WHIM Syndrome Research Articles

Circulating B-Cell Precursor Cells As Potential Diagnostic and Therapeutic Biomarker in Patients with WHIM Syndrome

Background Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a combined immunodeficiency caused in most known cases by gain-of-function mutations in the CXCR4 gene. The wide spectrum of multisystem clinical presentations, often with incomplete penetrance and expressivity, can pose a diagnostic challenge. WHIM syndrome has historically been a clinicopathologic diagnosis, with myelokathexis being the pathognomonic feature of WHIM. As genetic testing is becoming readily available, sequencing of the CXCR4 gene is often used to confirm a clinical suspicion of WHIM syndrome by finding genetic variants. There is a need for additional easily accessible and noninvasive biomarkers for diagnosing WHIM syndrome. We hypothesize that B-cell compartment might also show pathognomonic features of WHIM syndrome. Patients and Methods We analyzed peripheral blood B-cell subsets in 13 patients diagnosed with WHIM syndrome from 8 families with pathogenic CXCR4 mutations using 9-multicolor flow cytometry after informed consent approval. Samples from 2 patients who received plerixafor were collected at baseline before and after treatment. The duration of treatment was approximately 6 months. Results Using multicolor flow cytometry, we analyzed circulating B-cell subsets (CD10,CD19,CD21,CD27,CD38,IgA,IgD,IgG and IgM) in 13 patients (pediatric, n=7; adult, n=6) with WHIM syndrome (9 patients with p.Arg334Stop mutation, 2 patients with p.Val320Glufs*23 mutation, 1 patient with p.Ser338Stop mutation, and 1 patient with p.Ser346Stop mutation). We observed severe peripheral B-cell lymphopenia, accompanied by the presence of a consistent and specific scarce CD19+CD10+CD38+IgM-IgD-CD21-CD27- B-cell subpopulation. A B-cell subpopulation with an equivalent marker expression pattern was not found in healthy controls (n=50) or in non-WHIM disease controls (n=20). The rare B-cell population found in WHIM syndrome was further characterized as belonging mostly to the pre-B-cell stage revealed by intracellular expression of IgM, CD179a (VpreB chain), and CD179b (lambda 5). The presence of these early B-cell precursors was independent of age and was stable over time in patients with WHIM syndrome. The number of these peripheral B-cell precursors found specifically in patients with WHIM syndrome was greatly reduced when the patients were treated with granulocyte colony-stimulating factor (G-CSF) or plerixafor; counts of the precursor cells returned to baseline after the patients discontinued plerixafor treatment. Conclusions Taken together, we identify a unique CD19+CD10+CD38+IgM-IgD-CD21-CD27- B-cell subpopulation in patients with WHIM syndrome that might be used as a noninvasive biomarker to help identify patients with this rare primary combined immunodeficiency. In addition, these circulating B-cell precursors may be used to monitor response to therapy in patients with WHIM syndrome. The observed impact of G-CSF on the B-cell precursor population may be attributed to downregulation of CXCL12/CXCR4, which influences myelokathexis and impairs egression of the precursor population from the bone marrow but needs further experimental validation.

Mavorixafor for Patients with Chronic Neutropenic Disorders: Results from a Phase 1b, Open-Label, Multicenter Study

