Decitabine Priming Enhances Mucin 1 Inhibition Mediated Disruption of Redox Homeostasis in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a hematologic malignancy trophic to the skin with diverse patterns of disease presentation and clinical outcome. Although therapy of localized disease is highly effective, therapeutic options for patients with advanced disease remains limited. Patients with advanced stages of mycosis fungoides (MF) and Sezary syndrome (SS), the two most common subtypes of CTCL, have estimated 5-year survival rates of 20% to 40% (1, 2). Even more challenging has been the treatment of patients who experience relapsed or refractory (R/R) disease. Better understanding of the tumor biology of CTCL has yielded an opportunity to change the treatment paradigm with the potential for cure.

The Mucin 1 C-terminal subunit (MUC1-C) oncoprotein plays a pivotal role in the survival of malignant cells including self-renewal, proliferation, and resistance to apoptosis(3). A primary mediator of these effects is the capacity of MUC1-C to regulate cellular levels of reactive oxygen species (ROS; ref. 4). Notably, maintenance of redox balance appears to be a critical factor in protecting CTCL cells from apoptosis in comparison with normal T cells (5, 6). We recently demonstrated that MUC1-C is overexpressed in CTCL cells (7). We have developed a clinical grade cell-penetrating peptide that disrupts homodimerization of the MUC1-C subunit necessary for nuclear translocation and downstream signaling (ref. 8; Supplementary Fig. S1E and S1F). Treatment of CTCL cells with the MUC1-C inhibitor (GO-203) was associated with increased oxidative stress resulting in cell death in the context of late apoptosis and necrosis. These findings indicated that MUC1-C contributes to redox balance in CTCL and thereby is a novel target for its treatment. However, disease response in in vivo models was incomplete highlighting the need to explore synergistic combinations of the MUC1-C inhibitor with other agents that can enhance redox disruption mediated cytotoxicity of the CTCL cells.

Histone deacytelase and hypomethylating agents (HMA) were recently shown to exhibit efficacy in preclinical T-cell lymphoma models (9, 10). Of note, CTCL demonstrates abnormal patterns of methylation and expression of tumor suppressor genes correlating with the clinical disease presentation (11, 12). In addition, decitabine has also been shown to induce ROS accumulation in acute myeloid leukemia models (13). As such, we hypothesized that CTCL would demonstrate enhanced sensitivity to combination therapy with agents that increase oxidative stress by modulating the epigenome.

In this study, we demonstrate that exposure of CTCL cells to decitabine and the MUC1-C inhibitor at minimal cytotoxic concentrations results in a marked increase in ROS levels and depletion of NADP, NADPH, and glutathione (GSH). The combination also induced greater downregulation of the TP53-induced glycolysis and apoptosis regulator (TIGAR), critical to protection from oxidative stress. Further, we demonstrate that GO-203 exerts an independent effect on hypomethylation via its suppression of DNA methytransferases (DNMT) in CTCL. Accordingly, the combination of GO-203 and decitabine was associated with greater downregulation of DNMT 1 and 3b and this resulted in both a global and gene-specific decrease in DNA methylation in CTCL cells. A corresponding increase in expression of the ROS inducing enzymes, NADPH oxidase 4 (Nox4), and dual oxidase 2 (Duox2) potentially epigenetically regulated methylation in CTCL was seen both at mRNA and protein level. This increase in oxidative stress led to phosphorylation of Smad2 and 3 proteins, activation of Smad signaling and consequently downregulation of c-Myc. In concert with these findings, combination therapy resulted in a synergistic and dose-dependent increase in late apoptosis/necrosis across CTCL cells and in primary patient samples. In a murine xenograft model of CTCL, treatment with GO-203 and decitabine resulted in significant reduction of tumor volume as compared to either agent alone. Collectively, these results provide a strong and diverse molecular rationale for the potential synergy of this combination in CTCL.

