Todd M. Wilson

Clonal analysis of NRAS activating mutations in KIT-D816V systemic mastocytosis

Cooperating genetic events are likely to contribute to the phenotypic diversity of KIT-D816V systemic mastocytosis. In this study, 44 patients with KIT-D816V systemic mastocytosis were evaluated for coexisting NRAS, KRAS, HRAS or MRAS mutations. Activating NRAS mutations were identified in 2 of 8 patients with advanced disease. NRAS mutations were not found in patients with indolent systemic mastocytosis. To better understand the clonal evolution of mastocytosis, we evaluated the cell compartments impacted by the NRAS and KIT mutations. Clonal mast cells harbored both mutations. KIT-D816V was not detected in bone marrow CD34+ progenitors, whereas the NRAS mutation was present. These findings suggest that NRAS mutations may have the potential to precede KIT-D816V in clonal development. Unlike other mature lineages, mast cell survival is dependent on KIT and the presence of these two activating mutations may have a greater impact on the expansion of this cell compartment and in resultant disease severity. (Clinicaltrials.gov identifier: NCT00044122, NCT00001756)

mTORC1 and mTORC2 differentially regulate homeostasis of neoplastic and non-neoplastic human mast cells

Increased mast cell burden is observed in the inflamed tissues and affected organs and tissues of patients with mast cell proliferative disorders. However, normal mast cells participate in host defense, so approaches to preferentially target clonally expanding mast cells are needed. We found that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) are up-regulated in neoplastic and developing immature mast cells compared with their terminally differentiated counterparts. Elevated mTOR mRNA was also observed in bone marrow mononuclear cells of patients exhibiting mast-cell hyperplasia. Selective inhibition of mTORC1 and mTORC2 through genetic and pharmacologic manipulation revealed that, whereas mTORC1 may contribute to mast-cell survival, mTORC2 was only critical for homeostasis of neoplastic and dividing immature mast cells. The cytostatic effect of mTORC2 down-regulation in proliferating mast cells was determined to be via inhibition of cell-cycle progression. Because mTORC2 was observed to play little role in the homeostasis of differentiated, nonproliferating, mature mast cells, these data provide a rationale for adopting a targeted approaching selectively inhibiting mTORC2 to effectively reduce the proliferation of mast cells associated with inflammation and disorders of mast cell proliferation while leaving normal differentiated mast cells largely unaffected.

Activity of imatinib in systemic mastocytosis with chronic basophilic leukemia and a PRKG2-PDGFRB fusion

Systemic mastocytosis with an associated hematologic non-mast cell lineage disease is a rare subtype of systemic mastocytosis. This study describes a patient with systemic mastocytosis associated with chronic basophilic leukemia and a PRKG2-PDGFRB fusion gene. This patient had a complete response to imatinib mesylate. See related perspective on page 6. Background Translocations involving region 5q31-32 (PDGFRB) have been reported in a variety of myeloproliferative diseases and are often associated with significant peripheral eosinophilia. We report an unusual case of a patient presenting with peripheral basophilia and systemic mastocytosis in whom cytogenetic analysis revealed a t(4;5)(q21.1;q31.3). Design and Methods We used molecular analyses to determine the role of PDGFRB in this case. The patient was treated with imatinib. Results Fluorescence in situ hybridization (FISH) documented a breakpoint in PDGFRB. In agreement with this, the patient responded very well to imatinib with resolution of clinical symptoms, basophilia, and mast cell disease. Molecular analyses revealed that PDGFRB, encoding an imatinib-sensitive tyrosine kinase, was fused to PRKG2. The fusion gene incorporates the first two exons of PRKG2 fused to the truncated exon 12 of PDGFRB, resulting in the disruption of its juxtamembrane domain. Functional studies confirmed that the activity and transforming properties of PRKG2-PDGFRβ were dependent on the disruption of the auto-inhibitory juxtamembrane domain. Conclusions Our results identify a second case of the PRKG2-PDGFRB fusion and confirm the unusual PDGFRB breakpoint associated with this fusion. This work also illustrates the use of imatinib for the treatment of specific cases of systemic mastocytosis.

Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number

Elevated basal serum tryptase levels are present in 4–6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase–encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.

Alpha 2-macroglobulin capture allows detection of mast cell chymase in serum and creates a reservoir of angiotensin II-generating activity.

Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by α2-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that α2-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used α2-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of ∼1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. α2-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to α2-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that α2-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.

IL-5 receptor α levels in patients with marked eosinophilia or mastocytosis

BACKGROUND IL-5 plays a central role in the development and maintenance of eosinophilia (EO) and eosinophil activation in a wide variety of eosinophilic disorders. Although IL-5, IL-3, and GM-CSF can modulate the expression of IL-5 receptor α (IL-5Rα) on eosinophils in vitro, little is known about soluble and surface IL-5Rα levels in vivo. OBJECTIVE To assess soluble and surface IL-5Rα levels in patients with EO and/or mastocytosis. METHODS Surface IL-5Rα expression was assessed by flow cytometry in blood and/or bone marrow from subjects with EO (n = 39) and systemic mastocytosis (n = 8) and from normal volunteers (n = 28). Soluble IL-5Rα (sIL-5Rα) level was measured in a cohort of 177 untreated subjects and correlated with EO, eosinophil activation, and serum tryptase and cytokine levels. RESULTS IL-5Rα expression on eosinophils inversely correlated with EO (r = -0.48; P < .0001), whereas serum levels of sIL-5Rα increased with the eosinophil count (r = 0.56; P < .0001) and serum IL-5 (r = 0.40; P < .0001) and IL-13 (r = 0.29; P = .004) levels. Of interest, sIL-5Rα level was significantly elevated in patients with systemic mastocytosis without EO. Although sIL-5Rα levels correlated with serum tryptase levels in these patients, eosinophil activation, assessed by CD69 expression on eosinophils and serum eosinophil-derived neurotoxin levels, was increased compared with that in normal subjects. CONCLUSIONS These data are consistent with an in vivo IL-5Rα regulatory pathway in human eosinophils similar to that described in vitro and involving a balance between soluble and surface receptor levels. This may have implications with respect to the use of novel therapeutic agents targeting IL-5 and its receptor in patients with EO and/or mastocytosis.

