Cutaneous T-cell Lymphoma Cells Research Articles

RuBi-Ruxolitinib: A Photoreleasable Antitumor JAK Inhibitor.

We describe the synthesis and biological testing of ruthenium-bipyridine ruxolitinib (RuBiRuxo), a photoreleasable form of ruxolitinib, a JAK inhibitor used as an antitumoral agent in cutaneous T-cell lymphomas (CTCL). This novel caged compound is synthesized efficiently, is stable in aqueous solution at room temperature, and is photoreleased rapidly by visible light. Irradiation of RuBiRuxo reduces cell proliferation and induces apoptosis in a light- and time-dependent manner in a CTCL cell line. This effect is specific and is mediated by a decreased phosphorylation of STAT proteins. Our results demonstrate the potential of ruthenium-based photocompounds and light-based therapeutic approaches for the potential treatment of cutaneous lymphomas and other pathologies.

XPO1 blockade with KPT-330 promotes apoptosis in cutaneous T-cell lymphoma by activating the p53–p21 and p27 pathways

Dysregulated nuclear-cytoplasmic trafficking has been shown to play a role in oncogenesis in several types of solid tumors and hematological malignancies. Exportin 1 (XPO1) is responsible for the nuclear export of several proteins and RNA species, mainly tumor suppressors. KPT-330, a small molecule inhibitor of XPO1, is approved for treating relapsed multiple myeloma and diffuse large B-cell lymphoma. Cutaneous T-cell lymphoma (CTCL) is an extranodal non-Hodgkin lymphoma with an adverse prognosis and limited treatment options in advanced stages. The effect of therapeutically targeting XPO1 with KPT-330 in CTCL has not been established. We report that XPO1 expression is upregulated in CTCL cells. KPT-330 reduces cell proliferation, induces G1 cell cycle arrest and apoptosis. RNA-sequencing was used to explore the underlying mechanisms. Genes associated with the cell cycle and the p53 pathway were significantly enriched with KPT-330 treatment. KPT-330 suppressed XPO1 expression, upregulated p53, p21WAF1/Cip1, and p27Kip1 and their nuclear localization, and downregulated anti-apoptotic protein (Survivin). The in vivo efficacy of KPT-330 was investigated using a bioluminescent xenograft mouse model of CTCL. KPT-330 blocked tumor growth and prolonged survival (p < 0.0002) compared to controls. These findings support investigating the use of KPT-330 and next-generation XPO1 inhibitors in CTCL.

Matrine Enhances the Antitumor Efficacy of Chidamide in CTCL by Promoting Apoptosis.

Cutaneous T-cell Lymphoma (CTCL) is a rare group of non-Hodgkin lymphoma originating from the skin, which is characterized by T-cell lymphoproliferative disorders. Chidamide, a Chinese original antineoplastic agent with independent intellectual property rights, and matrine, an extract of Chinese herbal medicine, both have been reported to exert effects on the treatment of tumors individually. However, chidamide combined with matrine has not been tested for the treatment of CTCL. Both HH and Hut78 CTCL cell lines were treated with chidamide (0.4 μmol/L), matrine (0.6 g/L), or chidamide combined with matrine for 24, 48, and 72 h. Cell viability was estimated by MTS assay at each time point. Flow cytometry was then conducted to detect cell apoptosis. The exact mechanism of chidamide combined with matrine on CTCL cells was detected by Western blotting and further validated in xenograft models of NOD/SCID mice. Compared to the single drug, chidamide combined with matrine showed a more significant effect on proliferation inhibition and apoptosis induction on CTCL cells both in vitro and in vivo. The results from the in vitro and in vivo studies suggested that matrine could enhance the anti-tumor effect of chidamide by increasing the protein expression of cleaved caspase- 3 and decreasing the expression of E-cadherin, NF-κB, p-Bad, and Bcl-2 to activate apoptosis. Our data have demonstrated chidamide combined with matrine to exhibit elevated antitumor activity in both CTCL cells and xenograft models of NOD/SCID mice, which may be a potential treatment option for CTCL.

