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The extrafollicular immune response is essential to generate a rapid but transient wave of protective antibodies during infection. Despite its importance, the molecular mechanisms controlling this first response are poorly understood. Here, we demonstrate that enhanced Cxcr4 signaling caused by defective receptor desensitization leads to exacerbated extrafollicular B-cell response. Using a mouse model bearing a gain-of-function mutation of Cxcr4 described in 2 human hematologic disorders, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and Waldenström macroglobulinemia, we demonstrated that mutant B cells exhibited enhanced mechanistic target of rapamycin signaling, cycled more, and differentiated more potently into plasma cells than wild-type B cells after Toll-like receptor (TLR) stimulation. Moreover, Cxcr4 gain of function promoted enhanced homing and persistence of immature plasma cells in the bone marrow, a phenomenon recapitulated in WHIM syndrome patient samples. This translated in increased and more sustained production of antibodies after T-independent immunization in Cxcr4 mutant mice. Thus, our results establish that fine-tuning of Cxcr4 signaling is essential to limit the strength and length of the extrafollicular immune response.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare primary immunodeficiency caused by gain-of-function mutations in the CXCR4 gene. We report the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary efficacy of mavorixafor from a phase 2 open-label dose-escalation and extension study in 8 adult patients with genetically confirmed WHIM syndrome. Mavorixafor is an oral small molecule selective antagonist of the CXCR4 receptor that increases mobilization and trafficking of white blood cells from the bone marrow. Patients received escalating doses of mavorixafor, up to 400 mg once daily. Five patients continued on the extension study for up to 28.6 months. Mavorixafor was well tolerated with no treatment-related serious adverse events. At a median follow-up of 16.5 months, we observed dose-dependent increases in absolute neutrophil count (ANC) and absolute lymphocyte count (ALC). At doses ≥300 mg/d, ANC was maintained at >500 cells per microliter for a median of 12.6 hours, and ALC was maintained at >1000 cells per microliter for up to 16.9 hours. Continued follow-up on the extension study resulted in a yearly infection rate that decreased from 4.63 events (95% confidence interval, 3.3-6.3) in the 12 months prior to the trial to 2.27 events (95% confidence interval, 1.4-3.5) for patients on effective doses. We observed an average 75% reduction in the number of cutaneous warts. This study demonstrates that mavorixafor, 400 mg once daily, mobilizes neutrophil and lymphocytes in adult patients with WHIM syndrome and provides preliminary evidence of clinical benefit for patients on long-term therapy. The trial was registered at www.clinicaltrials.gov as #NCT03005327.

Introduction: WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome is an inborn error of immunity characterized as a primary immunodeficiency with neutropenia-but the acronym does not reflect the broad spectrum of disease manifestations that patients may experience. A WHIM syndrome diagnosis may be confirmed clinically by the presence of myelokathexis, the retention of white blood cells in the bone marrow, or by identification of a known pathogenic gain-of-function mutation in the CXCR4 gene coding for the CXCR4 receptor. Diagnosis of WHIM syndrome is thought to be frequently missed because of low disease awareness, missed identification of myelokathexis, and lack of routine genetic testing (Al Ustwani O, et al. Br J Haematol. 2014:164;15-23; Dotta L, et al. Curr Mol Med. 2011;11:317-325; Heusinkveld L, et al. Exp Opin Orphan Drugs. 2017;5(10):813-825). The prevalence of WHIM syndrome has never been systematically studied and is unknown. Determination of prevalence via insurance claims data is hindered by the absence of an International Classification of Diseases (ICD)-10 code for WHIM syndrome as well as inconsistent coding for key symptoms of WHIM syndrome, which are variably penetrant. This study applied an artificial intelligence (AI)/machine learning (ML) model to estimate the potential prevalence of WHIM syndrome using a large US insurance claims database. Methods: A deidentified, longitudinal, patient-level US claims database of >300 million lives was used for this study. Thirty-two patients with genetically confirmed WHIM syndrome were identified from the claims database by linking deidentified patients to known physicians and matching clinical and demographic features. Using this group as a positive training class, an AI/ML model was deployed to identify patients with WHIM look-alike clinical phenotypes in the database. Patients were further filtered based on clinical features to generate low (presence of warts, history of infections, and hypogammaglobulinemia) and high (presence of warts, history of infections, and coding associated with immunodeficiency) prevalence estimates; a final prevalence number for the US was projected to account for incomplete coverage of the US population in the claims database. Finally, insurance codes for disease symptoms, treatments, and management were analyzed to investigate the burden of disease in patients identified by the model. Results: The model showed a high predictive value for distinguishing patients with known WHIM syndrome from a random sample of age-matched patients in the database (area under the curve [AUC] of receiver operating characteristic [ROC] plot, >0.99) as well as a control group of patients with ICD-10 codes defining immunodeficiency conditions (AUC of ROC plot, 0.99). The model generated estimates ranging from 1803 (low) to 3718 (high) patients with WHIM look-alike phenotype in the US. Analysis of medical history in the high-estimate WHIM look-alike group revealed symptomatic and severe disease, as evidenced by ≥1 instance of use of granulocyte colony-stimulating factor (41%) or intravenous immunoglobulin (46%) therapy (both Conclusions: The methodology used here provides an approach to explore the prevalence of rare diseases that are often mis- or under-diagnosed and are not captured with a unique ICD-10 code. This study estimates a prevalence of 1803 to 3718 WHIM look-alike patients in the US, supporting the possibility that there may be ≤~3700 patients with either diagnosed or undiagnosed WHIM syndrome in the US. An analysis of the medical history of the WHIM look-alike patients revealed a history of symptomatic and severe disease and a high unmet medical need. Since it is not feasible to definitively confirm a WHIM diagnosis in the look-alike group, it is possible that some of these look-alike patients may have diagnosed or undiagnosed WHIM syndrome, while others may have a clinical phenotype consistent with WHIM syndrome without meeting its classic diagnostic criteria. Disclosures Garabedian: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Neri: X4 Pharmaceuticals: Current Employment. Seng: X4 Pharmaceuticals: Current Employment. Jones: Real Chemistry (Formerly Swoop/IPM): Current Employment, Other: I was paid salary to perform secondary research project work for client "X4" which resulted in this publication.

