ITF2357, a Novel Histone-Deacetylase Inhibitor, Is Effective against Peripheral T-Cell Lymphomas in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4981-4981
Author(s):  
Pier Paolo Piccaluga ◽  
Maura Rossi ◽  
Aurora Esposito ◽  
Pier Luigi Tazzari ◽  
Francesca Ricci ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Cell Viability ◽  
Cell Lines ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Phase Ii Trials ◽  
T Cell Lymphomas ◽  
Peripheral T Cell Lymphomas

Abstract Background. Recently, gene expression profiling (GEP) indicated histone-deacetylases (HDAC) as potential therapeutic targets in peripheral T-cell lymphomas (PTCL) not otherwise specified (NOS), the commonest PTCL type. Consistently, phase II trials demonstrated the efficacy of some HDAC inhibitors (HDACi), including SAHA, which was approved for cutaneous T-cell lymphomas (CTCL) treatment. Aims and methods. We investigated the anti-tumour effects of ITF2357 (Italfarmaco, Italy), a novel hydroxamic acid HDACi, on PTCL primarily-cultured cells and cell lines (HH and FEDP), and in a xenografted mouse-model of CTCL. Cultured cells were incubated with different dosages of ITF2357 and SAHA (ranging from 0.5 to 2.5 mM). Cell viability, assessed by trypan-blue exclusion assay, cell-cycle progression, assessed by bromodeossiuridine assay, and apoptotic rate, determined by flow-cytometry analysis of annexin-V binding populations were determined at 48, 72 and 120 hours. Nude mice, injected with HH cells, received ITF2357 (10–20mg/Kg, per os) for 14 days. Micro-PET scan was adopted for disease measurement and treatment response evaluation. Finally, GEP of cell lines exposed to ITF2357 and SAHA were generated to elucidate their mechanisms of action. Results. Cell viability of HH cells treated with ITF2357 ranged from 50% (0.5 mM, at 48 h), to <10% (0.5–2.5 mM, at 72–120 h), in comparison to untreated cells. Differently, cell viability of HH cells treated with SAHA ranged from 80% (0.5 mM, at 48–120 h) to 25% (2.5 mM at 48 h). Analogue effects were documented in FEDP and primarilycultured PTCL cells. Conversely, viability of normal T-lymphocyte was not significantly affected. Interestingly, exposure to ITF2357 was associated to G0/G1 cell-cycle arrest and apoptosis induction. Finally, ITF2357 determined significant reduction of tumoral masses and survival benefit in a xenografted mice-model inoculated with HH cells. Conclusion. Taken together, these data demonstrate that ITF2357 is effective against PTCLs ex vivo and in vivo, by nominating it for clinical evaluation in this setting.

PIM Kinases Inhibition, a Rational Strategy in Peripheral T-Cell Lymphomas,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3494-3494
Author(s):  
Esperanza Martin-Sanchez ◽  
Socorro M. Rodriguez-Pinilla ◽  
Margarita Sanchez-Beato ◽  
Beatriz Dominguez-Gonzalez ◽  
Magdalena B. Wozniak ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Gene Set Enrichment Analysis ◽  
Compensatory Mechanism ◽  
Pim Kinases ◽  
Cycle Arrest ◽  
T Cell Lymphomas ◽  
Peripheral T Cell Lymphomas