Antibody targeting of surface P-selectin glycoprotein ligand 1 leads to lymphoma apoptosis and tumorigenesis inhibition.

Lymphomas are a heterogeneous group of diseases that originate from T, B or natural killer cells. Lymphoma treatment is based on chemotherapy, radiotherapy, and monoclonal antibody (mAb) or other immunotherapies. The P-selectin glycoprotein ligand 1 (PSGL-1) is expressed at the surface of hematological malignant cells and has been shown to have a pro-oncogenic role in multiple myeloma and lymphoma. Here, we investigated the expression and therapeutic potential of PSGL-1 in T and B cell lymphomas. By flow cytometry analysis, we found that PSGL-1 was expressed in both T and B cell-derived lymphoma cell lines but generally at higher levels in T cell lymphoma cell lines. For most T and B cell-derived lymphoma cell lines, in vitro targeting with the PL1 mAb, which recognizes the PSGL-1 N-terminal extracellular region and blocks functional interactions with selectins, resulted in reduced cell viability. The PL1 mAb pro-apoptotic activity was shown to be dose-dependent, to be linked to increased ERK kinase phosphorylation, and to be dependent on the MAP kinase signaling pathway. Importantly, anti-PSGL-1 treatment of mice xenografted with the HUT-78 cutaneous T-cell lymphoma cell line resulted in decreased tumor growth, had no effect on in vivo proliferation, but increased the levels of apoptosis in tumors. Anti-PSGL-1 treatment of mice xenografted with a Burkitt lymphoma cell line that was resistant to anti-PSGL-1 treatment in vitro, had no impact on tumorigenesis. These findings show that PSGL-1 antibody targeting triggers lymphoma cell apoptosis and substantiates PSGL-1 as a potential target for lymphoma therapy.

Development of a tetrahydroindazolone-based HDAC6 inhibitor with in-vivo anti-arthritic activity

Histone deacetylase 6 (HDAC6) induces the expression of pro-inflammatory cytokines in macrophages; therefore, HDAC inhibitors may be beneficial for the treatment of macrophage-associated immune disorders and chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis. Structure-activity relationship studies were conducted on various phenyl hydroxamate HDAC6 inhibitors with indolone/indazolone-based bi- or tricyclic ring moieties as the cap group aiming to develop novel anti-arthritic drug candidates. Several compounds exhibited nanomolar activity and HDAC6 selectivity greater than 500-fold over HDAC1. Compound 21, a derivative with the tetrahydroindazolone cap group, is a potent HDAC6 inhibitor with an IC50 of 18 nM and 217-fold selectivity over HDAC1 and showed favorable oral bioavailability in animals. Compound 21 increases the acetylation level of tubulin without affecting histone acetylation in cutaneous T-cell lymphoma cells and inhibits TNF-α secretion in LPS-stimulated macrophage cells. The anti-arthritic effects of compound 21 were evaluated using a rat adjuvant-induced arthritis (AIA) model. Treatment with compound 21 significantly reduced the arthritis score, and combination treatment with methotrexate showed a synergistic effect in AIA models. We identified a novel HDAC6 inhibitor, compound 21, with excellent in vivo anti-arthritic efficacy, which can lead to the development of oral anti-arthritic drugs.

ATF5 promotes malignant T cell survival through the PI3K/AKT/mTOR pathway in cutaneous T cell lymphoma.