The CTCL cell lines, H9 (HTB-176), HuT-78 (TIB-161), and HH (CRL-2105) were obtained from ATCC 1 month before start of experiments in July 2015, and authentication was performed using short tandem repeat (STR) profiling. CTCL cell lines, Myla, and SeAx was obtained from Dr Robert Gniadecki (University of Copenhagen, Denmark) and authenticated by STR before use. All cell lines were grown as previously described (7). All cell lines were tested for mycoplasma detection with the MycoSEQ Mycoplasma Detection Assay from Applied Biosystems prior to use. Cell lines were in passage for three months between thawing and use in described experiments.

Decitabine was purchased from Selleckchem (S1200). Genus Oncology LLC provided GO-203. N-acetyl cysteine (NAC) was purchased from Sigma.

Briefly, H9, HuT-78, Myla and primary CTCL cells were incubated with 5 μmol/L carboxy-H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) for 30 minutes at 37°C to assess ROS-mediated oxidation to the fluorescent compound 2′,7′-dichlorofluorescein (DCF), which was measured by excitation at 480 nm and emission at 590 nm. The experiment was done in triplicate for the cell lines and repeated thrice.

H9 and HuT-78 cells were incubated with propidium iodide (PI)/annexin V-fluorescein isothiocyanate (BD BioSciences) for 15 minutes at room temperature and then analyzed by flow cytometry. Each experiment was done in triplicate and repeated thrice.

Intracellular NADPH concentrations were measured using the NADP/NADPH-Glo Assay Kit (Promega G9081). Intracellular GSH concentrations were measured using GSH-Glo Glutathione Assay Kit (Promega V6911) as per manufacturer's instructions. Each experiment was done in triplicate and repeated thrice.

Cell lysates were prepared as described (7). Soluble proteins were analyzed by immunoblotting with anti-DNMT1, 3b (Abcam), anti-Histone 3 (tri methyl K27; Abcam), anti-TIGAR (Abcam), anti-phospho-p38 (Abcam), anti-Total p38 (Abcam), anti-phospho JNK (Abcam), anti-Total JNK (Abcam), anti-Nrf2 (Abcam), anti-phospho Smad2, and Smad3 (Cell Signaling Technology), anti-Total Smad2 and Smad3 (Cell Signaling Technology), anti-c-Myc (Abcam), and anti-GAPDH (Cell Signaling Technology).

Untreated and treated H9 cells were harvested. Following bisulfite conversion of DNA, direct pyrosequencing was performed to quantify methylation levels in 4 CpG positions of the LINE-1 element as a global measure of DNA methylation. Using the same technique promoter specific methylation of 8 CpG positions in the human Nox4 gene and 7 CpG positions in the human Duox2 gene was performed and quantified. Experiment was done in triplicate in conjunction with EpigenDx, Inc.

RNA was extracted from H9 and HuT-78 cells under described experimental conditions using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RT-PCR primers for human Nox4 and Duox2 were purchased from Sigma-Aldrich. Real-time quantitative PCR analysis was performed using Bio-Rad's iTaq Universal One-step RT-qPCR Kit per manufacturer's instructions.

H9, HuT-78, Myla, SeAx, and HH lines were resuspended at a concentration of 1.0 to 1.5 × 10⁴ cells per well in a 96-well plate (CoStar) and incubated at 37°C in a 5% CO₂ humidified incubator for 96 hours. Decitabine was added at concentrations from 10 to 40 nmol in 100 μL as a single dose, selected to approximate the IC₅–IC₁₀ (concentration at which 5% to 10% cells are dead), GO-203 was added daily for 3 days at concentrations ranging from 1.5 to 3 μmol in 100 μL selected to approximate the IC₁₀–IC₂₅ (concentration at which 10–25% cells are dead). When combining decitabine and GO-203, identical drug concentrations and schedule was used. At 96 hours, the cells were evaluated for viability using the CellTiter-Glo reagent from Promega (G7572) as previously described (7). Each experiment was done in triplicate and repeated thrice and viability compared with untreated cells.

