Cell Peripheral T-cell Lymphoma Research Articles

Pyroptotic macrophages promote proliferation and chemotherapy resistance of peripheral T-cell lymphoma via TLR4 signaling pathway.

Peripheral T-cell lymphoma (PTCL) is a highly aggressive type of non-Hodgkin's lymphoma with a poor prognosis. Pyroptosis is a newly discovered procedural cell death mode, which has been implicated to occur in both tumor cells and immune cells. However, the occurrence and effect of pyroptosis on PTCL remain unclear. Here, we found that pyroptosis occurred in interstitial macrophages of PTCL rather than in tumor cells. In clinical specimens, macrophage pyroptosis was associated with a poor prognosis of PTCL. Invitro experiments and gene sequencing results showed that pyroptotic macrophages could upregulate the expression of TLR4 through secreting inflammatory cytokines IL-18. Upregulated TLR4 activated its downstream NF-κB anti-apoptotic signaling pathway, thus leading to malignant proliferation and chemotherapy resistance of tumor cells. Moreover, the expression of factors such as XIAP in the NF-κB anti-apoptotic pathway was downregulated after the knockdown of TLR4, and the malignant promotion effect of pyroptotic macrophages on PTCL cells was also reversed. Our findings revealed the mechanism of pyroptotic macrophages promoting the malignant biological behavior of PTCL and elucidated the key role of TLR4 in this process. In-depth analysis of this mechanism will contribute to understanding the regulatory effect of PTCL by the tumor microenvironment and providing new ideas for the clinical treatment of PTCL.

Abstract 1772: A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified

Abstract Despite being the most common subtype of human peripheral T-cell lymphoma (PTCL), PTCL-not otherwise specified (PTCL-NOS) remains a poorly understood diagnosis of exclusion with poor survival times and treatment responses. Canine PTCL clinically and immunophenotypically resembles human PTCL-NOS, and PTCL is more common in dogs than humans, leading to their interest as a naturally occurring preclinical model. Canine models of cancer offer several unique advantages: in contrast to traditional rodent models, these are spontaneous tumors in genetically diverse and immunocompetent animals living in a shared environment with humans, but relative to their human counterparts, they offer accelerated lifespans with shorter clinical courses of disease. Here, we analyzed the gene expression profile of 96 canine CD4+ PTCLs to determine similarities to human PTCL-NOS and investigate a possible cell of origin. Bulk RNA-seq was performed on lymph node aspirates of 96 cases of canine CD4+ PTCL diagnosed by flow cytometry and sorted CD4+ nodal lymphocytes and CD4+ thymocytes from healthy control dogs. Raw reads were aligned to the Ensembl canine reference genome (ROS_Cfam_1.0) and tabulated. Data normalization and differential expression analysis was conducted with DESeq2, and differentially expressed genes were compared to human PTCL-NOS and various stages of T-cell development via gene set enrichment analyses (GSEA). Global gene expression in canine CD4+ PTCL was analogous to that of human PTCL-NOS (NES = 2.3, p < 0.001). Additionally, canine CD4+ PTCL had increased expression of GATA3 (log2fc = 1.9, padj = 6.5e-07) and was enriched for gene signatures associated with downregulation of PTEN (NES = 1.7, p = 0.0001) and upregulation of PI3K/AKT/mTOR signaling (NES = 1.5, padj = 0.0007), resembling the more aggressive human GATA3-PTCL subtype. Canine CD4+ PTCL was also enriched for human and murine gene signatures associated with early thymocyte progenitor cells and had increased expression of genes of immaturity, including CD34 (log2fc = 7.8, padj = 2.6e-14), KIT (log2fc = 3.3, padj = 7.7e-05), and CCR9 (log2fc = 4.8, padj = 3.9e-11), although surface CD34 expression is not detected by flow cytometry. These findings were confirmed when we compared the canine PTCL gene expression programs to a canine single cell transcriptomic atlas of hematopoietic precursors and T cells across canine bone marrow, thymic, and lymph node tissues. Canine CD4+ PTCL cells were significantly (padj < 0.05) enriched for gene signatures associated with early thymic and bone marrow precursors (NES 1.3-2.1), and negatively enriched for gene signatures associated with naïve nodal CD4+ T cells (NES = -3.2, padj = 0.006). In conclusion, the gene expression profile of canine CD4+ PTCL resembles human PTCL-NOS, supporting its potential as a naturally occurring preclinical model, and a subset of these neoplasms may arise from a thymic precursor cell of origin. Citation Format: Eileen Owens, Adam Harris, Anne Avery. A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1772.

Abstract 3201: Preclinical development of a novel Romidepsin nanoparticle for the treatment of T-Cell lymphoid malignancies

Abstract The histone deacetylase inhibitors (HDACi) have long been regarded as an important class of drugs for treating T-cell lymphoid malignancies (TCL). Romidepsin (Romi), a bicyclic peptide, was approved for patients with relapsed or refractory cutaneous (CTCL) and peripheral T-cell lymphomas (PTCL). Unfortunately, the approval of Romi for relapsed/refractory PTCL was withdrawn due to a negative Phase 4 post-marketing requirement. Given its potent activity against Class I HDACs, Romi is considered one of the most effective HDAC inhibitors studied and used in patients with TCL. Herein, we report on a first-in-class nanoparticle of Romi (NanoRomi) with a superior safety and efficacy profile compared to the free drug in preclinical models of PTCL and large granular lymphocyte leukemia (LGLL). We developed a first-in-class NanoRomi using our proprietary amphiphilic di-block copolymer-based nanochemistry platform. NanoRomi was characterized in cell viability, flow cytometry, and western blotting (WB) assays to determine the concentration-effect relationships with regard to the induction of apoptosis and histone acetylation in cell lines representative of TCL and LGLL, as well as primary LGLL patient PBMC samples. In addition, we performed extensive in vivo modeling using murine xenografts of CTCL, including repeated dose toxicity, biodistribution, efficacy, and survival studies. Cytotoxicity assays demonstrated superior/comparable activity of NanoRomi (IC50: 1.1-2.3 nM) compared to free Romi (IC50: 4.8-10.6 nM) across all PTCL and LGLL cell lines studied, while in primary LGLL samples, NanoRomi exhibited superior cytotoxicity (2-fold) compared to Romi. WB analysis showed that NanoRomi induced apoptosis and increased acetylation of histone H3 and H4 in a time and concentration-dependent manner in cell lines representing CTCL, ALK+-ALCL, and LGLL. Repeat dose toxicity studies in CTCL xenografts confirmed that the overall tolerability of NanoRomi was substantially better than Romi. Tracking studies with a fluorescent dye (DiO) in the NanoRomi demonstrated preferential localization of NanoRomi in tumors of mice compared to the normal organs after 6 hours of drug administration. NanoRomi exhibited superior efficacy in the murine CTCL xenograft models, including a significant improvement in overall survival compared to Romi. Collectively, these data show that NanoRomi exhibits markedly superior pharmacokinetics, tolerability, and efficacy in models of TCL. We anticipate that the improved activity and tolerability of NanoRomi relate to the altered pharmacologic profile and superior bioavailability of the drug, which optimizes the effects of the drug for its influence on epigenetic biology. This unique platform offers the prospect of improving the tolerability and efficacy of epigenetic treatment strategies in these challenging diseases. Citation Format: Ipsita Pal, Anuradha Illendula, Andrea Joyner, Tess M. Deddens, Deepthi P. Damera, Todd E. Fox, Marya E. Dunlap-Brown, Kallesh D. Jayappa, Thomas P. Loughran, David J. Feith, Owen A. O'Connor. Preclinical development of a novel Romidepsin nanoparticle for the treatment of T-Cell lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3201.

