Bcr-Abl1 Kinase-Independent Upregulation of FcγRIIb Mediates Malignant Signaling That Is Critical for Leukemogenesis

Chronic myeloid leukemia (CML) is provoked by the chromosomal translocation t(9;22) within the hematopoietic stem cell (HSC) compartment, and this mutation gives rise to the oncogenic tyrosine kinase Bcr-Abl1. Although tyrosine kinase inhibitor (TKI) therapy results in enormous clinical success, TKIs fail to eradicate the disease initiating leukemic stem cell population (LSC) in the majority of CML patients. In a transgenic CML mice (SCLtTA/Bcr-Abl), which we have previously demonstrated to serve as an excellent model to study Bcr-Abl1 independent stem cell persistence, we identified the ITIM carrying Fc gamma receptor IIb (FcγRIIb; CD32) to be 2.8-fold upregulated in Bcr-Abl1⁺ versus control bone-marrow (BM) derived LSK (lin^-;Sca-1⁺;c-kit⁺) cells, using microarray and qRT-PCR. Aiming to evaluate the role of FcγRIIb function in Bcr-Abl1 mediated leukemogenesis, we previously retrovirally infected 5-FU treated SCLtTA/Bcr-Abl BM cells (CD45.1⁺) using FcγRIIb shRNA or scrambled control and transplanted these cells into FVB/N wildtype (WT) CD45.2⁺ recipients. Spleen weight in recipients receiving shRNA transduced stem and progenitor cells was significantly reduced (352 ± 59.1 mg), as compared to scrambled controls (568.1 ± 101.7 mg). Stem cell compartment analysis revealed decreased leukemic cells of shRNA versus scrambled control transplanted mice in the BM LSK compartment (lin^-, c-kit⁺, Sca-1⁺, CD45.1⁺, GFP⁺, 1.38-fold, p≤0.05). In addition, these effects were even more pronounced upon total depletion of FcγRIIb using the retroviral Bcr-Abl1 model. In these mice, complete loss of FcγRIIb decreased malignant BM LSK cells by 3.5-fold (p<0.05) as compared to Bcr-Abl1 transduced FcγRIIb^+/+recipients. Here, we have now studied the oncogenic mechanisms of FcγRIIb expression using the transgenic mice, a retroviral CML mouse model, as well as human cells.

Ectopic Bcr-Abl1 expression in human TF-1 cells increased FcγRIIb levels by 4-fold (p<0.001). In lin^- SCLtTA/Bcr-Abl BM cells, FcγRIIb was increased 1.4-fold (p<0.001), and receptor upregulation was thus higher in the LSK compartment (2.8-fold) as compared to lin^- cells. We then studied the effect of TKI treatment on FcγRIIb expression in Bcr-Abl1 transduced lin^- BM and lin^-SCLtTA/Bcr-Abl cells using imatinib or dasatinib. However, both TKIs did not reduce FcγRIIb levels, suggesting that persistent receptor upregulation is regulated independently of the Bcr-Abl1 kinase. Next, we used HoxB8-immortalized progenitor cells from FcγRIIb^-/- vs wt BM and virally introduced Bcr-Abl1 or ev. CFU (colony forming unit) potential in Bcr-Abl1 positive FcγRIIb ^-/- cells was significantly decreased 4-fold (p<0.01) as compared to Bcr-Abl1 transduced wt cells. Interestingly, re-introduction of FcγRIIb into Bcr-Abl1 positive FcγRIIb^-/- cells significantly increased CFU capacity again. CFU reduction was also observed upon overexpression of an SH2-SHIP1 domain in leukemic cells, as SHIP1 is the predominant FcγRIIb binding partner upon activation. Furthermore, depletion of the receptor, using shRNAs, decreased the proliferation rate in CML cells (2.3-fold on day 4, p<0.01) and this was accompanied by an increase in G0/G1 phase and decrease in S and G2/M phase in CML cells lacking the receptor. Along the same line, expression of p16, p19 and p21 were significantly increased in FcγRIIb depleted CML cells. CFU potential as well as proliferation was likewise decreased (10-fold, p<0.01; 2.8-fold p<0.01) in shRNA-mediated FcγRIIb knock-down immortalized SCLtTA/Bcr-Abl CML cells. Western-Blot analysis showed that FcγRIIb is phosphorylated in leukemic cells, and analysis of downstream signal pathways revealed decreased levels of p-ERK and p-p38 among others in FcγRIIb^-/-, compared to FcγRIIb^+/+ Bcr-Abl1 transduced cells. Taken together, we demonstrate that the FcγRIIb upregulation on LSC from transgenic CML mice, progenitor cells and human cells critical for CML cell biology. Genetic depletion or knock-down of the receptor reduces CFU capacity and cell growth which is mediated due to impaired leukemic downstream signaling. As FcγRIIb inactivation significantly impairs CML development and LSC burden in vivo and is upregulated independent of the Bcr-Abl1 kinase this receptor represents an attractive novel target.