T-cell Lymphoma Cell Lines Research Articles

Cytotoxicity and molecular activity of fenretinide and metabolites in T-cell lymphoid malignancy, neuroblastoma, and ovarian cancer cell lines in physiological hypoxia

All-trans-N-(4-hydroxyphenyl)retinamide or fenretinide (4-HPR) acts by reactive oxygen species (ROS) and dihydroceramides (DHCers). In early-phase clinical trials 4-HPR has achieved complete responses in T-cell lymphomas (TCL) and neuroblastoma (NB) and signals of activity in ovarian cancer (OV). We defined the activity of 4-HPR metabolites in N-(4-methoxyphenyl)retinamide (MPR), 4-oxo-N-(4-hydroxyphenyl)retinamide (oxoHPR), and the 4-HPR isomer 13-cis-fenretinide (cis-HPR) in NB, OV, and TCL cell lines cultured in physiological hypoxia. We compared the effect of 4-HPR, cis-HPR, oxoHPR, and MPR on cytotoxicity, ROS, and DHCers in a panel of TCL, NB, and OV cell lines cultured in bone marrow level physiological hypoxia (5% O2), utilizing a fluorescence-based cytotoxicity assay (DIMSCAN), flow cytometry, and quantitative mass spectrometry. 4-HPR (10 µmol/l) achieved more than three logs of cell kill in nine of 15 cell lines. Cytotoxicity of 4-HPR and oxoHPR was comparable; in some cell lines, cis-HPR cytotoxicity was lower than 4-HPR, but additive when combined with 4-HPR. MPR was not cytotoxic. ROS and DHCers were equivalently increased by 4-HPR and oxoHPR in all cell lines (P<0.01), to a lesser extent by cis-HPR (P<0.01), and not increased in response to MPR (P>0.05). Mitochondrial membrane depolarization, caspase-3 cleavage, and apoptosis (TUNEL) were all significantly increased by 4-HPR and oxoHPR (P<0.01). Cytotoxic and pharmacodynamic activity was comparable with 4-HPR and oxoHPR, lower with cis-HPR, and MPR was inactive. Neither MPR or cis-HPR antagonized 4-HPR activity. These data support focusing on achieving high 4-HPR exposures for maximizing antineoplastic activity.

045 - Metabolite profile of extracellular vesicles derived of a cutaneous T-cell lymphoma (CTCL) cell line

045 - Metabolite profile of extracellular vesicles derived of a cutaneous T-cell lymphoma (CTCL) cell line

The Novel Tumor Suppressor SAMHD1 Is Differentially Expressed and Partly Regulated By MYC in Peripheral T-Cell Lymphomas (PTCL)

Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which has been initially described to restrict human immunodeficiency virus type 1 (HIV-1) in certain cell types through depletion of intracellular dNTP substrates required for HIV-1 reverse transcription. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS) and have been identified as putative drivers of chronic lymphocytic leukemia resulting in decreased SAMHD1 mRNA and protein levels. More recently, SAMHD1 mutations have been reported in T-prolymphocytic leukemia (T-PLL). Based on these findings and the fact that SAMHD1 limits the dNTP pool in the cell, it may play a role in oncogenesis as a tumor suppressor. In addition, SAMHD1 may confer resistance to nucleoside-based chemotherapies by hydrolysing their active triphosphate metabolites, with cytarabine in acute myeloid leukemia being an example (Herold et al, Nat Med 2017; 23(2):256-263). The expression patterns and the potential role of SAMHD1 in the pathogenesis of peripheral T-cell lymphomas (PTCL) are not yet known.Methods: The patient cohort included 64 PTCLs of various histologic types which were diagnosed and treated at Karolinska University Hospital (Sweden). A control group of 4 reactive lymph nodes and 2 reactive tonsils was included in the study for comparison. All tissue samples were obtained prior to therapy. SAMHD1 expression was assessed by immunohistochemistry performed on a PTCL tissue microarray (TMA) with duplicate tumor cores from each case or full tissue sections using dual immunostaining (SAMHD1 / CD68) and a monoclonal antibody against SAMHD1 (Bethyl Laboratories, San Antonio, TX). At least 500 lymphoma cells were counted to calculate the percentage of SAMHD1-positive tumor cells. Overall survival (OS) was defined as time from diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from diagnosis to relapse, death, or last follow-up. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models. Two T-cell lymphomas cell lines (Mac1, Mac2A) were used as an in vitro system. As our preliminary findings from in silico analysis revealed potential binding sites for MYC on the SAMHD1 gene promoter, we hypothesized that MYC might regulate SAMHD1 expression. Therefore, the T-cell lymphoma cell lines were treated with the selective BET / MYC inhibitor JQ-1 or transiently transfected with a MYC-overexpressing plasmid or MYC gene-specific siRNA constructs, respectively. Western blot analysis was used to assess the protein levels.Results: SAMHD1 protein was expressed in reactive T-cells and histiocytes (CD68+) in all reactive lymphoid tissues (lymph nodes and tonsils) with strong staining intensity. SAMHD was differentially expressed among PTCL subtypes generally with weaker staining intensity as compared to normal T-cells and histiocytes, thus being positive in all (100%) angioimmunoblastic T-cell lymphomas (AILT), 67% PTCL-NOS, 45% ALK+ ALCL, 20% of ALK+ ALCL, and none (0%) of T-lymphoblastic lymphomas (p=0.0017, chi-square test). Among the SAMHD1- positive cases, the percentage of positive lymphoma cells ranged from 0 to 100% and its highest median was observed in AILT. SAMHD1 expression inversely correlated with CD30 expression (% CD30+ positive lymphoma cells) (p=0.0025, Mann-Whitney test). No significant associations between SAMHD1 levels and other clinicopathologic parameters or clinical outcome (EFS or OS) were found, however, the number of patients analyzed in each histologic subtype was limited. Inhibition of MYC activity by JQ-1 or MYC gene silencing with specific siRNA resulted in a substantial increase in the SAMHD1 protein level in T-cell lymphoma cell lines. Inversely, transient transfection of the cell lines with a MYC overexpressing plasmid resulted in decreased levels of SAMHD1. Taken together, the in vitro data suggest a possible MYC-associated regulation (repression) of SAMHD1 gene expression in T-cell lymphoma.Conclusions: SAMHD1 is shown for the first time to be differentially expressed among PTCL types and its regulation may involve MYC. Preliminary survival analysis shows no significant associations of SAMHD1 expression with EFS and OS in this cohort of PTCL, however, analysis of a larger PTCL study group is underway to draw definite conclusions. DisclosuresÖsterborg:Gilead: Consultancy, Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Abbvie: Research Funding.

