PTCL Cells Research Articles

Abstract 4605: AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-cell lymphoma models

Abstract Peripheral T-cell Lymphoma (pTCL) makes up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor prognosis. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E. Here we examine the efficacy of CDK9 inhibition for the treatment of pTCL. CDK9 regulates transcription through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor; acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are often overexpressed in hematologic tumors. It has been shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression of BCL-2 family members is highly heterogeneous in pTCL, with MCL-1 being the most universally expressed. Using MCL-1 inhibitor AZD5991, we showed statistically significant benefit in survival when combined with CHOP in MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition, we have previously reported the importance of the anti-apoptotic protein BFL-1 in cell survival in NHL, including a subset of pTCL (Boiko et al 2021). AZD4573 treatment showed a range of activity across the panel of 18 human pTCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, with EC50 values less than 100 nM, and 13 reached a max caspase activation of over 40%. pTCL subtypes that responded to AZD4573 treatment included ALCL, NK-TCL, and PTCL-NOS and CTCL. Acute AZD4573 treatment resulted in decreased pSer2-RNAP2 and reduced protein levels of c-MYC and MCL-1. To determine what was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 for viability after KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that efficacy is mediated through MCL-1 in ALCL pTCL cell lines assessed. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and measured viability after 24-72 hours using RealTime-Glo after 24hr exposure to CHOP with AZD4573 added for the last 6hrs. Adding AZD4573 to CHOP in vitro increased the efficacy in pTCL ALCL cell lines with MCL-1 dependency, but not in CTCL cell line with BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels but not MCL-1, suggesting that combination benefit may be driven through c-MYC. This data suggests that treatment with AZD4573 as a monotherapy or in combination with CHOP regimen would be an effective therapeutic strategy in pTCL. Citation Format: Danielle Sinead Potter, India Ott, Jamal Saeh, Richard Olsson, Stephen Fawell, Lisa Drew, Justine Roderick. AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-cell lymphoma models [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 4605.

The gene expression profile and cell of origin of canine peripheral T-cell lymphoma

BackgroundPeripheral T-cell lymphoma (PTCL) refers to a heterogenous group of T-cell neoplasms with poor treatment responses and survival times. Canine PTCL clinically and immunophenotypically resembles the most common human subtype, PTCL-not otherwise specified (PTCL-NOS), leading to interest in this canine disease as a naturally occurring model for human PTCL. Gene expression profiling in human PTCL-NOS has helped characterize this ambiguous diagnosis into distinct subtypes, but similar gene expression profiling in canine PTCL is lacking.MethodsBulk RNA-sequencing was performed on tumor samples from 33 dogs with either CD4+ (26/33), CD8+ (4/33), or CD4-CD8- (3/33) PTCL as diagnosed by flow cytometry, and sorted CD4+ and CD8+ lymphocytes from healthy control dogs. Following normalization of RNA-seq data, we performed differential gene expression and unsupervised clustering methods. Gene set enrichment analysis was performed to determine the enrichment of canine CD4+ PTCL for human PTCL-NOS, oncogenic pathways, and various stages of T-cell development gene signatures. We utilized gene set variation analysis to evaluate individual canine CD4+ PTCLs for various human and murine T-cell and thymocyte gene signatures. Cultured canine PTCL cells were treated with a pan-PI3K inhibitor, and cell survival and proliferation were compared to DMSO-treated controls. Expression of GATA3 and phosphorylated AKT was validated by immunohistochemistry.ResultsWhile the canine CD4+ PTCL phenotype exhibited a consistent gene expression profile, the expression profiles of CD8+ and CD4-CD8- canine PTCLs were more heterogeneous. Canine CD4+ PTCL had increased expression of GATA3, upregulation of its target genes, enrichment for PI3K/AKT/mTOR signaling, and downregulation of PTEN, features consistent with the more aggressive GATA3-PTCL subtype of human PTCL-NOS. In vitro assays validated the reliance of canine CD4+ PTCL cells on PI3K/AKT/mTOR signaling for survival and proliferation. Canine CD4+ PTCL was enriched for thymic precursor gene signatures, exhibited increased expression of markers of immaturity (CD34, KIT, DNTT, and CCR9), and downregulated genes associated with the T-cell receptor, MHC class II associated genes (DLA-DQA1, DLA-DRA, HLA-DQB1, and HLA-DQB2), and CD25.ConclusionsCanine CD4+ PTCL most closely resembled the GATA3-PTCL subtype of PTCL-NOS and may originate from an earlier stage of T-cell development than the more conventionally posited mature T-helper cell origin.

Targeting the Cgas-Sting Pathway Inhibits the Progression and Enhances the Chemotherapy Efficacy of Peripheral T-Cell Lymphoma

