High-throughput sequencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients

Abstract

Transforming mutations in NRAS and KRAS are thought to play a causative role in the development of numerous cancers, including myeloid malignancies. Although mutations at amino acids 12, 13, or 61 account for the majority of oncogenic Ras variants, we hypothesized that less frequent mutations at alternate residues may account for disease in some patients with cancer of unexplained genetic etiology. To search for additional, novel RAS mutations, we sequenced all coding exons in NRAS, KRAS, and HRAS in 329 acute myeloid leukemia (AML) patients, 32 chronic myelomonocytic leukemia (CMML) patients, and 96 healthy individuals. We detected 4 "noncanonical" point mutations in 7 patients: N-Ras(G60E), K-Ras(V14I), K-Ras(T74P), and K-Ras(A146T). All 4 Ras mutants exhibited oncogenic properties in comparison with wild-type Ras in biochemical and functional assays. The presence of transforming RAS mutations outside of positions 12, 13, and 61 reveals that alternate mechanisms of transformation by RAS may be overlooked in screens designed to detect only the most common RAS mutations. Our results suggest that RAS mutations may play a greater role in leukemogenesis than currently believed and indicate that high-throughput screening for mutant RAS alleles in cancer should include analysis of the entire RAS coding region.

Figure 1

RAF-1 pull-down assay reveals that novel mutations confer increased Ras-GTP. (A) Wild-type NRAS and KRAS as well as each mutant were expressed in HEK 293T/17 cells for 48 hours. Whole-cell extracts were incubated with RAF-1 peptides conjugated with agarose beads for precipitation of Ras-GTP. Precipitates were subjected to SDS-PAGE analysis and immunoblotted with an antibody specific for total Ras. (B) Densitometry was performed on blots in panel A. Densitometric units for Ras-GTP were normalized to their respective total Ras counterparts, and all Ras-GTP/total Ras ratios were divided by the ratio for WT N-Ras or K-Ras. Values represent mean plus or minus SEM (n = 3).

Figure 2

Immunoblotting reveals that novel mutations confer increased signaling of downstream Ras pathways. (A) Wild-type NRAS and KRAS as well as each mutant were expressed in HEK 293T/17 cells for 48 hours. Whole-cell extracts were subjected to SDS-PAGE analysis and immunoblotted with antibodies specific for total and phospho-MEK and ERK as well as β-actin. (B) Densitometry was performed on blots in panel A. Densitometric units for phosphorylated proteins were normalized to their respective total protein counterparts, and all phospho/total ratios were divided by the ratio for WT N-Ras or K-Ras. Values represent mean plus or minus SEM (n = 3).

Figure 3

RAS mutant alleles confer growth factor hypersensitivity to murine bone marrow cells. (A) Murine bone marrow cells were infected with retrovirus-expressing empty vector, wild-type KRAS, as well as each KRAS mutant allele. Cells were plated in triplicate in methylcellulose containing various doses of GM-CSF (0-10 ng/mL). After 7 days, colonies were quantified on a bright-field microscope. Values represent mean plus or minus SEM. (B) Murine bone marrow cells were infected with retrovirus-expressing empty vector, wild-type NRAS, and each NRAS mutant allele. Cells were plated in triplicate in methylcellulose containing various doses of GM-CSF (0-10 ng/mL). After 7 days, colonies were quantified on a bright-field microscope. Values represent mean plus or minus SEM.