Leukemia Gene Expression Research Articles

Analysis of class I and II histone deacetylase gene expression in human leukemia

Histone deacetylase (HDAC) inhibitors are well-characterized anti-leukemia agents and HDAC gene expression deregulation has been reported in various types of cancers. This study sought to characterize HDAC gene expression patterns in several types of leukemia. To do so, a systematic study was performed of the mRNA expression of all drug-targetable HDACs for which reagents were available. This was done by real-time PCR in 24 leukemia cell lines and 39 leukemia patients, which included AML, MDS and CLL patients, some of whom received HDAC inhibitor treatment. Among the samples analyzed, there was no discernible pattern in HDAC expression. HDAC expression was generally increased in CLL patients, except for HDAC2 and HDAC4. HDAC expression was also generally increased in VPA-treated MOLT4 cells. However, this increased expression was not seen in AML patients treated with vorinostat. In summary, increased HDAC expression was noted in CLL patients in general, but the HDAC expression patterns in myeloid malignancies appear to be heterogeneous, which implies that the role of HDACs in leukemia may be related to global expression or protein function rather than specific expression patterns.

Drug screen in patient cells suggests quinacrine to be repositioned for treatment of acute myeloid leukemia.

To find drugs suitable for repositioning for use against leukemia, samples from patients with chronic lymphocytic, acute myeloid and lymphocytic leukemias as well as peripheral blood mononuclear cells (PBMC) were tested in response to 1266 compounds from the LOPAC1280 library (Sigma). Twenty-five compounds were defined as hits with activity in all leukemia subgroups (<50% cell survival compared with control) at 10 μM drug concentration. Only one of these compounds, quinacrine, showed low activity in normal PBMCs and was therefore selected for further preclinical evaluation. Mining the NCI-60 and the NextBio databases demonstrated leukemia sensitivity and the ability of quinacrine to reverse myeloid leukemia gene expression. Mechanistic exploration was performed using the NextBio bioinformatic software using gene expression analysis of drug exposed acute myeloid leukemia cultures (HL-60) in the database. Analysis of gene enrichment and drug correlations revealed strong connections to ribosomal biogenesis nucleoli and translation initiation. The highest drug–drug correlation was to ellipticine, a known RNA polymerase I inhibitor. These results were validated by additional gene expression analysis performed in-house. Quinacrine induced early inhibition of protein synthesis supporting these predictions. The results suggest that quinacrine have repositioning potential for treatment of acute myeloid leukemia by targeting of ribosomal biogenesis.

Study on Leukemia Cell Differentiation and Drug Resistance By SATB1 Gene

Background and objective:In this article, four types of lymphoma cell lines U937, Raji, Hut-102, Akata, and three types of leukemia cell lines K562, HL-60, Jurkat were used the expression of SATB1 in leukemia cells, Through cell differentiation induced by all-TRANS Retinoic acid and DMSO, finds the SATB1 gene expression is reduced, suggest that the SATB1 gene may be involved in cell differentiation. By gene silencing, reduce the SATB1 gene expression, sensitivity to chemotherapy drugs is increased, suggest that the SATB1 gene may be associated with drug resistance.Methods:1. Western Blot Assay for detection of SATB1 gene expression in cell lines.2. DMSO and ATRA to HL-60, Jurkat cells for different times, SATB1 gene expression in the cells are detected by Western Blot.3. Building SATB1-shRNA, transfected Jurkat cells and to HL-60.4. Testing inhibition rate of chemotherapy drugs on HL-60, HL-60-SATB1-ctr, HL-60-SATB1-sh, Jurkat, Jurkat-SATB1-ctr, Jurkat-SATB1-sh cell, suggesting its relationship with the drug resistance.Results:1. SATB1 gene was expression in all the plant cells.2. The expression of SATB1 in U937, Raji, Hut-102, Akata four types of lymphoma cell line is low.3. It was highly expression in HL-60, Jurkat cells, but low in K562.4. DMSO and ATRA treat HL-60, Jurkat cells for 48 and 96 hours, HL-60 cells become larger, rounded, Jurkat cells into smaller, is more obvious for 96-hour.5. For HL-60, Jurkat cells, after DMSO and ATRA treated, the expression of SATB1 was decreased, more obvious for 96-hour.6. HL-60-SATB1-sh1, Jurkat-SATB1-sh1 cell compared with the control group, the SATB1 protein content decreased significantly, successfully building SATB1-shRNA HL-60 and Jurkat cell line.7. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity has improved significantly.Conclusion:1. The SATB1 gene expression in leukemia and lymphoma cells, and higher expression in leukemia cells.2. After DMSO and ATRA treatment, the SATB1 expression significantly reduced, suggest that SATB1 may be involved in cell differentiation.3. Successfully built SATB1-shRNA, and successfully transferred to HL-60 and Jurkat cell lines.4. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity increased significantly, prompting SATB1 may be related to drug-resistance. DisclosuresNo relevant conflicts of interest to declare.

