Loucy Cell Research Articles

Abstract 1574: Homoharringtonine is effective in treating acute lymphoblastic leukaemia via modulation of apoptotic pathway

Abstract Acute lymphoblastic leukaemia (ALL) is an aggressive and highly lethal malignancy caused by clonal mutations in lymphoid progenitor cells. The prognosis for adult ALL remains poor, with only 30-40% of adult patients achieving long-term remission. Therefore, novel therapies are needed. Homoharringtonine (HHT) is a natural-derived plant alkaloid from tree of Cephalotaxus. FDA approved the use of HHT in the treatment of chronic myeloid leukaemia (CML) in 2012. Limited pre-clinical or clinical studies were conducted for therapeutic efficacy and molecular mechanisms of HHT on ALL to the best of our knowledge. To fill this knowledge gap, here we investigated the antileukemic activity of HHT in T lymphoblastic leukaemia (T-ALL) and B lymphoblastic leukaemia (B-ALL). In this study, T-ALL cell lines namely JURKAT, MOLT-4, LOUCY, CCRF-CEM and B-ALL cell clines namely PALL-2, SUP-B15 were used. Cell viability was reduced upon treatment of HHT at time and dose-dependent manner. Percentage of apoptotic cells that determined by flow cytometric analysis of PI and Annexin V double staining was increased upon treatment of HHT. Moreover, HHT was able to induce apoptosis in both T-ALL and B-ALL cell lines. Western blot analysis showed that MCL-1 protein level was reduced upon treating with HHT for 24 hours. The above findings provide conclusive evidence for the therapeutic effect of HHT on various ALL cell lines by modulating the apoptotic pathway. Citation Format: Anan Jiao, Kei Ching Yuen, Rahman Ud DIN, On Yi Phoebe Wong, Sin Chun-Fung. Homoharringtonine is effective in treating acute lymphoblastic leukaemia via modulation of apoptotic pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1574.

JAK/BCL2 inhibition acts synergistically with LSD1 inhibitors to selectively target ETP-ALL

ETP-ALL (Early T cell Progenitor Acute Lymphoblastic Leukemia) represents a high-risk subtype of T cell acute lymphocytic leukemia (T-ALL). Therapeutically, ETP-ALL patients frequently relapse after conventional chemotherapy highlighting the need for alternative therapeutic approaches. Using our ZEB2Tg ETP-ALL mouse model we previously documented the potential utility of the catalytic LSD1 inhibitor (GSK2879552) for treating mouse/human ETP-ALL. However, this approach proved to be inefficient, especially in killing human LOUCY cell ETP-ALL xenografts in vivo. Here we have revealed the novel involvement of ZEB2/LSD1 complexes in repressing the intrinsic apoptosis pathway by inhibiting the expression of several pro-apoptotic proteins such as BIM (BCL2L11) as a major driver for ETP-ALL survival. Treatment with LSD1i (particularly with the steric inhibitor SP2509) restored the expression of ZEB2/LSD1 pro-apoptotic BIM (BCL2L11) target. In combination with a JAK/STAT pathway inhibitor (JAKi, Ruxolitinib) or with a direct inhibitor of the anti-apoptotic BCL2 protein (BCL2i, ABT-199) resistance of human and mouse ETP-ALL to LSD1i was reversed. This new combination approach efficiently inhibited the growth of human and mouse ETP-ALL cells in vivo by enhancing their differentiation and triggering an apoptotic response. These results set the stage for novel combination therapies to be used in clinical trials to treat ETP-ALL patients.

Abstract 5337: Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL)

Abstract Background: Inhibitors of apoptosis proteins (IAPs) are overexpressed in ALL leading to resistance to apoptosis and chemo-resistance. Tolinapant, a non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis (cIAP1/2 and XIAP) being evaluated in Phase 1/2 clinical trial [NCT02503423] showed single-agent activity in T-cell lymphoma. Objectives: Herein, we analyzed the preclinical activity of tolinapant in combination with ABT199 and Dexamethasone (DEX) in T-ALL (in vitro and ex-vivo patient derived xenografts (PDX). Results: Using a panel of 8 T-ALL cell lines we analyzed the single agent activity of tolinapant. Loucy and SUP-T11 were most sensitive (IC50 = 190-309 nM). CCRF-CEM and ALL-SIL showed moderate sensitivity (IC50 = 10-18 µM) while Jurkat, MOLT16, MOLT4 and PF382 were resistant. Western blotting (WB) showed decreased levels of cIAP1and cIAP2 with no change in XIAP in response to tolinapant as a single agent. We next investigated the efficacy of tolinapant with Bcl2 inhibitor ABT199. The combination of ABT199 and ASTX660 was synergistic in Loucy cell line with combination index (CI) of 0.14. Cell death was increased to 64±3% in combination v/s 27±0.9% in ABT199. WB showed decreased cIAP2 and increased levels of cleaved caspase 7 and cleaved caspase 9 in combination compared to single agents, suggesting increased apoptosis. Treatment of PDX-derived cells with tolinapant and ABT199 was more effective than monotherapy in inducing apoptosis in CD45+ bulk (46±0.7% to 63±6%, p<0.0001) and leukemia initiating cells (LICs, CD45+ CD7+ CD19- CD34+) (39±3% to 54±8%, p=0.003). Next, we checked the effect of combination of DEX with tolinapant in T-ALL cell lines. CCRF-CEM cell line is from a relapsed patient and is resistant to DEX. The combination was synergistic with CI of 0.26 and cell death of 50±4% compared to 20±3% by DEX. A triple combination of DEX, ABT199 and tolinapant increased apoptotic response to 82.9±1%, compared to DEX+ABT199 dual combination where apoptosis was induced to 52.4±2%. Strong synergy in terms of both cytoreduction as well as apoptosis induction was observed in SUP-T11 cells. Tolinapant sensitized SUP-T11 cells to DEX with ED50 value of 542 nM in combination compared to ~2 µM with DEX treatment. Simultaneous analysis of cell proliferation, stress response and DNA damage using single-cell proteomics analysis showed downregulation of proliferation (Ki-67), stress response (ATF4, LC3B) and increased levels of cleaved PARP, cleaved caspase 3 suggesting increased apoptosis in combination of tolinapant with DEX. Treatment of PDX-derived cells with tolinapant enhanced the cytotoxic effect of DEX in CD45+ bulk (58±2% to 71±0.1%, p<0.0001) and LICs (62±2% to 78±0.9%, p<0.0001). Conclusion: Tolinapant synergizes with the anti-leukemic activity of ABT199 and DEX, establishing a therapeutic rationale for IAP antagonist in T-ALL. Citation Format: Priyanka Sharma, Sujan Piya, Huaxian Ma, Natalia Baran, Muharrem Muftuoglu, Mahesh Basyal, Vivian Ruvolo, George Ward, Tomoko Smyth, Martin J. Sims, Michael Andreeff, Gautam Borthakur. Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5337.