Introduction Chronic neutropenia (CN) encompasses different blood disorders all characterized by low levels of neutrophils (absolute neutrophil count [ANC] <1500/µl for >3 months) and is associated with increased risk of recurrent and/or severe infections [Donadieu J. Exp RevHematol. 2021]. The most common chronic neutropenic disorders (CNDs) are chronic idiopathic neutropenia (CIN), which is not attributable to drugs or a specific infection or inflammatory, malignant, or genetic cause [Dale DC. Curr Opin Hematol. 2017]; cyclic neutropenia (CYN), characterized by recurrent episodes of neutropenia approximately every 21 days [Donadieu J. Exp Rev Hematol. 2021]; and severe congenital neutropenia (SCN), caused by genetic variants [Skokowa J. Nat Rev Dis Primers. 2018]. The current management strategy for CN is long-term treatment with injectable granulocyte colony-stimulating factor (G-CSF). Although typically effective in the short-term, G-CSF comes with many adverse effects such as bone pain and, especially for patients with SCN, an increased risk for hematologic malignancy [Newburger PE. Semin Hematol. 2013; Dale DC. Blood Advances. 2022]. Mavorixafor is an investigational oral antagonist of CXCR4 in clinical development for treatment of patients with multiple CNDs, including Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome [Dale DC. Blood. 2020]. Herein we report results of phase 1b, open-label, multicenter trial (NCT04154488) evaluating the safety, tolerability, and serving as a proof of concept for efficacy, of mavorixafor alone or with concurrent G-CSF use across several CNDs. Methods Patients aged ≥12 years with diagnosis of CIN, congenital neutropenia, or CYN ≥6 months prior and ANC <1000/µL at screening visits were included. Patients on G-CSF with ANC >1000/µL were also included. All patients with congenital neutropenia without documented genetic variants underwent genetic screening via a 642-gene comprehensive immune and cytopenia panel. Patients received ≥1 dose of mavorixafor (>50 kg, 400 mg; ≤50 kg, 200 mg), regardless of current treatment with or without G-CSF. Patients were monitored for hematologic parameters such as total white blood cell (WBC) and subset counts, including ANC, absolute lymphocyte count (ALC), and absolute monocyte count (AMC), over the course of 6 to 8 hours 1 day before receiving mavorixafor (day -1) and on the first day of mavorixafor treatment (day 1) and were followed for 30 days for safety and tolerability. Fold change in total peripheral WBC count, ANC, ALC, and AMC from day -1 levels was assessed. Results As of June 2022, 15 patients (CIN, n=11; congenital neutropenia, n=3; CYN, n=1) were enrolled and dosed. The mean (SD) age in years was 39.7 (17.2); 40% were male, and 60% were on G-CSF. All patients received ≥1 dose of mavorixafor once daily. The mean (SD) maximum fold change in ANC from day -1 to day 1 across all included patients was 4.72 (2.74) at peak over the course of 6 to 8 hours after dose 1. Table 1 shows fold change in ANC for individual participants, and Figure 1, by type of neutropenia. The mean (SD) maximum fold change in other hematologic parameters, including total WBC count, ALC, and AMC, from day -1 to day 1 was 3.14 (0.65), 3.05 (0.62), and 2.64 (0.80), respectively. For the subgroup of patients on G-CSF at day -1, the mean (SD) maximum fold change in total WBC count, ANC, ALC, and AMC from day -1 to day 1 was 2.87 (0.58), 3.67 (1.29), 3.07 (0.73), and 2.44 (0.59), respectively. For patients not on G-CSF, the mean (SD) maximum fold change in total WBC count, ANC, ALC, and AMC from day -1 to day 1 was 3.54 (0.52), 6.28 (3.45), 3.01 (0.47), and 2.95 (0.95), respectively. Treatment-emergent adverse events (AEs; n=11) were all grade 1; no serious AEs were reported. Conclusions Meaningful increases in ANC and all other hematologic parameters evaluated were observed on day 1 in all patients treated with ≥1 dose of mavorixafor. Responses were observed regardless of concurrent G-CSF use or type of CN. Mavorixafor treatment was well tolerated overall, with only mild AEs reported. This study supports the potential therapeutic use of mavorixafor in the treatment of CNDs beyond WHIM syndrome and underscores the possibility for mavorixafor to become the first oral treatment for CN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Morphologic Spectrum of Myelokathexis in WHIM Syndrome and Germline CXCR4 Variants: New Insights into Cellular Changes in the Bone Marrow and Peripheral Blood

Introduction WHIM syndrome (WHIMs) is a rare disorder caused by germline CXCR4 mutations, classically characterized by warts, hypogammaglobulinemia, infection, and neutrophil retention in the bone marrow (BM), with distinctive morphology termed myelokathexis. Demonstration of myelokathexis by BM evaluation plays a key role in disease recognition and diagnosis. However, the spectrum of BM morphologic features and correlation with specific CXCR4 mutations are not well described. Moreover, diagnostically useful peripheral blood (PB) smear findings and morphologic changes after CXCR4 antagonist therapy are largely unknown. Methods We conducted a multinational, retrospective review of clinicopathologic features of patients with established WHIMs (n=20) or germline CXCR4 variants considered likely pathogenic or of uncertain significance (n=3), including 14 BM specimens and 10 PB smears. Assessments to date were performed by 2 observers in 6 cases and a single observer in the remainder. BM morphology was compared to other neutropenia disorders (Shwachman-Diamond syndrome, n=5; GATA2 deficiency, n=5; severe congenital neutropenia with ELANE variants, n=4) and patients without hematologic disorders (n=10), together "controls." Correlations and contingency analysis (2-tailed Fisher exact test) were performed using Prism v.9.4.0 (GraphPad Software). Results Median age at WHIMs diagnosis was 18.5 years (range, 0.1-53 years), with female:male ratio of 1.1:1. Recurrent infections, warts, and hypogammaglobulinemia were present in 100%, 53%, and 40% of cases, respectively. BM aspirate smears contained neutrophils with long intranuclear chromatin strands and hypermature condensed chromatin ("myelokathectic neutrophils") in all WHIMs cases, making up 32% to 80% (median 66%) of total neutrophils (Table). In comparison, neutrophils with myelokathectic changes were seen in 0% to 4% of control BMs. Myeloid hyperplasia (myeloid:erythroid ratio >4:1) was present in 50% of WHIMs and 4% of controls (P=.0028); vacuolated neutrophils in 92% of WHIMs (≤29% of total neutrophils) and 17% of controls (P<.0001); apoptotic neutrophils in 58% of WHIMs (1%-4% of total neutrophils) and 8% of controls (P=.0025); and nonparatrabecular neutrophil clusters on core biopsy in 3/3 WHIMs and 5% of controls (P=.0017). Vacuolated eosinophils and myeloid shift toward hypermaturity were present in WHIMs. There was no correlation between percentage of BM myelokathectic neutrophils and degree of CXCR4 truncation. BM in 2 patients with CXCR4 p.S341Y showed myeloid hyperplasia, increased neutrophil apoptosis, and infrequent myelokathectic neutrophils (5%-10%). Neutropenia was present in all untreated WHIMs. Myelokathectic neutrophils were present in PB of all patients with known WHIMs and accounted for 8% to 30% (median, 18%) of total neutrophils. Percentage of PB myelokathectic neutrophils showed an inverse correlation with absolute neutrophil count (ANC) (r=-0.783; P=.0074). Neutrophil apoptosis, neutrophil vacuolization, dysmorphic (hypogranular, hyperlobated, vacuolated, or apoptotic) eosinophils, and/or dysmorphic basophils were present in 80%, 30%, 70%, and 60% of WHIMs cases, respectively (Figure). PB in 1 patient with CXCR4p.D84H showed neutrophil hypolobation without myelokathectic morphology. CXCR4 antagonist therapy with plerixafor (n=4) [Dale et al. Blood. 2011] or mavorixafor (n=1) (interval, 4-12 h) resulted in increased white blood cell (WBC) count and ANC, with a similar proportion of myelokathectic neutrophils as before therapy. Conclusions In WHIMs BM specimens, the proportion of myelokathectic neutrophils is variable and may not be prominent; the classic finding of myeloid hyperplasia is only present in a subset of cases, which may result in underrecognition of the disease and delayed diagnosis. Neutrophil vacuolization and apoptosis, eosinophil vacuolization, and prominent neutrophil clusters may also prompt disease recognition. We establish characteristic PB morphologic features which, in a patient with unexplained neutropenia, may suggest a diagnosis of WHIMs, including myelokathectic neutrophils, neutrophil vacuolization or apoptosis, and dysmorphic eosinophils and basophils. CXCR4 antagonist therapy led to an increase of WBC/ANC, while pretreatment morphologic changes persisted after a short interval. Analysis of additional cases is ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genotype–phenotype correlations in WHIM syndrome: a systematic characterization of CXCR4WHIM variants

Warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome is a rare primary immunodeficiency predominantly caused by heterozygous gain-of-function mutations in CXCR4 C-terminus. We assessed genotype–phenotype correlations for known pathogenic CXCR4 variants and in vitro response of each variant to mavorixafor, an investigational CXCR4 antagonist. We used cell-based assays to analyze CXCL12-induced receptor trafficking and downstream signaling of 14 pathogenic CXCR4 variants previously identified in patients with WHIM syndrome. All CXCR4 variants displayed impaired receptor trafficking, hyperactive downstream signaling, and enhanced chemotaxis in response to CXCL12. Mavorixafor inhibited CXCL12-dependent signaling and hyperactivation in cells harboring CXCR4WHIM mutations. A strong correlation was found between CXCR4 internalization defect and severity of blood leukocytopenias and infection susceptibility, and between AKT activation and immunoglobulin A level and CD4+ T-cell counts. This study is the first to show WHIM syndrome clinical phenotype variability as a function of both CXCR4WHIM genotype diversity and associated functional dysregulation. Our findings suggest that CXCR4 internalization may be used to assess the pathogenicity of CXCR4 variants in vitro and also as a potential WHIM-related disease biomarker. The investigational CXCR4 antagonist mavorixafor inhibited CXCL12-dependent signaling in all tested CXCR4-variant cell lines at clinically relevant concentrations.

Disease Progression of WHIM Syndrome in an International Cohort of 66 Pediatric and Adult Patients

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS) is a combined immunodeficiency caused by gain-of-function mutations in the C-X-C chemokine receptor type 4 (CXCR4) gene. We characterize a unique international cohort of 66 patients, including 57 (86%) cases previously unreported, with variable clinical phenotypes. Of 17 distinct CXCR4 genetic variants within our cohort, 11 were novel pathogenic variants affecting 15 individuals (23%). All variants affect the same CXCR4 region and impair CXCR4 internalization resulting in hyperactive signaling. The median age of diagnosis in our cohort (5.5 years) indicates WHIM syndrome can commonly present in childhood, although some patients are not diagnosed until adulthood. The prevalence and mean age of recognition and/or onset of clinical manifestations within our cohort were infections 88%/1.6 years, neutropenia 98%/3.8 years, lymphopenia 88%/5.0 years, and warts 40%/12.1 years. However, we report greater prevalence and variety of autoimmune complications of WHIM syndrome (21.2%) than reported previously. Patients with versus without family history of WHIM syndrome were diagnosed earlier (22%, average age 1.3 years versus 78%, average age 5 years, respectively). Patients with a family history of WHIM syndrome also received earlier treatment, experienced less hospitalization, and had less end-organ damage. This observation reinforces previous reports that early treatment for WHIM syndrome improves outcomes. Only one patient died; death was attributed to complications of hematopoietic stem cell transplantation. The variable expressivity of WHIM syndrome in pediatric patients delays their diagnosis and therapy. Early-onset bacterial infections with severe neutropenia and/or lymphopenia should prompt genetic testing for WHIM syndrome, even in the absence of warts.

