Mutations in the let-7 binding site - a mechanism of RAS activation in juvenile myelomonocytic leukemia?

Juvenile myelomonocytic leukemia (JMML) is a rare hematologic malignancy in childhood and accounts for less than 3% of all childhood hematologic malignancies.¹ The role of hyperactive RAS in JMML is underlined by the fact that approximately 80% of JMML develop due to gain-of-function mutations in NRAS, KRAS, PTPN11 and SOS1 or homozygous loss-of-function mutations in NF1 or c-CBL.^2–4 These genes are all components of the RAS/ERK signaling network, implicating deregulation of this signaling pathway in JMML pathogenesis. Therefore, it may be speculated that JMML lacking known mutations of genes playing a role in RAS signaling may carry other mutations, which result in activation of this pathway. Recently, germline mi-RNA gene variations were proposed to affect the expression levels of tumor suppressor or oncogenes and, thereby, familial/hereditary cancer risk.⁵ The let-7 mi-RNA family targets many important genes including cell cycle regulators such as CDC25A and CDK6, a number of early embryonic genes including HMGA2, Mlin-41 and IMP-1 and promoters of growth including RAS and C-MYC. In Caenorhabditis elegans let-7 mutant seam cells fail to exit the cell cycle and to terminally differentiate, thus demonstrating continuous proliferation, a hallmark of cancer.⁶ Human RAS expression was also shown to be regulated by let-7.⁷ Evidence of a role of let-7 in cancer came from the observation that lung tumor tissues display significantly reduced let-7 levels and significantly increased RAS protein levels relative to normal lung tissue.⁸ Recently, an SNP (rs61764370) in a let-7 complementary site in the KRAS 3’ UTR, which leads to KRAS overexpression, was shown to increase non-small cell lung cancer risk.⁹ It is still unknown whether alterations of the let-7 binding site in target genes or if mutations of let-7 mi-RNAs play a role in the development and progression of JMML. To address this question, we sequenced the 3’UTR of N-RAS and K-RAS in bone marrow cells from 10 JMML patients who had no other known RAS pathway mutations. The let-7 complementary sites LCS1-LCS9 in the 3′ UTR of NRAS and the complementary sites LCS1-LCS8 of KRAS were amplified and PCR products were directly sequenced.⁷ No sequence alterations were found in the 3′ UTR of NRAS or KRAS. However, in KRAS, the SNP rs61764370 T>G in the LCS6 was observed in one case. So far, only a few studies have investigated whether germline mutations in genomic mi-RNA sequences predispose to development of certain types of cancer or other diseases.^10,11 Recently, a germline mutation in mature miR-125a was found to be highly associated with breast cancer tumorigenesis, suggesting that miR-125a is likely to function as a tumor suppressor gene in human cancer.¹² Sequencing of let-7a-1 (MI0000060) as well as the neighboring let-7f-1 (MI0000067) genomic sequences in the 10 JMML cases did not identify any sequence alterations in these genomic regions.

In summary, we found no evidence that mutations in let-7 or in binding sites of let-7 mRNA targets lead to an upregulation of RAS genes in JMML. Yet, we cannot rule out that other mi-RNAs known to bind to NRAS- or KRAS-UTR or other let-7 family miRNAs may play a role in the development of JMML.