Abstract
BackgroundWe have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for NRAS-related cancers. For KRAS-driven cancer, although much research has been focused on the KRAS4B splice variant, which does not undergo palmitoylation, KRAS4A has recently been shown to play an essential role in the development of carcinogen-induced lung cancer in mice and to be widely expressed in human cancers. However, the role of palmitoylation in KRAS4A tumorigenesis is not clear.MethodsThe expression of KRAS4A in KRAS-mutated leukemia cell lines and acute myeloid leukemia (AML) cells were checked using western blotting and reverse transcriptions-quantitative polymerase chain reaction (RT-qPCR) analysis, respectively. The leukemogenic potentials of oncogenic KRAS4A and its palmitoylation-defective mutants were examined by a mouse bone marrow transduction and transplantation model and the in vitro transformation assays. The activation of the RAS downstream signaling pathways and the membrane localizations of the KRAS4A and its mutants were analyzed via western blot analysis and confocal microscopy, respectively.ResultsWe show here that KRAS4A is expressed in human leukemia cell lines and in AML cells harboring KRAS mutations and that mutation at the palmitoylation site of oncogenic KRAS4A significantly abrogates its leukemogenic potential. However, unlike NRAS, palmitoylation-defective KRAS4A still induces leukemia in mice, albeit with a much longer latency. Using NRAS/KRAS4A chimeric constructs, we found that the KIKK motif of KRAS4A contributes to the transforming activity of KRAS4A. Mutations at both palmitoylation site and the KIKK motif abolish the ability of oncogenic KRAS4A to induce leukemia in mice.ConclusionsOur studies suggest that therapies targeting RAS palmitoylation may also be effective in treating KRAS4A associated malignancies and that interfering the KIKK membrane-targeting motif would enhance the therapeutic effectiveness.
Highlights
We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for NRAS-related cancers
To determine whether KRAS4A is expressed in blood cancer cells harboring KRAS mutations, we checked the KRAS4A protein levels with a KRAS4A specific antibody in acute myeloid leukemia cell lines SHI-1 and NB4, T cell acute lymphoblastic leukemia cell line CCRF-CEM, as well as diffuse large B cell lymphoma cell line Toledo
To confirm our observation in the hematologic malignant cells from patients, we examined the relative abundance of KRAS4A and KRAS4B mRNA in bone marrow samples from patients suffering acute myeloid leukemia (AML) with oncogenic KRAS mutations by reverse transcriptions-quantitative polymerase chain reaction
Summary
We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for NRAS-related cancers. For KRAS-driven cancer, much research has been focused on the KRAS4B splice variant, which does not undergo palmitoylation, KRAS4A has recently been shown to play an essential role in the development of carcinogen-induced lung cancer in mice and to be widely expressed in human cancers. Mammalian RAS family includes three RAS genes, which encode four highly homologous proteins: HRAS, NRAS, KRAS4A, and KRAS4B. The latter two are alternative splicing isoforms differing only at the carboxyl terminus. These isoforms possess over 90 % identity in the first 166 amino acid residues (G domain, including switch loops and the binding surfaces for downstream effectors) and are mainly diverse in the carboxyl terminal hypervariable region (HVR). HRAS mutation is relatively rare (3 %) [4]
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