The RNA sequencing results revealed the expression of different genes and signaling pathways during chemotherapy resistance in peripheral T-cell lymphoma

BackgroundPeripheral T-cell lymphoma (PTCL) is a subtype of non-Hodgkin's lymphoma that occurs primarily at extranodal sites and is commonly treated using chemotherapy and radiotherapy. PTCL is more malignant than other lymphoid tumors, resulting in a poor prognosis.The 5-year recurrence rate remains high, and there is a lack of standard treatment for patients with relapse-resistant disease. However, the molecular mechanisms underlying the resistance of peripheral T-cell lymphoma cells to chemotherapeutic drugs, as well as identifying strategies to overcome drug resistance remains unclear. In this study, we aimed to identify pivotal genes and signaling pathways associated with chemotherapy resistance in PTCL.MethodsIn this study, a total of 5 healthy controls and 7 clinical patients were enrolled; 4 patients were classified as chemotherapy sensitive, and 3 patients were classified as chemotherapy resistant. Peripheral blood samples were collected from each participant, and total RNA was extracted from the white blood cells. RNA sequencing was conducted on the Illumina HiSeq platform to obtain comprehensive gene expression profiles. Subsequently, the expression patterns of the DEGs associated with the most enriched signaling pathways, with a special focus on cancer-related genes, were validated using quantitative real-time polymerase chain reaction (qRT–PCR) in peripheral TCL patients.ResultsRNA sequencing (RNA-seq) analysis revealed 4063 differentially expressed genes (DEGs) in peripheral T-cell lymphoma specimens from patients with chemotherapy resistance, of which 1128 were upregulated and 2935 were downregulated. Subsequent quantitative gene expression analysis confirmed a differential expression pattern in all the libraries, with 9 downregulated genes and 10 upregulated genes validated through quantitative real-time PCR in 6 clinical specimens from patients with chemotherapy resistance. KEGG pathway analysis revealed significant alterations in several pathways, with 6 downregulated pathways and 9 upregulated pathways enriched in the DEGs. Notably, the TNF signaling pathway, which is extensively regulated, was among the pathways that exhibited significant changes. These findings suggest that DEGs and the TNF signaling pathway may play crucial roles in chemotherapy resistance in peripheral T-cell lymphoma.ConclusionOur study revealed that the expression of specific genes, including TNFRSF1B, TRADD2, and MAP3K7, may play an important role in chemotherapy resistance in peripheral T-cell lymphoma. Moreover, we identified the downregulation of the TNF signaling pathway, a crucial pathway involved in cell survival, death, and differentiation, as a potential contributor to the development of chemotherapy resistance in peripheral T-cell lymphoma. These findings provide valuable insights into the molecular mechanisms underlying chemotherapy resistance and highlight potential targets for overcoming treatment resistance in this challenging disease.

Genome-Wide CRISPR Screen Identifies ZNF451 Regulating Sensitivity to Topoisomerase 2 Inhibitors in Peripheral T-Cell Lymphoma Cells

Introduction: Despite the recent therapeutic developments for peripheral T-cell lymphomas (PTCLs), their prognosis remains dismal. Topoisomerase 2 inhibitors (TOP2i), which increase the amount of TOP2-DNA covalent complex and block DNA double-strand break repair, have long been used as key drugs in the treatment of PTCLs. The molecular mechanisms underlying sensitivity to TOP2i remain to be fully elucidated. Methods: To elucidate the cell-intrinsic genes affecting TOP2i sensitivity in PTCL cells, we performed whole-genome loss-of-function CRISPR screenings in adult T-cell leukemia/lymphoma (ATLL) cell lines. ST1 and KK1 cells were transduced with the Brunello CRISPR knockout pooled library targeting 19,114 genes (4 sgRNAs/gene) followed by puromycin selection. The transduced cells were then treated with etoposide (ETP), doxorubicin (DOX) or DMSO control for 8 days. The MAGeCK algorithm was used to rank the sgRNAs responsible for TOP2i sensitivity. Among the top 500 enriched genes, we further selected the those with at least 2 sgRNAs with a log2 fold change (TOP2i/DMSO) < -0.5 or >0.5 in both ST1 and KK1 cells as genes whose knockout conferred TOP2i sensitivity or resistance, respectively. Results: Using the whole genome loss-of-function CRISPR screen, we identified 3 genes and 2 genes whose knockout conferred ETP sensitivity and resistance, respectively. TOP2A, a known ETP target, was ranked as a top gene for ETP resistance, confirming the reliability of our screenings (Fig. 1). In the same way we also extracted 3 genes whose knockout conferred DOX sensitivity. Interestingly, zinc finger protein 451 (ZNF451) was identified as an overlapping gene whose knockout conferred sensitivity in both screens for ETP and DOX. ZNF451 is an E3 SUMO ligase and was previously reported to be an endogenous DNA repair factor for TOP2i-induced DNA double-strand breaks (Schellenberg et al. Science 2017). The role of ZNF451 in PTCL cells has not been previously reported, prompting us to focus on ZNF451 in further analyses. For a confirmatory experiments, we transduced sgRNAs targeting ZNF451 (sgZNF451) in ATLL cell lines. To see the knockout efficiencies of the sgZNF451, we performed immunoblot analysis by using anti-ZNF451 antibody and found three bands with different levels of reduction. To ensure whether the bands detected by the anti-ZNF451 antibody represented endogenous ZNF451 protein, we engineered the ATLL cell line with an HA-tag at the 5' UTR of the endogenous ZNF451 gene locus by CRISPR-mediated knock-in. Immunoblot analysis of sgZNF451-transduced engineered cells expressing HA-tag-ZNF451 by using anti-HA antibody showed almost the same pattern of the bands as shown in the blot by anti-ZNF451 antibody. Thus, we confirmed that sgZNF451 worked efficiently and concluded that post-transcriptional modification affected the stability of ZNF451 protein in ATLL cells. Next, we investigated the role of ZNF451 on cell viability. While sgZNF451-transduced ATLL cells did not differ from control sgRNA-transduced cells in terms of cellular proliferation under normal culture conditions, sgZNF451-transduced ATLL cells showed increased sensitivity to both ETP and DOX compared to control cells with statistical significance, confirming our screening results (Fig. 2). Importantly, sgZNF451-mediated sensitization to ETP and DOX was found not only in ATLL cells but also in other PTCL cell lines including ALK-positive and ALK-negative anaplastic large cell lymphoma (ALCL). Mechanistically, apoptosis and G2/M cell cycle arrest were induced at significantly higher frequencies by ETP treatment in ZNF451 knocked out cells compared to control cells. Given the role of ZNF451 in TOP2i sensitivity in PTCL cells, we investigated whether ZNF451 expression levels stratify the prognosis of PTCL patients treated with CHOP-like regimens. To test this, FFPE samples from 45 PTCL-NOS patients were used for Q-PCR analysis of ZNF451 mRNA but we did not find any correlation between the ZNF451 expression and prognosis. Conclusions: We performed unbiased genome-wide CRISPR/Cas9 library screens and identified ZNF451 as an important regulator of sensitivity to TOP2i in PTCL cells. Further studies are needed to determine whether measuring ZNF451 protein levels can be used as a biomarker in PTCL patients who are treated with TOP2i.