Bortezomib Is Effective in Treating T-ALL, Inducting G2/M Cell Cycle Arrest and WEE1 Downregulation

T lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy with poor outcome, especially for relapse/refractory disease. Early T- cell precursor acute lymphoblastic leukaemia (ETP-ALL) is a recently identified subtype of T-ALL with worse treatment outcome compared with other subtypes of T-ALL and treatment options are limited. T-ALL frequently harbors genetic aberrations leading to cell cycle dysregulation and it is one of the major molecular pathogenesis of T-ALL. WEE1 is a protein kinase that is responsible for inhibiting mitosis with unrepaired damaged DNA via inactivating CDK1. WEE1 is highly express in adult T-ALL and its overexpression is associated with adverse prognosis in various cancers. Inhibiting WEE1 expression is a novel approach of therapy. Bortezomib is a 26S proteosome inhibitor and it is FDA approved for treating plasma cell myeloma and mantle cell lymphoma. Bortezomib had been demonstrated therapeutic efficacy in clinical setting for relapse/refractory paediatric T-ALL and B-ALL when combined with chemotherapy. Despite its therapeutic efficacy in clinical studies, the mechanism of action of Bortezomib in T-ALL remain uncertain. The role of Bortezomib in cell cycle modulation had not been established in T-ALL. Moreover, it had not been demonstrated that the effect of Bortezomib in WEE1 expression in T-ALL. Here, we present our study that demonstrated the therapeutic efficacy of Bortezomib in treating T-ALL via cell cycle modulation and downregulation of WEE1 by Bortezomib.T-ALL cell lines including MOLT16, MOLT4, LOUCY and CEM were used in the study. Cell viability was measured by trypan blue. Apoptosis and cell cycle analysis were measured by flow cytometry. Western blot of WEE1, p53, cyclin B1, p21 and p27 were performed.Our result showed that Bortezomib reduce the cell viability of T-ALL cell lines in dose and time-dependent manner. Bortezomib was also sensitive towards LOUCY, a T-ALL cell line with ETP-ALL phenotype. It implied that Bortezomib could be a promising therapy for ETP-ALL. Bortezomib also triggered apoptosis in various T-ALL and the effect of apoptosis was more pronounced after 72 hours of treatment when compared with 24-hour. Again, Bortezomib was able to induce apoptosis in LOUCY cell line. G2/M cell cycle arrest was observed in various T-ALL upon treatment of Bortezomib. The effect on cell cycle modulation was also observed in LOUCY cell line. The protein expression of p21 and p27 were increased after the treatment of Bortezomib. The level of cyclin B1 was increased also. There was upregulation of p53 after Bortezomib treatment. Strikingly, the protein expression level of WEE1 was reduced. The findings of WEE1 downregulation by Bortezomib is a novel findings. We also showed that Bortezomib downregulate WEE1 mRNA expression by quantitative PCR.Our study showed that Bortezomib is active against T-ALL cell lines, including ETP-ALL cell line, LOUCY and modulates cell cycle with G2/M arrest. Bortezomib had been shown to increase the level of p21, p27 and cyclin B1 and induced G2/M cell cycle arrest in glioblastoma cells. However, studies on cell cycle modulation by Bortezomib in T-ALL are scarce. Here, we demonstrated Bortezomib stabilized p21, p27 and upregulation of cyclin B1 in T-ALL as well, which could account for the G2/M cell cycle arrest. We first showed that downregulation of WEE1 after treatment with Bortezomib, in protein level as well as in mRNA level. Recent study showed that inhibition of WEE1 is a novel target of therapy in T-ALL. WEE1 is upregulated in T-ALL to prevent entry of mitosis with unrepaired damaged DNA. The downregulation of WEE1 by Bortezomib as showed by our study could reverse its effect and leads to apoptosis of leukaemic cells.In summary, our study provides the insight on mechanism of action of Bortezomib in modulating cell cycle in T-ALL. Moreover, it is the first study to demonstrate WEE1 downregulation by Bortezomib in T-ALL. These findings not only enhance our understanding of mechanism of action of Bortezomib in T-ALL, but also rationalized the use of certain synergistics combination therapy with Bortezomib in treating T-ALL, e.g., chemotherapeutic agents, PARP inhibitors which could damage DNA of leukaemic cells. Further research is needed to explore those combination therapy in T-ALL and molecular mechanism of downregulation of WEE1 by Bortezomib in T-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Personalized Therapy Design for Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) Using Computational Biology Modeling with in Vitro Validation

