΔ/Δ mice also exhibited increased tumor cell killing capacity in vivo, based on clearance of NK-sensitive Yac-1 cells. NK cell-mediated killing of Yac-1 cells occurred in a FasL-dependent manner, which was partially dependent upon the presence of CXCR4MyeΔ/Δ neutrophils. Furthermore, enhanced NK cell activity in CXCR4MyeΔ/Δ mice was also associated with increased production of IL18 by specific leukocyte subpopulations. These data suggest that CXCR4-mediated signals from myeloid cells suppress NK cell-mediated tumor surveillance and thereby enhance tumor growth. Systemic delivery of a peptide antagonist of CXCR4 to tumor-bearing CXCR4WT mice resulted in enhanced NK-cell activation and reduced tumor growth, supporting potential clinical implications for CXCR4 antagonism in some cancers. Cancer Immunol Res; 6(10); 1186-98. ©2018 AACR.

Epithelial Smad4 Deletion Up-Regulates Inflammation and Promotes Inflammation-Associated Cancer.

Background & Aims: Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor ß (TGFß) family signaling through SMAD4 in colonic epithelial cells. Methods: The Smad4 gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFß1 and bone morphogenetic protein 2. Results: Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic Smad4 expression, with resulting tumors bearing striking resemblance to human colitis-associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis-associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of Smad4 increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFß signaling inhibited the epithelial inflammatory response to proinflammatory cytokines. Conclusions: TGFß suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082.

Development of Aggressive Pancreatic Ductal Adenocarcinomas Depends on Granulocyte Colony Stimulating Factor Secretion in Carcinoma Cells.

The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48TGFßRIIKO), we discovered an effect of TGFß signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G+ cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b+ cells. Deletion of csf3 in PDAC cells or use of a G-CSF-blocking antibody decreased tumor growth. Anti-G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G+ cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. Cancer Immunol Res; 5(9); 718-29. ©2017 AACR.

Switching off CD73: a way to boost the activity of conventional and targeted antineoplastic therapies.

Over the past few years, several preclinical studies have highlighted the value of CD73 (ecto-5'-nucleotidase) as a potential therapeutic target for cancer therapy. Indeed, the pharmacological blockade of CD73, via monoclonal antibodies or small molecules, has promise in counteracting cancer development, growth and spread. Synergistic combinations of anti-CD73 drugs with conventional cancer treatments (i.e., chemotherapy, radiation therapy, immunotherapy, targeted therapy) have increased therapeutic potential. In this review, we discuss the potential synergistic effects of CD73 blockers and conventional antineoplastic therapies in the treatment of cancer.

PI3K Inhibition Reduces Mammary Tumor Growth and Facilitates Antitumor Immunity and Anti-PD1 Responses.

Purpose: Metastatic breast cancers continue to elude current therapeutic strategies, including those utilizing PI3K inhibitors. Given the prominent role of PI3Kα,ß in tumor growth and PI3Kγ,δ in immune cell function, we sought to determine whether PI3K inhibition altered antitumor immunity.Experimental Design: The effect of PI3K inhibition on tumor growth, metastasis, and antitumor immune response was characterized in mouse models utilizing orthotopic implants of 4T1 or PyMT mammary tumors into syngeneic or PI3Kγ-null mice, and patient-derived breast cancer xenografts in humanized mice. Tumor-infiltrating leukocytes were characterized by IHC and FACS analysis in BKM120 (30 mg/kg, every day) or vehicle-treated mice and PI3Kγnull versus PI3KγWT mice. On the basis of the finding that PI3K inhibition resulted in a more inflammatory tumor leukocyte infiltrate, the therapeutic efficacy of BKM120 (30 mg/kg, every day) and anti-PD1 (100 µg, twice weekly) was evaluated in PyMT tumor-bearing mice.Results: Our findings show that PI3K activity facilitates tumor growth and surprisingly restrains tumor immune surveillance. These activities could be partially suppressed by BKM120 or by genetic deletion of PI3Kγ in the host. The antitumor effect of PI3Kγ loss in host, but not tumor, was partially reversed by CD8+ T-cell depletion. Treatment with therapeutic doses of both BKM120 and antibody to PD-1 resulted in consistent inhibition of tumor growth compared with either agent alone.Conclusions: PI3K inhibition slows tumor growth, enhances antitumor immunity, and heightens susceptibility to immune checkpoint inhibitors. We propose that combining PI3K inhibition with anti-PD1 may be a viable therapeutic approach for triple-negative breast cancer. Clin Cancer Res; 23(13); 3371-84. ©2016 AACR.

Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome.

Alveolar epithelial cell (AEC) dysfunction underlies the pathogenesis of pulmonary fibrosis in Hermansky-Pudlak syndrome (HPS) and other genetic syndromes associated with interstitial lung disease; however, mechanisms linking AEC dysfunction and fibrotic remodeling are incompletely understood. Since increased macrophage recruitment precedes pulmonary fibrosis in HPS, we investigated whether crosstalk between AECs and macrophages determines fibrotic susceptibility. We found that AECs from HPS mice produce excessive MCP-1, which was associated with increased macrophages in the lungs of unchallenged HPS mice. Blocking MCP-1/CCR2 signaling in HPS mice with genetic deficiency of CCR2 or targeted deletion of MCP-1 in AECs normalized macrophage recruitment, decreased AEC apoptosis, and reduced lung fibrosis in these mice following treatment with low-dose bleomycin. We observed increased TGF-ß production by HPS macrophages, which was eliminated by CCR2 deletion. Selective deletion of TGF-ß in myeloid cells or of TGF-ß signaling in AECs through deletion of TGFBR2 protected HPS mice from AEC apoptosis and bleomycin-induced fibrosis. Together, these data reveal a feedback loop in which increased MCP-1 production by dysfunctional AECs results in recruitment and activation of lung macrophages that produce TGF-ß, thus amplifying the fibrotic cascade through AEC apoptosis and stimulation of fibrotic remodeling.

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression.

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality, yet antistromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor-ß (TGF-ß) signaling have high epithelial STAT3 activity and develop stiff, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several KRAS-driven mouse models, both the loss of TGF-ß signaling and elevated ß1-integrin mechanosignaling engaged a positive feedback loop whereby STAT3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial STAT3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-ß signaling. In PDAC patient biopsies, higher matricellular protein and activated STAT3 were associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors and highlight STAT3 and mechanics as key drivers of this phenotype.

Multivalent Forms of the Notch Ligand DLL-1 Enhance Antitumor T-cell Immunity in Lung Cancer and Improve Efficacy of EGFR-Targeted Therapy.

Activation of Notch signaling in hematopoietic cells by tumors contributes to immune escape. T-cell defects in tumors can be reversed by treating tumor-bearing mice with multivalent forms of the Notch receptor ligand DLL-1, but the immunologic correlates of this effect have not been elucidated. Here, we report mechanistic insights along with the efficacy of combinational treatments of multivalent DLL-1 with oncoprotein targeting drugs in preclinical mouse models of lung cancer. Systemic DLL-1 administration increased T-cell infiltration into tumors and elevated numbers of CD44(+)CD62L(+)CD8(+) memory T cells while decreasing the number of regulatory T cells and limiting tumor vascularization. This treatment was associated with upregulation of Notch and its ligands in tumor-infiltrating T cells enhanced expression of T-bet and phosphorylation of Stat1/2. Adoptive transfer of T cells from DLL1-treated tumor-bearing immunocompetent hosts into tumor-bearing SCID-NOD immunocompromised mice attenuated tumor growth and extended tumor-free survival in the recipients. When combined with the EGFR-targeted drug erlotinib, DLL-1 significantly improved progression-free survival by inducing robust tumor-specific T-cell immunity. In tissue culture, DLL1 induced proliferation of human peripheral T cells, but lacked proliferative or clonogenic effects on lung cancer cells. Our findings offer preclinical mechanistic support for the development of multivalent DLL1 to stimulate antitumor immunity.