Mefloquine causes selective mast cell apoptosis in cutaneous mastocytosis lesions by a secretory granule-mediated pathway.

Mastocytosis is a KIT-related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult-onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 µM. Flow cytometric analysis of purified dermal MCs showed that mefloquine-induced cell death was mainly due to apoptosis and accompanied by caspase-3 activation. However, caspase inhibition provided only partial protection against mefloquine-induced cell death, indicating predominantly caspase-independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug-induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule-localised serine proteases play major roles in the execution of mefloquine-induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.

Exploration of novel candidate genes involved in epidermal keratinocyte differentiation and skin barrier repair in man.

A proper skin barrier function requires constant formation of stratum corneum, i.e. the outermost layer of epidermis composed of terminally differentiated keratinocytes. The complex process of converting proliferative basal keratinocytes into corneocytes relies on programmed changes in the activity of many well-established genes. Much remains however to be investigated about this process, e.g. in conjunction with epidermal barrier defects due to genetic errors as in ichthyosis. To this end, we re-analyzed two sets of microarray-data comparing altered gene expression in differentiated vs. proliferating keratinocytes and in the skin of patients with autosomal recessive congenital ichthyosis (ARCI) vs. healthy controls, respectively. We thus identified 24 genes to be upregulated in both sets of array and not previously associated with keratinocyte differentiation. For 10 of these genes (AKR1B10, BLNK, ENDOU, GCNT4, GLTP, RHCG, SLC15A1, TMEM45B, TMEM86A and VSNL1), qPCR analysis confirmed the array results and subsequent immunostainings of normal epidermis showed superficial expression of several of the proteins. Furthermore, induction of keratinocyte differentiation using phorbol esters (PMA) resulted in increased expression of eight of the genes, whereas siRNA silencing of PPARδ, a transcription factor supporting differentiation, had the opposite effect. In summary, our results identify ten new candidate genes seemingly involved in human epidermal keratinocyte differentiation and possibly important for epidermal repair in a genetic skin disease characterized by barrier failure.

Patients with congenital ichthyosis and TGM1 mutations overexpress other ARCI genes in the skin: Part of a barrier repair response?

Autosomal recessive congenital ichthyosis (ARCI) is a group of monogenic skin disorders caused by mutations in any of at least 12 different genes, many of which are involved in the epidermal synthesis of ω-O-acylceramides (acylCer). AcylCer are essential precursors of the corneocyte lipid envelope crosslinked by transglutaminase-1 (TGm-1), or a yet unidentified enzyme, for normal skin barrier formation. We hypothesized that inactivating TGM1 mutations will lead to a compensatory overexpression of the transcripts involved in skin barrier repair, including many other ARCI-causing genes. Using microarray, we examined the global mRNA expression profile in skin biopsies from five ARCI patients with TGM1 mutations and four healthy controls. There were a total of 599 significantly differentially expressed genes (adjusted P < 0.05), out of which 272 showed more than 1.5 log2fold-change (FC) up- or down-regulation. Functional classification of the latter group of transcripts showed enrichment of mRNA encoding proteins mainly associated with biological pathways involved in keratinocyte differentiation and immune response. Moreover, the expression of seven out of twelve ARCI-causing genes was significantly increased (FC = 0.98-2.05). Also, many of the genes involved in keratinocyte differentiation (cornified envelope formation) and immune response (antimicrobial peptides and proinflammatory cytokines) were upregulated. The results from the microarray analysis were also verified for selected genes at the mRNA level by qPCR and at the protein level by semi-quantitative immunofluorescence. The upregulation of these genes might reflect a compensatory induction of acylCer biosynthesis as a part of a global barrier repair response in the patient's epidermis.

Quantitative image analysis of protein expression and colocalisation in skin sections.

Immunofluorescence (IF) and in situ proximity ligation assay (isPLA) are techniques that are used for in situ protein expression and colocalisation analysis, respectively. However, an efficient quantitative method to analyse both IF and isPLA staining on skin sections is lacking. Therefore, we developed a new method for semi-automatic quantitative layer-by-layer measurement of protein expression and colocalisation in skin sections using the free open-source software CellProfiler. As a proof of principle, IF and isPLA of ichthyosis-related proteins TGm-1 and SDR9C7 were examined. The results indicate that this new method can be used for protein expression and colocalisation analysis in skin sections.

Identification and characterization of VEGF-A-responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans.

Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d(+)VEGFR1(high)CXCR4(high) was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk(-/-), tsad(-/-)), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d(+) neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d(-) neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens.

Ablation of human skin mast cells in situ by lysosomotropic agents.

