Cutaneous T-cell Lymphoma Cell Lines Research Articles

Sensitivity of Cutaneous T-Cell Lymphoma Cells to the Mcl-1 Inhibitor S63845 Correlates with the Lack of Bcl-w Expression.

Long-term, curative treatment of cutaneous T-cell lymphomas (CTCL) remains a major challenge. Therapy resistance is often based on apoptosis deficiency, and may depend on antiapoptotic Bcl-2 proteins, such as Bcl-2, Bcl-xL, Bcl-w and Mcl-1. For their targeting, several antagonists have been generated, which mimic the Bcl-2 homology domain 3 (BH3 mimetics). As dysregulation and overexpression of Mcl-1 has been reported in CTCL, the use of Mcl-1 inhibitors appears as an attractive strategy. Here, we investigated the effects of the selective Mcl-1 inhibitor S63845 in a series of four CTCL cell lines, in comparison to ABT-263 and ABT-737 (inhibitors of Bcl-2, Bcl-xL and Bcl-w). In two cell lines (HH, HuT-78), S63845 resulted in significant apoptosis induction, decrease in cell viability, loss of mitochondrial membrane potential and caspase activation, while two other cell lines (MyLa, SeAx) remained completely resistant. An inverse correlation was found, as S63845-resistant cells were highly sensitive to ABT-263/-737, and S63845-sensitive cells showed only moderate sensitivity to ABTs. Combinations of S63845 and ABT-263 partially yielded synergistic effects. As concerning Bcl-2 protein expression, weaker Mcl-1 expression was found in S63845-resistant MyLa and SeAx, while for Bcl-2 and Bcl-xL, the lowest expression was found in the highly sensitive cell line HH. The most striking difference between S63845-resistant and -sensitive cells was identified for Bcl-w, which was exclusively expressed in S63845-resistant cells. Thus, CTCL may be efficiently targeted by BH3 mimetics, providing the right target is preselected, and Bcl-w expression may serve as a suitable marker.

TCL-318 Evaluation of the Antitumor Activity of Amiloride in Peripheral T-Cell Lymphomas Using In-Vitro Models.

Peripheral T-cell lymphomas (PTCL) have an aggressive clinical behavior and poor response to treatment; therefore, the search for new drugs is required. We have previously demonstrated the antitumor activity of amiloride in multiple myeloma. This prompted us to investigate the possibility of a cytotoxic effect of amiloride in this kind of T-cell lymphoma. To evaluate the antitumor activity of amiloride in different PTCL using in-vitro assays and identify the possible mechanisms involved. Two anaplastic large-cell lymphoma (ALCL) cell lines, KARPAS-299 and SR-786, and two cutaneous T-cell lymphoma cell lines, HH and HuT-78 (Sézary syndrome), were used. Cell viability and apoptosis were studied after exposure to different increasing concentrations of the drug at 24, 48, and 72 hours by colorimetric MTT and annexin V/propidium iodide double-labeling assays, respectively. Amiloride significantly inhibited cell growth in the PTCL cell lines, depending on increasing drug concentration and time. Inhibition was statistically significant (P<0.05) from 0.15 mM for KARPAS-299, SR-786, and HH at 24, 48, and 72 hours and from 0.3 mM for HuT-78 at 48 and 72 hours. KARPAS-299 and SR-786 were the most sensitive cell lines to amiloride exposure, whereas HuT-78 cells were less sensitive, although with the highest concentration of amiloride, its cell viability decreased to levels below 50% and 40% at 48 and 72 hours, respectively. Induction of cell death by apoptosis was detected in all cell lines, with apoptosis levels up to 25% at 24 hours and more than 80% at 48 and 72 hours using intermediate doses of the drug. 1) Amiloride significantly decreased the cell viability of PTCL. 2) ALCL cell lines were the most sensitive to amiloride exposure. 3) The cytotoxic effect induced by amiloride in PTCL was mediated by apoptosis. Ongoing Objectives: To evaluate apoptosis as the possible mechanism of cell death induction through the analysis of caspases, synergism with drugs commonly used in the treatment of PTCL, and changes in gene and protein expression induced by amiloride. Regional Health Management of Castilla y León (GRS 2142/A/2020);); Carlos III Health Institute European Union (PI19/00674); AECC (PROYE20047GUTI).

Concomitant Inhibition of FASN and SREBP Provides a Promising Therapy for CTCL.