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma characterized by skin infiltration of malignant T cells. The biological overlap between malignant T cells and their normal counterparts has brought obstacles in identifying tumor-specific features and mechanisms, limiting current knowledge of CTCL pathogenesis. Transcriptional dysregulation leading to abnormal gene expression profiles contributes to the initiation, progression and drug resistance of cancer. Therefore, we aimed to identify tumor-specific transcription factor underlying CTCL pathology. We analyzed and validated the differentially expressed genes (DEGs) in malignant T cells based on single-cell sequencing data. Clinical relevance was evaluated based on progression-free survival and time to next treatment. To determine the functional importance, lentivirus-mediated gene knockdown was conducted in two CTCL cell lines Myla and H9. Cell survival was assessed by examining cell viability, colony-forming ability, in-vivo tumor growth in xenograft models, apoptosis rate and cell-cycle distribution. RNA sequencing was employed to investigate the underlying mechanisms. Activating transcription factor 5 (ATF5) was overexpressed in malignant T cells and positively correlated with poor treatment responses in CTCL patients. Mechanistically, ATF5 promoted the survival of malignant T cells partially through the PI3K/AKT/mTOR pathway, and imparted resistance to endoplasmic reticulum (ER) stress-induced apoptosis. These findings revealed the tumor-specific overexpression of the transcription factor ATF5 with its underlying mechanisms in promoting tumor survival in CTCL, providing new insight into the understanding of CTCL's pathology.

Mogamulizumab Multimodality Therapy with Systemic Retinoids, Interferon, or Extracorporeal Photopheresis for Advanced Cutaneous T-Cell Lymphoma

Mogamulizumab is a novel monoclonal antibody designed to target CC chemokine receptor 4 (CCR4), which is expressed by tumor cells in cutaneous T-cell lymphoma (CTCL). In clinical trials, mogamulizumab monotherapy has demonstrated efficacy in reducing tumor burden in advanced CTCL, particularly in the peripheral blood. In clinical practice at our center, mogamulizumab has been combined with other agents such as systemic interferon, systemic retinoids, and extracorporeal photopheresis for an enhanced synergistic effect. There is limited published research on mogamulizumab combination therapy. This study evaluates the clinical efficacy of mogamulizumab in combination with systemic interferon, retinoids, and/or extracorporeal photopheresis for therapy of CTCL. Nineteen patients on this regimen at our academic center were identified. Treatment response rates, progression-free survival, duration of response, and adverse effects were characterized by retrospective chart review. All patients on this regimen at our center had an initial partial or complete response. Moreover, responses were durable, and the regimen was well-tolerated with few grade 3 adverse effects. These results demonstrate the utility of a multimodality approach to the therapy of CTCL with mogamulizumab and warrant further confirmation.