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome is a rare primary immunodeficiency associated with broad cytopenia, including neutropenia. It is caused by gain-of-function mutations in C XCR4, leading to dysregulated immune cell trafficking with retention of neutrophils, lymphocytes, and monocytes in the bone marrow and in some cases, hypogammaglobulinemia. As a result, patients with WHIM syndrome have recurrent bacterial and viral infections, and unusual susceptibility to human papillomavirus infection predisposing individuals to recalcitrant warts and malignancy (McDermott D, et al. Immunol Rev. 2019;91-102). Therapeutic options are limited and do not address the underlying pathogenic mechanism of WHIM syndrome. The investigational oral CXCR4 antagonist mavorixafor directly targets the underlying cause of disease and has been shown to increase absolute neutrophil, lymphocyte, and monocyte counts, and to decrease annualized infection rate, and reduce cutaneous wart burden in a phase 2 trial of adults with WHIM syndrome (NCT03005327; Dale D, et al. Blood. 2020;136: 2994-3003). Findings from an ongoing long-term extension of this study support a sustained clinical benefit of long-term mavorixafor treatment in patients with WHIM syndrome. Here, we describe the design of a global phase 3 registrational trial evaluating the safety and efficacy of mavorixafor in WHIM syndrome in participants aged ≥12 years while reporting on preliminary baseline characteristics of the enrolled population. Methods: This phase 3 trial (4WHIM; NCT03995108) is a randomized, double-blind, placebo-controlled study with open-label extension (OLE) and planned enrollment of 18 to 28 patients from sites in Asia, Australia, Europe, Israel, and the United States. Patients aged ≥12 years with a confirmed CXCR4 mutation consistent with WHIM syndrome phenotype and a screening absolute neutrophil count (ANC) ≤400 cells/µL without clinical evidence of active systemic infection are eligible for enrollment. Patients are randomized 1:1 to receive mavorixafor (400 mg in adults, and adolescents weighing >50 kg; 200 mg in adolescents weighing ≤50 kg) or matching placebo once daily for a total of 52 weeks (≥9 patients per group). Patients who complete the randomized period or are granted early release due to recurrent infections requiring treatment (≤2 requiring hospitalization or 4 requiring intravenous antibiotic or granulocyte colony-stimulating factor) are eligible to enroll in the OLE and receive mavorixafor until commercial availability or study termination. The primary end point is the number of hours above ANC threshold of 500 cells/µL over a 24-hour period, assessed prior to treatment, and 4 times (every 3 months) over the 52-week randomized period. Secondary end points include infection rates adjudicated by a blinded, independent committee, change from baseline in cutaneous warts also with blinded assessment, number of hours above absolute lymphocyte count (ALC) of 1000 cells/µL over a 24-hour period, and patient-reported outcomes such as work/school absence and quality of life assessment using age-appropriate questionnaires. Patient Demographics and Characteristics: As of July 15 2021, the first 18 patients have enrolled from 10 countries and 15 sites. Of these, 56% are pediatric patients, 56% are males, 56% of the patients have warts, 94% have nonsense and 6% have frameshift CXCR4 mutations. All patients had severe neutropenia (ANC ≤400 cells/μL) and majority had significant lymphopenia (ALC ≤1000 cells/μL). Conclusions: Evidence supporting efficacy of current therapies in WHIM syndrome is lacking, and approved therapies targeting the underlying molecular mechanisms and hence, able to address the full clinical spectrum of this condition, are needed. The 4WHIM study is the first double-blind, placebo-controlled, randomized trial in patients with this syndrome and is an important next step in the clinical development of a novel, orally bioavailable targeted therapy for WHIM syndrome and potentially for other individuals with related cellular immunodeficiencies. This robust study will build on the findings of the phase 2 trial that suggested a clinical benefit of mavorixafor for WHIM syndrome by incorporating a broader, global population of patients observed for a longer period of time. Full phase 3 trial results are anticipated in late 2022. Disclosures Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding. Azar: X4 Pharmaceuticals: Research Funding. Badolato: Angelini: Consultancy; X4 Pharmaceuticals: Consultancy; Janssen: Consultancy; SOBI (IDMC): Other. Bhandari: X4 Pharmaceuticals: Current Employment. Belschner: X4 Pharmaceuticals: Current Employment. Cadavid: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dickerson: Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Hoffman: Zomagen: Research Funding; Novartis: Consultancy, Speakers Bureau; Takeda: Research Funding; Jecure: Research Funding. Jiang: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kang: Cartexell: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Korea: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen Korea: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kulagin: Roche: Speakers Bureau; Sanofi: Speakers Bureau; Generium: Speakers Bureau; Biocad: Research Funding; Apellis: Research Funding; Alexion: Research Funding; X4 Pharmaceuticals: Research Funding; Novartis: Speakers Bureau; Johnson & Johnson: Speakers Bureau; Pfizer: Speakers Bureau. Langguth: RCPA: Membership on an entity's Board of Directors or advisory committees. MacLeod: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sharathkumar: Amgen: Research Funding; BMS: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; X4 Pharmaceuticals: Research Funding; NIH Trials of Pediatric VTE: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Shcherbina: X4 Pharmaceuticals: Speakers Bureau. Tang: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Vossen: X4 Pharmaceuticals: Other: PI in WHIM Trial; Brothers of St. John: Consultancy; Austrian National Bank: Research Funding; Menarini: Honoraria; Gilead: Honoraria; Astro Pharma: Honoraria.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a genetic disease characterized by neutropenia, lymphopenia, susceptibility to infections, and myelokathexis, which describes degenerative changes of mature neutrophils and hyperplasia of bone marrow myeloid cells. Some patients present with hypogammaglobulinemia and/or refractory warts of skin and genitalia. Congenital cardiac defects constitute uncommon manifestations of the disease. The disorder, which is inherited as an autosomal dominant trait, is caused by heterozygous mutations of the chemokine receptor CXCR4. These mutations lead to an increased sensitivity of neutrophils and lymphocytes to the unique ligand CXCL12 and to an increased accumulation of mature neutrophils in the bone marrow. Despite greatly improved knowledge of the disease, therapeutic choices are insufficient to prevent some of the disease outcomes, such as development of bronchiectasis, anogenital dysplasia, or invasive cancer. The available therapeutic measures aimed at preventing the risk for infection in WHIM patients are discussed. We critically evaluate the diagnostic criteria of WHIM syndrome, particularly when WHIM syndrome should be suspected in patients with congenital neutropenia and lymphopenia despite the absence of hypogammaglobulinemia and/or warts. Finally, we discuss recent results of trials evaluating plerixafor, a selective antagonist of CXCR4, as a mechanism-oriented strategy for treatment of WHIM patients.

Background: WHIM syndrome (warts, hypogammaglobulinemia, immunodeficiency, myelokathexis) is a very rare autosomal dominant immunodeficiency disorder attributable to mutations in CXCR4. Neutropenia, lymphocytopenia and monocytopenia occur because the CXCR4 mutation impairs normal leukocyte trafficking. This disease is of special interest with the finding that CXCR4 antagonists can increase blood leukocytes and may be a therapeutic option for these patients. (Dale DC et al EHA abstract, Jun 16, 2018; McDermott DH et al., N Engl J Med 2019; 380: 163). Methods: Through the Severe Chronic Neutropenia International Registry (SCNIR) we identified 9 WHIM families in the US with 24 affected members: 13 female, 11 male, median age 31.9 years, mean 35.2 +/- 21.6 SEM (range 0.4 to 82), with mutations p.R334x (16), p.E343x (3), p.fs344x (3), or p.fs365x (2). As expected, family members had the same CXCR4 mutation. In these families, the diagnosis of WHIM syndrome usually followed from a patient seeing a physician because of recurrent fevers and infections and having a CBC revealing a very low WBC. Findings: We investigated the diversity of clinical manifestations and outcomes in these patients. Immunoglobulin levels were low but not extremely low, e.g., IgG mean of 683 mg/dL +/- 53.1, median 668 mg/dL, range 443-1188 mg/dL, and only 2 patients were treated with long-term IVIG treatment. There was no apparent difference in outcomes. Most patients have received routine vaccinations and appear to mount normal or near normal IgG responses to tetanus, rubella, rubeola and H. influenza type B, based on serial observations in 6 patients. By contrast, response to human papilloma virus vaccine in these same patients appeared to be much weaker and unsustained. Off all therapies and absent infection, profound leukopenia (WBC mean 1.83 x 109/L +/- 0.21, median 1.48 x 109/L, range 0.30 - 7.90 x 109/L, neutropenia mean 0.42 x 109/L +/- 0.08, median 0.21 x 109/L, range 0.00 - 6.48 x 109/L, lymphocytopenia mean 1.18 x 109/L +/- 0.20, median 0.84, range 0.04 - 4.14) was a consistent finding across ages and mutations. Fifteen patients received low dose G-CSF (median dose 1.56 mcg/kilogram/day) most intermittently but one patient was on G-CSF for 28 years. G-CSF elevated neutrophils but did not increase other leukocyte counts. Intermittent bacterial or presumed bacterial infections (otitis, sinusitis, pharyngitis, bronchitis, skin abscesses and cellulitis) were relatively common. Several patients were known to have hearing loss secondary to repeated episodes of otitis media. Serious infections requiring hospitalizations were rare, estimate at less than one per year. Warts were also quite variable within families living in the same household. There was no apparent genotype/phenotype association of warts but they did tend to accumulate with age and did not regress spontaneously. In this cohort, 4 patients developed malignancies: 1 AML (death age 12.3 years), 2 HPV-associated cervical cancer (death ages 46 and 73), and 1 lymphoma (death age 48). Cervical dysplasia is a frequent problem and concern. Conclusions: Leukopenia, neutropenia and lymphocytopenia are the most consistent features of WHIM syndrome. Hearing loss, disfiguring and disabling warts and HPV associated malignancies are its most serious complications. The variability of warts, respiratory and skin infections may be due to environmental factors. The promise of novel therapies should lead to more frequent diagnosis and early treatment to prevent the numerous serious complications of this syndrome. Disclosures Dale: Athelas: Equity Ownership; Amgen: Consultancy, Research Funding; Hospira: Consultancy; Prolong: Consultancy; Beheringer/Ingelheim: Consultancy; Cellerant: Other: Scientific Advisory Board; Coherus: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Consultancy, Honoraria.