ETP-ALL is a high-risk subtype of T-ALL with poor prognosis and high risk of relapse in patients treated with standard of care (SOC). ETP-ALL patients have more complex cytogenetic profiles and a unique immunophenotype compared to other T-ALL cases. ETP-ALL is characterized by the expression of myeloid and hematopoietic stem cell (HSC) surface proteins, as well as gene expression profiles similar to myeloid progenitors and HSCs, including frequent mutations in myeloid oncogenes. Targeted therapies for ETP-ALL are limited because identified gene mutations are not actionable by currently available drugs. Recently we developed a predictive simulation modeling method that projects cancer patient-specific protein network maps based on the patient's unique cancer genomics (PMID 27855285). Here, we present a computational biology approach to analyze the genomic profile of ETP-ALL and identify novel treatment strategies using available FDA approved drugs. In this study, we used the LOUCY cell line as a model of ETP-ALL due to its high expression of characteristic ETP-ALL markers HHEX, LYL1, MEF2C and ZEB2.Aim: To identify and validate novel drug or drug combinations for ETP-ALL using an integrated genomics and computational biology modeling (CBM) approach.Methods: The LOUCY cell line was used as a model of ETP-ALL. Publicly available genomic profiling data (cBioPortal, Sanger) were collected. All available genomic data for the LOUCY cell line were entered into a CBM program (Cellworks Group) that generates a disease-specific protein network map using PubMed and other online resources. Disease-specific biomarkers unique to the LOUCY cell line were identified within the protein network maps. A digital drug library of FDA approved therapies and drugs in clinical development was developed and digitally simulated in the computational model at varying doses. Drug impact was quantitatively assessed by a cell growth score, which is a composite of cell proliferation, viability, apoptosis, along with the impact on disease-specific biomarkers. This CBM's predictions for drug response were compared with publicly available in vitro experimental data. Novel drug options and combinations to treat ETP-ALL were also identified by CBM. The novel drugs and drug combinations identified by CBM were then tested on the LOUCY cells at varying concentrations to calculate IC50 values. Drug activity was measured by XTT assay, using the Jurkat cell line as a non-ETP-ALL control and the KG-1 AML cell line as a non-ALL control.Results: CBM predicted extensive dysregulation of multiple protein signaling pathways in the LOUCY cell line: Cell cycle upregulation was predicted due to copy number loss of CDKN1B, CDKN2B and CDKN2A genes. BCL2 expression was predicted to be increased due to the presence of a NUP214 - SET fusion protein, activating the BCL2 transcription factor HOXA9. Knockdown of PTEN and TSC2 was predicted to activate the AKT-mTOR pathway, while knockdown of ETV6 and SOCS1 was predicted to activate the JAK-STAT pathway (Fig 1). The response predictions of 31 drugs were compared with data from in vitro experiments. LOUCY cells were predicted to respond to 9 of the drugs, and in vitro testing showed sensitivity to 8 out of 9 drugs (88.8%). LOUCY cells were predicted to show no response to 22 of the drugs, and in vitro testing showed resistance to all 22 drugs (100%) (Table 1). Additionally, CBM predicted everolimus, nelfinavir, and palbociclib as novel therapies for the ETP-ALL representative LOUCY cell line, and were validated (100%) by additional in vitro XTT assays (Fig 2). Key statistical correlations for drug efficacy were observed (Table 2).Conclusions: Predictive simulation modeling of the unique genomic abnormalities found in the ETP-ALL LOUCY cell line led to the creation of an ETP-ALL protein network map that shows multiple interacting and cooperative protein network disturbances including cell cycle, anti-apoptosis, AKT-mTOR and JAK-STAT pathways. Digital drug screening using this distinctive protein network map led to the discovery of new drugs with predicted efficacy. In vitro studies validated the computational method with high accuracy. Results from this work inform future clinical trials for patients with ETP-ALL. This CBM technology may be a useful tool for clinicians to personalize treatment for patients with relapsed/refractory ETP-ALL. [Display omitted] DisclosuresKumar:Cellworks: Employment. K:Cellworks: Employment. Kamath:cellworks: Employment. Nair:cellworks: Employment. Raju:cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

Computational Biology Model of ETP-ALL Used to Accurately Classify ETP-ALL Vs Non ETP-ALL Patients with Genomic Input

Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive hematological malignancy with poor prognosis after chemotherapy compared to other ALLs. ETP-ALL cases typically display a high burden of chromosomal copy number variations (CNVs) and myeloid gene mutations. To enhance the understanding of this disease, a computational biological modeling approach was developed to classify ETP-ALL vs. non-ETP-ALL patient based on genomic abnormalities and protein biomarkers associated with ETP-ALL and non ETP-ALL profiles.Methods: Genomic profiling data for whole exome sequencing (WES), array CGH (Agilent) and karyotype data of 69 ETP- ALL and Non-ETP-ALL patient were used for this study. The genomic data for each patient were entered into a computational biology model (CBM) (Cellworks Group), which generates a disease-specific protein network map using PubMed and other online resources. CBM was used for a) Classification between ETP-ALL and Non-ETP ALL model, b) Identification of unique patient characteristics and c) Drug response.For the CBM approach, we divided the data into training and validation data sets. 40 patients were selected for training data and 39 patients selected for the validation data set. In a training dataset, genomic data from 22 ETP-ALL patients were used from a GEO database (GSE28703) (N=11) and a prior genomics report (PMID:22237106) (N=11). The training dataset also included non-ETP-ALL patients from a GEO database (GSE28703) (N=18). Each patient's data was analyzed by CBM, to translates genomic variants into protein networks.To validate these findings, genomic data from 15 ETP-ALL patients, different than training set patients, were used from a GEO database (GSE28703) (N=1), Moffitt Cancer Center (IRB201600284) (N=6), a prior genomics report (PMID: 22237106) (N=7), and an ETP-ALL LOUCY cell line with known genomic aberrations. The validation dataset also included Non-ETP-ALL patients different than training set patients, from a GEO database (GSE28703) (N=14).Results: Prediction accuracy for classification between ETP-ALL and Non ETP ALL patient was 89.65%. PPV value was 86.67% and NPV value was observed 92.86%. Sensitivity and Specificity of prediction outcome was observed as 92.86% and 86.67% respectively.The simulation model identified six protein biomarkers which predicted increased activity in ETP-ALL profiles compared to non-ETP-ALL cases: STAT3, RUNX1, HIF1A, FOXM1, CEBPA, mTORC1.Key genomic differences were also identified between ETP-ALL and non ETP-ALL patients (Table1).Conclusions: ETP-ALL is distinct from other T-ALL with respect to genomic and protein network abnormalities. Computational biology model can accurately classify between ETP-ALL and Non ETP-ALL patients. CBM also predicted unique biomarkers between ETP-ALL and Non-ETP-ALL patients. [Display omitted] DisclosuresKumar:Cellworks: Employment. K:Cellworks: Employment. Vasista:Cellworks Research India: Employment. Tyagi:Cellworks: Employment. Rajagopalan:Cellworks: Employment. Alam:Cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

Digging deeper into the immunopeptidome: characterization of post-translationally modified peptides presented by MHC I