Objective Peripheral T-cell lymphoma (PTCL) is a highly heterogeneous group of mature T-cell malignancies. Owing to its complex clinicopathological characteristics and diagnostic difficulties, the underlying molecular mechanism leading to the high invasiveness and rapid progression remains significantly unidentified. The efficacy of current first-line treatment is dismal, novel agents and therapeutic strategies are urgently needed to improve the prognosis of PTCL patients, especially for relapsed/refractory (RR) ones. Recent studies have indicated a close association between the cGAS-STING pathway and tumor promotion, which may provide new therapeutic targets. Herein, we investigated the role of the cGAS-STING pathway and its underlying mechanism in PTCL progression. Methods Firstly, we conducted single-cell RNA sequencing (scRNA-seq) to examine cellular identity and investigate the expression heterogeneity of cGAS-STING pathway. The cGAS-STING pathway score (CSPS) was calculated based on the expression of seven marker genes of this pathway. Immunofluorescence was performed to detect cGAS and STING expression in PTCL tissue microarray containing 35 PTCL samples and 8 normal lymph node biopsies. To assess the tumor-killing activity of cGAS-STING pathway inhibitors, here we tested five drugs including cGAS inhibitors G150 and G140, STING inhibitor H151, TANK binding kinase-1 (TBK1) inhibitor TBK1/Ikkε-IN-5 and signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic. Protein mass spectrometry was used to evaluate the protein changes in PTCL cell lines after G150 treatment. Next, we established a xenograft model to test the anti-tumor effect of G150 and TG003 (an inhibitor of CLK1) single-agent or combination therapy in vivo. Furthermore, we applied single-cell dynamic RNA sequencing to investigate the mechanism on the synergistic effect of TG003 and G150 in inhibition of cell proliferation. CCK8 and flow cytometric analysis were performed to detected the cellular viability and the apoptotic rate with the drug combination compared with single-drug treatment. Results scRNA-seq and showed the cGAS-STING pathway was overexpressed in PTCL with high heterogeneity and could be used as a prognostic marke (Fig. A-D). cGAS expression was positively correlated with DNA repair. cGAS inhibition effectively suppressed tumor growth via cGAS-STING-RELB-STAT3 signaling pathway and impairing DNA damage repair in PTCL. G150 treatment led to a concentration-dependent DNA damage (Fig. E-G). Moreover, single-cell dynamic RNA sequencing revealed that TG003 functions through inducing the reduction of proliferation-related nascent RNAs on the basis of G150 treatment (Fig. H,I). CLK1 played a critical role in the survival of residual tumor cells after treatment with cGAS inhibitors, and CLK1 suppression could enhance the sensitivity to cGAS inhibitors (Fig. J). In first-line therapy, chemotherapy-induced DNA damage activated the cGAS-STING pathway, which may ultimately lead to relapse or progression. cGAS inhibitors could synergize with chemotherapeutic agents in tumor killing (Fig. K-N). Fig. O summarized the oncogenic role of cGAS-STING pathway and the interaction between Tfh cells and B cells in AITL. Conclusion Herein, we demonstrated for the first time that the activated cGAS-STING pathway plays an oncogenic role in PTCL progression, in part by enhancing DNA repair and proliferation, which may also trigger chemotherapy resistance and ultimate relapse. CLK1 may serve as an indicator for the sensitivity to cGAS inhibitors. This may partly unveil the deficiency of first-line therapy and lay foundation for clinical application of cGAS-STING pathway-associated inhibitors.

AZD4573 in Combination with CHOP Increases Combination Benefit in Preclinical Peripheral T-Cell Lymphomas Models

Peripheral T-cell Lymphoma (pTCL) make up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor outcomes to standard of care therapy. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E and for CD30-postive pTCLs, brentuximab vedotin is approved. CDK9 regulates transcription elongation through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor, acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are frequently overexpressed in haematologic tumours. It has been previously shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression and dependencies of the anti-apoptotic BCL-2 family members is highly heterogeneous in pTCL; however MCL-1 is the most universally expressed BCL-2 family member. Using the MCL-1 inhibitor AZD5991, we have shown statistically significant benefit in survival when combined with CHOP in 2 MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition to MCL-1, we have previously reported the importance of the anti-apoptotic protein BFL-1 in mediating cell survival in NHL, including a subset of pTCL (Boiko et al 2021). Consistent with these findings, AZD4573 treatment exhibited a range of activity across the panel of 18 human TCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, having EC 50 values < 100 nM, and 13 reached a max caspase-3/7 activation of greater than 40%. pTCL cell lines that responded to AZD4573 treatment including ALCL, NK-TCL, and PTCL-NOS and CTCL subtypes. Acute AZD4573 treatment of pTCL cell lines resulted in decreased p-SerRNApoll II and reduced levels of c-MYC and MCL-1 protein levels. To determine if MCL-1 or c-MYC was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 after long term KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that AZD4573 efficacy is mediated through MCL-1, in ALCL pTCL cell lines tested. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and assessed for viability after 24-72 hours using RealTime-Glo after 18hr exposure to CHOP and AZD4573 added for the last 6hrs. Adding AZD4573 treatment to CHOP schedule in vitro increased the combination benefit in pTCL ALCL cell lines that show MCL-1 dependency, but not in CTCL cell line which show a BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels at 24-48 hours, but not MCL-1 suggesting that combination benefit may be driven through decreased c-MYC levels, as KO of c-MYC resulted is loss of cell viability in these models. These data suggested that treatment with AD4573 either as a monotherapy or in combination with CHOP, would be an effective therapeutic strategy in pTCL. AZD4573 monotherapy is currently being evaluated in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK in patients with relapsed/refractory pTCL.

The CD38/CD3xCD28 Trispecific Antibody (SAR442257) Potentially Represents a Novel Therapeutic Strategy for Peripheral T-Cell Lymphomas

Introduction Peripheral T-cell lymphomas (PTCL) remain an unmet medical need and novel therapeutic options inducing longstanding responses are needed. Immunotherapies with humanized monoclonal antibodies targeting cell surface receptors have dramatically improved the prognosis of many cancers. However, so far, only two antibodies targeting CCR4 and CD30 have been approved for the treatment of subsets of PTCL patients. Currently, several bi- and tri-specific antibodies are under pre-clinical or clinical evaluation to treat lymphomas. TSAR442257, a trispecific antibody (TriAb) which targets and binds CD38, CD3 and CD28 to engage T-cells against CD38-expressing tumor cells and enhance their CD3-triggered activation via CD28 co-stimulation. Here, we performed a preclinical investigation to evaluate the potential therapeutic value of the SAR442257 to treat PTCL, knowing that both CD28 and CD38 might represent targets in tumor T cells. Material and methodsCD28 and CD38 expression was studied in tumor cells of 268 PTCL patients. In situ semi-quantitative evaluation was done using immunohistochemistry on FFPE samples (n=250), and quantitative evaluation was performed using flow cytometry (FCM) on PBMCs (n=18) and PTCL lines (n=6). Multiplex immunofluorescence stainings with tumor cell marker were also performed on biopsies sections of selected cases. In vitro cytotoxic assays were performed by using 24h co-cultures of PTCL cell lines stained with a viability dye with allogeneic blood cells from healthy donors (HD) and SAR442257 or control TriAbs with null mutations in CD28 (D28), CD38 (D38) or both recognition domains at various concentrations (0-25000 pM) at 10:1 effector-to-target ratio. For primary PTCL cells, similar experiments were done with appropriate panels of surface markers aiming to identify tumor cells. Activation assays, measuring expression of CD25 and CD69, were done on PTCL lines and PBMCs from HD using the afore mentioned TriAbs at similar concentrations. ResultsWe found a recurrent but variable expression of CD28 and CD38 among the different PTCL entities (Figure 1). Overall, CD38 expression by tumor cells was found in 42% of PTCL samples in situ and 67% of PBMCs (ranging from 2.5% to 65%; data not shown). CD28 was found in neoplastic T cells in 56% of PTCL samples by IHC and 100% of PBMCs with a strong heterogeneity ranging from 30% to 99.4% (data not shown). CD38 and CD28 showed a range of expression among PTCL categories, with CD38 being more frequently expressed (Avg. IHC score: 250-300)) in PTCL diseases with a cytotoxic profile postulated to derive from innate immune/cytotoxic cells whereas CD28 was more frequently expressed (Avg. IHC score: 150-200) in PTCLs known to derive from helper T-cells (p<0.001, Figure 2). Co-expression of both targets was rare. Multiplex stainings confirmed co-expression of CD38 and/or CD28 targets in tumor cells but consistent with the FCM analysis, CD28 and CD38 were variably expressed in tumor T-cells of TFH PTCLs. The SAR442257 induced efficient killing of all CD28+/CD38+ and CD28-/CD38+ PTCL lines (with maximum killing reaching about 35%), while the D28 and D38 control TriAbs showed less efficient cytotoxic activities in six different cell lines. SAR442257 also induced CD25 and CD69 expression on normal T-cells suggesting efficient T-cell activation, (data not shown). Conclusion Altogether, this study shows that 1) most PTCL cells express at least CD28 or CD38, and 2) SAR442257 can efficiently kill malignant PTCL cells, while ensuring effective T-cell activation; In view of these results, clinical investigation of SAR442257 in PTCL is warranted.