Genome-Wide CRISPR Screen Identifies MAD2L1BP, ANAPC15 and SLC39A7 As Intrinsic Regulators for Brentuximab Vedotin Sensitivity in Peripheral T-Cell Lymphoma Cells

Introduction: Brentuximab Vedotin (BV), an antibody-drug conjugate that specifically delivers the potent cytotoxic drug monomethyl auristatin E (MMAE) to CD30-positive cells, has been widely used as one of the key drugs for the treatment of peripheral T-cell lymphomas (PTCLs). BV shows relatively high response rate, however, the prognosis of PTCLs is not still satisfactory. The molecular mechanisms concerning sensitivity to BV remain to be clarified. Methods: To assess the cell-intrinsic genes affecting susceptibility to BV in PTCL cells, we performed a whole-genome loss-of-function CRISPR screen in DEL, an anaplastic large cell lymphoma (ALCL), ALK-positive cell line. DEL cells were transduced with the Brunello CRISPR knockout pooled library targeting 19,114 genes (4 sgRNAs/gene) followed by puromycin selection. Then, the transduced cells were treated with BV or DMSO control for 6 days. To rank the sgRNAs responsible for BV sensitivity, MAGeCK algorithm was used. Among the top 20 enriched genes, we further selected ones having at least 2 sgRNAs with a log2 fold change (BV/DMSO) < -0.5 or >0.5 as genes whose knock out confer BV sensitivity or resistance, respectively. Results: Through the whole-genome loss-of-function CRISPR screen, we identified 17 genes or 15 genes whose knock out confer BV sensitivity or resistance, respectively. TNF receptor superfamily member 8 (TNFRSF8, also known as CD30), a known BV target, was ranked as a top gene for BV resistance, confirming the reliability of our screening. Among the 17 enriched genes whose knock out confer BV sensitivity, endogenous mitotic checkpoint complex (MCC) inhibitors, namely MAD2L1BP and ANAPC15 were identified. As a confirmation, we knocked out MAD2L1BP or ANAPC15 in PTCL cell lines by CRISPR and found the enhanced susceptibility to BV of the cells. MAD2L1BP and ANAPC15, the endogenous MCC inhibitors, are known to facilitate anaphase-promoting complex/cyclosome (APC/C) activation. Therefore, we evaluated whether APC/C inhibition sensitize the PTCL cells to BV or not. Importantly, a combination use of proTAME, an APC/C inhibitor, and BV significantly suppressed cell proliferation of multiple PTCL lines, including ALCL, ALK-positive and adult T-cell leukemia/lymphoma (ATLL) (Fig. 1), compared to each single agent. In cell cycle analyses, PTCL lines treated with the combination demonstrated increased percentages of cells in G2/M phase, compared to each single agent. On the other hand, we found SLC39A7, a zinc channel in endoplasmic reticulum (ER), among 15 enriched genes whose knock out confer BV resistance. Since SLC39A7 has been reported to regulate TNFR1 cell surface expression (Fauster et al. Cell Death & Differentiation 2019), we hypothesized SLC39A7 could also affect the CD30 cell surface expression levels. In fact, flow cytometry analyses revealed downmodulation of CD30 cell surface expression in SLC39A7 knocked out PTCL lines (Fig. 2). We further evaluated the amount of CD30 protein expression by immunoblotting and found that SLC39A7 knocked out cells reduced the amount of mature form of CD30, which was consistent with the findings in flow cytometry. Intriguingly, we observed increasement of lower molecular weight band, which was considered as immature precursor CD30, in SLC39A7 knocked out cells, suggesting that impared glycobiology in ER might be a cause for surface CD30 downregulation in SLC39A7 knocked out PTCL cells. Finally, we treated PTCL lines with NVS-ZP7-4, an SLC39A7 inhibitor and the similar results were obtained regarding to CD30 expression by flow cytometry and immunoblotting. Conclusions: We performed an unbiased genome-wide CRISPR/Cas9 library screen and identified cell-intrinsic regulators for BV sensitivity in PTCLs. Our screening results suggested that the disruption of MAD2L1BP/ANAPC15 or APC/C inhibition would be a potential strategy to enhance therapeutic activity of BV in PTCLs. We also identified SLC39A7 downregulation could confer BV resistance by downregulating CD30 cell surface expression.

AFM13 in Combination with Allogeneic Natural Killer Cells (AB-101) in Relapsed or Refractory Hodgkin Lymphoma and CD30 + Peripheral T-Cell Lymphoma: A Phase 2 Study (LuminICE)

