Cutaneous T-cell Lymphoma Cell Lines 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.

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.

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.

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.

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

Abstract 345: Cutaneous T-cell lymphoma: ectopic expression of gametocyte specific factor 1 (GTSF1) stimulates the Th phenotype

Abstract Diagnosis of Cutaneous T-Cell Lymphomas (CTCL) can take up to 6 years, increasing the risk of disease progression in these patients. Transcriptomic analyses have helped identify several diagnosis biomarkers, such as TOX, KIRD3DL2, GTSF1 and others. Although not officially a Cancer-Testis (CT) antigen, ectopic expression of Gametocyte Specific Factor 1 (GTSF1), presents the same advantages than CT antigens. Its germline-cell restricted expression potentially makes this gene easily identifiable by the immune system and a therapeutic target with less side effects. The role of GTSF1 has been elucidated in mice, where it controls retrotransposon expression during germ-cell development. Here, we investigate the molecular function of GTSF1 in CTCL. Using shRNA-mediated GTSF1 knockdown in CTCL cell lines, our current work demonstrates an unexpected role of GTSF1 regulating the Th phenotype of CTCL cells. With multiple approaches (qRT-PCR, Western blotting and a dual-luciferase based retrotransposition assay) we concluded that GTSF1 knockdown does not lead to retrotransposon reactivation. Transcriptome analysis with RNA-Seq, followed by differential gene expression analysis with DESeq2 and pathway enrichment analysis with GO, KEGG, Reactome and WikiPathways, suggested an involvement of GTSF1 in the Th phenotype of CTCL cells. Differential gene expression analysis revealed 12,496 differentially expressed genes (p adjusted < 0.05). Pathway enrichment analysis demonstrated an enrichment of several pathways including response to stimulus, immune response, response to cytokine and inflammatory response. More specifically, GTSF1 knockdown leads to an upregulation of the key cytokines associated with a Th1 phenotype, IFNG and TNF. Disease progression of CTCL is associated with a decrease in Th1 cytokine production and an enhanced Th2 cytokine production by malignant T cells. These findings shed light on the molecular function of GTSF1 as a regulator of the Th phenotype in CTCL and paves the way for the development of a valuable treatment target for CTCL patients. Citation Format: Amelia Martínez Villarreal, Ivan Litvinov. Cutaneous T-cell lymphoma: ectopic expression of gametocyte specific factor 1 (GTSF1) stimulates the Th phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 345.

Blockade of the Immune Checkpoint CD47 by TTI-621 Potentiates the Response to Anti−PD-L1 in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is an incurable and cosmetically disfiguring disease associated with microenvironmental signals. We investigated the effects of CD47 and PD-L1 immune checkpoint blockades, as a strategy for targeting both innate and adaptive immunity. CIBERSORT analysis identified the immune-cell composition in the CTCL tumor microenvironment and the immune checkpoint expression profile for each immune-cell gene cluster from CTCL lesions. We investigated the relationship between MYC and CD47 and PD-L1 expression and found that MYC short hairpin RNA knockdown and MYC functional suppression by TTI-621 (SIRPαFc) and anti-PD-L1 (durvalumab) in CTCL cell lines reduced the expression of CD47 and PDL1 mRNA and protein as measured by qPCR and flow cytometry, respectively. Invitro, blockade of the CD47-SIRPα interaction with TTI-621 increased the phagocytic activity of macrophages against CTCL cells and enhanced CD8+ T-cell-mediated killing in a mixed leucocyte reaction. Moreover, TTI-621 synergized with anti-PD-L1 in macrophages reprogram to M1-like phenotypes and inhibited CTCL cell growth. These effects were mediated by cell death-related pathways, including apoptosis, autophagy, and necroptosis. Collectively, our findings show that CD47 and PD-L1 are critical regulators of immune surveillance in CTCL and that dual targeting of CD47 and PD-L1 will provide insight into tumor immunotherapy for CTCL.

TMEM244 Is a Long Non-Coding RNA Necessary for CTCL Cell Growth.

Transmembrane protein 244 (TMEM244) was annotated to be a member of the TMEM family, which are is a component of cell membranes and is involved in many cellular processes. To date, the expression of the TMEM244 protein has not been experimentally confirmed, and its function has not been clarified. Recently, the expression of the TMEM244 gene was acknowledged to be a diagnostic marker for Sézary syndrome, a rare cutaneous T-cell lymphoma (CTCL). In this study, we aimed to determine the role of the TMEM244 gene in CTCL cells. Two CTCL cell lines were transfected with shRNAs targeting the TMEM244 transcript. The phenotypic effect of TMEM244 knockdown was validated using green fluorescent protein (GFP) growth competition assays and AnnexinV/7AAD staining. Western blot analysis was performed to identify the TMEM244 protein. Our results indicate that TMEM244 is not a protein-coding gene but a long non-coding RNA (lncRNA) that is necessary for the growth of CTCL cells.