Simple SummaryThe biosynthesis of fatty acids catalysed by FASN plays an important oncogenic role in various malignancies but has not been reported in CTCL yet. Here, we show that FASN is highly expressed in both cell lines and primary cells from CTCL patients. The inhibition of FASN impairs cell viability, survival, and proliferation. FASN expression is partly controlled by SREBP, and dual inhibition of FASN and SREBP enhances the impairment of cell proliferation. Overall, our data suggest that the combination of FASN and SREBP inhibitors could be a promising novel strategy in CTCL therapy.Cutaneous T cell lymphoma (CTCL) is a group of non-Hodgkin’s primary cutaneous T cell lymphomas, with Mycosis Fungoides and Sézary syndrome (SS) being the two most common subtypes. Fatty acid synthase (FASN) is a crucial enzyme that catalyses the biosynthesis of fatty acids, which has been reported to play an oncogenic role in various malignancies but not in CTCL so far. Herein, we show that FASN is highly expressed in CTCL cell lines and in peripheral blood mononuclear cells (PBMCs) from CTCL patients, while it is not in PBMCs from healthy individuals. The inhibition of FASN in CTCL cell lines impairs cell viability, survival, and proliferation, but, interestingly, it also increases FASN expression. However, inhibiting sterol regulatory element binding protein (SREBP), a transcription factor that promotes the expression of FASN, partially reversed the upregulation of FASN induced by FASN inhibitors. Thus, the combination of FASN and SREBP inhibitors enhanced the effects on both CTCL cell lines and PBMCs from SS patients, where a valid inhibition on cell proliferation could be verified. Importantly, compared to non-malignant cells, primary malignant cells are more sensitive to the inhibition of FASN and SREBP, making the combination of FASN and SREBP inhibitors a promising novel therapeutic strategy in CTCL.

Genomic Aberrations Generate Fusion Gene FOXK2::TP63 and Activate NFKB1 in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a severe lymphoid malignancy with a worse prognosis lacking curative treatment regimens. Several gene mutations and deregulated pathways, including NFkB signaling, have been implicated in its pathogenesis. Accordingly, CTCL cell line HUT-78 reportedly contains mutated NFKB2, which is constitutively activated via partial gene deletion, also demonstrating that genomic rearrangements cause driving mutations in this malignancy. Here, along with HUT-78, we analyzed CTCL cell line HH to identify additional aberrations underlying gene deregulation. Karyotyping and genomic profiling of HH showed several rearrangements worthy of detailed investigation. Corresponding to the established karyotype, RNA-seq data and PCR analysis confirmed the presence of t(3;17)(q28;q25), generating a novel fusion gene, FOXK2::TP63. Furthermore, chromosomal rearrangement t(1;4)(p32;q25) was connected to amplification at 4q24–26, affecting aberrant NFKB1 overexpression thereat. Transcription factor binding-site analysis and knockdown experiments demonstrated that IRF4 contributed to NFKB1 expression. Within the same amplicon, we identified amplification and overexpression of NFkB signaling activator CAMK2D (4q26) and p53-inhibitor UBE2D3 (4q24). Genomic profiling data for HUT-78 detailed a deletion at 10q25 underlying reported NFKB2 activation. Moreover, amplifications of ID1 (20q11) and IKZF2 (2q34) in this cell line drove overexpression of these NK cell differentiation factors and possibly thus formed corresponding lineage characteristics. Target gene analysis for NFKB1 via siRNA-mediated knockdown in HH revealed activation of TP63, MIR155, and NOTCH pathway component RBPJ. Finally, treatment of HH with NFkB inhibitor demonstrated a role for NFkB in supporting proliferation, while usage of inhibitor DAPT showed significant survival effects via the NOTCH pathway. Collectively, our data suggest that NFkB and/or NOTCH inhibitors may represent reasonable treatment options for subsets of CTCL patients.

The role of immune checkpoint receptors in the malignant phenotype of cutaneous T cell lymphoma.