Pathobiology and Targeting of CD38 in Cutaneous T-Cell Lymphoma

Introduction: Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature CD4+ T-cells that primarily affects the skin. Despite a wide variety of topical and systemic therapies for patients, CTCL remains difficult to treat. Because of its high likelihood of relapse and resistance, new therapeutic approaches are needed. We recently showed strong CD38 expression on neoplastic T-cells from patients with mature T-cell neoplasms, including CTCL. In this study, we evaluated i) the role of CD38 in CTCL pathogenesis ii) strategies to enhance CD38 expression in CTCL and iii) tested efficacy of combination therapy in vivo. Methods: Skin and peripheral blood samples from patients and healthy donors were evaluated for CD38 expression by immunohistochemistry (N=7), microarray (N=82), single-cell RNA-sequencing (N=11), and flow cytometry (N=14). To investigate the role of CD38 in CTCL, we generated CD38 knockout cell lines (H9, HH, and HuT-78) using CRISPR/Cas9-mediated genomic deletion. The CD38 wild-type (CD38 WT) and knockout (CD38 KO) cells were purified, cultured, and used for downstream functional analysis. We evaluated the lines for growth, metabolic capacities (XFe24 Seahorse assay), and engraftment potential in vivo. For in vivo therapeutic studies, we transduced firefly-luciferase gene into H9 CD38 WT and CD38 KO cells, and engrafted them intravenously into immunodeficient NOD Rag -/-γc -/- mice. Mice were treated with anti-CD38 antibody daratumumab or IgG control and disease progression was monitored over time using an in vivo imaging system. For combination studies, we evaluated CD38 expression by flow cytometry in H9 cells treated with increasing doses of HDAC inhibitor, panobinostat (5nM, 10nM, 25nM) for 24, 48, and 72 hours. Next, we tested co-treatment with panobinostat and daratumumab in vivo in mice engrafted with H9 CD38 WT cells with four treatment groups (vehicle/IgG, panobinostat/IgG, vehicle/daratumumab, and panobinostat/daratumumab). Results: Patient skin biopsies showed increased CD38 expression at both protein and RNA levels (log fold change 4.8; p&amp;lt;0.0001 by Mann-Whitney test). Across nine days of tracking under standard cell culture conditions, CD38 KO CTCL cells showed minimal growth differences compared to CD38 WT (p=0.22 by linear regression). However, CD38 KO CTCL cells showed significantly increased basal and maximum oxygen consumption rates (OCR) via seahorse assay when compared to CD38 WT cells (basal OCR 2.26 fold increase, p&amp;lt;0.0001; max OCR 1.83 fold increase, p&amp;lt;0.0001 by Mann-Whitney test). Using IVIS luminescent imaging, we found significantly increased in vivo growth of CD38 KO CTCL cells compared to CD38 WT cells (CD38 WT=2.2e8 photons/sec average total flux, N=7; CD38 KO=1e9 photons/sec, N=5; p=0.003 by Mann-Whitney test). These data suggest that the loss of CD38 in CTCL cells can accelerate the development of tumors in mice. In order to study the effectiveness of a targeted drug against CD38 in CTCL, we used anti-CD38 antibody daratumumab in our H9 xenograft model. Daratumumab treatment had a significant therapeutic effect on CD38 WT tumors compared to isotype control, reducing tumor burden by 84% (daratumumab average total flux=1.4e7 photons/sec, N=4; IgG average total flux=9.0e7 photons/sec, N=3; p=0.0002). Additionally, we explored methods to increase CD38 expression on CTCL cells. We found that panobinostat significantly increased CD38 expression on H9 CTCL cells in a dose dependent manner across multiple time points (max 85% increase in CD38 expression with 25nM panobinostat vs. DMSO at 72 hours; p&amp;lt;0.0001 by 2way ANOVA). In vivo testing demonstrated a statistically significant improved survival benefit in mice treated with the combination of panobinostat and daratumumab compared to mice that received daratumumab alone (vehicle median survival 23 days, N=4; daratumumab alone median survival 32 days N=4; panobinostat alone median survival 27.5 days, N=4; combination median survival 39 days, N=4; p=0.01 by log-rank test). The prolonged average survival benefit amounted to approximately one quarter of their entire overall lifespan. Conclusion: Our studies demonstrate strong evidence for further study into how CD38 regulates the growth and survival of CTCL cells. Our data also provide preliminary evidence for the clinical usefulness of combination therapies that increase CD38 expression to enhance tumor cell immunotherapeutic targeting.

C17ORF58 Is Upregulated in Peripheral T-Cell Lymphomas and Encodes a Protein Critical for Survival of PTCL Cells