Background and Significance Limited treatment options are available for patients with relapsed or refractory (R/R) Hodgkin lymphoma (HL) and no standard-of-care therapy is established for R/R peripheral T-cell lymphoma (PTCL); novel therapies to improve outcomes in these patients are required. AFM13 is a tetravalent, bispecific innate cell engager that binds CD16A on natural killer (NK) cells and CD30 expressed (CD30 +) on HL and PTCL cells, enhancing NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). AFM13 monotherapy trials in patients with R/R HL and PTCL have shown promising clinical activity and a tolerable safety profile (Sasse et al. Blood 2020; Kim et al. Cancer Res 2023). Recently, a Phase 1/2 study of AFM13 in combination with cord blood (cb)-derived NK cells in patients with R/R CD30 + lymphomas treated at the recommended phase 2 dose (n=35) achieved an objective response rate (ORR) of 94% and a complete response (CR) rate of 71% (Nieto et al. Blood 2022, oral presentation). AB-101 is a non-genetically modified, allogeneic, cryopreserved, off-the-shelf, cb-derived NK cell product optimized for enhanced ADCC through selection for the KIR-B haplotype and the CD16 F158V polymorphism. AB-101 has demonstrated potent killing of tumor cell lines in vitro and in vivo and preliminary results of a Phase 1/2 trial of AB-101 alone and in combination with rituximab in patients with R/R B cell non-Hodgkin lymphoma demonstrated AB-101 is well tolerated (Khanal et al. J Clin Oncol 2023). Combining AFM13 with AB-101 has the potential to synergistically enhance and redirect antitumor immune responses to target HL and CD30 + PTCL cells. Study Design and Methods This Phase 2, open-label, multi-center, multi-cohort study (NCT05883449) aims to evaluate the efficacy and safety of AFM13 in combination with AB-101 in patients with R/R HL and certain R/R CD30 + PTCL subtypes. PTCL subtypes permitted are PTCL not-otherwise specified, angioimmunoblastic T-cell lymphoma, and ALK-positive and -negative anaplastic large cell lymphoma (ALCL). Patients aged ≥18 years are planned for enrolment and patients with R/R HL must have received at least two prior lines of therapy including prior combination chemotherapy, brentuximab vedotin (BV) and a checkpoint inhibitor. Patients with R/R PTCL must have confirmed CD30 expression of ≥1% by immunohistochemistry and have received at least one prior line of combination chemotherapy; patients with ALCL must have received or been intolerant to BV. Prior autologous or allogeneic hematopoietic stem cell transplant is permitted. Exclusion criteria include treatment with any anti-cancer agent ≤21 days prior to enrolment, continuing toxicity from a prior therapy, central nervous system involvement, or previous treatment with AFM13 or NK cells. The primary objective is to determine the ORR (complete and partial responses) by Independent Radiology Committee (IRC) based on PET-CT per Lugano classification. Secondary objectives include safety and immunogenicity, complete response rate, duration of response and progression free survival. Treatment will be given intravenously (IV) over 48-day cycles for up to 3 cycles. A run-in phase will assess two dose levels of AFM13 and AB-101 in 4 cohorts (Figure). A standard lymphodepletion regimen of fludarabine (30 mg/m 2/day) and cyclophosphamide (300 mg/m 2/day) will be administered IV from Day −5 to Day −3 at the start of each treatment cycle. Following this, AFM13 (200 mg or 300 mg once weekly) will be given, with AB-101 (dose level 1 or 2, see Figure) given 1 hour later per cycle. Patients will also receive 6 ×10 6 IU of IL-2 subcutaneously at least 1 hour after each AB-101 dose. Cohorts 1 and 2 will enrol in parallel; cohorts 3 and 4 will start only if cohorts 1 and 2 are cleared per protocol safety criteria. After cycle 1 has completed for each subject enrolled in all 4 cohorts, an analysis of the safety and clinical responses will be performed to determine the two dose levels to be evaluated in the main study. In addition, an exploratory cohort (cohort 5) will begin enrolment of patients with CD30 + PTCL. Disease and efficacy assessments will be conducted at screening and on Day 43 (± 3 days) of each cycle.

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 and Characterization of a Novel Murine Peripheral T-Cell Lymphoma Cell Line Which Closely Mimics Human Disease

Peripheral T-cell lymphoma (PTCL) represents a heterogenous group of aggressive non-Hodgkin lymphomas with poor prognostic outcomes and limited treatment options. Despite the advances in genomic sequencing and molecular characterization, the complex and elusive nature of PTCL's underlying biology has proven difficult to fully understand and manage effectively. The development and refinement of therapeutic strategies for PTCL are impeded by a paucity of reliable preclinical models that accurately mimic the disease's pathophysiology. There is a dire need for more physiologically relevant models for PTCL. Improved models allow for a better understanding of the disease's molecular and cellular mechanisms, facilitate the development of innovative therapeutic strategies, and ultimately improve patient outcomes. Here we described a new T-cell lymphoma cell line (LM-23) derived from a 12-week-old female Balb/cJ mouse which developed diffuse adenopathy and splenomegaly. Tumors from this mouse were cultured in cRPMI + 10% fetal bovine serum for 4 weeks without supplementation of additional cytokines and passaged 3-4 times per week where we found the cells to be non-adherent. Immunophenotyping of LM-23 demonstrated positivity for CD90, CD3, TCR-beta, CD4(partial/subset), CD8, PD1 (positive), ICOS (positive) and negative for B-cell and myeloid lineage markers (Fig. 1B). This immunophenotype most closely resembles PTCL-NOS in humans at an immunophenotypic characterization level. Whole exome sequencing is in progress and will be presented. Intravenous administration of this cell line to syngeneic Balb/cJ mice resulted in rapid establishment of diffuse adenopathy, splenomegaly, and liver metastases. Bulky adenopathy was observable within 14 days of administration. Histologically, these tumors consisted of sheets of medium to large sized lymphocytes with clumped chromatin and prominent nucleoli, similar to human T-cell lymphomas (Fig. 1A). Subcutaneous administration demonstrated rapid tumor growth with mice reaching terminal endpoints (>2,000mm 3) within 21 days following administration. As CHOP-based (cyclophosphamide, doxorubicin, vincristine, and prednisone) therapies are the primary backbone for treatment of PTCL, we tested whether CHOP therapy alone had anti-tumor activity to LM-23. Tumor bearing mice (~150mm 3) were treated with CHOP and monitored for tumor growth. All mice treated were non-responsive to CHOP therapy with no difference in tumor growth compared to untreated mice. Additionally, LM-23 bearing mice treated with anti-PD1 showed signs of hyperprogression, which has been observed in human patients in clinical trials (Gao Y et al. JCI Insight 2023, Bennani NN et al. Blood Supplement 2019). Additional therapeutic target testing is underway and will be presented. In conclusion, the newly established LM-23 T-cell lymphoma cell line provides a novel, physiologically relevant preclinical model for the study of PTCL. Its phenotypic characteristics, rapid growth, and metastatic behavior in syngeneic mice, along with its resistance to CHOP therapy and hyperprogression upon anti-PD1 treatment, mimicking the clinical scenarios observed in some human PTCL patients. These features highlight LM-23's potential as a valuable tool for understanding the biological mechanisms underlying PTCL and for the development and testing of innovative therapeutic strategies. We hope that the LM-23 cell line will contribute significantly to these efforts, ultimately improving outcomes for patients afflicted with PTCL.

Dependence of peripheral T-cell lymphoma on constitutively activated JAK3: Implication for JAK3 inhibition as a therapeutic approach.

Peripheral T-cell lymphoma (PTCL) is a clinically heterogeneous group that represents 10%-15% of all lymphomas. Despite improved genetic and molecular understanding, treatment outcomes for PTCL have not shown significant improvement. Although Janus kinase-2 (JAK2) plays an important role in myeloproliferative neoplasms, the critical role of JAK isoforms in mediating prosurvival signaling in PTCL cells is not well defined. Immunohistochemical analysis of PTCL tumors (n=96) revealed high levels of constitutively active JAK3 (pJAK3) that significantly (p<0.04) correlated with the activation state of its canonical substrate STAT3. Furthermore, constitutive activation of JAK3 and STAT3 positively correlated, at least in part, with an oncogenic tyrosine phosphatase PTPN11. Pharmacological inhibition of JAK3 but not JAK1/JAK2 significantly (p<0.001) decreased PTCL proliferation, survival and STAT3 activation. A sharp contrast was observed in the pJAK3 positivity between ALK+ (85.7%) versus ALK-negative (10.0%) in human PTCL tumors and PTCL cell lines. Moreover, JAK3 and ALK reciprocally interacted in PTCL cells, forming a complex to possibly regulate STAT3 signaling. Finally, combined inhibition of JAK3 (by WHI-P154) and ALK (by crizotinib or alectinib) significantly (p<0.01) decreased the survival of PTCL cells as compared to either agent alone by inhibiting STAT3 downstream signaling. Collectively, our findings establish that JAK3 is a therapeutic target for a subset of PTCL, and provide rationale for the clinical evaluation of JAK3 inhibitors combined with ALK-targeted therapy in PTCL.

Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells

BackgroundPatients with relapsed/refractory T-cell malignancies have limited treatment options. The use of chimeric antigen receptor (CAR)-T cell therapy for T-cell malignancies is challenging due to possible blast contamination of autologous...

PTGDS PROMOTES TUMORIGENESIS OF PERIPHERAL T CELL LYMPHOMA THROUGH REGULATING IRON METABOLISM

Introduction: Accumulating evidence indicates that ferroptosis, an iron-dependent cell death, is involved in the development of tumors, and targeting ferroptosis offers great potential in tumor therapy. However, the regulatory networks of ferroptosis in peripheral T cell lymphoma (PTCL) remain unclear. Methods: Paraffin-embedded tissues of 112 PTCL patients and 38 reactive hyperplasia cases were collected with informed consents. Lentiviral vectors were stably transfected into PTCL cell lines. Tandem mass tag (TMT)-mass spectrometry and RNA-sequencing (RNA-seq) were performed for downstream analysis. Co-IP and confocal immunofluorescence were performed to verify protein–protein interaction. Xenograft models were established using SCID Beige mice. Results: Firstly, both mRNA and protein levels of PTGDS were found to be higher in PTCL cells and patients’ tissue (Figure 1A–C). Survival analysis revealed that the positive expression of PTGDS was closely associated with worse prognosis in PTCL patients (Figure 1D). PTGDS overexpression could significantly promote the proliferation of PTCL cells while PTGDS knockdown inhibited it in vitro and in vivo (Figure 2A,B). Besides, PTCL cells with PTGDS knockdown displayed a higher apoptosis rate (Figure 2C). PTGDS specific inhibitor AT56 could significantly inhibit the proliferation of PTCL cells, in a dose- and time-dependent manner (Figure 2D). Moreover, AT56 treatment induced the cell cycle arrest at G0/G1 phase in PTCL cells (Figure 2E). Analysis based on TMT-mass spectrometry found that AT56 treatment significantly regulated the expression of ferroptosis-related molecules (Figure 3A) in PTCL cells. The anti-tumor effects of sorafenib and erastin, classic ferroptosis inducers, was significantly enhanced by AT56 treatment, and Fer-1, ferroptosis inhibitor, rescued the inhibitory role of AT56 on cell proliferation (Figure 3B,C). Moreover, AT56 promoted erastin/sorafenib-induced accumulation of lipid ROS (Figure 3D), a classic biomarker of ferroptosis. Further in-vivo experiments observed that AT56 treatment enhanced the anti-tumor effects of sorafenib (Figure 3E). Mechanically, structure analysis of differentially expressed molecules in TMT-mass spectrometry identified HMOX1, a regulator of heme catabolism, as PTGDS interactive protein. Co-IP and confocal immunofluorescence assays verified their co-localization and interaction in PTCL cells (Figure 4A,B). Moreover, AT56 treatment increased the level of intracellular iron, and HMOX1 knockdown could rescue it (Figure 4C). Keywords: aggressive T-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, molecular targeted therapies The research was funded by: National Natural Science Foundation (No. 82270200, No. 82170189, No. 82070203, No. 81800194, No. 81770210); Key Research and Development Program of Shandong Province (No. 2018CXGC1213); China Postdoctoral Science Foundation (No. 2021T1404223); Translational Research Grant of NCRCH (No. 2021WWB02, No. 2020ZKMB01); Shandong Provincial Natural Science Foundation (ZR2021YQ51); Taishan Scholars Program of Shandong Province; Shandong Provincial Engineering Research Center of Lymphoma; Academic Promotion Programme of Shandong First Medical University (No. 2019QL018). No conflicts of interests pertinent to the abstract.

The mTOR kinase inhibitor everolimus synergistically enhances the anti‐tumor effect of the Janus kinase 1 (JAK1) inhibitor AZD4205 on Peripheral T cell lymphoma

Introduction: Peripheral T-cell lymphoma (PTCL) constitute a heterogenous group of non-Hodgkin lymphomas, which is distinguished by their aggressive courses and poor clinical outcomes. Multiple researches have indicated that the JAK/STAT pathway acts as an important role in the mechanism of TCL pathogenesis. Preliminary results from related clinical studies showed that JAK inhibitors have a high remission rate and durable response in PTCL patients. AZD4205 is a potent JAK1-selective inhibitor currently evaluated in phase II clinical studies in PTCLs, which could reduce the side effects caused by JAK2 blockade by selectively targeting JAK1. Therefore, the aim of this study is to explore the efficacy and appropriate combination regimen of the novel JAK1 inhibitor AZD4205 in PTCL and to decipher the underlying anti-tumor mechanism. Methods: We analyzed the expression of JAK/STAT pathway in PTCL tissues via bioinformatics analysis and IHC. In vitro cell viability was assessed by using Cell Titer-Glo Luminescent assay. Induction of cell apoptosis was measured by flow cytometry. Western blot was utilized to identify the influence of AZD4205 on the intracellular relevant signaling pathways. The effects of AZD4205 on the transcriptome of PTCL cells were analyzed via transcriptome sequencing technology. High-throughput compounds screening was applied to explore potential combination therapeutic targets of AZD4205. Tumor-bearing mouse models were established to validate the efficacy and safety of AZD4205 and the combination regimen. Results: High expression of phosphorylated JAK1 in PTCL tissues are potential poor prognostic factors. The JAK1 inhibitor AZD4205 inhibited the proliferation of PTCL cells and down-regulated the activation of JAK/STAT signaling pathway. In addition, AZD4205 induced apoptosis in PTCL cells, and showed anti-tumor effects in vivo. However, AZD4205 also induced adverse effects of hepatic impairment and dyslipidemia in vivo. Transcriptome sequencing analysis revealed that AZD4205 treatment upregulated the expression of mTOR pathway-related genes. Moreover, high-throughput screening assay showed that the mTOR inhibitor everolimus significantly enhanced the inhibitory efficacy of AZD4205. We further confirmed that AZD4205 combined with the mTOR inhibitor everolimus exerted synergistic anti-PTCL efficacy both in vitro and in vivo, and the combination regimen significantly reduced AZD4205-induced adverse effects. The RNA-seq results suggested that the combination treatment affected the cholesterol homeostasis pathway and a further validation of the combined anti-PTCL effect of mevastatin and AZD4205 was confirmed in mouse models. Keywords: Aggressive T-cell non-Hodgkin lymphoma, Combination Therapies, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Identifying the mechanistic differences between hypomethylating agents for the treatment of peripheral T cell lymphoma