Mast cells are known to have a detrimental impact on numerous types of inflammatory skin diseases such as contact dermatitis, atopic eczema and cutaneous mastocytosis. Regimens that dampen skin mast cell-mediated activities can thus offer an attractive therapeutic option under such circumstances. As mast cells are known to secrete a large array of potentially pathogenic compounds, both from preformed stores in secretory lysosomes (granules) and after de novo synthesis, mere inhibition of degranulation or interference with individual mast cell mediators may not be sufficient to provide an effective blockade of harmful mast cell activities. An alternative strategy may therefore be to locally reduce skin mast cell numbers. Here, we explored the possibility of using lysosomotropic agents for this purpose, appreciating the fact that mast cell granules contain bioactive compounds prone to trigger apoptosis if released into the cytosolic compartment. Based on this principle, we show that incubation of human skin punch biopsies with the lysosomotropic agents siramesine or Leu-Leu methyl ester preferably ablated the mast cell population, without causing any gross adverse effects on the skin morphology. Subsequent analysis revealed that mast cells treated with lysosomotropic agents predominantly underwent apoptotic rather than necrotic cell death. In summary, this study raises the possibility of using lysosomotropic agents as a novel approach to targeting deleterious mast cell populations in cutaneous mastocytosis and other skin disorders negatively influenced by mast cells.

Patient-Specific Measurable Residual Disease Markers Predict Outcome in Patients With Myelodysplastic Syndrome and Related Diseases After Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clinical relapse is the major threat for patients with myelodysplastic syndrome (MDS) undergoing hematopoietic stem-cell transplantation (HSCT). Early detection of measurable residual disease (MRD) would enable preemptive treatment and potentially reduced relapse risk. METHODS: Patients with MDS planned for HSCT were enrolled in a prospective, observational study evaluating the association between MRD and clinical outcome. We collected bone marrow (BM) and peripheral blood samples until relapse, death, or end of study 24 months after HSCT. Patient-specific mutations were identified with targeted next-generation sequencing (NGS) panel and traced using droplet digital polymerase chain reaction (ddPCR). RESULTS: Of 266 included patients, estimated relapse-free survival (RFS) and overall survival (OS) rates 3 years after HSCT were 59% and 64%, respectively. MRD results were available for 221 patients. Relapse was preceded by positive BM MRD in 42/44 relapses with complete MRD data, by a median of 71 (23-283) days. Of 137 patients in continuous complete remission, 93 were consistently MRD-negative, 39 reverted from MRD+ to MRD-, and five were MRD+ at last sampling. Estimated 1 year-RFS after first positive MRD was 49%, 39%, and 30%, using cutoff levels of 0.1%, 0.3%, and 0.5%, respectively. In a multivariate Cox model, MRD (hazard ratio [HR], 7.99), WHO subgroup AML (HR, 4.87), TP53 multi-hit (HR, 2.38), NRAS (HR, 3.55), and acute GVHD grade III-IV (HR, 4.13) were associated with shorter RFS. MRD+ was also independently associated with shorter OS (HR, 2.65). In a subgroup analysis of 100 MRD+ patients, presence of chronic GVHD was associated with longer RFS (HR, 0.32). CONCLUSION: Assessment of individualized MRD using NGS + ddPCR is feasible and can be used for early detection of relapse. Positive MRD is associated with shorter RFS and OS (ClinicalTrials.gov identifier: NCT02872662).

BTK and PLCG2 remain unmutated in one-third of patients with CLL relapsing on ibrutinib.

Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet need. Though Bruton tyrosine kinase (BTK) and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL on ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital polymerase chain reaction (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32 of 49 relapsing cases (65%) carried at least 1 hotspot BTK and/or PLCG2 mutation(s); in 6 of 32, BTK mutations were only detected by ddPCR (variant allele frequency [VAF] 0.1% to 1.2%). BTK/PLCG2 mutations were also identified in 6 of 49 responding patients (12%; 5/6 VAF <10%), of whom 2 progressed later. Among the relapsing patients, the BTK-mutated (BTKmut) group was enriched for EGR2 mutations, whereas BTK-wildtype (BTKwt) cases more frequently displayed BIRC3 and NFKBIE mutations. Using an extended capture-based panel, only BRAF and IKZF3 mutations showed a predominance in relapsing cases, who were enriched for del(8p) (n = 11; 3 BTKwt). Finally, no difference in TP53 mutation burden was observed between BTKmut and BTKwt relapsing cases, and ibrutinib treatment did not favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of a real-world cohort failing ibrutinib, and propose additional mechanisms contributing to resistance.

Ultra-sensitive monitoring of leukemia patients using superRCA mutation detection assays.

Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses to therapy in clinical routine, and improved assay techniques are needed for broad adoption. We describe herein a highly sensitive and selective molecule amplification technology - superRCA assays - for rapid and highly specific detection of DNA sequence variants present at very low frequencies in DNA samples. Using a standard flow cytometer we demonstrate precise, ultra-sensitive detection of single-nucleotide mutant sequences from malignant cells against up to a 100,000-fold excess of DNA from normal cells in either bone marrow or peripheral blood, to follow the course of patients treated for acute myeloid leukemia (AML). We also demonstrate that sequence variants located in a high-GC region may be sensitively detected, and we illustrate the potential of the technology for early detection of disease recurrence as a basis for prompt change of therapy.

Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

Neuropilin-1 (NRP1) regulates developmental and pathological angiogenesis, arteriogenesis, and vascular permeability, acting as a coreceptor for semaphorin 3A (Sema3A) and the 165-amino acid isoform of vascular endothelial growth factor A (VEGF-A165). NRP1 is also the receptor for the CendR peptides, a class of cell- and tissue-penetrating peptides with a specific R-x-x-R carboxyl-terminal motif. Because the cytoplasmic domain of NRP1 lacks catalytic activity, NRP1 is mainly thought to act through the recruitment and binding to other receptors. We report here that the NRP1 intracellular domain mediates vascular permeability. Stimulation with VEGF-A165, a ligand-blocking antibody, and a CendR peptide led to NRP1 accumulation at cell-cell contacts in endothelial cell monolayers, increased cellular permeability in vitro and vascular leakage in vivo. Biochemical analyses, VEGF receptor-2 (VEGFR-2) silencing, and the use of a specific VEGFR blocker established that the effects induced by the CendR peptide and the antibody were independent of VEGFR-2. Moreover, leakage assays in mice expressing a mutant NRP1 lacking the cytoplasmic domain revealed that this domain was required for NRP1-induced vascular permeability in vivo. Hence, these data define a vascular permeability pathway mediated by NRP1 but independent of VEGFR-2 activation.

The endothelial adaptor molecule TSAd is required for VEGF-induced angiogenic sprouting through junctional c-Src activation.

Activation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by VEGF binding is critical for vascular morphogenesis. In addition, VEGF disrupts the endothelial barrier by triggering the phosphorylation and turnover of the junctional molecule VE-cadherin, a process mediated by the VEGFR2 downstream effectors T cell-specific adaptor (TSAd) and the tyrosine kinase c-Src. We investigated whether the VEGFR2-TSAd-c-Src pathway was required for angiogenic sprouting. Indeed, Tsad-deficient embryoid bodies failed to sprout in response to VEGF. Tsad-deficient mice displayed impaired angiogenesis specifically during tracheal vessel development, but not during retinal vasculogenesis, and in VEGF-loaded Matrigel plugs, but not in those loaded with FGF. The SH2 and proline-rich domains of TSAd bridged VEGFR2 and c-Src, and this bridging was critical for the localization of activated c-Src to endothelial junctions and elongation of the growing sprout, but not for selection of the tip cell. These results revealed that vascular sprouting and permeability are both controlled through the VEGFR2-TSAd-c-Src signaling pathway in a subset of tissues, which may be useful in developing strategies to control tissue-specific pathological angiogenesis.

NRP1 presented in trans to the endothelium arrests VEGFR2 endocytosis, preventing angiogenic signaling and tumor initiation.

Neuropilin 1 (NRP1) modulates angiogenesis by binding vascular endothelial growth factor (VEGF) and its receptor, VEGFR2. We examined the consequences when VEGFR2 and NRP1 were expressed on the same cell (cis) or on different cells (trans). In cis, VEGF induced rapid VEGFR2/NRP1 complex formation and internalization. In trans, complex formation was delayed and phosphorylation of phospholipase Cγ (PLCγ) and extracellular regulated kinase 2 (ERK2) was prolonged, whereas ERK1 phosphorylation was reduced. Trans complex formation suppressed initiation and vascularization of NRP1-expressing mouse fibrosarcoma and melanoma. Suppression in trans required high-affinity, steady-state binding of VEGF to NRP1, which was dependent on the NRP1 C-terminal domain. Compatible with a trans effect of NRP1, quiescent vasculature in the developing retina showed continuous high NRP1 expression, whereas angiogenic sprouting occurred where NRP1 levels fluctuated between adjacent endothelial cells. Therefore, through communication in trans, NRP1 can modulate VEGFR2 signaling and suppress angiogenesis.

The role of differential VE-cadherin dynamics in cell rearrangement during angiogenesis.

Endothelial cells show surprising cell rearrangement behaviour during angiogenic sprouting; however, the underlying mechanisms and functional importance remain unclear. By combining computational modelling with experimentation, we identify that Notch/VEGFR-regulated differential dynamics of VE-cadherin junctions drive functional endothelial cell rearrangements during sprouting. We propose that continual flux in Notch signalling levels in individual cells results in differential VE-cadherin turnover and junctional-cortex protrusions, which powers differential cell movement. In cultured endothelial cells, Notch signalling quantitatively reduced junctional VE-cadherin mobility. In simulations, only differential adhesion dynamics generated long-range position changes, required for tip cell competition and stalk cell intercalation. Simulation and quantitative image analysis on VE-cadherin junctional patterning in vivo identified that differential VE-cadherin mobility is lost under pathological high VEGF conditions, in retinopathy and tumour vessels. Our results provide a mechanistic concept for how cells rearrange during normal sprouting and how rearrangement switches to generate abnormal vessels in pathologies.