Proteasome Inhibitors Interact Synergistically with BCL2, Histone Deacetylase, BET, and Jak Inhibitors against Cutaneous T-Cell Lymphoma Cells

Cutaneous T-cell lymphoma (CTCL) is often refractory to treatment at advanced stages with blood involvement ( Dummer et al., 2021 Dummer R. Vermeer M.H. Scarisbrick J.J. Kim Y.H. Stonesifer C. Tensen C.P. et al. Cutaneous T cell lymphoma. Nature Reviews Disease Primers. 2021; 7: 61 Crossref PubMed Scopus (22) Google Scholar ). We have previously revealed the synergistic cytotoxic effects of inhibiting BCL2, HDAC, BET, and/or JAK in patient-derived CTCL cells and CTCL cell lines, suggesting potential advantages of a spectrum of combination treatment strategies for this disease ( Cyrenne et al., 2017 Cyrenne B.M. Lewis J.M. Weed J.G. Carlson K.R. Mirza F.N. Foss F.M. et al. Synergy of BCL2 and histone deacetylase inhibition against leukemic cells from cutaneous T-cell lymphoma patients. Blood. 2017; 130: 2073-2083 Crossref PubMed Scopus (36) Google Scholar , Kim et al., 2018 Kim S.R. Lewis J.M. Cyrenne B.M. Monico P.F. Mirza F.N. Carlson K.R. et al. BET inhibition in advanced cutaneous T cell lymphoma is synergistically potentiated by BCL2 inhibition or HDAC inhibition. Oncotarget. 2018; 9: 29193-29207 Crossref PubMed Scopus (35) Google Scholar , Yumeen et al., 2020 Yumeen S. Mirza F.N. Lewis J.M. King A.L.O. Kim S.R. Carlson K.R. et al. JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL. Blood Adv. 2020; 4: 2213-2226 Crossref PubMed Scopus (19) Google Scholar ). Proteasome inhibitors target multiple pathways including protein degradation to cause cytotoxicity and have been utilized in other hematologic malignancies. They have demonstrated efficacy as a single agent in a small phase II trial of patients with relapsed or refractory CTCL ( Zinzani et al., 2007 Zinzani P.L. Musuraca G. Tani M. Stefoni V. Marchi E. Fina M. et al. Phase II Trial of Proteasome Inhibitor Bortezomib in Patients With Relapsed or Refractory Cutaneous T-Cell Lymphoma. Journal of Clinical Oncology. 2007; 25: 4293-4297 Crossref PubMed Scopus (236) Google Scholar ), as well as in limited combinations during preclinical studies ( Yumeen et al., 2020 Yumeen S. Mirza F.N. Lewis J.M. King A.L.O. Kim S.R. Carlson K.R. et al. JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL. Blood Adv. 2020; 4: 2213-2226 Crossref PubMed Scopus (19) Google Scholar ) and early clinical trials ( Holkova et al., 2017 Holkova B. Yazbeck V. Kmieciak M. Bose P. Ma S. Kimball A. et al. A phase 1 study of bortezomib and romidepsin in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma, indolent B-cell lymphoma, peripheral T-cell lymphoma, or cutaneous T-cell lymphoma. Leuk Lymphoma. 2017; 58: 1349-1357 Crossref PubMed Scopus (15) Google Scholar ). Herein, we share our expanded preclinical assessment of the synergistic activity of proteasome inhibitors when used with the BCL2 inhibitor venetoclax, HDAC inhibitor vorinostat, BET inhibitor mivebresib, or JAK inhibitor fedratinib. We reveal that the cytotoxic effects from combination treatment were greater than additive when assessed in patient-derived CTCL cells. Furthermore, we demonstrate an increase in apoptosis pathway activation with combination treatment and explore the gene expression changes underlying these synergistic effects.

