T-cell Lymphoma Cell Research Articles

DUSP22 Rearrangements Identify a Molecularly Distinct Type of Anaplastic Large Cell Lymphoma Characterized By DNA Hypomethylation and Lack of Activated STAT3

Background: Anaplastic large cell lymphomas (ALCLs) comprise a group of T-cell non-Hodgkin lymphomas that share common pathologic features such as CD30 expression, but demonstrate marked heterogeneity in clinical presentation, genetic features, and outcomes. Current classification of ALCLs is based on site of presentation (systemic or localized) and ALK status (positive or negative). Rearrangement of the DUSP22 locus is seen in ~30% of ALK-negative ALCLs and is considered a prognostically favorable biomarker, but the molecular features of these cases are incompletely understood. Specifically, their relationship to convergent mechanisms that activate STAT3 in both ALK-positive and ALK-negative ALCL has not been studied.Methods: Gene expression profiling (GEP) data from 31 frozen ALCL samples were analyzed using hierarchical clustering and gene-set enrichment analysis (GSEA). Immunohistochemistry for phosphorylated (p) STAT3 (Tyr705) was performed on whole-tissue paraffin sections and scored in a blinded fashion based on percentage of tumor cell nuclei staining. Reduced representation bisulfite sequencing (RRBS) was performed on DNA extracted from the 31 frozen ALCLs and differentially methylated regions (DMRs) were identified among genetic subtypes using methylKit software. RRBS and RNA sequencing also was performed on 6 T-cell lymphoma cell lines treated with the demethylating agent decitabine or vehicle alone, and gene expression and methylation profiles were evaluated.Results: Unsupervised clustering of GEP data identified two main groups of ALCLs, Cluster 1 (n=10) and Cluster 2 (n=21). All DUSP22- rearranged ALCLs (n=7) were in Cluster 1 whereas all ALK-positive ALCLs (n=8) were in Cluster 2. Both clusters contained ALK-negative ALCLs lacking DUSP22 rearrangements. GSEA identified significant depletion of IL6-JAK-STAT3 signaling pathway genes in Cluster 1 (NES=-2.05; p<0.001). Immunohistochemistry in 100 ALCLs confirmed low or absent pSTAT3 in DUSP22- rearranged cases (n=23; 9.2 ± 21.5 % versus 55.7 ± 35.2 % for ALCLs without DUSP22 rearrangements; p<0.0001). However, absent pSTAT3 alone could not explain the favorable prognosis in DUSP22- rearranged ALCLs, and the 3 ALK-negative ALCLs in Cluster 1 that lacked DUSP22- rearrangements showed aggressive clinical behavior. We previously found that DUSP22- rearranged ALCLs showed upregulation of cancer-testis antigen genes. Because these genes are regulated largely by methylation, we performed RRBS and found marked hypomethylation of DUSP22- rearranged ALCLs across all categories of gene-associated methylation sites (p≤0.0001). Furthermore, this hypomethylation could be recapitulated in vitro using decitabine, which led to upregulation of a DUSP22- specific gene expression signature in T-cell lymphoma cell lines.Conclusions: DUSP22- rearranged ALCLs are a molecularly distinct type of ALCL. These cases belong to a group of ALCLs that show down-regulation of STAT3-associated genes and lack activated STAT3 protein. Furthermore, DUSP22- rearranged ALCLs show global hypomethylation that contributes to their unique GEP signature, including up-regulation of cancer-testis antigens. These data suggest DUSP22- rearranged ALCLs may be less sensitive to therapies targeting JAK-STAT3 that have been proposed in ALCL but might benefit from immunotherapy approaches, which have efficacy in other cancers demonstrating hypomethylation and overexpression of cancer-testis antigens. DisclosuresAnsell:Celldex: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Merck: Research Funding. Cerhan:Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering .

Development and Characterization of the Novel Orally Bioavailable EZH1/2 Dual Inhibitor DS-3201

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27) and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 function as catalytic subunits of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Many reports suggest that inhibiting PRC2 activity through EZH2 knockdown gives rise to anti-cancer effect against hematological and solid cancers.To date, some EZH2 selective inhibitors have demonstrated anti-cancer potential in preclinical and clinical studies. In spite of this promise, there is concern that the efficacy anticipated with EZH2 selective inhibitors may be compromised due to compensation by EZH1 activity.Herein, we describe the novel, orally bioavailable EZH1/2 dual inhibitor DS-3201. This compound strongly and selectively inhibited the methyltransferase activity of both EZH1 and EZH2 with an IC50 value of 10 nM and 6.0 nM, respectively in cell-free enzymatic assays. This compound also suppressed trimethylation of H3K27 in cells (IC50: 0.55 nM) more potently than EZH2 selective inhibitors.In order to identify the anti-cancer spectrum of DS-3201, we tested its in vitro growth inhibition activity against a panel of cancer cell lines including a variety of hematological cancers. Among the panel, hematological cell lines showed particular sensitivity to DS-3201, with GI50 values less than 100 nM, including 1) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines harboring fusion genes, such as MLL-AF9, MLL-AF4, or AML1-ETO, 2) diffuse large B-cell lymphoma (DLBCL) cell lines having EZH2 gain-of-function (GOF) mutations, 3) most of the peripheral T-cell lymphoma and multiple myeloma cell lines tested. Our data thus shows this compound has broader anti-cancer activity than EZH2 selective inhibitors, especially in hematology cancers. We also evaluated the in vivo anti-tumor efficacy of DS-3201 against KARPAS-422, a DLBCL tumor xenograft with an EZH2 GOF mutation. A 100 mg/kg once daily oral administration of DS-3201 caused nearly complete tumor regression without any body weight loss and 25 mg/kg administration also showed clear tumor growth delay.Based on these results, we initiated clinical studies of DS-3201 for patients with lymphoma (Clinical trial information: NCT02732275) and for those with AML and ALL (NCT03110354). DisclosuresHonma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Kanno:Asubio Pharma Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Employment. Hirasawa:Daiichi Sankyo Co., Ltd.: Employment. Nosaka:Daiichi Sankyo Co., Ltd.: Employment. Shiroishi:Daiichi Sankyo Co., Ltd.: Employment. Takizawa:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Yasumatsu:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Horiuchi:Daiichi Sankyo Co., Ltd.: Employment. Nakao:Daiichi Sankyo Co., Ltd.: Employment. Suzuki:Daiichi Sankyo Co., Ltd.: Employment. Yamasaki:Daiichi Sankyo Co., Ltd.: Employment. Nakajima:Daiichi Sankyo Co., Ltd.: Employment. Hayakawa:Daiichi Sankyo Co., Ltd.: Employment. Yamazaki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo: Employment. Fujiwara:Daiichi Sankyo Co., Ltd.: Employment.

