Although anti-PD-1 therapy has improved clinical outcomes for select patients with advanced cancer, many patients exhibit either primary or adaptive resistance to checkpoint inhibitor immunotherapy. ...The role of the tumor stroma in the development of these mechanisms of resistance to checkpoint inhibitors remains unclear. We demonstrated that pharmacologic inhibition of the TGFβ signaling pathway synergistically enhanced the efficacy of anti-CTLA-4 immunotherapy but failed to augment anti-PD-1/PD-L1 responses in an autochthonous model of BRAF
melanoma. Additional mechanistic studies revealed that TGFβ pathway inhibition promoted the proliferative expansion of stromal fibroblasts, thereby facilitating MMP-9-dependent cleavage of PD-L1 surface expression, leading to anti-PD-1 resistance in this model. Further work demonstrated that melanomas escaping anti-PD-1 therapy exhibited a mesenchymal phenotype associated with enhanced TGFβ signaling activity. Delayed TGFβ inhibitor therapy, following anti-PD-1 escape, better served to control further disease progression and was superior to a continuous combination of anti-PD-1 and TGFβ inhibition. This work illustrates that formulating immunotherapy combination regimens to enhance the efficacy of checkpoint blockade requires an in-depth understanding of the impact of these agents on the tumor microenvironment. These data indicated that stromal fibroblast MMP-9 may desensitize tumors to anti-PD-1 and suggests that TGFβ inhibition may generate greater immunologic efficacy when administered following the development of acquired anti-PD-1 resistance.
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Myeloid cell receptor tyrosine kinases TYRO3, AXL, and MERTK and their ligands, GAS6 and PROTEIN S, physiologically suppress innate immune responses, including in the tumor microenvironment. Here, we ...showed that myeloid-derived suppressor cells (MDSC) dramatically upregulated TYRO3, AXL, and MERTK and their ligands monocytic MDSCs (M-MDSC)>20-fold, polymorphonuclear MDSCs (PMN-MDSC)>15-fold in tumor-bearing mice. MDSCs from tumor-bearing
, and
mice exhibited diminished suppressive enzymatic capabilities, displayed deficits in T-cell suppression, and migrated poorly to tumor-draining lymph nodes. In coimplantation experiments using TYRO3
, AXL
, and MERTK
MDSCs, we showed the absence of these RTKs reversed the protumorigenic properties of MDSCs
Consistent with these findings,
pharmacologic TYRO3, AXL, and MERTK inhibition diminished MDSC suppressive capability, slowed tumor growth, increased CD8
T-cell infiltration, and augmented anti-PD-1 checkpoint inhibitor immunotherapy. Mechanistically, MERTK regulated MDSC suppression and differentiation in part through regulation of STAT3 serine phosphorylation and nuclear localization. Analysis of metastatic melanoma patients demonstrated an enrichment of circulating MERTK
and TYRO3
M-MDSCs, PMN-MDSCs, and early-stage MDSCs (e-MDSC) relative to these MDSC populations in healthy controls. These studies demonstrated that TYRO3, AXL, and MERTK control MDSC functionality and serve as promising pharmacologic targets for regulating MDSC-mediated immune suppression in cancer patients.
Abstract
Tyro3, Axl and MerTK (TAM) are a family of receptor tyrosine kinases (RTKs) that play physiologic roles in dampening innate immune responses, preventing chronic inflammation and ...autoimmunity. Their roles in potentially promoting tolerance in the immunosuppressive tumor microenvironment (TME) in the context of neoplasia are unclear. Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis, a notoriously immune suppressive TME and limited treatment options. Here we evaluate the role of TAM RTKs in the PDAC TME using orthotopic injection of a pancreatic cell line into mertk-/-,axl-/- ,tyro3-/- and mertk-/-/tyro3 double knockout( DKO) mice. The loss of MerTK or Tyro3 in the TME substantially attenuates orthotopic PDAC growth. Moreover, pancreatic tumors implanted in the mertk-/-/tyro3-/- DKO tumors completely regress suggesting that these two RTKs have at least some non-redundant suppressive activities. Furthermore, loss of MerTK or Tyro3 overcame resistance to anti-PD-1 treatment, with most tumors completely regressed. Surprisingly none of these effects are seen in the axl-/- mouse and, in fact ,a metastatic assay showed increased KPC liver metastases in axl-/- mice as compared to WT. In this assay, liver metastases were decreased in tyro3-/- and mertk-/- mice compared to WT mice. This contrasts with data from our lab and others showing that knock down of Axl in the tumor cells themselves diminishes metastatic potential in wild type mice. These data suggest that inhibition of Axl in host infiltrating cells and the tumor cells may have divergent actions in PDAC. Mechanistically, we utilized NanoString to identify significant changes in genes associated with antigen processing and presentation as well as an overall increase in immune response in mertk-/- mice. However, NanoString RNA expression data highlighted distinct signaling pathways in tumor bearing-tyro3-/- mice, again suggesting that the similar endpoint phenotypes (attenuated growth) are brought about by at least some non-redundant mechanisms. The growth attenuation in the mertk-/- and tyro3-/- is lost with antibody depletion of CD8a cells indicating the final common mechanism for MerTK or Tyro3 loss is CD8 cell dependent. These data highlight the fact that while these three receptors are often thought of as similar in action, deletion or inhibition may have distinct mechanisms or effects in the TME. These data also suggest that MerTK or Tyro3 targeted therapy in combination with anti-PD-1 merits exploration in PDAC; those experiments are ongoing with UNC-developed MerTK-selective inhibitors.
