Dual targeting of interlocked PTK2B and MAPK signaling triggers synergistic ferroptosis via autophagic flux disruption in BRAFV600E glioma.

BACKGROUND

BRAFV600E mutation is one of the most common oncogenic drivers in gliomas, sharing the highest incidence in pediatric low-grade gliomas (~ 20%) and being frequently associated with poor prognosis. Although combination therapies targeting both BRAFV600E and downstream MAPK signaling have been developed, their efficacy is substantially limited by acquired drug resistance. Consequently, identifying the underlying mechanisms of resistance and novel therapeutic vulnerabilities remains an urgent need.

METHODS

Using patient gene expression profiles, immunohistochemistry on glioma samples, glioma cell lines, and a Drosophila glioma model, we identified Protein Tyrosine Kinase 2 Beta (PTK2B) as a druggable vulnerability in BRAFV600E glioma. Loss-of-function studies were achieved by PTK2B knockdown in DBTRG-05MG cells and Fak knockout in Drosophila, assessing effects on tumor survival/proliferation. The underlying signaling mechanisms were investigated using RNA sequencing, proteomics and a suite of assays (e.g., Western blot, immunofluorescence, flow cytometry). Finally, the reciprocal compensatory mechanism between PTK2B and MAPK signaling was established in both cellular and Drosophila models, and the synergistic effect of their co-inhibition was validated in DBTRG-05MG and AM-38 cells.

RESULTS

PTK2B and its Drosophila orthologue Focal adhesion kinase (Fak) are highly expressed in BRAFV600E/dRafGOF glioma, and their inhibition significantly suppresses tumor growth. Our studies revealed that PTK2B knockdown triggers potent ferroptosis in glioma cells through endoplasmic reticulum stress-induced autophagic flux disruption, a mechanism which is partially overlapped with the cell death induced by MAPK signaling inhibition. We uncovered the interlocking mechanism between highly expressed PTK2B and hyperactive MAPK signaling in BRAFV600E glioma, wherein suppression of either one prompts compensatory upregulation of the other, thereby attenuating tumor cell death under the stress. Simultaneous inhibition of PTK2B and MAPK signaling achieves strong synergistic anti-tumor effect, highlighting the therapeutic promise of this combination strategy.

CONCLUSIONS

Our findings establish PTK2B as a critical regulator and co-targetable vulnerability in BRAFV600E glioma. We delineate a novel interlocking mechanism wherein PTK2B and MAPK engage in reciprocal negative feedback regulation, enabling adaptive resistance. This work provides a compelling mechanistic rationale for co-targeting PTK2B and MAPK to disrupt this survival axis and overcome therapeutic resistance.