In Vitro andpre-Clinical In Vivo evidence Support MDMX/MDM2 As Common Vulnerabilities across TP53 -Wild-Type T-Cell Lymphomas That Are Targetable with the α-Helical p53 Stapled Peptide Alrn-6924

T-cell lymphomas (TCL) are a heterogeneous group of non-Hodgkin lymphomas with poor outcomes. Biological heterogeneity across TCLs has hampered the development of targeted therapies. Thus, we sought to identify common vulnerabilities that could be targeted across multiple TCL subtypes. We performed transcriptome and exome sequencing of 21 TCL cell lines and 8 TCL patient-derived xenografts (PDX), and a genome-wide CRISPR-Cas9 pooled screening in 7 TCL lines. Sequencing identified multiple genomic alterations of clinical significance and disease-specific recurrence, like activating JAK1 and STAT3 mutations in cell lines and a PDX-model of anaplastic large cell lymphoma (ALCL) or mutations of TET2, DNMT3A and RHOA in PDX-models of angioimmunoblastic TCL (AITL). Unsupervised hierarchical clustering of transcriptomes identified disease-specific gene-expression profiles across cell lines and PDX models. Ranking the genetic vulnerabilities of the CRISPR-Cas9 screen by z-score and recurrence across multiple cell lines, we identified both previously defined (e.g. IRF4) and novel vulnerabilities (e.g. PTPN2) within multiple TCL subtypes. The most consistent dependence in TP53- wild-type (WT) lines was the E3 ubiquitin ligase MDM2, which polyubiquitinates and targets p53 for degradation. Multiple TCL lines harbored copy number gains or amplifications of MDM2 and/or MDMX that correlated with high transcript levels. To address the therapeutic potential for targeting MDMX/MDM2 in TCL we tested ALRN-6924, a p53-derived stapled peptide that inhibits both MDMX and MDM2, and that is currently in multicenter phase I/Ib and phase II trials. ALRN-6924 was recently shown to induce objective responses in a variety of cancers as a single agent in phase 1 dose-escalation (Meric-Bernstam, F. et al, ASCO 2017). Our results showed that ALRN-6924 potently induced G1 cell cycle arrest and apoptotic cell death in WT TP53 TCL lines (n=8; IC50 100 nm-1 µM) while inducing expression of p21 and Annexin V. ALRN-6924 was inactive in TP53 -mutant lines (n=11; IC50 > 5 µM). Next, we performed a phase II-like trial in eight WT TP53 PDXs: hepatosplenic (HS-TCL), cutaneous (CTCL), NK/T, AITL (n=2), T-cell prolymphocytic leukemia (TPLL) and Alk+ ALCL (n=2). At the time of extensive lymphoma engraftment (e.g. >20% splenic involvement), mice were randomized to treatment with vehicle, ALRN-6924 or the 2nd line standard-of-care agent Romidepsin (n=3/arm/model) on days 1, 4 and 7, and sacrificed on day 8. ALRN-6924 had a favorable safety profile and significantly reduced tumor burden compared to vehicle in all 8 models, with superior efficacy to Romidepsin in the majority of models (Figure). Residual tumor cells collected from ALRN-6924-treated mice consistently had increased AAD/Annexin V staining and p21 induction, consistent with on-target p53 activation and apoptosis.In summary, we identified MDM2/MDMX as a targetable vulnerability that is effectively targeted by the stapled peptide ALRN-6924 in TP53 WT TCLs with promising activity across multiple subtypes, supporting further clinical development of ALRN-6924 in TCL for patients requiring 2nd or frontline treatment. A Phase IIa clinical trial of ALRN-6924 in PTCL patients is currently enrolling (NCT02264613). [Display omitted] DisclosuresHorwitz:Millenium/Takeda: Consultancy, Research Funding; HUYA: Consultancy; Forty-Seven: Consultancy, Research Funding; BMS: Consultancy; Seattle Genetics: Consultancy, Research Funding; Kyowa-Hakka-Kirin: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Mundipharma: Consultancy; ADCT Therapeutics: Research Funding; Aileron Therapeutics: Research Funding; Infinity/Verastem: Consultancy, Research Funding. Santiago:Aileron: Employment. Ren:Aileron: Employment. Guerlavais:Aileron: Employment. Annis:Aileron: Employment. Aivado:Aileron: Employment.