Peptides presented by MHC molecules on the cell surface, or the immunopeptidome, play an important role in the adaptive arm of the immune response. Antigen processing for MHC class I molecules is a ubiquitous pathway present in all nucleated cells which generates and presents peptides of both self and non-self-origin. Peptides with post-translational modifications represent one category of peptides presented by MHC class I molecules. However, owing to the complexity of self-peptides presented by cells, the diversity of peptides with post-translational modifications is not well-studied. In this study, we carried out MHC Class I immunopeptidomics analysis of Loucy T-cell leukemia and A375 malignant melanoma cell line to characterize the diversity of post-translational modifications of MHC class I-bound peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both cell lines using Bolt and Sequest search engines. The enrichment method was highly specific as ~ 90% of the peptides were of typical length (8–12 amino acids long) and the motifs were expected based on previously reported motifs for MHC I alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these PTMs compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that accounted for ~ 5–7% of MHC-bound peptides that were otherwise similar in their features as normal MHC-bound peptides. We validated the identity of several post-translationally modified peptides and spliced peptides through mass spectrometric analysis of synthetic peptides. Our study confirms post-translationally modified peptides to be present at low stoichiometric levels along with unusual spliced peptides through unbiased identification using high resolution mass spectrometry.

The Splenic Microenvironment Is a Novel Site of Relapse Following ABT-199 Treatment

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy arising from the transformation of immune T-cell lymphocytes. Early T-cell progenitor (ETP-ALL) is a subgroup particularly associated with a poor prognosis and a high risk for relapse. While the leukaemia initially develops in the thymus it spreads in the blood to the bone marrow, lymph nodes and often the spleen. Interestingly, splenomegaly was previously associated with a poor prognosis in leukemic patients. Recently, it was shown that ETP-ALL is dependent on the expression of the anti-apoptotic protein BCL-2, and is sensitive to inhibition with ABT-199, a BCL-2 specific BH3 mimetic. However, one issue with targeted agents, like ABT-199, is the development of resistance to treatment. Our aim was to determine potential in vivosites of resistance/relapse following ABT-199 treatment using a xenograft model of ETP-ALL. We confirmed that the ETP-ALL LOUCY cell line is BCL-2 dependent and then labelled it with luciferase to enable visualisation of the leukaemia in vivo. Following establishment of the leukaemia in NOD/SCID gamma mice, as assessed by hCD45+, the mice were randomised to receive vehicle control or 50 mg/kg ABT-199 by oral gavage daily for two weeks. While the mice were initially sensitive to ABT-199, the leukaemia started to progress while on treatment. Interestingly, there appeared to be a selective redistribution of the leukaemia to the spleen following ABT-199 treatment. Indeed, LOUCY cells isolated from the spleen of the mice had a reduced BCL-2 dependence, as assessed by BH3 profiling. The reduced BCL-2 dependence correlated with reduced BCL-2 expression at both the mRNA and protein level. Next, we confirmed that human splenic fibroblasts (HSF) co-cultured with the LOUCY cell line in vitro also altered BCL-2 dependence and expression using BH3 profiling and Western blotting. To identify potential splenic cytokines involved in the regulation of BCL-2 protein expression in ETP-ALL we performed a screening cytokine array. Upon co-culture of the LOUCY cells with HSF there was an increased expression of IL-6, this was confirmed using ELISA. Using an IL-6 receptor antibody we confirmed that blocking IL-6 receptor reversed the change in BCL-2 dependence in the presence of the splenic microenvironment. Lastly, we confirmed in a T-ALL patient-derived xenograft, that is BCL-2 dependent, that the splenic microenvironment alters the mitochondrial apoptotic threshold. Currently, there are reports in the literature of ETP-ALL patients being treated with ABT-199. While there have been numerous studies lately describing cell autonomous events leading to ABT-199 resistance, our novel finding that the splenic microenvironment is a site of relapse is potentially of great clinical importance for BCL-2 dependent leukemia's. Disclosures Ni Chonghaile: AbbVie: Research Funding.

Abstract 1314: MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia

Abstract Background: Early T-precursor acute lymphoblastic leukemias (ETP-ALL) account for 15% of pediatric T-cell ALL (T-ALL) cases and are characterized by an immature phenotype, resistance to therapy, and high relapse rates. MERTK receptor tyrosine kinase is ectopically expressed in ~50% of T-ALLs, particularly those with an immature T cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is specifically expressed in immature T cell precursors, is preferentially expressed in ETP-ALL compared to other T-ALLs, is essential for ETP-ALL cell survival, and is regulated downstream of MERTK in acute leukemia cells. Thus, combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL. Methods: MERTK and BCL-2 mRNA expression was assessed in T-ALL patient samples using publicly available data. Loucy and PEER ETP-ALL cell lines were cultured with vehicle or MRX-2843, a dual MERTK/FLT3 inhibitor, alone or in combination with the BCL-2 inhibitor venetoclax. Phosphorylated and total MERTK were assessed by immunoblot. Cells were stained with PoPro-1-iodide and propidium iodide dyes and analyzed by flow cytometry to assess cell death. Relative cell numbers were assessed using Presto Blue reagent. Orthotopic xenografts were established in NSG or NSGS mice using luciferase-expressing Loucy cells or an ETP-ALL patient sample and leukemia burden was monitored by bioluminescence imaging or flow cytometry. MRX-2843 or saline vehicle were administered orally once daily. Median survival was determined by Kaplan-Meier analysis. Results: MERTK and BCL-2 mRNAs were expressed at significantly higher levels in ETP-ALL patient samples relative to other T-ALLs. MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK and induced dose-dependent cell death (43.2% vs 16% in vehicle-treated cultures, p<0.01) in PEER cells. Moreover, in a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 reduced peripheral blood disease burden by 83% (p<0.001) and decreased spleen weight by 64% (p<0.001) compared to vehicle-treated mice. Furthermore, treatment with MRX-2843 prolonged median survival by 11 days compared to control mice (n=8/group, p=0.0016). In a cell line-derived xenograft model, MRX-2843 reduced disease burden by 60% (n=10/group, p<0.0001) compared to vehicle. Treatment with a combination of MRX-2843 and venetoclax decreased cell density more effectively than either single agent in cultures of the Loucy and PEER cell lines (p<0.05). Conclusions: MERTK and BCL-2 are preferentially expressed in ETP-ALL relative to T-ALL and MRX-2843 has robust therapeutic activity in cell culture and xenograft models of ETP-ALL. These data validate MRX-2843 as a novel agent with potential for clinical application in patients with ETP-ALL. Combined targeting of MERTK and BCL-2 may be particularly effective. Citation Format: Ryan J. Summers, Katherine A. Minson, Eleana Vasileiadi, Xiaodong Wang, Steven V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1314.

