Therapeutic inhibition of FcγRIIb signaling targets leukemic stem cells in chronic myeloid leukemia

Abstract

Despite the successes achieved with molecular targeted inhibition of the oncogenic driver Bcr-Abl in chronic myeloid leukemia (CML), the majority of patients still require lifelong tyrosine kinase inhibitor (TKI) therapy. This is primarily caused by resisting leukemic stem cells (LSCs), which prevent achievement of treatment-free remission in all patients. Here we describe the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing Fc gamma receptor IIb (FcγRIIb, CD32b) for being critical in LSC resistance and show that targeting FcγRIIb downstream signaling, by using a Food and Drug Administration-approved BTK inhibitor, provides a successful therapeutic approach. First, we identified FcγRIIb upregulation in primary CML stem cells. FcγRIIb depletion caused reduced serial re-plaiting efficiency and cell proliferation in malignant cells. FcγRIIb targeting in both a transgenic and retroviral CML mouse model provided in vivo evidence for successful LSC reduction. Subsequently, we identified BTK as a main downstream mediator and targeting the Bcr-Abl-FcγRIIb-BTK axis in primary CML CD34⁺ cells using ibrutinib, in combination with standard TKI therapy, significantly increased apoptosis in quiescent CML stem cells thereby contributing to the eradication of LSCs.. As a potential curative therapeutic approach, we therefore suggest combining Bcr-Abl TKI therapy along with BTK inhibition.

Fig. 1. FcγRIIb is upregulated in murine and human leukemic stem cells.

a FcγRIIb RNA and protein expression were analyzed in LSK⁺ cells (lin⁻;Sca-1⁺;c-kit⁺) from transgenic SCLtTA/Bcr-Abl mice that had been induced to express Bcr-Abl vs. controls. FACS sorted LSK⁺ from 3 weeks induced mice were analyzed using qRT-PCR (n = 3/3). The cell surface expression of FcγRIIb (CD32b) was assessed by FACS in mice that had been induced for 6 days (n = 3/3). b Lineage-depleted BM cells from C567B/L6 wild-type mice were virally transduced to express Bcr-Abl or empty vector (ev) control. Transduced cells were FACS sorted and analyzed for FcγRIIb expression using qRT-PCR (left) and western blot analysis (right). c FcγRIIb mRNA expression was analyzed in CD34⁺ healthy control (HC) and CML patient samples (n = 5/9). d Lineage negative (lin⁻) BM cells from transgenic SCLtTA/Bcr-Abl mice vs. controls were analyzed for FcγRIIb expression before and after 2 µM imatinib (IM) treatment for 18 h. e K562 and KCL-22 cells were treated for 18 h with 5 µM imatinib. f Public available microarray data from the leukemia MILE study (#GSE13159 and #GSE13164, probe set id: 207539_s_at) from BM biopsy of healthy control (HC) and CML patients at first diagnosis (CML) were analyzed for IL-4 expression (n = 73/76). Data are shown as mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001, and n.s. not statistically significant.

Fig. 2. FcγRIIb knockout reduces leukemic clonogenic potential and cell proliferation.

a Bcr-Abl and ev (empty vector) infected lin⁻ BM cells from FcγRIIb^+/+ and FcγRIIb^−/− (1000 cells/ml) were seeded in methylcellulose with or without cytokines. Numbers of colonies (colony-forming units, CFUs) were assessed after 7 days. Re-plating was performed using 1 × 10⁴ cells. CFU numbers were counted after 7 days. b Proliferation assay using FcγRIIb^+/+ and FcγRIIb^−/− Bcr-Abl⁺ cells was performed using Trypan blue exclusion method. c Cell cycle was assessed in FcγRIIb^+/+ and FcγRIIb^−/− Bcr-Abl⁺ cells. Data are shown as mean ± SD. n = 3, each, *p < 0.05, **p < 0.001, ***p < 0.0001, and n.s. not statistically significant.

Fig. 3. Loss or reduction of FcγRIIb attenuates CML development and reduces leukemic LSK cells in vivo.

a Schematic illustration of bone marrow transplantation approach using either FcγRIIb^+/+ and FcγRIIb^−/− C567B/L6 cells that were virally transduced to express Bcr-Abl (model A) or SCLtTA/Bcr-Abl FVB/N-derived BM cells that were transduced to express shRNA targeting FcγRIIb (model B). b Spleen weight of transplanted recipients receiving FcγRIIb^+/+:Bcr-Abl (n = 3) or FcγRIIb^−/−:Bcr-Abl (n = 5) BM cells. c Total leukemic GFP⁺ cells in BM, spleen, and BM LSK compartment. d Spleen weight of FVB/N recipient mice transplanted with SCLtTA/Bcr-Abl BM cells that were transduced with either shRNA:scr control or shRNA:FcγRIIb. e Total leukemic GFP⁺ cells in BM, spleen, and BM LSK compartment. Data are shown as mean ± SD. *p < 0.05 and **p < 0.001.

Fig. 4. FcγRIIb mediates malignant BTK activation in Bcr-Abl-positive cells.

a Western blot analyses of immortalized FcγRIIb^+/+ and FcγRIIb^−/− Bcr-Abl and ev (empty vector) BM cells were performed using the indicated antibodies. b FcγRIIb was retrovirally introduced into FcγRIIb^−/−:Bcr-Abl cells and protein lysates were analyzed for BTK and ERK1/2 activation. c CFU assay using FcγRIIb^+/+:ev, FcγRIIb^−/−:ev, FcγRIIb^+/+:Bcr-Abl, FcγRIIb^−/−:Bcr-Abl, or FcγRIIb^−/−:Bcr-Abl:FcγRIIb cells. Colony numbers were counted 7 days after seeding. Data are shown as mean ± SD. **p < 0.001 and ***p < 0.0001.

Fig. 5. Combined inhibition of Bcr-Abl and BTK impairs clonogenic potential, reduces proliferation, and enhances apoptosis in nondividing LSCs.

a K562 cells were treated with increasing concentrations of Ibr (Ibrutinib, 0.5–2.5 µM) or IM (Imatinib, 0.5–2.5 µM) for 18 h. Subsequently, protein lysates were analyzed for pBcr-Abl, pSTAT5, pBTK, Bcr-Abl, STAT5, BTK, and GAPDH. b Primary CML MNCs (10,000 cells/ml) were treated for 48 h (c) and CML CD34⁺ cells (1000 cells/ml) for 72 h with Ibr (0.5 µM, 2.5 µM), IM (2.5 µM, 5.0 µM), or the combination of both drugs. Cells were resuspended and plated using methylcellulose-containing cytokines. CFU numbers were counted after 7 days (n = 3, each). d CML CD34⁺ cells were treated for 72 h with 2.5 µM Ibr, 5.0 µM IM, and the combination of both drugs. Protein lysates were analyzed for pSTAT5, pBTK, pERK1/2, STAT5, BTK, and ERK1/2. e Evaluation of cell divisions in CML CD34⁺ cells upon treatment using CFSE combined with FACS analyses. For a clear presentation, the level of significance is given for Ibr + IM vs. IM only. f Annexin V⁺ staining of undivided CFSE^Max cells in treated CML CD34⁺ cells. g Proposed mechanism: elevated FcγRIIb expression in malignant cells persists despite BCR-ABL inhibition and this could be mediated via inflammatory cytokines in the CML microenvironment. FcγRIIb activity results in increased BTK expression and phosphorylation and dual targeting of BCR-ABL and BTK activity induces apoptosis in LSCs. Data are shown as mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001, and n.s. not statistically significant.