SUMMARY The brain tumor microenvironment (TME) is dominated by myeloid cell infiltrates which are mostly brain resident microglia and infiltrating glioblastoma-associated myeloid cells (GAMs). Despite this fact, most clinical trials for glioblastoma have been directed to T cell-based immunotherapies, which have failed to impact outcome for most patients. Targeting glioblastoma based on re-education of GAMS to enable immunological clearance is the goal of this clinical trial. The signal transducer and activator of transcription 3 (STAT3) is a negative regulator of both innate and adaptive immunity and this pathway is markedly up regulated in the TME. We have previously developed and tested, in the context of Phase I clinical trials, a first-in-man blood-brain-barrier penetrant small molecule inhibitor of STAT3, designated WP1066. The Phase I trial results have shown that WP1066 is well tolerated and is inhibiting the STAT3 target in immune cells. As a monotherapy, WP1066 treatment has demonstrated the ability to stimulate robust anti-tumor response in multiple preclinical studies that is further enhanced with radiation therapy, which is standard of care for newly diagnosed glioblastoma and is known to increase tumor immunogenicity. Unbiased nanostring analysis of treated tumors revealed that activation of antigen presentation in the TME with the combination treatment. For the research proposed here we want to extend our preclinical findings to newly diagnosed glioblastoma patients. In this clinical trial, we will test the hypothesis that the administration of WP1066 with radiation will induce T cell-dendritic cell cluster interactions in the glioblastoma microenvironment, and this induction, in turn, will increase progression free survival (PFS) for glioblastoma patients. In specific Aim 1, we conduct a Phase II trial with an expansion cohort for the combination of WP1066 and radiation in newly diagnosed MGMT unmethylated glioblastoma patients. In cohort 1, newly diagnosed MGMT unmethylated, IDH1 wild-type glioblastoma patients will be treated with 8 mg/kg of WP1066 while undergoing standard-of-care radiation therapy at a daily dose of 2 Gy. PFS will be used to ascertain if there is indication of treatment benefit. In cohort 2, patients for whom gross total resection was not achieved will be treated with the combination of radiation and WP1066 with the intent of obtaining treated tumor, if a surgically amenable lesion is present. Such tissue would allow for analysis of TME immune activation and cluster interactions, drug concentrations and target engagement in Aim 2. Aim 2 will also use longitudinal textural MRI analysis to correlate inflammatory responses in the TME by using our STAT3-specific multiplex immune fluorescent panel and complementary ex vivo flow cytometry and nanostring profiling. STAT3 target inhibition, including within specific immune cell populations, will be ascertained through use of the multiplex panel, which will also inform ...