Identification of Tipifarnib Sensitivity Biomarkers in T-Cell Tumor Cell Lines

BackgroundT-cell leukemia and lymphoma (TCL) is a very aggressive and heterogeneous group of hematological malignancies with poor prognosis and inadequate response to current therapies. The standard first-line treatments have resulted in unsatisfactory patient outcomes and unfortunately, targeted treatments are still at a preliminary phase. The RAS/MAPK pathway is crucial for TCR signaling of T-cells and it is deregulated in TCL. We aimed to determine the therapeutic value of farnesyl transferase inhibitor (FTIs) tipifarnib in TCL cell lines. Effects on cell viability, apoptosis, cell cycle and gene expression were evaluated. Tipifarnib blocks the localization of some farnesylated proteins, including some RAS family members, to cellular membranes, thereby inhibiting their activation. Tipifarnib is a potent and specific FTI with prominent anti-proliferative effects.MethodsWe tested tipifarnib in 18 T-cell leukemia and 4 T-cell lymphoma cell lines for in vitro sensitivity and for biomarker discovery, both genomic and immunohistochemical. We selected cell lines with available genomic data from COSMIC, CCLE or generated by our group. The MAPK, NFAT, NFKB and JAK/STAT pathways were tested by immunohistochemistry analysis under basal conditions. The range of drug concentrations to perform IC50 analysis was established between 0-10,000 nM (ten points), using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) following manufacturer´s instructions at 0h, 48h and 96h. All experiments were done in sextuplet and all numerical data were expressed as the average of the values ± the standard error of the mean. IC50 analyses were performed with GraphPad Prism v5. Clinically-relevant drug sensitivity was defined as IC50 <100nM at 96h (defined as sensitive). RNAseq was performed in 4 sensitive cell lines with tipifarnib and DMSO (vehicle) in three biological replicates at 72h. Differential gene expression (DE) analysis was carried out under these two conditions. Pathway analysis was done using Gene Set Enrichment Analysis (GSEA). Induction of apoptosis and cell viability were tested by flow cytometry in sensitive cell lines.Results59.1% (n=13) of cell lines were sensitive to tipifarnib at concentrations which are readily achievable in the clinic. RAS, RAS-guanine nucleotide exchange factors (GEFs) and RAS-GTPase activating proteins (GAPs) genes were mutated in 45.5%, 50% and 27.3%, of cell lines respectively, but their mutational status was not associated with drug sensitivity. We found TP53 and DNMT3 mutated in 80% and 33.3% in sensitive cell lines respectively, and in 63.6% and 0% in resistant cell lines. The mutational state of NOTCH1 was associated with tipifarnib sensitivity; 66.7% in sensitive cell lines versus 9.1% in resistant cell lines (p=0.005). ERK activation (MAPK pathway) was significantly associated with drug sensitivity (p=0.046). Conversely, RelB (NFκB pathway) was associated with drug resistance (p=0.007). GSEA analysis showed that tipifarnib downregulated cell cycle, protein localization to membranes, metabolism, and ribosome and mitochondrial activity. The effect of tipifarnib on cell cycle progression and apoptosis was validated by flow cytometry assays. Tipifarnib decreased cellular viability, increased apoptosis and blocked cell cycle progression in G1 phase. GSEA of molecular pathways regulated by tipifarnib identified downregulation of MYC targets and mTOR and MAPK pathways. MYC is a target of NOTCH1, mutated in sensitive cases and associated with tipifarnib sensitivity.ConclusionsIn vitro response to tipifarnib in T-cell lymphoma and leukemia cell lines can be predicted using several biomarkers. The mutational NOTCH1 status, together with p-ERK (MAPK activation) were associated with increased sensitivity while RelB expression was associated with resistance. DisclosuresGualberto:Kura Oncology: Employment, Equity Ownership. Piris:Takeda: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Kura: Honoraria.

Pevonedistat, a Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Induces Cell Cycle Deregulation, Anaphase Catastrophe, and Apoptosis in T-Cell Lymphoma Cells

