Pharmacological inhibition of EGFR signaling enhances G-CSF-induced hematopoietic stem cell mobilization

Abstract

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine granulocyte colony-stimulating factor (G-CSF) has become the preferred source of HSPCs for stem cell transplants. However, G-CSF fails to mobilize sufficient numbers of stem cells in up to 10% of donors, precluding autologous transplantation in those donors or substantially delaying transplant recovery time. Consequently, new regimens are needed to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in mice, we mapped the gene encoding the epidermal growth factor receptor (Egfr) to a genetic region modifying G-CSF-mediated HSPC mobilization. Amounts of EGFR in HSPCs inversely correlated with the cells' ability to be mobilized by G-CSF, implying a negative role for EGFR signaling in mobilization. In combination with G-CSF treatment, genetic reduction of EGFR activity in HSPCs (in waved-2 mutant mice) or treatment with the EGFR inhibitor erlotinib increased mobilization. Increased mobilization due to suppression of EGFR activity correlated with reduced activity of cell division control protein-42 (Cdc42), and genetic Cdc42 deficiency in vivo also enhanced G-CSF-induced mobilization. Our findings reveal a previously unknown signaling pathway regulating stem cell mobilization and provide a new pharmacological approach for improving HSPC mobilization and thereby transplantation outcomes.

Figure 1

Regulation of G-CSF–mediated mobilization is linked to a 5-Mbp interval on mouse chromosome 11 containing the Egfr locus. (a) Frequency of CFCs after G-CSF induced mobilization in C57BL/6 (B6, n = 10) and line G (n = 10) (0–36 Mbp on chromosome 11) mice. *P < 0.05 versus C57BL/6. PB, peripheral blood. (b) Genetic constitution of C57BL/6. D2 chromosome 11 (line G) and the new subcongenic lines generated from line G (B, C57BL/6 allele, D, DBA/2 allele). The column headings indicate the PCR markers that define the underlying SNPs. The square represents the 5-Mbp interval between 14.7 and 19.5 Mbp. (c) G-CSF induced mobilization in subcongenic lines 106 (D2 interval 8.9–36.7 Mbp) (n = 4), 338 (D2 interval 26.1–36.7 Mbp) (n = 7), 1023 (D2 interval 8.9–26.1 Mbp) (n = 4) and 1804 (D2 interval 14.7–19.5 Mbp) (n = 8) relative to C57BL/6 and line G. *P < 0.05 versus C57BL/6, ^#P < 0.05 versus line G. (d) Relative differences in expression in HPCs (Lin⁻c-Kit⁺ cells) from the bone marrow of C57BL/6 and line G mice of the indicated genes in the 5-Mbp interval represented on the MOE430 chip. The level of expression for C57BL/6 set to 1. Data are based on three independent hybridizations per genotype. *P < 0.05. Plek, plekstrin; Pno1 partner of NOB1 homolog; Wdr92, WD repeat domain 92; Etaa1, Ewing tumor-associated antigen-1; Meis1, myeloid ectropic viral integration-1. (e) Egfr expression by quantitative RT-PCR in bone marrow–derived HPCs (Lin⁻c-Kit⁺ cells) from C57BL/6, line G and line 1804 mice; for C57BL/6 and line G, n = 3 repeats per experimental group (four mice per group); for line 1804, n = 2 repeats per experimental group (four mice per group). Steady state refers to expression in HPCs from nonmobilized mice; mobilized refers to expression in HPCs from G-CSF–mobilized mice. *P < 0.05 versus C57BL/6 at steady state, ^#P < 0.05 versus C57BL/6 mobilized. Error bars represent the mean ± s.e.m., except for expression data in line 1804 in e, where they represent s.d.

Figure 2

EGF reduces G-CSF–induced mobilization of HSPCs. (a) Mobilization efficiency of C57BL/6 mice after a single dose of EGF on day 5 of the standard G-CSF regimen (n = 6, at least three mice per group), *P < 0.05 versus G-CSF only. (b) Schematic of the setup for competitive transplant experiments in c to measure repopulating units in peripheral blood using identical volumes of peripheral blood as donor tissue from mice treated with G-CSF (n = 3) or G-CSF and EGF (n = 4) in competition with identical numbers of C57BL/6 CD45.1⁺ bone marrow cells. BM, bone marrow. (c) Repopulating unit values based on donor chimerism measured by flow cytometry in peripheral blood 3 months after transplant. *P < 0.05. (d) Mobilization of line 1804 compared to C57BL/6 mice after G-CSF and EGF treatment. ^#P < 0.05 versus G-CSF alone, *P < 0.05 C57BL/6 versus line 1804 at the same dose of EGF. (e) Expression of known EGFR ligands in total bone marrow. RT-PCR was performed with specific primers for the genes encoding epidermal growth factor (EGF), TGF-α, HB-EGF and betacellulin (BTC) using cDNA isolated from total bone marrow (TBM) and lung (positive control). Error bars represent the mean ± s.e.m.

