Glioblastoma (GBM) is notorious for its immunosuppressive tumor microenvironment (TME) and is refractory to immune checkpoint blockade (ICB). Here, we identify calmodulin-dependent kinase kinase 2 (CaMKK2) as a driver of ICB resistance. CaMKK2 is highly expressed in pro-tumor cells and is associated with worsened survival in patients with GBM. Host CaMKK2, specifically, reduces survival and promotes ICB resistance. Multimodal profiling of the TME reveals that CaMKK2 is associated with several ICB resistance-associated immune phenotypes. CaMKK2 promotes exhaustion in CD8+ T cells and reduces the expansion of effector CD4+ T cells, additionally limiting their tumor penetrance. CaMKK2 also maintains myeloid cells in a disease-associated microglia-like phenotype. Lastly, neuronal CaMKK2 is required for maintaining the ICB resistance-associated myeloid phenotype, is deleterious to survival, and promotes ICB resistance. Our findings reveal CaMKK2 as a contributor to ICB resistance and identify neurons as a driver of immunotherapeutic resistance in GBM.

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Objective Glioblastoma (GBM) is the most lethal primary brain tumor in adults. Although immunotherapy has been successful in some cancers, it has shown limited success in GBM. This is partially due to poor T cell infiltration, and because many T cells found in tumors are either exhausted or Tregs. Re-analysis of publicly available data has shown that CaMKK2 is more highly expressed in advanced brain tumors, and high CaMKK2 expression results in worse survival outcomes in GBM. Additionally, in survival studies that we performed, CaMKK2−/− mice showed an impressive survival phenotype that was dependent on the presence of CD8+ tumor infiltrating lymphocytes (TILs). The objective of our study was to investigate what is different about T cells found in the TME of CaMKK2−/− mice, and if this can explain the observed survival benefit. Methods We performed flow cytometry to determine the identities of T cells found in the tumors of CaMKK2−/− and WT mice. Results/Conclusions Our experiments show that there was a greater percentage of CD4+ TILs relative to CD8+ TILs in CaMKK2−/− mice, and this increase in percentage was due to increased accumulation of CD4+ TILs. Additionally, we found that there was greater accumulation of CD4+ and CD8+ effector memory TILs and a greater ratio of effector memory TILs to Tregs in CaMKK2−/− mice. The increased accumulation of the CD8+ effector memory TILs is particularly interesting because it is not simply explained by an increased accumulation of cells, as is the case with the CD4+ TILs. The increased accumulation of CD4+ TILs and greater ratio of effector memory TILs to Tregs is expected to have an anti-tumor effect. These findings make CaMKK2 a promising target to treat GBM.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults, with the median survival time for GBM patients being only about 15 months. Although immunotherapy treatments have been successful in more immunogenic tumors, it has shown very limited success in GBM. This could be because it is difficult for T cells to infiltrate the tumor, and the tumors have a very immunosuppressive tumor microenvironment (TME) that can often consist of up to about 30 percent tumor associated macrophages (TAMs) by mass. Thus, two ways of possibly improving immunotherapy treatments on GBMs would be to increase the number of tumor infiltrating lymphocytes (TILs) present, and to target tumor-supportive cells such as TAMs. Calmodulin Dependent Protein Kinase Kinase 2 (CaMKK2) is a gene that has been shown to be highly expressed in myeloid cells and is known to alter their phenotype. Recent studies in breast cancer have shown that myeloid cells adopt a more immunostimulatory phenotype in murine knockout CaMKK2 models. However, it is not well understood how CaMKK2 causes this immunosuppressive effect and warrants further exploration. The data from our experiments help to answer this question. High-dimensional flow cytometry with 15 parameters was used in order to determine the identities of immune cells found in the tumor of wildtype and CaMKK2-/- mice. The results of our experiments show that there appear to be more CD4+ TILs present in the TME of knockout mice and that there is an increase in MHC II expression on TAMs found in knockout mice. Since CD4+ TILs recognize peptides presented on MHC II molecules, and there is an increase in the number of CD4+ TILs and expression of MHC II on TAMs in knockout mice, we hypothesize that a stronger immune response could be instigated against the tumor. Therefore, it can be concluded that a possible reason that knocking out CaMKK2 leads to a less immunosupressive TME is because MHC II expression on TAMs is upregulated, and the number of CD4+ TILs in the TME is increased. Whether or not the increase in MHC II expression causes the increase in the number CD4+ TILs requires further exploration. This makes CaMKK2 a promising therapeutic target to treat tumors with immunosuppressive TMEs such as GBM. Citation Format: Molly A. Chakraborty, William H. Tomaszewski, Jessica Waibl Polania, Lauren S. Riley, Luigi Racioppi, Luis A. Sanchez-Perez, John H. Sampson. CaMKK2 knockout in mice challenged by orthotopic GBM leads to upregulation of MHC II on TAMs and an increase in accumulation of CD4+ TILs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2667.