Introduction: Despite the significant progress of targeted therapies in B-cell malignancies, T-cell lymphomas remain an area of unmet medical need. Most patients are diagnosed at an advanced stage and have limited treatment options. Moreover, most patients who relapse following initial chemotherapy ultimately succumb to disease. Recent successes of targeting the proteasome (i.e., bortezomib) and E3 ligases (i.e., lenalidomide) identify the ubiquitin-proteasome system (UPS) as a tractable target in lymphoma. Pevonedistat, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE), interferes with activation of NEDD8, a ubiquitin-like modifier. This interference ultimately leads to decreased activity of cullin-RING (E3) ligases and accumulation of their substrates, including inhibitor of NFκB (IκB), the replication licensing protein Cdt1, and p27. We previously demonstrated that targeting NAE affected primary neoplastic B cells via several mechanisms: disruption of NFκB activity as well as induction of Cdt1, DNA damage, and cell cycle arrest. Here, we demonstrate that targeting NAE in T-cell lymphoma cells mediates apoptosis via cell cycle deregulation, accompanied by induction of Cdt1 and p27, and induction of anaphase catastrophe.Methods: Experiments were performed in T-cell lymphoma cell lines (SR, HH, Jurkat, and SUP-T1) as well as circulating primary cells from patients with peripheral T-cell lymphoma and Sezary syndrome. Pevonedistat (TAK-924) was obtained from Millennium Pharmaceuticals, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). Apoptosis was assessed by Annexin V staining.Results: SR (PTCL) cells and primary T-cell lymphoma cells were the most sensitive to pevonedistat (IC50of ~250nM at 24 hours); Jurkat and SUP-T1 cells demonstrated low/intermediate sensitivity, whereas HH (CTCL) cells were resistant. Targeting NAE disrupted cullin neddylation in a dose-dependent manner across all tested cell lines and primary neoplastic T cells, followed by accumulation of phospho-IκBα. Upregulation of phospho-IκBα was notable within 2 hours of pevonedistat treatment across both sensitive and resistant cell lines and primary cells. Concomitantly, we observed induction of p27 and Cdt1. Upregulation of Cdt1 was attenuated in HH cells compared with SR, consistent with the low proliferation rate of the former. Treatment of SR cells with pevonedistat led to DNA damage as evidenced by γH2AX and G2/M arrest.Chromosomal instability is a prominent feature in cancer and poorly studied as a therapeutic target. We have previously shown that cancer cells undergo multipolar anaphase in response to inhibition of cyclin-dependent kinase-2 (CDK2), an interphase CDK, followed by apoptosis and termed this event anaphase catastrophe (Hu et al., 2015; Danilov et al., 2016). As we observed robust accumulation of the endogenous CDK inhibitor p27 in cells treated with pevonedistat, an event presumed to lead to attenuated CDK2 activity, we studied anaphase catastrophe in this setting. We visualized anaphase catastrophe by immunofluorescent staining for nuclear material (DAPI) and γ-tubulin, and scored it in 50 cells per condition. NAE inhibition with pevonedistat induced anaphase catastrophe in SR and Jurkat cells. Upon 24-hour exposure to 250 nM pevonedistat, 9.8±6.0% of SR and 18±4.4% of Jurkat cells demonstrated multipolar anaphases, compared with 1±0.8% and 3.0±2.6% with vehicle control, respectively.Conclusions: Inhibiting NAE with pevonedistat induces apoptosis of T-cell lymphoma cells. We propose deregulation of Cdt1 and p27, followed by anaphase catastrophe, as a key mechanistic event implicated in pevonedistat-induced apoptosis in neoplastic T cells. Our work provides rationale to further investigate neddylation as a therapeutic target in T-cell lymphoma. DisclosuresDanilov:Verastem: Consultancy, Research Funding; TG Therapeutics: Consultancy; Genentech: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Consultancy, Research Funding; Astra Zeneca: Consultancy; Aptose Biosciences: Research Funding; Bayer Oncology: Consultancy, Research Funding.

GSK458 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in T Cell Lymphomas As a Single Agent and in Combination Therapy

Introduction: T-cell lymphoma (TCL) accounts approximately 15% of all Non-Hodgkin's lymphoma cases. TCL are often divided into either systemic or cutaneous TCL (CTCL). The management of TCL systemic or cutaneous is challenging because the emergence of chemotherapy resistance that lead to early death (systemic T-cell lymphomas) or chronic debilitating clinical course (CTCL). To improve the clinical outcomes and reduce treatment-related toxicity, research need to be done focusing on understanding and targeting the molecular mechanism driving TCL subtypes. Recently, studies showed that the PI3K AKT/mTOR pathway was activated in TCL. GSK458 is a potent oral dual inhibitor of pan PI3K (α, β, γ and δ) and mTOR (mTOR1 and mTOR2). Preclinical studies in B-cell lymphomas showed GSK458 had broad antitumor activity in vitro and in vivo. In 2016, Phase I clinical trial of GSK458 was competed at the maximal dosage of 2.5mg twice daily. However, the effects of GSK458 on T-cell malignancies remain utterly unknown. Here, we evaluated the activity of GSK458 in preclinical T cell lymphoma models. Methods: We used a panel of T-cell lymphoma cell lines representing PTCL (J45), T-cell lymphoblastic lymphoma (SupT-1), and Mycosis Fungoides (MF)(MJ, HH and H9). TCL cell lines were exposed to escalating doses of GSK458 (1nM-100µM) without or with chemotherapeutic agents (doxorubicin, cisplatin, carboplatin, and dexamethasone); Bcl-2 inhibitor (Venetoclax); proteasome inhibitors ( bortezomib, carfilzomib, Ixazomib); or HDAC inhibitors (SAHA) for 48 and 72 hrs. Differences in cell viability, ATP levels, low mitochondria potential, glucose update, apoptosis and cell cycle distribution were evaluated utilizing PrestoBlue, Cell-Titer Glo assays, DiOC6, 2-NDG, Annexin V and propidium iodide staining followed by flow cytometric analysis, respectively. IC50 was calculated by GraphPad. PI3K and mTOR downstream pathway phosphorylation status, such as p-AKT Ser473, p-AKT Thr308, p-mTOR and p-GSK3β were detected by internally staining of FITC conjugated-antibodies followed by flow cytometry. Apoptosis proteins (MCL-1, PARP, p53, XIAP etc.) were detected by western blot. The additive/synergistic activity of GSK458 was detected by presto blue assay and Coefficient of synergy was calculated using CalcuSyn. Results:In vitro exposure of TCL cell lines to GSK458 demonstrated a dose- and time-dependent cell death. The IC50 of the cells were ranged from 3nM to 1.05uM at 72 hours. At 72h, GSK458 10nM lowered cellular mitochondrial potential, ATP levels and glucose uptake. GSK458 induced apoptosis and arrested the cell cycle at G1. At molecular level, GSK458 reduced phosphorylation status of AKT ser473 and Thr308, mTOR and GSK3β. Interestingly, GSK458 inhibited Mcl-1 expression level. GSK458 exhibited synergistic activity when combined with doxorubicin and dexamethasone. To a lesser degree, GSK458 enhanced the anti-tumor activity of Venetoclax, proteasome and HDAc inhibitors. Conclusion: GSK458 is active as a single agent or in combination with chemotherapy agents or small molecule inhibitors in a variety of T-cell pre-clinical models representing forms of systemic or cutaneous T-cell lymphoma. GSK458 was able to inhibit phosphorylation of AKT, mTOR and GSK3β, which may be the mechanism to reduce ATP production and glucose uptake in the cancer cells. Moreover, GSK458 arrested cell cycle at G1 arrest. Our data supports the clinical evaluation of GSK458 in relapsed/refractory T-cell lymphoma patients. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) DisclosuresNo relevant conflicts of interest to declare.