Blood samples were obtained from three patients with leukemic CTCL (L-CTCL) seen at the Cutaneous Lymphoma Program at the Dana-Farber/Brigham and Women's Cancer Center under an IRB approved protocol for sample acquisition as previously described (7, 14). Briefly, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll centrifugation and incubated with mAb DF3 (anti-MUC1-N) followed by secondary labeling of the cells with FITC-conjugated goat anti-mouse IgG and malignant clone specific PE-conjugated anti-TCR Vβ mAbs as previously described(7, 14). The MUC1-N and TCR Vβ double positive population was FACS sorted on a Mo-Flo Legacy cell sorter (Beckman Coulter). Cells were counted and re-suspended at an approximate concentration of 1.0 × 10⁴ per well in a 96-well plate (Becton Dickinson Labware) and incubated at 37°C in a 5% CO₂ humidified incubator for up to 96 hours. Cells were left untreated, or a single dose of decitabine was added at a concentration of 40 nmol, or GO-203 was added daily for 3 days at 3 μmol or the combination. At 96 hours, the cells were evaluated for viability using the CellTiter-Glo reagent.

Five- to seven-week-old female NSG mice were injected with 2 million H9 cells in the flank via a subcutaneous route. Three-dimensional ultrasound imaging data sets were collected for each xenograft using a Vevo 2100 ultrasound microimaging system (VisualSonics Inc.) designed for small animal imaging. For analysis of ultrasound data, images were imported into Amira 5.2 (Visage Imaging) for volumetric analysis. Mice were imaged once a week for 3 weeks starting 4 days after inoculation of cells. After the xenograft tumors reached an average of 5 to 7 mm in diameter on imaging, mice were randomized to the four treatment groups of five animals each: (1) a control group treated with PBS alone; (2) a decitabine treated- group that received the drug at 0.4 mg/kg i.p. twice per week for 3 weeks; (3) GO-203-treated group that received the drug at 14 mg/kg by intradermal administration each day for 3 weeks and; (4) combination therapy group where mice received both drugs at the same concentration and schedule as single agents. All the mice were monitored twice a week and managed as per IACUC regulations mentioned above. All mice were sacrificed 1 week after completion of treatment and tumor tissue was harvested for immunohistochemical analysis. Formalin-fixed and paraffin-embedded 5 μm sections of tumor samples were analyzed for apoptotic cells by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining using the tumor TACS in situ Apoptosis Detection Kit (R&D Systems, Inc.) as per manufacturer instructions. Tumors were scored by the percentage of cells positive for necrosis and for TUNEL (as a measure of apoptosis). FFPE tumors were also analyzed for changes in the expression of DNMT1, DNMT3B, and H3K27me by IHC. Tumors were scored based on grading of intensity (1+ to 3+) of the stain per high power field (hpf). An average of the score along with a range is reported. All experiments were performed in accordance with IACUC approval.

The Student t test was used to assess statistical significance. The standard reference model of Bliss independence was used to assess synergy. Bliss predicts the combined response C for two single compounds with effects A and B is C = A + B – A × B, where each effect is expressed as fractional inhibition between 0 and 1. The difference between Bliss expectation and observed growth inhibition induced by the combination of agent A and B at the same dose is the “Bliss excess” (15, 16). For each of the experiments, synergy was confirmed by Calcusyn Version 2.0 software by determining combinational index (CI). Values < 1 represent synergistic effect of the two drugs, values equal to 1 indicate the mean additive effect of the drugs, and values >1 represent an antagonistic effect (17).