Abstract Abstract 3494 The search of an efficient therapy for Peripheral T-cell lymphomas (PTCL) patients is still a challenge, in part due to the very little knowledge about the PTCL pathogenesis, and the absence of appropriate models. This heterogeneous group of very aggressive malignancies can not be cured with conventional therapies; therefore, new therapeutic strategies are needed to improve the poor outcome in these patients. PIM family is composed of 3 kinases (PIM1, PIM2 and PIM3) which play an essential role in cell proliferation and survival. They are mainly activated through JAK/STAT signaling pathway, and are frequently altered in human malignancies by amplification, mutation and overexpression. The aim of this study is to determine the efficiency and the mechanism of action of PIM inhibition in PTCL. Gene expression profiling of twenty two PTCL cases and seven reactive lymph nodes was performed. We observed a strong overexpression of the three PIM family genes in PTCL cases, especially PIM2. In addition, Gene Set Enrichment Analysis identified an overexpression of STAT3 and IL-2 pathways in PTCL cases, probably responsible for the strong expression of PIMs we found. Furthermore, PIM genes expression was confirmed by quantitative RT-PCR in 6 PTCL-derived cell lines compared to normal T cells from healthy donors, highlighting again the relevance of PIM2. Genetic inhibition was carried out using small interference RNA to specifically abolish the expression of each PIM1, PIM2 and PIM3 in a panel of 6 PTCL cell lines belonging to different PTCL subgroups. Additionally, pharmacological inhibition with one PIM inhibitor (ETP-39010) was performed. Surprisingly, genetic inhibition of each of the PIM gene alone did not show any cellular effect, neither cell cycle arrest nor apoptosis. But interestingly, we found that specific inhibition of each of the PIM genes caused an increased expression of the other PIM family members, probably leading to a compensatory mechanism among these kinases balancing the lack of one of them, avoiding pro-apoptotic effects and allowing cell survival. Accordingly, a simultaneous inhibition of PIM1, PIM2 and PIM3 using the pharmacological pan-PIM inhibitor produced a decrease in cell viability and a strong induction of apoptosis in all cell lines, without cell cycle arrest. Several PIM inhibitor biomarkers have been identified at the mRNA level, involving the DNA damage response signaling. In conclusion, our results indicate that PIM kinases inhibition could be an effective therapeutic approach for PTCL. Disclosures: No relevant conflicts of interest to declare.

Expression of CYP3A4 as a predictor of response to chemotherapy in peripheral T-cell lymphomas

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3345-3351 ◽  
Author(s):  
Cristina Rodríguez-Antona ◽  
Susanna Leskelä ◽  
Magdalena Zajac ◽  
Marta Cuadros ◽  
Javier Alvés ◽  
...  
Keyword(s):  
T Cell ◽  
Clinical Response ◽  
Cell Lines ◽  
Current Therapy ◽  
Fish Analysis ◽  
Primary Tumors ◽  
Chemotherapy Drugs ◽  
T Cell Lymphomas ◽  
Response To Chemotherapy ◽  
Peripheral T Cell Lymphomas

Abstract Peripheral T-cell lymphomas (PTCLs) are aggressive tumors in which the current therapy based on multiagent chemotherapy is not successful. Since cytochrome P450 3A subfamily (CYP3A) enzymes are involved in the inactivation of chemotherapy drugs, we hypothesized that CYP3A and P-glycoprotein (MDR1) expression in these lymphomas could result in a poor clinical response. We measured tumoral CYP3A and MDR1 mRNA content in 44 T-cell lymphomas, finding a large variation in CYP3A expression. Multiplex polymerase chain reaction (PCR) analysis and fluorescence in situ hybridization (FISH) analysis showed genomic gains affecting CYP3A and MDR1 genes in T-cell lines and primary tumors, suggesting that this could be the mechanism underlying the tumoral expression variation. To test whether the tumoral expression of CYP3A and/or MDR1 could influence PTCL treatment outcome, their expression levels were compared with the clinical response and survival of the patients, finding that a high tumoral expression of CYP3A4 was significantly associated with a lower complete remission rate. This was further investigated with cell lines stably expressing CYP3A4 that exhibited an increased resistance to doxorubicin and etoposide. In conclusion, a high CYP3A4 tumoral expression could be useful to predict poor response to the standard PTCL chemotherapy; in these cases alternative chemotherapy combinations or doses should be explored.