Azacitidine and Romidepsin Synergize to Induce Expression of Cancer Testes Antigens (CTA) and Shift Peripheral T-Cell Lymphoma (PTCL) Cell Lines Toward a Th1-like Phenotype

Introduction: The PTCL appear to be exquisitely sensitive to epigenetic modifiers, including histone deacetylase inhibitors and hypomethylating agents. While the PTCL harbor recurring mutations in genes controlling CpG methylation, including TET2, IDH2 and DNMT3, it is not clear these genetic events portend sensitivity to these classes of drugs. In preclinical models of PTCL, HDACi and HMA exhibit marked class synergy in vivo and in vitro. Early Phase 1 and 2 experiences have confirmed compelling activity across the PTCL, with exceptionally high response rates in patients with angioimmunoblastic T-cell lymphoma. While the precise mechanism of synergy is under investigation, it is clear these drugs can induce a host of immunological effects on both the tumor cell proper as well as the host. We present details of these class effects in models of PTCL.Methods: We have explored combinations of azacitidine and romidepsin in a panel of 6 TCL cell lines. The cell viability was accessed and synergy was calculated using Excess Over Bliss modeling to determine the efficacy of the combination. We then performed RNAseq analysis on 4 TCL lines. We confirmed the protein expression of multiple targets using western blot to confirm to results obtained from sequencing. TCL lines were exposed to combination for 96 hours, supernatant was collected and assayed for proliferative properties using isolated PBMCs from healthy donors and anti-CD3/anti-CD28 beads.Results: The combination of azacitidine and romidepsin were potently synergic across all 6 cell lines. Determination of DNA methylation revealed a decrease in %MdC, as a function of concentration. A supervised analysis of the RNAseq revealed almost all of the genes perturbed by the single agent treatments were enhanced by the combination. There were 1215 genes affected by combination treatment with a p-value ≤0.05; 921 of the genes were not found in either single agent cohort. Of the 1215 genes, 936 were upregulated and 279 genes were downregulated. A gene set enrichment analysis (GSEA) of the RNAseq data displayed a significant down regulation in multiple genes involved in cholesterol biosynthesis in the combination, for example, HMGCR (-2.1 FC), FDPS (-2 FC) and IDI1 (-1.8 FC). The RNAseq analysis revealed a marked increase in expression levels in a variety cancer/testis antigens (CTA) that were most amplified by the combination, such as PRAME (4.9 FC) and MAGE-A1 (11.3 FC). As single agents, azacitidine alone was capable of inducing minor expression of some targets while romidepsin did not affect any. There was a radical change in genes involved in viral/interferon response and immune response observed in RNAseq. The expression of a handful of interleukins and interferons associated with viral/interferon and immune responses were induced by single agent azacitidine and amplified the most by the combination, such as IF16 (1.4 FC aza, 2.8 FC combo), IL18 (3.4 FC combo), IL34 (2.4 FC aza, 3.7 FC combo) and IL26 (5.2 combo FC). Noticeably, single agent romidepsin (3 FC) and the combination (6 FC) augmented the expression of CD274 (PD-L1).Most drastically intensified from this subset of immune response genes was TBX21 (6.9 FC) expression that was induced by the combination. We see from a western blot that the combination induces maximum protein expression levels of T-bet (transcription factor encoded by TBX21) in 4/6 TCL cell lines. STAT4, known to interact with T-bet to promote Th1-differentiation, protein expression was confirmed to increase with combination treatment in 4/6 TCL cell lines. Enhanced proliferation was seen when PBMCs were cultured with conditioned medium from 4 different TCL lines treated with the combination.Conclusions: The generated data presents a strong justification for the use of combination in addition to an immune checkpoint inhibitor, such as a PD-1/PD-L1 inhibitor. With the emergence of members of CTA family becoming a developing marker in immunotherapy, it is of absolute importance to narrow down the possible targets induced by combination therapy. Here we identify two members of the family that are uniquely expressed as a result from exposure to the combination. Interestingly, the combination is also able to induce a Th1-like phenotype that is often associated with a favorable prognosis. These data suggest that azacitidine and romidepsin in TCL is a promising platform treatment that undeniably proves itself advantageous in preclinical models. DisclosuresO'Connor:Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Celgene: Research Funding.

Oncolytic Reovirus Is an Effective Treatment for Histone Deacetylase Inhibitor Resistant T-Cell Lymphoma