WHIM syndrome is a rare congenital immunodeficiency disorder characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (neutropenia because of impaired egress from the BM); most patients also have severe panleukopenia. Because WHIM syndrome is caused by mutations in the chemokine receptor CXCR4 that result in increased agonist-dependent signaling, we hypothesized that the CXCR4 antagonist plerixafor (Mozobil [Genyzme Corporation], AMD3100), might be an effective treatment. To test this, we enrolled 3 unrelated adult patients with the most common WHIM mutation, CXCR4R334X, in a phase 1 dose-escalation study. Plerixafor increased absolute lymphocyte, monocyte, and neutrophil counts in blood to normal without significant side effects in all 3 patients. Peak responses occurred at 3-12 hours after injection and waned by 24 hours after injection which tracked the drug's pharmacokinetics. All 3 cell types increased in a dose-dependent manner with the rank order of responsiveness absolute lymphocyte > monocyte > neutrophil. These data provide the first pharmacologic evidence that panleukopenia in WHIM syndrome is caused by CXCL12-CXCR4 signaling-dependent leukocyte sequestration, and support continued study of plerixafor as mechanism-based therapy in this disease. This study is registered at http://www.clinicaltrials.gov as NCT00967785.

The Severe Chronic Neutropenia International Registry (SCNIR) in cooperation with Washington University has developed a next-generation sequencing assay for the genetic diagnosis of chronic neutropenia. Exome-based sequencing is performed, and genetic alterations in 27 genes implicated in the pathogenesis of severe congenital neutropenia (SCN) are reported. Of note, with consent, patients are enrolled in the SCNIR to collect detailed clinical data and to allow researchers access to the exome sequence data to identify new genetic alterations contributing to disease pathogenesis, severity, and progression to acute myeloid leukemia. Here, we report sequencing results for 61 cases of SCN, 31 cases of cyclic neutropenia, and 3 cases of idiopathic or unclassified neutropenia. The median age of this cohort was 17.2 years (range 0.13 to 78 years). Ninety-eight per cent of the SCN cases received G-CSF with a median dose of 1.85 mg/kg/day (range, 0-99.3), and 87% of cyclic neutropenia cases received G-CSF with a median dose of 0.34 mg/kg/day (range, 0-5.2). ELANE mutation status was previously determined for the majority of these cases. In total, 16 of 95 cases of neutropenia had a known ELANE mutation, and the remainder of the cases had no genetic diagnosis. Initial analyses focused on the following genes previously implicated in SCN pathogenesis: ELANE, AP3B1, CSF3R, CXCR2, CXCR4, G6PC3, GFI1, HAX1, LYST, LAMTOR2, RAB27A, SBDS, SLC37A4, SRP54, TCIRG1, VPS13B, VPS45, USB1, TAZ, and WAS. Heterozygous mutations of ELANE were identified in the 16 previously identified cases, but in no additional cases. Of the remaining 50 cases of SCN, probable pathogenic mutations were identified in 8 (16%). Surprisingly, in the 27 cases of cyclic neutropenia with wildtype ELANE, no probable pathogenic mutations were identified. We identified three cases of SCN (including two family members) with biallelic mutations of G6PC3 and single cases of SCN with N382S GFI or R184C TCIRG1 mutations. A novel homozygous R547X CSF3R mutation was seen in one case of SCN. This truncation mutation in the extracellular domain of the G-CSF receptor is predicted to result in a complete loss-of-function allele. Indeed, this patient was refractory to G-CSF therapy. We also identified a single case of SCN carrying a R338X CXCR4 mutation, the cause of WHIM syndrome. Of note, emerging data suggest that the neutropenia in WHIM syndrome can be effectively treated with CXCR4 antagonists. We identified two cases of SCN carrying heterozygous mutations of SRP54, G175E and T191I, respectively. These SRP54 mutations, although not previously reported, localize to the GTPase domain of SRP54 (similar to previously identified SRP54 mutations), and they have not been identified in over 60,000 individuals in the ExAC database. Conclusions: Extended genetic testing in patients with chronic neutropenia and wildtype ELANE, although identifying a pathogenic mutation in the minority of cases, may impact the choice of therapy. Analyses of the exome sequence data are underway to identify novel candidate genes contributing to neutropenia. Disclosures Dale: Athelas, Inc.: Equity Ownership; Amgen: Consultancy, Research Funding; Sanofi-Aventi: Consultancy, Honoraria; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer-Ingelheim: Consultancy; Coherus: Consultancy.

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) Syndrome is a serious, ultrarare genetic disorder caused by a gain-of-function mutation in CXCR4 that causes profound neutropenia and lymphopenia associated with increased rates of bacterial and viral infections. Long-term sequelae include hearing loss, bronchiectasis, and HPV-associated malignancies. The cancer risk in WHIM patients is estimated at 30% by age 40. There is currently no treatment that addresses the underlying mechanism of disease. G-CSF increases neutrophil counts but does not appear to reduce infection rates; use of parenteral gamma globulin has had mixed reports of benefit, but no controlled trials have been reported; Plerixafor, a CXCR4 antagonist, has been shown to increase leukocyte counts and reduce infection rates when absolute neutrophil counts (ANC)>600 mm-3 and absolute lymphocyte counts (ALC)>1000 mm-3, but is a parenteral agent that must be administered twice daily. X4P-001 is an orally available, allosteric inhibitor of CXCR4 that is being developed to treat WHIM. We report here the PK/PD data from the Phase 2 portion of Phase 2/3 trial X4P-001 MKKA (NCT03005327) in WHIM patients that led to selection of the dose for Phase 3 development. Methods: Study X4P-001 MKKA is a Phase 2/3 study. The primary objective of Ph2 portion was to assess the dose required to achieve a consistent increase in circulating neutrophils and lymphocytes. Adult patients with WHIM syndrome, with documented WHIM-associated genotype, and either ANC Results: Eight patients were enrolled and were evaluable for efficacy. 6 of 8 were female, and the mean (SD) age was 35.5 (13.37). Two patients started treatment with 50 mg, then received, 100, 150, 300 and 400 mg; 2 started at 100 and then received 200 and 300 mg; 2 started at 200 mg; 1 escalated to 300 and 400 mg while the other terminated after 1 week due to an adverse event (AE). Two started at 300 mg and did not escalate. Doses of at least 300 mg/day were needed to achieve success criteria for ANC, while at doses ≥100 mg/day, 50-100% met success criteria for ALC. (Figure 1) Two patients discontinued: 1 due to an AE of grade 1 rash, and 1 voluntarily withdrew. No serious AEs were reported. One patient had unrelated cholecystitis (grade 3). All other AEs were grade 1 or 2. Conclusions: Treatment of WHIM patients with X4P-001 results in meaningful increases in ANC at doses ≥300 mg, and in ALC at doses ≥100 mg per day for at least 5 weeks. These doses are anticipated to be associated with clinically meaningful outcomes in terms of infections and warts. X4P-001 appeared to be safe and well-tolerated at doses of 50-400 mg per day.4 Disclosures Dale: Athelas, Inc.: Equity Ownership; Amgen: Consultancy, Research Funding; Sanofi-Aventi: Consultancy, Honoraria; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer-Ingelheim: Consultancy; Coherus: Consultancy. Hartmann:Cetara: Employment; X4P Pharmaceuticals: Consultancy. Brown:Certara: Employment; X4P Pharmaceuticals: Consultancy. Ebrahim:X4Pharmaceuticals: Employment. Gorelick:X4 Pharma: Consultancy; PIN Pharma: Consultancy; Shire: Consultancy; NGN Capital: Consultancy, Other: Venture partner; IntraBio: Consultancy; Zymo Consulting Group LLC: Employment.

