scholarly journals A Novel Combination Therapy for Paediatric T Cell Acute Lymphoblastic Leukaemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3767-3767
Author(s):  
Ben Christopher Ede ◽  
Paraskevi Diamanti ◽  
Charlotte V. Cox ◽  
Allison Blair
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Combination Therapy ◽  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Nsg Mice ◽  
Gene And Protein Expression ◽  
Apoptotic Proteins

Abstract T cell acute lymphoblastic leukaemia (T-ALL) is a rare form of leukaemia that accounts for approximately 15% of paediatric ALL cases. Unfortunately, approximately 20% of patients do not achieve long term remission as a result of failure of therapy to eradicate the disease. T-ALL is a highly heterogeneous disease that displays a spectrum of immunophenotypes, chromosomal aberrations and gene expression profiles. This heterogeneity has prompted research into more targeted therapies, with the aim of overcoming drug resistance often found with standard chemotherapeutic regimens. Here, we build upon use of the drug Parthenolide (PTL), which has shown promise in treatment of T-ALL and other leukaemias such as BCP-ALL and AML, in combination with ABT-263, a BCL-2 family antagonising agent. Bone marrow samples from 10 T-ALL cases, taken at diagnosis, were treated with PTL in vitro for 24 hours then viability was assessed using the annexin V / PI flow cytometric assay. Variable cytotoxic effects were observed in samples treated with PTL (1-10µM), with half maximal inhibitory concentrations ranging from 2.6-10 µM. At the highest dose tested, the proportion of surviving cells ranged from 5.79-56% (median 35.33%). BM from 5 of these samples was used for whole genome microarray (WGA) analysis. We compared gene expression in bulk ALL and in specific subpopulations, known to have leukaemia initiating capacity in vivo; CD34+/CD7+, CD34+/CD7-, CD34-/CD7+ and CD34-/CD7- cells. WGA data demonstrated that CD34+/CD7- was the only subpopulation to express significantly lower levels (5.38 fold) of the pro-apoptotic gene Bcl-2L11 (BIM) compared to the unsorted bulk T-ALL cells, p=0.006. Interestingly, we have previously shown that CD34+/CD7- cells from a few patients were resistant to PTL treatment in vivo compared to unsorted cells. To validate these results, mRNA and relative protein quantification was performed by qPCR and western blotting in bulk material from 8 of the 10 samples, 3 of which had been analysed by microarray for BIM expression. We found that the gene and protein expression levels of BIM were negatively correlated with PTL resistance in vitro, p≤0.0001 and p=0.049 respectively. This suggests that reduced BIM expression is related to PTL resistance. We next evaluated the effects of combining PTL and ABT-263 on T-ALL cells in vitro. ABT-263 is a BH3 protein mimetic, like BIM it promotes apoptosis by blocking the inhibitory effects that BCL-2 anti-apoptotic proteins have on pro-apoptotic proteins. The effects of combining the drugs were assessed in 7 of the original 10 samples. Unsorted ALL cells were incubated with PTL and ABT-263 for 24 hours, before viability was analysed by flow cytometry and drug synergy was calculated via the Chou Talalay method. This drug combination showed enhanced cytotoxicity to T-ALL cells compared to PTL (p=0.0282) or ABT-263 (p=0.0358) alone. Moreover, the highest combined dose tested (2.5µM PTL with 0.25µM ABT-263) killed 86.1±9% cells cf 71.8±18% with ABT-263 alone and only 21.7±11% with PTL alone. The combination also showed synergism with a combination index value below 1 in all doses tested. Previous findings in our laboratory have shown that in vivo PTL treatment eliminated childhood leukaemia in NOD/LtSz-scid IL-2Rγc null (NSG) mice, in most cases tested. It may be possible to further enhance this toxicity using ABT-263 alone or in combination with PTL. NSG mice were inoculated with unsorted T-ALL cells and leukaemia was allowed to establish until levels in peripheral blood (PB) exceeded 0.1%. NSG mice were subsequently treated orally for 21 days with 100mg/kg of ABT-263 or placebo and leukaemia burden was monitored weekly in PB aspirates. Twenty-eight days following commencement of treatment, leukaemia burden in the placebo treated group was 80.73±2.94% and the animals were electively culled. In contrast, disease burden was significantly lower in the treated animals at this stage (35.2±2.1%, p=0.004). ABT-263 treatment has significantly improved survival of all xenografts to date, (P<0.014). In summary, we have shown that PTL resistance is related to the expression of BIM. By combining PTL with ABT-263, which mimics the pro-apoptotic action of BIM, the drugs work synergistically to enhance T-ALL cytotoxicity in vitro. Ongoing in vivo studies will assess the full potential of this combination therapy for paediatric T-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulation of c-Jun NH2-terminal Kinase ( Jnk) Gene Expression during T Cell Activation