PO-072 ω3-Polyunsaturated fatty acids inhibit cell growth and invasion of human hypopharyngeal carcinoma cells

IntroductionT-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy resulting from the transformation of T-cell progenitors. Early T-cell progenitor ALL (ETP-ALL) subgroup accounts for 5% to 10% of T-ALL cases and associated with a poor prognosis and a very high risk for relapse. T-ALL leukemias develop a dependence on anti-apoptotic BCL-2 family proteins that is linked to the maturation stage of the blasts. Typical T-ALL is mostly dependent on BCL-XL, whereas ETP-ALL is more dependent on BCL-2. In recent years, there has been the development of a series of specific BH3 mimetics (ABT-199, ABT-263, WEHI 539) that can inhibit the function of anti-apoptotic BCL-2 proteins and induce apoptosis. T-ALL patients have blasts disseminated in the blood, bone marrow and spleen. Each niche may provide distinct extrinsic signals that could offer a survival advantage to leukemic cells.Material and methodsWe aim to assess in both ETP-ALL (Loucy cell line) and typical T-ALL (CEM-CCRF cell line) if distinct microenvironments affect the BCL-2 family proteins expression and the sensitivity to BH3 mimetics. To study the anti-apoptotic BCL-2 dependencies we will use the cutting-edge technology mitochondrial BH3 profiling. In brief, BH3 profiling is a functional assay that measures the response of mitochondria to exposure of known concentrations of synthetic BH3 peptides by measuring loss of mitochondrial membrane potential or cytochrome c release.Results and discussionsWe found that ETP-ALL cell line was BCL-2 dependent and sensitive to ABT-199 (BCL-2 specific BH3 mimetic). While the Typical T-ALL cell line was BCL-XL dependent and sensitive to both ABT-263 (BCL-2,BCL-XL and BCL-w) and WEHI 539 (BCL-XL) BH3 mimetics. Interestingly, when the ETP–ALL cell line Loucy was co-cultured with the human splenic fibroblast (HSF) cell line we found a reduction in BCL-2 dependency, as assessed by BH3 profiling, and a reduced sensitivity to ABT-199 (BCL-2 specific BH3 mimetic). Remarkably, we found the inverse in the typical T-ALL cell line co-cultured with the HSF. We found an increase in BCL-2 dependence and an increase in sensitivity to ABT-199.ConclusionCurrently, we are trying to identify the signalling pathways which are activated to cause this switch in BCL-2 family dependence in the different subtypes of T-ALL. In addition, we are assessing the BCL-2 family dependence of a xenograft cell line isolated from the blood and spleen of animals to determine if this switiching of BCL-2 dependence occurs in vivo.

PO-068 Determining how distinct microenvironments alter BCL-2 family dependence in T-cell acute lymphoblastic leukaemia

IntroductionT-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy resulting from the transformation of T-cell progenitors. Early T-cell progenitor ALL (ETP-ALL) subgroup accounts for 5% to 10% of T-ALL cases and associated with a poor prognosis and a very high risk for relapse. T-ALL leukemias develop a dependence on anti-apoptotic BCL-2 family proteins that is linked to the maturation stage of the blasts. Typical T-ALL is mostly dependent on BCL-XL, whereas ETP-ALL is more dependent on BCL-2. In recent years, there has been the development of a series of specific BH3 mimetics (ABT-199, ABT-263, WEHI 539) that can inhibit the function of anti-apoptotic BCL-2 proteins and induce apoptosis. T-ALL patients have blasts disseminated in the blood, bone marrow and spleen. Each niche may provide distinct extrinsic signals that could offer a survival advantage to leukemic cells.Material and methodsWe aim to assess in both ETP-ALL (Loucy cell line) and typical T-ALL (CEM-CCRF cell line) if distinct microenvironments affect the BCL-2 family proteins expression and the sensitivity to BH3 mimetics. To study the anti-apoptotic BCL-2 dependencies we will use the cutting-edge technology mitochondrial BH3 profiling. In brief, BH3 profiling is a functional assay that measures the response of mitochondria to exposure of known concentrations of synthetic BH3 peptides by measuring loss of mitochondrial membrane potential or cytochrome c release.Results and discussionsWe found that ETP-ALL cell line was BCL-2 dependent and sensitive to ABT-199 (BCL-2 specific BH3 mimetic). While the Typical T-ALL cell line was BCL-XL dependent and sensitive to both ABT-263 (BCL-2,BCL-XL and BCL-w) and WEHI 539 (BCL-XL) BH3 mimetics. Interestingly, when the ETP–ALL cell line Loucy was co-cultured with the human splenic fibroblast (HSF) cell line we found a reduction in BCL-2 dependency, as assessed by BH3 profiling, and a reduced sensitivity to ABT-199 (BCL-2 specific BH3 mimetic). Remarkably, we found the inverse in the typical T-ALL cell line co-cultured with the HSF. We found an increase in BCL-2 dependence and an increase in sensitivity to ABT-199.ConclusionCurrently, we are trying to identify the signalling pathways which are activated to cause this switch in BCL-2 family dependence in the different subtypes of T-ALL. In addition, we are assessing the BCL-2 family dependence of a xenograft cell line isolated from the blood and spleen of animals to determine if this switiching of BCL-2 dependence occurs in vivo.

