Profound Signaling Abnormalities Due to Cytoplasmic Variants of CSF3R in Patients with Myelodysplastic/Myeloproliferative Syndromes Associated with CSF3R Mutations or Mutations of Splicing Factor U2AF1
Authors: Mehta Hrishikesh , Maskishima Hideki , Futami Muneyoshji , Kao Wei-Ming , Przychodzen Bartlomiej P , Cheng Chonghui , Maciejewski Jaroslaw P. , Corey Seth J. 
Published: October 2019

Abstract 3792

Myelodysplastic Syndromes (MDS) are a group of bone marrow disorders closely related to acute myeloid leukemia (AML). Even though a number of genetic mutations have been recently identified in patients with MDS, their contributions to MDS pathogenesis remains poorly understood. Some of these genetic mutations involve transcription factors, but they are also found in AML: TET2, EZH2, and ASXL1. One group of mutations distinct to MDS are those encoding proteins involved in RNA splicing (e.g. U2AF1/U2AF35, ZRSR2, SRSF2, SF3B1). Based on RNA-Seq of MDS/AML patients, we report exon skipping in the 3′ end of the CSF3R transcript, which encodes the granulocyte colony-stimulating factor receptor (GCSFR), in a patient carrying the S34F mutation in the U2AF1 gene. U2AF1 is one of the more recurrent genes affected by mutation in MDS, and it is associated with progression to secondary AML. The S34F mutation in U2AF1 is a gain of function mutation that promotes excess splicing and exon skipping. Alternative splicing of CSF3R results in 7 transcripts, of which the two most common are Class I and Class IV. There are putative splicing sequences within exon 17 of the CSF3R locus; GT (GU) at the 5′ site and AG at the 3′ site - a recognition sequence for U2AF1. In addition, we also identified mutations affecting CSF3R in two patients with chronic myelomonocytic leukemia (GCSFR T595I or Q726X), one patient with Refractory Cytopenias with Multilineage Dysplasia and Ring Sideroblasts (GCSFR W650L), and one patient with primary AML (GCSFR G659fs). In the last case, this mutation affects the Class III transcript of CSF3R, an alternatively spliced form expressed highly in the placenta. The Class IV isoform lacks much of the C-terminal domain, similar to the protein produced by nonsense mutations found in patients with severe congenital neutropenia who develop MDS/AML or the patient we identified.

Little is known about the signaling-phenotype relationship of a mutant or alternatively-spliced GCSFR. To address these questions, we first studied the expression patterns of Class I and Class IV GCSFR in the human NB4 promyelocytic leukemia cell line and primary human hematopoietic stem (CD34+) cells induced to differentiate into neutrophils. Quantitative PCR of Class I and Class IV transcripts showed a positive feedback loop for Class I. Expression of the Class IV transcript was downregulated during hematopoietic cell differentiation. Scatchard analysis showed no differences between the two receptors in high-affinity Kd (∼ 500 nM) for the GCSF-GCSFR. Because Class IV lacks the C-terminal di-leucyl motif that facilitates internalization, we measured internalization rates and found that indeed the Class IV internalized more slowly and less completely. Using an MTT assay to measure proliferation we observed Class IV isoform had lower proliferative capacity at lower GCSF concentrations (0.1 – 2 nM GCSF); however at higher GCSF dose (>100 nM) its proliferative response was greater than Class I. Using western blotting we observed that the Class IV isoform showed weaker signaling via the JAK/STAT and ERK1/2 pathways, but had higher Lyn activity when treated with 100 ng/ml GCSF. To determine effects on differentiation, we made chimeric human growth hormone receptor-GCSFR for transfection into murine 32D cells. 32D cells express low levels of murine GCSFR, thus we made the chimeric receptor. Expression of Class IV Receptor impaired their differentiation (as demonstrated by morphology and Gr-1 expression). We are now developing a mouse model of perturbed hematopoiesis due to dysregulated expression of Class IV GCSFR. Altogether, our studies show that S34F mutation of U2AF1 splicing gene is associated with exon skipping of CSF3R. This would result in expression of a C-terminal truncated GCSFR, similar to that observed in patients with nonsense mutations or alternative splicing. A C-terminal truncated GCSFR causes aberrant hematopoietic cell proliferation, altered post-receptor signaling events, and impaired myeloid differentiation.

Our findings and those involving GCSFR E785K in high-risk MDS (Wolfler et al, Blood 105:3731, 2005) strongly suggest that aberrant signaling by alterations in the C-terminus of the GCSFR contributes to the pathogenesis of MDS.

Disclosures:

Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.