2000 ◽  
Vol 191 (1) ◽  
pp. 139-146 ◽  
Author(s):  
Linda Weiss ◽  
Alan J. Whitmarsh ◽  
Derek D. Yang ◽  
Mercedes Rincón ◽  
Roger J. Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
T Cell Activation ◽  
Map Kinases ◽  
Cell Activation ◽  
Cell Receptor ◽  
Jnk Pathway ◽  
Gene And Protein Expression

The c-Jun NH2-terminal kinases (JNKs) are a group of mitogen-activated protein (MAP) kinases that participate in signal transduction events mediating specific cellular functions. Activation of JNK is regulated by phosphorylation in response to cellular stress and inflammatory cytokines. Here, we demonstrate that JNK is regulated by a second, novel mechanism. Induction of Jnk gene expression is required in specific tissues before activation of this signaling pathway. The in vivo and in vitro ligation of the T cell receptor (TCR) leads to induction of JNK gene and protein expression. TCR signals are sufficient to induce JNK expression, whereas JNK phosphorylation also requires CD28-mediated costimulatory signals. Therefore, both expression and activation contribute to the regulation of the JNK pathway to ensure proper control during the course of an immune response.

Investigating the Expression of the MRD Marker CD58 on Leukaemia Initiating Cells in Childhood Acute Lymphoblastic Leukaemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1887-1887 ◽  
Author(s):  
Charlotte Victoria Cox ◽  
Paraskevi Diamanti ◽  
Allison Blair
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Model Systems ◽  
Childhood All ◽  
Therapy Assessment ◽  
Childhood Acute Lymphoblastic Leukaemia

Abstract Abstract 1887 Further improvements in outcome for childhood acute lymphoblastic leukaemia (ALL) will require a better understanding of the underlying biology of this disease and the fundamental mechanisms of drug resistance. The discoveries that a few populations can initiate leukemia in mouse models and that new populations of leukaemia initiating cells (LIC) can be detected following an initial round of transplantation in these models raises important questions about the biology of the leukaemias. If several cell populations have LIC properties, what are the relationships of these populations to each other and which populations are most important to target with therapy? It will also be important to determine whether there is any correlation in the biological properties of LIC identified in the model systems with the response of the patients to therapy. Assessment of minimal residual disease (MRD) levels provides a sensitive measurement of early treatment response and permits detection of the in vivo selected drug resistant population. CD58 (leucocyte function-associated antigen 3; LFA-3) is a useful marker in MRD tracking of B cell precursor (BCP) ALL. CD58 is over expressed in these cases permitting discrimination of leukaemia blasts from normal B cells. In this study we investigated whether CD58 is expressed on LIC populations in childhood ALL. Expression of CD58 and CD34 was assessed in a cohort of 12 diagnostic samples with mixed prognoses and compared to levels detected in 11 normal bone marrow (NBM) samples. Levels of CD58 were significantly higher in the ALL cases (57.4±37.7%) than on NBM cells (21.1±12.2%; p=0.007). Likewise, the CD34+/CD58+ population was larger in ALL cases than in normal cells (22.2±34.7% and 0.25±0.25%, respectively; p=0.05). Cells from eight of the 12 patients, were sorted on the basis of expression or lack of expression of these markers and the functional ability of the sorted subpopulations was assessed in vitro and in vivo. On sorting, the majority of cells were CD34−/CD58− (43.7±39.2%), 20.7±30.7% were CD34−/CD58+, 19±14.3% were CD34+/CD58+ and the CD34+/CD58− population accounted for 16.6±35.3%. Unsorted cells and all 4 sorted populations were set up in long-term culture to assess proliferative capability and the in vivo propagating potential was assessed in NSG mice. All 4 sorted subpopulations proliferated over the 6 week period but the highest levels of expansion were observed in the cultures of CD34+/CD58+ (6–420 fold) and CD34+/CD58− (3–24 fold) cells. Cytogenetic analyses confirmed that leukaemia cells were maintained in the culture system. Results from the in vivo analyses on 5 cases to date indicate that all 4 subpopulations contain LIC. In these cases, higher levels of engraftment were observed with CD34+/CD58+ (up to 20%) and with CD34−/CD58− subpopulations (6.1-98%). Serial transplantation studies will determine whether there are differences in the repopulating and self-renewal abilities of these LIC. These findings suggest that using CD58 alone or in combination with CD34 would be insufficient to track disease progression in ALL. Incorporating additional markers that are commonly used in MRD panels will provide valuable information on LIC populations and facilitate development of improved disease monitoring. Disclosures: No relevant conflicts of interest to declare.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13569-e13569
Author(s):  
Enrica Marchi ◽  
Matko Kalac ◽  
Danielle Bongero ◽  
Christine McIntosh ◽  
Laura K Fogli ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Molecular Level ◽  
Cell Lymphoma ◽  
Single Agent ◽  
T Cell Lymphoma ◽  
Cytotoxicity Assays

e13569 Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.