Chemopreventive Actions of Blond and Red-Fleshed Sweet Orange Juice on the Loucy Leukemia Cell Line

Red-fleshed sweet orange juice (ROJ) comes from a new variety of citrus cultivated in Brazil that contains high levels of β-carotene and lycopene, and similar amounts of hesperidin (HSP) and nutrients, equivalently to blond orange juice (BOJ). Such bioactive compounds are associated with chemopreventive actions in several cancer cell lines. The purpose of this study was to examine the cytotoxicity, cell cycle, apoptosis, and cytokine secretion after BOJ, ROJ, and HSP treatment of a novel T acute lymphoblastic leukemia cell line, Loucy. Loucy cells were incubated for 24-h with BOJ, ROJ, and HSP, and the viability was measured using trypan blue. Cell cycling and apoptosis were assessed by propidium iodide (PI) and annexin V-FITC/PI flow cytometry, respectively. Secretion of cytokines IL-1α, IL1-β, IL-2, IL-4, IL-6, IL-10, IL-17A, IFNγ, TNFα, TGFβ, MIPα, and MIPβ was determined by ELISA array. BOJ and ROJ treatments promoted Loucy cell cytotoxicity. Additionally, BOJ induced cell cycle arrest in the G0/G1 phase, and decreased the cell accumulation in the G2/M. ROJ decreased only the G0/G1 fraction, while HSP did not change the cell cycle. BOJ led to apoptosis in a different fashion of ROJ, while the first treatment induced apoptosis by increase of late apoptosis and primary necrotic fractions, the second increased early and late apoptosis, and primary necrotic fraction compared to positive controls. HSP had no effect on apoptosis. IL-6 and IL-10 were abrogated by all treatments. Taking together, these results suggest potential chemopreventive effects of BOJ and ROJ on Loucy cells.

Repressed BMP signaling reactivates NKL homeobox gene MSX1 in a T-ALL subset

In T-cell acute lymphoblastic leukemia (T-ALL), several members of the NK-like (NKL) homeobox genes are aberrantly expressed. Here, we have analyzed the activity of NKL homeobox gene MSX1 using pediatric T-ALL in silico data, detecting overexpression in 11% of patients. Quantification of MSX1 transcripts in a panel of 24 T-ALL cell lines demonstrated overexpression in two examples. Comparative expression profiling indicated inhibition of the bone morphogenetic protein (BMP) signaling pathway, which was shown to inhibit MSX1 transcription. In the LOUCY cell line we identified conspicuous expression of CHRDL1 encoding a BMP inhibitor which mediated activation of MSX1. Promoter analyses demonstrated activation of CHRDL1 by oncogenic PITX1. Furthermore, knockdown and overexpression studies of hematopoietic transcription factors demonstrated that GATA2 and FOXC1 mediate activation and GATA3, LEF1, TAL1 and TOX repression of MSX1 transcription. Collectively, our findings suggest that MSX1 is physiologically restricted to lymphoid progenitors. The identification of deregulated BMP signaling may provide novel therapeutic options for the treatment of T-ALL.

Characterization of the Loucy cell line as a model for human peripheral γδ T cells (638.5)

As unique innate‐like lymphocytes, γδ T cells make up a small proportion of circulating T cells. Up to now, there is no widely accepted cell line model for human γδ T cell. Our aim was to investigate whether Loucy cell line shares similarities with γδ T cells derived from human peripheral blood mononuclear cells (PBMC). First, 95% of Loucy cells express the unique γδ T cell receptor (TCR). Second, most Loucy cells were double negative for CD4 and CD8, like most peripheral γδ T cells. Third, Loucy cells express TLR2, like peripheral γδ T cells. At the functional level, Loucy cells proliferated in response to isopentenyl pyrophosphate, a known phosphoantigen for γδ T cell proliferation. Activated Loucy cells express CD69 on cell surface, similar to γδ T cells in activated mixed PBMC cultures. Unlike peripheral γδ T cells, Loucy cell did not express NKG2D. TCR Vδ gene usage in Loucy cells is mainly Vδ3 and to a lesser extent Vδ1, while Vδ2Vγ9 T cells dominate in blood. In addition, 95% of Loucy cells were CD45RO+ (memory), whereas γδ T cells in PBMC were 45% CD45RO+ and 52% CD45RA+ (naive). The only cytokine detected from PHA‐L‐stimulated Loucy cells was TGF‐β, whereas peripheral γδ T cells produce many more cytokines. These results suggest that Loucy cells have many characteristics similar to γδ T cells in PBMC, and this cell line, for some aspects of γδ T cell function, may be useful as an in vitro model for human γδ T cells.Grant Funding Source: Funded in part by UF AES

BCL2-specific inhibitor ABT-199 synergizes strongly with cytarabine against the early immature LOUCY cell line but not more-differentiated T-ALL cell lines

BCL2-specific inhibitor ABT-199 synergizes strongly with cytarabine against the early immature LOUCY cell line but not more-differentiated T-ALL cell lines

ABT-737 Restores Prednisolone Sensitivity In Early T-Cell Precursor Acute Lymphoblastic Leukemia With High MEF2C Expression