Introduction: Peripheral T-cell lymphomas (PTCL) are a rare and heterogeneous group of malignancies that share a unifying feature of epigenetic dysregulation. Recent evidence suggests that PTCL, unlike other forms of cancer or even lymphoma, exhibits a marked vulnerability to hypomethylating agents (HMAs) alone and especially in combination with HDAC inhibitors. HMAs like azacitidine (AZA) and decitabine (DAC) have been shown to reverse transcriptional repression secondary to hypermethylation. Cladribine (CLAD) is FDA-approved for the treatment of hairy cell leukemia and is postulated to inhibit both DNA and histone methylation. Here, we sought to define the mechanistic differences between different DNA HMAs in order to inform a rationale on the optimal combinations that might exploit the epigenetic vulnerabilities of PTCL. Methods: Cell viability, caspase activity assay, and western blotting (WB) were performed on a panel of T-cell lymphoma cell lines to identify the effects of AZA/DAC/CLAD on cell survival, apoptosis, and DNA methyltransferase (DNMT) levels. Liquid chromatography-based mass spectrometry (LC-MS) was done to quantify 5-methyl cytosine (5-mC) levels. Results: Cell viability analysis confirmed that the dose and time-dependent sensitivity of the six PTCL cell lines differed following exposure to AZA/DAC/CLAD, where CLAD showed the lowest IC50 value (0.1–1 µM) across all cell lines, followed by AZA (IC50: 1–10 µM), and DAC had the least effect on cell viability (IC50: >100 µM, except T-ALL). A differential threshold of caspase activation was observed across the PTCL cell lines where CLAD showed the most superior caspase activity leading to the highest apoptotic potential. However, WB analysis showed that after 24 hours of treatment, DNMT1 and DNMT3A protein levels were significantly depleted at low concentrations of DAC (<0.01 µM) compared to AZA whereas CLAD did not affect the DNMT1 and DNMT3A levels. LC-MS-based quantification of 5-mC showed a similar relationship in methylation, with initial changes observed at doses >0.01 μM of DAC and >0.1 μM of AZA/CLAD. Taking the short half-life of AZA/DAC into account, cell viability assays with daily addition of AZA/DAC showed increased cell cytotoxicity for AZA/DAC treated samples, which was comparable to CLAD. Ongoing analyses of gene expression and metabolism of the universal methyl donor S-adenosylmethionine (SAM) will help to understand the mechanistic difference between these HMAs. Conclusions: These data suggest that the HMAs have a distinct mechanism of action for hypomethylation. While AZA/DAC induces hypomethylation by depletion of DNMTs, CLAD acts in a DNMT depletion-independent pathway. A mechanistic understanding of AZA/DAC/CLAD will inform how best to use them in clinic as well as assist in the strategic development of biological correlates to further support their therapeutic application. The research was funded by: Translational Orphan Blood Cancer Research Initiative Fund and RO1 # FD-R-006814-01. Keywords: Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Synthesis and Preclinical Development of a Promising Novel Romidepsin Nanoparticle for the Treatment of Peripheral T‐Cell Lymphoma (PTCL)

Introduction: The HDAC inhibitors are important drugs for the treatment of PTCL. While these drugs produce ORR of 25–30% with short PFS, responses can last over a year. There is still debate on the optimal ways to use HDAC inhibitors. Most HDAC inhibitors are oral with prolonged AUC of exposure. Romidepsin (R) is administered IV and exhibits a half-life of only 3 hours, though biological effects on chromatin may persist for 24–48 hours. A failed Phase 4 study led to the withdrawal of R for R/R PTCL. In an effort to try and create improved alternatives to R with better efficacy and safety, we deployed a proprietary nanochemistry platform to synthesize NanoRomidepsin (NR) particles, identifying a lead molecule that is comparable to or better than the predecessor R. Methods: Using a combinatorial strategy of solvent screening and polymer chemistry, we developed a first-in-class NR therapeutic with a unique and specific Drug/ Polymer/Surfactant ratio. We utilized dynamic Light Scattering (DLS), cryo-electron microscopy (EM) and liquid chromatography-mass spectrometry (LC-MS) to characterize the size, morphology, and drug encapsulation efficiency of the NR nanoparticles. Cell viability, flow cytometry and western blotting (WB) were performed to determine the effect of NR on cytotoxicity, apoptosis, and histone acetylation across a panel of PTCL cell lines. The maximum tolerated dose of NR was based on an in vivo single-dose and repeated dose toxicity study. Pharmacokinetic studies were performed in mice treated at ½ MTD with quantitation of plasma drug levels by LC-MS. Results: Cryo-EM and DLS confirmed that NR had a poly-dispersity index (PDI) between 0.16 to 0.27 and a z-average of 60–70 nm, indicating uniform size and homogenous particle distribution in the desired ranges. The cell viability assays demonstrated that R and NR produced comparable cytotoxicity (IC50 in range of 0.7–1.9 nM). Flow cytometry and WB analysis demonstrated that NR induced apoptosis and increased acetylation of H3/H4 in a time and concentration-dependent manner. In vivo single-dose and repeated dose toxicity assays revealed a clear correlation between dose and weight loss and/or clinical score in both R and NR treated groups. Pharmacokinetic analyses revealed a substantially greater AUC for NR, which was 3- and 25-fold higher compared to R for IP and IV routes of administration respectively. Interestingly, the markedly greater AUC for NR did not appear to be associated with increased in vivo toxicity. Conclusions: We have synthesized a lead nanotherapeutic of R that will be translated to the clinic. In keeping with other nanotherapeutic drugs, nanosizing drugs tend to increase their volume of distribution (Vd), AUC and markedly improve their tolerability. Large scale in vivo modeling of NR is underway. Data on the development and differentiation of our lead NR asset will be presented, as will the strategy for clinical development. The research was funded by: IVY grant, Translational Orphan Blood Cancer Research Initiative Fund Keyword: Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Synergistic anti-tumor activity of the mTOR inhibitor everolimus and gemcitabine for the treatment of peripheral T cell lymphoma.

e19538 Background: Patients with relapsed/refractory (R/R) peripheral T-cell lymphoma (PTCL) have dismal prognosis and limited treatment options. The mTOR inhibitor everolimus or gemcitabine as a single agent regimen had limited clinical efficacy in patients with R/R PTCL. Therefore, investigation of active combination therapies with everolimus for T cell lymphoma needs to be investigated. Methods: A retrospective analysis of 24 R/R PTCL patients were treated with everolimus and gemcitabine from December 2017 to March 2021. The primary endpoint was objective response rate (ORR). CellTiter Glo and AnnexinV FITC/PI were utilized to determine cell viability and apoptosis, respectively. Results: Here we demonstrated that combination treatment of everolimus and gemcitabine is effective in treating R/R PTCL patients, achieving an ORR of 70.8%, with 45.8% complete response rate. The median time to response was 1.7 months. With a median follow-up time of 22.3 months (95% confidence interval[CI], 5.7-39.0), the median PFS of all patients was 9.9 months (95%CI, 0-20.81), the median DOR was 16.8 months for responders. Patients with best response of CR and PR achieved superior PFS (18.7 vs. 1.2 months, P = 0.0007) and OS (30.9 vs. 5.8 months, P = 0.021) than that with SD and PD. Grade 3 or 4 neutropenia, thrombocytopenia and anaemia occurred in 13 (29.2% grade 3 and 25.0% grade 4), 11 (8.3% grade 3 and 37.5% grade 4) and 7 (16.6% grade 3 and 12.5% grade 4) patients, respectively. Functional in vitro studies showed that combination treatment synergistically inhibited cell viability and induced apoptosis in PTCL cell lines. Mechanistically, transcriptomic profiling indicated that the synergistic effect was induced through the abrogation of MYC pathway. Conclusions: Collectively, our study sheds light on synergistic antitumor effect of combinatorial therapy, providing a potential therapeutic strategy for patients with R/R PTCL.

