Screening Novel Agent Combinations to Expedite CTCL Therapeutic Development

Abstract

Cutaneous T-cell lymphoma (CTCL) is a group of non-Hodgkin lymphomas of skin-homing malignant T lymphocytes. In advanced stages, CTCL is incurable and often fatal (Arulogun et al., 2008Arulogun S.O. Prince H.M. Ng J. Lade S. Ryan G.F. Blewitt O. et al.Long-term outcomes of patients with advanced-stage cutaneous T-cell lymphoma and large cell transformation.Blood. 2008; 112: 3082-3087Crossref PubMed Scopus (135) Google Scholar), and blood involvement portends a poorer outcome (Agar et al., 2010Agar N.S. Wedgeworth E. Crichton S. Mitchell T.J. Cox M. Ferreira S. et al.Survival outcomes and prognostic factors in mycosis fungoides/Sézary syndrome: validation of the revised International Society for Cutaneous Lymphomas/European Organisation for Research and Treatment of Cancer staging proposal.J Clin Oncol. 2010; 28: 4730-4739Crossref PubMed Scopus (515) Google Scholar). Overall response rates to systemic therapies are 30–50% and generally not durable. High-throughput screening has emerged as a rapid method for identification and prioritization of novel therapeutic compounds. Analysis of natural compounds and agents in ongoing clinical trials may accelerate opportunities for drug repurposing, synergy testing, and preclinical assessment for CTCL therapeutic potential. Patients with CTCL at the Yale Cancer Center (New Haven, CT) were enrolled with written and informed consent in accordance with the Yale Human Investigational Review Board. For initial screening, malignant cells were isolated from the peripheral blood of four patients with CTCL, as previously reported (Cyrenne et al., 2017Cyrenne B.M. Lewis J.M. Weed J.G. Carlson K.R. Mirza F.N. Foss F.M. et al.Synergy of BCL2 and histone deacetylase inhibition against leukemic cells from cutaneous T-cell lymphoma patients.Blood. 2017; 130: 2073-2083Crossref PubMed Scopus (33) Google Scholar, Kim et al., 2018Kim S.R. Lewis J.M. Cyrenne B.M. Monico P.F. Mirza F.N. Carlson K.R. et al.BET inhibition in advanced cutaneous T cell lymphoma is synergistically potentiated by BCL2 inhibition or HDAC inhibition.Oncotarget. 2018; 9: 29193-29207Crossref PubMed Scopus (30) Google Scholar). High-throughput screening was conducted on a 1,348 agent panel rich in phosphatase and kinase inhibitors and natural compounds (Selleckchem Kinase Inhibitors, Enzo Phosphatase Inhibitors, and MicroSource Gen-Plus libraries) at the Yale Center for Molecular Discovery (New Haven, CT). The 4,000–6,000 sorted cells per well were cultured in 384-well plates (Corning 3764) and cell viability assessed (Cell Titer-Glo, Promega, Madison, WI) 72 hours following drug addition. Positive and negative controls were 20% and 0.1% DMSO, respectively, and effective kill was determined with the equation:EffectiveKill=1−luminescenceofdrugadditionwellluminescenceofnegativecontrolwell Mean and SD of positive and negative control wells were used to quantify signal-to-background and Z values for each screening plate to ensure assay robustness. Drug data were normalized to the mean values of the negative control (set as 0% effect) and positive control (set as 100% effect) wells within the plate. Samples from four unique patients with CTCL were used as biological replicates to minimize the risk of selecting agents on the basis of stochastic variation. For confirmatory and combination assessments, selected agents that demonstrated >80% effective kill in initial screening were further assessed using 13 patient-derived CTCL cells‒sorted malignant cell samples (5 female and 8 male), 8 normal (CD4+ sorted) controls (6 female and 2 male), and 5 established CTCL cell lines (HH, Hut78, Sez4, SeAx, and MyLa2059). MyLa (Myla2059) and SeAx were provided by E. Contassot (University Hospital, Zurich, Switzerland), HH and Hut78 were purchased from American Type Culture Collection, and Sez4 was provided by A. Rook (University of Pennsylvania, Philadelphia, PA). We have previously characterized genetic alterations in HH, Hut78, and Sez4 (Lin et al., 2012Lin W.M. Lewis J.M. Filler R.B. Modi B.G. Carlson K.R. Reddy S. et al.Characterization of the DNA copy-number genome in the blood of cutaneous T-cell lymphoma patients.J Invest Dermatol. 