scholarly journals In vitro drug resistance and prognostic impact of p16INK4A /P15INK4B deletions in childhood T-cell acute lymphoblastic leukaemia

2001 ◽  
Vol 112 (3) ◽  
pp. 680-690 ◽  
Author(s):  
N. L. Ramakers-van Woerden ◽  
R. Pieters ◽  
R. M. Slater ◽  
A. H. Loonen ◽  
H. B. Beverloo ◽  
...  
Keyword(s):  
Drug Resistance ◽  
T Cell ◽  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Prognostic Impact

scholarly journals Differentiation of T cell lymphokine gene expression: the in vitro acquisition of T cell memory.

1991 ◽  
Vol 173 (1) ◽  
pp. 25-36 ◽  
Author(s):  
S Ehlers ◽  
K A Smith
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Interleukin 2 ◽  
Experimental System ◽  
Functional Differentiation ◽  
Gene Repertoire ◽  
Anamnestic Response

A simple in vitro experimental system was devised to reflect the in vivo generation of a T cell anamnestic response so that T cell differentiation could be examined at the level of lymphokine gene expression. Comparison of neonatal and adult T cells revealed that both populations expressed the genes for interleukin 2 (IL-2) and its receptor, but only adult T cells were capable of transcribing mRNAs for IL-3, IL-4, IL-5, IL-6, interferon gamma, and granulocyte/macrophage colony-stimulating factor. However, neonatal T cells could be induced to undergo functional differentiation in vitro, thereby acquiring the capacity to express the lymphokine gene repertoire characteristic for adult T cells. These data suggest that the T cells generated from neonatal blood by a primary stimulation in vitro are functionally indistinguishable from the T cells in adult blood that presumably have undergone primary stimulation in vivo. Therefore, we propose that the term "memory cell" be applied to those T cells that can be identified by their differentiated state of inducible effector-lymphokine gene expression.

scholarly journals Rapid default transition of CD4 T cell effectors to functional memory cells

2007 ◽  
Vol 204 (9) ◽  
pp. 2199-2211 ◽  
Author(s):  
K. Kai McKinstry ◽  
Susanne Golech ◽  
Won-Ha Lee ◽  
Gail Huston ◽  
Nan-Ping Weng ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cd4 T Cell ◽  
Effector Memory ◽  
Memory Cells ◽  
Two Stages

The majority of highly activated CD4 T cell effectors die after antigen clearance, but a small number revert to a resting state, becoming memory cells with unique functional attributes. It is currently unclear when after antigen clearance effectors return to rest and acquire important memory properties. We follow well-defined cohorts of CD4 T cells through the effector-to-memory transition by analyzing phenotype, important functional properties, and gene expression profiles. We find that the transition from effector to memory is rapid in that effectors rested for only 3 d closely resemble canonical memory cells rested for 60 d or longer in the absence of antigen. This is true for both Th1 and Th2 lineages, and occurs whether CD4 T cell effectors rest in vivo or in vitro, suggesting a default pathway. We find that the effector–memory transition at the level of gene expression occurs in two stages: a rapid loss of expression of a myriad of effector-associated genes, and a more gradual gain of expression of a cohort of genes uniquely associated with memory cells rested for extended periods.

The Role of CD4+ T Cells in a Murine Model of Acute Lymphoblastic Leukaemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1838-1838
Author(s):  
Ahmed N. Hegazy ◽  
Mathias Wolenski ◽  
Karl Welte ◽  
Christoph Klein
Keyword(s):  
T Cells ◽  
T Cell ◽  
Acute Lymphoblastic Leukaemia ◽  
Adoptive Transfer ◽  
Lymphoblastic Leukaemia ◽  
Tumor Antigen ◽  
T Cell Responses ◽  
Interferon Γ ◽  
Cell Responses