A Novel and Potent EZH1/2 Dual Inhibitor, HM97662 Demonstrated a Wide Spectrum of Therapeutic Potential for Hematological Malignancies

Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homolog 2 (EZH2) and its homolog EZH1 are catalytic components of polycomb repressive complex 2 (PRC2), which tri-methylate histone H3 at lysine 27 (H3K27me3) to repress transcription of their target genes. Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677, and A687) has been associated with the progression of hematological malignancies including B-cell lymphoma, T-cell lymphoma, and multiple myeloma. Hence, it has been suggested that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Although the methyltransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. Moreover, EZH1 directly binds to chromatin and modulates its condensation. Therefore, dual inhibition of EZH1 and EZH2 can give better effect than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we presented a novel EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. SPR analysis of HM97662 on trimeric complexes (EZH1/EZH2, SUZ12, and EED) demonstrated that HM97662 binds both EZH1- and EZH2-PRC2 complexes. HM97662 potently repressed tri-methylation of H3K27 in KARPAS 422 DLBCL, HH PTCL, and MM.1S MM cell lines. Furthermore, HM97662 showed broader and stronger activities than EZH2 selective inhibitors in long-term proliferation assay against a variety of hematological cancer cell lines. HM97662 induced the differentiation of DLBCL to plasma cells with the increment of cell lineage specific markers (e.g. PRDM1 and CD38), and caused cell cycle G0/G1 arrest and apoptosis in KARPAS 422 DLBCL cells harboring EZH2 Y641N GOF mutation. Additionally, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH PTCL cell line. Two cell cycle repressors, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were significantly increased by HM97662. We further conducted a chromatin accessibility assay and identified that the chromatin structures of that genes were loosened and highly transcribed after the treatment of HM97662. As a result, HM97662 showed potent antitumor activity in various hematological cell lines xenograft mouse models including B-cell lymphoma, T-cell lymphoma, and multiple myeloma (e.g. KARPAS 422, HuT-102, and MM.1S). Once daily oral dosing of HM97662 greatly inhibited tumor growth in a dose-dependent manner without significant clinical signs compared to the known EZH2 selective or EZH1/2 dual inhibitor. In conclusion, the present preclinical studies demonstrated that HM97662, an EZH1/2 dual inhibitor, had a promising and wide spectrum of therapeutic potential for hematological malignancies. It is urgent to assess the effectiveness of HM97662 in further clinical trials.

AFM13 IN PATIENTS WITH CD30 POSITIVE RELAPSED OR REFRACTORY (R/R) PERIPHERAL T CELL LYMPHOMA (PTCL): RESULTS FROM THE PHASE 2 REDIRECT STUDY