PTEN deficiency in mast cells causes a mastocytosis-like proliferative disease that heightens allergic responses and vascular permeability

Kit regulation of mast cell proliferation and differentiation has been intimately linked to the activation of phosphatidylinositol 3-OH kinase (PI3K). The activating D816V mutation of Kit, seen in the majority of mastocytosis patients, causes a robust activation of PI3K signals. However, whether increased PI3K signaling in mast cells is a key element for their in vivo hyperplasia remains unknown. Here we report that dysregulation of PI3K signaling in mice by deletion of the phosphatase and tensin homolog (Pten) gene (which regulates the levels of the PI3K product, phosphatidylinositol 3,4,5-trisphosphate) caused mast cell hyperplasia and increased numbers in various organs. Selective deletion of Pten in the mast cell compartment revealed that the hyperplasia was intrinsic to the mast cell. Enhanced STAT5 phosphorylation and increased expression of survival factors, such as Bcl-XL, were observed in PTEN-deficient mast cells, and these were further enhanced by stem cell factor stimulation. Mice carrying PTEN-deficient mast cells also showed increased hypersensitivity as well as increased vascular permeability. Thus, Pten deletion in the mast cell compartment results in a mast cell proliferative phenotype in mice, demonstrating that dysregulation of PI3K signals is vital to the observed mast cell hyperplasia.

Targeting the KIT activating switch control pocket: a novel mechanism to inhibit neoplastic mast cell proliferation and mast cell activation

Activating mutations in the receptor tyrosine kinase KIT, most notably KIT D816V, are commonly observed in patients with systemic mastocytosis. Thus, inhibition of KIT has been a major focus for treatment of this disorder. Here we investigated a novel approach to such inhibition. Utilizing rational drug design, we targeted the switch pocket (SP) of KIT, which regulates its catalytic conformation. Two SP inhibitors thus identified, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. Autophosphorylation of both wild-type and, where also examined, KIT D816V activation was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines, and in CD34+-derived human mast cells activated by stem cell factor (SCF). Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Moreover, the SP inhibitors more selectively blocked SCF potentiation of FcɛRI-mediated degranulation. Overall, SP inhibitors represent an innovative mechanism of KIT inhibition whose dual suppression of KIT D816V neoplastic mast cell proliferation and SCF-enhanced mast cell activation may provide significant therapeutic benefits.

Characterization and function of histamine receptors in human bone marrow stromal cells

There are several clinical trials worldwide using bone marrow stromal cells (BMSCs) as a cellular therapy to modulate immune responses in patients suffering from various inflammatory conditions. A deeper understanding of the molecular mechanisms involved in this modulatory effect could help us design better, more effective protocols to treat immune mediated diseases. In this study, we demonstrated that human BMSCs express H1, H2, and H4 histamine receptors and they respond to histamine stimulation with an increased interleukin 6 (IL‐6) production both in vitro and in vivo. Using different receptor antagonists, we pinpointed the importance of the H1 histamine receptor, while Western blot analysis and application of various mitogen‐activated protein kinase inhibitors highlighted the role of p38, extracellular signal‐regulated kinase, and c‐Jun N‐terminal kinase kinases in the observed effect. When BMSCs were pretreated with either histamine or degranulated human mast cells, they exhibited an enhanced IL‐6‐dependent antiapoptotic effect on neutrophil granulocytes. Based on these observations, it is likely that introduction of BMSCs into a histamine‐rich environment (such as any allergic setting) or pretreatment of these cells with synthetic histamine could have a significant modulatory effect on the therapeutic potential of BMSCs. STEM CELLS 2012; 30:222–231.

A distinct biomolecular profile identifies monoclonal mast cell disorders in patients with idiopathic anaphylaxis

Background: Clonal mast cell disorders are known to occur in a subset of patients with systemic reactions to Hymenoptera stings. This observation has prompted the question of whether clonal mast cell disorders also occur in patients with idiopathic anaphylaxis (IA). Objective: We sought to determine the prevalence of clonal mast cell disorders among patients with IA, criteria to identify those patients who require a bone marrow biopsy, and whether the pathogenesis of IA involves a hyperresponsive mast cell compartment. Methods: We prospectively enrolled patients with IA (≥3 episodes/y) who then underwent a medical evaluation that included a serum tryptase determination, allele‐specific quantitative PCR (ASqPCR) for the KIT D816V mutation, and a bone marrow examination. Mast cells were cultured from peripheral blood CD34+ cells and examined for releasability after Fc&egr;RI aggregation. Results: Clonal mast cell disease was diagnosed in 14% of patients referred with IA. ASqPCR for the KIT D816V mutation was a useful adjunct in helping identify those with systemic mastocytosis but not monoclonal mast cell activation syndrome. A modified overall clonal prediction model was developed by using clinical findings, a serum tryptase determination, and ASqPCR. There was no evidence of a hyperresponsive mast cell phenotype in patients with IA. Conclusion: Patients with clonal mast cell disease can present as having IA. Distinct clinical and laboratory features can be used to select those patients more likely to have an underlying clonal mast cell disorder (monoclonal mast cell activation syndrome or systemic mastocytosis) and thus candidates for a bone marrow biopsy.