Abstract CT024: REDIRECT: A Phase 2 study of AFM13 in patients with CD30-positive relapsed or refractory (R/R) peripheral T cell lymphoma (PTCL)

Abstract PTCLs are aggressive hematologic malignancies often with poor prognoses; for patients with R/R PTCL there is no standard-of-care therapy and novel treatments are required. AFM13 is a tetravalent, bispecific Innate Cell Engager (ICE ® ) that binds CD30 when expressed on PTCL cells, and CD16A on innate effector cells, redirecting and enhancing the innate immune response to CD30+ tumor cells. Phase 1 clinical studies of AFM13 in patients with R/R Hodgkin lymphoma and cutaneous CD30+ lymphomas showed a tolerable safety profile and clinical activity; early correlative science data in a small group of patients with HL showed increased natural killer (NK) cell activity in responsive patients. A Phase 2, open-label, multi-cohort study (NCT04101331) assessing the efficacy of AFM13 in patients with R/R PTCL was initiated in 2019. Patients had histologically confirmed CD30 expression in ≥1% of tumor cells and had received ≥1 prior systemic therapy. The primary endpoint was to assess the overall response rate (ORR) based on FDG-PET per independent review committee. Secondary endpoints included safety, complete response rate (CRR), duration of response (DoR), pharmacokinetics, immunogenicity, and quality of life. Progression-free survival (PFS) and overall survival (OS) were exploratory endpoints. Patients received 200 mg AFM13 intravenously once weekly until disease progression, intolerable toxicity, termination at the investigator’s discretion, or withdrawal of consent. A total of 108 patients (age 21-93; 61% male) received AFM13, with a median (min, max) number of infusions of 9.0 (1, 116). Numbers of patients per PTCL subtype assessed were: PTCL not-otherwise-specified (PTCL-NOS), 41; angioimmunoblastic T cell lymphoma (AITL), 30; anaplastic large cell lymphoma (ALCL), 26; other, 11. Patients received a mean number of 2.7 prior treatment lines; 46.3% received prior brentuximab vedotin (BV), 17.6% received prior auto-transplant. The ORR was 32.4% (CRR was 10.2%); ORR in each subgroup was 22.0% (PTCL-NOS), 53.3% (AITL), 23.1% (ALCL), and 36.4% (other). Median DoR, PFS, and OS were 2.3 months, 3.5 months, and 13.8 months, respectively. Reported treatment emergent adverse events (TEAEs) were as anticipated based on previous studies. AFM13-related TEAEs occurred in 79/108 patients (73.1%), with 14 events in 9 patients (8%) considered serious. The most frequent TEAE was infusion-related reactions, observed in 34/108 patients (31.5%), including 12 Grade 3 events in 6 patients (5.7%). Neutropenia was the most frequent TEAE with Grade ≥3 occurring in 9.3% of patients. AFM13 monotherapy was well managed and showed robust clinical activity in selected R/R PTCL subtypes. These data, together with encouraging preliminary efficacy seen in AFM13 combination studies in HL, support further evaluation of AFM13 in combination with NK cells to augment the innate immune response to CD30+ tumors. Citation Format: Won Seog Kim, Jake Shortt, Pier Luigi Zinzani, Natalya Mikhaylova, Ana Marin-Niebla, Dejan Radeski, Vincent Ribrag, Eva Domingo Domenech, Ahmed Sawas, Karenza Alexis, Michael Emig, Linta Garcia, Andre Overesch, Kerstin Pietzko, Steven Horwitz. REDIRECT: A Phase 2 study of AFM13 in patients with CD30-positive relapsed or refractory (R/R) peripheral T cell lymphoma (PTCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT024.

AT56 Exerts Anti-Tumor Effects in Peripheral T Cell Lymphoma By Regulating HMOX1-Mediated Ferroptosis

Introduction: Accumulating evidence indicates that prostaglandin D2 synthase (PTGDS) plays important roles in various diseases, including tumor. Ferroptosis, a recently recognized form of cell death, has been found to participate in the development of hematologic tumor, especially lymphoma. Here, we investigated the effects of AT56, the specific inhibitor of PTGDS, and its regulatory role on ferroptosis in peripheral T cell lymphoma (PTCL). Methods: Lymph node biopsies from 112 de novo PTCL patients and 38 reactive hyperplasia cases. RNA-sequencing and Tandem mass tag (TMT)-mass spectrometry were conducted in PTCL cells treated with AT56. C11-BODIPY was used to detect the level of lipid ROS. Xenograft PTCL mice model was established to evaluate drug efficacy. Co-immunoprecipitation (Co-IP) and confocal immunofluorescence (IF) were performed to validate protein interaction. Plasmids of HMOX1-WT and HMOX1-H25A were synthesized and transfected into PTCL cells. Results: Firstly, we explored the expression and function of PTGDS in PTCL. The expression level of PTGDS in tumor tissue (n=112) was found to be higher than control group. Survival analysis revealed that elevated PTGDS expression predicted inferior prognosis in PTCL patients. Gene ontology (GO) analysis indicated that AT56 treatment was associated with biological processes involved in tumor progression, such as cell death, apoptosis, and cell cycle. Then, AT56 could inhibit the proliferation of PTCL cells in a dose- and time-dependent manner. Analysis based on TMT-mass spectrometry revealed the differential expression of ferroptosis-associated proteins after AT56 treatment. As erastin and sorafenib are ferroptosis inducers, AT56 significantly enhanced their effects on the accumulation of lipid ROS (Fig. 1), a classic biomarker of ferroptosis. Besides, ferrostatin-1(Fer-1), a specific ferroptosis inhibitor reversed AT56-induced lipid ROS accumulation and cell proliferation inhibition. Furthermore, the combined drug effects of AT56 and sorafenib was revealed in xenograft mice model, indicating the regulatory role of AT56 on ferroptosis in PTCL. As ferroptosis is characterized by its dependence on iron accumulation, reactive oxygen species (ROS) production, lipid peroxidation, we further explored the specific mechanism by which AT56 regulated ferroptosis process. In our study, ferroptosis-associated molecules (NRF2, KEAP1) and iron-associated molecules (NCOA4, FTH, FTL) were aberrantly expressed after AT56 treatment, indicating ferroptosis activation, while the expression of lipid and ROS-associated molecules (ACSL4, xCT, GPX4) displayed compensatory changes. As expected, deferoxamine (DFO), an iron chelator, rescued the effect of AT56 on cell proliferation and lipid ROS accumulation. Elevated level of Fe2+ was found in tumor tissue from mice receiving AT56, indicating that the role of AT56 on ferroptosis mainly depended on iron accumulation. High expression of NCOA4 indicated the involvement of autophagy in ferroptosis process induced by AT56. Chloroquine, a common autophagy inhibitor, could partly reverse AT56-induced accumulation of lipid ROS and Fe2+, and expression of Fe2+ storage protein, suggesting the dependence of AT56-induced ferroptosis on autophagy in PTCL cells. Structure analysis based on TMT-mass spectrometry identified HMOX1, a regulator of heme catabolism and autophagy, as PTGDS-interacting protein, and further Co-IP and IF assays validated their co-localization and interaction in PTCL cells. Besides, HMOX1 knockdown could rescue AT56-induced cell proliferation inhibition and Fe2+ accumulation (Fig. 2). Furthermore, point mutation of HMOX1 (H25A) was found to inhibit its regulatory role on ferroptosis. Conclusions: Taken together, our investigations identified for the first time that high expression of PTGDS was associated with poor prognosis in PTCL patients. AT56 exerted potent anti-tumor effects in PTCL via promoting ferroptosis process, which was mediated by HMOX1 and partly dependent on autophagy. Our in vitro and in vivo study revealed the synergistic effect and mechanism of AT56 and sorafenib, providing a novel AT56-based treatment paradigm for PTCL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