Introduction: PTCLs are aggressive hematologic malignancies often with poor prognoses; there is no standard of care therapy for patients with R/R PTCL. AFM13, a tetravalent, bispecific innate cell engager, binds CD30 when expressed on PTCL cells, and CD16A on innate effector cells, augmenting the innate immune response to CD30+ tumor cells. Phase 1 clinical trials of AFM13 in patients with R/R Hodgkin lymphoma (HL) and cutaneous CD30+lymphomas showed a tolerable safety profile and clinical activity; correlative science data in a small group of AFM13-responsive patients with HL showed increased natural killer (NK) cell activity. Methods: A Phase 2 study (NCT04101331) assessed the efficacy of AFM13 in patients with select R/R PTCL subtypes, exhibiting histologically confirmed CD30+expression in ≥1% tumor cells, who had received ≥1 prior systemic therapy. The primary endpoint was to assess the overall response rate (ORR) by FDG-PET per independent review committee. Secondary endpoints included safety, complete response rate (CRR), and duration of response (DoR). Progression-free survival (PFS) and overall survival (OS) were exploratory endpoints. Patients received 200 mg AFM13 intravenously once weekly until disease progression, intolerable toxicity, consent withdrawal or termination by the investigator. Results: Patients (n = 108; age 21–93; 61% male) received AFM13 with a median (min, max) number of infusions of 9.0 (1, 116). PTCL subtypes assessed (n) were: PTCL not-otherwise-specified (PTCL-NOS, 41); angioimmunoblastic T cell lymphoma (AITL, 30); anaplastic large cell lymphoma (ALCL, 26); other (11). Patients had received a mean 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 per subtype 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. AFM13-related treatment emergent adverse events (TEAEs) occurred in 79/108 patients (73.1%); 14 events in 9 patients (8.0%) were considered serious. The most frequent TEAE was infusion-related reactions, as reported by the investigator, seen in 34/108 patients (31.5%), including 12 Grade 3 events in 6 patients (5.7%). Neutropenia was the most frequent Grade ≥3 TEAE occurring in 9.3% of patients. Conclusion: AFM13 monotherapy demonstrated robust clinical activity in heavily pretreated patients with R/R PTCL. The safety profile of AFM13 was well managed and consistent with previously reported data from prior and ongoing clinical studies with AFM13. These data support future evaluation of AFM13 in combination with other immunotherapies, including allogeneic NK cells, to further potentiate anti-tumor immune responses to CD30+ lymphomas. Keyword: immunotherapy Encore Abstract—previously submitted to AACR 2023 The research was funded by: Affimed GmbH Conflicts of interests pertinent to the abstract W. S. Kim Research funding: Sanofi; BeiGene; Boryung; Roche; Kyowa Kirin Co., Ltd.; Dong-A Pharmaceutical Co., Ltd. J. Shortt Consultant or advisory role: Bristol Myers Squibb; Novartis; Otsuka; Astellis; Mundipharma; Pfizer Research funding: Astex; Amgen; Bristol Myers Squibb; Novartis P. L. Zinzani Consultant or advisory role: MSD; Eusapharma; Novartis; Celltrion Healthcare; Gilead; Janssen-Cilag; Bristol Myers Squibb; Servier; AstraZeneca; Takeda; Roche; Kyowa Kirin; Incyte; BeiGene; Secura Bio; Sandoz; ADC Therapeutics A. Marin-Niebla Other remuneration: Janssen; Gilead-Kite; Roche; Eli Lilly; Takeda; Kiowa-Kirin; AbbVie; AstraZeneca; BeiGene K. Alexis Employment or leadership position: Affimed Inc. Stock ownership: Affimed Inc. M. Emig Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH L. Garcia Employment or leadership position: Affimed Inc. Stock ownership: Affimed Inc. A. Overesch Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH K. Pietzko Employment or leadership position: Affimed GmbH Stock ownership: Affimed GmbH S. Horwitz Consultant or advisory role: Affimed; Daiichi Sankyo; Kyowa Kirin; ONO Pharmaceuticals; SecuraBio; Takeda; Shoreline Pharmaceuticals; Yingli Pharma; Abcuro Inc. Tublulis GmbH Research funding: ADC Therapeutics; Auxilius Pharma; Celgene; CRISPR Therapeutics; Millennium/Takeda; Seattle Genetics; C4 Therapeutics; Verastem/SecuraBio

T-Cell Receptor Dependent and Independent NF-kappa B Activation is a Prognostic Marker and a Therapeutic Target in Peripheral T-cell Lymphoma Not Otherwise Specified

Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) is the commonest subtype of PTCL. NF–kB related molecules have been found to be variably expressed in PTCL/NOS, suggesting a potential involvement of the NF–kB system in their pathogenesis. However, the actual contribution of NF–kB molecular programs to the PTCL/NOS landscape has not been investigated yet. In this study, we assessed in a large series of PTCL/NOS, the activation status of NF–kB programs and investigated the prognostic impact of such NF–kB expression. Moreover, we explored the possible role of NF–kB inhibitors. We studied the gene expression profiles of 180 PTCL cases and tested two different drugs, the IKK inhibitor BMS-345541 and the proteasome inhibitor Bortezomib, in four PTCL cell lines. We found that most cases (84%) presented with some degree of NF–kB activation, based on the expression of REL and RELA. Functionally, the latter was strictly related with TCR signaling activation, while REL was at least partially TCR independent. We also identified genes related with NF–kB activation in this setting that were mainly involved in cell proliferation and apoptosis inhibition. Further, by reverse engineering we defined the transcriptional network of both REL and RELA in PTCLs that only partially overlapped. On the clinical ground, we found that RELA expression was related to a significantly poorer overall survival, with similar trends for REL. However, most remarkably, when all the three genes were considered together, cases with at least one gene over-expressed, showed a dramatically inferior overall survival (28.67 vs. 56.018 months; p = 0.004). Finally, we showed that NF–kB pharmacological inhibition was associated with cell cycle arrest and cell death in NF–kB positive PTCL cells. In conclusion, we extensively explored NF–kB activation in PTCL/NOS, documenting its negative prognostic role. Further, we showed that NF–kB inhibition might represent a rational therapeutic approach in selected cases.