Gemcitabine/Clofarabine/Busulfan: A New Myeloablative Reduced-Toxicity Conditioning Regimen with High Activity for Allogeneic Stem-Cell Transplant (alloSCT) in Aggressive Lymphomas

INTRODUCTION:Stem-cell transplantation (SCT) is standard treatment of relapsed lymphomas. Patients in refractory relapse or relapsed after autoSCT are often considered for an alloSCT, aiming at a graft-vs.-lymphoma (GVL) effect. While reduced intensity conditioning regimens have substantially contributed safety to alloSCT, aggressive lymphomas often progress rapidly afterwards, partly due to tumor cell growth outpacing GVL. Outcomes are particularly poor for patients with active disease at the time of alloSCT. Thus, development of more efficacious conditioning regimens for aggressive lymphomas, avoiding mucotoxic drugs (such as melphalan), is a major need. We previously showed that the combination of 2 nucleoside analogues [gemcitabine (GEM) + clofarabine (CLO)] followed by busulfan (BU) is highly synergistic against B- and T-cell lymphoma cell lines, through inhibition of DNA damage repair. Those observations led us to clinically study a novel high-dose regimen of GEM/CLO/BU with alloSCT for aggressive lymphomas.METHODS:Eligibility included age 12-65; refractory DLBCL, T-cell and Hodgkin lymphomas; acceptable end-organ function, and an 8/8 HLA-matched (A, B, C and DRB1) sibling (MSD) or unrelated donor (MUD). GEM was escalated from dose level 1 to 3; it was given on days -6 (d-6) and d-4 as a loading dose of 75 mg/m2 followed by infusion at 10 mg/m2/min over 40-90 min (total daily dose 475, 675 or 975 mg/m2), to be followed by CLO (40 mg/m2/day x 4, d-6 to d-3) and BU (pharmacokinetically targeted to an average daily AUC of 4,000 μMol•min x 4, d-6 to d-3). AlloSCT was on d0. Patients receiving a MUD transplant received rabbit ATG (0.5, 1.5 and 2 mg/Kg on d-3, d-2, and d-1, respectively). Patients with CD20+ tumors received rituximab (375 mg/m2) on d-14, d-7, d+1 and d+8. The GVHD prophylaxis included tacrolimus (tapered after d+100) and mycophenolate mofetil (from d0 to d+60). Dose limiting toxicities (DLT) were defined as any G4-5 nonhematological organ toxicity, or as G3 skin or G3 mucositis lasting >3 days at peak severity. Dose escalation of gemcitabine followed a Bayesian design targeting a maximal DLT probability of 25%. Non-relapse mortality (NRM), relapse incidence, G II-IV or III-IV acute GVHD or chronic GVHD were calculated with the cumulative incidence method with consideration of competing risks.RESULTS:Between 11/12 and 06/17, 48 patients were enrolled: 25 DLBCL (5 double hit), 13 T-NHL and 10 HL (Table); 27 MSD, 20 MUD.All patients received PBPC. The MTD of gemcitabine was level 3. DLT was observed at the following frequencies: level 1: 12.5% (1/8), level 2: 9% (2/9), and level 3: 25% (4/16). Regimen-related toxicities were manageable, including mucositis (47% G2, 42% G3), self-limited transaminitis (21% G2, 49% G3), hyperbilirubinemia (no cases of VOD) (30% G2, 19% G3), and dermatitis (13% G2). There were no cardiac, pulmonary, renal or CNS toxicities. Neutrophils dropped to <0.1 in all patients on median d0 (d-3 to +2), and engrafted on median d+10 (d+9 to +20). Platelets engrafted on median d+12 (d+8 to +38).All patients engrafted; full donor chimerism was established early: a median of 100% (50-100%) on d+28, and 100% (98%-100%) on d+100. The rates of G II-IV and III-IV acute GVHD were 29.8% (35% MSD, 25% MUD) and 10.6% (19% MSD, 0% MUD), respectively. The rates of moderate-severe / severe chronic GVHD were 8.5% / 6.4%. 1/3 HL pts with prior exposure to anti-PD1 developed steroid-responsive hepatic chronic GVHD.The γ-H2AX levels in PBMNC (N=19) increased throughout treatment, to a median of 8.5 fold from baseline (range, 0.4-46.3) (P=0.0001) and PARP-1 decreased to 0.45 fold (0.2-6.3) (P=0.4), indicative of activation of DNA damage response and apoptosis, respectively. The overall response rates on d+30 (in 34 patients with measurable disease) are 77% (DLBCL), 100% (T-NHL), and 75% (HL). Their respective CR rates are 69%, 100% and 75%. The d+100 / 1-year NRM rates were 6.25% / 14.5%. NRM causes were infection (6) and GVHD (1). At median follow-up of 18 months (1-53) the EFS / OS rates of the entire population are 44% / 56%. Outcomes appear especially promising in the DLBCL (EFS / OS, 54% / 54%) and the T-NHL (62% / 69%) subgroups.CONCLUSIONS:GEM/CLO/BU is a new, myeloablative, reduced-toxicity alloSCT regimen with high activity in this population of heavily pretreated and refractory aggressive lymphomas, which warrants further study in disease-specific trials [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Jak-STAT Inhibition Mediates Romidepsin and Mechlorethamine Synergism in Cutaneous T-Cell Lymphoma

Sézary syndrome is an aggressive and disseminated form of cutaneous T-cell lymphoma associated with dismal prognosis in which the histone deacetylase inhibitor romidepsin has shown remarkable activity as a single agent. However, clinical responses to romidepsin are typically transient, highlighting the need for more effective therapies. In this study, we show synergistic antilymphoma effects of romidepsin in combination with mechlorethamine, an alkylating agent, in cutaneous T-cell lymphoma cell lines and primary samples with strong antitumor effects in an invivo model of Sézary syndrome. Mechanistically, gene expression profiling points to abrogation of Jak/signal transducer and activator of transcription (STAT) signaling as an important mediator of this interaction. Consistently, the combination of mechlorethamine plus romidepsin resulted in downregulation of STAT5 phosphorylation in romidepsin-sensitive cell lines and primary Sézary syndrome samples, but not in romidepsin-resistant tumors. Moreover, in further support of Jak/STAT signaling as a modulator of romidepsin activity in cutaneous T-cell lymphoma, treatment with romidepsin in combination with Jak inhibitors resulted in markedly increased therapeutic responses. Overall, these results support a role for romidepsin plus mechlorethamine in combination in the treatment of cutaneous T-cell lymphoma and uncover a previously unrecognized role for Jak/STAT signaling in the response to romidepsin and romidepsin-based combination therapies in Sézary syndrome.

