(AS1) Large Tumor Suppressor Kinase 2, a Key Mapk-associated Effector of the Hippo Pathway, Modulates Response to anti-pd-1 Immunotherapy in Glioblastoma

Introduction: The immunosuppressive properties of the tumor microenvironment in glioblastoma (GBM) have posed a significant challenge to the use of anti-PD-1 immunotherapy (aPD-1) as an efficacious treatment strategy for GBM patients. Previously, we reported that GBMs responsive to PD-1 blockade exhibit mitogen-activated protein kinase (MAPK) pathway activation. Here, we investigate downstream targets of MAP kinases and hypothesize that inhibition of the large tumor suppressor kinase 2 (LATS2), a key MAPK-associated kinase modulator of the Hippo pathway, enhances the immune response of aPD-1 in these brain tumors.

Methods: We employed an in vivo kinome CRISPR knock-out screen in immunocompetent mice with intracranial implantation of glioma cells that were subsequently treated with aPD-1. In this model, we analyzed the depletion and enrichment of single-guide RNAs (sgRNAs) for each gene in the kinome following PD-1 blockade. Furthermore, single-cell RNA sequencing data of human GBMs were analyzed to evaluate the cell populations contributing to the expression of the depleted/enriched genes found in the screen.

Results: The in vivo kinome knock-out CRISPR screen identified LATS2 as a glioma-intrinsic, MAPK-associated kinase that confers resistance to aPD-1 in gliomas. Administration of aPD-1 to glioma-bearing C57BL/6 mice demonstrated a significant depletion of sgRNAs targeting LATS2 from day 0 to day 74, an advantage that was not seen in mice with CD8 knock-out, suggesting a CD8+ T-cell mediated selection in glioma-bearing mice with intact adaptive and cellular immunity. Furthermore, single-cell RNA sequencing analysis of 28 GBM patients demonstrated distinct phenotypes associated with differential LATS2 expression in the glioma cell population.

Conclusion : This study demonstrates that loss of LATS2, a key MAPK-associated kinase effector of the Hippo pathway, enhances the response of gliomas to aPD-1 immunotherapy.