Aberrant gene expression plays a pivotal role during tumorigenesis and cancer progression. The acetylation status of histones is an important determinant of gene expression and is controlled by two opposing classes of enzymes: histones acetyl transferases (HATs) and histone deacetylases (HDACs). The deacetylation of histones is associated with repression of key tumor suppressor genes and has been linked to HDAC overexpression in multiple forms of cancer including lymphomas. Several HDAC inhibitors have been FDA approved for T-cell lymphoma (TCL) therapy including belinostat, vorinostat, and romidepsin. Despite the promising anti-lymphoma activity of HDAC inhibitors as a drug class, resistance is a significant clinical issue. Identification of new strategies that are more effective in the drug resistant patient population is a high priority, but the mechanisms underlying HDAC inhibitor-induced cell death and the development of drug resistance are not completely understood. Elucidating the molecular mechanisms driving HDAC inhibitor resistance and/or the specific targets that are altered in drug-resistant cells may facilitate the development of strategies that overcome drug resistance and are effective for refractory patients. To pursue this goal, we generated novel TCL cell line models of acquired HDAC inhibitor resistance through repeated exposure to belinostat. The sensitivity of parental and resistant TCL cells to belinostat and other clinically relevant HDAC inhibitors was initially characterized using cell viability and flow cytometric assays. Notably, belinostat-resistant cells displayed significant cross-resistance to other HDAC inhibitors including vorinostat, romidepsin, panobinostat, and ricolinostat. This indicates that TCL patients that fail one HDAC inhibitor regimen may not benefit significantly from subsequent treatment with other drugs of this class. Consistent with a lack of sensitivity to HDAC inhibitors, the resistant cells failed to induce increased acetylated histones, tubulin acetylation (an HDAC6 target), and exhibited reduced upregulation of the CDK inhibitor p21 following belinostat treatment. In agreement with the absence of these hallmark characteristics of HDAC inhibition, belinostat also failed to cleave caspase-3 in the belinostat-resistant cells. Comprehensive transcriptome analysis was conducted to further characterize these new drug-resistant models and identify potential mechanisms of resistance and targets for second-line treatment. Drug resistant cells featured significantly increased basal levels of the reovirus receptor junctional adhesion molecule-A (JAM-A) as well as decreased JAK/STAT activity, a key antiviral response pathway. Based on these findings, we investigated the efficacy of the proprietary clinical formulation of reovirus (Reolysin) in parental and drug-resistant models. Our investigation revealed that belinostat-resistant cells displayed enhanced sensitivity to oncolytic reovirus-induced cell death compared to their parental counterparts. The increased benefit of Reolysin in resistant models was linked to elevated viral loads and more efficient endoplasmic reticular stress-mediated apoptosis. The heightened sensitivity of HDAC-resistant TCL cells to Reolysin was further validated in parental and belinostat-resistant T-cell lymphoma xenograft models. Collectively, these data demonstrate that oncolytic reovirus may be a novel therapeutic approach to treat T-cell lymphoma patients that are relapsed/refractory to HDAC inhibitors. We are currently planning an early phase clinical trial to further test the safety and benefit of this new approach. DisclosuresPersky:Morphosys (IDMC): Consultancy; Merck: Research Funding; Spectrum: Research Funding; Genentech: Honoraria.

Targeted Inhibition of CD47-Sirp Alpha Requires Fc-Fc Gamma Receptor Interactions to Maximize Phagocytosis in T-Cell Lymphomas

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Monoclonal antibodies (mAbs) that inhibit engagement of the “don't eat me” signal CD47 with SIRPα on myeloid cells may induce phagocytosis and have exhibited promise in multiple cancers. We dissected the role of CD47 blockade in opsonization and phagocytosis-induction through Fc-Fc gamma receptor (FcγR) interactions. CD47 was expressed at higher levels on 22 TCL cell lines by flow cytometry compared to quiescent T cells from 10 healthy donors (mean fluorescent intensity, 176.9 versus 69.85; Welch t-test p=0.002). We determined tumor cell-specific protein expression of CD47 by calculation of H scores in a tissue microarray (TMA) of 68 primary TCLs. The common subtypes (PTCL-NOS (n=16), AITL (n=19), and ALCL (n=12)) each had heterogeneous CD47 expression that was not associated with progression-free or overall survival. We utilized B6H12, an anti-CD47 mAb to assess apoptosis, antibody-dependent cell mediated-cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) of TCL cells (HuT-78, HH, Myla, SU-PM2, KIJK, MAC-2A, SMZ1 and K-299) and no increase was observed except for ADCC by murine FcγRIII-expressing engineered Jurkat cells. Upon addition of B6H12 or SRF231, a fully human IgG4 mAb against CD47 that is currently in a Phase I clinical trial (NCT03512340), phagocytosis of CFSE+ TCL cells with human monocyte-derived macrophages (hMDMs) and murine bone-marrow derived macrophages (mBMDMs) was increased. In contrast, induction of phagocytosis of resting T cells from healthy donors was tested using B6H12 and no increase was observed (22.7 and 19.5%). A flow cytometry-based receptor occupancy assay demonstrated a linear correlation between receptor binding and phagocytosis induction in Jurkat T-cell lymphoblastic leukemia cells. hMDMs failed to induce phagocytosis of TCL cells with F(ab')2 portion of B6H12 suggesting complete dependence on Fc receptors. In contrast, mBMDM-mediated phagocytosis of TCL cells with the F(ab')2 portion of B6H12 (mean, 21.5%) was increased compared to negative control but reduced compared to full length (mean, 33.4%), suggesting both Fc-dependent and Fc-independent mechanisms. mBMDMs from FcγR knockout mice demonstrated a statistically significant decrease in phagocytosis (compared to wild-type mBMDMs) of Jurkat, HuT-78, MAC2A and K-299 cells with B6H12, confirming a role for Fc-FcγR engagement. Phagocytosis of tumor cells during CD47 blockade may depend on the ability of SLAMF7 to interact with the integrin Mac-1 (Chen J, Nature 2017). Phagocytosis assays of TCL cells with mBMDMs from Mac-1 knockout mice with full length and F(ab')2 B6H12 demonstrated a reduction in phagocytosis using Mac-1 knockout macrophages (compared to wild-type) only with full-length B6H12. Thus, the Mac-1 contribution to phagocytosis induced by B6H12 in this context is Fc-dependent. We tested SRF231 in vivo using patient-derived xenograft (PDX) models of systemic hepatosplenic (HSTL), transformed cutaneous, and NKTCL. After 10 days, SRF231-treated mice had remarkable reductions in tumor burden compared to untreated mice, across multiple compartments. SRF231 treatment resulted in prolonged survival despite a short course of therapy (Figure 1). Bone marrow from xenografted mice, collected at day 11 post-treatment with SRF231, had significantly increased cytokines known to promote monocyte recruitment. Ex-vivo phagocytosis assays using mBMDMs and hMDMs, enhanced engulfment of HSTL, T-PLL and anaplastic large cell-lymphoma PDX harvested cells upon exposure to SRF231. Finally, we observed that in our HSTL PDX model, depletion of macrophages with clodronate completely abrogated the antitumor efficacy of SRF231 while depletion of neutrophils with anti-Ly6G Ab had no effect, highlighting macrophages as the essential effectors of phagocytosis in this context. In summary, monoclonal antibodies that disrupt CD47-SIRPα have striking in vitro and in vivo preclinical efficacy but require interactions with human FcγR on macrophages to maximize efficacy. [Display omitted] DisclosuresPeluso:Surface Oncology: Employment, Equity Ownership. O'Connor:Surface Oncology: Employment, Equity Ownership. Paterson:Surface Oncology: Employment, Equity Ownership. Holland:Surface Oncology: Employment, Equity Ownership. Weinstock:Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties; Travera: Equity Ownership; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Genentech/Roche, Monsanto: Consultancy; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy.