P793: SCREENING OF NATURALLY OCCURRING CXCR4 VARIANTS FOR IDENTIFICATION OF NOVEL PATHOGENIC MUTATIONS FOR WHIM SYNDROME

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare, autosomal-dominant primary immunodeficiency marked by neutropenia and lymphopenia. Classically, WHIM syndrome pathogenesis is causally linked to a variety of heterozygous gain-of-function mutations in the C-terminus of chemokine receptor CXCR4, a master regulator of immune cell trafficking and homeostasis. As of February 2022, 18 variants in CXCR4 have been implicated in WHIM syndrome, while a substantial number remain unexplored for their association with the disease. Using in vitro functional assays, we previously found that impaired receptor internalization and enhanced chemotaxis are conserved in all CXCR4WHIM variants and that defective internalization correlates with neutropenia, the most penetrant phenotype of patients with WHIM syndrome. We used this approach to confirm functional defects in primary cells isolated from patients harboring a novel variant, suggesting that the in vitro profiling approach can potentially drive identification of pathogenic variants. Aims: We aimed to functionally characterize multiple previously uncharacterized variants of CXCR4 using in vitro assays. We also examined allele frequencies of these novel variants to estimate the potential number of individuals harboring these variants with a long-term goal of determining the actual prevalence of WHIM syndrome. Methods: The CXCR4-negative K562 cell line was used as a model system to express 53 novel CXCR4 variants identified in patient and population databases (ClinVar, Ensembl, CentoMD, GnomAD) and genetic screening initiatives (Invitae PATH4WARD) found in the C-terminus (hotspot of CXCR4WHIM mutations) as well as throughout the protein. The effects of these mutations on CXCR4 internalization and chemotaxis were studied in cells stimulated with the C-X-C chemokine ligand 12 (CXCL12). The in vitro functional parameters were compared with known pathogenic CXCR4 variants and were used to assign a cumulative score (impaired internalization + enhanced chemotaxis) of functional defect severity. Results: Out of 53 selected variants, 42 were missense (ms), 6 were frameshift (fs), 3 were nonsense (ns), and 2 were transcript variants (tv). Eighteen of the screened variants led to a decreased internalization of CXCR4 in comparison to the wild-type (WT) receptor upon stimulation with CXCL12 while 20 variants demonstrated enhanced chemotaxis of cells toward CXCL12. Overall, 34 variants showed defects in at least one CXCR4-dependent marker. Correlation analysis and scoring of the functional parameters in cells expressing mutated or CXCR4WT revealed 17 variants (9 ms, 5 fs, 2 ns, 1 tv) that co-segregated with the known pathogenic WHIM variants. Finally, white blood cell counts in 3 patients harboring novel variants showed that CXCR4 internalization defects in vitro correlated with the severity of neutropenia. Summary/Conclusion: This is the first study characterizing functional impairments in naturally occurring CXCR4 variants via a pipeline of assays intended to predict pathogenicity. Seventeen CXCR4 variants were identified (several outside the C-terminus of CXCR4) that caused in vitro functional defects resembling those exhibited by known WHIM variants. Many (10/17) of these newly identified variants are present at high frequencies (4.4 × 10-4–8.0 × 10-6) in genomic databases. Potential association of these variants with WHIM syndrome is under investigation and is likely to expand current estimates of WHIM syndrome prevalence.

Can we identify WHIM in infancy? Opportunities with the public newborn screening process.

Newborn screening (NBS) for severe combined immunodeficiency (SCID) utilizing T-cell receptor excision circles (TRECs) has been implemented in all 50 states as of December 2018 and has been transformative for the clinical care of SCID patients. Though having high sensitivity for SCID, NBS-SCID has low specificity, therefore is able to detect other causes of lymphopenia in newborns including many inborn errors of immunity (IEIs). In a recent study, three of six newborns later diagnosed with Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome were found to have a low TRECs and lymphopenia at birth. This presents an opportunity to increase the detection and diagnosis of WHIM syndrome by NBS-SCID with immunological follow-up along with a combination of flow cytometry for immune cell subsets, absolute neutrophil count, and genetic testing, extending beyond the conventional bone marrow studies. Coupled with emerging technologies such as next-generation sequencing, transcriptomics and proteomics, dried blood spots used in NBS-SCID will promote earlier detection, diagnosis, and therefore treatment of IEIs such as WHIM syndrome.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate β-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of β-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.

Increased bone resorption in mice bearing WHIM Syndrome mutations does not rely on increased intrinsic OCL differentiation capacity

Increased bone resorption in mice bearing WHIM Syndrome mutations does not rely on increased intrinsic OCL differentiation capacity

A Sherlock Approach to a Kindred With a Variable Immunohematologic Phenotype

Given the ubiquity of leukopenia and sinopulmonary infections in childhood, differentiating patients with inborn errors of immunity (IEI) from otherwise healthy patients can be challenging. The diagnostic complexity is further exacerbated in disorders with wide phenotypic variability such as warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome. However, using a Sherlock approach with careful attention to details in the patient's medical history and physical examination coupled with a comprehensive family history can heighten the index of suspicion for underlying IEI. Subsequent iterative and deductive reasoning incorporating results from laboratory interrogation, response (or lack thereof) to standard therapy, and emergence of new symptoms can further aid in a timely diagnosis of IEI. Herein, we detail a WHIM syndrome kindred with marked phenotype variability, identified after the presentation of a child with intermittent neutropenia and sinopulmonary infections. The complexity of this kindred highlights the utility of an interspecialty, collaborative Sherlock approach to diagnosis, and care. In addition, the genetic underpinnings, diagnostic approaches, clinical features, supportive care options, and management of WHIM syndrome are reviewed.

EP174: A novel variant of CXCR4 gene in a case of WHIM syndrome

Warts, hypergammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an extremely rare, autosomal dominant immunodeficiency disorder. It is caused by gain of function variant that truncate the C-terminus of CXCR4, an important chemokine receptor associated with inflammation, immunity, cancer and development. Myelokathexis refers to neutropenia resulting from retention of mature neutrophils and increased neutrophil apoptosis in the bone marrow. A subset of patients can have hypogammaglobulinemia with poor response to vaccines.

Consideration of underlying immunodeficiency in refractory or recalcitrant warts: A review of the literature.

