Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens.

Background The receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR) is overexpressed and an important therapeutic target in Head and Neck cancer (HNC). Cetuximab is currently the only EGFR-targeting agent approved by the FDA for treatment of HNC; however, intrinsic and acquired resistance to cetuximab is a major problem in the clinic. Our lab previously reported that AXL leads to cetuximab resistance via activation of HER3. In this study, we investigate the connection between AXL, HER3, and neuregulin1 (NRG1) gene expression with a focus on understanding how their interdependent signaling promotes resistance to cetuximab in HNC. Methods Plasmid or siRNA transfections and cell-based assays were conducted to test cetuximab sensitivity. Quantitative PCR and immunoblot analysis were used to analyze gene and protein expression levels. Seven HNC patient-derived xenografts (PDXs) were evaluated for protein expression levels. Results We found that HER3 expression was necessary but not sufficient for cetuximab resistance without AXL expression. Our results demonstrated that addition of the HER3 ligand NRG1 to cetuximab-sensitive HNC cells leads to cetuximab resistance. Further, AXL-overexpressing cells regulate NRG1 at the level of transcription, thereby promoting cetuximab resistance. Immunoblot analysis revealed that NRG1 expression was relatively high in cetuximab-resistant HNC PDXs compared to cetuximab-sensitive HNC PDXs. Finally, genetic inhibition of NRG1 resensitized AXL-overexpressing cells to cetuximab. Conclusions The results of this study indicate that AXL may signal through HER3 via NRG1 to promote cetuximab resistance and that targeting of NRG1 could have significant clinical implications for HNC therapeutic approaches.

Background The tyrosine kinase receptors Axl and MerTK are highly overexpressed in head and neck cancer (HNC) cells, where they are critical drivers of survival, proliferation, metastasis, and therapeutic resistance. Methods We investigated the role of Axl and MerTK in creating an immunologically “cold” tumor immune microenvironment (TIME) by targeting both receptors simultaneously with a small molecule inhibitor of Axl and MerTK (INCB081776). Effects of INCB081776 and/or anti‐PDL1 on mouse oral cancer (MOC) cell growth and on the TIME were evaluated. Results Targeting Axl and MerTK can reduce M2 and induce M1 macrophage polarization. In vivo, INCB081776 treatment alone or with anti‐PDL1 appears to slow MOC tumor growth, increase proinflammatory immune infiltration, and decrease anti‐inflammatory immune infiltration. Conclusions This data indicates that simultaneous targeting of Axl and MerTK with INCB081776, either alone or in combination with anti‐PDL1, slows tumor growth and creates a proinflammatory TIME in mouse models of HNC.

The tyrosine kinase receptors Axl and MerTK, known for their role on macrophages in regulating clearance of apoptotic cells, are highly overexpressed in head and neck cancer (HNC). Previous studies in our laboratory have shown that Axl is a critical driver of survival, proliferation, metastasis, and therapeutic resistance in HNC, and that MerTK is functionally redundant to Axl. In this study, we investigated the cooperative role of Axl and MerTK in creating an immunologically cold tumor immune microenvironment (TIME) by targeting both receptors simultaneously with a small molecule inhibitor of Axl and MerTK (INCB081776). Because Axl and MerTK are expressed on both macrophages and HNC cancer cells, we examined the effect of INCB081776 treatment on each cell type. In macrophages, Axl and MerTK signaling leads to M2-type polarization, an anti-inflammatory state that leads to the resolution of inflammation and, in cancer settings, promotes tumor growth. Our experiments suggest that treatment with INCB081776 can reduce M2 polarization and increase M1-type polarization, a pro-inflammatory state that promotes inflammation and tumor cell killing. Next, to determine the efficacy of INCB081776 on HNC cancer cells, mouse oral cancer (MOC) tumors were implanted in syngeneic mice and treated with INCB081776 alone or in combination with a monoclonal antibody against PDL1 (anti-PDL1), thereby mimicking current standard-of-care immune checkpoint inhibitor treatment. The results showed a marked effect of INCB081776 single-agent treatment on MOC tumor growth and an increase in several pro-inflammatory cell types (M1 macrophages, CD8+ T cells, total infiltrating leukocytes), suggesting that INCB081776 treatment can create an immunologically hot TIME. Further, in-depth analysis of tumor infiltrating leukocytes following INCB081776 treatment in both immunologically hot (MOC1) and cold (MOC2) HNC tumors suggested that INCB081776 has a greater effect in cold tumors. In cold tumors, levels of pro-inflammatory cells (CD8+ T cells, M1 macrophages, etc.) increased, and levels of anti-inflammatory cells (M2 macrophages, regulatory T cells, etc.) decreased, both to a greater extent than in hot tumors. Finally, the combination of INCB081776 and anti-PDL1 was superior to either treatment alone in slowing tumor growth. Together, these studies indicate that INCB081776 cooperates with anti-PDL1 in a syngeneic mouse model of HNC to slow tumor growth and create a pro-inflammatory environment, especially in immunologically cold tumors, thereby highlighting the potential clinical benefit of this therapeutic combination. Citation Format: Kourtney L. Kostecki, Mari Iida, Anne L. Wiley, Seungpyo Hong, Ravi Salgia, Paul M. Harari, Deric L. Wheeler. Simultaneous inhibition of Axl and MerTK enhances anti-PDL1 efficacy and creates a pro-inflammatory tumor immune microenvironment in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3535.

