EHA Library - The official digital education library of European Hematology Association (EHA)

Chronic myeloid leukemia (CML) is provoked by the chromosomal translocation t(9;22) that gives rise to the oncogenic tyrosine kinase Bcr-Abl. Implementation of tyrosine kinase inhibitor (TKI) therapy resulted in significant clinical success but with TKIs failing to eradicate the disease initiating leukemic stem cell population (LSC), this treatment is not curative in the vast majority of patients. By using a transgenic CML mouse model, we previously showed that LSC persist despite complete Bcr-Abl kinase inhibition due to a lack of oncogene-addiction. Subsequently, we identified the ITIM carrying Fc gamma receptor IIb (FcγRIIb; CD32) to be 2.8-fold upregulated in Bcr-Abl⁺ versus control LSK (lin^-;Sca-1⁺;c-kit⁺) cells using microarray and qRT-PCR.

Bcr-Abl increased FcγRIIb mRNA (13.2-fold, p≤0.001) and protein expression in primary murine lineage negative (lin^-) BM cells. Reduction of FcγRIIb in immortalized SCLtTA/Bcr-Abl progenitor cells significantly reduced CFU potential by nearly 10-fold (p≤0.01) and it impaired the proliferation rate in these cells (2.27-fold, p≤0.001). Moreover, transplantation of SCLtTA/Bcr-Abl-shRNA:FcγRIIb BM cells (CD45.1⁺) into FVB/N wildtype (WT) CD45.2⁺ recipients reduced spleen weight (352 ± 59.13 mg), as compared to scrambled shRNA (568.1 ± 101.72 mg). FACS analysis revealed a decrease in GFP⁺;CD45.1⁺ BM cells (1.43-fold, p≤0.001) upon FcγRIIb knock down. Likewise, donor-derived Gr-1⁺ cells (Gr-1⁺;CD45.1⁺;GFP⁺) were reduced in the BM (1.28-fold, p≤0.01) of these mice. Flow-cytometric analysis of the stem cell compartment revealed decreased leukemic BM LSK cells (lin^-, c-kit⁺, Sca-1⁺, CD45.1⁺, GFP⁺, 1.38-fold, p≤0.05) in mice transplanted with shRNA:FcγRIIb vs scrambled control. We previously observed similar effects upon FcγRIIb depletion (FcγRIIb^-/-) vs wildtype (FcγRIIb^+/+), combined with virally induced Bcr-Abl expression. Interestingly, Bcr-Abl signaling induces FcγRIIb phosphorylation in leukemic cells. Analysis of downstream signal pathways showed decreased levels of p-ERK, p-BTK, p-PLCγ1 in FcγRIIb^-/-, compared to FcγRIIb^+/+ Bcr-Abl transduced immortalized primary murine BM cells.

FcγRIIb is upregulated in LSC derived from transgenic CML mice upon Bcr-Abl expression. Complete depletion or knock down of the receptor reduces CFU capacity and cell growth in CML cells and significantly impairs CML development and LSC burden in vivo, presumably due to impaired leukemic downstream signaling. Our data demonstrate that FcγRIIb is critical and disease specific making it a potential novel therapeutic target in CML stem cells.