CXCR4 is a G protein–coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypo-gammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of CXCR4-SDF1 interactions in regulating neutrophil motility in vivo is not known. Here, we use the optical transparency of zebrafish to visualize neutrophil trafficking in vivo in a zebrafish model of WHIM syndrome. We demonstrate that expression of WHIM mutations in zebrafish neutrophils induces neutrophil retention in hematopoietic tissue, impairing neutrophil motility and wound recruitment. The neutrophil retention signal induced by WHIM truncation mutations is SDF1 dependent, because depletion of SDF1 with the use of morpholino oligonucleotides restores neutrophil chemotaxis to wounds. Moreover, localized activation of a genetically encoded, photoactivatable Rac guanosine triphosphatase is sufficient to direct migration of neutrophils that express the WHIM mutation. The findings suggest that this transgenic zebrafish model of WHIM syndrome may provide a valuable tool to screen for agents that modify CXCR4-SDF1 retention signals.

The chemokine receptor CXCR4 and its functional ligand, CXCL12, are essential regulators of development and homeostasis of hematopoietic and lymphoid organs. Heterozygous truncating mutations in the CXCR4 intracellular tail cause a rare genetic disease known as WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), whose pathophysiology remains unclear. We report CXCR4 function in 3 patients with WHIM syndrome carrying heterozygous truncating mutations of CXCR4. We show that CXCR4 gene mutations in WHIM patients do not affect cell surface expression of the chemokine receptor and its internalization upon stimulation with CXCL12. Moreover, no significant differences in calcium mobilization in response to CXCL12 are found. However, the chemotactic response of both polymorphonuclear cells and T lymphocytes in response to CXCL12 is increased. Furthermore, immunophenotypic analysis of circulating T and B lymphocytes reveals a decreased number of memory B cells and of naive T cells and an accumulation of effector memory T cells associated with a restricted T-cell repertoire. Based on our results, we suggest that the altered leukocyte response to CXCL12 may account for the pathologic retention of mature polymorphonuclear cells in the bone marrow (myelokathexis) and for an altered lymphocyte trafficking, which may cause the immunophenotyping abnormalities observed in WHIM patients. (Blood. 2004;104:444-452)

In this issue of Blood , Balabanian and colleagues describe a mouse model of WHIM syndrome, a combined myeloid and lymphoid immunodeficiency disorder.[1][1] The results provide a high-definition histopathologic picture of the effects of WHIM mutation CXCR41013 on immune organs and suggest complex

Background: WHIM syndrome (WHIMs; Warts, Hypogammaglobulinemia, Infections, Myelokathexis) is a rare autosomal dominant immunodeficiency disease attributable to mutations in CXCR4 . Most patients have severe neutropenia and lymphocytopenia with total white blood cell counts (WBC) Methods: We have begun the phase 2, dose-titration portion of a phase 2/3 study of a novel, orally bioavailable, selective, CXCR4 antagonist, X4P-001, in adult patients with genetically confirmed WHIMs to evaluate safety and tolerability of X4P-001 and determine the dose for a phase 3 study. All patients were required to be off chronic immunoglobulin and/or G-CSF treatment while on study. Patients receive increasing doses of X4P-001 ranging from 50 mg to 300 mg orally once daily with periodic monitoring of serial blood counts and intra-patient dose escalation is based on 24-hour serial area-under-the-curve (AUC) measurements of absolute neutrophil counts (ANC) and absolute lymphocyte counts (ALC) with prespecified thresholds. Results: As of July 10, 2017, 4 patients (2 females, 2 males; age range 19 to 57 years; 3 with R334X and 1 with E343X mutation) have been enrolled. The study entry screening WBC was 0.775 x 109/L +/-0.16 (mean +/- SEM); ANC was 0.125 x 109/L +/- 0.027; and ALC was 0.588 x 109/L +/- 0.155. All patients had mild hypogammaglobulinemia; the mean levels were: Immunoglobulin G 766 +/- 130 (SEM), IgA 50 +/- 17, IgM 74 +/- 13. In the 6 months prior to study entry, these adult patients had up to 6 episodes of infections, primarily upper respiratory and skin infections. Patients 1 and 2 have escalated through 50 mg/day and 100 mg/day to 150 mg/day over a total exposure period of 169 days. Patients 3 and 4 have escalated from 100 mg/day to 200 mg/day with an exposure period of 62 and 54 days, respectively. All patients have demonstrated a dose-dependent increase in ANC and ALC from screening values, with ALC increasing in greater proportion than ANC. At the 100 mg dose level, the pre-dose WBC was 1.24 x 109/L +/- 0.469 (mean +/- SEM) with peak levels of 2.8 x 109/L +/- 0.721 (increase=2.3 fold), the pre-dose ANC was 0.385 x 109/L +/- 0.178) with peak levels of 0.58 x 109/L +/- 0.202 (increase=1.5 fold) and pre-dose ALC was 0.728 x 109/L +/- 0.276 with peak levels of 2.143 x 109/L +/-0.513 (increase=2.9 fold). The increases in the pre-dose levels of both neutrophils and lymphocytes above the study entry screening values after 1-3 months on treatment suggest a continuing effect of daily exposure to this drug. However, the ANC and ALC responses thus far are below the targeted AUC for ANC and ALC, so dose escalation continues. Thus far, X4P-001 has been well tolerated and the most common treatment-related adverse events are mild dyspepsia and dry eyes. Patients have not required antibiotic treatment for infections during the cumulative 15 months on X4P-001, except for Patient 1 who had an infection at the start of the study. Conclusion: Preliminary data suggests X4P-001 is a promising oral agent for treating WHIM syndrome. Disclosures Dale: Sanofi Aventis: Consultancy, Editor, Current Opinions in Hematology, Honoraria; Cellerant: Membership on an entity's Board of Directors or advisory committees; Genzyme (now owned by Sanofi-Aventis): Consultancy, Patents & Royalties, Research Funding; Genkyotex: Other: DSMB (work completed 6/2015); Hospira: Consultancy; Prolong: Consultancy; Boheringer-Ingelheim: Consultancy; Coherus: Consultancy; Omeros: Other: DSMB; Shire: Other: Independent Review Board; X4Pharma: Research Funding; Philips: Research Funding; Wolter Kluwer: Other: Editor, Current Opinions in Hematology; WedMD/Medscape: Membership on an entity's Board of Directors or advisory committees; National Institutes of Health: Research Funding; University of Washington: Employment, Research Funding; American College of Physicians: Other: Editor and author; GlaxoSmithKline: Equity Ownership; Johnson&Johnson: Equity Ownership; Amgen: Consultancy, Research Funding. Johnson: X4 Pharmaceuticals: Employment. Gan: X4 Pharmaceuticals: Employment, Equity Ownership. Parasuraman: X4 Pharmaceuticals: Employment, Equity Ownership.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare immunodeficiency disorder caused by gain-of-function mutations in the G protein-coupled chemokine receptor CXCR4. The CXCR4 antagonist plerixafor, which is approved by the US Food and Drug Administration (FDA) for stem cell mobilization in cancer and administered for that indication at 0.24 mg/kg, has been shown in short-term (1- to 2-week) phase 1 dose-escalation studies to correct neutropenia and other cytopenias in WHIM syndrome. However, long-term safety and long-term hematologic and clinical efficacy data are lacking. Here we report results from the first long-term clinical trial of plerixafor in any disease, in which 3 adults with WHIM syndrome self-injected 0.01 to 0.02 mg/kg (4% to 8% of the FDA-approved dose) subcutaneously twice daily for 6 months. Circulating leukocytes were durably increased throughout the trial in all patients, and this was associated with fewer infections and improvement in warts in combination with imiquimod; however, immunoglobulin levels and specific vaccine responses were not fully restored. No drug-associated side effects were observed. These results provide preliminary evidence for the safety and clinical efficacy of long-term, low-dose plerixafor in WHIM syndrome and support its continued study as mechanism-based therapy in this disease. The ClinicalTrials.gov identifier for this study is NCT00967785.