Citation Format: Nancy Kren, Alisha Holtzhausen, Douglas Graham, Yuliya Pylayeva-Gupta, H SHELTON EARP. Loss of MerTK and Tyro3, but not Axl, substantially reverses the immune-suppressive tumor microenvironment in a syngeneic pancreatic cancer model abstract. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B45.
The tumor-intrinsic NOD-, LRR- and pyrin domain-containing protein-3 (NLRP3) inflammasome-heat shock protein 70 (HSP70) signaling axis is triggered by CD8
T cell cytotoxicity and contributes to the ...development of adaptive resistance to anti-programmed cell death protein 1 (PD-1) immunotherapy by recruiting granulocytic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into the tumor microenvironment. Here, we demonstrate that the tumor NLRP3-HSP70 axis also drives the accumulation of PMN-MDSCs into distant lung tissues in a manner that depends on lung epithelial cell Toll-like receptor 4 (TLR4) signaling, establishing a premetastatic niche that supports disease hyperprogression in response to anti-PD-1 immunotherapy. Lung epithelial HSP70-TLR4 signaling induces the downstream Wnt5a-dependent release of granulocyte colony-stimulating factor (G-CSF) and C-X-C motif chemokine ligand 5 (CXCL5), thus promoting myeloid granulopoiesis and recruitment of PMN-MDSCs into pulmonary tissues. Treatment with anti-PD-1 immunotherapy enhanced the activation of this pathway through immunologic pressure and drove disease progression in the setting of
amplification. Genetic and pharmacologic inhibition of NLRP3 and HSP70 blocked PMN-MDSC accumulation in the lung in response to anti-PD-1 therapy and suppressed metastatic progression in preclinical models of melanoma and breast cancer. Elevated baseline concentrations of plasma HSP70 and evidence of NLRP3 signaling activity in tumor tissue specimens correlated with the development of disease hyperprogression and inferior survival in patients with stage IV melanoma undergoing anti-PD-1 immunotherapy. Together, this work describes a pathogenic mechanism underlying the phenomenon of disease hyperprogression in melanoma and offers candidate targets and markers capable of improving the management of patients with melanoma.
Tyro3, Axl, Mer (TAM) receptor tyrosine kinases reduce inflammatory, innate immune responses. We demonstrate that tumor-secreted protein S (Pros1), a Mer/Tyro3 ligand, decreased macrophage M1 ...cytokine expression in vitro and in vivo. In contrast, tumor cells with CRISPR-based deletion of Pros1 failed to inhibit M1 polarization. Tumor cell-associated Pros1 action was abrogated in macrophages from Mer- and Tyro3-but not Axl-KO mice. In addition, several other murine and human tumor cell lines suppressed macrophage M1 cytokine expression induced by IFN-gamma and LPS. Investigation of the suppressive pathway demonstrated a role for PTP1b complexing with Mer. Substantiating the role of PTP1b, M1 cytokine suppression was also lost in macrophages from PTP1b-KO mice. Mice bearing Pros1-deficient tumors showed increased innate and adaptive immune infiltration, as well as increased median survival. TAM activation can also inhibit TLR-mediated M1 polarization. Treatment with resiquimod, a TLR7/8 agonist, did not improve survival in mice bearing Pros1-secreting tumors but doubled survival for Pros1-deleted tumors. The tumor-derived Pros1 immune suppressive system, like PD-L1, was cytokine responsive, with IFN-gamma inducing Pros1 transcription and secretion. Inhibition of Pros1/TAM interaction represents a potential novel strategy to block tumor-derived immune suppression.