Peripheral T-cell lymphomas (PTCL) constitute a group of heterogeneous T- and NK-cell malignancies, accounting for about 15% of Non-Hodgkin lymphomas (NHL). Most PTCL subtypes exhibit an aggressive disease presentation, carrying a worse prognosis than B-cell lymphomas. PTCL - Not Otherwise Specified (PTCL-NOS), the largest subgroup, is categorized by exclusion, with a diagnosis given if the patient does not meet the diagnostic criteria of other PTCL subtypes. PTCL-NOS is a heterogeneous group with diverse cells of origin, varied cytogenic, molecular, and morphological phenotypes, and antigen variability. Despite the increase in specific T-cell lymphoma treatment options, such as belinostat, romidepsin, pralatrexate, and brentuximab vedotin, alongside conventional non-targeted chemotherapy like CHOP and R-CHOP, the overall 5 year survival remains only at 20-30%. A deeper understanding of the pathways and genes critical for PTCL-NOS survival is urgently needed. In our quest to better understand the molecular landscape, we performed a gene expression analysis of ten PTCL-NOS lymphomas and five normal samples using RNAseq analysis. Our study identified ~400 genes consistently upregulated in lymphoma samples, indicating potential involvement in tumor maintenance. Following the exclusion of common essential genes, we performed a shRNA-mediated knockdown screen of the remaining candidates in the human PTCL-NOS cell line T8ML-1. This approach unveiled several lethal genes, including MYBL2, TRIP13, KIF18B, C17ORF58, and others. Cell cycle analysis demonstrated that while knockdown of MYBL2, TRIP13, and KIF18B induced G2-M arrest, downregulation of C17ORF58 primarily induced apoptosis. Notably, downregulation of C17ORF58 did not impact the proliferation of human embryonic kidney fibroblasts HEK 293T, suggesting that normal cells might not require this protein for growth. However, its downregulation in additional hematologic cell lines, such as Cutaneous T-cell lymphoma cell lines (HH, Hut78) and T-cell lymphoblastic leukemia (Jurkat), also induced apoptosis, which suggests a key role for C17ORF58 in T-cell malignancies. Analysis of publicly available RNAseq data revealed overexpression of C17ORF58 in the majority of PTCL-NOS, Anaplastic Large Cell, Hepatosplenic T-cell, and malignant NK/T-cell lymphomas, relative to normal T-cells, thus highlighting its possible role in tumor development and maintenance. C17ORF58 encodes a predicted protein of 339 amino acids, with an estimated molecular weight of 37 kDa. This protein features two putative domains: the PRK07764 domain, which is homologous to DNA polymerase III subunits gamma and tau, and the netrin module, a 130 amino acid residue domain found within the C-terminal sections of various proteins, including netrins, complement proteins C3-C5, secreted frizzled-related proteins, and type I procollagen C-proteinase enhancer proteins. The predicted promoter has binding sites for Oct-B1-3, Pax-2, POU2F1-2, 2B, and 2C, suggesting that the C17ORF58-encoded protein may participate in normal development. In conclusion, our data indicate that upregulation of C17ORF58 may contribute to the progression and maintenance of PTCL and could serve as a potential target for treating T-cell malignancies. Investigations into its physiological role in normal T-cells and pathways deregulated upon its inactivation are ongoing.

Development of Bexarotene Analogs for Treating Cutaneous T-Cell Lymphomas.

Bexarotene, a drug approved for treatment of cutaneous T-cell lymphoma (CTCL), is classified as a rexinoid by its ability to act as a retinoid X receptor (RXR) agonist with high specificity. Rexinoids are capable of inducing RXR homodimerization leading to the induction of apoptosis and inhibition of proliferation in human cancers. Numerous studies have shown that bexarotene is effective in reducing viability and proliferation in CTCL cell lines. However, many treated patients present with cutaneous toxicity, hypothyroidism, and hyperlipidemia due to crossover activity with retinoic acid receptor (RAR), thyroid hormone receptor (TR), and liver X receptor (LXR) signaling, respectively. In this study, 10 novel analogs and three standard compounds were evaluated side-by-side with bexarotene for their ability to drive RXR homodimerization and subsequent binding to the RXR response element (RXRE). In addition, these analogs were assessed for proliferation inhibition of CTCL cells, cytotoxicity, and mutagenicity. Furthermore, the most effective analogs were analyzed via qPCR to determine efficacy in modulating expression of two critical tumor suppressor genes, ATF3 and EGR3. Our results suggest that these new compounds may possess similar or enhanced therapeutic potential since they display enhanced RXR activation with equivalent or greater reduction in CTCL cell proliferation, as well as the ability to induce ATF3 and EGR3. This work broadens our understanding of RXR-ligand relationships and permits development of possibly more efficacious pharmaceutical drugs. Modifications of RXR agonists can yield agents with enhanced biological selectivity and potency when compared to the parent compound, potentially leading to improved patient outcomes.

New JAK3-INSL3 Fusion Transcript-An Oncogenic Event in Cutaneous T-Cell Lymphoma.