WHIM syndrome is a rare, autosomal dominant, immunodeficiency disorder so-named because it is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (defective neutrophil egress from the BM). Gain-of-function mutations that truncate the C-terminus of the chemokine receptor CXCR4 by 10-19 amino acids cause WHIM syndrome. We have identified a family with autosomal dominant inheritance of WHIM syndrome that is caused by a missense mutation in CXCR4, E343K (1027G → A). This mutation is also located in the C-terminal domain, a region responsible for negative regulation of the receptor. Accordingly, like CXCR4R334X, the most common truncation mutation in WHIM syndrome, CXCR4E343K mediated approximately 2-fold increased signaling in calcium flux and chemotaxis assays relative to wild-type CXCR4; however, CXCR4E343K had a reduced effect on blocking normal receptor down-regulation from the cell surface. Therefore, in addition to truncating mutations in the C-terminal domain of CXCR4, WHIM syndrome may be caused by a single charge-changing amino acid substitution in this domain, E343K, that results in increased receptor signaling.

Desensitization controls G protein–dependent signaling of chemokine receptors. We investigate the physiologic implication of this process for CXCR4 in a mouse model harboring a heterozygous mutation of the Cxcr4 gene, which engenders a desensitization-resistant receptor. Such anomaly is linked to the warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, a human rare combined immunodeficiency. Cxcr4+/mutant(1013) mice display leukocytes with enhanced responses to Cxcl12 and exhibit leukopenia as reported in patients. Treatment with CXCL12/CXCR4 antagonists transiently reverses blood anomalies, further demonstrating the causal role of the mutant receptor in the leukopenia. Strikingly, neutropenia occurs in a context of normal bone marrow architecture and granulocyte lineage maturation, indicating a minor role for Cxcr4-dependent signaling in those processes. In contrast, Cxcr4+/1013 mice show defective thymopoiesis and B-cell development, accounting for circulating lymphopenia. Concomitantly, mature T and B cells are abnormally compartmentalized in the periphery, with a reduction of primary follicles in the spleen and their absence in lymph nodes mirrored by an unfurling of the T-cell zone. These mice provide a model to decipher the role of CXCR4 desensitization in the homeostasis of B and T cells and to investigate which manifestations of patients with WHIM syndrome may be overcome by dampening the gain of CXCR4 function.

WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelokathexis) is a rare autosomal dominant immunodeficiency disorder that is caused by a gain in function in cysteine-X-cysteine chemokine receptor 4 (CXCR4) gene. Affected individuals have recurrent infections of the respiratory tract and soft tissues, and marked susceptibility to warts caused by human papilloma viruses (HPV). Patients have an increased risk of malignancy (lymphoma) and HPV-associated cancers. The optimal therapy for WHIM syndrome has not been defined and treatment has been with supportive care using intravenous immunoglobulin (IVIG) and granulocyte colony stimulating factor (G-CSF). New CXCR4 antagonist therapies are still in either phase I trials (plerixafor) or considered experimental (Chlacone-4). A successful umbilical cord blood transplant has been reported by Kriven et al. Here we describe a successful matched unrelated allogeneic stem cell transplant (SCT) in a girl with WHIM syndrome caused by a known mutation in the CXCR4 gene. This patient was diagnosed at birth with familial congenital neutropenia as findings were similar to those for the patient's mother and maternal grandmother. A complete blood count showed pancytopenia (WBC= 1.03 x103/µL, hemoglobin= 9.2 g/dL, platelet count= 74,000/µL, ANC= 70). Despite G-CSF and IVIG therapy, the patient had recurrent infections with sinusitis and pneumonia, along with progressive organomegaly with progressive pancytopenia. At the age of 4, the patient underwent matched unrelated SCT with a fully ablative regimen [busulfan 0.8 mg/kg/dose for 16 doses (days -9, -8, -7, and -6), cyclophosphamide 50 mg/kg/dose for 4 doses (days -5, -4, -3, and -2) and alemtuzumab 5 mg/day (weight-based dosage) for 3 doses (days -5, -4, and -3)]. Tacrolimus and mini methotrexate (on days +1, +3, +6 and +11) were given for GVHD prophylaxis. Neutrophil and platelet engraftment occurred on days + 21 and + 45, respectively. The patient is 100% donor chimeric. Post-transplant course has been complicated by grade II skin GVHD treated successfully with oral and topical steroids. The patient also developed adenovirus, BK virus and HHV-6 viral reactivations but without disease. The patient is now approximately 2 years post SCT with durable engraftment, normal humoral immune reconstitution and no chronic GVHD. Thus, stem cell transplant following myeloablative conditioning can be accomplished for patients with WHIM syndrome. Furthermore, transplantation performed at the patient's young age likely prevented complications of recurrent infections and warts. Disclosures Allen: NovImmune: Consultancy, Other: unpaid; Roche: Consultancy, Other: unpaid. Heslop:Cell Medica: Other: Licensing Agreement; Celgene: Other: Collaborative research agreement.

