PROJECT SUMMARY: Glioblastoma (GBM) is the most common primary malignant brain tumor, with a median survival of up to 20 months. Males have a 1.6-fold higher incidence of GBM compared to females and worse disease outcomes. Immunotherapies, which are currently in clinical trials, have had limited success in improving patient outcomes. An immunosuppressive microenvironment facilitating tumor progression and restricting anti- tumor immune response likely underlies therapeutic resistance. Although the accumulation of myeloid-derived suppressor cells (MDSCs) plays a critical role in the establishment of this immunosuppressive milieu, the mechanisms by which individual MDSC subsets promote tumorigenesis and are regulated remain poorly defined. We previously demonstrated that monocytic MDSCs (mMDSCs) infiltrated male tumors at higher rates in preclinical models and patient specimens. In contrast, granulocytic MDSCs (gMDSCs) expand in the peripheral circulation of female animal models and the gMDSC gene signature is associated with poor prognosis in female patients. This variation in MDSC subsets also informed sex-specific therapeutic responses to fludarabine and anti-IL-1β in preclinical models. More recently, we observed that mMDSCs and gMDSCs have a distinct epigenetic landscape, which is also informed by biological sex. While this highlights the potential role of epigenetic regulation of MDSC subset activity, there is limited insight into the mechanisms driving distinct MDSC subset functions. Our preliminary results suggested that the complement pathway could be one such mechanism as complement component 1q (C1q) was highly expressed by gMDSCs and elevated in females. Based on these observations, we hypothesize that epigenetic regulation of C1q informs sex-specific behavior of MDSC subsets and epigenetic reprogramming of MDSCs will improve the efficacy of T cell-activating strategies. Specific Aim 1 will test the hypothesis that the unique epigenetic signatures of MDSC subsets make them susceptible to different histone modifiers that can be combined with checkpoint modulators. Sub-Aim 1A will examine the efficacy of histone lysine demethylase inhibitors on MDSC activity in vitro and in vivo, while Sub-Aim 1B will attempt to achieve durable anti-tumor immune response by combining lead inhibitors with anti-PD-1, anti-CTLA- 4, and anti-OX40. Specific Aim 2 will test the hypothesis that the C1q locus is differentially accessible between male versus female MDSC subsets. Sub-Aim 2A will use ATAC-seq and CUT & RUN to evaluate the epigenetic landscape of complement proteins and specific histone mark occupancy based on the differential histone lysine demethylase expression profile. Sub Aim 2B will use pharmacological inhibitors to test the sex-specific effect of histone lysine demethylases on complement regulation. These studies lay the foundation for the development of epigenetic modifiers for GBM immunotherapies by addressing variations in anti-tumor immuni...