Immune checkpoint receptors (ICR) modulate the immune response and are critical hubs for immunotherapy. However, data on their role in T lymphoid malignancies, such as cutaneous T cell lymphoma (CTCL), is sparse. We aimed to explore the role of ICR in the malignant features of transformed T lymphocytes and evaluate the effect of ICR-targeting monoclonal antibodies, often used as immunotherapy for solid tumors. We used the CTCL cell line HH and the Sézary cell line Hut78 to examine ICR expression and the effects of ICR inhibition on cell viability and proliferation. Despite their shared T cell progeny, the different CTCL cell lines exhibit markedly different ICR expression profiles. Programmed cell death-ligand 1 (PD-L1) was expressed by both cell lines, while programmed death-1 (PD-1) was expressed only by the HH cell line. Common to all malignant T cells was an autonomous hyper-proliferative state that did not require T cell receptor stimulation. A monoclonal antibody blocking PD-1 had a small but statistically significant augmenting effect on T cell proliferation. Of note, when the cells were exposed to ionizing radiation, healthy lymphocytes and those derived from the HH cell line were salvaged by anti-PD-L1. We show a regulatory role of ICR, mainly PD-1 and its ligand PD-L1, on cutaneous T cell malignancy.

A novel mouse model of cutaneous T-cell lymphoma revealed the combined effect of mogamulizumab with psoralen and ultraviolet a therapy.

Mycosis fungoides (MF) is a subtype of cutaneous T-cell lymphoma (CTCL). Topical or systemic treatment with psoralen, such as 8-methoxypsoralen (8-MOP), followed by ultraviolet A (UVA) irradiation (PUVA therapy) is an effective phototherapy for early-stage MF. However, the efficacy of PUVA therapy for advanced-stage MF is not satisfactory, and the ideal combination partner for PUVA therapy has not yet been found. In this study, we developed a new mouse model of CTCL in which efficacy of PUVA was detected and further evaluated the efficacy of combination treatment of PUVA and mogamulizumab, an anti-CCR4 monoclonal antibody. Cytotoxicity of PUVA therapy against HH cells, a CTCL cell line, was observed in vitro. The cytotoxicity was dependent on both 8-MOP and UVA. Using HH cells, we developed a mouse model in which HH cells were subcutaneously inoculated in the ear. In this model, PUVA therapy suppressed tumour growth with statistical significance, while 8-MOP or UVA alone did not. Combination therapy of PUVA and mogamulizumab showed greater antitumor activity than either monotherapy with statistical significance. In the histological analysis of the tumour tissue, PUVA accelerated tumour necrosis and then induced the infiltration inflammatory cells in the necrotic area, suggesting that these cells served as effector cells for mogamulizumab. This combination therapy is expected to be a beneficial option for CTCL therapy.

Abstract 5196: CD47 Blockade potentiates immunotherapy of durvalumab against cutaneous T cell lymphoma

Abstract Immune checkpoint (IC) blockade for programmed cell death 1 (PD1) and PD-Ligand 1 (PD-L1) has shown promising and durable therapeutic outcomes in cutaneous T-cell lymphoma (CTCL), but resistance and/or relapses are common. While research focused on improving adaptive immune functions in CTCL, we investigated the role of 1) the “do not eat me signal” (CD47), and PD-L1, as a dual (innate and adaptive IC)-targeting strategy in CTCL. RNA sequencing and flow cytometric analysis showed that CTCL tumor cells and CTCL cell lines (MyLa, Hut78, HH, and H9 cells) overexpressed CD47 and PD-L1 compared with healthy control, and CD47 positively correlated with PD-L1 in CTCL patients. Overexpression of CD47 and PD-L1 was induced by high MYC expression in CTCL tumor cells lines. In addition, the signal-regulatory protein (SIRP)α receptor for CD47 and PD-L1 were significantly more abundant on macrophages, dendritic cells and natural killer cells in CTCL patients than healthy control by flow cytometric analysis. Furthermore, the RNA sequencing data indicated that the M2 macrophages, immature dendritic cells, and inhibitory receptors expressed natural killer cells in CTCL patients were higher than in healthy control. Notably, TTI-621 (SIRPαFc) treatment decreased M2 macrophage, immature dendritic cells, and inhibitory receptors expressed natural killer cells in CTCL patients at the end of the treatment, compared to baseline. In vitro, TTI-621 treatment increased the macrophage phagocytic activity compared to untreated control. Moreover, TTI-621 synergized with an anti-PD-L1 antibody (durvalumab) to reprogram M2-like TAMs, induced by MyLa supernatant, to M1-like phenotypes. The simultaneous blockade of both CD47 and PD-L1 inhibited growth of CTCL cell lines more potently than each single antibody alone or blank control in vitro. Enhanced CD8+ T cell mediated killing was detected using a chromium-release assay following dual blockade of in CTCL cell lines, suggesting anti-CD47 and anti-PD-L1 may synergistically facilitate elimination of CTCL tumor cells through specific pathways. RNA-sequencing analysis indicated that these effects were mediated by cell death related pathways such as apoptosis, autophagy, and necroptosis. Collectively, our findings demonstrated that CD47 and PD-L1 are critical regulators of innate and adaptive immune surveillance in CTCL and that dual targeting of CD47 and PD-L1 will provide insight into tumor immunotherapy to improve tumor control in CTCL. Citation Format: Zhen Han, Mingye Feng, Xiwei Wu, Chingyu Su, Yate-Ching Yuan, Hanjun Qin, James F. Sanchez, Jasmine Zain, Steven T. Rosen, Christiane Querfeld. CD47 Blockade potentiates immunotherapy of durvalumab against cutaneous T cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5196.