Introduction: Warts, Hypogammaglobulinemia, Infections and Myelokathexis Syndrome (WHIMS) is an autosomal dominant immunodeficiency resulting from gain-of-function mutations in the chemokine receptor CXCR4. We recently described a unique WHIMS patient who underwent spontaneous genetic and phenotypic reversion at approximately age 30 after being severely affected as a child. Her reversion was due to a single catastrophic genetic event known as chromothripsis (chromosome shattering) resulting in the deletion of one copy of 163 genes in addition to her mutant copy of CXCR4 on chromosome 2. This event was traced to a hematopoietic stem cell (HSC) that had spontaneously repopulated her bone marrow; however, which of the genes was responsible and the mechanism required further investigation. Methods: Mouse models of CXCR4 haploinsufficiency (Cxcr4+/o) and WHIMS (Cxcr4+/S338X) were used in competitive bone marrow repopulation experiments transplanting whole bone marrow cells or purified HSC. Recipient mice were treated with / without lethal irradiation prior to transplant. Genome editing with TALENs and CRISPR-Cas9 technology was used to target CXCR4 for deletion in human cell lines. Results: Cxcr4 haploinsufficiency markedly enhanced HSC engraftment potential in recipient WHIM mice whether the donor HSC were purified from whole bone marrow cells or not, and whether the recipient was conditioned by lethal irradiation or not. Enhanced engraftment by Cxcr4 haploinsufficient donor HSC also occurred in wild-type mouse recipients, but to a lesser extent, and was also HSC intrinsic. Genome editing experiments have been successful at deleting one or both copies of CXCR4 in human cell lines in up to 40% of treated cells, and in reducing CXCR4 surface expression. Conclusion: While CXCR4 was already understood to be important in HSC biology, this patient and subsequent murine experiments have proven that the gene dosage of CXCR4 is a critical factor affecting HSC engraftment. Genome editing is a promising technology for deleting one copy of CXCR4, ideally the WHIM allele,in autologous HSC as a strategy to cure WHIM syndrome. Disclosures McDermott: US National Institutes of Health: Employment, Patents & Royalties: pending. Jacobs:US National Institutes of Health: Employment, Patents & Royalties: pending. Liu:US National Institutes of Health: Employment, Patents & Royalties: pending. Gao:US National Institutes of Health: Employment, Patents & Royalties: pending. Murphy:US National Institutes of Health: Employment, Patents & Royalties: pending.

Introduction: WHIM syndrome is a rare autosomal dominant, primary immunodeficiency disease characterized by warts, hypogammaglobulinemia, infections and myelokathexis (impaired neutrophil egress from bone marrow resulting in neutropenia). Mutations in the C-terminus of chemokine receptor CXCR4 cause almost all known cases of WHIM syndrome (4 truncations, 3 frame-shifts and 1 single amino acid charge-change), and result in impaired ligand-induced receptor internalization and desensitization. This enhances receptor signaling, and exaggerates normal CXCR4 function to retain neutrophils and other leukocytes in bone marrow, resulting in myelokathexis and increased susceptibility to certain infections. Methods: A 2 year old boy with recurrent infections and neutropenia was referred to the NIH Clinical Center. Four anonymized healthy donors were recruited as homozygous CXCR4WT controls. Genomic DNA was isolated from peripheral blood and the CXCR4 gene was sequenced using the Sanger method. The eukaryotic expression vector pcDNA3.1(-) was used to make 3 recombinant plasmids encoding the wild type gene, (CXCR4WT) the most common disease mutation (CXCR4R334X) and the index patient's mutation (CXCR4L329fs). K562 cells were transfected with plasmids by nucleofection. Blood leukocyte subsets and CXCR4 expression on PBMCs from healthy donors and the patient and stably transfected K562 cells with or without CXCL12 stimulation were examined by FACS analysis. Calcium flux in response to CXCL12 was analyzed in receptor-transfected and parental cell lines as well as in primary leukocytes. Results: The boy had recurrent infections, hypogammaglobulinemia, panleukopenia and myelokathexis, consistent with WHIM syndrome but both parents were healthy. Genetic analysis revealed a novel heterozygous mutation in CXCR4, designated CXCR4L329fs, involving a 5 base pair deletion from nucleotides 986-990 in the open reading frame. This deletion causes a frame-shift at codon 329 that introduces 12 missense amino acids and deletes the conserved C-terminal 24 amino acids. Interestingly a different mutation that causes a frame-shift at the same codon has been recently described as a somatic mutation in one case of Waldenström's macroglobulinemia, which deletes nucleotide 984-986 and inserts a T. The CXCR4L329fs mutation in our patient is the largest truncation mutant reported to date in WHIM syndrome, and was not identified in unaffected family members or in the general population, suggesting that it is a de novo mutation. As with the most common WHIM mutation, CXCR4R334X, this new mutation also caused decreased receptor downregulation in response to CXCL12 in both PBMCs from the patient as well as in K562 cells transfected with the cloned receptor and increased CXCR4 calcium flux signaling in stably transfected K562 cells. Conclusion: CXCR4L329fs is a novel de novo gain-of-function mutation of the C-terminus of CXCR4 that causes WHIM syndrome. Disclosures Liu: US National Institutes of Health: Employment, Patents & Royalties: pending. Murphy:US National Institutes of Health: Employment, Patents & Royalties: pending. McDermott:US National Institutes of Health: Employment, Patents & Royalties: pending.

WHIM syndrome is a rare congenital immunodeficiency disorder characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (neutropenia because of impaired egress from the BM); most patients also have severe panleukopenia. Because WHIM syndrome is caused by mutations in the chemokine receptor CXCR4 that result in increased agonist-dependent signaling, we hypothesized that the CXCR4 antagonist plerixafor (Mozobil [Genyzme Corporation], AMD3100), might be an effective treatment. To test this, we enrolled 3 unrelated adult patients with the most common WHIM mutation, CXCR4R334X, in a phase 1 dose-escalation study. Plerixafor increased absolute lymphocyte, monocyte, and neutrophil counts in blood to normal without significant side effects in all 3 patients. Peak responses occurred at 3-12 hours after injection and waned by 24 hours after injection which tracked the drug's pharmacokinetics. All 3 cell types increased in a dose-dependent manner with the rank order of responsiveness absolute lymphocyte > monocyte > neutrophil. These data provide the first pharmacologic evidence that panleukopenia in WHIM syndrome is caused by CXCL12-CXCR4 signaling-dependent leukocyte sequestration, and support continued study of plerixafor as mechanism-based therapy in this disease. This study is registered at http://www.clinicaltrials.gov as NCT00967785.

