Three annotated splice variants for CSF3R, the gene encoding the receptor for granulocyte colony-stimulating factor (G-CSF) that critically regulates neutrophil production, have been described. Splice variant 1 (CSF3R-V1, also known as wild-type) is the most highly expressed and widely studied. Splice variant 4 (CSF3R-V4) is generated through use of cryptic 5' and 3' splice sites in the last exon of CSF3R, and produces a frame shift that results in early termination of the receptor. Increased expression of this splice variant has been reported in some patients with de novo AML. This splice variant induces a hyperproliferative phenotype and delayed receptor internalization by a mechanism similar to that reported in patients with severe congenital neutropenia transforming to AML with acquired CSF3R mutations. Although expression of the CSF3R-V3 splice variant was reported some 20 years ago in placenta, nothing is currently known about its expression in primary human hematopoietic cells. The CSF3R-V3 splice variant is generated through use of an alternate 3' splice acceptor site in the last intron that results in an in-frame insertion of 27 amino acids. Lack of a suitable assay to detect the CSF3R-V3 splice variant has limited studies of this variant.
In order to detect and quantitate the three annotated CSF3R splice variants, we developed and validated a digital PCR method that permitted absolute quantitation of each splice variant. Using this assay, we initially quantitated expression of each variant in purified CD34+ cells, neutrophils, and monocytes from healthy donors. As shown in Table 1, CSF3R-V3 is expressed along with the CSF3R-V1 and CSF3R-V4 splice variants, and it comprises a significant proportion of the total CSF3R receptor in normal myeloid and progenitor cells. We next investigated the effect of co-expression of CSF3R-V3 and CSF3R-V1 to reproduce the in vivo situation. Ba/F3 clones expressing both the CSF3R-V3 and CSF3R-V1 variants were generated and G-CSF dose response and proliferation assays performed. Co-expression of both variants in the same cell induced G-CSF hypersensitivity and increased cell proliferation relative to cells expressing only CSF3R-V1.
We next examined expression of the three CSF3R splice variants in primary cells from patients with AML. We were particularly interested in determining the effect of mutations in the SRSF2 gene that encodes a splicing factor frequently mutated in myeloid neoplasms. Using our digital PCR assay, expression of the three CSF3R splice variants was measured in purified CD34+ cells isolated from 11 patients with AML lacking mutations in splicing factor genes and compared to CD34+ cells isolated from 6 healthy donors. No significant differences were detected in the ratio of V3/V1 or the ratio of V4/V1 in AML cells compared to normal cells. Notably, when digital PCR was performed on CD34+ selected cells from 4 patients with AML with mutations in SRSF2, a statistically significant increase in the V3/V1 ratio was observed compared to CD34+ cells from patients with AML lacking mutations in splicing factor genes. These results suggest that SRSF2 regulates CSF3R splicing. Knockout of SRFS2 in HL-60 cells using CRISPR/Cas9 also resulted in an increased V3/V1 ratio, lending further support for a role of SRSF2 in CSF3R splicing and isoform expression.
Collectively, we demonstrate for the first time expression of the CSF3R-V3 splice variant in primary human myeloid cells, and show that SRSF2 modulates CSF3R splicing. The observation that expression of CSF3R splice variants is altered in cells from patients with AML and SRSF2 mutations provides novel insights into the pathophysiologic mechanisms by which SRSF2 mutations contribute to aberrant cell growth and differentiation in AML.
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