Abstract To assess CD4-mediated anti-tumor immunity in a murine acute lymphoblastic leukaemia model system, we have generated a series of BCR-ABL positive pre-B cell lines expressing the surrogate tumor antigen ovalbumine. Upon intravenous injection, PKH-26-labeled leukaemia cells were taken up by splenic CD8+ but not by CD8− dendritic cells (DC). In comparison to PBS-injected DCs, CD8+ DCs also showed increased expression of CD40, CD80, and CD86. Purified DCs from leukemic mice stimulated transgenic DO11.10 T cells recognizing OVA323–339 in the context of I-Ad, suggesting efficient presentation of the surrogate tumor antigen. Next, we utilized adoptive transfer of DO11.10 T cells to measure tumor-specific T cell responses in vivo. OVA-expressing BM185 cells were unable to directly stimulate DO11.10 T cells, as shown by 3H-thymidine incorporation. In contrast, DO11.10 T cells were activated in vivo in spleen and lymph nodes, as shown by upregulation of CD44 and CFSE staining, suggesting that DC effectively present tumor antigens to DO11.10 T cells in vivo. However, despite of detectable T cell activation and proliferative T cell responses, all animals succumbed to progressive leukemia. Furthermore, adoptive transfer of naïve DO11.10 cells did not induce protective anti-leukemia immunity. Interestingly, in vivo primed DO11.10 T cells did not express interferon-γ. We therefore hypothesized that inefficient in vivo priming of TH1 cells may contribute to immune evasion of ALL cells. To address this question, we primed DO11.10 T cells in vitro prior to adoptive transfer. In this setting, DO11.10 T cells expressed interferon-γ and induced regression of preestablished leukaemia. This effect was dependent on CD8 cells, as shown by in vivo depletion experiments. Our experimental system supports the concept of CD4-dependent antitumor immunity and provides a platform to assess immunological mechanisms of novel strategies to therapeutically enhance antileukaemic immune responses.

Context Dependent Role of Polycomb Repressive Complex 2 in Acute Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 610-610
Author(s):  
Etienne Danis ◽  
Taylor Yamauchi ◽  
Kristen Echanique ◽  
Jessica Haladyna ◽  
Huafeng Xie ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Acute Leukemia ◽  
Murine Models ◽  
Polycomb Repressive Complex ◽  
Cell Precursor ◽  
Polycomb Repressive Complex 2