CTCL cell lines H9 and HuT-78 overexpress MUC1-C in comparison to other cell lines SeAx and HH and hence these were chosen to perform in vitro and in vivo experiments (7). Treatment of H9 and HuT-78 cells with minimal inhibitory concentrations of decitabine in combination with the MUC1-C inhibitor, GO-203, was associated with a marked increase in ROS levels (Fig. 1A and B) as compared to either agent alone. This increase was at least partially reversed by concurrent exposure of the cells to the antioxidant NAC that promotes the scavenging of ROS undermining greater disruption of redox balance with the combination. Representative flow cytometry histograms are presented in Supplementary Fig. S1A and S1B. Similar results were confirmed in Myla, another MUC1-C expressing CTCL cell line (Supplementary Fig. S2A and S2B). We subsequently investigated the impact of GO-203 in combination with decitabine on pro-apoptotic proteins such as p38 MAPK and JNK characteristically induced by increased oxidative stress. A marked increase in the ratio of phospho-p38 and phospho-JNK to Total-p38 and Total-JNK, respectively, was observed in H9 cells with dual exposure to decitabine and GO-203 (Figs. 1C and D).

Consistent with above findings, exposure to decitabine and GO-203 resulted in a marked reduction of the ROS scavenging molecules, NADP, NADPH, and GSH in H9 cells as compared to cells treated with either agent alone (Fig. 2A and 2B). Similar findings were observed in HuT-78 cells (Fig. 2C and D). TIGAR is a potent regulator of glycolysis, redox potential, and apoptosis whose expression is modulated by p53 expression. We have previously demonstrated that MUC1-C inhibition was associated with decrease in the expression of TIGAR that escalates ROS expression. In this study, we demonstrate that combination of GO-203 and MUC1-C inhibition is associated with further decrease in TIGAR expression potentially through impact on p53 expression (Fig. 2E and F). This attenuation of ROS scavenging process provides a potential mechanistic explanation for redox deregulation and associated synergistic induction of ROS by the combination. These results highlight that decitabine in combination with MUC1-C inhibition intensifies ROS generation and decreases ROS-scavenging capacity leading to disturbance in intracellular redox homeostasis.

Having observed a diminution in ROS scavenging abilities, we investigated the effects of the combination on ROS generating mechanisms. Decitabine has been demonstrated to disrupt redox homeostasis through its modulation of ROS generative enzymes such as Nox. Accordingly, we assessed the impact of the combined therapy on hypomethylation and the resultant transcriptional changes that could increase ROS levels in this setting. In this study, we demonstrate that exposure of CTCL cells to GO-203 downregulates DNMT 1 and 3b expression. Consistent with this finding, combined therapy with GO-203 and decitabine markedly reduced the expression of these proteins compared to either agent alone (Fig. 3A and B). We hypothesized that the resulting hypomethylation would increase expression of proteins such as trimethyl histone 3 (H3K27me) and NADPH oxidases, a family of oxidases that induces superoxide production with two of its isoforms having putative CpG islands in its promoter (13). Consistent with our hypothesis, we demonstrated that combined therapy with decitabine and GO-203 increased expression of H3K27me and isoforms Nox4 and Duox2 providing a mechanistic link between combined hypomethylating effect of these agents and subsequent ROS-mediated killing of CTCL cells. To validate the effects of the increased oxidative stress exerted by the combination on established downstream signaling changes that promote apoptosis, we investigated the effect on Smad signaling pathway. We also observed an increase in expression of Nrf2, which prevents excessive cellular damage produced by oxidative stress under homeostatic conditions, supporting previous observations. ROS is known to regulate the expression of the Smad family of transcription factors (18). Accordingly, increased phosphorylation of Smad2 and Smad3 proteins leading to their activation was observed with the combination. Further, expression of the c-Myc oncoprotein, a known downstream target gene of the Smad and MUC1-C signaling pathways was downregulated in the combination treated cells (19). These results highlight the potential mechanisms by which ROS is induced by the combination and the role of redox disruption in altering expression of key transcriptional proteins regulating proliferation and cell cycle.