Preclinical analysis of an autologous CD4-targeted chimeric antigen receptor T-cell (CAR-T) immunotherapy for relapsed or refractory peripheral T-cell lymphoma (PTCL) or cutaneous T-cell lymphoma (CTCL).

e14510 Background: CD4 is highly and uniformly expressed T-cell lymphomas (TCL) including PTCL and CTCL, suggesting its potential as a surface target for CAR-T therapy. However, there is a significant risk of potential antigen masking by CAR introduced into tumor cells, which thereby leads to escape of tumor cells from recognition by CAR-T cells. For example, Ruella M et al ( Nat Med, 2018) reported that contaminating malignant B cells transduced with anti-CD19 CAR (called CAR-B cells) in the manufacturing process led to the CD19 antigen masking by CAR molecules. Hence, CAR-B tumor cells could not be recognized by CAR-T cells, which resulted in relapse of CAR expressing B lymphoma cells. A similar antigen masking effect might occur for the anti-CD4 CAR-T product. Methods: To identify single-chain variable fragment (scFv) without antigen-masking effect, large panels of fully human monoclonal antibodies were converted to format of CAR modality with the scFv-4-1BB-CD3z structure and introduced into CD4+ and CD8+ T cells. After lentiviral transduction, residual CD4+ T cells were quantified and the CARs with complete elimination of CD4+ T cells were selected for further validation. The selected CAR constructs were then introduced into CD4+ TCL cells HH (CAR-HH cells) to mimic the potential risk of introducing CAR into contaminating malignant T cells in the manufacturing process. Thereafter, CAR-HH cells were subject to in vitro killing assay by LB1901. Last, identified CAR constructs were further tested for in vitro and in vivo anti-tumor efficacy and off-target binding and killing. Results: Introduction of LB1901 CAR into CD4+ and CD8+ T cells led to complete elimination of CD4+ T cells, suggesting no masking effect of CAR on CD4 antigen. Furthermore, the introduction of CAR into CD4+ HH cells did not protect HH cells from being recognized and eliminated by LB1901, further confirming that the CAR modality of LB1901 does not mask CD4 antigen. An in vivo anti-tumor efficacy study showed that LB1901 exhibited dose-dependent anti-tumor activity without significant adverse effect. As low as 0.3 million CAR+ cells completely suppressed tumor growth, suggesting the potent anti-tumor activity by LB1901. Immunohistochemical analysis of normal tissues with LB1901 scFV binder showed no off-target binding. Furthermore, no killing toward CD4- cell lines and primary cells derived from vital organs or antigen-independent cytokine release was observed in vitro. Conclusions: Altogether, the in vitro and in vivo studies showed that LB1901 did not “mask” the CD4 antigen but exhibited potent anti-tumor activity without off-target effects. A phase 1 study of LB1901 CAR-T in patients with relapsed or refractory PTCL or CTCL is ongoing in the US to assess the safety and tolerability of LB1901 CAR-T (NCT04712864).

Cell membrane permeability and defective G2/M block as factors potentially contributing to increased cell chemosensitivity. SeAx cell line as an example

BackgroundImmortalized mammalian cell lines are a valuable research tool, though they represent a highly simplified model. Due to accumulated mutations they may not reflect characteristics of the disease or even the tissue they derive from. ObjectiveWe aim to pinpoint factors distinguishing SeAx cells from two other cutaneous T-cell lymphoma (CTCL) cell lines, namely Hut78 and MyLa2000. Of note, these factors may influence cell sensitivity in an unspecific way and therefore should be taken under consideration. MethodsWe evaluated transcriptional levels of drug transporters across cell lines, cell membrane permeability, functionality of pathways related to DNA damage response and activation of G2/M block. ResultsAnalysis of the transcriptional levels of genes coding drug efflux pumps indicated that they are not consistently down-regulated in SeAx. However, we noted that SeAx cell membrane is markedly more permeable than Hut78 and MyLa2000, which may contribute to increased chemosensitivity in an unspecific way.Moreover, though DNA damage response seemed to be at least partly functional in SeAx cells, they fail to activate G2/M block in response to psoralen + UVA treatment. Any DNA damage should be repaired before cells enter mitosis, in order to uphold genome integrity. Thus, a defective cell cycle block may contribute to cell sensitivity. ConclusionsWe believe that factors such as increased membrane permeability or defective cell cycle block should be accounted for when comparing sensitivity of cell line panels to chemotherapeutics of interest. It is worth to exclude a simple, indiscriminative mechanisms of cell resistance or sensitivity before attempting comparisons. Cell lines that are indiscriminately sensitive to a broad range of chemicals may contribute to overestimating the cytotoxic potential of tested compounds if used in cytotoxicity studies.

Therapeutic Potential of Novel Rexinoids in Prevention and Treatment of Cancer and Alzheimer's Disease

Bexarotene is a synthetic rexinoid that binds to the retinoid X receptor (RXR) causing a signaling cascade that results in transcription of RXR target genes to control cellular differentiation, apoptosis, and proliferation. Bexarotene is an FDA-approved therapeutic used in the treatment of cutaneous T-cell lymphoma (CTCL) and non-small cell lung cancer. Off-label use has been extended to breast cancer, although significant side effects like hyperlipidemia and hypothyroidism can occur because bexarotene not only modulates retinoid-X-response element (RXRE)-regulated transcription but it also acts as an RXR heterodimer-selective agonist resulting in activation of other RXR-containing heterodimers. Bexarotene has also shown promise to reverse neurodegeneration, improve cognition and decrease levels of amyloid-β in transgenic mice expressing familial Alzheimer's disease mutations. The RXR heterodimers with the liver-X-receptors (LXR) and with peroxisome proliferator-activated receptor-gamma (PPARγ) control cholesterol efflux, inflammation, and transcriptionally regulate the production of apolipoprotein (ApoE) in the brain. In the present study, we assessed the potential of bexarotene and a group of novel rexinoids to induce RXR dimerization using a mammalian two-hybrid system (M2H). RXRE-mediated reporter assays were also employed to assess the transcriptional efficiency of these compounds. LXRE-mediated luciferase assays were performed to analyze the role of the novel compounds as activators of LXRE-directed transcription, and as inducers of ApoE mRNA in U87 glial cells. Results from these multiple assays indicate that our panel of RXR analogs contain compounds with an assortment of activities, some of which bind to RXR with higher affinity than others, and in some cases can upregulate ApoE expression to a greater extent than bexarotene. These results suggest that minor modifications to the bexarotene core chemical structure may yield more potent novel analogs which allows for their therapeutic use at lower concentrations, thereby potentially reducing the corresponding side effects associated with high-dose bexarotene therapy. Moreover, these results indicate that some of the novel analogs possess a greater capacity to activate RXR and may be useful as therapeutic agents against both cancers as well as neurodegenerative disorders including Alzheimer's disease.