DNMT3A Mutations Identify a Prognostic Subgroup in Peripheral T-Cell Lymphoma

Background Mutational profiling of angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) has revealed recurrent mutations in DNMT3A, a de novo methyltransferase. DNMT3A catalyzes the conversion of cytosine to 5-methylcytosine (5-mC) while interacting with histones and transcription factors to influence gene expression. While the DNMT3A mutational profile in PTCL entities indicates loss-of-function, hotspot change-of-function mutations (e.g., DNMT3AR882H/C) have been observed with their frequency differing between PTCL entities. Despite the high occurrence of DNMT3A mutations in PTCLs (~30% of cases), their functional consequences have not been extensively studied. Herein, we examined DNMT3A mutations in AITL and the novel molecular subgroups of PTCL-NOS (i.e., PTCL-TBX21 and PTCL-GATA3) and observed distinct biological and prognostic significance associated with DNMT3A mutations in the PTCL-TBX21 subgroup. Methods PTCL-NOS cases (n = 141) were utilized following PTCL-TFH exclusion. Using previously described molecular classification methods, cases were classified as PTCL-TBX21 (n = 80) or PTCL-GATA3 (n = 61). A separate cohort of AITL cases (n = 176) were included for comparative purposes. Clinical outcome data were assessed with the Kaplan-Meier method. Mutation data were generated from DNA-sequencing (n = 224) or RNA-sequencing methods (n = 46). Gene expression comparisons were conducted using BRB-ArrayTools. Immune-cell signatures were generated from the CIBERSORT and/or xCell computational tools. 5-mC DNA immunoprecipitation sequencing (MeDIP-Seq) was performed on available PTCL-TBX21 cases (n = 7) or healthy tonsil controls (n = 2). Four of these cases carried DNMT3A mutations (n = 3 DNMT3AR882, n = 1 DNMT3AQ886) while the remaining cases (n = 3) were wild type for DNMT3A. In vitro analyses of ectopic expression of the DNMT3AR882H mutant or DNMT3A knockdown were conducted using healthy-donor CD3+ T-cells or the CD8+ T8ML1 PTCL cell line. Following corrections for false discoveries, p-values < 0.05 were considered significant. Results DNMT3A-mutated PTCL-TBX21 cases had an inferior overall survival, with DNMT3A mutated residues skewed toward the methyltransferase domain. In contrast to the DNMT3A mutation profile seen in AITL, PTCL-TBX21 featured DNMT3AR882H/C mutations at a frequency (30%) similar to other hematological malignancies. Gene expression profiling revealed that DNMT3A-mutant PTCL-TBX21 cases were enriched for activated CD8+ T-cell gene signatures and showed association with the previously described TH1/αβ cytotoxic T-cell lymphoma subgroup. Following MeDIP-Seq, assessment of differentially methylated regions comparing DNMT3AR882/Q886 PTCL-TBX21 cases to wild type found hypomethylation in pathways associated with T-cell activation, TCR signaling, and TH1 responses. In vitro analyses demonstrated that ectopic expression of the DNMT3AR882H mutant or DNMT3A knockdown lead to enhanced proliferation and NF-κB signaling in T8ML1 cells in comparison to control vectors. In primary CD3+ T-cell cultures, ectopic expression of the DNMT3AR882H mutant protein resulted in the preferential outgrowth of CD8+ T-cells. Conclusions Taken together, our findings establish mutations in DNMT3A as a novel prognostic marker in PTCL-TBX21. The integrated expression, methylation, and in vitro findings suggest that disruption of DNMT3A leads toward an activated and cytotoxic phenotype and could potentially drive oncogenic TCR signaling. Clinically, as these cases were associated with the TH1/αβ cytotoxic T-cell lymphoma subgrouping, these findings should be taken into consideration for future treatment strategies regarding PTCL-NOS patients as current standard-of-care treatments may be particularly inadequate in the treatment of PTCLs with cytotoxic phenotype. Disclosures No relevant conflicts of interest to declare.

Interleukin-2–inducible T-cell kinase inhibitors modify functional polarization of human peripheral T-cell lymphoma cells

Key Points ITK inhibitors perturb functional changes due to polarizing culture conditions in normal human tonsil CD4+ T cells. Primary human PTCL cells alter their functional properties in culture and ITK inhibitors modify these changes.

Exploring PRMT5 As a Potential Therapeutic Target in Canine Lymphomas

Non-Hodgkin lymphoma (NHL) represents approximately 4 percent of all cancer diagnoses in the United States, with diffuse large B-cell lymphoma (DLBCL) accounting for approximately 40 percent of all new cases. There are numerous histologic (GC, NGC) and genetic (double hit/expressor) subtypes of DLBCL and the overall outcome of patients who receive standard therapy is heterogeneous. Lymphoma is also a common, malignancy in dogs, and while initial responses to combination chemotherapy show clinical responses, remission time is short and cures rare. Protein arginine methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase (PRMT) enzyme capable of driving symmetric demethylation of arginine residues on histones (H3(me2)R8; H4(me2)R3) and other proteins such as P53, and the NFkB subunit p65. PRMT5 is overexpressed and dysregulated in both solid and hematologic tumors and plays a key role in the driver activity of MYC, CYCLIND1 and NOTCH in lymphoma models. Experimental therapeutic strategies aimed at targeting PRMT5 activity have led to numerous, highly selective inhibitors that are currently being translated into the clinic with several clinical trials underway or in development. Here, we characterized patterns of PRMT5 expression and correlated these with histologic subtype in canine lymphoma tissue microarrays (TMAs, n=337 lymphoma specimens). We characterized expression of PRMT5 and its symmetric dimethylation histone marks in three canine lymphoma-derived cell lines, CLBL-1, 17-71, and OSW. Treatment of PRMT5 with a highly selective small molecule PRMT5 inhibitor (CMP220) was performed to determine the dose-dependent effects (1nM-10uM range) on proliferation (MTS assay), viability (annexin V/PI flow cytometry) and biomarkers of PRMT5 activity (SDMA, ADMA, histone symmetric demethylation). CMP220 showed PRMT5 inhibitory activity that was highly selective in a methyltransferase screening assay with 37 enzymes/complexes. TMAs of all histologic subtypes of lymphoma showed over-expression of PRMT5 in 96% of specimens. Canine DLBCL showed variable over-expression in the cytoplasmic compartment (48.8% strong, 50.0% weak, n = 165) compared to negative or weak staining in normal and hyperplastic lymph nodes (n = 40). Nuclear staining was observed primarily in lymphomas of T cell lineage (PTCL, and pre T lymphoblastic lymphomas). Primary and cell line lymphoma samples showed PRMT5 over expression by Western blot and RT-PCR. PRMT5 inhibition showed a dose-dependent decrease in symmetric dimethylarginine (SDMA) and symmetric demethylation of histone H4 arginine-3 (H4(me2s)R3) with no effect on asymmetric dimethylarginine (ADMA). Interestingly, in contrast to PRMT5 inhibition in human lymphomas, symmetric demethylation of histone H3 arginine-8 (H3(me2s)R8) showed no change with PRMT5 inhibition. The PRMT5 small molecule inhibitor CMP220 inhibited growth of CLBL-1 (IC50 of 123.2 nM at 6d) and 17-71 (IC50 of 100 nM at 6d). The OSW PTCL cell line showed profound resistance to PRMT5 inhibition with little cell death observed at 10 uM. Ex-vivo canine patient samples showed suppression of growth in a time and dose-dependent fashion. We have demonstrated that PRMT5 is expressed in canine B cell primary lymphomas and cell lines and that PRMT5 inhibition leads to growth suppression and induction of apoptosis. We are currently exploring genome-wide recruitment of PRMT5 on chromatin and examining chromatin and whole transcriptome changes that occur in canine lymphoma cell lines treated with PRMT5 inhibitors. This data provides justification for incorporating the canine lymphoma model into the preclinical development of PRMT5 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3123: Peripheral T cell lymphoma-associated fibroblasts promote tumor growth in an in vivo model