Based on downregulation of DNA methyltransferases associated with combination treatment, we investigated the effects of the combination on DNA methylation patterns. Indeed, in the combination treated H9 and HuT-78 cells, there was a significant decrease in the percentage of DNA methylation at each of the four CpG positions in the long interspersed nucleotide element (LINE-1) repeats, a surrogate marker of global DNA methylation (Fig. 4A and D). This supports a role for the combination in inducing greater global DNA methylation compared to the single agents. Having observed a global decrease in methylation and increase in protein expression of Nox4 and Duox2, we investigated the effects of the combination on methylation of the Nox4 and Duox2 promoter. In line with our hypothesis, we noted a statistically significant decrease in the percentage of DNA methylation at each of the eight CpG positions in the Nox4 promoter (positions −386 to −296; Fig. 4B and E) and seven CpG sites in the Duox2 promoter (positions −902 to −860) (Fig. 4C and F). Locations of the CpG sites that were analyzed in the promoter of these genes with respect to the remaining to the structure of the genes are represented in Supplementary Figs. S3 and S4. The present results extend the importance of the combination in enhancing global and gene-specific DNA demethylation including epigenetic changes in redox homeostatic genes.

To investigate whether the combination regulates transcription of Nox4 and Duox2 following promoter demethylation, qRT-PCR was performed. A statistically significant increase in Nox4 and Duox2 mRNA levels was observed in the combination treated H9 and HuT-78 CTCL cells compared with control and single agents (Supplementary Fig. S5).

In keeping with increase in oxidative stress, a significant increase in cells manifesting late apoptosis/necrosis was observed following exposure to decitabine and GO-203 as compared to either agent alone as determined by flow cytometric analysis of PI/annexin V–FITC staining (Supplementary Fig. S6A and S6B). Representative flow cytometry plots are demonstrated in Supplementary Fig. S6C and S6D. The induction cell death was reversed by exposure of the cells to NAC highlighting the contribution of redox disruption in inducing cytotoxicity.

To formally assess synergy of cytotoxic killing by decitabine and GO-203, H9 and HuT-78 cells were treated with a single dose of decitabine in nanomolar concentrations corresponding to IC₅–IC₁₀ (10–40 nmol) and MUC1-C inhibitor, corresponding to IC₁₀–IC₂₅ (1.5–3 μmol). The combination demonstrated marked synergy with respect to induction of cytotoxic death over a range of concentrations in both cell lines (Supplementary Fig. S7A and S7B). The validated “Excess over Bliss Independence Model” was used to assess synergistic interaction. Values >10 represent synergy. Excess over Bliss (EOB) scores and combinational index values presented in the adjoining tables (Supplementary Fig. S7C and S7D) indicated increasing synergy with escalation of doses across the cell lines. Similar results were confirmed in another MUC1-C expressing cell line, Myla (Supplementary Fig. S2C and S2D). SeAx and HH are two CTCL cell lines with low to negative expression of MUC1-C as previously demonstrated by us (7). The combination failed to induce cytotoxic killing in both these cell lines, providing support for cytotoxic specificity of the combination in MUC1-C positive CTCL cells (Supplementary Fig. S1C and S1D).

The malignant clonal population was isolated from three primary CTCL samples by FACS of PBMCs for the double positive cells (TCR Vβ and MUC1-N) as previously described. FACS plots of all patients are presented in Fig. 5D. Sorted cells were treated with single minimally cytotoxic dose of decitabine (40 nmol) and daily GO-203 (3 μmol) for 3 days. Exposure to decitabine and GO-203 resulted in a substantial decrease in viability at 96 hours as compared to untreated and cells treated with either agent alone (Fig. 5A–C). EOB scores presented below confirmed synergy. An increase in ROS levels with the combination that was partially reversed by NAC was also confirmed in primary CTCL cells and is presented in Supplementary Fig. S8.