The Prognostic Impact of High MEL1 Gene Expression in Pediatric Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia (AML) is a complex disease caused by mutations, epigenetic modifications, and deregulated expression of genes, leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. Although, many prognosis-associated gene alterations have been identified in adult AML, genetic alterations responsible for an adverse outcome in most patients with pediatric AML have remained obscure.In our previous study, we have identified NUP98-NSD1 fusion gene in 6 of 124 pediatric AML patients. As a result of gene expression profile using microarray, all these 6 patients have showed high expression of HOXA9, HOXA10, HOXB3, HOXB5, HOXB6, and a zinc finger transcription factor gene MDS1-EVI1-like-1 (MEL1), and clustered a distinct poor prognostic subgroup [4-year-overall survival (OS); 33%]. Furthermore, this subgroup has also formed the bigger poor prognostic subgroup involving the 18 patients without NUP98-NSD1 fusion gene. We defined this subgroup as NUP98-NSD1 signature consisting of 24 of 124 patients (19%), 4-year-OS; 37.5%. MEL1 gene expression represented the specific characteristics of this subgroup.To verify the significance of MEL1 gene expression and the relationship with other poor prognostic markers such as FLT3-ITD, MLL-PTD, and NPM1, we investigated MEL1 expression by real-time PCR in 369 pediatric AML cases other than acute promyelocytic leukemia (APL) and Down syndrome-associated AML, because these subtypes formed genetically different unique entities and treated by other protocols. Interestingly, MEL1 is highly homologous to MDS1/EVI1 gene, which is an alternatively spliced transcript of the EVI1 genes. As EVI1 high expression has been reported as a poor prognostic marker in pediatric AML patients, especially in patients with MLL rearrangement, we also measured and examined the EVI1 gene expression.MethodsBetween 2006 and 2010, 485 de novo pediatric AML patients participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Among them, 369 samples were available in this study. Quantitative RT-PCR analysis was performed in these patients using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to EVI1 and MEL1, ABL1 was also evaluated as a control gene. We investigated the correlations between these gene expressions and other genetic alterations, and clarified the prognostic impact of MEL1 gene.ResultsA total of 84 of 369 patients (22.8%) showed high expression of MEL1 gene. The number of patients with MEL1 high expressions were gradually enriched in order of the intermediate risk (IR; 34 of 142 patients, or 23.9%), high risk (HR; 21 of 48 patients, or 43.8%), and non-complete remission (non-CR; 20 of 39 patients, or 51.3%), but were almost all absent in patients with a low risk (LR) cytogenetic profile (4 of 120 patients, or 3.3%) consisting of t(8;21) and inv(16). The absolute values of MEL1 gene expression have also gradually increased in order of LR < IR < HR < non-CR. The overall survival among patients with MEL1 high expressions was significantly lower than that among patients without such gene aberrant expression (50% vs. 82%, P<0.001). Remarkably, MEL1 gene expression is extremely useful to stratify the AML patients with FLT3-ITD (4-year OS; high 30% vs. low 75%, P<0.001) and normal karyotype (4-year OS; high 48% vs. low 84%, P<0.001), whom we could not exactly stratify into the appropriate risk so far. On the other hand, a total of 58 of 369 patients (15.7%) have showed high expression of EVI1 gene. The patients with high expression of EVI1 gene have been mainly found in the IR group, especially in patients with MLL rearrangement. Furthermore, MEL1 high group and EVI1high group have the tendency of mutual exclusive in this study.ConclusionsMEL1 high expression was highly recurrent in de novo pediatric AML patients with high/intermediate-risk cytogenetic profiles and was independently associated with a poor outcome. Combined with MEL1expression and genetic alterations enable to clarify the genetic background of pediatric AML profiles. The data in this study showed a way in which integrated mutational profiling of a clinical trial cohort can advance our understanding of the biologic characteristics of AML, improve current prognostic models, and inform prospective therapeutic decisions. DisclosuresNo relevant conflicts of interest to declare.

Design, Assessment, and in vivo Evaluation of a Computational Model Illustrating the Role of CAV1 in CD4(+) T-lymphocytes.

