Genetic Depletion of Fc Gamma Receptor 2b Affects CML Stem Cell Biology

Chronic myeloid leukemia (CML) is caused by the acquisition of the Philadelphia chromosome t(9;22) at the hematopoietic stem cell (HSC) level. The resulting Bcr-Abl protein loses the Abl autoinhibitory function and gains the Bcr oligomerization domain resulting in the constitutive activation of the Abl tyrosine kinase. The implementation of Bcr-Abl tyrosine kinase inhibitors (TKI) has greatly improved therapy outcome of CML patients. However, various mechanisms such as the acquisition of tyrosine kinase domain mutations or kinase-independent alterations have been described to result in therapy resistance. Using an inducible transgenic SCLtTAxBcr-Abl CML mouse model, we previously demonstrated that Bcr-Abl inhibition leads to eradication of mature leukemic cells but spares disease-driving leukemic stem cells (LSC) due to a lack of oncogene-addiction (Schemionek et al., BLOOD 2010; Hamilton, Helgason, Schemionek et al., BLOOD 2012). Aiming to identify new Bcr-Abl targets that could overcome therapy resistance of stem cells, we previously performed a microarray analysis using murine LSC. Expression of FcγRIIb (Fc gamma receptor 2b; CD32) was significantly increased 2.79-fold in Bcr-Abl positive vs. control Lin^-Sca1⁺c-kit⁺(LSK) cells. The FcγRIIb contains an immuno receptor tyrosine-based inhibitory motif (ITIM) and inhibits intracellular signaling upon activation. Phosphorylation of the receptor is mainly mediated via Src kinase Lyn and enables the receptor to recruit Src homology 2 domain-containing phosphatases.

Using qRT-PCR, we confirmed 2.83-fold upregulation of FcγRIIb within the LSK population of CML mice. Retroviral infection of 32D cells and primary BM cells with Bcr-Abl significantly increased FcγRIIb expression level (90.3-fold and 13.2-fold, resp., p<0.005 each vs. emtpy vector). TKI treatment of Bcr-Abl positive 32D cells did not affect FcγRIIb mRNA levels or surface expression. Bcr-Abl infection of lineage-depleted BM cells decreased growth factor independent colony forming capability (5.5-fold, p<0.05) and the proliferation potential of FcγRIIb^-/- lineage negative bone marrow (cells) compared to wild type (wt) in the absence of cytokines. Transplantation of Bcr-Abl infected FcγRIIb^-/- BM cells significantly reduced white blood cell count (WBC) of the recipients as compared to wt Bcr-Abl positive cells by 2.4-fold (p<0.05). Interestingly, WBC of Bcr-Abl positive FcγRIIb^-/- mice was similar to FcγRIIb^-/- empty vector control animals (p=0.875) as well as wt control mice (p=0.241). CML development was analyzed upon autopsy by FACS of BM and spleen cells and showed a 2.0-fold reduction (p<0.05) of leukemic c-kit positive cells as well as a reduction in spleen weight in recipients of Bcr-Abl transduced FcγRIIb^-/- cells (293mg ± 30) vs. Bcr-Abl transduced wt controls (381mg ± 13; p<0.05). Moreover, total numbers of leukemic LSK cells were significantly decreased by 3.5-fold in BM and 3-fold in spleen of Bcr-Abl transduced FcγRIIb^-/- BM recipients compared to recipients of Bcr-Abl transduced wt cells. Transplantation of empty vector transduced FcγRIIb^-/- vs. empty vector transduced wt control cells did not result in a decrease of knock-out cells showing that the observed effects were specific for impairment of Bcr-Abl transforming activity by loss of FcγRIIb but not due to FcγRIIb depletion alone.

In conclusion, we show that FcγRIIb is upregulated in the stem cell compartment of CML mice and that this is due to Bcr-Abl expression. The absence of FcγRIIb impairs leukemic cell growth in vitro and in vivo. Moreover, genetic depletion of the receptor decreases LSC numbers and affects CML development in vivo suggesting that FcγRIIb plays an important role in Bcr-Abl leukemogenesis, making it an attractive novel stem cell specific target for CML therapy. (*Authors contributed equally)