2012; 132: 188-197Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Cells were tested for mycobacterial contamination by PCR in November 2019. Cells were exposed to (i) seven agents with previous Food and Drug Administration approval for non-CTCL indications or available over the counter as natural compounds (sanguinarine, pyrvinium pamoate, quinacrine, ciclopirox, salinomycin, gentian violet, and ceritinib) and (ii) two agents in phase II clinical trials for other indications (BIIB021 and NVP-BGT226) individually and in combination. Mean inhibitory concentrations were calculated using GraphPad Prism (version 8.2.0). Specificity index (half-maximal inhibitory concentration for control divided by half-maximal inhibitory concentration for patients) and synergy combination index (Chou-Talalay method [Chou, 2010Chou T.C. Drug combination studies and their synergy quantification using the Chou-talalay method.Cancer Res. 2010; 70: 440-446Crossref PubMed Scopus (3268) Google Scholar]) or fold potentiation were determined using Stata/SE (version 15). Normality was formally assessed using the Shapiro–Wilk test, and on the basis of the results, either a parametric or nonparametric one-way ANOVA test (with a correction for multiple comparisons by the Benjamini‒Hochberg method) was applied to assess for statistical significance of agents showing clinical promise. The primary screen yielded 95 agents that demonstrated over 80% effective kill (available on Mendeley). Of these, nine agents were selected with previous Food and Drug Administration approval for non-CTCL indications or in advanced clinical trials. Single-agent titrations identified the anthelmintic pyrvinium pamoate (x̄ = 0.027 μM), antimicrobial gentian violet (x̄ = 0.068 μM), NVP-BGT226, a phosphoinositide 3-kinase/mTOR dual inhibitor (x̄ = 0.101 μM), and the antibiotic salinomycin (x̄ = 0.405 μM) as having the lowest half-maximal inhibitory concentration values (i.e., greatest per molar cytotoxicity) against patient-derived CTCL cells (Figure 1a) and that patient-derived CTCL cells were significantly more sensitive to salinomycin (specificity index = 6.380) and gentian violet (specificity index = 3.265) than control CD4+ cells (Figure 1b). Patient-derived CTCL cells were relatively susceptible to the antimalarial quinacrine (x̄ = 2.662 μM) and relatively resistant to the HSP-90 inhibitor BIIB021 (x = 29.970 μM) compared with CTCL cell lines (P ≤ 0.05). When these agents were tested in combination, the average Chou-Talalay indices revealed synergy in the patient-derived CTCL cells for salinomycin plus either ciclopirox, gentian violet, or pyrvinium pamoate and for gentian violet plus sanguinarine (Figure 2a). For gentian violet plus sanguinarine, a statistical trend was observed in patient-derived CTCL cells compared with normal CD4+ control cells (P = 0.059). Patient-derived CTCL cells also showed greater potentiation with salinomycin or gentian violet plus NVP-BGT226 than normal control cells (p = 0.022 or 0.034, respectively) (Figure 2b).Figure 2Patient samples, normal control CD4+ samples, and CTCL cell lines demonstrate variable synergism and potentiation to combination drug pairs. Isolated malignant patient-derived CTCL cells (n = 13) and normal control cells (n = 8) were incubated with a range of concentrations of combinations of sanguinarine, BIIB021, pyrvinium, ciclopirox, salinomycin, gentian violet, and NVP-BGT226 for 72 hours, and combination indices and fold change were calculated. (a) Combination Indices. For patient-derived CTCL cells, synergism is observed for salinomycin plus ciclopirox, gentian violet, or pyrvinium and gentian violet plus sanguinarine (C.I. < 1). Patient-derived cells marginal differential synergism for gentian violet and sanguinarine. Combinations of sanguinarine or gentian violet with pyrvinium or ciclopirox were antagonistic or showed very minimal synergy. (b) Fold Changes. The combination of salinomycin or gentian violet plus NVP-BGT226 showed more potentiation for patient-derived CTCL cells than for normal control cells. C.I., synergy combination index; CTCL, cutaneous T-cell lymphoma.