Abstract Survival rates for acute lymphoblastic leukemia (ALL), the most common childhood cancer, are now close to 90%, but survivors of childhood cancer are at an increased risk for long term cognitive deficits, particularly affecting executive function (e.g., attention, planning, inhibitory control, cognitive flexibility). The chemotherapeutic agent methotrexate (MTX) is used to treat most ALL patients, and is closely associated with executive function deficits. Thus a pressing need exists to define the mechanisms that link MTX exposure to cognitive dysfunction, to guide development of intervention strategies to protect the developing brain, reduce symptoms and optimize quality of life in ALL survivors, during childhood, adolescence, young and full adulthood. To that end, we have developed a translationally relevant mouse model of leukemia survival that combines cancer exposure (mouse leukemic cell line (L1210 cells) with contemporary chemotherapeutic drugs (vincristine and MTX, with leucovorin rescue) administered during early life. PFC development extends through adolescence, which renders this area of the brain particularly vulnerable to early life chemotherapy. Providing a solid premise for the proposed experiments, our mouse model recapitulates executive function deficits observed in ALL patients. Additionally, in response to early life cancer + chemotherapy, we have found an increase in the proinflammatory molecules IL-1 and CCL2, as well as a decrease in white matter associated genes within the PFC. In Aim 1, single cell RNA sequencing will be used to define the effects of cancer and/or chemotherapy on the transcriptional profile of the PFC. MTX disrupts folate metabolism to inhibit cell growth, but this disruption also leads to increased levels of the proinflammatory metabolite, homocysteine (HCY), in both plasma and cerebrospinal fluid. Increased HCY can drive inflammation, oxidative stress and is associated with both white and gray matter damage, as well as cognitive impairment. Further executive function deficits have also been linked to altered synaptic function, and microglia, a brain resident immune cell, can contribute to synapse elimination via the CD11b(CR3)-C3 phagocytic pathway. Therefore, we hypothesize that MTX-driven increased HCY levels will lead to neuroinflammation and oxidative stress, leading to gray and white matter damage, and altered synaptic pruning in the prefrontal cortex, which underlie deficits in executive function. To test this hypothesis, HCY-lowering strategies (folate and B vitamin supplementation, or the antioxidant, N-acetylcysteine amide) will be evaluated in Aim 2. Aim 3 will test the necessity of IL-1 activity while Aim 4 will test the necessity of microglia in mediating the chemotherapy-associated cognitive deficits, as well as neuroinflammation and oxidative stress, leading to gray and white matter damage, and altered synaptic pruning in the prefrontal cortex