Targeting Cutaneous T-Cell Lymphoma Cells by Ingenol Mebutate (PEP005) Correlates with PKCδ Activation, ROS Induction as Well as Downregulation of XIAP and c-FLIP.

New therapeutic strategies are needed for cutaneous T-cell lymphoma (CTCL), and the plant extract ingenol mebutate (PEP005) may be considered. PEP005 has been approved for actinic keratosis, and proapoptotic activities were described in different cancer cells. Here, we aimed to investigate its efficacy in four CTCL cell lines and its mode of action. While HuT-78 and HH responded with induced apoptosis as well as with loss of cell viability and cell proliferation, MyLa and SeAx remained resistant. Interestingly, both sensitive and resistant cells showed caspase-8 activation and enhanced levels of reactive oxygen species (ROS), while final caspase-3 activation was restricted to sensitive cells. Apoptosis induction was prevented by the caspase inhibitor QVD-Oph as well as by the antioxidant vitamin E. Caspase activation by PEP005 may be explained to some extent by the downregulation of the caspase antagonistic proteins c-FLIP and XIAP in sensitive cells, whereas both proteins were strongly expressed in resistant cells. Finally, PEP005 resulted in the activation of proapoptotic PKCδ, and the PKC inhibitor bisindolylmaleimide I reduced apoptosis, caspase-3 processing and ROS production, as well as restored cell viability. In conclusion, PKCδ appeared as a central player in apoptosis regulation in CTCL cells, also suggesting its therapeutic targeting.

053 The inhibitory role of ganglioside GD3 on the functional activities of benign T cells in cutaneous T-cell lymphoma

053 The inhibitory role of ganglioside GD3 on the functional activities of benign T cells in cutaneous T-cell lymphoma

Ganglioside GD3 May Suppress the Functional Activities of Benign Skin T Cells in Cutaneous T-Cell Lymphoma.

In cutaneous T-cell lymphoma (CTCL), which arises from skin-tropic memory T cells, malignant T cells and benign T cells are confined in the same skin lesions. It is thus difficult to evaluate the phenotypic characteristics and functional activities of benign T cells in CTCL. Disialoganglioside with three glycosyl groups (GD3) is increasingly expressed on the surface of solid malignant tumor cells and takes part in tumor progression and suppression of tumor immunity. However, the role of GD3 in CTCL is not well-understood. In this study, the malignant and benign T cells in CTCL skin lesions were distinguished by flow cytometry and their phenotypic characteristics were compared with those of T cells from control skin specimens. In CTCL skin lesions, the benign T cells included limited resident memory T cells (TRM), which are sessile in skin and known to exert strong antitumor function. The benign T cells showed diminished Th17 property, and the expression of GD3 was high in the malignant T cells. The expression of GD3 in the malignant T cells inversely correlated with IL-17A production from the benign CD4 T cells. GD3 from the malignant T cells was implied to be involved in suppressing the Th17 activity of the benign T cells independent of the regulation of TRM differentiation in CTCL. Revealing the role of GD3 in inhibiting the production of IL-17A in CTCL would aid the understanding of the suppressive mechanism of the antitumor activity by malignant tumor cells.

Overexpression of Interleukin-1α Suppresses Liver Metastasis of Lymphoma: Implications for Antitumor Effects of CD8+ T-cells.

Interleukin (IL)-1 plays a key role in carcinogenesis, tumor progression, and metastasis. Although IL-1 may enhance the expansion of CD8+ T-cells, the pathological contribution of IL-1-activated CD8+ T-cells to tumor metastasis remains unclear. This study used a liver metastasis model of the EL4 T-cell lymphoma cells transplanted into human IL (hIL)-1α conditional transgenic (hIL-1α cTg) mice. Overproduction of hIL-1α suppressed both macroscopic and histological liver metastasis of EL4 T-cell lymphoma. The hIL-1α-induced inflammatory state increased the number of CD8+ T-cells both within and around metastatic tumors. Moreover, larger numbers of CD8+ T-cells showed greater infiltration of liver blood vessels in hIL-1α cTg mice than in control wild-type mice. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining of liver tissue from hIL-1α cTg mice indicated increased apoptosis of cells in the tumor. Localization of apoptosis cells resembled that of CD8+ T-cells. In addition, cytotoxicity assay showed that CD8+ T-cell counts from tumor-bearing hIL-1α cTg mice correlated with cytotoxicity against EL4. In summary, IL-1α suppresses lymphoma metastasis, and IL-1α-activated CD8+ T-cells may play important roles in inhibiting both tumor metastasis and metastatic tumor growth.

Plasma 25-hydroxyvitamin D and the inflammatory response in canine cancer.