PI3KCA and PIM Inhibitors in Peripheral T-Cell Lymphomas

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4917-4917
Author(s):  
Esperanza Martin-Sanchez ◽  
Socorro M. Rodriguez-Pinilla ◽  
Luis Lombardia ◽  
Margarita Sanchez-Beato ◽  
Beatriz Dominguez-Gonzalez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
T Cell ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Gene Expression Signature ◽  
Dependent Manner ◽  
Expression Signature ◽  
Cycle Arrest ◽  
T Cell Lymphomas

Abstract Abstract 4917 T-cell lymphomas (TCL) are a heterogeneous group of aggressive malignancies lacking specific and efficient therapy. Unfortunately, there are neither animal models nor representative cell lines for most TCL types, making functional and pharmacogenomics studies even more difficult. PI3K and PIM are kinases involved in cell proliferation, frequently altered in human cancer that seems to play a critical role in T-cell development and activation. Genomic studies have identified PIK3CD subunit to be significantly associated with in activation of CD40, NF-kB and TCR-pathways. The aim of this project is to determine the efficiency of PI3K inhibitors (PI3Ki) and PIM inhibitors (PIMi) in TCL, looking for biomarkers of their mechanism of action and to identify markers that could identify responders from non-responders. Twenty PTCL and seven reactive lymph nodes were studied using gene expression microarrays. We performed an in silico analysis using the Connectivity Map program to identify drugs that could potentially reverse PTCL gene expression signature. Among them, several PI3K/mTOR inhibitors were found. A panel of 6 TCL cell lines belonging to different TCL subgroups were treated with 3 PI3Ki (LY294002, ETP-45658, GDC-0941) and one PIMi (ETP-39010). Functional studies were also done to establish the role of each of the targeted genes. In vitro studies showed that PI3Ki induced G1 cell cycle arrest in all cell lines, and apoptosis in a portion of them, in a time/dose-dependent manner. We also observed a decrease in the levels of pAKT(S473), pGSK3B(S9) and p-p70S6K(T389) after treatment. In addition, both the analysis of the PTCL gene expression signature as well as western blot studies on TCL cell lines has shown overexpression of PIM family genes, A decrease in cell viability, and a strong induction of apoptosis in all cell lines was seen after PIM inhibition, without cell cycle arrest. Several diagnostic and pharmacodynamic biomarkers of PIMi have been identified at the mRNA and protein level in both cell lines In conclusion, our results indicate that PI3Ki and PIMi are effective therapeutic approaches for TCLs, identifying potential markers for patient's stratification and pharmacodynamic assessment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals STX11 Acts As a Novel Tumor Suppressor Gene in Peripheral T-Cell Lymphomas

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1615-1615
Author(s):  
Noriaki Yoshida ◽  
Shinobu Tsuzuki ◽  
Kennosuke Karube ◽  
Miyuki Katayama ◽  
Taishi Takahara ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Nk Cell ◽  
Suppressor Gene ◽  
Rt Pcr ◽  
T Cell Lymphomas ◽  
T Cell Lines ◽  
Peripheral T Cell Lymphomas

