Methylation of dual-specificity phosphatase 4 controls cell differentiation.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

Darusentan is a potent inhibitor of endothelin signaling and function in both large and small arteries.

Endothelin is a potent vasoconstrictor often up-regulated in hypertension. Endothelin vasoconstriction is mediated via the G-protein coupled endothelin A (ETA) receptor present on vascular smooth muscle. Endothelin receptor antagonists (ERAs) have been shown to antagonize ET-induced vasoconstriction. We describe the primary pharmacology of darusentan, a propanoic acid based ERA currently in phase 3 clinical trials for resistant hypertension. Darusentan was tested in membrane-, cell-, and tissue-based assays to determine its biochemical and functional potency. Rat aortic vascular smooth muscle cells (RAVSMs) were characterized using flow cytometry. RAVSM membrane fractions tested in saturation experiments exhibited moderate endothelin receptor density. Receptor counting revealed that >95% of the endothelin receptors in these fractions were the ETA subtype. (S)-Darusentan competed for radiolabeled endothelin binding in RAVSM membranes with single-site kinetics, exhibiting a Ki = 13 nmol/L. (R)-Darusentan exhibited no binding activity. In cultured RAVSMs, endothelin induced increases in inositol phosphate and Ca2+ signaling, both of which were attenuated by (S)-darusentan in a concentration-dependent manner. In isolated endothelium-denuded rat aortic rings, (S)-darusentan inhibited endothelin-induced vascular contractility with a pA2 = 8.1 +/- 0.14 (n = 4 animals; mean +/- SD). (R)-Darusentan had no effect. The vasorelaxant potency of (S)-darusentan did not change when determined in isolated denuded rat mesenteric arterioles, suggesting a similar mode of action in both conductance and resistance arteries. In vascular smooth muscle, (S)-darusentan is an ERA with high affinity for the ET receptor, which in this preparation is predominantly ETA receptors. (S)-Darusentan inhibits endothelin-induced signaling related to pro-contractile activity and is a potent inhibitor of vasoconstriction in large and small arteries.

A phase I study of oral ARRY-614, a p38 MAPK/Tie2 dual inhibitor, in patients with low or intermediate-1 risk myelodysplastic syndromes.

PURPOSE: Data suggest that activity of p38 MAPK and Tie2 kinases is dysregulated in myelodysplastic syndromes (MDS) and may be targets for novel therapies. A phase I study of ARRY-614, an oral dual inhibitor of p38 MAPK and Tie2, was conducted in patients with low or intermediate-1 International Prognostic Scoring System risk MDS to evaluate safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary responses by International Working Group 2006 criteria. EXPERIMENTAL DESIGN: Forty-five patients received ARRY-614 either once daily or twice daily in dose escalation (400, 600, 900, or 1,200 mg once daily; 200 or 300 mg twice daily) or expansion cohorts. RESULTS: The 300 mg twice daily schedule was not tolerated, and an MTD was not reached for once daily dosing. Treatment-related adverse events were primarily grade 1-2, with the most common being rash, diarrhea, dry skin, fatigue and anorexia. Interpatient PK variability was high, although exposure was sufficient to achieve reduction in p38 MAPK activation in bone marrow and in the levels of circulating biomarkers. Disease responses were observed in 14 of 44 (32%) evaluable patients, 13 (93%) of whom had previously been treated with a hypomethylating agent. Responses were observed in all lineages, with 5 patients experiencing bilineage responses. Three of 25 red blood cell transfusion-dependent (TD) patients achieved transfusion independence (TI) and 5 of 7 platelet TD patients achieved TI. CONCLUSIONS: ARRY-614 was well tolerated and has sufficient activity to warrant further evaluation in this patient population. We recommend 1,200 mg once daily as the optimal dose for further study.

Impact of common epidermal growth factor receptor and HER2 variants on receptor activity and inhibition by lapatinib.

The goal of this study was to characterize the effects of non-small cell lung carcinoma (NSCLC)-associated mutations in epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2) on interactions with the dual tyrosine kinase inhibitor lapatinib. Biochemical studies show that commonly observed variants of EGFR [G719C, G719S, L858R, L861Q, and Delta746-750 (del15)] are enzyme activating, increasing the tyrosine kinase V(max) and increasing the K(m)((app)) for ATP. The point mutations G719C and L861Q had minor effects on lapatinib K(i)s, whereas EGFR mutations L858R and del15 had a higher K(i) for lapatinib than wild-type EGFR. Structural analysis of wild-type EGFR-lapatinib complexes and modeling of the EGFR mutants were consistent with these data, suggesting that loss of structural flexibility and possible stabilization of the active-like conformation could interfere with lapatinib binding, particularly to the EGFR deletion mutants. Furthermore, EGFR deletion mutants were relatively resistant to lapatinib-mediated inhibition of receptor autophosphorylation in recombinant cells expressing the variants, whereas EGFR point mutations had a modest or no effect. Of note, EGFR T790M, a receptor variant found in patients with gefitinib-resistant NSCLC, was also resistant to lapatinib-mediated inhibition of receptor autophosphorylation. Two HER2 insertional variants found in NSCLC were less sensitive to lapatinib inhibition than two HER2 point mutants. The effects of lapatinib on the proliferation of human NSCLC tumor cell lines expressing wild-type or variant EGFR and HER2 cannot be explained solely on the basis of the biochemical activity or receptor autophosphorylation in recombinant cells. These data suggest that cell line genetic heterogeneity and/or multiple determinants modulate the role played by EGFR/HER2 in regulating cell proliferation.