Although the exact mechanisms have yet to be elucidated, it is clear that cellular immunity plays a role in clearance of human papillomavirus (HPV) infections as it relates to the development of warts. Patients with extensive, recalcitrant, or treatment‐refractory warts may have an underlying immune system impairment at the root of HPV susceptibility. Early recognition of genetic disorders associated with immunologic defects that allow for recalcitrant HPV infection may expedite appropriate treatment for patients. Early recognition is often pivotal in preventing subsequent morbidity and/or mortality that may arise from inborn errors of immunity, such as WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome. Among these, cervical cancer is one of the most common malignancies associated with HPV, can be fatal if not treated early, and is seen more frequently in patients with underlying immune deficiencies. A review of diseases with susceptibility to HPV provides clues to understanding the pathophysiology of warts. We also present diagnostic guidance to facilitate the recognition of inborn errors of immunity in patients with extensive and/or recalcitrant HPV infections.

Effects Of Mavorixafor On Functional Impairments Due To A Novel Missense CXCR4 Mutation In A Patient With WHIM-Like Syndrome

WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare primary immunodeficiency mostly caused by gain-of-function mutations in the C-terminus of the C-X-C chemokine receptor 4 (CXCR4). We described a case of clinical WHIM-like syndrome in a patient with a novel heterozygous mutation in the CXCR4 transmembrane region (CXCR4D84H). Here, we further evaluate the patient’s peripheral blood leukocytes and transfected cells expressing CXCR4D84H for their in vitro response to CXCR4 antagonist mavorixafor, currently in phase 3 clinical development.

Characterization of a new WHIM syndrome mutant reveals mechanistic differences in regulation of the chemokine receptor CXCR4

WHIM syndrome is a rare immunodeficiency disorder that is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. While several gain-of-function mutations that lead to C-terminal truncations, frame shifts and point mutations in the chemokine receptor CXCR4 have been identified in WHIM syndrome patients, the functional effect of these mutations are not fully understood. Here, we report on a new WHIM syndrome mutation that results in a frame shift within the codon for Ser339 (S339fs5) and compare the properties of S339fs5 with wild-type CXCR4 and a previously identified WHIM syndrome mutant, R334X. The S339fs5 and R334X mutants exhibited significantly increased signaling compared to wild-type CXCR4 including agonist-promoted calcium flux and extracellular-signal-regulated kinase activation. This increase is at least partially due to a significant decrease in agonist-promoted phosphorylation, β-arrestin binding, and endocytosis of S339fs5 and R334X compared with wild-type CXCR4. Interestingly, there were also significant differences in receptor degradation, with S339fs5 having a very high basal level of degradation compared with that of R334X and wild-type CXCR4. In contrast to wild-type CXCR4, both R334X and S339fs5 were largely insensitive to CXCL12-promoted degradation. Moreover, while basal and agonist-promoted degradation of wild-type CXCR4 was effectively inhibited by the CXCR4 antagonist TE-14016, this had no effect on the degradation of the WHIM mutants. Taken together, these studies identify a new WHIM syndrome mutant, CXCR4-S339fs5, which promotes enhanced signaling, reduced phosphorylation, β-arrestin binding and endocytosis, and a very high basal rate of degradation that is not protected by antagonist treatment.

Oral Administration of Mavorixafor, a CXCR4 Antagonist, Increases Peripheral White Blood Cell Counts across Different Disease States

Oral Administration of Mavorixafor, a CXCR4 Antagonist, Increases Peripheral White Blood Cell Counts across Different Disease States

Characterization of a Novel Missense CXCR4 Mutation in a Patient with WHIM-like Syndrome