To assess in vivo antitumor activity of the combination, H9 cells were inoculated subcutaneously in the flank of NSG mice. After the xenograft tumors reached a volume of approximately 100 mm³ as determined by 3D ultrasonography, cohorts of animals were assigned to control (treated with PBS); treatment with decitabine alone; GO-203 or combined therapy. Tumor volume was quantified by serial assessment by ultrasonography. Rapid tumor growth was observed in the control cohort that was blunted modestly in animals treated with either decitabine or GO-203 alone. In contrast, combination therapy resulted in a statistically significant inhibition of tumor growth after completion of 3 weeks of therapy (Figs. 6A and B). One week after completion of therapy, mice were sacrificed for histological examination of the xenograft tumors and stained for TUNEL and trimetyl histone 3 analysis. Consistent with the in vitro studies, tumors isolated from mice treated with decitabine and GO-203 demonstrated higher levels of apoptotic cells and trimethyl histone as compared to animals treated with either agent alone (Figs. 6C–F). P values <0.05 represent statistical significance.

We have demonstrated that the MUC1-C oncoprotein is selectively expressed by CTCL cells as compared to normal T cells and plays a vital role in protecting tumor cells from ROS-mediated cell death. Consistent with these findings, inhibition of MUC1-C signaling using a cell penetrating peptide that block dimerization, nuclear translocation, and downstream signaling by the MUC1-C subunit results in increased ROS levels and apoptotic cell death in in vitro and in vivo models. Although it demonstrated therapeutic efficacy, treatment with the MUC1-C inhibitor alone did not fully eradicate disease in a xenogeneic mouse model, suggesting that its effect in promoting ROS-mediated cell death is incomplete. As such, we have investigated strategies to further enhance MUC1-C inhibition-mediated cytotoxicity in an effort to develop effective therapies without detrimental effects for this challenging T-cell lymphoma. The hypomethylating agent, decitabine, modulates redox balance in leukemia models (13). Based on our recent observation that MUC1 contributes to regulation of oxidative stress in CTCL cells, we sought to systematically explore the effect of the MUC1-C inhibitor with decitabine on redox signaling. To minimize toxicity of combination treatments, we specifically chose minimal inhibitory concentrations of both compounds. Herein, we demonstrate that exposure to decitabine and GO-203 results in increased ROS levels and increased expression of proapoptotic proteins such as phospho-p38 and phospho-JNK as compared to either agent alone. Given the pleiotropic effects of MUC1-C targeting and HMAs, we hypothesized that the combination may have an effect on ROS-scavenging ability. In this context, the combination led to a decrease in levels of the scavenger molecules, NADP, NADPH, and GSH. The downregulation of TIGAR resulted in its inability to inhibit glycolysis and protect from increase in intracellular ROS levels. To investigate the mechanistic basis for enhanced generation of ROS with the combination, we first examined the impact of MUC1-C inhibition on DNMT 1 and 3b, essential for maintaining DNA methylation. We observed that targeting MUC1-C affects methylation patterns via its regulation of DNMT expression. We hypothesized that combining MUC1 inhibition with decitabine would further increase the degree of hypomethylation, resulting in enhanced transcription of genes that could modulate redox signaling. Indeed, we found that targeting MUC1-C in combination with decitabine resulted in the near abrogation of DNMT 1 and 3b levels supporting our hypothesis. The combination induced marked decrease in global DNA methylation as reflected in the reduction of DNA methylation percentage of all CpG sites in the LINE-1 element. Further, the combination treatment was associated with a greater diminution in DNA methylation of Nox4 and Duox2 promoter that play a critical role in cellular redox balance. These results provide evidence for synergistic effect of the combination in inducing DNA demethylation and varied molecular effects leading to the intensified antitumor activity of the combination. Decrease in the expression of other proteins such DNMT1 and 3b was also seen in the xenograft tumors but was not statistically significant probably due to heightened apoptosis mediating clearance of tumor cells.

