WHIM syndrome myelokathexis reproduced in the NOD/SCID mouse xenotransplant model engrafted with healthy human stem cells transduced with C-terminus–truncated CXCR4

Abstract

IntroductionWHIM syndrome is characterized by warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis (severe chronic neutropenia with marrow hyperplasia and inappropriate apoptosis of mature myeloid cells in the bone marrow [BM]). [1][2][3][4][5] Many but not all cases of WHIM syndrome have been linked to autosomal dominant mutations in CXC chemokine receptor 4 (CXCR4), all of which cause truncations of the carboxy-terminus of CXCR4. Specific mutations identified in some families with myelokathexis include R334X, S339fs342X, E343X, and G335 X. 2,6,7 CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1; also known as CXCL12), play a central role in BM homing and trafficking of hematopoietic progenitor cells, mobilization of lymphocytes, and release of developing neutrophils from bone marrow. 1,[8][9][10] Mice lacking CXCR4 demonstrate defective hematopoiesis and cerebellar and cardiovascular development and die perinatally, highlighting the importance of CXCR4 in these organ systems. 11 There is 1 report of a cardiac malformation in a patient who may have had WHIM syndrome. 12 In vitro studies of WHIM variants of CXCR4 (mutated CXCR4) have provided evidence for increased agonist-dependent signaling by the mutant receptor, suggesting that the clinical manifestations may be due to hyperfunction of these receptors in vivo. In particular, we previously reported that transduction of the R334X WHIM variant into healthy human CD34 ϩ peripheral blood mobilized stem cells (PBSCs) results in enhanced chemotactic and calcium flux responses of the cells to SDF-1, and found that this effect was associated with and presumably caused by a failure of the mutant receptor to downregulate and internalize, leading to prolongation of activation. 13 In the current study we explore the mechanism of myelokathexis in WHIM syndrome using the NOD.CB17-Prkdc scid /J mouse (nonobese diabetic/severe combined immunodeficiency [NOD/SCID] mouse) xenotransplantation model engrafted with healthy human mobilized CD34 ϩ PBSCs that had been transduced with internal ribosome entry site (IRES)-containing bifunctional retrovirus vectors encoding mutated CXCR4 or wild-type (wt) CXCR4 together with a green fluorescent protein (GFP) construct or with vector-encoding GFP construct only. We will demonstrate that expression of mutated CXCR4 does not itself induce apoptosis in transduced myeloid cells differentiated in culture from the transduced PBSCs, but does result in a WHIM-type myelokathexis pattern of the transduced human cells in the in vivo xenotransplantation model (myeloid apoptosis in marrow and decreased release of cells from the marrow to the circulation). An Inside Blood analysis of this article appears at the front of this issue.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. Materials and methods G-CSF-mobilized CD34 ؉ PBSCsFollowing informed cons...

WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4

“…26 Neutropenia and myelokathexis have been recapitulated in mice and zebrafish genetically modified to express CXCR4 R334X . 27,28 Moreover, patients with WHIM syndrome due to CXCR4 R334X have shown significant improvement in circulating leukocyte numbers after brief treatment with plerixafor, a CXCR4 antagonist approved by the US Food and Drug Administration for hematopoietic stem cell mobilization with G-CSF for transplantation of multiple myeloma or non-Hodgkin lymphoma patients after cytoreductive chemotherapy. 4,29 The C-tail of CXCR4 has been shown by truncation mutagenesis studies to be important for ligand-induced receptor internalization, desensitization, ubiquitination, degradation, and endocytosis.…”

The CXCR4 antagonist plerixafor corrects panleukopenia in patients with WHIM syndrome

“…Consistent with this, gene transfer of CXCR4 R343X phenocopies myelokathexis in both zebrafish and Nod-scid (ie, nonobese diabetes/severe combined immunodeficiency) mice. 9,10 Current treatments for WHIM syndrome include G-CSF (filgrastim [Neupogen; Amgen Inc]) and intravenous immunoglobulin but are nonspecific, expensive, difficult to administer, and only partially effective. 4 Recently, a small molecule CXCR4 antagonist named plerixafor (trade name Mozobil [Genyzme Corporation] and formerly known as AMD3100) was approved by the Food and Drug Administration (FDA) for mobilizing HSCs from BM to blood with G-CSF for the purpose of autologous transplantation after cytoreductive therapy in patients with non-Hodgkin lymphoma or multiple myeloma.…”

Severe congenital neutropenia resulting from G6PC3 deficiency with increased neutrophil CXCR4 expression and myelokathexis

“…Hyperfunctional chemokine receptor CXCR4 mutations, which cause WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelokathexis syndrome), result in myelokathexis (defective neutrophil egress from bone marrow) resulting from increased CXCR4-dependent bone marrow retention of neutrophils. 16 Recently, Boztug et al described a new SCN syndrome in 12 children from Europe and the Middle East that was associated with increased susceptibility to bacterial infections and cardiovascular abnormalities, including prominent ectactic superficial veins resulting from mutations in the gene G6PC3. 17 Consistent with this, G6pc3 knockout mice had previously been reported to have neutropenia and increased susceptibility to Escherichia coli infection.…”