BackgroundEarly T-cell precursor-acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subtype of pediatric T cell acute lymphoblastic leukemia (T-ALL). ETP-ALL is characterized by lack of expression of CD1a and CD8, weak or absent expression of CD5 and aberrant expression of myeloid and hematopoietic stem cell markers, such as CD13, CD33, CD34 and CD117. Because conventional chemotherapy is not fully effective for this subtype, potential therapeutic target should be explored. Here, we assessed gene expression pattern of the transcriptional factors related to differentiation of lymphoid and myeloid cells in ETP-ALL to determine the potential therapeutic target for this particular subtype. Patients and MethodsThirty-one patients with T-ALL treated in Kyoto Prefectural University of Medicine and affiliated hospitals were classified in ETP-ALL or non- ETP ALL based on the results of surface marker analysis by FACS. To compare the gene expression pattern of transcriptional factors related to differentiation of myeloid and lymphoid cells in ETP-ALL with those in non-ETP-ALL, real time RT-PCR (RQ-PCR) was performed to determine expression level of C/EBPa, ID2, NOTCH1, LYL1, IL7R, LMO2, MEF2C, PU.1, and FLT3 in primary leukemic cells from twenty-one patients with T-ALL (ETP-ALL: 9, non ETP-ALL: 12) based on the availability of appropriate RNA. To assess the relationship between high MEF2C expression and prednisolone (PSL) /Bcl2 inhibitor (ABT-737) sensitivity, cell proliferation of Jurkat (not expressing MEF2C) and Loucy (expressing MEF2C) cells cultured with serial concentration of PSL and/or ABT-737 was measured with the WST-1 assay to determine the 50% growth inhibitory concentration (IC50) of these agents. BaF3 cells expressing MEF2C (BaF3-MEF2C) was also generated using retrovirus gene transduction system (MSCV) and cultured with serial concentration of PSL/ABT-737 to determine the IC50 of PSL/ABT-737. Apoptosis was determined by Annexin V/PI staining. Viability of primary leukemic blasts of ETP-ALL and non-ETP-ALL, which were co-cultured with murine stromal cell(MS-5 cell) and treated with PSL and/or ABT-737, was also determined by Annexin V/PI staining. ResultsThirty-one patients diagnosed with T-ALL were classified in ETP-ALL (N=10) or non- ETP ALL (N=21). Although 5 patients in ETP-ALL experienced induction failure (IF), none of the patients in non-ETP-ALL experienced IF (p<0.05). The 5-year EFS in ETP-ALL was significantly inferior than that in non-ETP-ALL ( 40±15.5% vs 76.1±9.2%, log-rank p<0.05). However, the 5-year OS was not significantly different in two groups (67±15.7% in ETP-ALL vs 81±8.5% in non-ETP ALL). RQ-PCR analysis determined that MEF2C and FLT3 was expressed more in ETP-ALL than in non-ETP ALL (MEF2C: p=0.039, FLT3: p=0.014). Because MEF2C augments Bcl2 activity to inhibit the apoptosis, we explored whether high expression of MEF2C was associated with PSL poor response and Bcl2 inhibitor restored the sensitivity to PSL. Combination treatment of PSL and ABT-737 resulted in the significant reduction of IC50 of PSL in Loucy cell (combination index, CI: 0.39). In addition, apoptotic cells increased when Loucy cells were treated with PSL (25mM) in combination with ABT-737 (10nM) compared to be treated with PSL (25mM) alone. In contrast, combination treatment did not reduce significantly IC50 of PSL in Jurkat cell (CI: 1.12). The combination treatment of PSL and ABT-737 also resulted in the significant reduction of IC50 of PSL in BaF3-MEF2C compared to BaF3 cells. Finally, treatment of ABT-737(10nM) in combination with PSL (50 or 200 mM) reduced viability of primary leukemic blasts of ETP-ALL with high expression of MEF2C more profoundly than the treatment of PSL (50 or 200 mM) alone. ConclusionsThese findings suggest that high expression of MEF2C is associated with PSL poor response and Bcl2 inhibitor restores PSL sensitivity in ETP-ALL in vitro. Thus, Bcl2 inhibitor might be therapeutic option for ETP-ALL with high expression of MEF2C. Disclosures:No relevant conflicts of interest to declare.

The Potent PI3K-δ,γ Inhibitor, IPI-145, Exhibits Preclinical Activity In Murine and Human T-Cell Acute Lymphoblastic Leukemia

Abstract Phosphoinositide-3 kinases (PI3Ks) are key cellular signaling proteins that act as a central node for relaying signals from cell surface receptors to downstream mediators, such as AKT. The PI3K-δ and PI3K-γ isoforms are preferentially expressed in normal and malignant leukocytes where they play critical roles in cell differentiation, migration, and proliferation. Constitutive activity of the PI3K pathway is common in T-cell acute lymphoblastic leukemia (T-ALL) and frequently involves the deletion of PTEN, the phosphatase that negatively regulates the PI3K pathway. An important role for the PI3K-δ and PI3K-γ isoforms has been demonstrated in the Pten-deleted genetically engineered murine model of T-ALL in conjunction with PI3K-δ and/or PI3K-γ gene knock outs (Subramanian et al, Cancer Cell, 2012). IPI-145 is a potent inhibitor of PI3K-δ and PI3K-γ currently being studied in a Phase 1 trial (IPI-145-02) in patients with advanced hematologic malignancies, including T-ALL (ClinicalTrials.gov NCT01476657). We performed in vitro studies to address the sensitivity of human and murine T-ALL cell lines to IPI-145 and to additional PI3K inhibitors with defined isoform selectivity. The human T-ALL cells examined were from cell lines that lack PTEN protein expression (Loucy, MOLT-4, CCRF-CEM, CEM/C2, p12 Ichikawa, and Karpas-45) and cell lines that express PTEN protein (MOLT13 and MOLT16). In addition, two murine cell lines derived from a Pten-deleted model of T-ALL (LPN049 and LPN236) were studied. The expression levels of class I PI3K isoforms were determined by western blotting and quantitative RT-PCR, which revealed varying levels of protein and RNA expression across the cell lines. In vitro treatment of human T-ALL cells with IPI-145 resulted in variable degrees of growth inhibition, with the PTEN-deficient Loucy cell line demonstrating the greatest sensitivity with an IC50 of 245 nM. In the cell lines tested, growth inhibition to IPI-145 was only seen in PTEN-deficient human cell lines, whereas all PTEN-expressing human T-ALL cell lines were resistant to IPI-145 (IC50 &gt; 10 uM). However, not all PTEN-deficient human T-ALL cells demonstrated sensitivity to IPI-145 (e.g., CEM/C2), indicating that loss of PTEN does not confer sensitivity to PI3K inhibition in all settings. Inhibition of phospho-AKT (pAKT) correlated with growth inhibition, with an IC50 of 286 nM in the Loucy cell line. Studies to evaluate the mechanism of growth inhibition revealed that IPI-145 treatment resulted in apoptosis of sensitive cells as measured by 7-AAD and Annexin V staining. Cell lines derived from the Pten-deleted murine T-ALL model were also sensitive to IPI-145 with IC50s in the 300-600 nM range as measured by MTT assay. In addition, IPI-145 led to apoptosis, as measured by cleaved Caspase 3 and 7-AAD/Annexin V. Interestingly, Pten-deleted murine T-ALL cell lines showed down-regulation of pAKT and c-MYC expression with IPI-145 in a dose responsive manner that corresponded with increasing activated Caspase-3 expression. In NOTCH1-expressing murine T-ALL cell lines, down regulation of NOTCH1 and activated NOTCH1 was also observed in parallel with c-MYC down-regulation. To explore further the individual contributions of the varying PI3K isoforms on T-ALL cell growth, the effect of IPI-145 on tumor cell growth was compared with PI3K-δ, PI3K-δ,γ, and PI3K-β selective compounds in the Loucy PTEN-deficient T-ALL cell line. These experiments support an anti-leukemic effect for both PI3K-δ and PI3K-γ inhibition and indicate that the greatest effect is seen with combined PI3K-δ and PI3K-γ inhibition. A role for PI3K-β in T-ALL cell survival was not observed. Evaluation of the in vivo activity of IPI-145 on Loucy xenografts, as well as PTEN-expressing MOLT-13 xenografts, is ongoing. Together, these data provide a strong rationale for combined targeted inhibition of PI3K-δ and PI3K-γ in T-ALL. Disclosures: Huang: Infinity Pharmaceuticals, Inc.: Research Funding. Proctor:Infinity Pharmaceuticals, Inc.: Employment. Yang:Infinity Pharmaceuticals, Inc.: Research Funding. Gao:Infinity Pharmaceuticals, Inc.: Research Funding. Zhang:Infinity Pharmaceuticals, Inc.: Research Funding. Huang:Infinity Pharmaceuticals, Inc.: Research Funding. Changelian:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. You:Infinity Pharmaceuticals, Inc.: Research Funding.

