Differentiation-Defective Class IV GCSF Receptor Promotes Resistance to Apoptosis Through Induction of Anti-Apoptotic Bcl-2 Family Proteins

Granulocyte colony stimulating factor (GCSF) drives the production, proliferation and differentiation of myeloid progenitor and precursor cells via the GCSF Receptor (GCSFR). The Class I GCSFR constitutes the full-length, wild type receptor. Alternative splicing events create a truncated isoform, the Class IV GCSFR, which lacks 3 of the 4 distal tyrosine residues in the C-terminal domain, and results in impaired differentiation. A role for the Class IV receptor in human myeloid leukemia has been suggested from two clinical observations: 1) Bone marrow cells from patients with monosomy 7 overexpress the Class IV receptor and demonstrate increased proliferative response to GCSF; 2) Increased relapse rates were found in children and adolescents with AML whose blasts overexpress the Class IV receptor. Additionally, multiple splicing factors have recently been discovered to be mutated in myelodysplastic syndrome (MDS). We have discovered that the S34F mutation in the splicing factor U2AF1 is associated with exon skipping of the GCSFR. Yet, little is known about the biological properties of this Class IV isoform.

The IL-3 dependent murine Ba/F3 cell line was transfected with cDNA to express either human Class I (Ba/F3 GRI) or Class IV (Ba/F3 GRIV) GCSFR. Proliferation of Ba/F3 GRI and GRIV cells was measured using MTT assay after 48 hours of exposure to varying doses of GCSF. Cells were serum- and cytokine-starved for 0, 4, 8 and 16 hours. Cell count and viability were determined by trypan blue exclusion. Members of the mitochondrial apoptosis pathway were also evaluated by western blot analysis. Time-dependent changes in GCSF-mediated phosphoprotein signaling events were studied by western blotting. Samples were also stained with propidium iodide and used for cell cycle analysis by flow cytometry or used to perform AnnexinV apoptosis assay.

Flow cytometry results demonstrated a G0/G1 cell cycle arrest at 8 hrs for both Ba/F3 GRI and GRIV cells with a similar magnitude of effect. However, longer starvation of 16 hrs resulted in increased cell death, more pronounced in the GRI than GRIV cells (46% v. 9% in the sub-G1 phase of the cell cycle, respectively). This pattern of apoptotic resistance in the GRIV cells was also seen in cell number and viability (viability of GRI and GRIV cells at 16 hrs was 67% ± 4.9% v. 90% ± 4%, respectively). These results were validated by flow cytometry using the AnnexinV apoptosis assay: proportion of GRI and GRIV cells staining positive for both AnnexinV and 7-AAD was 71% and 32%, respectively. To determine the molecular basis for this resistance to apoptosis, we performed western blotting to analyze Bcl-2, Mcl-1, Puma, GSK3ß, Bcl-x_Land other Bcl-2 family members. Of these, the most prominent differences were seen in Bcl-2 and Mcl-1. The Class IV cells demonstrated increased levels of Bcl-2 throughout 16 hrs of starvation. The Class IV cells also displayed persistence of Mcl-1 for a longer duration than the GRI cells, correlating with the observed pattern of viability and cell cycle data. When these starved cells were stimulated with GCSF, there was a greater time dependent increase of phopho-p38 MAPK in the GRI cells.

Here, we show that the Class IV GCSFR isoform confers both a proliferative and survival or anti-apoptotic advantage. Increased levels of both Mcl-1 and Bcl-2 may contribute to the apoptotic resistance seen in the Ba/F3 GRIV cells. Because there were no differences in activation state of GSK3ß, an alternate pathway for regulating Mcl-1 levels must be operative. The p38 MAPK stress response has been implicated in affecting downstream Mcl-1 and Bcl-2 regulation. p38 activation is important for chemotherapy induced cancer cell death, and chemotherapeutic resistance has been linked to overexpression of Bcl-2 or Mcl-1 in various cancer types. It is intriguing that Class IV expression has been linked to increased relapse rates in AML patients. Thus, we speculate that there may be a synergistic, protective effect of decreased p38 activation coincident with enhancement of the anti-apoptotic Bcl-2 and Mcl-1 proteins in the Class IV cells. Further elucidation of the mechanism for apoptotic resistance conferred by the Class IV GCSFR will help define its contribution to MDS transformation and provide candidate targets for novel therapeutics or prevention of disease progression in patients with primary or secondary MDS/AML.

No relevant conflicts of interest to declare.