Caveolin-1 (CAV1) is a vital scaffold protein heterogeneously expressed in both healthy and malignant tissue. We focus on the role of CAV1 when overexpressed in T-cell leukemia. Previously, we have shown that CAV1 is involved in cell-to-cell communication, cellular proliferation, and immune synapse formation; however, the molecular mechanisms have not been elucidated. We hypothesize that the role of CAV1 in immune synapse formation contributes to immune regulation during leukemic progression, thereby warranting studies of the role of CAV1 in CD4+ T-cells in relation to antigen-presenting cells. To address this need, we developed a computational model of a CD4+ immune effector T-cell to mimic cellular dynamics and molecular signaling under healthy and immunocompromised conditions (i.e., leukemic conditions). Using the Cell Collective computational modeling software, the CD4+ T-cell model was constructed and simulated under CAV1+/+, CAV1+/−, and CAV1−/− conditions to produce a hypothetical immune response. This model allowed us to predict and examine the heterogeneous effects and mechanisms of CAV1 in silico. Experimental results indicate a signature of molecules involved in cellular proliferation, cell survival, and cytoskeletal rearrangement that were highly affected by CAV1 knock out. With this comprehensive model of a CD4+ T-cell, we then validated in vivo protein expression levels. Based on this study, we modeled a CD4+ T-cell, manipulated gene expression in immunocompromised versus competent settings, validated these manipulations in an in vivo murine model, and corroborated acute T-cell leukemia gene expression profiles in human beings. Moreover, we can model an immunocompetent versus an immunocompromised microenvironment to better understand how signaling is regulated in patients with leukemia.

Homeobox gene expression in acute myeloid leukemia is linked to typical underlying molecular aberrations.

BackgroundAlthough distinct patterns of homeobox (HOX) gene expression have been described in defined cytogenetic and molecular subsets of patients with acute myeloid leukemia (AML), it is unknown whether these patterns are the direct result of transcriptional alterations or rather represent the differentiation stage of the leukemic cell.MethodTo address this question, we used qPCR to analyze mRNA expression of HOXA and HOXB genes in bone marrow (BM) samples of 46 patients with AML and sorted subpopulations of healthy BM cells. These various stages of myeloid differentiation represent matched counterparts of morphological subgroups of AML. To further study the transcriptional alterations of HOX genes in hematopoiesis, we also analyzed gene expression of epigenetic modifiers in the subpopluations of healthy BM and leukemic cells.ResultsUnsupervised hierarchical clustering divided the AMLs into five clusters characterized by the presence of prevalent molecular genetic aberrations. Notably, the impact of genotype on HOX gene expression was significantly more pronounced than that of the differentiation stage of the blasts. This driving role of molecular aberrations was best exemplified by the repressive effect of the PML-RARa fusion gene on HOX gene expression, regardless of the presence of the FLT3/ITD mutation. Furthermore, HOX gene expression was positively correlated with mRNA levels of histone demethylases (JMJD3 and UTX) and negatively correlated with gene expression of DNA methyltranferases. No such relationships were observed in subpopulations of healthy BM cells.ConclusionOur results demonstrate that specific molecular genetic aberrations, rather than differentiation per se, underlie the observed differences in HOX gene expression in AML. Moreover, the observed correlations between epigenetic modifiers and HOX ex pression that are specific to malignant hematopoiesis, suggest their potential causal relationships.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0094-0) contains supplementary material, which is available to authorized users.

Binding of a Smad4/Ets-1 complex to a novel intragenic regulatory element in exon12 of FPGS underlies decreased gene expression and antifolate resistance in leukemia.

Polyglutamylation of antifolates catalyzed by folylpoly-γ-glutamate synthetase (FPGS) is essential for their intracellular retention and cytotoxic activity. Hence, loss of FPGS expression and/or function results in lack of antifolate polyglutamylation and drug resistance. Members of the TGF-β/Smad signaling pathway are negative regulators of hematopoiesis and deregulation of this pathway is considered a major contributor to leukemogenesis. Here we show that FPGS gene expression is inversely correlated with the binding of a Smad4/Ets-1 complex to exon12 of FPGS in both acute lymphoblastic leukemia cells and acute myeloid leukemia blast specimens. We demonstrate that antifolate resistant leukemia cells harbor a heterozygous point mutation in exon12 of FPGS which disrupts FPGS activity by abolishing ATP binding, and alters the binding pattern of transcription factors to the genomic region of exon12. This in turn results in the near complete silencing of the wild type allele leading to a 97% loss of FPGS activity. We show that exon12 is a novel intragenic transcriptional regulator, endowed with the ability to drive transcription in vitro, and is occupied by transcription factors and chromatin remodeling agents (e.g. Smad4/Ets-1, HP-1 and Brg1) in vivo. These findings bear important implications for the rational overcoming of antifolate resistance in leukemia.

