The Novel Organic Arsenical Darinaparsin Induces MAPK-Mediated and SHP1-Dependent Cell Death in T-cell Lymphoma and Hodgkin Lymphoma Cells and Human Xenograft Models

Abstract

Purpose Darinaparsin (Zio-101) is a novel organic arsenical compound with encouraging clinical activity in relapsed/refractory T-cell lymphoma (TCL) and Hodgkin lymphoma (HL), however little is known regarding its mechanism of action. Experimental Design TCL cell lines (Jurkat, Hut78, and HH) and HL cell lines (L428, L540, and L1236) were examined for in vitro cell death by MTT assay and Annexin-V based flow cytometry. Jurkat and L540-derived xenografts in SCID mice were examined for in vivo tumor inhibition and survival. Biological effects of darinaparsin on MAPK pathway were investigated using pharmacological inhibitors, RNA interference (RNAi) and transient transfection for overexpression for SHP1 and MEK. Results Darinaparsin treatment resulted in dose-dependent cytotoxicity and apoptosis in all TCL and HL cell lines. Additionally, darinaparsin had more rapid, higher, and sustained intracellular arsenic levels compared with arsenic trioxide via mass spectrometry. In vivo experiments with Jurkat (TCL) and L540 (HL)-derived lymphoma xenografts showed significant inhibition of tumor growth and improved survival in darinaparsin-treated SCID mice. Biologically, darinaparsin caused phosphorylation of ERK (and relevant downstream substrates) primarily by decreasing the inhibitory SHP1 phosphatase and co-immunoprecipitation showed significant ERK/SHP1 interaction. Furthermore, ERK shRNA knockdown or constitutive overexpression of SHP1 resulted in increased apoptosis, while co-treatment with pharmacologic MEK inhibitors resulted in synergistic cell death. Conversely, SHP1 blockade (via pharmacologic inhibition or RNAi) as well as MEK constitutive activation decreased darinaparsin-related cell death. Conclusions Altogether, these data show that darinaparsin is highly active in HL and TCL and its activity is dependent primarily on MAPK mechanisms.

Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel

“…Next, we sought to determine if these mutants or variants detected in cTCL were reflected in abnormal or aberrant protein expression. It should be highlighted that MET, KDR (VEGFR) STK11 and BRAF are all upstream signaling components in the MAPK pathway [ 19 – 22 ] and aberrant MAPK signaling is a known oncogenic mechanism in lymphoma [ 23 ]. Somatic mutations and copy number alterations of these genes have been reported to activate MAPK and NFκB signaling pathways in human TCL [ 22 ] suggesting that the observed mutations, if functional, could result in activation of MAPK and NFκB signaling in cTCL.…”

Proteasomal Inhibition by Ixazomib Induces CHK1 and MYC-Dependent Cell Death in T-cell and Hodgkin Lymphoma

“…For MTT assay and Annexin V apoptosis detection, performed as described before (22). IC 50 values and combination indices for drug treatment were derived using Calcusyn Version 2.1 Software (Biosoft, Ferguson, MO).…”

“…For examination of the in vivo effect of ixazomib, human lymphoma xenografts derived from Jurkat (TCL) or L540 (HL) grown in SCID mouse models as described before,(22) were treated with either saline (control), or ixazomib (0.36 or 0.72mg/kg) by intraperitoneal ( i.p. ) injections daily for 5 days for three weeks consisting eight mice per group.…”

“…injections daily for 5 days for three weeks consisting eight mice per group. Animals observation, tumor and body weight measurements, and statistical analysis using GraphPad Prism 5.0, (GraphPad Software, Inc. La Jolla, CA), were performed as described before (22). To quantify tumor growth, we used nonlinear regression to fit the tumor growth phase data with curves of the form V= be ct , where V is tumor volume and t is time (23).…”

“…Preparation of protein lysates and Western blot was performed, as previously described (22). Primary antibodies against Myc, total or phospho Chk1 (Ser 345), total or acetylated Histone H3, β-actin, HDAC3, p62, Cathepsin D total and cleaved caspase-3, 8, 9, PARP and β-actin were purchased from Cell Signaling Technology (Beverly, MA).…”