BP4. Dasatinib reverses myeloid derived suppressor cell (MDSC) inhibition of T-cell proliferation in a triple negative breast cancer model

MDSC are expanded in cancer patients and inhibit immunity via the release of nitric oxide (NO) and reactive oxygen species. MDSC levels are associated with failure of immune checkpoint blockade. We investigated if dasatinib, an FDA-approved small-molecule tyrosine kinase inhibitor could disrupt MDSC NO production and reinvigorate immune responses.


Methods:

Expression of genes related to nitric oxide production, inflammation, immunosuppression (Nos2, Cox2, and IL-6) were measured by PCR in myeloid cell lines (RAW macrophages and MSC2) and in splenic MDSC from tumor-bearing mice or in melanoma patient peripheral blood. MDSC NO output was measured with the Greiss reagent. Dasatinib’s ability to reverse MDSC suppression of T cell proliferation was measured via flow cytometry of activated, fluorescent-labeled T cells. The murine triple negative breast cancer cell (TNBC) line 4T1 was injected into the mammary glands of BALB/c mice and dasatinib (20 mg/kg/day via oral gavage) was given for 8 days. Levels of tumor-infiltrating lymphocytes and myeloid cells were measured by spectral flow cytometry.


Results: Dasatinib treatment significantly reduced mRNA expression of Nos2, Cox2, and IL-6 expression by RAW macrophages (P=0.0014, P=0.001, P< 0.001) and the MDSC-like cell line MSC2. Similar trends were seen in MDSC from the blood of patients with stage IV melanoma. Dasatinib significantly reduced MDSC NO output. Murine CD8+ T cell proliferation was significantly inhibited in the presence of MDSC in a co-culture experiment. But 24 hr pre-treatment of MDSC with 100 nM dasatinib reversed this inhibition compared to DMSO controls (44.43% vs 21.43%, P=0.0141). This effect was significant in CD8+ T cells but not in CD4+ T cells. Compared to controls, expression of Nos2, Cox2, and IL-6 was significantly reduced in splenic MDSC from dasatinib-treated 4T1 tumor bearing mice (P=0.020, P=0.044, P=0.035, respectively). Dasatinib reduced 4T1 tumor growth. Flow cytometric analysis of intra-tumoral immune cells revealed significantly fewer monocytic-like MDSC (P=0.0083), PMN-like MDSC (P< 0.001), and pro-tumor CD206+ M2 macrophages relative to anti-tumor CD206- M1 macrophages (P< 0.001) as compared to vehicle control. Levels of CD8+ T cells were unaffected by dasatinib.


Conclusions: Dasatinib modulates MDSC function and reduces their presence in the tumor microenvironment. This strategy could reverse immune suppression in the setting of TNBC checkpoint inhibitor therapy.