RESUMEN
Background: Glioblastoma (GB) remains a formidable challenge in neuro-oncology, with immune checkpoint blockade (ICB) showing limited efficacy in unselected patients. We previously recently established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GB. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain poorly understood. Method: We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T cell responses and performed survival studies to validate the most relevant genes. Additionally, paired single cell RNA-sequencing (scRNA-seq) with p-ERK staining, spatial transcriptomics on GB samples, and ex-vivo slice culture of a BRAFV600E mutant GB tumor treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. Results: CRISPR/Cas9 screens identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to CD8+ T cells, and anti-PD-1 across all kinases. Experimentally-induced ERK phosphorylation in gliomas enhanced survival with ICB treatment, led to durable anti-tumoral immunity upon re-challenge and memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells correlated with increased interferon responses, antigen presentation and T cell infiltration in GB. Moreover, spatial transcriptomics and scRNA-seq analysis revealed the modulation of interferon responses by the MAPK/ERK pathway in BRAFV600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAFV600E mutant GB. Conclusion: Our data indicate that the MAPK/ERK pathway is a critical regulator of GB cell susceptibility to anti-tumoral immunity, modulating interferon responses, and antigen-presentation in glioma cells, as well as tumor cell interaction with microglia. These findings not only elucidate the mechanistic underpinnings of immunotherapy resistance in GB but also highlight the MAPK/ERK pathway as a promising target for enhancing immunotherapeutic efficacy in this challenging malignancy.
RESUMEN
Many vaccines, including those using recombinant antigen subunits, rely on adjuvant(s) to enhance the efficacy of the host immune responses. Among the few adjuvants clinically approved, QS-21, a saponin-based immunomodulatory molecule isolated from the tree bark of Quillaja saponaria (QS) is used in complex formulations in approved effective vaccines. High demand of the QS raw material as well as manufacturing scalability limitation has been barriers here. We report for the first-time successful plant cell culture production of QS-21 having structural, chemical, and biologic, properties similar to the bark extracted product. These data ensure QS-21 and related saponins are broadly available and accessible to drug developers.