Downregulation of miR-26 promotes invasion and metastasis via targeting interleukin-22 in cutaneous T-cell lymphoma.

It has been reported that certain microRNAs (miRNA) are associated with the pathogenesis of lymphoma. We have previously demonstrated that histone deacetylase inhibitors restore tumor‐suppressive miRNAs, such as miR‐16, miR‐29, miR‐150, and miR‐26, in advanced cutaneous T‐cell lymphoma (CTCL). Among these, the function of miR‐26 remains unclear. In this study, we aimed to reveal the function of miR‐26 in CTCL oncogenesis. First, we confirmed that the miR‐26 family was markedly dysregulated in CTCL cell lines and primary samples. In vivo analysis using miR‐26a‐transduced CTCL cells injected into immunodeficient NOG mice demonstrated the significant prolonged survival of the mice, suggesting that the miRNA had a tumor‐suppressive function. We performed gene expression assays and identified 12 candidate miR‐26 targets, namely RGS13, FAM71F1, OAF, SNX21, CDH2, PTPLB, IL22, DNAJB5, CASZ1, CACNA1C, MYH10, and CNR1. Among these, IL22 was the most likely candidate target because the IL‐22–STAT3–CCL20–CCR6 cascade is associated with tumor invasion and metastasis of advanced CTCL. In vitro analysis of IL22 and IL22RA knockdown and miR‐26 transduction demonstrated inhibited CTCL cell migration. In particular, IL22 knockdown induced cell apoptosis. Finally, we conducted in vivo inoculation analysis of mice injected with shIL22‐transfected CTCL cells, and found no tumor invasion or metastasis in the inoculated mice, although the control mice showed multiple tumor invasions and metastases. These results, along with our previous data, demonstrated that miR‐26 is a tumor suppressor that is associated with tumor invasion and the metastasis of advanced CTCL by regulating the IL‐22–STAT3–CCL20 cascade. Therefore, a IL‐22‐targeting therapy could be a novel therapeutic strategy for advanced CTCL.

Roles of OX40 and OX40 Ligand in Mycosis Fungoides and Sézary Syndrome.

Mycosis fungoides (MF) and Sézary syndrome (SS), the most common types of cutaneous T-cell lymphoma (CTCL), are characterized by proliferation of mature CD4+ T-helper cells. Patients with advanced-stage MF and SS have poor prognosis, with 5-year survival rates of 52%. Although a variety of systemic therapies are currently available, there are no curative options for such patients except for stem cell transplantation, and thus the treatment of advanced MF and SS still remains challenging. Therefore, elucidation of the pathophysiology of MF/SS and development of medical treatments are desired. In this study, we focused on a molecule called OX40. We examined OX40 and OX40L expression and function using clinical samples of MF and SS and CTCL cell lines. OX40 and OX40L were co-expressed on tumor cells of MF and SS. OX40 and OX40L expression was increased and correlated with disease severity markers in MF/SS patients. Anti-OX40 antibody and anti-OX40L antibody suppressed the proliferation of CTCL cell lines both in vitro and in vivo. These results suggest that OX40–OX40L interactions could contribute to the proliferation of MF/SS tumor cells and that the disruption of OX40–OX40L interactions could become a new therapeutic strategy for the treatment of MF/SS.