Constitutively activated tyrosine kinase JAK3 is implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCL). The mechanisms of constitutive JAK3 activation are unknown although a JAK3 mutation was reported in a small portion of CTCL patients. In this study, we assessed the oncogenic roles of a newly identified JAK3-INSL3 fusion transcript in CTCL. Total RNA from malignant T-cells in 33 patients with Sézary syndrome (SS), a leukemic form of CTCL, was examined for the new JAK3-INSL3 fusion transcript by RT-PCR followed by Sanger sequencing. The expression levels were assessed by qPCR and correlated with patient survivals. Knockdown and/or knockout assays were conducted in two CTCL cell lines (MJ cells and HH cells) by RNA interference and/or CRISPR/Cas9 gene editing. SS patients expressed heterogeneous levels of a new JAK3-INSL3 fusion transcript. Patients with high-level expression of JAK3-INSL3 showed poorer 5-year survival (n = 19, 42.1%) than patients with low-level expression (n = 14, 78.6%). CTCL cells transduced with specific shRNAs or sgRNAs had decreased new JAK3-INSL3 fusion transcript expression, reduced cell proliferation, and decreased colony formation. In NSG xenograft mice, smaller tumor sizes were observed in MJ cells transduced with specific shRNAs than cells transduced with controls. Our results suggest that the newly identified JAK3-INSL3 fusion transcript confers an oncogenic event in CTCL.

Embelin inhibits viability of cutaneous T cell lymphoma cell lines HuT78 and H9 by targeting inhibitors of apoptosis

Cutaneous T cell lymphoma (CTCL) is a varied group of neoplasms that affects the skin. Acquired resistance against chemotherapeutic drugs and associated toxic side effects are limitations that warrant search for novel drugs against CTCL. Embelin (EMB) is a naturally occurring benzoquinone derivative that has gained attention owing to its anticancer pharmacological actions and nontoxic nature. We assessed the anticancer activity of EMB against CTCL cell lines, HuT78, and H9. EMB inhibited viability of CTCL cells in a dose-dependent manner. EMB activated extrinsic and intrinsic pathways of apoptosis as shown by the activation of initiator and executioner caspases. EMB-induced apoptosis also involved suppression of inhibitors of apoptosis, XIAP, cIAP1, and cIAP2. PARP cleavage and upregulation of pH2AX indicated DNA damage induced by EMB. In conclusion, we characterized a novel apoptosis-inducing activity of EMB against CTCL cells, implicating EMB as a potential therapeutic agent against CTCL.

MAb14, a Monoclonal Antibody against Cell Surface PCNA: A Potential Tool for Sezary Syndrome Diagnosis and Targeted Immunotherapy.

Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common types of primary cutaneous T-cell lymphoma (CTCL). Proliferating cell nuclear antigen (PCNA) is expressed on the cell surface of cancer cells (csPCNA), but not on normal cells. It functions as an immune checkpoint ligand by interacting with natural killer (NK) cells through the NK inhibitory receptor NKp44, leading to the inhibition of NK cytotoxicity. A monoclonal antibody (mAb14) was established to detect csPCNA on cancer cells and block their interaction with NKp44. In this study, three CTCL cell lines and peripheral blood mononuclear cells (PBMCs) from patients with SS and healthy donors were analyzed for csPCNA using mAb14, compared to monoclonal antibody PC10, against nuclear PCNA (nPCNA). The following assays were used: immunostaining, imaging flow cytometry, flow cytometry, cell sorting, cell cycle analysis, ELISA, and the NK-cell cytotoxic assay. mAb14 successfully detected PCNA on the membrane and in the cytoplasm of viable CTCL cell lines associated with the G2/M phase. In the Sézary PBMCs, csPCNA was expressed on lymphoma cells that had an atypical morphology and not on normal cells. Furthermore, it was not expressed on PBMCs from healthy donors. In the co-culture of peripheral blood NK (pNK) cells with CTCL lines, mAb14 increased the secretion of IFN-γ, indicating the reactivation of pNK activity. However, mAb14 did not enhance the cytotoxic activity of pNK cells against CTCL cell lines. The unique expression of csPCNA detected by mAb14 suggests that csPCNA and mAb14 may serve as a potential biomarker and tool, respectively, for detecting malignant cells in SS and possibly other CTCL variants.