Abstract Introduction: Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas noted for their poor prognosis. Their molecular pathogenesis has not been entirely elucidated. We previously found that primary thyroid T-cell lymphoma (PTTL) is a distinct entity among heterogeneous PTCLs and that this disease is characterized by the genomic loss of 6q24 (Br J Haematol., 161:214-223). In this study, we extended the analysis to other types of PTCLs and performed functional assays to identify causative genes located on 6q24. Methods: Focusing on chromosome 6q loss, we reexamined previous comparative genomic hybridization data from 267 PTCL cases comprising 6 PTTLs, 51 PTCLs-not otherwise specified (NOS), 62 adult T-cell leukemias/lymphomas, 35 natural killer (NK)-cell lymphomas, 39 angioimmunoblastic T-cell lymphomas (Genes Chromosomes Cancer, 46:37-44), and 74 anaplastic large cell lymphomas (Br J Haematol., 140:516-526). Gene expression levels were determined by using published gene expression profiling (GEP) data (GSE6338 and GSE19069) and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Subsequently, we established Tet-Off cell lines belonging to several lineages (6 T-cell lines, 1 NK-cell line, 4 B-cell lines, 1 myeloid cell line, and 3 epithelial cell lines) for functional analyses. Results: Genomic loss of 6q24 was observed in 8% (n = 267) of PTCL cases, and it occurred most frequently in PTTL cases (67%; n = 6). All the genomic losses were heterozygous; homozygous loss of this region was not observed in our analysis. The smallest region of deletion, observed in a PTTL case, was considered the minimal common region (MCR) of 6q24 loss. The MCR contained 2 known coding genes, STX11 and UTRN. Combined GEP data and quantitative RT-PCR analyses showed that the expression of STX11, but not UTRN, was markedly lower in PTCL than in normal T-cells. We therefore regarded STX11 as the most probable candidate gene located in 6q24. Syntaxin 11, encoded by STX11, is a t-SNARE protein that plays a role in binding vesicles to cell membranes, and alteration of STX11 in the germline causes familial hemophagocytic syndrome type 4. To further evaluate genomic alteration of STX11, mutation analysis was performed on PTCL-NOS and PTTL cases as well as T-cell lines, for which adequate DNA was available. This revealed STX11 mutations in 2 cases (1 PTCL-NOS case and 1 T-cell line). Wild-type STX11 expression suppressed the proliferation of T-cell lines bearing genomic alterations at the STX11 locus only, and it did not show suppressive effects on other lineage cell lines (Fig. 1). Expression of STX11 induced cellular apoptosis in the cell line, although the number of apoptotic cells induced was relatively small. Interestingly, expression of a novel STX11 mutant (p.Arg78Cys), observed in a T-cell line, did not exert suppressive effects on the induced cell lines suggesting that there was a loss-of-function mutation (Fig. 2). Finally, we evaluated the clinical impact of STX11 alteration in PTTL and PTCL-NOS cases where data were available. This showed that PTCL-NOS cases with genomic alterations of STX11 tended to have a poorer prognosis than those without (Fig. 3; P = 0.069). Conclusion: In the present study, we examined the MCR of 6q24 loss and showed that STX11 acts as a tumor suppressor gene in PTCLs only. These findings provide a novel approach for understanding the molecular pathogenesis of PTCLs, and they may contribute to the future development of new drugs for the treatment of PTCLs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Selective STAT3 Degraders Dissect Peripheral T-Cell Lymphomas Vulnerabilities Empowering Personalized Regimens

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 865-865
Author(s):  
Giuseppina Astone ◽  
Luca Vincenzo Cappelli ◽  
William Chiu ◽  
Clarisse Kayembe ◽  
Rui Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
T Cell ◽  
Growth Inhibition ◽  
Research Funding ◽  
Granzyme B ◽  
Publicly Traded ◽  
T Cell Lymphomas ◽  
Peripheral T Cell Lymphomas