Cytokine profile alterations in Cutaneous T-cell Lymphoma cell lines after Lenalidomide and/or Bortezomib treatment

Cytokine profile alterations in Cutaneous T-cell Lymphoma cell lines after Lenalidomide and/or Bortezomib treatment

Apoptosis of Murine T-Cell Lymphoma EL4 Cells Induced by Murine Cytomegalovirus and Its Mechanism

To detect whether the murine T-cell lymphoma cell line EL4 could be infected by murine cytomegalovirus (MCMV), and to observe the morphological changes and apoptosis of El4 cells before and after infection. EL4 cells were infected with MCMV smith strain with 1, 10 and 100 multiplicity of infection (MOI) respectively. The morphology of the cells was observed by light microscopy and Wright's-Giemsa staining. The survival rate was calculated by trypan blue staining. RT-PCR-based assay was used to detect the copy number of MCMV-DNA in the infected ELA cells. Flow cytometry was used to detect apoptosis. RT-qPCR was used to detect the mRNA expression levels of P53, P21, cFlip and Caspase 8. The protein expression levels of Caspase8, P53, BAX, BCL-2 and Cleaved Caspase3 proteins were detected by Western blot. After Wright-Giemsa staining, it was found that the infected EL4 cells displayed larger volume, irregular nuclei and the folded twist under light microscopy. Compared with the normal control group, the survival rate of EL4 cells decreased, and the apoptosis rate statistically significantly (P<0.05) increased with the increasing MOI and the infected time in each group. While, the level of apoptosis protein P53, BAX/BCL-2, Cleaved-caspase3 and Caspase8 were up-regulated. And the survival rate, apoptosis rate and the apoptosis protein level of infected EL4 cells with MOI=10 were the most obvious at the 5thday. Compared with MCMV infection group (MOI=60), the content of MCMV DNA in EL4 cells was decreased in MCMV+GGV group ［MOI=60, GCV 25 (g/ml)］, and the cell apoptosis rate and apoptosis protein expression of P53, Caspase8, BAX/BCL-2 were decreased (P<0.05). Murine T-cell lymphoma cell line EL4 can be infected by MCMV and displayes an obvious apoptosis phenomenon. MCMV may up-regulate the expression levels of apoptosis protein P53, BAX-BCL-2, Cleaved-caspase3 and Caspase8 in EL4 cells. The drug ganciclovir reduces the copy mumber of MCMV DNA in infected EL4 cells and inhibited the killing effect of MCMV on EL4 cells.

Genome-Wide Analysis of Interchromosomal Interaction Probabilities Reveals Chained Translocations and Overrepresentation of Translocation Breakpoints in Genes in a Cutaneous T-Cell Lymphoma Cell Line.

In classical models of tumorigenesis, the accumulation of tumor promoting chromosomal aberrations is described as a gradual process. Next-generation sequencing-based methods have recently revealed complex patterns of chromosomal aberrations, which are beyond explanation by these classical models of karyotypic evolution of tumor genomes. Thus, the term chromothripsis has been introduced to describe a phenomenon, where temporarily and spatially confined genomic instability results in dramatic chromosomal rearrangements limited to segments of one or a few chromosomes. Simultaneously arising and misrepaired DNA double-strand breaks are also the cause of another phenomenon called chromoplexy, which is characterized by the presence of chained translocations and interlinking deletion bridges involving several chromosomes. In this study, we demonstrate the genome-wide identification of chromosomal translocations based on the analysis of translocation-associated changes in spatial proximities of chromosome territories on the example of the cutaneous T-cell lymphoma cell line Se-Ax. We have used alterations of intra- and interchromosomal interaction probabilities as detected by genome-wide chromosome conformation capture (Hi-C) to infer the presence of translocations and to fine-map their breakpoints. The outcome of this analysis was subsequently compared to datasets on DNA copy number alterations and gene expression. The presence of chained translocations within the Se-Ax genome, partly connected by intervening deletion bridges, indicates a role of chromoplexy in the etiology of this cutaneous T-cell lymphoma. Notably, translocation breakpoints were significantly overrepresented in genes, which highlight gene-associated biological processes like transcription or other gene characteristics as a possible cause of the observed complex rearrangements. Given the relevance of chromosomal aberrations for basic and translational research, genome-wide high-resolution analysis of structural chromosomal aberrations will gain increasing importance.

Checkpoint molecule expression by B and T cell lymphomas in dogs.

Immunotherapies targeting checkpoint molecule programmed cell death 1 (PD-1) protein were shown to be effective for treatment of non-Hodgkin lymphoma in people, but little is known about the expression of PD-1 or its ligand PD-L1 by canine lymphoma. Therefore, flow cytometry was used to analyse expression of PD-1 and PD-L1 in canine lymphoma, using fine-needle aspirates of lymph nodes from 34 dogs with B cell lymphoma (BCL), 6 dogs with T cell lymphoma (TCL) and 11 dogs that had relapsed. Furthermore, fine-needle aspirates were obtained from 17 healthy dogs for comparison. Lastly, the impact of chemotherapy resistance on expression of PD-1 and PD-L1 was assessed in vitro. These studies revealed increased expression of PD-L1 by malignant B cells compared to normal B cells. In the case of TCL, tumour cells and normal T cells both showed low to negative expression of PD-1 and PD-L1. In addition, tumour infiltrating lymphocytes from both BCL and TCL had increased expression of both PD-1 and PD-L1 expression compared to B and T cells from lymph nodes of healthy animals. In vitro, chemotherapy-resistant BCL and TCL cell lines exhibited increases in both PD-1 and PD-L1 expression, compared to non-chemotherapy selected tumour cells. These findings indicate that canine lymphomas exhibit upregulated checkpoint molecule expression, though the impact of checkpoint molecule expression on tumour biological behaviour remains unclear.