O-109 - Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor

O-109 - Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor

P-156 - Sensitivity to brentuximab vedotin depends on CD30-expression levels in different subtypes of cutaneous T-cell lymphoma cell lines

P-156 - Sensitivity to brentuximab vedotin depends on CD30-expression levels in different subtypes of cutaneous T-cell lymphoma cell lines

Skin infiltrating NK cells in cutaneous T-cell lymphoma are increased in number and display phenotypic alterations partially driven by the tumor.

Cutaneous T-cell lymphomas (CTCL) are characterized by focal infiltration of malignant T cell clones in solitary skin lesions. Many CTCL patients experience an indolent disease, but some progress to advanced disease with high fatality. We hypothesized that natural killer (NK) cells participate in local control of tumor growth in CTCL skin. Immunohistochemistry and flow cytometry analysis of the density, localization, phenotype and function of NK cells in twenty-nine fresh or formalin-fixed skin biopsies from twenty-four CTCL patients and twenty-three biopsies from twenty healthy controls highlighted higher numbers of CD56+CD3- NK cells in CTCL skin. A reduced fraction of CTCL skin NK cells expressed the maturation marker CD57, the cytotoxic protein granzyme B and the activation marker CD69, indicating reduced tumor-killing abilities of the NK cells. Retained expression of immune checkpoint proteins or inhibitory proteins including PD1, TIM3, LAG3, CD73 and NKG2A and the activating receptors CD16 and NKp46 indicated maintained effector functions. Indeed, the capacity of NK cells to produce anti-tumor acting IFNγ upon PMA+ionomycin stimulation was similar in cells from CTCL and healthy skin. Co-cultures of primary human NK cells or the NK cell line NKL with CTCL cells resulted in reduced levels of granzyme B and CD69, indicating that close cellular interactions with CTCL cells induced the impaired functional NK cell phenotype. In conclusion, increased numbers of NK cells in CTCL skin exhibit a partially impaired phenotype in terms of activity. Enhancing NK cell activity with NK cell activating cytokines such as IL-15 or immune checkpoint blockade therefore represents a potential immunotherapeutic approach in CTCL.

Dupilumab aggravates Sézary syndrome: The importance of accurate pathological diagnosis

