Mutant CXCR4-Induced Aberrant Retention and Apoptosis of Hematopoietic Cells in Myelokathexis and Normalization of Cell Survival and Mobilization with Specific Inhibitors.
Authors: Makaryan Vahagn , Penate Oscar , Dale David C. , Aprikyan Andrew A 
Published: October 2019

Abstract
Abstract 237
Myelokathexis (MK), also known as WHIM syndrome, is a rare congenital autosomal dominant stem cell disorder characterized by aberrant retention of hematopoietic cells in the bone marrow and severe neutropenia and lymphopenia. MK patients may evolve to develop Acute Myeloid Leukemia (AML) or fatal B-cell lymphoma. We and others reported that severe leukopenia in MK is due to accelerated apoptosis of bone marrow CD34+ stem cells and CD33+ myeloid progenitors. Impaired cell survival was associated with reduced Bcl-X expression, which, at least partially is restored by treatment of patients with G-CSF. Most of MK patients harbor heterozygous mutations in the CXCR4 gene product, which interacts with SDF-1 ligand and plays a key role in controlling homing and mobilization of hematopoietic stem cells. Mutant CXCR4 exhibits reduced internalization, enhanced calcium flux and results in elevated chemotaxis of human CD34+ stem cells toward SDF-1. However, the mechanisms of mutant CXCR4-induced severe neutropenia in MK and its malignant evolution remain unknown. We examined 11 patients from 6 unrelated families affected with MK and identified heterozygous CXCR4 mutations in these patients. Expression of different CXCR4 mutants in human HL-60 myeloid progenitor cells cultured in the presence of FBS triggers accelerated apoptosis comparable to that observed in MK. Particularly, the rate of apoptotic annexin V-positive cells was ∼2-fold higher in mutant CXCR4-expressing cells compared with control cells transfected with wild type CXCR4 (24±2% for wt CXCR4 vs 50±6% and 51±5% for different CXCR4 mutants). Similar increase in apoptosis was observed regardless whether the cells were cultured in the presence or absence of serum, SDF-1 or G-CSF, thus suggesting that increased apoptosis of bone marrow cells in MK is due to intrinsic CXCR4-activated pro-apoptotic signal transduction pathway. Accelerated apoptosis appeared to stem from enhanced dissipation of mitochondrial membrane potential as determined by FACS analysis of DIOC6-labeled cells expressing mutant CXCR4 compared with control CXCR4 (p<0.02). Similar to enhanced chemotaxis of patients' mononuclear cells, expression of CXCR4 mutants, but not the normal CXCR4 triggered a robust increase in directional motility of the cells to SDF-1 (p<0.01).
These data indicate that this cellular model with mutant CXCR4 expression appears to closely recapitulate the MK phenotype with accelerated apoptosis and impaired mobilization of bone marrow stem cells and myeloid progenitors. Accelerated apoptosis, but not enhanced chemotaxis triggered by mutant CXCR4 was normalized by treatment with caspase-specific inhibitor zVAD-fmk. Interestingly, the mutant CXCR4-induced increase in chemotaxis was restored with treatment with protein kinase C (PKC) inhibitor. The observed normalization of mutant CXCR4-triggerred increase in chemotaxis in response to PKC inhibitor was similar to that observed in response to a treatment with CXCR4 antagonist AMD3100. Interestingly, combined treatment with both PKC inhibitor and AMD3100 had no synergistic effect, suggesting that these compounds may utilize the same signaling pathway. Noteworthy, neither the treatment with AMD3100 nor with PKC inhibitor affected apoptosis, suggesting that observed accelerated apoptosis and increased chemotaxis in MK are two independent pathways triggered by CXCR4 mutants. Importantly, treatment of patients' blood mononuclear cells with PKC inhibitor restored the abnormal chemotactic properties to near normal levels. Thus, our data suggest that PKC inhibitor and AMD3100 may be effective for treatment of MK patients and for controlling the aberrant retention of bone marrow stem and myeloid cells in this stem cell disorder.

Disclosures:
Dale: Amgen: Consultancy, Research Funding, Speaker; Genzyme: Research support pending.