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare primary immunodeficiency with a heterogeneous presentation of symptoms defining its acronym as well as panleukopenia. The majority of cases are inherited in an autosomal dominant manner, with gain-of-function mutations in the C-terminus of the C-X-C chemokine receptor 4 (CXCR4) (McDermott D, et al. Immunol Rev. 2019;287:91-102). To our knowledge, there are no literature reports implicating CXCR4WHIM mutations outside the CXCR4 C-terminus. Here we report the clinical presentation of a patient with the novel mutation CXCR4D84H and characterize the functional effects of the mutation on CXCR4 trafficking and chemotaxis in in vitro and ex vivo assays. Further, CXCR4D84H is reported in 3 population genetic databases (average allele frequency 3.8 × 10 -5) and is also found in 5 unrelated patients in another rare disease database (CentoMD).C ase Report: A 40-year-old female presented with a history of recurrent vulvovaginal and anal dysplasia and carcinoma in situ requiring multiple surgeries starting at age 20 years. Cytopenia was first documented at age 15 years in the context of mononucleosis, which resolved, although cytopenias persisted through adulthood. To date, the patient has decreased absolute neutrophil counts (600-1100/mm 3 range), WBC counts ~2000/mm 3, and normal immunoglobulin levels. Bone marrow biopsy revealed bilobed neutrophils and granulocyte precursors. The patient has no history of recurrent infections besides HPV/EBV and no family history of warts, immunodeficiency, or squamous cell carcinoma. Genetic testing revealed a heterozygous mutation in CXCR4 (c.250G>C, D84H) that has not been previously reported. This missense mutation occurs in the transmembrane domain of CXCR4 in proximity of the residues involved in signal initiation, and hence, may alter signaling responses.Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from this patient and 2 healthy controls and analyzed for functionality ex vivo including CXCR4 internalization assays and chemotaxis to exogenously added CXCR4-ligand CXCL12. The CXCR4-negative K562 cell line was transiently transfected to express CXCR4D84H, CXCR4WT, and known CXCR4WHIM mutations for further comparison of their functional responses to CXCL12.Results: Flow cytometry data shows abnormal peripheral leukocyte frequency and count. Lymphocyte subpopulations showed 33%-57% reduced CXCR4 internalization in response to CXCL12 compared to controls. Chemotactic response to CXCL12 was increased in PBMC subsets including natural killer T (NKT) cells compared to healthy donor cells. These observations are comparable to previously published responses to pathogenic CXCR4WHIM variants, but, chemotactic responses of NKT cells is a novel finding. In K562-transfected cells, CXCR4D84H demonstrated impaired CXCL12-induced internalization compared to CXCR4WT at a level comparable to the CXCR4E343K variant, the only CXCR4 missense variant known to date to cause WHIM syndrome. Chemotaxis of cells expressing CXCR4D84H in response to CXCL12 is increased by 1.25- to 2.4-fold compared to CXCR4WTdepending on CXCL12 concentration, in line with the observation of increased chemotaxis of cells expressing known CXCR4WHIM variants. Overall, the CXCR4D84H mutation recapitulates the phenotypes exhibited by known CXCR4WHIM variants in both internalization and chemotaxis assays.Conclusions: We show the functional effects of a novel missense mutation CXCR4D84H found in a patient with clinical WHIM syndrome recapitulates the internalization and chemotaxis findings of other CXCR4WHIM mutations. This is the first report of a missense mutation in CXCR4 outside of the C-terminus causing a WHIM phenotype, and additionally highlights defective chemotaxis in NKT cells, which may be relevant in this patient's HPV-associated carcinoma in situ phenotype. Based on an analysis of population databases, and assuming a conservative 5-10% penetrance, there are potentially ~1250-2500 individuals in the United States with disease due to the p.D84H variant alone, highlighting the under-recognition of WHIM syndrome. Patients who have similar clinical presentation as described in this case study should be evaluated for WHIM syndrome and other primary immunodeficiencies by using genetic screening for CXCR4 and diagnostic assays described. DisclosuresPawar: X4 Pharmaceuticals: Current Employment. Zmajkovicova: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Wiest: X4 Pharmaceuticals: Current Employment. Nguyen: X4 Pharmaceuticals: Current Employment. Monticelli: X4 Pharmaceuticals: Current Employment. Maier-Munsa: X4 Pharmaceuticals: Current Employment. Sondheimer: X4 Pharmaceuticals: Consultancy. Walter: Octapharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmig: Consultancy, Membership on an entity's Board of Directors or advisory committees. Taveras: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Badarau: X4 Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Tarrant: ThermoFisher Scientific: Consultancy; X4 Pharmaceuticals: Consultancy, Research Funding; Department of Justice: Consultancy; Abbvie: Research Funding; Viela Bio: Research Funding.

CXCR4 Drives Lympho-Myeloid Fate of Hematopoietic Progenitors Via mTOR and Mitochondrial Metabolic Pathways

Blood production is a tightly regulated process that starts with hematopoietic stem cells (HSCs). In adults, HSCs are unique in their capacity to self-renew and replenish the entire blood system through production of a series of increasingly committed progenitor cells within the bone marrow (BM) microenvironment. HSCs form a rare, quiescent population that displays a metabolism skewed towards anaerobic glycolysis at the expense of mitochondrial oxidative phosphorylation (OXPHOS) to preserve its quiescent state and long-term reconstitution capacity. However, when HSCs differentiate, they undergo a metabolic switch from anaerobic glycolysis to mitochondrial OXPHOS, a process that is in part mediated by the metabolic sensor mTOR. It is well-established that HSCs in the BM adapt the production of myeloid and lymphoid cells depending on the needs of the body and that metabolic plasticity is a critical driver of HSC fate decisions. This has never been assessed for multipotent progenitors (MPPs) which constitute the stage at which the major divergence of lymphoid and myeloid lineages occurs. In mice, common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) are generated from phenotypically and functionally distinct subpopulations of lineage-biased MPPs, i.e. MPP2 and MPP3 are reported as distinct myeloid-biased MPP subsets that operate together with lymphoid-primed MPP4 to control blood leukocyte production. This question is thus of paramount importance to understand how the lympho-myeloid specification process is regulated.Signaling by the G protein-coupled receptor CXCR4 on MPPs in response to stimulation by its natural ligand, the chemokine CXCL12, produced by BM perivascular stromal cells constitutes a key pathway through which the niches and MPPs communicate. However, the mechanisms whereby CXCR4 signaling regulates MPP specification are still unknown. We addressed this point using BM samples of patients with WHIM Syndrome (WS), a rare immunodeficiency caused by inherited heterozygous autosomal gain-of-CXCR4-function mutations affecting desensitization of CXCR4 and characterized by chronic lympho-neutropenia, as well as a unique WS mouse model which phenocopies severe pan-leukopenia.We unraveled myeloid skewing of the hematopoietic stem and progenitor cell (HSPC) compartment in BM of patients with WS and of WS mice. This relied on CXCR4 signaling strength that controls the output of the lymphoid and myeloid lineages by coordinating the composition and molecular identity of the MPP compartment. The fate of the lymphoid-biased MPP4 subset was central in such a process. Indeed, CXCR4 signaling termination was required for efficient generation and maintenance of the MPP4 pool, while regulating intrinsically their cell cycle status and lymphoid-myeloid gene landscape. In fact, we demonstrated for the first time that enhanced mTOR signaling, accumulation of damaged mitochondria and overactive OXPHOS-driven metabolism promoted cell-autonomous molecular changes that reprogram mutant MPP4 away from lymphoid differentiation. Consistent with this, in vivo chronic treatment with the CXCR4 antagonist AMD3100/Plerixafor or the mTOR inhibitor Rapamycin normalized mitochondrial metabolism and MPP4 differentiation.Thus, our study shows that CXCR4 signaling acts through the mTOR pathway as an essential gatekeeper for integrity of the mitochondrial machinery, which in turn controls lymphoid potential of MPP4. DisclosuresNo relevant conflicts of interest to declare.

