Khaket, T. P.; Gosh, C.; Yang, Z.; Myriem, M. B.; Hu, J.; Alamaw, E. D.; O'Neill, M.; Andresson, T.; Zhang, Y.-Q.; Shen, M.; Haileselassie, B.; Kebebew, E.

PDPK1 functions downstream of PI3K and is essential for activating AKT and other AGC kinases. Although PDPK1 has a central role in the PI3K/AKT/mTOR signaling pathway, there has been limited evaluation of it as a target for cancer therapy. Anaplastic thyroid cancer (ATC) has one of the highest mortality rates of all human malignancies. Although combined BRAF and MEK inhibition in BRAF V600E mutant ATC (45% of cases) results in response, resistance is common, and there is no curative treatment. The majority (up to 95.8%) of ATC cases have activation in the PI3K/AKT/mTOR and RAS/RAF/MEK/MAPK pathways due to genetic alterations (including driver mutations and genomic gains or losses), involved in these pathways. In this study, we investigated PDPK1 as a therapeutic target for ATC. We used in vitro, ex vivo, and in vivo ATC models to evaluate the effect of targeting PDPK1 (BX795) alone and in combination with mutated BRAF V600E inhibition (dabrafenib), and the mechanism of action that resulted in ATC cell death. BX795 monotherapy significantly reduced ATC cell proliferation, invasion, colony formation, and spheroid size. The combination of BX795 with dabrafenib produced strong synergistic antitumor activity in BRAF V600E mutant ATC models. Dual inhibition led to simultaneous and sustained suppression of PDPK1/AKT and MAPK signaling, preventing the compensatory pathway reactivation observed with single-agent treatment. This integrated blockade induced pronounced oxidative stress, DNA damage, and G2-phase cell-cycle arrest, accompanied by mitochondrial dysfunction and robust activation of apoptotic cascades. These effects translated into marked tumor regression in in vitro, ex vivo, and in vivo experimental systems. Our findings identify PDPK1 as a critical and therapeutically tractable vulnerability in anaplastic thyroid cancer. Co-targeting PDPK1 and BRAF V600E produces potent synergistic antitumor activity by shutting down convergent oncogenic signaling nodes and amplifying apoptotic stress responses. These data support PDPK1 inhibition alone and in combination with BRAF blockade acts as a promising strategy to improve outcomes for patients with BRAF V600E mutant ATC.