OPN is a multifunctional phosphoglycoprotein expressed in a wide range of cells, including osteoclasts, osteoblasts, neurons, epithelial cells, T, B, NK, NK T, myeloid, and innate lymphoid cells. OPN plays an important role in diverse biological processes and is implicated in multiple diseases such as cardiovascular, diabetes, kidney, proinflammatory, fibrosis, nephrolithiasis, wound healing, and cancer. In cancer patients, overexpressed OPN is often detected in the tumor microenvironment and elevated serum OPN level is correlated with poor prognosis. Initially identified in activated T cells and termed as early T cell activation gene, OPN links innate cells to adaptive cells in immune response to infection and cancer. Recent single cell RNA sequencing revealed that OPN is primarily expressed in tumor cells and tumor-infiltrating myeloid cells in human cancer patients. Emerging experimental data reveal a key role of OPN is tumor immune evasion through regulating macrophage polarization, recruitment, and inhibition of T cell activation in the tumor microenvironment. Therefore, in addition to its well-established direct tumor cell promotion function, OPN also acts as an immune checkpoint to negatively regulate T cell activation. The OPN protein level is highly elevated in peripheral blood of human cancer patients. OPN blockade immunotherapy with OPN neutralization monoclonal antibodies (mAbs) thus represents an attractive approach in human cancer immunotherapy.
Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16–25.91 μM for human ASAH2 and 18.6–30.2 μM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.
Human colorectal cancers are mostly microsatellite-stable with no response to anti-PD-1 blockade immunotherapy, necessitating the development of a new immunotherapy. Osteopontin (OPN) is elevated in human colorectal cancer and may function as an immune checkpoint. We aimed at elucidating the mechanism of action of OPN and determining the efficacy of OPN blockade immunotherapy in suppression of colon cancer. We report here that OPN is primarily expressed in tumor cells, myeloid cells, and innate lymphoid cells in human colorectal carcinoma. Spp1 knock out mice exhibit a high incidence and fast growth rate of carcinogen-induced tumors. Knocking out Spp1 in colon tumor cells increased tumor-specific CTL cytotoxicity in vitro and resulted in decreased tumor growth in vivo. The OPN protein level is elevated in the peripheral blood of tumor-bearing mice. We developed four OPN neutralization monoclonal antibodies based on their efficacy in blocking OPN inhibition of T cell activation. OPN clones 100D3 and 103D6 increased the efficacy of tumor-specific CTLs in killing colon tumor cells in vitro and suppressed colon tumor growth in tumor-bearing mice in vivo. Our data indicate that OPN blockade immunotherapy with 100D3 and 103D6 has great potential to be further developed for colorectal cancer immunotherapy and for rendering a colorectal cancer response to anti-PD-1 immunotherapy.
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