High expression of CC chemokine receptor 4 (CCR4) has been identified as a hallmark gene in ATLL, an aggressive peripheral T-cell neoplasm. CCR4 is a chemokine receptor, which has a critical role in immune cell trafficking including Th2, T-reg and skin-homing memory T-cells. CCR4 ligands, CCL17 and CCL22, were produced in lymph nodes and skin from dendritic cells, macrophages and Langerhans cells. Most ATLL cases express surface CCR4 (90%) and infiltrate to lymph nodes and skin. These observations suggest that CCR4 could have a role in ATLL biology, but it is still unclear whether dysregulation of CCR4 function contributes to ATLL pathogenesis. We performed RNA-Seq for two primary ATLL cases and discovered recurrent non-sense mutations in CCR4. Though an extended analysis using Sanger sequencing, CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxy-terminus. We noticed that the location of the CCR4 mutations in ATLL were reminiscent of mutations affecting the chemokine receptor CXCR4 in the WHIM syndrome, a human immunodeficiency disease. Most CXCR4 mutations in the WHIM syndrome are nonsense or frameshift, resulting in carboxy-terminal truncation of the protein and conferring a gain-of-function phenotype with respect to chemotaxis towards the CXCR4 ligand SDF1. We therefore hypothesized that mutant CCR4 isoforms might enhance chemotaxis of the affected cells to CCR4 ligands. Chemotaxic assay using 32Db, a mouse myeloid cell line, and ED40515(+), an ATLL cell line, clarified that the ectopic expression of CCR4-Q330* enhanced the chemotactic ability of the transduced cells toward CCL17 and CCL22 rather than CCR4-WT transduced cells with statistical significance. To understand the mechanism of this enhanced chemotactic ability, we studied the change in surface CCR4 levels after CCL22 exposure in CCR4-WT-and CCR4-Q330*-reconstituted ED40515(+) cells. Compared with CCR4-WT, CCR4 internalization in CCR4-Q330*-reconstituted cells was significantly impaired. Thus, the ATLL CCR4 mutants impair desensitization by ligand, which likely contributes to the enhanced chemotaxis of cells bearing these mutants. We explored the influence of the ATLL CCR4 mutants on PI(3) kinase (PI3K)-dependent activation of AKT since it has been reported that binding of CCL22 to CCR4 activates AKT in CEM leukemic T-cells and in human Th2 cells. CCR4-Q330*-reconstituted ED40515(+) showed strong activation of AKT with CCL22 ligation compared with CCR4-WT-reconstituted cell. The AKT activation was cancelled with pan-PI3K inhibitor, BKM120, indicating that CCR4-mediated AKT activation was PI3K dependent. Lastly, we tested whether the acquisition of CCR4 mutations by ATLL cells imparts a selective growth advantage relative to cells with wild type CCR4. CCR4-Q330*-reconstituted cells had a selective growth advantage in the presence of CCL22, supporting at least in part the hypothesis that CCR4 mutation are able to provide the affected cells a positive selection pressure through CCL22 ligation and contributes to ATLL pathogenesis. We discovered for the first time recurrent somatic mutations in CCR4 in ATLL. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxy-terminus. Functionally, the CCR4-Q330* was a gain-of-function since it increased cell migration towards the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation following receptor engagement by CCL22 in ATLL cells, and conferred a growth advantage in in vitro cultures. Our findings provide a rationale to test whether inhibition of CCR4 signaling might have a therapeutic potential for patients with ATLL. Disclosures No relevant conflicts of interest to declare.

CXCR4 is a G protein–coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypo-gammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of CXCR4-SDF1 interactions in regulating neutrophil motility in vivo is not known. Here, we use the optical transparency of zebrafish to visualize neutrophil trafficking in vivo in a zebrafish model of WHIM syndrome. We demonstrate that expression of WHIM mutations in zebrafish neutrophils induces neutrophil retention in hematopoietic tissue, impairing neutrophil motility and wound recruitment. The neutrophil retention signal induced by WHIM truncation mutations is SDF1 dependent, because depletion of SDF1 with the use of morpholino oligonucleotides restores neutrophil chemotaxis to wounds. Moreover, localized activation of a genetically encoded, photoactivatable Rac guanosine triphosphatase is sufficient to direct migration of neutrophils that express the WHIM mutation. The findings suggest that this transgenic zebrafish model of WHIM syndrome may provide a valuable tool to screen for agents that modify CXCR4-SDF1 retention signals.

Introduction WHIM (Warts, Hypogammaglobulinemia, Infections and Myelokathexis) syndrome is a hereditary disorder caused by activating mutation in CXCR4 receptor. CXCR4 receptor is expressed in many cells including myeloid and lymphoid cells. Dasatanib is aTKI inhibitor and very effective drug in treating Ph+ve acute lymphoblastic leukaemia (Ph+ALL). Dasatinib has many off target effects which are not completely known as yet. Dasatinib effect on CXCR4 is not well described. In Chronic myeloid leukaemia it causes reacquisition CXCR4 activation leading to hiding of myeloid leukaemia cells inside the bone marrow. However some work has been done on effects of Dasatinib on mature neutrophil indicates adhesion defect and inhibition of pro-inflammatory effect. Here we describe two children who developed acquired WHIM syndrome like features while on Dasatinib therapy. Methods Two girls aged 9 and 11 year old with Ph+ALL were treated initially as per BFM95 protocol and Imatinib 375 mg/m2 daily. Both went in remission and later achieved complete molecular remission. However during maintenance therapy first patient at 19 months from diagnosis and second at 12 months from diagnosis started having rising value of quantitative BCR-ABL values. As both children had no siblings and no matched unrelated donor was available so in both Imatinib was stopped and after taking informed consent of parents Dasatinib was started at a dose of 100/mg/m2/day in two divided doses and maintenance therapy of oral 6-Mercaptopurine (6-MP) and Methotrexate (MTX) was continued. They both achieved reduction in BCR-ABL quantitative PCR after 3 months and molecular remission at 6 and 9 months respectively after starting Dasatinib. Results During therapy with dasatinib and oral 6-MP and MTX both children started having recurrent respiratory tract infections with neutropenia and warts appeared on face and body. Serum immunoglobulin G levels were lower than 200 mg/dl in both. We felt that these symptoms resembled WHIM syndrome and possibly could be due to Dasatinib. So we gave Intravenous immunoglobulin to both the children and IV antibiotics and stopped both Dasatinib and chemotherapy for a week. Neutropenia recovered within 72 hr after stopping of dasatinib. However, we did not perform bone marrow examination in both the cases to prove myelokathexis. Both children did well when Dasatinib was restarted at a lower dose of 50 mg/m2/day once daily and oral 6-MP and MTX. Warts disappeared over next 4-6 months and did not have any further episodes of fever or febrile neutropenia needing IV antibiotics. Their repeat serum immunoglobulin G levels stayed normal over next 6-12 months. Conclusion Both our children on dasatinib developed WHIM syndrome like features which resolved on reducing dose of Dasatinib. Dasatinib possibly mediates this side effect through CXCR4 receptor or downstream pathway. More data are needed to prove this association. Disclosures: No relevant conflicts of interest to declare.

[Figure][1] A 43-year-old man with a known history of WHIM syndrome (warts, hypogammaglobulinemia [IgG 271 mg/dL, IgM 39 mg/dL], infecions with chronic neutropenia [0.72 × 109/L], and myelokathexis, with a documented CXCR4 mutation) had previously been treated for B-cell lymphoma and

Abstract 2715 Introduction: Waldenstrom's Macroglobulinemia (WM) is an indolent non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells (LPC) in the bone marrow. Using paired normal/WM lymphoplasmacytic cell paired tissues and whole genome sequencing (WGS), we identified somatic mutations in the CXC chemokine receptor 4 (CXCR4) gene which were present in 16/55 (29%) WM patients. CXCR4 is a G-protein-coupled receptor, together with its ligand, the stromal cell- derived factor-1(CXCL12/SDF-1), play an important role in leukocyte and lymphocyte hematopoiesis and trafficking. Upon SDF-1 stimulation, CXCR4 is phosphorylated and interacts with b-arrestins, which then trigger extracellular signal-regulated kinase (ERK) MAPKs and chemotaxis. CXCR4 signaling is then terminated through receptor internalization which is mediated via phosphorylation of its C-terminal tail. Methods: Sanger sequencing was used to validate WGS results. To clarify the functional significance of one of the most common somatic mutation identified (C1013G), cloning by PCR was undertaken from CD19+ bone marrow cells from a WM patient with the C1013G CXCR4 (C1013G-CXCR4) mutation. Wild type (WT) and C1013G-CXCR4 cDNAs were subcloned into plenti-IRES-GFP vector, and transduced using an optimized lentiviral based strategy for WM cells into BCWM.1 WM cells. Five days after transduction, GFP positive cells were sorted and used for functional studies. Surface expression of CXCR4 was determined by FACS using PE-conjugated anti-CXCR4 monoclonal antibody 12G5. CXCR4 internalization was studied by comparing CXCR4 surface expression before and after SDF-1 stimulation. Chemotaxis studies were performed using a transwell assay. The expression of phosphorylated ERK1/2 and total ERK2 was determined by western blot. Results: Validated somatic mutations in CXCR4 were all present in the C-terminal domain and included premature stop codons (C1013A; C1013G), and frameshift mutations. Importantly, the identified mutations are similar to those reported in the germline of patients with WHIM (Warts, Hypogammaglobulinemia, Infections and Myelokathexis) Syndrome, a dominant autosomal genetic disorder caused by tonic CXCR4 activation by impairment of CXCR4 internalization, and stimulation of G-protein-dependent responses, and chemotaxis. Consistent with such a role, SDF-1a stimulation showed decreased internalization of CXCR4 in C1013G-CXCR4 versus WT CXCR4 transduced BCWM.1 WM cells. SDF-1a stimulated ERK1/2 phosphorylation was also more robust C1013G-CXCR4 versus WT CXCR4 expressing cells. Lastly, C1013G-CXCR4 transduced cells displayed stronger migratory response toward SDF-1a versus WT CXCR4 expressing BCWM.1 cells. Conclusions: C-terminal domain somatic mutations are common in WM and overlap with germline mutation identified in WHIM syndrome. Moreover, the most common of these mutations (C1013G) confers gain of function including decreased CXCR4 internalization, more robust ERK ½ phosphorylation, and chemotaxis. The findings provide new insights into the pathogenesis of WM, and a framework for the study of CXCR4 inhibitors for WM therapy. Disclosures: Treon: Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees.