Abstract Introduction: Peripheral T-cell lymphomas (PTCLs) include heterogeneous entities of rare and aggressive neoplasms. The improved understanding of the biological/molecular mechanisms driving T-cell transformation and tumor maintenance has powerfully propelled new therapeutic programs. However, despite this progress, PTCLs remain an unmet medical need. Recurrent aberrations and the deregulated activation of distinct signaling pathways have been mapped and linked to selective subtypes. The JAK/STAT signaling pathway's deregulated activation plays a pathogenetic role in PTCL, including ALCL subtypes. STATs regulate the differentiation/phenotype, survival and cell-growth, metabolism, and drug resistance of T-cell lymphomas as well as host immunosuppressive microenvironments. Although many drugs' discovery programs were launched, a plethora of compounds has failed. Methods: We have discovered heterobifunctional molecules by an iterative medicinal chemistry SAR campaign that potently and selectively degrade STAT3 in a proteasome-dependent manner. Conventional PTCL cell lines and Patient Derived Tumor Xenograft (PDTX) and/or derived cell lines (PDTX-CL), carrying either WT- or mutated-STAT3, were exposed to increasing amounts (50nM⇒5µM) of STAT3-degraders. Proteins and mRNA transcripts (2⇒144hrs) were assessed by deep-proteomics and paired-end RNA sequencing, combined with WB/flow cytometry and qRT-PCR. Cell-titer-glo, cell titer blue, Annexin-V and S-cell cycle analyses were used as readouts. Chromatin accessibility, STAT3 DNA binding, 3D chromosomal architecture reorganization and 5-hmC profiling were assessed by ATACseq, CHIPseq and Hi-C and H3K27ac Hi-CHIP and mass-spectrometry. Drug testing/discovery combinations (96-well-plate) were performed using a semi-automated flow-cytometry. A battery of PTCL PDTX models were tested in pre-clinical trials. Results: Treatment of ALK+ ALCL (SU-DHL1) led to the rapid (~6hrs.) and profound down-regulation of STAT3 followed by the loss of canonical STAT3-regulated proteins (SOCS3, MYC, Granzyme B, GAS1, and IL2RA), without appreciable changes for other STAT family members (STAT1, STAT5b). In vitro, cytoplasmic, nuclear, and mitochondrial STAT3 downregulation was maintained up to 144 hrs. Loss of STAT3 in ALK+/- ALCL and BIA-ALCL cells was associated with major transcriptional changes (7116-10615 and 15114 DEGs in ALK- and ALK+ ALCL, respectively), underscoring public/shared as well as private time-dependent signatures. Main down-regulated pathways included JAK-STAT, MAPK, NF-kB, PI3K, TGFb, and TNFa. Comparison of STAT3 shRNA (ALK+ ALCL) and STAT3 degrader (ALK-/ALK+ ALCL) signatures demonstrated a substantial and concordant gene modulation (24hrs) among all models with the highest overlaps between ALK+ ALCL (Figure 3). To identify direct STAT3 gene targets, we analyzed CHIPseq peaks and predicted bindings sites, demonstrating that canonical genes, i.e., SOCS3, Granzyme B, GAS1, IL2RA, STAT3, and CD30, were significantly downregulated. Conversely, CD58, CD274, and MCH-I/II were upregulated at late time points. By mapping the STAT3 binding sites in ALK+ and ALK- ALCL, we have identified 1077 and 2763 STAT3 peaks within promoter/5'-/3'- and distant intergenic regions, corresponding to both coding and non-coding genes. Therapeutically, in vitro treatments led to cell cycle arrest and profound growth inhibition, and over time increased cell death of PTCL cells, including ALCL. Accordingly, growth inhibition of ALCL xenograft and PDTX tumors was also achieved (Figure 2). To identify drugs that could synergize withSTAT3-degrader activity, we tested a compound library (40) targeting pro-tumorigenic PTCL pathways as well as FDA-approved compounds. Ongoing studies are in progress. Conclusion: We have discovered selective STAT3 degraders which control PTCL growth. STAT3 degraders are powerful tools to define the STAT3 pathogenetic mechanisms and dissect genes/pathways to be targeted for T-cell lymphoma eradication. These data provide additional rationale for testing STAT3 degraders in the clinic for the treatment of aggressive malignancies including PTCL/ALCL. Figure 1 Figure 1. Disclosures Yang: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: Owkin: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Johnson and Johnson: Research Funding; Eli Lilly: Research Funding; Janssen: Research Funding; Champions Oncology: Consultancy; Freenome: Consultancy, Other: Current equity holder in a privately-held company; One Three Biotech: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding. Horwitz: Affimed: Research Funding; Aileron: Research Funding; ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; Celgene: Research Funding; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding. DeSavi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3493-3493
Author(s):  
Esperanza Martin-Sanchez ◽  
Socorro M. Rodriguez-Pinilla ◽  
Luis Lombardia ◽  
Beatriz Dominguez-Gonzalez ◽  
Margarita Sanchez-Beato ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Gene Set Enrichment Analysis ◽  
Dependent Manner ◽  
Pi3k Inhibitors ◽  
T Cell Lymphomas ◽  
Pi3k Inhibition ◽  
Peripheral T Cell Lymphomas ◽  
Pi3k Delta

Abstract Abstract 3493 Peripheral 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.

