Authors
Yang Zhang, Jun Ma, Hua Wang, Pan Gao, Yang Han, Peipei Li, Changqing Zhen, Zhumei Zhan, Kang Lu, Xianghua Wang, Wenbo Zhao, Dongwei Kang, Xiaosheng Fang, Ya Zhang.
Background
Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous malignancy characterized by altered metabolic dependencies, including an increased reliance on fatty acid oxidation (FAO). While the oncogenic role of KRAS mutations is well-established in solid tumors, their functional impact in CLL remains unclear. The G13D mutation in KRAS (KRAS^G13D) is particularly understudied in hematologic malignancies. The present study aimed to elucidate the functional consequences of KRAS^G13D in CLL pathogenesis, focusing on its regulation of fatty acid metabolism via the PI3K/AKT/mTOR pathway, and to assess the therapeutic efficacy of co-targeting KRAS and PI3K.
Methods
KRAS mutation patterns were analyzed in 3,205 CLL patients who underwent whole-genome or next-generation. Functional studies were performed by transfecting CLL cell lines (MEC1, EHEB) and primary CLL cells with KRAS^WT or KRAS^G13D plasmids. Cell proliferation, FAO, and mitochondrial respiration were evaluated with functional assays. Molecular mechanisms were investigated using transcriptomic profiling, qRT-PCR, Western blotting, and bioinformatics. A direct protein interaction was validated through molecular docking and co-immunoprecipitation. Drug sensitivity to KRAS (BI-2865) and PI3K (MIPS-21335, idelalisib) inhibitors was tested to assess combinatorial treatment strategies.
Results
Among 3,205 CLL patients, 119 (3.71%) harbored KRAS mutations. Notably, KRAS^G13D accounted for 29.13% of all KRAS-mutant cases, making it the most prevalent variant. Functional assays demonstrated that KRAS^G13D significantly enhanced proliferation in MEC1, EHEB, and primary CLL cells compared to KRAS^WT controls (p < 0.05). KRAS^G13D-expressing cells also exhibited increased sensitivity to the pan-KRAS inhibitor BI-2865, with dose-dependent suppression of proliferation. Transcriptomic and biochemical analyses revealed that KRAS^G13D upregulated key fatty acid oxidation genes, including CPT1A, CPT2, and ACOX1, while increasing cellular oxygen consumption and FAO activity (p < 0.01). Mechanistically, KRAS^G13D induced significant upregulation of the class II PI3K gene PIK3C2A, with concurrent activation of AKT and mTOR signaling components at both total and phosphorylated protein levels. Bioinformatic enrichment highlighted strong associations with lipid metabolism and PI3K binding pathways. Molecular docking and Co-IP studies confirmed a direct interaction between KRAS^G13D and PI3K-C2α, establishing a mechanistic link to downstream PI3K/AKT/mTOR pathway activation. Pharmacologic inhibition of KRAS^G13D suppressed expression of PI3K-C2α, p-AKT, and p-mTOR, corroborating its central role in signaling activation. KRAS^G13D mutant cells exhibited relative resistance to PI3K inhibitors MIPS-21335 and idelalisib. However, co-treatment with BI-2865 restored sensitivity to PI3K inhibition, producing a synergistic anti-proliferative effect that overcame resistance in mutant cells. This therapeutic synergy was consistent across both CLL cell lines and primary patient samples.
Conclusions
The KRAS^G13D mutation represents a functionally significant driver in a subset of CLL patients, promoting disease progression through enhanced fatty acid oxidation and activation of the PI3K/AKT/mTOR axis. Our findings establish a direct mechanistic link wherein KRAS^G13D binds and upregulates PI3K-C2α, thereby connecting oncogenic signaling with metabolic reprogramming. The present study provides a compelling rationale for the co-inhibition of KRAS and PI3K as a precision therapy to overcome drug resistance in patients with KRAS^G13D-mutant CLL.
Keywords : Chronic lymphocytic leukemia, KRAS mutation, PI3K/AKT signaling pathway, fatty acid metabolism
Please indicate how this research was funded. : This study was supported by the National Natural Science Foundation (No. 82000195); Shandong Provincial Natural Science Foundation (No. ZR2024MH050, ZR2020QH094); China Postdoctoral Science Foundation (No. 2022M721981); Taishan Scholars Program of Shandong Province (No. tsqn201909184); Shandong Provincial Health Construction Program of Medical Science and Technology Innovation Team; Academic Promotion Programme of Shandong First Medical University (No.2020RC007); Shandong First Medical University Young Scientists Foundation Cultivation Support Program(No.202201-071) ; Research Promotion Program of Shandong Provincial Hospital.
Please indicate the name of the funding organization.: National Natural Science Foundation of China; Shandong Provincial Natural Science Foundation; China Postdoctoral Science Foundation; Taishan Scholars Program of Shandong Province; Shandong Provincial Health Construction Program of Medical Science and Technology Innovation Team; Shandong First Medical University; Shandong Provincial Hospital.