Abstract Checkpoint inhibitors, specifically anti–PD-1, have shown success in treating metastatic melanoma; however, some patients develop resistance. Dendritic cells (DCs) play a key role in ...initiating an immune response but in certain circumstances they become ineffective. We investigated the role of MerTK, a receptor tyrosine kinase responsible for myeloid cell clearance of dead cells, in the regulation of DC function and metabolism in the tumor microenvironment. Tumors resistant to anti–PD-1 exhibited increased levels of MerTK+ DCs. Treating wild-type DCs with apoptotic dead melanoma cells in vitro resulted in increased MerTK expression, elevated mitochondrial respiration and fatty acid oxidation, and reduced T-cell stimulatory capacity, all characteristics of dysfunctional DCs. In contrast, dead cells had only limited effect on the metabolism of MerTK-deficient DCs, which instead maintained an antigen presenting, stimulatory phenotype. The efficacy of anti–PD-1 to slow tumor progression and induce specific T-cell infiltration was markedly increased in mice with selective ablation of MerTK in the DC compartment, suggesting the possibility of therapeutically targeting MerTK to modulate DC metabolism and function and enhance anti–PD-1 therapy.
Cancers subvert the host immune system to facilitate disease progression. These evolved immunosuppressive mechanisms are also implicated in circumventing immunotherapeutic strategies. Emerging data ...indicate that local tumor-associated DC populations exhibit tolerogenic features by promoting Treg development; however, the mechanisms by which tumors manipulate DC and Treg function in the tumor microenvironment remain unclear. Type III TGF-β receptor (TGFBR3) and its shed extracellular domain (sTGFBR3) regulate TGF-β signaling and maintain epithelial homeostasis, with loss of TGFBR3 expression promoting progression early in breast cancer development. Using murine models of breast cancer and melanoma, we elucidated a tumor immunoevasion mechanism whereby loss of tumor-expressed TGFBR3/sTGFBR3 enhanced TGF-β signaling within locoregional DC populations and upregulated both the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in plasmacytoid DCs and the CCL22 chemokine in myeloid DCs. Alterations in these DC populations mediated Treg infiltration and the suppression of antitumor immunity. Our findings provide mechanistic support for using TGF-β inhibitors to enhance the efficacy of tumor immunotherapy, indicate that sTGFBR3 levels could serve as a predictive immunotherapy biomarker, and expand the mechanisms by which TGFBR3 suppresses cancer progression to include effects on the tumor immune microenvironment.
The mechanisms underlying tumor immunosurveillance and their association with the immune-related adverse events (irAEs) associated with checkpoint inhibitor immunotherapies remain poorly understood. ...We describe a metastatic melanoma patient exhibiting multiple episodes of spontaneous disease regression followed by the development of several irAEs during the course of anti-programmed cell death protein 1 antibody immunotherapy. Whole-exome next-generation sequencing studies revealed this patient to harbor a pyrin inflammasome variant previously described to be associated with an atypical presentation of familial Mediterranean fever. This work highlights a potential role for inflammasomes in the regulation of tumor immunosurveillance and the pathogenesis of irAEs.
Conventional dendritic cells (DCs) are essential mediators of antitumor immunity. As a result, cancers have developed poorly understood mechanisms to render DCs dysfunctional within the tumor ...microenvironment (TME). After identification of CD63 as a specific surface marker, we demonstrate that mature regulatory DCs (mregDCs) migrate to tumor-draining lymph node tissues and suppress DC antigen cross-presentation in trans while promoting T helper 2 and regulatory T cell differentiation. Transcriptional and metabolic studies showed that mregDC functionality is dependent on the mevalonate biosynthetic pathway and its master transcription factor, SREBP2. We found that melanoma-derived lactate activates SREBP2 in tumor DCs and drives conventional DC transformation into mregDCs via homeostatic or tolerogenic maturation. DC-specific genetic silencing and pharmacologic inhibition of SREBP2 promoted antitumor CD8
T cell activation and suppressed melanoma progression. CD63
mregDCs were found to reside within the lymph nodes of several preclinical tumor models and in the sentinel lymph nodes of patients with melanoma. Collectively, this work suggests that a tumor lactate-stimulated SREBP2-dependent program promotes CD63
mregDC development and function while serving as a promising therapeutic target for overcoming immune tolerance in the TME.
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