MicroRNA Regulation of T-Cell Exhaustion in Cutaneous T Cell Lymphoma

Cutaneous T cell lymphoma (CTCL) is characterized by a background of chronic inflammation, where malignant CTCL cells escape immune surveillance. To study how microRNAs (miRs) regulate T-cell exhaustion, we performed miR sequencing analysis, qRT-PCR, and in situ hybridization on 45 primary CTCL samples, three healthy skin samples, and CTCL cell lines, identifying miR-155-5p, miR-130b-3p, and miR-21-3p. Moreover, miR-155-5p, miR-130b-3p, and miR-21-3p positively correlated with immune checkpoint gene expression in lesional skin samples and were enriched in the IL-6/Jak/signal transducer and activator of transcription signaling pathway by gene set enrichment analysis. Further gene sequencing analysis showed decreased mRNA expression of the major negative regulators of Jak/signal transducer and activator of transcription signaling: SOCS, PIAS, and PTPN. Transfection of MyLa and HuT78 cells with anti-miR-155-5p, anti‒miR-21-3p, and anti‒miR-130b revealed a considerable increase in SOCS proteins along with a significant decrease in the levels of activated signal transducer and activator of transcription 3 and immune checkpoint surface protein expression as well as decreased cell proliferation. Downregulation of miR-155, miR-130, and miR-21 in CTCL cell lines decreased CTCL cell growth and facilitated CD8+ T-cell-mediated cytotoxic activity, with concordant production of IFN-γ and CD107a expression. Our results describe the mechanisms of miR-induced T-cell exhaustion, which provide a foundation for developing synthetic anti-miRs to therapeutically target the tumor microenvironment in CTCL.

A Novel Antibody Drug Conjugate (ADC) Targeting Cell Surface Heat Shock Protein 70 (csHSP70) Is Active Against Pre-Clinical Models of Peripheral T-Cell Lymphoma (PTCL)

A Novel Antibody Drug Conjugate (ADC) Targeting Cell Surface Heat Shock Protein 70 (csHSP70) Is Active Against Pre-Clinical Models of Peripheral T-Cell Lymphoma (PTCL)

ICOS Is Widely Expressed in Cutaneous T-Cell Lymphoma and Its Targeting Promotes Potent Killing of Malignant Cells

Background: Advanced cutaneous T-cell lymphomas (CTCLs) remain an unmet medical need. Brentuximab vedotin (BV), an anti-CD30 antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), do not deliver significant long-term improvements in patient outcomes. More recently, mogamulizumab and anti-KIR3DL2 provided encouraging results but new targeted therapies are needed. Inducible Co-Stimulator (ICOS), a T-cell costimulatory receptor involved in the development of CTCLs, arouses interest.Methods: We used immunohistochemistry to study ICOS expression in skin biopsies of 23 patients with early-stage mycosis fungoides (MF), 12 with transformed MF (TMF) and 17 with Sézary Syndrome (SS), at diagnosis or in relapse. Skin samples from 12 patients with B-cell lymphomas, 14 with CD30 + lymphoproliferative disease (LPD), 12 with primary cutaneous CD4+ small/medium T-cell lympho-proliferative disorder and 13 with angioimmunoblastic T-cell lymphoma (AITL) were used as controls. ICOS expression by circulating Sézary cells and regulatory T cells (Tregs) in patients with SS was evaluated using flow cytometry and compared to healthy donors (HD) lymphocytes. In 5 patients with SS, we also analyzed concomitant biopsies from involved nodes.Then, we investigated the efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS 314.8 monoclonal antibodies with MMAE and pyrrolobenzodiazepine (PBD), in comparison to BV. We used ICOS + CTCL cell lines (MyLa, MJ and HUT78), murine xenograft models with MyLa and ICOS + Patient Derived Xenografts (PDXs) from patients with SS and AITL.In order to identify the best anti-ICOS clone that we should develop for a clinical trial, we evaluated the affinity of the antibody on the receptor, the internalization capacity of the antibody using pHAb Reactive Dyes kit (Promega®), and the ability of the antibody to act as an ADC using a secondary conjugate (Mab-ZAP kit, Advanced Targeting Systems®).Results: ICOS was highly expressed by the cutaneous atypical lymphocytic infiltrates in respectively 61%, 75% and 88% of patients with early-stage MF, TMF and SS, such as in all the involved nodes. Double staining experiments which were performed in both skin and lymph node revealed that ICOS expression appears mainly restricted to neoplastic CD4 + T-cells, with rare ICOS +CD8 + T-cells in the tumor micro-environment. ICOS expression by circulating Sézary cells was strong: 69 ± 7.3% versus 38.8 ± 7.1% of non-tumoral CD4 + cells (p<0.009; CI95%: 8.7-51.6); and 31 ± 3.2% of CD4+ cells in HD (p<0.0001; CI95%: 20.3-46.3). Percentages of ICOS + Tregs were significantly higher in patients with SS than in HD.In CTCL cell lines, we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS-MMAE (IC50 = 8.2ng/mL) and anti-ICOS-PBD (IC50 = 1.2ng/mL) ADCs. In a mouse xenograft model (MyLa), anti-ICOS-MMAE ADCs provided a longer overall survival (OS) than BV (HR=15.2; CI95%: 3.2-71.1; p<0.0006). Finally, in ICOS + PDXs, anti-ICOS-MMAE ADCs significantly improved OS, and reduced the number of tumor cells in the blood, spleen, and bone marrow. No evidence of ADC toxicity was observed in treated mice.Among 8 different anti-ICOS clones, clone 314.8 had the best affinity on MyLa and MJ cell lines. Clones 53.3, 293.1, 92.17, 88.2 and 279.1 also had good affinity to receptor, whereas clones 145.1 and 121.4 had poor affinity. Using the internalization pHAb reactive dyes kit, we found that clones 314.8, 53.3, 92.17, 88.2 internalized significantly better and faster than the other ones. In order to verify if there is a correlation between internalization capacity and ADC effect, clones 53.3, 92.17 and 145.1 were coupled to MMAE. While anti-ICOS-53.3 and anti-ICOS-92.17 ADCs had similar efficacy to anti-ICOS-314.8 ADCs on MyLa, anti-ICOS 145.1 ADCs resulted in significantly lower cell death. Finally, all clones showed good ability to act as ADCs with Mab-ZAP, excluding clones 279.1, 145.1 and 121.4.Discussion: ICOS is a promising therapeutic target because it is expressed both by tumor T-cells and regulatory T-cells. We report for the first time the strong and frequent expression of ICOS in CTCLs, as well as the preclinical efficacy of anti-ICOS ADCs on CTCL cell line and PDXs. These results could be extended to the spectrum of follicular variant peripheral T-cell lymphomas.Conclusion: Collectively, our findings provide the preliminary basis for a therapeutic trial [Display omitted] DisclosuresLopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Functional Depletion of HSP72 by siRNA and Quercetin Enhances Vorinostat-Induced Apoptosis in an HSP72-Overexpressing Cutaneous T-Cell Lymphoma Cell Line, Hut78.