462 Gametocyte Specific Factor 1 contributes to the Th phenotype in Cutaneous T-Cell Lymphomas

Ectopic expression of Gametocyte Specific Factor 1 (GTSF1) in Cutaneous T-Cell Lymphoma (CTCL) patients has been reported by several groups. Moreover, it has been associated with a worse prognosis and late-stage CTCL. GTSF1 is a gamete gene that participates in the piRNA pathway to silence retrotransposons on the genome. Despite the established association with a worse prognosis the molecular function of GTSF1 expression remains unknown. Therefore, we established shRNA-mediated GTSF1 knockdown CTCL cell lines. Using qRT-PCR, Western blot and a retrotransposition assay, we demonstrated that GTSF1 knockdown does not lead to mobilization of the retrotransposon LINE-1. These data suggest GTSF1 has a novel role in CTCL. Interestingly, after GTSF1 knockdown in the Mac2A cell line, we observed striking growth inhibition, suggesting this gene is a key player in CTCL carcinogenesis. To further explore this role, we performed RNA-Seq, comparing GTSF1 knockdown to control cells. Differential gene expression analysis using DESeq2, revealed 12496 differentially expressed genes (p adjusted <0.05). Pathway enrichment analysis was performed via GO, KEGG, Reactome and WikiPathways. We observed an enrichment of several pathways including response to stimulus, immune response, response to cytokine and inflammatory response. More specifically, transcriptome analysis showed an increased expression of cytokine induced genes, such as XAF1, IFI27 and IFI44L, and genes associated with a Th1 phenotype, IFNG and TNF. Research shows that CTCL progression leads to defects in Th1 cytokine production (IFN-γ and TNF-α) by antitumor immune cells and an enhanced Th2 cytokine production (IL-4 and IL-5) by malignant T cells. The chronic production of Th2 cytokines by malignant cells leads to immune deficiency in patients, allowing malignant cells to proliferate and the disease to progress. Taken together, these data suggest GTSF1 as a novel regulator of the Th phenotype on CTCL, thereby placing it as a potential treatment target for regulating disease progression.

Blockade of the Immune Checkpoint CD47 By TTI-621 Potentiates the Response to Anti-PD-L1 in Cutaneous T Cell Lymphoma

Background: Blockade of PD1/PD-L1 (NCT03011814) and CD47/SIRPα (NCT02890368) have shown promising and durable therapeutic outcomes in cutaneous T-cell lymphoma (CTCL), but resistance and/or relapses are seen in a subset of patients. To improve outcomes of immunotherapy regimens for CTCL patients, we investigated the effects of dual CD47 and PD-L1 blockade as a strategy for targeting both the innate and adaptive immune system. Methods: We performed RNA sequencing, gene expression deconvolution (CIBERSORT) and flow cytometry analysis to assess the immune cell composition, and CD47, SIPRα and PD-L1 expression in 45 CTCL patient samples and CTCL cell lines. CTCL specimens at baseline and during treatment with anti-CD47/SIRPα (TTI621) and anti-PD-L1 (durvalumab) were used to analyze immune cell gene expression. We performed functional assays utilizing our macrophage and CTCL cell line culture systems to assess macrophage polarization, phagocytic activity of macrophages against CTCL cells, T cell-mediated cytotoxicity using a chromium release assay, and CTCL cell line proliferation before and after CD47/SIRPα (TTI-621) and anti-PD-L1 antibody (durvalumab) treatment. Results: Our data showed that CTCL cells and CTCL cell lines overexpress CD47 and PD-L1 compared with healthy control cells, and overexpression of CD47 and PD-L1 was associated with high MYC expression in CTCL cell lines. CIBERSORT analysis of CTCL samples compared to healthy control indicated that the fraction of M2 tumor-associated macrophages (TAMs) was increased, while M0/1 were significantly lower. Our NanoString data showed decreased M2 genes, immature DC-related genes, and the NK cell inhibitory receptor related genes, but up-regulation of M1 genes and the NK cell stimulatory receptors associated genes for CTCL patients after treatment with TTI-621 compared to baseline. In vitro, treatment with TTI-621 increased the phagocytic activity of macrophages against CTCL cells and enhanced CD8+ T-cell-mediated killing. Moreover, TTI-621 synergized with anti-PD-L1 (durvalumab) to reprogram M2-like tumor-associated macrophages to M1-like phenotypes by inhibiting MYC expression. Simultaneous exposure to TTI-621 and anti-PD-L1 inhibited growth of CTCL cell lines more potently than either antibody alone. RNA sequencing analysis indicated these effects were mediated by cell‒death-related pathways, such as apoptosis, autophagy, and necroptosis. Conclusions: Collectively, our findings demonstrate that CD47 and PD-L1 are critical regulators of the immune microenvironment in CTCL and that dual targeting of CD47 and PD-L1 may potentiate anti-tumor responses in CTCL.