Triple negative breast cancer (TNBC) cells lack estrogen, progesterone, and HER2 receptors. TNBC tends to be more aggressive than most types of breast cancer and is more likely to recur even after surgery and treatment. Recently, a few targeted therapies were approved for TNBC, but additional molecular targeting agents are still needed. Previous studies have found that activated MerTK is associated with many types of human cancers, including breast cancer. In this study, we focused on MerTK expression and its tumorigenesis and immunoregulatory functions in TNBC. We first examined MerTK expression levels in human TNBC tissue samples by analyzing tissue microarrays (TMA) using immunohistochemistry (IHC). IHC revealed that ~40% of TNBC samples expressed high levels of MerTK. We further examined MerTK expression levels in ten TNBC cell lines by immunoblot analysis. Three cell lines (BT549, MDAMB231 and MDAMB436) showed strong MerTK expression levels. Next, to evaluate MerTK’s effect on cell proliferation, we treated MDAMB231 cells with MerTK siRNA, which inhibited cell proliferation by 20% compared to siNon-target (siNT). These data indicate that TNBC cells remain dependent on MerTK-activated signaling pathways for proliferation and survival. To further investigate the impact of MerTK expression in TNBC cells, we stably overexpressed MerTK in the TNBC cell line SUM102, which naturally has very low MerTK levels. The results indicated that overexpression of MerTK in SUM102 cells led to: 1) increased proliferation in vitro 2) robust tumor growth, 3) marked migration potential and 4) increased metastasis in mice. Collectively, these results demonstrate that activation of a MerTK-driven pathway could be involved in tumorigenesis in TNBC. To understand the role of MerTK in cell signaling and pro-tumor immunity, we utilized a cytokine array to examine which molecules were impacted in MerTK-overexpressing SUM102 cells. The results showed that production of CXCL4, CD74, and IL-1α was increased, and production of CXCL10 was decreased in MerTK-overexpressing SUM102 cells as compared to control cells. Interestingly, PD-L1 was upregulated and MHC class I was downregulated in MerTK-overexpressing SUM102 cells as compared to control cells. These preliminary findings suggested that MerTK-overexpressing TNBC could be creating a favorable immune microenvironment for proliferation of tumor cells. To further explore the cytokine, PD-L1, and MHC class I results, we overexpressed MerTK in 4T1 murine TNBC-like cells. Immunoblot analysis showed that PD-L1 was upregulated in MerTK-overexpressing 4T1 cells. We will utilize this cell line to evaluate tumor growth and immunosuppression in the tumor microenvironment in BALB/C mice. Citation Format: Mari Iida, Kourtney Kostecki, Carlene A. Kranjac, Christine Glitchev, David T. Yang, Deric L. Wheeler. MerTK tumorigenesis and immune evasion mechanisms in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1334.