Background Melanoma remains a daunting clinical challenge. Immunotherapy remains the mainstay in treatment of metastatic disease. Yet, nearly 50% of patients fail to respond to immunotherapy, due to multiple potential mechanisms of ICI resistance but acquired and innate. One key mechanism of resistance is the suppressive nature of the tumor microenvironment (TME), driven in large part to the highly inflamed nature of melanoma metastases. Though beneficial to ICI efficacy, prolonged inflammation is co-opted by the tumor as a pro-growth factor and leads to localized immunosuppression. This is most pronounced in the TME of melanoma brain metastases (MBM). Our group has previous shown that a key marker of this inflammation is the myddosomal pathway and specifically IRAK-4 activation. We have previously shown that inhibition of IRAK-4 through the oral small molecule inhibitor CA-4948 in murine models of melanoma increased tumor infiltrating lymphocyte penetration into tumors and when given in combination with anti-PD-1 therapy improves overall survival over anti-PD-1 therapy alone. In this current study we show this is driven in large part to changes in tumor associated macrophage phenotype and activation in the TME, with decreased PD-L1 expression and phenotypic skewing to an M1 phenotype by flow cytometry. These data support the combination of CA-4948 with ICI therapy as a novel strategy in overcoming ICI resistance.

Methods B16.F10 cells were implanted into the flank of C57BL/6 mice followed 3 days post implant with intracranial implantation by stereotaxis. Following tumor establishment for 5 days mice were started on treatment with either oral CA-4948 (100mg/kg) qD, anti-PD-1 therapy (10mg/kg) q72hrs, a combination of both agents, or an oral vehicle control plus IgG control and followed for 7 days prior to euthanasia. Flank and intracranial tumors were then removed and flow cytometry performed for mouse M1/M2 phenotyping and PD-L1 expression.

Results Mice treated with CA-4948 plus anti-PD-1 therapy have increased expression of macrophages in the TME displaying a classical M1 phenotype vs either monotherapy alone and vehicle control treated mice. CA-4948 treatment also downregulated PD-L1 expression in the TME in both flank and MBM.

Conclusions IRAK-4 inhibition through the oral small molecule inhibitor CA-4948 reduces TME expression of PD-L1 and alters TME associated macrophage phenotype skewing to the activate M1 phenotype, contributing to an improved anti-tumor response to ICI therapy in murine models of melanoma metastases including MBM.

Ethics Approval All experiments were performed following approval and in accordance with the policies and procedures of the University of Florida IACUC.