Retinoic Acid Receptor Alpha Expression Drives Cell-Cycle Progression and Is Associated with Increased Sensitivity to Retinoids in Peripheral T-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1749-1749
Author(s):  
Rebecca L Boddicker ◽  
Xueju Wang ◽  
Surendra Dasari ◽  
Grzegorz S. Nowakowski ◽  
Konstantinos N Lazaridis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
T Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Research Funding ◽  
Retinoic Acid Receptor ◽  
Wild Type ◽  
T Cell Lymphomas

Abstract Background: Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with marked clinical, pathological, and molecular heterogeneity. Outcomes following standard therapy generally are poor; however, few candidate therapeutic targets have been identified for precision medicine approaches. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to natural or synthetic retinoids. Retinoids have been used successfully to treat acute promyelocytic leukemia and some cutaneous T-cell lymphomas (CTCLs). However, the function of RARA and the action of retinoids in PTCL have not been defined. Methods:Based on identification of a PTCL patient with a non-synonymous point mutation, RARA R394Q, identified in the Mayo Clinic Center for Individualized Medicine, we sought to characterize the role of RARA in PTCL cells. To investigate the role of wild-type and mutant RARA, we constructed expression vectors containing either wild-type RARA or RARA R394Q coding sequences, and also used siRNAs targeting RARA to study the role of native RARA expression. Cell lines derived from post-thymic T-cell malignancies were used for in vitro studies, including HuT78 and Mac-1 (both derived from circulating tumor cells from CTCL patients) and Karpas 299 (from an ALK-positive anaplastic large cell lymphoma). Following RARA overexpression or knockdown, we measured cell growth, cell cycle regulation, and sensitivity to synthetic retinoids. In addition, RNA sequencing and pathway analysis were performed to profile the transcriptomic response to retinoids in malignant T cells. Results:In two RARAlow cell lines, Karpas 299 and HuT78, overexpression of wild-type RARA or RARA R394Q significantly increased cell growth (p<0.001), with a greater increase observed from mutant versus wild-type RARA in Karpas 299 (136% of control versus 122%; p=0.04). Accordingly, knockdown of wild-type RARA in the RARAhigh cell line, Mac-1, resulted in a 22% inhibition of cell growth (p=0.0002). This inhibition specifically was associated with G1 cell cycle arrest (120% of control; p=0.004) and decreased protein expression of the G1-S-associated cyclin-dependent kinases, CDK2, CDK4, and CDK6. These kinases were up-regulated by overexpression of RARA in RARAlow HuT78 cells. The relatively RARA-specific retinoid, AM80 (tamibarotene), and the less specific retinoid, all-trans retinoic acid (ATRA), resulted in RARA protein degradation, cell growth inhibition that was both dose-dependent and proportional to baseline RARA expression, G1 arrest, and CDK protein up-regulation. Gene-set enrichment analysis (GSEA) of transcriptome data confirmed that genes down-regulated by AM80 were highly enriched for regulators of cell cycle and particularly G1-S transition. Finally, overexpressing RARA in RARAlow Karpas 299 and HuT78 cell lines significantly increased the ability of AM80 to inhibit CDK2/4/6 expression and cell growth (16% to 23% greater growth inhibition than control; p<0.05). Conclusions:RARA drives cyclin-dependent kinase expression and G1-S transition in malignant T cells, and promotes cell growth. These functions may be enhanced by specific RARA gene mutations. Synthetic retinoids inhibit these functions in a dose-dependent fashion, and are most effective in cells with high RARA expression. These data suggest RARA as a candidate therapeutic target in some PTCL patients. Disclosures Nowakowski: Celgene: Research Funding; Morphosys: Research Funding; Bayer: Consultancy, Research Funding.

scholarly journals GSK458 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in T Cell Lymphomas As a Single Agent and in Combination Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5378-5378 ◽  
Author(s):  
Juan Gu ◽  
Lianjuan Yang ◽  
Dennis C Gaughan ◽  
Ling He ◽  
Weina Shen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Antitumor Activity ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
T Cell Lymphoma ◽  
Synergistic Activity ◽  
T Cell Lymphomas