Abstract Peripheral T-cell lymphomas (PTCLs) represent a rare and heterogeneous group of aggressive non-Hodgkin's lymphomas comprising different entities. Anthracycline-based regimens (usually CHOP, cyclophosphamide/doxorubicin/vincristine/prednisone) followed by stem cell transplantation are considered the standard of care in the front-line setting. However, long-term disease control can be achieved only in 30-40% of young patients. Increasing evidences highlight the role of tumor microenvironment in sustaining tumor progression and aggressiveness. Among microenvironment components, a central role is played by cancer associated fibroblast (CAFs). Recent studies demonstrated that this is applicable not only to solid tumors but also to haematological malignancies. Here, we report for the first time the isolation and characterization of PTCL-CAFs and their role in promoting cell proliferation. We kept in culture a skin biopsy of a cutaneous localization of a peripheral T cell lymphoma. Fibroblast-like cells grown out from the biopsy were isolated, cultivated for few passages and then characterized. Flow cytometric analyses revealed that the cell population entirely expressed cell-surface antigens specific for fibroblasts, such as CD140b and CD90, while they did not express CD45 and CD31, thus excluding hematopoietic or endothelial origin. Biochemical analyses confirmed that all cells were positive for vimentin, while αSMA expression highlighted the activated status of the cells isolated. We then assessed the in vitro effect of CAFs on PTCL cell growth by co-colture experiments. For this purpose, we coltured for 8 days the PTCL cell line OCI-Ly12 in fresh medium or in medium previously conditioned by CAFs or normal skin fibroblasts (NFs). PTCL cell growth was not affected by co-colture either with CAFs or with NFs. Moreover, the presence of conditioned medium did not alter the response of OCI-Ly12 cells to CHOP treatments. We then assessed the in vivo effect of PTCL-CAFs. We subcutaneously injected CAFs alone, OCI-Ly12 cells alone and OCI-Ly12 together with CAFs (ratio 1:1) in NOD/SCID mice and monitored tumor growth for 20 days. In contrast to the effect observed in vitro, co-injection of CAFs with cancer cells substantially promoted tumor growth (tumor volume OCI-Ly12 0,87±0,47 cm3; OCI-Ly12+CAFs 2,30±0,26 cm3). As expected, CAFs alone did not develop a measurable mass. Subsequent immunohistochemical staining with CD31 revealed the presence of more vascular structures in tumors originated by OCI-Ly12+CAFs co-injection than by OCI-Ly12 alone, suggesting that CAFs presence sustains tumor growth by promoting angiogenesis. In summary, we show that PTCL-derived CAFs promote cancer cell growth in vivo, while leaving unaffected cell growth in vitro and the response to chemotherapy. This is the first evidence of the role played by the microenvironment in promoting malignant cell proliferation in PTCLs. Citation Format: Martina Magni, Sara Rizzitano, Alessio Pellegrinelli, Paolo Corradini, Cristiana Carniti. Peripheral T cell lymphoma-associated fibroblasts promote tumor growth in an in vivo model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3123.

Abstract 363: STAT5 inhibition induces apoptosis in peripheral T cell lymphoma

Abstract Peripheral T cell lymphoma (PTCL) is a heterogeneous malignancy with extremely poor prognosis. Five-year overall survival is <40% and can be as low as 12% for specific subtypes. There is, therefore, an urgent need to identify and develop novel therapeutic targets and approaches. A number of recent sequencing studies, including a whole exome sequencing analysis of primary PTCL patients conducted by our laboratory, have revealed recurrent oncogenic driver mutations in the common gamma chain/JAK/STAT signaling pathway, resulting in the aberrant activation of the transcriptional regulator STAT5. STAT5 has additionally been found to be upregulated or contain activating mutations in 30-40% of PTCL cases of specific subtypes. These findings highlight STAT5 as an oncogenic driver of PTCL and suggest a role for it in novel therapeutic approaches. Based on these data, we aimed to evaluate STAT5 mechanistically as a therapeutic target in PTCL. The drug, pimozide, which is an FDA approved neuroleptic agent, was recently identified by drug screen to be a STAT5 inhibitor. We assessed the efficacy of pimozide in PTCL using resazurin dye assay, Fluorescence Activated Cell Sorting (FACS), and immunoblot analysis. Our data demonstrate a concentration dependent reduction in STAT5 activity and the number of viable cells in PTCL cell lines after culture with pimozide. This is shown to be due to an increase in cell death by apoptosis. To verify that pimozide’s efficacy is due to its inhibition of STAT5 activity, we transduced PTCL cell lines with anti-STAT5 shRNA to knock down STAT5 and assessed PTCL cell survival and proliferation. We show that STAT5 knockdown results in a three-fold reduction in PTCL cell viability, which occurs due to an increase in apoptosis. Furthermore, our data supports a TRAIL dependent mechanism for induction of apoptosis shown by an upregulation of TRAIL by PTCL cells after STAT5 knockdown or culture with pimozide. Mitochondrial membrane potential is also disrupted. These findings have potential clinical implications, as we further demonstrate that pimozide inhibits STAT5 phosphorylation, reduces cell viability, and induces apoptosis in primary PTCL patient samples ex vivo. This research supports further exploration of STAT5 as a therapeutic target in PTCL and the development of STAT5 inhibition in the treatment of PTCL. To facilitate the development of non-chemotherapeutic dependent therapeutic approaches for PTCL, we are now assessing STAT5 inhibition in combination with small molecule inducers of apoptosis including TRAIL pathway and PARP inhibitors. PARP inhibitors have shown efficacy in malignancies with DNA damage response pathway mutations, which have been identified in up to half of cases of certain subtypes of PTCL. We also aim to further investigate the use of JAK inhibitors, kinases responsible for STAT5 activation, in combination in PTCL, as several have demonstrated clinical efficacy in other hematologic malignancies. Citation Format: Haley M. Simpson, Aki Furusawa, Kavitha Sadashivaiah, Arnob Banerjee. STAT5 inhibition induces apoptosis in peripheral T cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 363. doi:10.1158/1538-7445.AM2017-363

Integrated mate-pair and RNA sequencing identifies novel, targetable gene fusions in peripheral T-cell lymphoma

AbstractPeripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a “wastebasket” category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

Epstein- Barr Virus: Clinical and Epidemiological Revisits and Genetic Basis of Oncogenesis.