FFA Promoting Proliferation and Migration Via Classical Wnt Signaling Pathway in Peripheral T Cell Lymphoma

Background: Peripheral t cell lymphoma (PTCL) is an important component of NHL, the overall prognosis is worse than B Cell Lymphoma. In the hyperlipidaemic microenvironment, exploration of the relationship between de novo adipose synthesis and the proliferation and migration capacity of PTCL cells will help to discover therapeutic targets that interfere with tumor cell growth. This study attempts to explore that abnormally high levels of free fatty acids in the tumor microenvironment may promote PTCL migration and proliferation through the classical Wnt signaling pathway Methods: The proliferation activity of EL4 cells treated with high fatty acids（OA:OP=2:1） was detected by CCK8 method, and the proportion of cells with tumor-stemness was detected by flow cytometry and Hoechst33342 stain; A C57BL FGF21-/-mouse model of non-alcoholic steatohepatitis (Nash) was constructed with the MCD method (Cholesterol & Cholate Methionine/choline deficient); PTCL tumor-bearing mice model was established by injecting EL4 cell into spleen in situ; WB method was used to detect the activation of WNT signal pathway in vitro and vivo; migration was evaluated by transwell assay. Results: 1. In Vitro, in the OP+ EL4 group, Axin-1 and p-β-catenin were both significantly decreased (P<0.001, P=0.005), additionally treated with XAV939 (WNT inhibitor) or LiCl (WNT agonistor), Axin-1 and p-β-catenin both showed a significant increase (P<0.001, P=0.002) or degradation of GSK3 β/p-β-catenin (P=0.033, P<0.001). Suggesting that OP could activate the Wnt pathway in EL4 cells. 2. In Vivo, compared with the control group, the FGF21-/-MCD Group had the most number of nodules on the surface of the liver (FGF21-/-MCD vs WT-CD, P<0.001, vs WT-MCD, P=0.009, vs FGF21-/-CD, P<0.001), In FGF21-/-MCD Group, β-Catenin was increased in tumor tissues and adjacent tissues, especially in tumor tissues (P<0.05 in each group). Suggested that the migration enhancer in gross, and the mechanism may related with Wnt signaling pathway. 3. The co-culture assay showed that OP+El4 enhanced its migration ability, which could be decreased by Xav939 and enhanced by Licl(P<0.05 in each group). 4. in Vivo, After treatment with PPARA agonist, β-catenin EPCAM decreased in all MCD groups (P<0.05). It is suggested that fenofibrate can improve the tumor outcome by correcting abnormal lipid metabolism and weakening the activation of Wnt pathway. Conclusion: High free fatty acids are associated with a poor prognosis in PTCL. Possibly mechanism is the activation of the canonical Wnt pathway by free fatty acids, which promote enhanced tumor cell proliferation and migration capacity. Effective Hypolipidemic therapy may be a new combined therapeutic target for PTCL patients with hyperlipidemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Evidence Based Reporting System for Molecular Tumorboards Identifies Novel Therapeutics for T-Cell Lymphomas

Introduction Peripheral T-cell lymphomas (PTCLs) exhibit significant diagnostic and therapeutic challenges. Following current treatment guidelines, the majority of patients either do not achieve a remission or experience an early relapse with fatal outcome. Hence, development of innovative therapeutic strategies targeting the underlying biology of PTCLs is highly demanded. To identify novel therapeutic options for PTCL, we here compiled a comprehensive database of genetic alterations in T-cell lymphomas and utilized bioinformatic methodology for evidence-based reporting of treatment options in the context of molecular tumorboards. Methods Utilizing newly developed bioinformatic methodology for molecular tumorboards1,2, genetic data from >1800 patients with T-cell lymphomas (TCL) compiled from public available sources were analyzed and queried for potential therapeutics addressing genetic alterations in TCL. Based on the results of this approach, a broad in vitro drug screening was performed to assess the activity of potential therapeutics in genetically and transcriptionally characterized cell lines of TCL and address potential biomarkers of response and resistance. Furthermore, functional apoptosis profiling, combination drug screening and mechanistic work up was performed to identify and mechanistically explore novel drug combinations. Results Integration of genetic alterations and drug sensitivity screens revealed novel therapeutic strategies for PTCL. Notably, multiple components of DNA damage response pathways were suggested as therapeutic targets, most prominently the cell cycle regulator WEE1. Indeed, the clinical-grade WEE1 kinase inhibitor, adavosertib, potently induced premature mitotic entry, accumulation of DNA damage and induction of apoptosis in PTCL cell lines, predominantly in cell line models harboring genetic alterations in the ATR signaling pathway. To further enhance the therapeutic effects of WEE1 inhibition, we explored potential combination strategies through mechanistic studies and functional apoptosis profiling. Here, we identified synergistic effects in DNA damage response pathways, cell cycle regulation and modulation of anti-apoptotic proteins through combined WEE1 and JAK/STAT inhibition. Indeed, the combination of adavosertib and ruxolitinib showed marked synergistic cytotoxicity in vitro in genetically characterized subsets of T-cell lymphoma. Conclusions Our results identified WEE1 as a promising therapeutic target for genetically defined PTCL subgroups. Furthermore, mechanistic studies identified concepts for rational combination strategies of WEE1 and JAK/STAT inhibition through modulation of DNA-damage response and priming for apoptosis.