Abstract Introduction: T-cell lymphoma (TCL) accounts approximately 15% of all Non-Hodgkin's lymphoma cases. TCL are often divided into either systemic or cutaneous TCL (CTCL). The management of TCL systemic or cutaneous is challenging because the emergence of chemotherapy resistance that lead to early death (systemic T-cell lymphomas) or chronic debilitating clinical course (CTCL). To improve the clinical outcomes and reduce treatment-related toxicity, research need to be done focusing on understanding and targeting the molecular mechanism driving TCL subtypes. Recently, studies showed that the PI3K AKT/mTOR pathway was activated in TCL. GSK458 is a potent oral dual inhibitor of pan PI3K (α, β, γ and δ) and mTOR (mTOR1 and mTOR2). Preclinical studies in B-cell lymphomas showed GSK458 had broad antitumor activity in vitro and in vivo. In 2016, Phase I clinical trial of GSK458 was competed at the maximal dosage of 2.5mg twice daily. However, the effects of GSK458 on T-cell malignancies remain utterly unknown. Here, we evaluated the activity of GSK458 in preclinical T cell lymphoma models. Methods: We used a panel of T-cell lymphoma cell lines representing PTCL (J45), T-cell lymphoblastic lymphoma (SupT-1), and Mycosis Fungoides (MF)(MJ, HH and H9). TCL cell lines were exposed to escalating doses of GSK458 (1nM-100µM) without or with chemotherapeutic agents (doxorubicin, cisplatin, carboplatin, and dexamethasone); Bcl-2 inhibitor (Venetoclax); proteasome inhibitors ( bortezomib, carfilzomib, Ixazomib); or HDAC inhibitors (SAHA) for 48 and 72 hrs. Differences in cell viability, ATP levels, low mitochondria potential, glucose update, apoptosis and cell cycle distribution were evaluated utilizing PrestoBlue, Cell-Titer Glo assays, DiOC6, 2-NDG, Annexin V and propidium iodide staining followed by flow cytometric analysis, respectively. IC50 was calculated by GraphPad. PI3K and mTOR downstream pathway phosphorylation status, such as p-AKT Ser473, p-AKT Thr308, p-mTOR and p-GSK3β were detected by internally staining of FITC conjugated-antibodies followed by flow cytometry. Apoptosis proteins (MCL-1, PARP, p53, XIAP etc.) were detected by western blot. The additive/synergistic activity of GSK458 was detected by presto blue assay and Coefficient of synergy was calculated using CalcuSyn. Results:In vitro exposure of TCL cell lines to GSK458 demonstrated a dose- and time-dependent cell death. The IC50 of the cells were ranged from 3nM to 1.05uM at 72 hours. At 72h, GSK458 10nM lowered cellular mitochondrial potential, ATP levels and glucose uptake. GSK458 induced apoptosis and arrested the cell cycle at G1. At molecular level, GSK458 reduced phosphorylation status of AKT ser473 and Thr308, mTOR and GSK3β. Interestingly, GSK458 inhibited Mcl-1 expression level. GSK458 exhibited synergistic activity when combined with doxorubicin and dexamethasone. To a lesser degree, GSK458 enhanced the anti-tumor activity of Venetoclax, proteasome and HDAc inhibitors. Conclusion: GSK458 is active as a single agent or in combination with chemotherapy agents or small molecule inhibitors in a variety of T-cell pre-clinical models representing forms of systemic or cutaneous T-cell lymphoma. GSK458 was able to inhibit phosphorylation of AKT, mTOR and GSK3β, which may be the mechanism to reduce ATP production and glucose uptake in the cancer cells. Moreover, GSK458 arrested cell cycle at G1 arrest. Our data supports the clinical evaluation of GSK458 in relapsed/refractory T-cell lymphoma patients. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) Disclosures No relevant conflicts of interest to declare.