Epstein-Barr virus (EBV) is classified as a member in the order herpesvirales, family herpesviridae, subfamily gammaherpesvirinae and the genus lymphocytovirus. The virus is an exclusively human pathogen and thus also termed as human herpesvirus 4 (HHV4). It was the first oncogenic virus recognized and has been incriminated in the causation of tumors of both lymphatic and epithelial nature. It was reported in some previous studies that 95% of the population worldwide are serologically positive to the virus. Clinically, EBV primary infection is almost silent, persisting as a life-long asymptomatic latent infection in B cells although it may be responsible for a transient clinical syndrome called infectious mononucleosis. Following reactivation of the virus from latency due to immunocompromised status, EBV was found to be associated with several tumors. EBV linked to oncogenesis as detected in lymphoid tumors such as Burkitt's lymphoma (BL), Hodgkin's disease (HD), post-transplant lymphoproliferative disorders (PTLD) and T-cell lymphomas (e.g. Peripheral T-cell lymphomas; PTCL and Anaplastic large cell lymphomas; ALCL). It is also linked to epithelial tumors such as nasopharyngeal carcinoma (NPC), gastric carcinomas and oral hairy leukoplakia (OHL). In vitro, EBV many studies have demonstrated its ability to transform B cells into lymphoblastoid cell lines (LCLs). Despite these malignancies showing different clinical and epidemiological patterns when studied, genetic studies have suggested that these EBV- associated transformations were characterized generally by low level of virus gene expression with only the latent virus proteins (LVPs) upregulated in both tumors and LCLs. In this review, we summarize some clinical and epidemiological features of EBV- associated tumors. We also discuss how EBV latent genes may lead to oncogenesis in the different clinical malignancies

The oncogenic transcription factor IRF4 is regulated by a novel CD30/NF-κB positive feedback loop in peripheral T-cell lymphoma

AbstractPeripheral T-cell lymphomas (PTCLs) are generally aggressive non-Hodgkin lymphomas with poor overall survival rates following standard therapy. One-third of PTCLs express interferon regulatory factor-4 (IRF4), a tightly regulated transcription factor involved in lymphocyte growth and differentiation. IRF4 drives tumor growth in several lymphoid malignancies and has been proposed as a candidate therapeutic target. Because direct IRF4 inhibitors are not clinically available, we sought to characterize the mechanism by which IRF4 expression is regulated in PTCLs. We demonstrated that IRF4 is constitutively expressed in PTCL cells and drives Myc expression and proliferation. Using an inhibitor screen, we identified nuclear factor κB (NF-κB) as a candidate regulator of IRF4 expression and cell proliferation. We then demonstrated that the NF-κB subunits p52 and RelB were transcriptional activators of IRF4. Further analysis showed that activation of CD30 promotes p52 and RelB activity and subsequent IRF4 expression. Finally, we showed that IRF4 transcriptionally regulates CD30 expression. Taken together, these data demonstrate a novel positive feedback loop involving CD30, NF-κB, and IRF4; further evidence for this mechanism was demonstrated in human PTCL tissue samples. Accordingly, NF-κB inhibitors may represent a clinical means to disrupt this feedback loop in IRF4-positive PTCLs.

Abstract 5275: miRNA profiling of peripheral T-cell lymphomas.

Abstract Background: Peripheral T cell lymphomas not otherwise specified (PTCLs/NOS) are rare and aggressive tumors, whose molecular pathogenesis is poorly known and whose diagnosis is quite difficult. In fact, both morphology and phenotype largely overlap with those of other nodal PTCLs, including angioimmunoblastic T cell lymphoma (AITL) and anaplastic large cell lymphomas (ALCLs). Here, we performed an extensive miRNA profiling of PTCLs in order to identify differentially expressed miRNA, either involved in their pathogenesis or potentially useful for their differential diagnosis. Methods: We studied by miRNA profiling (TaqMan Array MicroRNA Cards A) 60 samples including PTCLs/NOS (N=25), AITLs (N=10), ALCLs (N=12) and normal T cells (N=13); in addition, 40 independent PTCL cases were studied by qRT-PCR as validation. Further, to assess the functional impact of deregulated miRNAs, we performed a global gene expression profile (GEP) analysis in the same cases by using the Illumina Whole Genome DASL Assay. ANOVA and Student t-test were performed to find genes and miRNAs differentially expressed between PTCLs and normal T cells or among PTCL subtypes, respectively. Differentially expressed miRNAs were further filtered based on their predicted impact on the GEP using Linear Discriminant Analysis, Spearman correlation and Gene Set Enrichment Analysis. A discriminant function based on linear combination of GEP signatures was then used to better distinguish different PTCLs subtypes. Finally, the functional effects of selected miRNA were assessed by transfection of specific mimic and inhibitors into PTCL cells and then evaluating the GEPs (functional GEP). Findings: First, unsupervised analyses clearly discriminated PTCLs and normal T cells based on the miRNA expression patterns. Second, supervised analysis identified 256 miRNA differentiating the two groups (p<0.05, FC ≥2, Bonferroni correction). To identify those miRNA more likely to impact the GEP of the tumors, we then performed a multi-step analysis integrating miRNA and GEP data. We identified 4 highly discriminant miRNA inversely correlated with different potential target genes. All 4 miRNA were confirmed by qPCR in a validation set, while their target genes were assessed by functional GEP confirming their impact. Thereafter, we sought miRNAs differentially expressed in the PTCL subtypes, to be used as diagnostic markers. Based on ANOVA and subsequent linear discriminant analysis we identified 3 sets of miRNA able to efficiently distinguish PTCL/NOS vs. ALK-/ALCL (N=5 miRNA), ALK+/ALCL vs. ALK-/ALCL (N=4), and PTCL/NOS vs. AITL (N=8). Noteworthy, this novel molecular diagnostic tool was validated by qPCR in independent cases, demonstrating a remarkable diagnostic accuracy ranging between 85% and 95%. In conclusion, miRNA profiling allowed to identify miRNA possibly involved in PTCL pathogenesis and to develop a novel diagnostic tool for the differential diagnosis of the commonest subtypes. Citation Format: Maria Antonella Laginestra, Fabio Fuligni, Maura Rossi, Davide Gibellini, Maria Rosaria Sapienza, Anna Gazzola, Claudia Mannu, Elena Sabattini, Francesco Bacci, Claudio Agostinelli, Stefano Aldo Pileri, Pier Paolo Piccaluga. miRNA profiling of peripheral T-cell lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5275. doi:10.1158/1538-7445.AM2013-5275

Genome-wide analysis reveals recurrent structural abnormalities of TP63 and other p53-related genes in peripheral T-cell lymphomas

AbstractPeripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.