STAT3 Is a Potential Therapeutic Target for Histone Deacetylase Inhibitor-Resistant Cutaneous T-Cell Lymphoma

Background: Histone deacetylase inhibitors (HDACis), such as vorinostat and romidepsin, are effective treatment agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, and adult T-cell lymphoma/leukemia. Despite the promising anti-lymphoma activity of HDACis, drug resistance remains a significant clinical problem. Therefore, novel therapeutic strategies are required to overcome HDACi resistance. In this study, we generated HDACi-resistant CTCL cell lines and investigated the mechanisms of HDACi resistance. Aims: This study aimed to identify the mechanism of resistance to HDACis in CTCLs and to explore new therapeutic targets. Methods: The CTCL cell lines MyLa, MJ, and Hut78 were used in this study. Vorinostat-resistant cell lines, namely MyLa resistant, HUT78 resistant, and MJ resistant, were prepared by repeatedly exposing cells to increasing concentrations of vorinostat. To maintain drug resistance, the cells were continuously cultured in a medium containing 2.5 μM vorinostat. Following this, we conducted a comprehensive gene expression analysis using a microarray platform to investigate the deregulated genes in the resistant cell lines. Results: CTCL cell lines resistant to vorinostat were also resistant to romidepsin, suggesting that vorinostat-resistant cell lines showed cross-resistance to other HDACis. Next, we selected genes whose expression was upregulated more than 1.5 times in the three resistant cell lines and performed pathway analysis. Pathway analysis revealed that the upregulated genes were significantly enriched in the cytokine-mediated signaling pathways. Among the upregulated genes, we focused on interleukin-6 signal transducer (IL6ST)/glycoprotein (gp) 130, which forms a heterogeneous complex with IL6 and IL6 receptors, and activates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Furthermore, flow cytometric analysis revealed that surface gp130 expression was significantly upregulated in the resistant cell lines. Moreover, the MyLa-resistant and MJ-resistant cell lines showed increased sensitivity to a gp130 inhibitor (SC144). Gp130 activation could activate the JAK/STAT pathway; therefore, we examined the expression level of the activated forms of STAT3 and p-STAT3 and found that p-STAT3 was significantly upregulated in the resistant cell lines. To investigate whether p-STAT3 upregulation is involved in the development of vorinostat resistance, we performed STAT3 knockdown using siRNA. We confirmed that STAT3 knockdown significantly decreased the survival of the resistant cell lines. Moreover, resistant cell lines showed increased sensitivity to the STAT3-selective inhibitor C188-9. However, compared to the controls, the resistant cell lines showed no difference in their sensitivity towards the JAK1/2 inhibitor ruxolitinib. This suggests that the STAT3 activation occurs not only through the JAK/STAT pathway but also through a pathway that is not mediated by JAK. We also examined the expression level of p-STAT5; however, it was downregulated in the resistant cell lines, which was in contrast to that of p-STAT3. These findings suggest that STAT3 activation, which is mediated by HDAC inhibition, could contribute to the resistance of CTCL to HDACis. Finally, we examined the gp130 and p-STAT3 protein expression in CTCL skin samples, which were obtained from the same patients before and after vorinostat treatment. We found that the gp130 and p-STAT3 expression was significantly increased in the specimens during relapse after vorinostat treatment. Conclusion: Activation of STAT3 in HDACi-resistant CTCL cells was enhanced by gp130, which contributed to their resistance. These results suggest that STAT3 could be the new therapeutic target in HDACi-resistant CTCL and that gp130/STAT3 inhibition could be an important therapeutic strategy. To further elucidate the detailed mechanism of HDACi resistance, analysis of the pathways activating STAT3 without JAK and the target proteins of STAT3 is warranted.

Receptor Targeted Delivery of the p38γ Inhibitor PIK-75 By Organic-Core Templated Lipid Nanoparticles in Cutaneous T Cell Lymphoma