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our results build upon preclinical data demonstrating that gentian violet enhances CTCL apoptosis and impedes tumor cell proliferation in vitro (Wu and Wood, 2018Wu J. Wood G.S. Analysis of the effect of gentian violet on apoptosis and proliferation in cutaneous T-cell lymphoma in an in vitro study.JAMA Dermatol. 2018; 154: 1191-1198Crossref PubMed Scopus (7) Google Scholar) and has shown some potential as a novel, inexpensive topical therapy for recalcitrant lesions (Cowan et al., 2019Cowan N. Coman G. Duffy K. Wada D.A. Treatment of recalcitrant mycosis fungoides with topical gentian violet.JAAD Case Rep. 2019; 5: 413-415Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Herein, we identify another agent, salinomycin, that demonstrates almost twice of gentian violet’s specificity for patient-derived CTCL cells as a single agent. We also observe greater synergy and potentiation in patient-derived malignant CTCL cells than in normal controls for combinations of (i) salinomycin plus NVP-BGT226 and (ii) gentian violet plus sanguinarine or NVP-BGT226. The largest animal study on gentian violet to date helps reassure us of its safety and prompted its approval as an over-the-counter agent in the USA (Arbiser, 2009Arbiser J.L. Gentian violet is safe.J Am Acad Dermatol. 2009; 61: 359Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Of note, both gentian violet and salinomycin are antibacterial agents with reported activity against Staphylococcus aureus. Because anti-Staphylococcus antibiotic treatment has been shown to decrease malignant T cells in the lesional skin of patients with CTCL, these agents may have clinical benefit in addition to the direct cancer cell cytotoxicity effects reported here (Lindahl et al., 2019Lindahl L.M. Willerslev-Olsen A. Gjerdrum L.M.R. Nielsen P.R. Blümel E. Rittig A.H. et al.Antibiotics inhibit tumor and disease activity in cutaneous T-cell lymphoma.Blood. 2019; 134: 1072-1083Crossref PubMed Scopus (57) Google Scholar). The responses of five established CTCL cell lines to single and combination agents tested were diverse, confirming our earlier observation that cell lines do not consistently recapitulate the pathophysiology of patients’ CTCL cells (Yumeen et al., 2020Yumeen S. Mirza F.N. Lewis J.M. King A.L.O. Kim S.R. Carlson K.R. et al.JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL.Blood Adv. 2020; 4: 2213-2226Crossref PubMed Scopus (11) Google Scholar). Overall, our results suggest several individual drugs and combinations that may warrant further preclinical and clinical investigation as novel therapies for CTCL. Datasets related to this article can be found hosted at Mendeley (https://doi.org/10.17632/hpvnkrshdg.1). Fatima N. Mirza: http://orcid.org/0000-0003-1299-6258 Sara Yumeen: http://orcid.org/0000-0002-5729-5909 Julia M. Lewis: http://orcid.org/0000-0002-9897-1950 Amber Loren O. King: http://orcid.org/0000-0002-0302-2913 Sa Rang Kim: http://orcid.org/0000-0002-0345-7028 Kacie R. Carlson: http://orcid.org/0000-0003-2705-9689 Sheila Umlauf: http://orcid.org/0000-0002-8671-7744 Yulia V. Surovtseva: http://orcid.org/0000-0001-6079-9246 Francine M. Foss: http://orcid.org/0000-0001-7843-3162 Michael Girardi: http://orcid.org/0000-0003-1887-9343 FMF is a Seattle Genetics speakers bureau and consultant for Acrotech, Miragen, Daiichi Sanyo, and Mallinckrodt. The remaining authors state no conflict of interest. This work was supported by R. S. Foundation and Martin and Dorothy Spatz Charitable Foundation. This work was made possible in part by the Yale Clinical and Translational Science Award #TL1 TR001864 from the National Center for Advancing Translational Science and the Yale Center for Clinical Investigation . Conceptualization: FNM, SY, JML, MG; Data Curation: FNM, SY, JML, MG; Formal Analysis: FNM, SY, JML, MG; Funding Acquisition: MG; Investigation: FNM, SY, JML, ALOK, SU, YVS; Methodology: FNM, SY, JML, MG; Resources: KRC, FMF, MG; Supervision: MG; Validation: FNM, SY, JML, MG; Visualization: FNM, SY, JML, MG; Writing - Original Draft Preparation: FNM, SY, JML, MG; Writing - Review and Editing: FNM, SY, JML, ALOK, SRK, KRC, SU, YVS, FMF, MG