Abstract 3684: Targeting Aurora kinase in peripheral T-cell non-Hodgkin's lymphomas

Abstract Aurora kinases (A and B) are oncogenic serine/threonine (S/T) kinases that play central roles in the mitotic phase of the eukaryotic cell cycle. Over-expression of Auroras during dysregulated cell cycling and their aberrant activation over-rides mitotic and spindle check points leading to anueploidy in many human cancers make Auroras attractive therapeutic targets. Gene expression profiling in mantle cell lymphoma [MCL] have shown the Auroras are over-expressed and is a poor prognostic marker within the ‘proliferative’ signature. PTCL (NOS) are a heterogeneous group of rare non-Hodgkin's lymphomas (NHL) derived from activated CD4+ or CD8+ T cells. PTCL and anaplastic large cell lymphoma (ALCL) both of T-cell phenotype and ALK negative have an aggressive clinical course with an inferior event free and overall survival relative to B-cell NHL counterparts. The biological explanation for the difference is unknown. Patients with PTCL have a poor prognosis due to a very aggressive disease course coupled to a lack of effective therapies. Therefore, the development of novel and effective treatments based on biologically validated targets is urgently needed for this disease. We hypothesized and investigated that (1). Auroras are over-expressed in human PTCL-NOS, (2). Aurora ATP-site small molecule inhibitor (SMI) is effective in inhibiting cell proliferation and promoting apoptosis in cell culture and (3). Aurora SMI will be safe and effective in treating patients with relapsed aggressive T-cell NHL in early phase clinical trials. In this study, we demonstrate that both aurora A and B are highly over-expressed in PTCL patients and cell lines. MLN8237 (alisertib), an ATP-site small molecule inhibitor of Aurora A had potent activity in inhibiting phosphorylation of Aurora A and histone H3 in PTCL cell lines. Cells treated with MLN8237 exhibited endoreduplication confirming the mechanism of action of an Aurora inhibitor. Also, treatment of PTCL cell lines with MLN8237 induced apoptosis in a dose and time dependent manner (flow cytometry and PARP-cleavage) and inhibited cell proliferation with an IC50 < 0.1µM. Preliminary data from a phase II clinical trial using single-agent MLN8237 in patients with relapsed refractory aggressive NHL has demonstrated notable activity with 4 confirmed responses in 6 evaluable PTCL patients. Cytokine profiling on day 1 (pre-treatment) and day 8 (post-treatment) showed a profile consistent with aurora A activity. Together the data suggest inhibition of Auroras offers a promising treatment strategy for patients with aggressive T-cell NHL [Funded by the Lymphoma SPORE, P50 CAB0805501A1]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3684. doi:1538-7445.AM2012-3684

PI3K Inhibition As a Potential Therapeutic Strategy in Peripheral T-Cell Lymphomas,

Abstract 3493Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of very aggressive malignancies lacking efficient therapy. Unfortunately, there are neither animal models nor representative cell lines for most PTCL types, making functional and pharmacodynamic studies even more difficult. PI3K signaling is essential for cell proliferation and survival, is frequently altered in human cancer and seems to play a critical role in T-cell development and activation. The aim of this work is to determine the efficiency of PI3K inhibition in PTCL, looking for pharmacodynamic biomarkers, and to identify markers that could distinguish responders from non-responders.Twenty two PTCL cases and seven reactive lymph nodes were studied using gene expression profiling. We performed an in silico analysis using the Connectivity Map program to identify drugs that could potentially reverse the PTCL gene expression signature. Among them, several PI3K/mTOR inhibitors were found.Moreover, genomic studies using Gene Set Enrichment Analysis identified PIK3CD gene (encoding for the delta isoform of PI3K) to be the only one significantly correlated to the activation of CD40, NF-kB and TCR pathways. Quantitative RT-PCR confirmed the strong overexpression of PIK3CD in 6 PTCL-derived cell lines compared to normal T cells from healthy donors. Sequence analyses for the coding region of the PIK3CD gene identified a point mutation in one of these cell lines, described as activating in solid tumors.A panel of 6 PTCL cell lines belonging to different PTCL subgroups was treated with 3 PI3K inhibitors (LY294002, ETP-45658, GDC-0941). Moreover, genetic inhibition was also carried out using small interference RNA to specifically abolish the expression of alpha and delta isoforms of PI3K (PIK3CA and PIK3CD genes, respectively).In vitro studies showed very similar results with the three pharmacological PI3K inhibitors we used: they induced G1 cell cycle arrest in all cell lines, and apoptosis in some of them, in a time/dose-dependent manner. We also observed a decrease in the levels of pAKT(S473) in all cell lines, while pGSK3B(S9) and p-p70S6K(T389) were reduced after treatment only in sensitive cell lines.Our results indicate that genetic inhibition of PI3K delta isoform could induce apoptosis in those PTCL cell lines that were sensitive to PI3K inhibitors, but not in the resistant cell lines; while genetic inhibition of PI3K alpha isoform did not display such effects.Taken together these results could highlight the relevance of PI3K delta isoform in at least a subset of PTCL, indicating that PI3K inhibition, especially delta isoform, could be an effective therapeutic approach for PTCL and identifying potential markers for patients' stratification and pharmacodynamic assessment. Disclosures:No relevant conflicts of interest to declare.