Severe chronic neutropenia is a general term used to organize and study the inherited and acquired diseases causing blood neutrophils to be less than 0.5 × 109/L for months, years or a lifetime. The inherited or congenital disorders include severe congenital neutropenia (SCN) (the autosomal recessive form often called Kostmann syndrome), cyclic neutropenia, Shwachman-Diamond syndrome, Barth syndrome, myelokathexis or WHIM syndrome, and several other rare conditions. The genetic bases for many of these conditions are now known and the molecular, cellular and pathophysiological mechanisms are rapidly being discovered. The acquired disorders are less well characterized and include the immune, autoimmune and idiopathic neutropenias of childhood and adults. These disorders are often confused with the inherited disorders, particularly early in life. In 1994, the Severe Chronic Neutropenia International Registry (SCNIR) was established to study the causes, consequences, and treatment of severe chronic neutropenia with offices in Seattle WA, USA and Hannover, Germany. The total enrollment in the SCNIR-Seattle is currently 1100; 289 children (age less than 33 months at enrollment) and 811 patients (age greater than 33 months at enrollment). Of the 289 children, 119 were diagnosed with either idiopathic or autoimmune neutropenia. This analysis describes twenty children (8 male, 12 female, current age 3.6 to 16.6 years) with clinical diagnoses of idiopathic (11), autoimmune (5), cyclic (3), and congenital (1). These patients were enrolled in the SCNIR, followed with or without treatment of granulocyte colony stimulating factor (G-CSF), and spontaneously normalized their blood neutrophil counts. The median ANC for this population prior to G-CSF treatment was 0.263 × 109/L (range 0.061 to 0.926). All 20 patients had a pre G-CSF bone marrow examination read by a referring hematologist or pathologist and normal cytogenetics. In 15 patients there was normal maturation of the myeloid line. Five patients had “maturation arrest” at the myelocyte (1), metamyelocyte (1), or band (3) stage of neutrophil development. None were described as having early stage “maturation arrest” as is characteristically observed in SCN patients. Neutrophil recovery was defined by an increase in blood neutrophils to 2.0 × 109/L. Two patients had only mild symptoms and did not receive G-CSF before recovery occurred. The other 18 patients were treated with G-CSF, (median dose 1.375 mcg/kg/day, range 0.192 to 4.839 mcg/kg/day; duration median months 24.18, range 0.07 to 102.27 months). Before recovery, these 18 patients responded to G-CSF with an increase in the median ANC to 3.151 × 109/L, (range 0.120 to 14.553 × 109/L, all counts on G-CSF). All were reported to have diminution of recurrent fevers, mouth ulcers, and infections with G-CSF therapy. The daily dose during the first year of G-CSF treatment was positively correlated to the length of treatment: A low initial daily dose corresponded to less time to resolution (r=0.529; p=0.024). A similar relationship existed between a narrower dose range and the length of time a patient was neutropenic. Variability of dosing for this population ranged from 0.045 to 3.354 mcg/kg/day. Low variability between the highest and lowest doses was correlated to less time to resolution (r=0.66; p=0.0029). After recovery, the median ANC for 12 of the 18 patients was 3.337 × 109/L (range 2.181 to 9.282 × 109/L); the referring physicians reported normalization of blood cell counts in the other 6 cases, but did not submit documentation of these counts. No relapses and no evolution to myelodysplasia or leukemia (MDS/AML) have been reported with up to 8 years of follow-up for patients in this report. Similarly, the other 103 young children in the SCNIR with a diagnosis of idiopathic or autoimmune neutropenia have not transformed to MDS/AML. A retrospective review suggests that the patients in this report who were identified as having a diagnosis of cyclic or congenital neutropenia were probably misdiagnosed. These data confirm previous reports that spontaneous remissions may occur in children with idiopathic and autoimmune neutropenia. The data suggest that a normal marrow, normal cytogenetic analysis and response to a low dose of G-CSF are predictors of future recovery. In this patient group, treatment with G-CSF should be individualized and reserved for patients with a history of recurrent fevers and infections.

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

[Comment on Kawai et al, page 78][1]In this issue of Blood , Kawai and colleagues report careful and detailed studies on the effects of expression of mutant CXCR4 in mobilized human CD 34+ cells (peripheral blood progenitor cells or PBSCs) studied in ex vivo cultures and with engraftment into NOD/

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

Severe congenital neutropenia (CN) is a general term for a group of disorders characterized by extremely low blood neutrophil counts (ANC < 0.5 x 109 ), early stage maturation arrest of myelopoiesis, and recurrent bacterial infections. More than 90% of CN patients respond to daily G-CSF treatment with a sustained neutrophil increase associated with a significant reduction of infections and improved quality of life. However, prolonged survival unmasks an increased risk of leukemic transformation in some, but not all, subcategories of CN patients. The Severe Chronic Neutropenia International Registry (SCNIR) has collected longitudinal data on on more than 400 patients with various causes of CN. This unique resource allows classification of different subtypes of CN and estimation of the relative frequency of these conditions. Our new classification scheme is as follows: By inheritance - autosomal dominant severe congenital neutropenia (ADCN), autosomal recessive (ARCN, Kostmann syndrome), sporadic CN. By genetic aberrations - ELA2 related CN, SBDS related CN-(Shwachman-Diamond syndrome), G 4.5 on Xq28related neutropenia (Barth syndrome), CXCR4 related neutropenia (myelokathexis and WHIM syndrome). By clinical phenotype - associated symptoms (e. g. metabolic disorders, such as Shwachman-Diamond syndrome, glycogen storage disease 1b, Barth syndrome), G-CSF responsive or non-responsive CN, with or without G-CSF receptor mutations, with or without osteoporosis, or dysplastic features (e. g. organ abnormalities). By ethnic origin - e.g. CN in consanguineous Kurdish families, recessive neutropenia in Swedish family (Kostmann syndrome). Recent research and the work of the SCNIR now permit a much improved classification of CN. The identification of subtypes of CN, their distinctive risks of malignant transformation, and their responses to treatment has contributed substantially to our general understanding of the problem of neutropenia. This knowledge also now allows clinicians to give patients and families much improved prognostic information and better guidelines for therapy.