Abstract 516: Obinutuzumab (GA101): A novel type II glycoengineered CD20 antibody exhibits enhanced cell death against Rituximab-resistant and -sensitive cell lines in B-cell non-Hodgkin lymphoma

Abstract Background: Rituximab (RTX), an antibody targeting the B-cell antigen CD20, in combination with chemotherapy, is safe, well tolerated and associated with &amp;gt; 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). However, RTX resistance has evolved as one of the limiting factors of such therapy and the exact cause of this resistance is not well understood. C-terminal deletion mutations of CD20 may be one possible reason such resistance or for relapse (Mishima Y. et al, Blood Cancer Journal 2011). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glyco-engineered Fc region has shown to cause significant enhanced ADCC (Mössner et al, Blood 2010; Niederfellner G. et al, Blood 2011). The objective of this study was to evaluate the efficacy of GA101 as compared to RTX against RTX resistant cell lines (Raji-2R and Raji-4RH, generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY) in-vitro. Methods: We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines (RRCL), Raji-2R and Raji-4R (Barth M. et al, ASH 2010). All cell lines were cultured in RPMI with 10% FBS and incubated with dose escalation of GA101 (1-100 µg/ml) for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD using a standard kit (BD Biosciences) and flow-cytometry. B-Cell Leukemia-Lymphoma (BLL) U-698-M cells (CD20+) (DSMZ, Germany) were used as the positive control; whereas, Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) were used as the negative control. Results: At 100 µg/ml of GA101 when incubated for 24 hrs, Raji and U-698-M demonstrated 26.5±0.42% and 32.4±1.9% cell death, respectively. Cell death of the RRCL, Raji-2R and Raji-4RH, was 16.3±3.4% and 17.5±0.07% respectively, at 100 µg/ml of GA101 at 24 hrs. Significant cell death was demonstrated with RSCL as compared to the RRCL, Raji vs Raji-2R (p&amp;lt;0.05) and Raji vs Raji-4RH (p&amp;lt;0.01). There was no significant change in cell death with any concentration of GA101 with the Loucy cell line. Conclusion: Based on these findings, GA101 is a promising novel CD20 antibody for the treatment of RTX resistant B-Cell Lymphomas and related B-Cell malignancies. Further studies are aimed to identify novel candidates that may increase the efficacy of GA101 against RTX resistant cell lines in-vitro and in-vivo. One such approach is to combine GA101 with activated NK cells or a chemotherapeutic agent. 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 516. doi:1538-7445.AM2012-516

Biological Effects of Rabbit Anti-Thymocyte Immunoglobulin (rATG, Thymoglobulin®) in T-Cell Non-Hodgkin's Lymphoma Cell Lines.

Monoclonal antibodies (mAbs) have emerged as powerful adjuncts in the treatment of patients with B-cell lymphoproliferative disorders. While the treatment of B-cell lymphomas has incorporated mAbs and other biological agents into standard chemotherapy regimens, the treatment options for patients with T-cell lymphomas remain relatively limited. There exists a dire need to develop targeted therapies for T-cell lymphomas. Thymoglobulin® (rATG) is a rabbit polyclonal antibody targeting various receptors present on T-cell lymphocytes. When administered at high doses, rATG is known to deplete various subsets of T-cell lymphocytes and induce tolerance in solid organ or bone marrow transplant settings. Using several pre-clinical models, we evaluated the biological effects of rATG against various T-cell lymphoma cell lines. Experiments were conducted in HH, H9, Loucy and HT102 cell lines. A B-cell mantle cell lymphoma cell line was used as a control (MJ). rATG-induced cell-growth inhibition was measured by [3H]-Thymide incorporation assays and measured at 24 and 48 hours. Induction of apoptosis in T-cell lines following rATG exposure was determined by annexin-V/propidium iodine staining and quantified by flow cytometric analysis. Standard functional assays for ADCC/CMC were performed using rATG (5 or 25mg/ml) in 51Cr-labeled T-cells. We found that rATG inhibited DNA synthesis in all the T-cell lines tested. No biological effect was observed in the B-cell mantle cell lymphoma line. Treatment with rATG at either 5 or 25mg/ml resulted in a 30 to 50% growth inhibition when compared to isotype or vehicle controls (P<0.05). Induction of apoptosis was demonstrated in 30 to 40% of T-cell lymphoma cells 24 hrs following exposure to ATG. Biological effects of rATG were dose-dependent. In addition, rATG induced significant ADCC and CMC in T-cell lymphoma cell lines. In conclusion, our data demonstrate that rATG is active against a variety of T-cell lymphoma cell lines in vitro. Anti-tumor effects of rATG are mediated by induction of direct signaling and via the activation of the innate immune system. Additional in vivo studies using T-cell lymphoma are underway and will be presented at the annual meeting.