The Nox family of which there are currently seven identified isoforms (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2) produce ROS (13, 20). They use NADPH as an electron donor and are widely distributed in cancer cells. Nox4 and Duox2 have putative CpG islands around its promoter region invoking the possibility of hypomethylation by DNMT inhibitors such as decitabine. We found that the combination of decitabine and MUC1-C inhibitor is associated with upregulation of Nox4 and Duox2 expression potentially through decrease in their CpG methylation sites of the promoters and greater mRNA levels. In line with previous observations, the intracellular redox disruption associated with the combination led to activation of Nrf2, activation of Smad signaling pathway, which in turn resulted in downregulation of c-Myc, a critical oncogene. These data collectively produce diverse cellular evidence for the remarkable synergy observed with the combination in CTCL cells. In summary, we believe that the combination exerts pleiotropic antitumor effects by disrupting redox balance and modifying the expression of key regulatory proteins involved in apoptosis, cell adhesion, and cell cycle. Of note, our studies do not exclude the possibility that mechanisms outside of redox disruption can provide alternative molecular basis for the synergy observed. Transcription of tumor suppressive microRNAs with hypermethylated promoters unique to CTCL could be one such possibility and will be the focus of subsequent studies (11, 12).

It is worth noting the low concentrations of decitabine and MUC1-C inhibitor that were specifically selected for the experiments. The translational relevance of this observation raises the attractive prospect of combining these agents to improve therapeutic index without adverse events. It is also interesting to see that many of the profound molecular changes incited by the combination were modestly modified by the single agents and could be potentially related to the low concentrations of both agents and single treatment with decitabine. We believe that this argues favorably in the synergy invoked by this combination.

In conclusion, the present results support the potential of targeting MUC1-C and DNMT inhibitors in CTCL. The combination (1) impairs ROS repair mechanism through decrease of NADPH, GSH, and TIGAR (2) is associated with down regulation of DNMT's, decrease in global and gene-specific promoter DNA methylation and upregulation of Nox4 and Duox2 protein expression (3) collectively these lead to increase in overall cellular ROS levels and, (4) ROS-mediated late apoptosis/necrosis through modulation of different genes including oncoproteins and tumor suppressors. MUC1-C inhibitor has entered phase I/II evaluation in patients with AML (https://clinicaltrials.gov/ct2/show/NCT02204085) and based on the above results warrants an early phase study in CTCL patients. Efficacy of this regimen in the clinic will provide the impetus needed to combine agents with robust scientific rationale and preclinical data in this poorly understood neoplasm. Our observations have led us to use this combination as a backbone in developing novel strategies targeting redox regulation in CTCL.

Donald Kufe has ownership interest (including patents) for Genus Oncology; and also is a consultant/advisory board member in Genus Oncology. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S. Jain, A. Washington, R.K. Leaf, T.S. Kupper, D. Kufe, D. Avigan

Development of methodology: S. Jain, P. Bhargava, D. Avigan

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Jain, A. Washington, R.K. Leaf, R.A. Clark, D. Stroopinsky, L. Cole, A. Apel, D. Avigan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Jain, A. Washington, P. Bhargava, D. Stroopinsky, M. Nahas, A. Apel, D. Avigan

Writing, review, and/or revision of the manuscript: S. Jain, A. Washington, P. Bhargava, M. Nahas, A. Apel, J. Rosenblatt, J. Arnason, D. Kufe, D. Avigan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Jain, T.S. Kupper, D. Avigan

Study supervision: S. Jain, D. Avigan

Other (samples): R.A. Clark

Other (review of the manuscript): A. Pyzer

The authors would like to thank LLS, grant #6139-14. This work was also supported by R01 AR063962NIH/NIAMS (to R.A. Clark), R01 AR056720NIH/NIAMS (to R.A. Clark), a Damon Runyon Clinical Investigator Award (to R.A. Clark), the SPORE in Skin Cancer P50 CA9368305NIH/NCI (to T.S. Kufe) and R01 AI097128 NIAID (to T.S. Kufe and R.A. Clark). Imaging studies were performed in collaboration with the Small Animal Imaging Shared Resource within the Beth Israel Deaconess Medical Center.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.