Accelerated Apoptosis and Aberrant Retention of Bone Marrow Cells in Myelokathexis Are Two Independent Pathways Triggered by Mutant CXCR4 and Normalized upon Treatment with Specific Inhibitors.
Authors: Makaryan Vahagn , Penate Oscar , Dale David , Aprikyan Andrew 
Published: October 2019

Abstract
Myelokathexis (MK) is a rare congenital hematopoietic disease characterized by hypercellular marrow and severe neutropenia. In some patients there is an association of Warts, Hypogammaglobulinemia, and severe Infections with Myelokathexis (WHIM syndrome). We and others reported accelerated apoptosis of bone marrow myeloid cells in MK evidenced by electron microscopy and flow cytometry studies. The impaired cell survival in MK was associated with reduction in Bcl-X expression at least partially restored by G-CSF treatment. Heterozygous mutations in the CXCR4 gene were reported in most of the families with WHIM syndrome. Interaction of CXCR4, a G-protein coupled chemokine receptor with SDF-1 ligand plays a key role in homing and mobilization of CXCR4-expressing hematopoietic cells. It has been reported that expression of mutant CXCR4 leads to reduced receptor internalization, increased calcium flux and enhanced chemotaxis of transduced CD34+ cells toward SDF1. However, the mechanism of mutant CXCR4-triggered neutropenia in MK remains largely unknown. We examined 11 patients representing 6 unrelated families with MK and found heterozygous CXCR4 mutations in affected but not healthy family members. We identified truncation and deletion mutations including a novel mutation resulting in a deletion of the last 29 amino acids in the cytoplasmic domain of CXCR4. Expression of deletion and truncation mutants in human promyelocytic cells triggered apoptosis similar to that observed in MK patients. Specifically, the rate of apoptotic annexin-positive cells was approximately 2-fold higher in cells transfected with CXCR4 mutants compared with control cells transfected with normal CXCR4. Accelerated apoptosis appeared to stem from a significantly increased dissipation of mitochondrial membrane potential as determined by flow cytometry analysis of DIOC6-labeled cells expressing CXCR4 mutants compared with controls with normal CXCR4 (p<0.02). Similar to enhanced chemotaxis of mononuclear cells observed in MK patients, expression of CXCR4 mutants but not the wild type, resulted in a significant increase in directional motility of cells to SDF-1 (p<0.01). These data indicate that our cellular model appears to closely recapitulate the myelokathexis phenotype. Accelerated apoptosis, but not enhanced chemotaxis induced by mutant CXCR4 was reduced to near-normal level by zVAD-fmk-caspase-specific inhibitor. Interestingly, the mutant CXCR4-triggered increase in chemotaxis to SDF-1 was normalized by treatment with protein kinase C inhibitor. This reduction in mutant CXCR4-mediated increase in chemotaxis in response to the treatment with PKC inhibitor was similar to that observed in response to the treatment with AMD3100. Interestingly, combined treatment with both AMD3100 and PKC inhibitor did not result in a synergistic effect, suggesting that these agents may utilize the same signaling pathway. Of note, neither the treatment with PKC inhibitor not with AMD3100 affected accelerated apoptosis, suggesting that accelerated apoptosis and enhanced chemotaxis are two independent pathways triggered by mutant CXCR4. Importantly, treatment of myelokathexis patients’ blood mononuclear cells with PKC inhibitor restored the abnormal chemotactic properties to near normal levels. Thus, our data suggest that AMD3100 and PKC inhibitor may be effective for treatment of myelokathexis patients with aberrant retention of hematopoietic cells in the bone marrow.