Introduction: Patients with relapse/refractory cutaneous T cell lymphomas (CTCL) have limited therapeutic options. p38γ, part of the mitogen-activated protein kinases (MAPK) pathway, has been identified as a critical driver of CTCL. PIK-75, a known p110α inhibitor, was found to inhibit p38γ and induced cell death in CTCL cells in vitro and in vivo tumor xenograft models. However, PIK-75 is very hydrophobic and difficult to systemically deliver and achieve clinical efficacy. Therefore, designing a carrier to formulate and enable targeted cellular delivery of PIK-75 is a logical next step to facilitate translation. We, and others, have shown that scavenger receptor type B1 (SR-B1) is often overexpressed in hematologic malignancies and can be targeted by synthetic lipid nanoparticles for efficient drug delivery. Our group pioneered the use of inorganic and organic core (oc) "templates” to synthesize lipid nanoparticles (LNP) that target SR-B1. The ocLNPs are an ideal construct for the delivery of hydrophobic drugs to malignant cells. Therefore, we hypothesized that ocLNPs could be adapted to formulate PIK-75 and deliver this drug via SR-B1 to CTCL cells to induce cell death. Methods: A suite of organic core molecules were synthesized and tested for their ability to support the synthesis of PIK-75 loaded ocLNPs. The PIK-75 ocLNPs were synthesized by combining an organic core with PIK-75, phospholipids, and apolipoprotein A-I. The drug loaded ocLNPs were purified by dialysis and filtration. PIK-75 ocLNPs were characterized for size (size exclusion chromatography, dynamic light scattering and electron microscopy), surface charge (zeta potential) and drug loading. SR-B1 expression in primary CTCL samples was assessed by RNA sequencing. SR-B1 positive CTCL cell lines (HH and HuT78), and SR-B1 positive normal cells, HepG2 (hepatocytes) and THP-1 (macrophages), as well as SR-B1 negative PIK-75-sensitive U266B1 (myeloma) cells were used for these studies. Expression of SR-B1 in all cell lines was confirmed by western blot and flow cytometry. Cell viability was measured using the MTS assay. An SR-B1 blocking antibody was used to demonstrate the necessity of SR-B1 for PIK-75 ocLNP targeting and drug delivery. Results: Nineteen out of 49 primary patient lesional CTCL skin samples (38.8%) demonstrated SR-B1 over-expression, defined as an expression level greater than two standard deviations above the mean expression measured in normal, CD3+ peripheral T cells using RNAseq data. Fourteen of the 19 samples (74%) were from patients with advanced disease (stage III and IV). PIK-75 was successfully incorporated into ocLNPs. Data showed that ~16 PIK-75 molecules were loaded per ocLNP. PIK-75 ocLNPs induced significant cell death in SR-B1 positive CTCL cell lines HH (IC50 = 3.95 nM) and HuT 78 (IC50 = 6.33 nM), while treatment of an PIK-75 sensitive, SR-B1 negative myeloma cell line (U266B1) with PIK-75 ocLNPs resulted in minimal cell death. Treatment of HH cells with PIK-75 ocLNPs for a 2 hour "pulse” followed by 72 hours of additional culture was completed to test active SR-B1 targeting. Data reveal significant cell death, which was significantly reduced by the addition of an SR-B1 blocking antibody (10 nM PIK-75 ocLNPs, viability 31.6% ± 4.3% w/o blocking antibody vs viability 83.6% ± 8.4% w/ blocking antibody). Treatment of hepatocytes and macrophages, both known to express SR-B1, with PIK-75 ocLNPs revealed no cell death even after 72 hours of culture. Conclusions: ocLNPs can be successfully loaded with drug cargo, in this case hydrophobic PIK-75, enabling active delivery via SR-B1 in CTCL. PIK-75 ocLNPs potently induced cell death in CTCL cells while having minimal negative effects on hepatocytes or macrophages. These results provide proof-of-principle that ocLNPs can be successfully leveraged to deliver hydrophobic small molecule drugs by actively targeting SR-B1.

DOT1L inhibition does not modify the sensitivity of cutaneous T cell lymphoma to pan-HDAC inhibitors in vitro.

Cutaneous T-cell lymphomas (CTCLs) are a subset of T-cell malignancies presenting in the skin. The treatment options for CTCL, in particular in advanced stages, are limited. One of the emerging therapies for CTCL is treatment with histone deacetylase (HDAC) inhibitors. We recently discovered an evolutionarily conserved crosstalk between HDAC1, one of the targets of HDAC inhibitors, and the histone methyltransferase DOT1L. HDAC1 negatively regulates DOT1L activity in yeast, mouse thymocytes, and mouse thymic lymphoma. Here we studied the functional relationship between HDAC inhibitors and DOT1L in two human CTCL cell lines, specifically addressing the question whether the crosstalk between DOT1L and HDAC1 observed in mouse T cells plays a role in the therapeutic effect of clinically relevant broad-acting HDAC inhibitors in the treatment of human CTCL. We confirmed that human CTCL cell lines were sensitive to treatment with pan-HDAC inhibitors. In contrast, the cell lines were not sensitive to DOT1L inhibitors. Combining both types of inhibitors did neither enhance nor suppress the inhibitory effect of HDAC inhibitors on CTCL cells. Thus our in vitro studies suggest that the effect of commonly used pan-HDAC inhibitors in CTCL cells relies on downstream effects other than DOT1L misregulation.

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.