Project Summary Low-grade gliomas (LGGs), a heterogeneous group of primary central nervous system tumors, are one of the most common solid tumors in the adolescent and young adult (AYA) population. LGGs can progress to high- grade gliomas (HGGs) via a process known as malignant transformation (MT), resulting in dismal prognoses. The mechanisms driving MT of LGG remain poorly understood. In contrast to LGGs, we and others have shown that HGGs have increased levels of bone marrow-derived myeloid cells (BMDMs) and myeloid-derived suppressor cells (MDSCs) in peripheral circulation, with an intra-tumoral enrichment of tumor-associated macrophages (TAMs) and a paucity of CD8+T and natural killer (NK) cells. Although myeloid cells are known to accumulate during glioma progression, it remains unclear if these cells have a causal role in driving MT. Central hypothesis: Reductions in anti-tumor reactive CD8+T and NK cell-dependent immune surveillance are responsible for LGG to HGG malignant transformation in AYA patients and these reductions are dictated by increased infiltration and immune suppressive activity of tumor infiltrating BMDMs. We have identified two independent myeloid associated pathways that are responsible for these BMDM phenotypes and we propose to test the central hypothesis through the following two aims: Aim 1. Delineate the dependency of BMDM cell differentiation on inhibitor of DNA binding protein 2 (ID2). Using AYA RCAS/tv-a glioma mouse models, we will knockdown ID2 in BMDMs before and during MT. We will examine effects on pro-tumoral myeloid cells and evaluate intra-tumoral T and NK cells and mobilization in blood, spleen, and bone marrow at various times with scRNAseq, mass cytometry, and fluorescent-activated cell sorting. We will also leverage digital spatial transcriptomics (DSP) in ID2 modulated tumors and validate ID2 signaling in human paired LGG /HGG samples to dissect the expression of myeloid, NK, T cell markers, and checkpoint molecules to illuminate ROIs critical to MT. Results will illuminate ID2 mechanisms of BMDM cell differentiation to pro-tumoral myeloid cells. Aim 2. Determine if the CD74/macrophage migration inhibitory factor (MIF) axis regulates BMDM cell differentiation during LGG malignant transformation. We will investigate CD74’s role in MT using pharmacological treatments or transplant wild-type or CD74 KD/KO bone marrow cells in a murine RCAS/tv-a glioma model. We will analyze peripheral blood and the glioma TME using scRNAseq, mass cytometry, and FACS. We will validate findings with bulk RNA sequencing data on paired LGG/HGGs from AYA patients and investigate how CD74/MIF signaling drives reduced T and NK cells which supports LGG MT. We will also leverage DSP in CD74/MIF modulated tumors and validate CD74 signaling in human paired LGG/HGG samples to dissect the expression of myeloid, NK, T cell markers, and checkpoint molecules to illuminate ROIs critical to MT that will indicate potentially targetable vu...