Combination of Epigenetic Agents Synergistically Reverse the Malignant Phenotype in Models of T-Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2727-2727 ◽  
Author(s):  
Enrica Marchi ◽  
Matko Kalac ◽  
Danielle C Bongero ◽  
Christine M McIntosh ◽  
Laura K Fogli ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Molecular Level ◽  
Cell Lymphoma ◽  
Single Agent ◽  
T Cell Lymphoma ◽  
Combination Group ◽  
T Cell Lymphomas

Abstract Abstract 2727 CHOP and CHOP-like chemotherapy remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) are presently approved for the treatment of relapsed or refractory cutaneous T- cell lymphomas (CTCL) and peripheral T-cell lymphomas (PTCL) given their marked single agent activity in these diseases. The interaction between the HDACIs (depsipeptide (R) and belinostat (B)) and a DNMT inhibitor (decitabine (D)) was investigated in vitro, in vivo and at the molecular level in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (P12, PF-382). For all cytotoxicity assays, a luminescence based cell viability assay was used (CellTiter-Glo™) followed by acquisition on a Biotek Synergy HT. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR<1 are defining synergism). Apoptosis was assessed by staining with Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition and analyzed using FlowJo. The IC50s for B, R, vorinostat (V), panobinostat (P), D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours in all the cell lines. For the combination experiment we selected the most active DNMTI, decitabine. In the cytotoxicity assays, the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines studied. The RRRs for all the combinations were between 0.0007 and 0.9. When H9, HH, P12 and PF382 cell lines were treated with D and B or R for 72 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. Table 1 displays the range of apoptosis induction for B, R ± D and the RRR value for the most significant data.Table 1:BDB + DRRR(% Apoptotic + Dead Cells)H9100 nM (22.9%)500 nM (17.9%)51.5%0.7HH100 nM (42.9%)1 uM (46.9%)61.3%0.8P 12150 nM (16%)1 uM (42.7%)80.1%0.4PF 382100 nM (8.3%)1 uM (27.9%)40.1%0.8RDR + DH92 nM (22.2%)500 nM (17.9%)63.6%0.5HH2 nM (80%)1 uM (46.9%)89.7%0.6P 122 nM (9.9%)10 uM (58.7%)98%0.03PF 3822 nM (54.5%)500 nM (17.9%)88.7%0.2 An in vivo xenograft study in 6–8 weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells was performed. Mice were separated into different cohorts and treated i.p. for 3 cycles with D or B or their combination according to the following schedules: D at 1.5 mg/kg on days 1, 3, 5; B at 40 mg/kg/day for 10 days (I cycle); D at 1.5mg/kg on days 15,17,19,21; B at 65 mg/kg/day for 10 days (II cycle); D at 1.5 mg/kg on days 29,31,33,35,37,39,41,43; B at 100mg/kg for 19 days (III cycle). Statistically significantly tumor growth inhibition was observed in the combination cohort compared to all the other cohorts (analysis on day 42, 45). We analyzed the molecular basis for this synergistic effect by evaluating gene expression patterns using the Illumina Human HT-12 v4 Expression BeadChip microarrays. These analyses revealed differentially expressed genes and modulated pathways for each of the single treatment conditions and the combination. As shown in Figure 1, a set of genes (A) is down-regulated by both drugs. Other genes (B) are up-regulated by D and the effect is maintained in the combination. Other genes (C+E) are slightly up-regulated by R, though not significantly modified by D, and more strongly up-regulated in the combination group. Similarly, genes to some extent up-regulated by D but not by R (D+F) appeared to be more significantly affected by the combination. As shown in Figure 2, the effects of the two drugs are largely different (only 39 genes modified in common by all the treatment groups). Most of the effects induced by the single agent treatment are maintained in the combination group (174 genes out of 191 for romidepsin and 211 genes out of 221 for decitabine). Interestingly, an additional 944 genes appeared to be modulated uniquely by the combination treatment strongly supporting the hypothesis of synergism also at the molecular level. Collectively, the data suggest that the combination of a DNMTI and HDACIs is synergistic in in vitro and in vivo model of T-cell lymphoma and is able to synergistically reverse the malignant signature at the molecular level. These data may constitute the basis for future phase I-II clinical trials. Disclosures: O'Connor: celgene: Consultancy, Research Funding; merck: Research Funding; Novartis: Research Funding; spectrum: Research Funding.

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