Figure 3

Genetic and pharmacological inhibition of EGFR activity enhances G-CSF–mediated mobilization. (a) Schematic of the experimental setup of the transplants in b to determine mobilization of wa2/+ bone marrow cells. (b) CFCs in peripheral blood from WT and wa2/+ recipient mice (n = 6 per group) mobilized by G-CSF. *P < 0.05 versus WT. (c) Schematic of the experimental setup for competitive transplant experiments in d and e. (d) Donor chimerism in hematopoietic cells in peripheral blood of recipients before mobilization. (e) Percentage of Ly5.2⁺ donor colonies after mobilization (determined by flow cytometry of at least 30 individual CFCs per mouse), n = 6 mice per group. *P < 0.05 versus before mobilization and versus WT. (f) Mobilization in response to G-CSF or G-CSF and erlotinib treatment (2.5–10.0 μg per g body weight, administered on days 3, 4 and 5 of the G-CSF regimen). *P < 0.05 versus G-CSF. (g) Schematic of the setup for competitive transplant experiments in h to measure hematopoietic stem cell frequency in peripheral blood after mobilization by G-CSF or G-CSF plus erlotinib (5 μg per g body weight) in competition with identical numbers of C57BL/6 CD45.1⁺ bone marrow cells, n = 3 repeats per experimental group (three recipient mice per group). (h) Repopulating units based on donor chimerism determined by flow cytometry in peripheral blood 3 months after transplant. *P < 0.05 versus G-CSF. Error bars represent the mean ± s.e.m.

Figure 4

Cdc42 regulates G-CSF–mediated mobilization in response to EGFR signaling. (a) Representative immunoblot showing increased amounts of activated Cdc42 in LDBM cells from G-CSF–mobilized C57BL/6 mice after EGF treatment (n = 3 independent experiments, at least three mice per group). The ratio of activated Cdc42 to actin in response to EGF treatment was normalized relative to the ratio of activated Cdc42 to actin in mice treated with only G-CSF. (b) Representative immunoblot showing decreased amounts of activated Cdc42 in LDBM cells from G-CSF mobilized C57BL/6 mice in response to erlotinib (n = 3 independent experiments, at least three mice per group). The ratio of activated Cdc42 to actin in response to erlotinib treatment was normalized relative to the ratio of activated Cdc42 to actin in mice treated with only G-CSF. (c) Quantification of the amount of the activated (GTP-bound) form of Cdc42 in LDBM cells upon G-CSF– or G-CSF plus erlotinib (5.0 μg per g body weight)-induced mobilization. Statistical analyses are based on three independent western blots from three independent experiments with three mice in each group. *P < 0.05. (d) Quantification of progenitor cell adhesion to a layer of FBMD-1 stromal cells after G-CSF or G-CSF and EGF (200 ng ml⁻¹) treatment, n = 4 experiments. *P < 0.05 versus PBS, ^#P < 0.05 versus G-CSF. (e) Quantification of progenitor cell adhesion to a layer of FBMD-1 stromal cells after G-CSF or G-CSF plus erlotinib (10 μM) treatment (data represent at least three separate experiments). *P < 0.05 versus PBS, ^#P < 0.05 versus G-CSF. (f) Frequency of CFCs in peripheral blood in WT mice (littermates) mobilized with G-CSF and treated with EGF (0.8 μg per g body weight) on day 5. n = 3 experiments, at least three mice per group, *P < 0.05. (g) Frequency of CFCs in peripheral blood of wa2/+ mice mobilized with G-CSF and treated with EGF (0.8 μg per g body weight) on day 5. n = 3 experiments, at least three mice per group. (h) Frequency of CFCs in peripheral blood of WT-reconstituted C57BL/6.SJL(BoyJ) mice in response to G-CSF or G-CSF plus EGF after treatment with polyI:C. n = 3 experiments, at least three mice per group, *P < 0.05 versus G-CSF. (i) Frequency of CFCs in peripheral blood of mice reconstituted with Cdc42^−/− hematopoietic cells and treated with polyI:C (n = 12 mice per group). P = 0.4715 G-CSF versus G-CSF plus EGF. (j) Representative immunoblots showing the amount of activated Cdc42 in LDBM cells in response to G-CSF in ‘poor mobilizer’ C57BL/6 mice and the ‘better mobilizer’ line 1804 (representative of two individual experiments with three mice per group). The ratio of activated Cdc42 to actin was normalized relative to the ratio of activated Cdc42 to actin in PBS (control)-treated C57BL/6 mice. Error bars represent the mean ± s.e.m.