Histone deacetylase inhibitors (HDACis) are one of the therapeutic options for cutaneous T-cell lymphoma (CTCL), but they have limited effects. We previously demonstrated that HSP72 overexpression is associated with chemoresistance to HDACis in lymphoma cells. The purpose of this study was to investigate whether the functional depletion of HSP72 enhances the effect of the HDACi vorinostat. First, we established a stable HSP72-knockdown CTCL cell line and confirmed the influence of HSP72 reduction on the antitumor effects of vorinostat. Next, we studied the effect of quercetin, an inhibitor of HSP72, on the antineoplastic effects of vorinostat. In five CTCL cell lines examined, HSP72 expression was highest in Hut78 cells, and HSP72 knockdown enhanced vorinostat-induced apoptosis in these cells. Low-dose quercetin reduced HSP72 expression, increased HDAC activity, and enhanced vorinostat-induced suppression of Hut78 cell proliferation. A single low dose of quercetin induced G2 arrest and only slightly increased the sub-G1 cell fraction. Quercetin also significantly enhanced vorinostat-induced apoptosis, caspase-3, caspase-8, and caspase-9 activity, and the loss of mitochondrial membrane potential. HSP72 knockdown enhanced vorinostat-induced apoptosis in an HSP72-overexpressing CTCL cell line, and thus, quercetin may be a suitable candidate for combination therapy with vorinostat in clinical settings.

Prec-P-01 - Preclinical investigation of new targeted combination therapies in cutaneous T-cell lymphoma

Prec-P-01 - Preclinical investigation of new targeted combination therapies in cutaneous T-cell lymphoma

Bio-P-09 - Combination of ruxolitinib with resminostat exerts antitumor effects in a chick embryo metastasis model

Bio-P-09 - Combination of ruxolitinib with resminostat exerts antitumor effects in a chick embryo metastasis model

Hist-P-02 - CD74 overexpression in cutaneous T-cell lymphoma and its inhibition as a potential therapeutic approach