Spectrum of Pathogenic Genetic Variants in a Large Cohort of North American Congenital and Cyclic Neutropenia Patients: A Report from the Severe Chronic Neutropenia International Registry

▪Severe congenital neutropenia (SCN) is characterized by persistent neutropenia and risk of invasive, life-threatening infection as well as transformation to hematopoietic malignancy. The closely related syndrome cyclic neutropenia is characterized by recurrent episodic neutropenia accompanied by symptoms including infection. Understanding the genetic etiology of congenital neutropenia can help to direct therapy, guide surveillance and health maintenance strategies, and contribute to our understanding of basic neutrophil biology. Although mutations in ELANE are the most frequent cause of congenital neutropenia, there is a wide and ever-growing list of additional causative variants. Additionally, there appears to be regional genetic variability. For example, mutations in HAX1 are rarely if ever observed in North America while they are more common in Europe. We undertook exome sequencing of a large cohort from the Severe Chronic Neutropenia International Registry (SCNIR) of North America in an effort to define the genetic spectrum of congenital neutropenia and aid in the discovery of new pathogenic variants.We expanded our previously reported study of whole exome sequencing to include 152 cases of chronic neutropenia, comprised of 94 cases of SCN and 58 cases of cyclic neutropenia. We selected cases in which ELANE testing was negative, or in a small minority of cases, where ELANE testing had not yet been performed. Indeed, exome sequencing only identified 7 cases (5 SCN and 2 cyclic) carrying pathogenic ELANE mutations in this cohort. In the remaining 145 cases, we analyzed exomes for the presence of variants in genes previously associated with congenital neutropenia including AK2, AP3B1, CD40LG, CLPB, CSF3R, CXCR2, CXCR4, DNAJC21, DNM2, DOCK2, EFL1, EIF2AK3, ELANE, G6PC3, GATA1, GATA2, GFI1, GINS1, HAX1, IRAK4, JAGN1, KAT6A, KRAS, LAMTOR2, LYST, MYD88, PGM3, PSTPIP1, RAB27A, RAC2, SBDS, SEC61A1, SLC37A4, SMARCD2, SRP54, STK4, TAZ, TCIRG1, TCN2, TLR8, USB1, VPS13B, VPS45, WAS, WDR1 and WIPF1. Pathogenic heterozygous mutations of CLPB that localize to the ATP-binding pocket were identified in 7 cases, making it the second most common cause of congenital neutropenia in North America. We additionally identified 4 cases with G6PC3 pathogenic variants, and one case each with pathogenic variants in JAGN1, CXCR4 (the cause of WHIM syndrome), germline homozygous CSF3R, and GFI1.Interestingly, we identify 2 unrelated individuals (one with SCN and one with cyclic) and 2 siblings with SCN all of whom possess a recently described heterozygous variant in SRP54 (p.T117del). We collected genomic DNA from the affected mother of the 2 siblings, an additional unaffected sibling, and the unaffected grandparents. Through this kinship, we can confirm the de novo appearance of this variant in the second generation and demonstrate that it tracks with disease status (Figure 1). We also identified 2 unrelated individuals with SRP54 variants affecting residue 175 also located within the GTPase domain (p.G175E or p.G175del). Both variants are absent from the gnomAD database, and studies are underway to demonstrate de novo acquisition.In summary, we have defined the spectrum of mutations present in ELANE-wildtype chronic neutropenia cases in North America. Pathogenic or likely pathogenic variants were identified in 26 out of 145 (18%) cases. The most frequently mutated genes were of CLPB, SRP54, and G6PC3, while mutations in HAX1 were not seen. Importantly, some of these mutations are associated with genetic syndromes with extra-hematopoietic findings (for example, CLPB and SRP54) that would warrant additional evaluations and targeted health maintenance. These findings emphasize the importance of sending large panels for genotyping, rather than targeted ELANE testing. [Display omitted] DisclosuresBolyard: X4 Pharmaceuticals: Research Funding. Makaryan: Emendo Biotherapeutic: Research Funding. Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding.