Decreased circulating 25-hydroxyvitamin D (25[OH]D) and increased inflammatory marker concentrations have been reported separately in canine cancer. Correlations between the two exist in humans, but little work has examined links in dogs. This study aimed to determine plasma 25(OH)D and inflammatory marker concentrations in healthy dogs and dogs with cancer and to assess correlations in each group. Newly diagnosed dogs with B-cell lymphoma (B-cell, n=25), T-cell lymphoma (T-cell, n=9), osteosarcoma (OSA, n=21), and mast cell tumour (MCT, n=26) presenting to a tertiary oncology centre, and healthy dogs (n=25), were enrolled. Plasma samples were analysed for 25(OH)D, C-reactive protein (CRP), haptoglobin (HP), serum amyloid A (SAA), alpha-1-acid glycoprotein (AAG), and 13 chemokines and cytokines. Dogs with B-cell had decreased plasma 25(OH)D (P=.03), and increased plasma CRP, AAG, HP, KC-like and MCP-1 concentrations (P < =.001, .011, <.001, .013 and .009, respectively) compared with healthy dogs. Plasma CRP, HP and SAA concentrations were increased in dogs with OSA compared with healthy dogs (P=.001, .010 and .027, respectively). No differences were noted in dogs with T-cell and MCT. Negative correlations were observed between plasma 25(OH)D concentrations and: AAG concentrations in dogs with T-cell (Rs =-0.817, P=.007); GM-CSF concentrations (Rs =-0.569, P=.007) in dogs with OSA; and IL-7 concentrations (Rs =-0.548, P=.010) in dogs with OSA. Decreased 25(OH)D concentrations and increased concentrations of multiple inflammatory markers were observed in B-cell patients, supporting an association between 25(OH)D and inflammation. The cross-sectional study design meant the timing of changes could not be determined. Prospective cohort studies are warranted.

Programmed Cell Death Protein 1/Programmed Cell Death Ligand-1 Axis activates Intracellular ERK Signaling in Tumor Cells to Mediate Poor Prognosis in T-cell Lymphoma.

Purpose: To investigate the prognostic significance of programmed cell death ligand-1 (PD-L1) and phosphorylated ERK (p-ERK) and their interactions in T-cell lymphoma (TCL).Methods: The mRNA levels of PD-L1 and ERK in TCL samples were analyzed. Formalin-fixed paraffin-embedded tissues from 69 TCL patients were collected to detect the expression of PD-L1 and p-ERK by multiplexed immunofluorescence staining. The total PD-L1 and p-ERK was measured by western blotting, and membrane PD-L1 was determined using flow cytometry.Results: PD-L1 and ERK mRNA levels were significantly upregulated in TCL. The expression rates of PD-L1 and p-ERK were 52.2% and 27.5%, respectively. PD-L1 expression correlated with stage (R=0.304, P=0.011) and IPI score (R=0.313, P=0.009), and p-ERK expression correlated with stage (R=0.330, P=0.006) and IPI score (R=0.376, P=0.002). PD-L1 expression positively correlated with p-ERK expression (R=0.355, P=0.003). Patients with co-expression of PD-L1 and p-ERK had the worst overall survival (P=0.007). In three TCL cell lines with PD-L1 expression, we demonstrated that the expression of p-ERK was upregulated after stimulation with PD-1, suggesting that ERK signaling was activated.Conclusions: The PD-1/PD-L1 axis activates intracellular ERK signaling in tumor cells and that PD-L1, p-ERK or their combination are potential biomarkers for predicting the prognosis in TCL patients.

Angioimmunoblastic T-cell lymphoma with fever, arthritis and skin pigmentation: A case report

Angioimmunoblastic T-cell lymphoma is a rare T-cell lymphoma. The clinical manifestations are not specific. In addition to the common clinical manifestations of lymphomas such as fever, weight loss, night sweats and lymphadenopathy, it may also have skin rashes, arthritis, multiple serous effusions, eosinophilia and other systemic inflammatory or immune symptoms. The lymphoma cells of angioimmunoblastic T-cell lymphoma originates from follicular helper T cells, and the follicular structure of lymph nodes disappears. In the tumor microenvironment, in addition to tumor cells, there are a large number of over-activated immune cells, such as abnormally activated B cells, which produce a series of systemic inflammation or immune-related symptoms. This disease is rare and difficult to diagnose. This article reports a 36-year-old female. She got fever, joint swelling and pain, skin pigmentation, accompanied by hepatomegaly, splenomegaly, lymphadenopathy, anemia and other multiple-systems manifestations. The clinical manifestations of this patient were similar to autoimmune diseases such as adult onset Still's disease, rheumatoid arthritis, and systemic sclerosis, which made the diagnosis difficult. At the beginning of the disease course, the patient got arthritis and fever. And her white blood cells were significantly increased. Adult onset Still's disease should be considered, but her multiple-systems manifestations could not be explained by adult onset Still's disease. And her arthritis of hands should be distinguished with rheumatoid arthritis. However, the patient's joint swelling could get better within 3-7 days, and there was no synovitis and bone erosion on joint imaging examination. The rheumatoid factor and anti-CCP antibody were negative. The diagnostic evidence for rheumatoid arthritis was insufficient. The patient's skin pigmentation and punctate depigmentation were similar to those of systemic sclerosis. But the patient had no Raynaud's phenomenon, and her sclerosis-related antibody was negative. The diagnostic evidence for systemic sclerosis was also insufficient. After 3 years, she was finally diagnosed with angioimmunoblastic T-cell lymphoma by lymph node biopsy aspiration. This case suggests that the clinical manifestations of angioimmunoblastic T-cell lymphoma are diverse, and some symptoms similar to immune diseases may appear. When the patient's clinical symptoms are atypical and immune diseases cannot explain the patient's condition, and further evidence should be sought to confirm the diagnosis.

Photochemotherapy Induces Interferon Type III Expression via STING Pathway.

DNA-damaging cancer therapies induce interferon expression and stimulate the immune system, promoting therapy responses. The immune-activating STING (Stimulator of Interferon Genes) pathway is induced when DNA or double-stranded RNA (dsRNA) is detected in the cell cytoplasm, which can be caused by viral infection or by DNA damage following chemo- or radiotherapy. Here, we investigated the responses of cutaneous T-cell lymphoma (CTCL) cells to the clinically applied DNA crosslinking photochemotherapy (combination of 8–methoxypsoralen and UVA light; 8–MOP + UVA). We showed that this treatment evokes interferon expression and that the type III interferon IFNL1 is the major cytokine induced. IFNL1 upregulation is dependent on STING and on the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS). Furthermore, 8–MOP + UVA treatment induced the expression of genes in pathways involved in response to the tumor necrosis factor, innate immune system and acute inflammatory response. Notably, a subset of these genes was under control of the STING–IFNL1 pathway. In conclusion, our data connected DNA damage with immune system activation via the STING pathway and contributed to a better understanding of the effectiveness of photochemotherapy.