Abstract Polycomb Repressive Complex 2 (PRC2) is a multi-protein complex with important roles in development and cancer. Both hyper- and hypoactivity of PRC2 are associated with blood-related malignancies. Activating mutations of the PRC2 methyltransferase EZH2 have been found in human B-lineage lymphomas. Inactivating mutations of PRC2 components EZH2, EED and SUZ12 have been described in early T-cell Precursor ALL (ETP-ALL) and inactivating PRC2-alterations are found in Myelodysplastic and Myeloproliferative Syndromes. The mechanisms underlying this paradox are incompletely understood. We here investigate the context dependent role of PRC2 in murine models. We initially studied PRC2 in normal hematopoiesis: Chip-seq analysis of the PRC2-mediated H3K27me3 chromatin mark demonstrates that many genes highly expressed in immature hematopoiesis gain H3K27me3 in the developmental transition from more immature Lin-Sca1+Kit+ (LSK) cells to lineage committed Granulocyte Macrophage Progenitors (GMPs). Transcription of these genes is enriched in EZH2ko GMP compared to EZH2ffGMP by Gene Set Enrichment Analysis (GSEA). These data suggest that PRC2 is important for the silencing of immature gene expression programs in the developmental transition from LSK to GMP. We next analyzed the role of PRC2 in two murine models of acute leukemia: MLL-AF9 driven leukemia, and a model of early T-cell precursor T-ALL (ETP-ALL). In MLL-AF9 leukemia, we previously found that inactivation of Eed completely abrogate leukemogenesis in vitro and in vivo. We now report that genetic inactivation of the tumor suppressor Cdkn2a (a canonical PRC2 target) partially rescued MLL-AF9 mediated leukemia in vitro and in vivo. However, Cdkn2akoEEDko MLL-AF9 leukemia remained compromised. In vitro growth was reduced to approximately 10% of Eedff controls. While control MLL-AF9 leukemia developed in vivo in 100% of the recipients, Cdkn2akoEEDkoMLL-AF9 leukemia developed with significantly prolonged latency and incomplete penetrance (25%). RNAseq analysis revealed that high level expression of genes with established roles in MLL-AF9 leukemia such as HoxA9, Cdk6 and Jmjd1c unexpectedly depends on Eed. These data are in keeping with the absence of alterations in PRC2-components in human MLL-rearranged leukemia. In contrast, PRC2 core components (EZH2/EED/SUZ12) are deleted or mutated in > 40% of ETP-ALL. ETP-ALL also often has direct or indirect activation of the RAS-pathway, and carries frequent deletions of the CDKN2A locus. To model the effects of EED and EZH2-inactivation in ETP-ALL, we established Cdkn2akoEedff vs Cdkn2akoEedko, and Cdkn2akoEzh2ff vs Cdkn2akoEZH2koleukemias by transduction with NRASQ61K followed by expansion on OP9DL1 stroma cells to activate T-lineage differentiation via Notch-signaling. Cdkn2ako NRASQ61K leukemia showed an immunophenotype similar to human ETP-ALL (positive for c-Kit, CD5 and myeloid markers and mostly negative for CD4/8). Inactivation of Eed or Ezh2 in this model led to a shortening of latency (p=0.03 for Eed, p=0.0001 for Ezh2). RNAseq revealed enrichment of genes associated with murine DN1 thymocytes and with human ETP-ALL in Eedko vs Eedff Cdkn2ako NRASQ61K leukemia. These genesets showed even more pronounced enrichment in Ezh2kocompared to Ezh2ff Cdkn2ako NRASQ61K leukemia. Genes highly expressed in early hematopoiesis were enriched in Eedko and Ezh2ko cells in both, the MLL-AF9 and NRASQ61K leukemia models. However, there was an opposing effect on HoxA9 gene expression, with PRC2 inactivation leading to decreased HoxA9 expression in MLL-AF9, and increased HoxA9 expression in Cdkn2ako NRASQ61K leukemia. Decreased HoxA9 has been shown to impair MLL-AF9 leukemia growth. To test the functional significance of elevated HoxA9-levels in the Eedko and EZH2koNRASQ61K leukemias, we co-expressed HoxA9 and NRASQ61K in the presence of intact Eed and Ezh2 loci. Preliminary data suggest that HoxA9 accelerates leukemia development in this setting. Alterations in chromatin modifiers, including PRC2, are frequent in leukemia and lymphoma. Our data demonstrate that manipulation of PRC2 can have opposite effects on leukemia phenotype and expression of key PRC2-repressed genes such as HoxA9 in the context of different tumors. We are currently characterizing the mechanisms leading to divergent outcomes of PRC2 manipulation in MLL-AF9 leukemia compared to NRASQ61K ETP-like leukemia. Disclosures Armstrong: Epizyme : Consultancy.

scholarly journals Thyroid hormone receptors are down-regulated in skeletal muscle of patients with non-thyroidal illness syndrome secondary to non-septic shock

2010 ◽  
Vol 163 (5) ◽  
pp. 765-773 ◽  
Author(s):  
J Lado-Abeal ◽  
A Romero ◽  
I Castro-Piedras ◽  
A Rodriguez-Perez ◽  
J Alvarez-Escudero
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Septic Shock ◽  
Thyroid Hormone ◽  
Hormone Receptors ◽  
Serum Samples ◽  
Pathway Activation ◽  
Gene And Protein Expression

AimNon-thyroidal illness syndrome (NTIS) is related to changes in thyroid hormone (TH) physiology. Skeletal muscle (SM) plays a major role in metabolism, and TH regulates SM phenotype and metabolism. We aimed to characterize the SM of non-septic shock NTIS patients in terms of: i) expression of genes and proteins involved in TH metabolism and actions; and ii) NFKB's pathway activation, a responsible factor for some of the phenotypic changes in NTIS. We also investigated whether the patient's serum can induce in vitro the effects observed in vivo.MethodsSerum samples and SM biopsies from 14 patients with non-septic shock NTIS and 11 controls. Gene and protein expression and NFKB1 activation were analyzed by quantitative PCR and immunoblotting. Human SM cell (HSkMC) cultures to investigate the effects of patient's serum on TH action mediators.ResultsPatients with non-septic shock NTIS showed higher levels of pro-inflammatory cytokines than controls. Expression of TRβ (THRB), TRα1 (THRA), and retinoid X receptor γ (RXRG) was decreased in NTIS patients. RXRA gene expression was higher, but its protein was lower in NTIS than controls, suggesting the existence of a post-transcriptional mechanism that down-regulates protein levels. NFKB1 pathway activation was not different between NTIS and control patients. HSkMC incubated with patient's serum increased TH receptor and RXRG gene expression after 48 h.ConclusionsPatients with non-septic shock NTIS showed decreased expression of TH receptors and RXRs, which were not related to increased activation of the NFKB1 pathway. These findings could not be replicated in cultures of HSkMCs incubated in the patient's serum.

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