Multicenter Experience of Hematopoietic Stem Cell Transplantation in WHIM Syndrome

PurposeWHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a rare disease, caused by CXCR4 gene mutations, which incorporates features of combined immunodeficiency, congenital neutropenia, and a predisposition to human papillomavirus infection. Established conventional treatment for WHIM syndrome does not fully prevent infectious complications in these patients. Only single case reports of hematopoietic stem cell transplantation (HSCT) efficacy in WHIM have been published.MethodsTo summarize current information on HSCT efficacy in disease treatment, seven pediatric patients with WHIM syndrome who underwent allogeneic HSCT were identified in five centers worldwide.ResultsAll patients presented early after birth with neutropenia. Two of seven patients exhibited severe disease complications: poorly controlled autoimmunity (arthritis and anemia) in one and progressive myelofibrosis with recurrent infections in the other. The remaining patients received HSCT to correct milder disease symptoms (recurrent respiratory infections, progressing thrombocytopenia) and/or to preclude severe disease course in older age. All seven patients engrafted but one developed graft rejection and died of infectious complications after third HSCT. Three other patients experienced severe viral infections after HSCT (including post-transplant lymphoproliferative disease in one) which completely resolved with therapy. At last follow-up (median 6.7 years), all six surviving patients were alive with full donor chimerism. One patient 1.4 years after HSCT had moderate thrombocytopenia and delayed immune recovery; the others had adequate immune recovery and were free of prior disease symptoms.ConclusionHSCT in WHIM syndrome corrects neutropenia and immunodeficiency, and leads to resolution of autoimmunity and recurrent infections, including warts.

The Effect of the MK0339 Neutrophil Elastase Inhibitor on Proliferation and Maturation of iPSC Derived from Patients with ELANE, TCIRG1 and Whim Syndrome Associated Neutropenia

Genetic neutropenias are heterogeneous group of hematological diseases characterized by impaired survival and maturation of myeloid precursors in bone marrow. G-CSF and HSCT are currently the only effective treatment options. MK-0339 is a potent, cell permeable, orally absorbed inhibitor of neutrophil elastase (NE), previously investigated in preclinical and clinical studies by Merck/DuPont as a potential anti-inflammatory drug. We have previously reported that MK-0339 increases neutrophil proliferation in cellular models of ELANE associated neutropenia (2016 [ASH Annual Meeting Abstracts] Blood. 2016;128:406; 2017 J Leuk Biol [in press]). To examine the specificity of these responses, we investigated the effects of MK-0339 on iPSCs from healthy volunteers and compared its effects on differentiation of iPSC derived from patients with ELANE, TCIRG-1 and CXCR4 associated neutropenia.We generated induced pluripotent stem cells (iPSC) from dermal fibroblasts from healthy volunteers and neutropenic patients harboring ELANE, TCIRG1 and CXCR4 mutations and developed a feeder free, reproducible and efficient protocol for myeloid differentiation.In our initial experiments, we observed that iPSC lines derived from patients with ELANE mutations (P139L and G214R) consistently grew much slower and formed much smaller colonies than the controls using both mTeSR1/matrigel and DEF-CS feeder free culture medium systems. We observed more than 3-fold growth reduction of patient derived lines compared to healthy volunteers.We next tested the effects of MK-0339 under the same culture conditions. Growth of control and ELANE patient cell lines was enhanced, and the cells appeared more robust, after addition of 1uM MK-0339. We were surprised to see a similar proliferative effect (~40% increase) with Ramos (human Burkitt's lymphoma cell line) cells cultured in the presence of 1uM MK-0339, although neither the Ramos cells nor the ELANE iPSCs had detectable levels of ELANE expression. The enhanced growth of control and patient iPSCs and the results with Ramos cells suggested that the effects of MK-0339 are not solely mediated via inhibition of NE.We next compared myeloid differentiation and CD34+ cell proliferation of iPSC from patients with ELANE, TCIRG1 and CXCR4 associated neutropenia in HPC expansion medium, using granulocytic differentiation markers and flow cytometry. Myeloid maturation was impaired and inefficient for both ELANE (P139L and G214R) patient derived cell lines. Myeloid differentiation of the TCIRG1 patient derived was also impaired. Introduction of MK-0339 restored the impaired myeloid differentiation capacity of both ELANE and TCIRG1 positive patient derived cell lines. An iPSC line derived from a WHIM (CXCR4 mutation) patient appeared to have normal maturation and proliferation. Western blot analysis revealed elevated levels of endoplasmic reticulum chaperone GRP78/BiP in ELANE and TCIRG1 mutant cell lines which was corrected by MK-0339 treatment.These studies demonstrate heretofore unreported proliferative effects of MK-0339 on cell lines not expressing neutrophil elastase. They suggest important mechanisms beyond enzyme inhibition to account for these findings. Although preliminary, we believe that this work suggests that MK-0339 may be a novel, oral therapy for both ELANE and non-ELANE associated neutropenia. DisclosuresDale:University of Washington: Employment, Research Funding; National Institutes of Health: Research Funding; WedMD/Medscape: Membership on an entity's Board of Directors or advisory committees; Wolter Kluwer: Other: Editor, Current Opinions in Hematology; Philips: Research Funding; X4Pharma: Research Funding; Shire: Other: Independent Review Board; Omeros: Other: DSMB; Coherus: Consultancy; Boheringer-Ingelheim: Consultancy; Prolong: Consultancy; Hospira: Consultancy; Genkyotex: Other: DSMB (work completed 6/2015); Genzyme (now owned by Sanofi-Aventis): Consultancy, Patents & Royalties, Research Funding; Cellerant: Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy, Editor, Current Opinions in Hematology, Honoraria; Amgen: Consultancy, Research Funding; American College of Physicians: Other: Editor and author; GlaxoSmithKline: Equity Ownership; Johnson&Johnson: Equity Ownership.