GATA3 Gene - a Potential New Biomarker Associated with Adverse Outcomes in ALK1-Negative Anaplastic Large Cell Lymphoma: Preliminary Results from a Retrospective Cohort of 80 South American Cases of Nodal Peripheral T Lymphoma

Introduction: Nodal peripheral T-cell lymphomas (nPTCLs) comprise a heterogeneous group of mature and aggressive T-cell lymphoid malignancies, including peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS), angioimmunoblastic T-cell lymphoma (AITL) and anaplastic large cell lymphomas (ALCL) ALK1-positive and ALK1-negative. The differential diagnosis of nPTCL can be very challenging in clinical practice and it has a markedly heterogeneous prognosis. Accurate biomarkers to distinguish the different histological subtypes of nPTCL and to stratify its prognosis are essential to improve the therapeutic approach. The aim of this study was to assess the prognostic impact of GATA3 gene expression, as well as its ability to discriminate between different histopathological subtypes of nPTCLs. Methods: In this observational, retrospective and single-center study, we analyzed clinical-epidemiological data, outcomes and molecular characteristics of 80 nPTCL patients treated at the largest Brazilian cancer center from January 2000 to December 2017. Analysis of GATA3 gene expression was assessed by quantitative real-time polimerase chain reaction (qRT-PCR) of tumor tissue biopsies fixed in formalin and embedded in paraffin (FFPE) at the time of diagnosis. The association of relative expression of the GATA3 gene with pathological variants of nPTCL was determined by the Kruskal-Wallis test and the Dunn's post-hoc test. The cutoff value of GATA3 expression capable of differentiating nPTCLs variants was determined by constructing receiver-operator-characteristic (ROC) curves. Overall survival (OS) and progression-free survival (PFS) curves were estimated using the Kaplan-Meier method. Results: The clinical and epidemiological characteristics of the 80 nPTCL patients are summarized in Table 1. Median age was 49 years (IqR 34-59), 43-80 (53.7%) of patients were male. Of these, 36.3% were classified as PTCL-NOS, 31.2% as ALK-negative ALCL, 21.2% as ALK-positive ALCL and 11.3% as AITL. Most of the cases had an advanced stage (III and IV Ann Arbor). With a median follow-up of 1.72 years, the estimated OS at 2 years and PFS were 52.2% and 39.5%, respectively. The median level of GATA3 gene expression was 0.49% (range 0 - 7.07%) in the global cohort, being 0.11% for ALK-positive ALCL, 0.46% for ALK-negative ALCL, 0.86% for PTCL, NOS and 0.67% for AITL. The difference in expression of the GATA3 gene between different nPTCL variants was statistically significant (p &amp;lt;0.001) - Figure 1. The levels of expression of the GATA3 gene ≥ 0.71% discriminated PTCL, NOS from ALK-negative ALCL and AITL with a sensitivity of 62% and specificity of 80.3%, contributing to the differential diagnosis of these neoplasms, particularly in cases of ALK-negative ALCL versus PTCL, NOS CD30-positive. Overexpression of GATA3 ≥ median was associated with poor 2-year OS for PTCL, NOS (46.7% x 21.4%, p = 0.04) and for ALK-negative ALCL (85.7% x 54.5%, p = 0.04) - Figures 2 and 3. Conclusion: Despite the relatively small number of patients in our cohort, preliminary results suggest that overexpression of the GATA3 gene may be an important biomarker associated with poor prognosis in PTCL, NOS and ALK-negative ALCL. Our results corroborate the findings of Iqbal et al, 2014, reinforcing the adverse prognostic impact of GATA3 gene expression in PTCL/NOS, and we show that its overexpression may be a potential novel biomarker related to poor prognosis for ALK-negative ALCL. In addition, it has been demonstrated here that GATA3 can play an auxiliary role in discriminating different subtypes of nPTCLs, aiding the differential diagnosis of these neoplasms, which often have overlapping clinical and pathological aspects. Other studies with larger series of patients should confirm our findings. Disclosures No relevant conflicts of interest to declare.

A Multi-Center, Open Label, Phase I/II Study to Assess the Safety and Efficacy of Tenalisib Given in Combination with Romidepsin in Patients with Relapsed/Refractory T-Cell Lymphoma

A Multi-Center, Open Label, Phase I/II Study to Assess the Safety and Efficacy of Tenalisib Given in Combination with Romidepsin in Patients with Relapsed/Refractory T-Cell Lymphoma

A Phase I/II Study (JACKPOT8) of DZD4205, a Selective JAK1 Inhibitor, in Refractory or Relapsed Peripheral T- Cell Lymphoma