Expression and clinical significance of centrosomal protein 55 in T-cell lymphoma.

T-cell lymphomas (TCLs) have been heterogeneous lymphoid malignancies with aggressive clinical phenotype and poor prognosis. Centrosomal protein 55 (CEP55) played a critical role in cytokinesis and served as a centrosome- and midbody-associated protein. Previous studies have reported the overexpression and clinical significance of CEP55 in various human malignancies, but the exact biological roles of CEP55 in TCLs remained unclear. In this study, we aimed to evaluate the CEP55 expression in patients with TCL and reactive hyperplasia of lymph nodes. The correlation between CEP55 levels and clinical characteristics was also explored for TCL patients. For further investigation, the cell viability of TCL cell lines after CEP55 inhibition was also assessed. Immunohistochemistry was applied to assess the elevated level of CEP55 in TCLs. After siRNA treatment, cell viability and apoptotic rate of TCL cell lines were observed with CCK-8 assay and flow cytometry, respectively. The Pearson's Chi-square test or Fisher's exact test was applied to analyze the correlations between CEP55 overexpression and clinical characteristics. All statistical tests were two-sided, and P < 0.05 was considered to be statistically significant. CEP55 was upregulated in TCL patients and significantly correlated with Ki-67 label index. Consistently, cell viability was decreased, and apoptosis was increased after the suppression of CEP55 in TCL cell lines. These results suggested that target CEP55 would be a novel therapeutic strategy for the TCL.

Histone Deacetylase Inhibitors Downregulate CCR4 Expression and Decrease Mogamulizumab Efficacy in CCR4-Positive Mature T-Cell Lymphomas

Background: Histone deacetylase inhibitors (HDACis) are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and adult T-cell lymphoma/leukemia (ATLL). CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against CTCL, PTCL, and ATLL. However, their combined effects and interactions have not been examined thus far. We previously showed that CCR6, a chemokine receptor, is overexpressed in cutaneous T-cell lymphomas (Ito et al., 2014 Blood). Moreover, we recently demonstrated that HDACis downregulate CCR6 expression in advanced cutaneous T-cell lymphomas (Abe et al., 2017 Oncotarget). These reports lead us to hypothesize that HDACis might also downregulate CCR4 in various T-cell lymphomas. In this study, we clarify the effect of the combined use of mogamulizumab and HDACis on various T-cell and NK-cell lymphomas. Based on our findings, we discuss what benefits or adverse effects might be assumed for patients if these molecular targeting agents are used in clinical practice.Methods: We evaluated changes in CCR4 expression and antibody-dependent cell-mediated cytotoxicity (ADCC) activities against mogamulizumab- and HDACi-treated T-cell and NK-cell lymphoma lines and primary cases. To determine which HDAC mainly regulated CCR4 expression, we used isoform-specific HDACis and induced knockdown of respective HDACs for T-cell lymphoma cell lines. To examine the effect of CCR4 downregulation by HDACis in clinical cases, we examined the CCR4 expression of CTCL skin samples, which were obtained from the same patients before and after HDACi treatment (n = 6).Results: We first examined the expression of CCR4 for 15 T-cell and NK-cell lymphoma cell lines and a peripheral blood mononuclear cell (PBMC) sample derived from healthy donors to investigate the effect of vorinostat, a pan-HDACi, on CCR4 expression. The expression of CCR4 was mostly expressed in the (11 out of 15) cell lines: ATLL (MT-1, MT-2, MT-4, and TL-Su), CTCL (My-La, HH, and MJ), and NK/T-cell lymphoma cell lines (Kai3, SNK6, HANK1, and SNK10). We found that vorinostat decreases mRNA expression and surface expression of CCR4 except for the cell lines without CCR4 expression. Next, we used isoform-specific HDACis to examine which isoform of HDAC is involved in the regulation of CCR4. We used the following class-specific HDACis: romidepsin as a class I selective HDACi, CI-994 as an HDAC1/HDAC2-selective inhibitor, RGFP966 as an HDAC3-selective inhibitor, ricolinostat as an HDAC6-selective inhibitor, and PCI-34051 as an HDAC8-selective inhibitor. When these drugs were exposed to T-cell lymphoma cells, romidepsin and CI-994 strongly suppressed CCR4 expression. These results suggest that class I HDACs might controls CCR4 expression. We further performed knockdown experiments using siRNAs against HDAC1, HDAC2, and HDAC3. When we compared the expression change of CCR4 in HDAC-knockdown cells, HDAC2 knockdown cells showed the most significantly decreased expression of CCR4. These results suggest that class I HDACs, especially HDAC2, might be deeply involved in CCR4 expression regulation. When we examined the CCR4 expression in skin samples from primary CTCL, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after vorinostat treatment. Finally, when we conducted an ADCC assay with mogamulizumab by using various lymphoma cell lines and primary T-cell lymphoma samples, we found that the efficacy of mogamulizumab was significantly reduced by pre-treatment with vorinostat.Conclusion: Our results suggest that the primary use of HDACis before treatment of mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results provide potential implications for optimal therapeutic sequences in various CCR4 positive T-cell and NK-cell lymphomas. DisclosuresKitadate:Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Eisai: Research Funding; Otsuka: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding; Toyama kagaku: Research Funding. Abe:Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Toyama Kagaku: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Eisai: Research Funding. Tagawa:TaNeDS (Daiichi Sankyo): Research Funding.