We report a case of Sézary syndrome aggravated by dupilumab. Although a diagnosis of atopic dermatitis was made previously, our retrospective diagnosis was cutaneous T-cell lymphoma. A 72-year-old woman was referred for evaluation of erythema over her entire body. She had received a diagnosis of atopic dermatitis (AD) based on skin biopsy elsewhere 7 years earlier. Since her symptoms were resistant to topical corticosteroids and oral antihistamines, dupilumab was started. She noticed peripheral lymph node enlargement 11 months after the commencement of dupilumab. Physical examination revealed generalized erythroderma with desquamation (Figure 1A,B) and lymphadenopathy in the neck, axilla, and inguinal region. A biopsy from her abdomen showed dense infiltrations of atypical lymphoid cells in the upper dermis with epidermotropism (Figure 1C,D). Immunohistochemically, the infiltrating cells were positive for CD3 and CD4; CD8 was almost negative. There was a 50% deletion of CD7 (Figure 1E–H). Her peripheral blood (PB) white blood cell count was 7660/μL, with 35% abnormal lymphocytes (2681/μL). Although CD4+/CD26− or CD4+/CD7− cells were not assessed, CD4/CD8 ratio was 10.59.1, 2 Human T-lymphotropic virus-1 antibody was negative. Monoclonal gene rearrangement of T-cell receptor β-chain was identified in her PB by Southern blot analysis. Inguinal lymph node biopsy showed partial effacement by atypical lymphocytes. The diagnosis was Sézary syndrome (SS), stage IVA2 (T4N3MXB2). Taking into account the risk of tumor lysis syndrome due to high tumor burden, we selected conventional multiagent chemotherapy as an initial therapy before using a molecular-targeted drug. Dupilumab was discontinued; cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) were started. After two cycles of CHOP, biweekly intravenous mogamulizumab was introduced. Her skin lesions gradually improved. The PB abnormal lymphocyte count decreased. Her disease was well controlled with mogamulizumab for 17 months. We reviewed the previous biopsy specimen. Dense dermal infiltrates with atypical cells and epidermotropism were seen (Figure 1I,J). These infiltrates were positive for CD3 and CD4. CD8-positive cells were sparse. There was a 30% deletion of CD7 (Figure 1K–N). The atypical cells were CCR4+ and CD68−. Although a diagnosis of AD was made previously, our retrospective diagnosis was cutaneous T-cell lymphoma (CTCL). Dupilumab is a monoclonal antibody which inhibits both IL-4 and IL-13 signaling pathways by binding specifically to the α subunit of the IL-4 receptor. Several cases of CTCL that developed or worsened after dupilumab use have been reported. A proposed mechanism of CTCL due to dupilumab is the involvement of IL-13 receptor α2 (IL-13Rα2), which is expressed on CTCL cells and promotes tumor progression. When dupilumab blocks IL-4 and IL-13 receptors, serum IL-13 levels increase. Because dupilumab inhibits IL-13Rα1 but not IL-13Rα2 signaling, increased IL-13 levels due to dupilumab stimulate IL-13Rα2, leading to the activation and proliferation of lymphoma cells.3 To our knowledge, there are 36 cases3-19 of CTCL worsened by dupilumab based on the diagnosis of AD or eczema have been reported. Of these 36 patients, 18 underwent skin biopsy prior to dupilumab therapy; no evidence of CTCL was described in 16 patients.3-10 In one remaining patient, the authors re-evaluated the biopsy specimen taken before dupilumab treatment and revised the diagnosis to early CTCL-not otherwise specified.10 In another patient, the first skin biopsy taken before dupilumab was compatible with AD; however, the diagnosis of mycosis fungoides was made by the second biopsy taken on the first day of dupilumab treatment. After 6 months of dupilumab treatment, the third biopsy turned out CD30-positive anaplastic large-cell lymphoma.11 When using dupilumab for AD, clinicians should be alert to the development or worsening of CTCL. Skin biopsy with immunohistochemical examination is strongly recommended before dupilumab therapy, especially in patients with erythroderma and elderly patients. The authors declare no conflict of interest. Approval of the research protocol: N/A. Informed Consent: N/A. Registry and the Registration No.: N/A. Animal Studies: N/A.

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

Dimethyl fumarate treatment in relapsed and refractory cutaneous T-cell lymphoma: a multicenter phase 2 study

AbstractTargeted therapies for cutaneous T-cell lymphoma (CTCL) are limited and curative approaches are lacking. Furthermore, relapses and drug induced side effects are major challenges in the therapeutic management of patients with CTCL, creating an urgent need for new and effective therapies. Pathologic constitutive NF-κB activity leads to apoptosis resistance in CTCL cells and, thus, represents a promising therapeutic target in CTCL. In a preclinical study we showed the potential of dimethyl fumarate (DMF) to block NF-κB and, specifically, kill CTCL cells. To translate these findings to applications in a clinical setting, we performed a multicentric phase 2 study evaluating oral DMF therapy in 25 patients with CTCL stages Ib to IV over 24 weeks (EudraCT number 2014-000924-11/NCT number NCT02546440). End points were safety and efficacy. We evaluated skin involvement (using a modified severity weighted assessment tool [mSWAT]), pruritus, quality of life, and blood involvement, if applicable, as well as translational data. Upon skin analysis, 7 of 23 (30.4%) patients showed a response with >50% reduction in the mSWAT score. Patients with high tumor burden in the skin and blood responded best to DMF therapy. Although not generally significant, DMF also improved pruritus in several patients. Response in the blood was mixed, but we confirmed the NF-κB–inhibiting mechanism of DMF in the blood. The overall tolerability of the DMF therapy was very favorable, with mostly mild side effects. In conclusion, our study presents DMF as an effective and excellently tolerable therapeutic option in CTCL to be further evaluated in a phase 3 study or real-life patient care as well as in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT02546440.

1584 Effects of MALT1 Inhibitors on cutaneous T-cell lymphoma cells

1584 Effects of MALT1 Inhibitors on cutaneous T-cell lymphoma cells