Mutant CXCR4-Triggered Apoptosis and Impaired Mobilization of Bone Marrow Cells in Myelokathexis Are Normalized upon Treatment with Specific Inhibitors.

Myelokathexis (MK) is a rare congenital disorder characterized by hypercellular marrow and severe chronic neutropenia. In some, but not all patients there is an association of Warts, Hypogammaglobulinemia, and Infections with Myelokathexis (WHIM syndrome). We and others reported that bone marrow myeloid cells in MK exhibit characteristic apoptotic features such as condensed chromatin, cytoplasmic blebbing, and cellular fragmentation. FACS analysis also demonstrated an impaired survival of myeloid cells in MK that was associated with reduced level of Bcl-X expression. Several heterozygous mutations in the CXCR4 gene have been identified in most of the families with autosomal dominant WHIM syndrome. CXCR4 is a G-protein coupled chemokine receptor and its interaction with SDF-1 ligand plays an important role in homing and mobilization of hematopoietic cells. Recently, it has been demonstrated that expression of mutant CXCR4 in human CD34+ cells results in reduced receptor internalization, enhanced calcium flux and enhanced migration of transduced CD34+ cells. However, the mechanism of mutant CXCR4-mediated neutropenia in MK remains largely unknown.

We examined 6 patients representing 4 unrelated families with MK and found that all affected family members harbor heterozygous mutations in the CXCR4 gene including a previously reported R334ter mutation. We identified two novel mutations that result in frame shifts and a premature stop codon in the cytoplasmic domain of CXCR4. Expression of CXCR4 mutants in human promyelocytic HL-60 cells resulted in massive apoptosis similar to that reported for bone marrow-derived myeloid cells from MK patients. Specifically, more than 50% of HL-60 cells transfected with previously reported R334ter and novel truncation mutants were positive for annexin V, whereas 20±6% of apoptotic annexin V-positive cells were observed in control cells transfected with normal CXCR4. Impaired cell survival appeared to be due to aberrant dissipation of mitochondrial membrane potential as flow cytometry analysis revealed significantly increased DIOC6-staining in cells expressing CXCR4 mutants compared with control cells expressing normal CXCR4 (p<0.02). Moreover, the expression of CXCR4 mutants but not of the wild type form led to a significant increase in directional motility of myeloid cells towards SDF-1 (p<0.01), similar to the enhanced chemotaxis of blood mononuclear cells observed in our MK patients. These data indicate that our cellular model closely recapitulates the myelokathexis phenotype. Premature apoptosis, but not enhanced chemotaxis triggered by mutant CXCR4 was reduced to near-normal level by caspase-specific inhibitor zVAD-fmk. Interestingly, the mutant CXCR4-induced increase in directional motility to SDF-1, but not accelerated apoptosis was normalized upon treatment with protein kinase Cξ-specific inhibitor. These data suggest that accelerated apoptosis and increased chemotaxis are two independent pathways activated by mutant CXCR4. Importantly, treatment of primary blood mononuclear cells from 3 MK patients with PKCξ inhibitor restored the abnormal chemotactic properties to the levels comparable to that of treated control cells from healthy volunteers. Further studies needed for assessing the therapeutic potential of PKCξ-specific inhibitor for normalizing the impaired mobilization of bone marrow cells in MK. Our data also suggest that the PKCξ-specific inhibitor may be effective for mobilization of human hematopoietic stem cells.