Abstract 5708: Dual targeting of PDPK1 and mutated BRAFV600E is synthetically lethal

Abstract Anaplastic thyroid cancer (ATC) exhibits near-uniform activation of the MAPK and PI3K/AKT/mTOR pathways, driving resistance to BRAF-targeted therapies. PDPK1, a key AGC-kinase activator downstream of PI3K, integrates multiple oncogenic and stress-response pathways and represents a critical resistance node. We investigated PDPK1 inhibition using BX795 alone and with BRAF inhibition (dabrafenib) in BRAFV600E mutant in vitro (8505C, SW1736), ex vivo (patient-derived ATC spheroids ATC-01, ATC-02), and orthotopic xenograft models. BX795 monotherapy reduced cellular proliferation and invasion, and in combination with dabrafenib produced strong synergistic anticancer activity (Combination Index <1), and led to ∼55% tumor volume reduction in vivo without toxicity. Mechanistically, dual blockade of PDPK1Ser241 and MEK/ERK phosphorylation, prevented the compensatory upregulation of PI3K/AKT and MAPK pathways seen with monotherapy. The synthetic lethality of dual targeting of PDPK1 and BRAFV600E was due to induction of extensive DNA damage (γ-H2AX↑, ATM/CHK2↑), G2/M cell-cycle arrest through suppression of CDC25C, CDK1, and cyclin A2, and triggering of mitochondrial hyperpolarization with impaired oxidative phosphorylation and increased ROS generation. Elevated mitochondrial ROS amplified DNA-damage signaling, culminating in BAD dephosphorylation, caspase-3 activation, and PARP cleavage. ROS scavengers (N-acetylcysteine, MitoQ) and CHK2 inhibition partially reversed apoptosis and cell cycle arrest, confirming a ROS-CHK2-dependent cell death mechanism. Together, these findings reveal that combined PDPK1 and BRAF inhibition is synthetically lethal in BRAFV600E-mutant cancer. PDPK1 represents a targetable vulnerability for enhancing BRAFV600E-targeted cancer therapy and in other MAPK/PI3K-coactivated cancers. Citation Format: Tejinder Pal Khaket, Chandrayee Gosh, Zhongyue Yang, Electron Kebebew, . Dual targeting of PDPK1 and mutated BRAFV600E is synthetically lethal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5708.