Background: Peripheral T-Cell Lymphoma (PTCL) is an aggressive malignancy which lacks effective treatment. Emerging data suggests that the JAK-STAT pathway may play an important role in mediating pathogenesis of PTCL. DZD4205 (AZD4205) is an orally available, potent and highly selective JAK1 inhibitor. In T-cell lymphoma cell lines, DZD4205 modulates pSTAT3 pathway and suppresses cell proliferation, and in tumor xenograft models, DZD4205 exhibits dose-dependent anti-tumor activities, with good correlation with drug exposure and modulations of pSTAT3 in tumor tissues. A phase I/II study (JACKPOT8) was initiated to assess the safety, tolerability, pharmacokinetics and anti-tumor efficacy of DZD4205 in patients with refractory/relapsed (r/r) PTCL. Methods: The JACKPOT8 study (ClinicalTrials.gov Identifier: NCT04105010) included two parts: Part A, dose escalation and Part B, dose expansion. In Part A, patients with r/r PTCL who have progressed on or were refractory to systemic therapy will be enrolled and treated with DZD4205 at two different dose levels, 150 mg or 250 mg once daily. In Part B, patients will receive DZD4205 treatment at a defined dose. The primary objective is to assess the safety and tolerability of DZD4205, and the secondary objectives include anti-tumor efficacy and pharmacokinetics. Evaluation of safety/tolerability and tumor response will be based on the CTCAE version 5.0 and 2014 Lugano classification, respectively. Patients will be treated until disease progression, intolerance to adverse events, or withdrawal of consent. Results: As of June 30, 2020, a total of 23 patients with r/r PTCL were enrolled and received DZD4205 at 150 mg (n = 19) or 250 mg (n = 4) once daily. Patient characteristics: median age (range): 65.0 years (34-79); median prior systemic therapies (range): 2 lines (1-8). Four patients had undergone prior hematopoietic stem cell transplantation (HSCT). Six patients had bone marrow involvement at the study entry. Histological subtypes included peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) (n = 10, 43.5%), angioimmunoblastic T-cell lymphoma (AITL) (n = 10, 43.5%), ALK-negative anaplastic large cell lymphoma (ALCL ALK-) (n = 2, 8.7%) and extra-nodal nasal NK/T-cell lymphoma (NKTCL) (n = 1, 4.3%) . Preliminary data showed that 20 patients experienced treatment emergent adverse events (TEAEs), among whom 13 (56.5%) had ≥ grade 3 TEAEs. Based on local investigators' assessment, eight patients (34.8%) had ≥ grade 3 TEAEs which were considered to be related to DZD4205. The most common (&amp;gt; 5% incidence) DZD4205-related TEAEs (any grade) included thrombocytopenia (n = 6, 26.1%), neutropenia (n = 4, 17.3%), decreased appetite (n = 3, 13.0%), nausea (n = 2, 8.7%), interstitial lung disease (n = 2, 8.7%) and pneumonia (n = 2, 8.7%). Most TEAEs were manageable with dose interruption and reduction. Preliminary PK data is available in 20 patients (n = 19 at 150 mg, n = 1 at 250 mg). Exposure at 250 mg is higher than 150 mg following a single and multiple dose. As expected from long t1/2 of DZD4205, accumulation of about 3 folds in AUC was observed. DZD4205 had flat PK profile with small difference between Css,max and Css,min after 22 days of once daily dosing, which is an optimal PK profile to maintain DZD4205 concentrations above effective levels throughout the dosing interval. As of June 30, 2020, 22 patients (n = 19 at 150 mg, n = 3 at 250 mg) had at least one post-treatment Lugano assessment. In 150 mg cohort, 8 out of 19 patients showed tumor response, with objective response rate (ORR) of 42%, among whom 4 (21%) had complete response (CR) and another 4 (21%) partial response (PR). In 250 mg cohort, 1 out of 3 patients showed tumor response, with ORR of 33%. Tumor response was observed in subtypes including AITL, PTCL-NOS, ALCL (ALK-) and NKTCL. At the data cut-off date, the longest duration on treatment was &amp;gt; 8 months. Patient enrolment is ongoing. Updated clinical data will be shared at the meeting. Conclusion: DZD4205 shows good tolerability, pharmacokinetic profiles and promising anti-tumor efficacy in patients with r/r PTCL, indicating its potential as a therapeutic option for this unmet medical need. Disclosures Kim: Donga: Research Funding; Kyowa-Kirin: Research Funding; Roche: Research Funding; Johnson&amp;Johnson: Research Funding; Celltrion: Research Funding; Mundipharma: Research Funding. Yoon:Celltrion: Honoraria; Samyang: Research Funding; Amgen, Chongkundang, Celgene, Astrazeneca: Consultancy. Wang:Dizal Pharmaceuticals: Current Employment. Li:Dizal Pharmaceuticals: Current Employment. Huang:Dizal Pharmaceuticals: Current Employment. Deng:Dizal Pharmaceuticals: Current Employment. Chen:Dizal Pharmaceuticals: Current Employment. Yang:Dizal Pharmaceuticals: Current Employment.

Abstract PO-12: Activity of ASTX660, a small-molecule IAP antagonist, reveals a novel, immune-based therapeutic approach in T-cell lymphoma

Abstract Introduction: ASTX660 is a potent, non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which is currently being tested in a first-in-human phase I-II study in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists are known regulators of cell death and have been reported to modulate immune responses (1). Herein, we describe the profile of ASTX660 in preclinical models with a specific focus on ASTX660's ability to harness a particular form of regulated cell death in T-cell lymphoma that directs an effective immune response and leads to sustained tumor regressions. Methods: ASTX660 was profiled across a panel of T-cell lymphoma lines, assessing apoptosis, necroptosis, and immunogenic cell death (ICD). ASTX660 was tested in vitro with or without caspase-8/RIPK1 inhibitors to demonstrate mechanism of action. Target engagement along with induction of apoptosis, necroptosis, and ICD markers were analyzed by Western blotting and flow cytometry. Murine tumor models in immunocompetent and immunocompromised mice were utilized to evaluate the efficacy of ASTX660 in the presence or absence of an adaptive immune response. Results: ASTX660 antagonized IAPs in cell lines, as indicated by a decrease in cIAP1 protein levels. In certain murine T-cell lymphoma cell lines, ASTX660 treatment was associated with an increase in cell death and release of biomarkers of ICD. Use of caspase and RIPK1 inhibitors indicated the cell death was via necroptosis. In a syngeneic model of T-cell lymphoma, administration of ASTX660 resulted in complete and sustained tumor regressions which were not seen in mice deficient in T and B cells. Mice cured of T-cell lymphoma by ASTX660 were resistant to a second inoculation of the same lymphoma. Conclusion: These data add to the description of ASTX660's mode of action and our ongoing understanding of the preliminary clinical efficacy in T-cell lymphoma (2). We describe a new role for ASTX660 as an immunomodulatory molecule with potential to deliver a novel immune-based therapeutic approach in T-cell lymphoma.