Key Points• We report a first-in-human dose-escalation study in relapsed/refractory B-cell malignancies with the potent BTK inhibitor ONO/GS-4059.• ONO/GS-4059 induced clinically durable responses in relapsed/refractory B-cell malignancies without significant toxicities.We report the results of a multicenter phase 1 dose-escalation study of the selective Bruton tyrosine kinase (BTK) inhibitor ONO/GS-4059 in 90 patients with relapsed/ refractory B-cell malignancies. There were 9 dose-escalation cohorts ranging from 20 mg to 600 mg once daily with twice-daily regimens of 240 mg and 300 mg. Twenty-four of 25 evaluable chronic lymphocytic leukemia (CLL) patients (96%) responded to ONO/GS-4059, with a median treatment duration of 80 weeks; 21 CLL patients remain on treatment. Lymph node responses were rapid and associated with a concurrent lymphocytosis. Eleven of 12 evaluable patients with mantle cell lymphoma (92%) responded (median treatment duration, 40 weeks). Eleven of 31 non-germinal center B-cell diffuse large B-cell lymphoma patients (35%) responded but median treatment duration was 12 weeks due to development of progressive disease. ONO/GS-4059 was very well tolerated with 75% of adverse events (AEs) being Common Toxicity Criteria for Adverse Events version 4.0 grade 1 or grade 2. Grade 3/4 AEs were mainly hematologic and recovered spontaneously during therapy. One CLL patient experienced a grade 3 treatment-related bleeding event (spontaneous muscle hematoma) but no clinically significant diarrhea, cardiac dysrhythmias, or arthralgia were observed. No maximal tolerated dose (MTD) was reached in the CLL cohort. In the non-Hodgkin lymphoma cohort, 4 patients developed a doselimiting toxicity, yielding an MTD of 480 mg once daily. ONO/GS-4059 has significant activity in relapsed/refractory B-cell malignancies without major drug-related toxicity. The selectivity of ONO/GS-4059 should confer advantages in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT01659255. (Blood. 2016;127(4):411-419)

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Previous studies on the refolding of calpain, a heterodimer comprising a catalytic 80 kDa subunit and a regulatory 30 kDa subunit, indicate that both subunits are required for the expression of full protease activity. We reexamined the conditions for refolding of calpain and found that under optimized conditions the renatured 80 kDa subunit has full enzyme activity even in the absence of the 30 kDa subunit. The 30 kDa subunit stabilizes the 80 kDa subunit rather than enhancing its activity. The theory that calpain functions as a dimer requires reexamination.

We identified an autoantibody that reacts with calpastatin [an inhibitor protein of the calcium-dependent neutral protease calpain (EC 3.4.22.17)]. In early immunoblot studies, sera from patients with rheumatoid arthritis (RA) recognized unidentified 60-, 45-, and 75-kDa proteins in HeLa cell extracts.

Development of p38α inhibitors for rheumatoid arthritis has been hindered by toxicity and limited efficacy. Therefore, we evaluated whether MKK6, an upstream kinase that regulates multiple p38 isoforms, might be an alternative therapeutic target in inflammatory arthritis. Wild-type (WT), MKK6−/−, and MKK3−/− mice were administered K/B×N serum to induce arthritis. Articular expression of activated kinases and cytokines was determined by Western blot, qPCR, ELISA, and multiplex analysis. Immunoprecipitation and confocal microscopy experiments were performed to determine the subcellular location of MKK6, P-p38, and MAPKAPK2 (MK2). Arthritis scores were significantly lower in MKK6−/− mice compared with WT mice. Joint destruction and osteoclast differentiation were lower in MKK6−/−, as were articular IL-6 and matrix metalloproteinase-3 expression. Phospho-p38 levels were modestly decreased in the joints of arthritic MKK6−/− mice compared with WT but were significantly higher than MKK3−/− mice. P-MK2 was low in MKK6−/− and MKK3−/− mice. Uncoupled p38 and MK2 activation was also observed in cultured, MKK6−/− FLS and confirmed using kinase assays. Immunoprecipitation assays and confocal microscopy showed that P-p38 and MK2 colocalized in activated WT but not MKK6−/− FLS. Distinct patterns of cytokine production were observed in MKK6−/− and MKK3−/− cells. MKK6 deficiency suppresses inflammatory arthritis and joint destruction, suggesting it might be a therapeutic target for inflammation. Although MKK3 and MKK6 activate the p38 pathway, they regulate distinct subsets of proinflammatory cytokines. MKK6 appears mainly to facilitate p38 and MK2 colocalization in the nucleus rather than to phosphorylate p38.