Cutaneous T-cell lymphomas form a heterogeneous group of non-Hodgkin lymphomas characterized by only poor prognosis in advanced stage. Despite significant progress made in the identification of novel genes and pathways involved in the pathogenesis of cutaneous lymphoma, the therapeutic value of these findings has still to be proven. CD74 is a type II membrane glycoprotein that plays an important role in MHC class II antigen presentation. It is expressed by a number of immune cells, including B cells and macrophages. CD74 conveys proliferative and provital signals. This can lead to procarcinogenic effects and thus increase the survival of malignant cells. So far, as far as is known, neither the analysis of CD74 expression nor a therapeutic approach via CD74 blockade in cutaneous T-cell lymphomas has been described. In the presented study, CD74 expression in cutaneous T-cell lymphomas (CTCL), including subentities, and its association with clinical features were analyzed. Here, we demonstrate by quantitative RT-PCR, Western blotting, FACS analyzes and immunohistochemistry in CTCL cell lines and primary patient samples that CD74 is highly overexpressed in tumor cells. Moreover, treatment with a specific CD74 inhibitor blocks cell viability. These new data provide a promising rationale for using CD74 inhibition as a therapeutic modality in CTCL.

Prec-O1-03 - Understanding the function of CD30 in cutaneous T-cell lymphoma: implications for therapy and prognosis

<h3>Introduction and objectives:</h3> Targeted therapies for cutaneous T-cell lymphoma (CTCL) are limited and curative approaches are still lacking. Relapses and drug-induced side effects are major challenges in the therapeutic management of CTCL patients, resulting in an urgent need for new, effective therapies. Expression of CD30 receptor is usually restricted to a small population of activated B- and T-cells, but is also found in hematologic malignancies, such as CTCL. In mycosis fungoides (MF) its expression seems to be associated with advanced disease stage and large-cell transformation. However, the functional role of CD30 in CTCL remains unknown. Consequently, this study aims to determine 1) CD30 and CD30 ligand (CD30L) expression in CTCL cell lines and primary CD4-positive cells from CTCL patients, 2) the dynamics of CD30 expression over time and after treatment, 3) CD30 function <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i>, 4) mechanisms involved in CD30 signaling. <h3>Materials and methods:</h3> The CTCL cell lines HH, SeAx and HUT-78 were used for <i>in vitro</i> experiments. For <i>ex vivo</i> analysis, primary CD4-positive T-cells isolated from fresh blood samples of patients with Sézary syndrome were used. Cells were challenged with dimethyl fumarate for 24 hours. CD30 was stimulated via its natural ligand. Methods used for expression and pathway analysis were FACS, western blot, PCR and luciferase assay. For <i>in vivo</i> experiments, CTCL cells were suspended in PBS/Matrigel (1:1, vol/vol) and injected intradermally into the right flank of female NOD/SCID/Il2Rg null (NSG) mice. After development of tumors, CD30L was injected into the lesions. <h3>Results:</h3> We found that CD30 activation can influence viability of CTCL cells <i>in vitro</i>, which is currently under investigation in an <i>in vivo</i> setting. Involved mechanisms include the MEK/ERK and NF-κB pathway. The highest CD30 expression was detected in the HH cell line. The primary CD4-positive cells analyzed so far were negative for CD30 surface expression. However, intracellular CD30 protein was detectable via western blot. CD30 levels were influenced by cell culture conditions in HH cells and significantly reduced upon therapeutic NFκB inhibition in HH, SeAx and HUT-78 cells. <h3>Conclusions:</h3> Our results indicate a therapeutic potential of CD30 receptor stimulation as a new targeted therapy in CD30-positive MF <i>in vitro</i>, which has to be proven <i>in vivo</i>. Furthermore, we found a dynamic nature of CD30 expression in CTCL, which might account for the described effectiveness of the antibody-drug conjugate brentuximab vedotin directed to CD30 also in patients diagnosed as CD30 low-expressing.

