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.

MEK1 is required for PTEN membrane recruitment, AKT regulation, and the maintenance of peripheral tolerance.

The Raf/MEK/ERK and PI3K/Akt pathways are prominent effectors of oncogenic Ras. These pathways negatively regulate each other, but the mechanism involved is incompletely understood. We now identify MEK1 as an essential regulator of lipid/protein phosphatase PTEN, through which it controls phosphatidylinositol-3-phosphate accumulation and AKT signaling. MEK1 ablation stabilizes AKT activation and, in vivo, causes a lupus-like autoimmune disease and myeloproliferation. Mechanistically, MEK1 is necessary for PTEN membrane recruitment as part of a ternary complex containing the multidomain adaptor MAGI1. Complex formation is independent of MEK1 kinase activity but requires phosphorylation of T292 on MEK1 by activated ERK. Thus, inhibiting the ERK pathway reduces PTEN membrane recruitment, increasing phosphatidylinositol-3-phosphate accumulation and AKT activation. Our data offer a conceptual framework for the observation that activation of the PI3K pathway frequently mediate resistance to MEK inhibitors and for the promising results obtained by combined MEK/PI3K inhibition in preclinical cancer models.

The Antifibrotic Activity of Prostacyclin Receptor Agonism Is Mediated through Inhibition of YAP/TAZ.

Idiopathic pulmonary fibrosis is a life-threatening progressive disease characterized by loss of alveolar epithelial cells, inflammation, and aberrant fibroblast activation. The two currently approved therapies do not halt or reverse tissue remodeling, and therefore novel disease-modifying mechanisms are needed. Our results describe YAP/TAZ inhibition through prostacyclin (IP) receptor activation as a novel mechanism that suppresses profibrotic (myo)fibroblast activity. We investigated the antifibrotic properties of the selective IP receptor agonist ACT-333679 using primary human lung fibroblasts. ACT-333679 prevented transforming growth factor ß1-induced fibroblast-to-myofibroblast transition, proliferation, extracellular matrix synthesis, and IL-6 and PAI-1 secretion, and exerted relaxant effects in cell contraction assays. ACT-333679 treatment also reverted an established myofibroblast phenotype. Unbiased analysis of ACT-333679-induced whole-genome expression changes in transforming growth factor ß1-treated fibroblasts identified significant attenuation of genes regulated by YAP/TAZ, two transcriptional cofactors that are essential for fibrosis. We then demonstrated that ACT-333679, via elevation of cAMP, caused YAP/TAZ nuclear exclusion and subsequent suppression of YAP/TAZ-dependent profibrotic gene transcription. In summary, we offer a rationale for further exploring the potential of IP receptor agonists for the treatment of idiopathic pulmonary fibrosis.

Identification of residues in the C-terminal domain of HIV-1 integrase that mediate binding to the transportin-SR2 protein.

Transportin-SR2 (TRN-SR2 and TNPO3) is a cellular cofactor of HIV replication that has been implicated in the nuclear import of HIV. TRN-SR2 was originally identified in a yeast two-hybrid screen as an interaction partner of HIV integrase (IN) and in two independent siRNA screens as a cofactor of viral replication. We have now studied the interaction of TRN-SR2 and HIV IN in molecular detail and identified the TRN-SR2 interacting regions of IN. A weak interaction with the catalytic core domain (CCD) and a strong interaction with the C-terminal domain (CTD) of IN were detected. By dissecting the catalytic core domain (CCD) of IN into short structural fragments, we identified a peptide (INIP(1), amino acids (170)EHLKTAVQMAVFIHNFKRKGGI(191)) retaining the ability to interact with TRN-SR2. By dissecting the C-terminal domain (CTD) of IN, we could identify two interacting peptides (amino acids (214)QKQITKIQNFRVYYR(228) and (262)RRKVKIIRDYGK(273)) that come together in the CTD tertiary structure to form an exposed antiparallel ß-sheet. Through site-specific mutagenesis, we defined the following sets of amino acids in IN as important for the interaction with TRN-SR2: Phe-185/Lys-186/Arg-187/Lys-188 in the CCD and Arg-262/Arg-263/Lys-264 and Lys-266/Arg-269 in the CTD. An HIV-1 strain carrying K266A/R269A in IN was replication-defective due to a block in reverse transcription, confounding the study of nuclear import. Insight into the IN/TRN-SR2 interaction interface is necessary to guide drug discovery efforts targeting the nuclear entry step of replication.

GPCR-induced YAP activation sensitizes fibroblasts to profibrotic activity of TGFß1.

Tissue fibrosis is a pathological condition characterized by uncontrolled fibroblast activation that ultimately leads to organ failure. The TGFß1 pathway, one of the major players in establishment of the disease phenotype, is dependent on the transcriptional co-activators YAP/TAZ. We were interested whether fibroblasts can be sensitized to TGFß1 by activation of the GPCR/YAP/TAZ axis and whether this mechanism explains the profibrotic properties of diverse GPCR ligands. We found that LPA, S1P and thrombin cooperate in human dermal fibroblasts with TGFß1 to induce extracellular matrix synthesis, myofibroblast marker expression and cytokine secretion. Whole genome expression profiling identified a YAP/TAZ signature behind the synergistic profibrotic effects of LPA and TGFß1. LPA, S1P and thrombin stimulation led to activation of the Rho-YAP axis, an increase of nuclear YAP-Smad2 complexes and enhanced expression of profibrotic YAP/Smad2-target genes. More generally, dermal, cardiac and lung fibroblast responses to TGFß1 could be enhanced by increasing YAP nuclear levels (with GPCR ligands LPA, S1P, thrombin or Rho activator) and inhibited by decreasing nuclear YAP (with Rho inhibitor, forskolin, latrunculin B or 2-deoxy-glucose). Thus, we present here a conceptually interesting finding that fibroblast responses to TGFß1 can be predicted based on the nuclear levels of YAP and modulated by stimuli/treatments that change YAP nuclear levels. Our study contributes to better understanding of fibrosis as a complex interplay of signalling pathways and proposes YAP/TAZ as promising targets in the treatment of fibrosis.