bioRxiv. 2024 Sep 18. pii: 2024.09.11.612571. [Epub ahead of print]
Kwang-Soo Kim,
Junyi Zhang,
Víctor A Arrieta,
Crismita Dmello,
Elena Grabis,
Karl Habashy,
Joseph Duffy,
Junfei Zhao,
Andrew Gould,
Li Chen,
Jian Hu,
Irina Balyasnikova,
Dhan Chand,
Dan Levey,
Peter Canoll,
Wenting Zhao,
Peter A Sims,
Raul Rabadan,
Surya Pandey,
Bin Zhang,
Catalina Lee-Chang,
Dieter Henrik Heiland,
Adam M Sonabend.
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 BRAF V600E 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 BRAF V600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAF V600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAF V600E 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.