Bio-O2-05 - Epigenetic modifiers can alter telomerase expression and clonogenic capacities of Sézary cells

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) for which current available treatments do not provide long-term responses. Two epigenetic-modulating agents belonging to the family of histone deacetylase inhibitors (HDACi), vorinostat and romidepsin are FDA approved for advanced stage CTCL. However, the rationale for using such therapies in SS and their biological impact are not well defined. The majority of cancer cells achieve proliferative immortality by activating or upregulating the normally silent human Telomerase Reverse Transcriptase (<i>hTERT</i>) gene. Sezary cells do not escape to this phenomenon. As the <i>hTERT</i> promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its re-expression. We analyzed <i>hTERT</i>-promoter methylation status in CTCL cells compared to healthy cells. Nine CTCL cell lines including, five SS cell lines were experimentally investigated (HuT78, the unique commercial SS cell line and four newly developed SS cell lines from our laboratory (PMID: 33106625). Gene-specific methylation analyses revealed a common promotor hypermethylation pattern exclusively observed in tumor cells. We explored <i>hTERT</i> promoter methylation status under the pressure of a demethylating agent approved for the treatment of several hematological disorders (5-azacytidine) by comparison with clinically approved HDACi (romidespin and vorinostat). Surprisingly, romidespin, vorinostat or 5-azacytidine could each reduce <i>hTERT</i> expression level without altering the methylation status of <i>hTERT</i> promoter. To go further, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells using the soft agar assay. Our data revealed that, besides <i>hTERT</i> downregulation, epidrugs reduced the proliferative and the tumor formation capacities in Sézary cells <i>in vitro</i>. Our first report on <i>hTERT</i> promotor methylation status in CTCL suggests that <i>hTERT</i> promotor methylation status is a hallmark of neoplastic CTCL cells. Our results also provide evidence for functional consequences of epidrugs treatments on tumor formation capacities in Sézary cells <i>in vitro</i>. Indeed, the present investigation states that Sézary cells' clonogenic capacity correlates well with <i>hTERT</i> expression level under epigenetic drugs pressure. The present findings represent a step forward towards a better understanding of the relationship between targeting epigenetic modifiers in a cell-context–dependent manner and the response generated at the cellular levels.

Bio-P-08 - Mode-of-action of HDAC inhibitor resminostat in CTCL

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma characterized by malignant skin-homing T cells with an increasing bias towards the T helper cell type 2 (Th2) during disease progression. Patients with advance CTCL have a high disease burden and the majority of CTCL patients suffer from severe itching (pruritus). A key challenge in the treatment of advanced CTCL is to maintain and to stabilize initial therapeutic responses after systemic treatment. Addressing this clinical need, the oral class I, IIb, IV HDAC inhibitor resminostat is currently under clinical evaluation for disease control after systemic therapy (RESMAIN, NCT02953301). Here, the mode-of-action of resminostat was explored. Several CTCL cell lines representing mycosis fungoides (MF) and Sézary syndrome (SS) were used in cell biological and molecular assays. HDAC inhibitors are epigenetic drugs that alter the chromatin landscape and the expression of genes. Indicating an interference with epigenetic processes, resminostat induced hyperacetylation on specific histone residues linked with transcriptional regulation. Whole transcriptome analysis upon resminostat treatment uncovered changes in oncogenic signaling pathways and in the expression of genes associated with the pathogenesis of CTCL. The expression of several skin-homing receptors, which mediate the infiltration of malignant T cells into the skin, was reduced by resminostat. Furthermore, resminostat up-regulated a gene expression signature representative of Th1 cell type and down-regulated genes of the Th2 cell type, thus favoring the beneficial Th1 T cell phenotype. In agreement with this observation, resminostat decreased the mRNA level and protein secretion of the Th2 and itch-mediating cytokine IL-31 suggesting that resminostat might improve pruritus. Moreover, resminostat treatment resulted in anti-proliferative and pro-apoptotic effects in both MF and SzS cells. In addition to counteracting intrinsic pathogenic pathways in malignant CTCL cells, we found that resminostat enhanced natural killer cell-mediated lysis, thus promoting the innate immune response towards malignant cells. In contrast to reports for other HDAC inhibitors, resminostat did not reduce the viability or function of NK cells but directly positively affected their cytolytic function. Moreover, resminostat up-regulated the expression NKG2D-ligands on cancer cells and, thereby, facilitates the recognition of cancer cells by NK cells. Interestingly, the combination of resminostat with opsonizing antibodies (e.g. Mogamulizumab, IPH4102) improved the lysis of CTCL cells in NK cell assays, suggesting a potential of resminostat's combinations with opsonizing antibodies. Taken together, these data corroborate that resminostat has the potential to counteract the malignant T cell population in CTCL. In conclusion, our preclinical data support the hypothesis that resminostat treatment will improve or stabilize CTCL and its symptoms.

Drug Repurposing Patent Applications April–June 2021

Drug Repurposing Patent Applications April–June 2021