PracticeUpdate Conference Series: IID 2018

Results of Two Preclinical Studies Point to Potential Therapeutic Targets inCutaneous T-Cell Lymphoma Combined inhibition of the BET family proteins + histone deacetylases, as well as the topic antimicrobial gentian violet, may hold promise in targeting cutaneous T-cell lymphoma, results of two preclinical studies show. Combined inhibition of the BET family proteins + histone deacetylases activation-induced cell death is an effective way to kill CTCL cells in vitro and ex vivo.

" …the superior antitumor effects of JQ1/SAHA in vitro and ex vivo provide a

Dr. Wu and colleagues set out to identify novel small molecules that induce extrinsic apoptosis in CTCL. They used the HH CTCL line in an enzyme- linked immunosorbent assay to detect cleaved caspase 8 induced by 1710 unique compounds in the National Institutes of Health Clinical Collection, Selleck Kinase Inhibitor Library and Prestwick Chemical Library. As assessed by annexin V/PI flow cytome- try of the HH cell line, the non-prescription topical antimicrobial gentian violet induced more total apoptosis than mechlorethamine. Mechlorethamine is approved as topical therapy for CTCL. Gentian violet induced approximately 4 to 6 times greater apoptosis in CTCL lines MyLa, SeAx, Hut78, HH, and SZ4 than in normal keratinocytes. This pronounced effect suggested a favorable topical toxicity profile. In addition to increasing caspase 8, gentian violet also upregulated death receptors DR4, DR5, and the TRAIL and FasL ligands. This upregulation was consistent with induction of extrinsic apoptosis via the TRAIL and Fas pathways. Increased phosphorylation of phospholipase C-γ 1, Ca++ influx, and reactive oxygen species were also detected in CTCL lines, which indicated that the mechanism of FasL upregulation involves key ele- ments of the activation-induced cell death pathway. Methotrexate increased gentian violet apop- tosis significantly in Sezary blood cells and all CTCL lines evaluated except Fas-null SeAx. This increase suggested the role of Fas in this process. In ex vivo studies, 1 μm gentian violet induced up to 90% CTCL apoptosis in Sezary blood cells. Gentian violet also reduced expression of anti-apoptotic MCL-1 and proproliferative NFkB components, while increasing IkB levels that pro- mote NFkB catabolism. Furthermore, gentian violet inhibited cell prolifera- tion and induced cell cycle arrest. Though purple at neutral pH, gentian violet can be rendered colorless by altering its pH. Dr. Wu concluded that these preclinical findings broaden the knowledge about the antineoplastic effects of gentian violet and provide a rationale for clinical studies of gentian violet as a novel, inexpensive topical therapy for CTCL.

Lei Zhao, PhD, of the Wisconsin School of Medicine and Public Health in Madison, Wisconsin, explained that advanced-stage cutaneous T-cell lymphoma (CTCL) is often a fatal malignancy despite optimal use of available treatments. Dr. Zhao and colleagues evaluated a novel therapy for CTCL in preclinical in vitro and ex vivo experiments. Their goal was to evaluate combination therapy with JQ1 (a prototypical BET bromodomain inhibitor (BET) + the HDAC inhibitor SAHA, approved to treat CTCL. In five CTCL lines, the IC50 was 0.5–1 µM for both compounds. The JQ1/SAHA combination proved to be uniformly synergistic against cell viability at 1 µM concentrations (Cl <1). The combination also induced G0/G1 cell cycle arrest. Propidium iodide/annexin V flowcytometry showed all CTCL lines exhibited significantly greater apop- tosis (approximately 40–90%) with combination treatment than with either agent alone. Relative to single agents, combination treatment showed significantly greater activation of apop- totic caspases 3, 8, and 9. This greater activation was confirmed by immunoblot analysis. Combination treatment induced significant increases in extrinsic apoptotic pathway death receptors and ligands (FasL, DR4, DR5, TRAIL, and tumor necrosis factor R1) that were more consistent across the CTCL lines than with the single agents. Ex vivo studies of leukemic CTCL cells obtained from four patients with Sezary syndrome showed the highest levels of apoptosis were in response to JQ1/SAHA combination treatment and ranged from 71% to 93%. In contrast, combination treatment of normal CD4- positive T cells induced only minimal increases in apoptosis (2–9%). Dr. Zhao concluded that the superior antitumor effects of JQ1/SAHA in vitro and ex vivo provide a rationale for clinical trials exploring BET inhibition/ HDAC inhibition in combination for CTCL. Gentian violet Jianqiang Wu, MD, PhD, also of the Wisconsin School of Medicine and Public Health, stated that in 2015 his team showed previously that triggering the extrinsic apoptotic pathway by upregulating

rationale for clinical trials exploring BET inhibition/ HDAC inhibition in combination for CTCL. "

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IID 2018 • PRACTICEUPDATE CONFERENCE SERIES