In Vitro, In Vivo, and Parallel Phase I Evidence Support the Safety and Activity of Duvelisib, a PI3K-δ,γ Inhibitor, in Combination with Romidepsin or Bortezomib in Relapsed/Refractory T-Cell Lymphoma

In Vitro, In Vivo, and Parallel Phase I Evidence Support the Safety and Activity of Duvelisib, a PI3K-δ,γ Inhibitor, in Combination with Romidepsin or Bortezomib in Relapsed/Refractory T-Cell Lymphoma

Targeting T-cell malignancies using anti-CD4 CAR NK-92 cells.

Peripheral T-cell lymphomas (PTCLs) are a group of very aggressive non-Hodgkin's lymphomas (NHLs) with poor prognoses and account for a majority of T-cell malignancies. Overall, the standard of care for patients with T-cell malignancies is poorly established, and there is an urgent clinical need for a new approach. As demonstrated in B-cell malignancies, chimeric antigen receptor (CAR) immunotherapy provides great hope as a curative treatment regimen. Because PTCLs develop from mature T-cells, these NHLs are commonly CD4+, and CD4 is highly and uniformly expressed. Therefore, CD4 is an ideal target for PTCL CAR immunotherapy. To that effect, we created a robust third-generation anti-CD4 CAR construct (CD4CAR) and introduced it into clonal NK cells (NK-92). CD4CAR NK-92 cells specifically and robustly eliminated diverse CD4+ human T-cell leukemia and lymphoma cell lines (KARPAS-299, CCRF-CEM, and HL60) and patient samples ex vivo. Furthermore, CD4CAR NK-92 cells effectively targeted KARPAS-299 cells in vivo that modeled difficult-to-access lymphoma nodules, significantly prolonging survival. In our study, we present novel targeting of CD4 using CAR-modified NK cells, and demonstrate efficacy. Combined, our data support CD4CAR NK cell immunotherapy as a potential new avenue for the treatment of PTCLs and CD4+ T-cell malignancies.

Histone deacetylase inhibitors downregulate CCR4 expression and decrease mogamulizumab efficacy in CCR4-positive mature T-cell lymphomas.

Histone deacetylase inhibitors are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma, peripheral T-cell lymphoma, and adult T-cell lymphoma/leukemia. CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against various T-cell lymphomas. In this study, we examined the in vitro synergistic effects of mogamulizumab and histone deacetylase inhibitors against various T-cell lymphomas. First, we examined the expression of CCR4 mRNA and surface CCR4 in various T-cell lymphoma cell lines and found that it was downregulated upon treatment with vorinostat, a pan-histone deacetylase inhibitor. Next, we used isoform-specific histone deacetylase inhibitors and short-interfering RNA to determine the histone deacetylase isoform involved in the regulation of CCR4, and demonstrated that romidepsin, a class I selective histone deacetylase inhibitor, reduced CCR4 most efficiently. Moreover, among class I histone deacetylases, histone deacetylase 2 knockdown led to a reduction of CCR4 in lymphoma cells, suggesting that CCR4 expression is mainly regulated by histone deacetylase 2. When we examined the CCR4 expression in skin samples from primary cutaneous T-cell lymphoma, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after treatment. Finally, when we conducted an antibody-dependent cell-mediated cytotoxicity assay with mogamulizumab by using various lymphoma cells, we found that the efficacy of mogamulizumab was significantly reduced by pretreatment with vorinostat. Altogether, our results suggest that the primary use of histone deacetylase inhibitors before treatment with mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results have potential implications for optimal therapeutic sequences in various CCR4-positive T-cell lymphomas.

AN-7, a butyric acid prodrug, sensitizes cutaneous T-cell lymphoma cell lines to doxorubicin via inhibition of DNA double strand breaks repair.

We previously found that the novel histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) had greater selectivity against cutaneous T-cell lymphoma (CTCL) than SAHA. AN-7 synergizes with doxorubicin (Dox), an anthracycline antibiotic that induces DNA breaks. This study aimed to elucidate the mechanism underlying the effect of AN-7 on Dox-induced double-strand DNA breaks (DSBs) in CTCL, MyLa and Hut78 cell lines. The following markers/assays were employed: comet assay; western blot of γH2AX and p-KAP1; immunofluorescence of γH2AX nuclear foci; Western blot of repair protein; quantification of DSBs-repair through homologous recombination. DSB induction by Dox was evidenced by an increase in DSB markers, and DSBs-repair, by their subsequent decrease. The addition of AN-7 slightly increased Dox induction of DSBs in MyLa cells with no effect in Hut78 cells. AN-7 inhibited the repair of Dox-induced DSBs, with a more robust effect in Hut78. Treatment with AN-7 followed by Dox reduced the expression of DSB-repair proteins, with direct interference of AN-7 with the homologous recombination repair. AN-7 sensitizes CTCL cell lines to Dox, and when combined with Dox, sustains unrepaired DSBs by suppressing repair protein expression. Our data provide a mechanistic rationale for combining AN-7 with Dox or other DSB inducers as a therapeutic modality in CTCL.

RARα/RXR synergism potentiates retinoid responsiveness in cutaneous T-cell lymphoma cell lines.

Retinoids, natural and synthetic derivatives of vitamin A, induce cellular changes by activating nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR). Although the ability of retinoids to govern gene expression is exploited clinically for cancer therapeutics, the full benefit of retinoid-based strategies is unrealized due to detrimental side effects. Delineating the receptors that prompt cellular outcomes is critical to advancing retinoid-based approaches. Here, we identify the receptors that evoke multiple responses in cutaneous T-cell lymphoma (CTCL). The data demonstrate that RARα drives integrin β7-dependent adhesion and CCR9-mediated chemotaxis in CTCL cells. Of note, concomitant activation of RARα and RXR nuclear receptors yielded synergistic increases in adhesion and migration at concentrations where single agents were ineffective. As the established paradigm of retinoid action in CTCL is apoptosis and growth arrest, the role of RARα/RXR in these events was studied. As with adhesion and migration, RARα/RXR synergism prompted apoptosis and dampened CTCL cell proliferation. Strikingly, RARα/RXR synergism induced responses from CTCL cell lines previously reported to be unresponsive to retinoids. These data provide a novel framework that may further refine a proven CTCL therapy.