Mouse models in the study of chronic lymphocytic leukemia pathogenesis and therapy

AbstractMouse models that recapitulate human malignancy are valuable tools for the elucidation of the underlying pathogenetic mechanisms and for preclinical studies. Several genetically engineered mouse models have been generated, either mimicking genetic aberrations or deregulated gene expression in chronic lymphocytic leukemia (CLL). The usefulness of such models in the study of the human disease may potentially be hampered by species-specific biological differences in the target cell of the oncogenic transformation. Specifically, do the genetic lesions or the deregulated expression of leukemia-associated genes faithfully recapitulate the spectrum of lymphoproliferations in humans? Do the CLL-like lymphoproliferations in the mouse have the phenotypic, histological, genetic, and clinical features of the human disease? Here we compare the various CLL mouse models with regard to disease phenotype, penetrance, and severity. We discuss similarities and differences of the murine lymphoproliferations compared with human CLL. We propose that the Eμ-TCL1 transgenic and 13q14-deletion models that have been comprehensively studied at the levels of leukemia phenotype, antigen-receptor repertoire, and disease course show close resemblance to the human disease. We conclude that modeling CLL-associated genetic dysregulations in mice can provide important insights into the molecular mechanisms of disease pathogenesis and generate valuable tools for the development of novel therapies.

Silencing of CHD5 Gene by Promoter Methylation in Leukemia

Chromodomain helicase DNA binding protein 5 (CHD5) was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. CHD5 is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of CHD5 down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that CHD5 was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the CHD5 promoter was hypermethylated in human leukemia cells and samples. Thus, CHD5 expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CHD5 expression in human leukemia cell lines. In vitro luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2) as a potential transcription factor involved in CHD5 expression and indicated that treatment with DAC increases the recruitment of AP2 to the CHD5 promoter. In vitro transcription-factor activity studies showed that AP2 over-expression was able to activate CHD5 promoter activity. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promoter.

P-Stat3 and bcr/abl gene expression in chronic myeloid leukemia and their relation to imatinib therapy

Flowcytometry analysis was carried out to evaluate the expression of the p-Stat3 in 50 CML patients and 20 age-matched healthy controls. p-Stat3 expression was increased in advanced stages of CML. Imatinib treatment was found to suppress the expression of p-Stat3 in bone marrow cells. The level of p-Stat3 was found to be higher in resistant cases than in responsive cases, which suggest the beneficial use of p-Stat3 as an indicator to follow the clinical course and the treatment response.

Ras-Pathway Inhibition With Targeted Therapies Abrogates Self-Renewal In Acute Myelogenous Leukemia

Hyperactivated Ras-pathways serve as oncogenic drivers in multiple human tumors including acute myelogenous leukemia (AML) (Ahearn et al. Nat Rev Mol Cell Biol 2011). The specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Recently, we have shown that NRASG12V–activated signaling pathways are critical to leukemia stem cell maintenance (Sachs et al. submitted). To elucidate which Ras-activated signaling molecules mediate self-renewal in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V) and develops AML (Kim et al. Blood 2009). Primary leukemia cells were treated with therapeutic agents targeting Ras-activated signaling pathways. We used PD325901 to inhibit the Mek-Erk pathway, GDC0941 to inhibit the Pi3k pathway, and RAD001 to inhibit the mTor pathway. Using MTS assays, we identified the IC50 dose for each of these agents. Inhibitor-treated leukemia cells were submitted for RNA sequencing in order to investigate the effects of these agents on leukemia gene expression. Previously, we identified a list of NRASG12V responsive genes in our model. In these studies, we identified that PD325901-treatment most closely recapitulates the effect of NRASG12V inhibition on this comprehensive list of RAS-responsive genes. However, when we study the effects of these inhibitors on the subset of RAS-responsive genes that mediate leukemia self-renewal, we find that both PD325901 and RAD001 independently recapitulate the effects of NRASG12V withdrawal on this subset of genes implicating the Mek and mTor pathways in leukemia self renewal. Next, we treated primary leukemia cells with the IC50 dose of each drug and plated them in colony forming assays. We found that Mek or mTor inhibition, but not Pi3k inhibition, abrogated secondary colony formation corroborating our gene expression analyses and showing that, at doses that have equivalent effects on cell growth, only the Mek and mTor pathways are important for leukemia cell stem cell maintenance. These studies provide potential targets for leukemia stem cell-specific therapies. Disclosures:Sachs:Silicon Valley Biosystems: Consultancy. Bendall:DVS Sciences: Consultancy. Nolan:SAB for DVS Sciences and Nodality: Chairman Other; Cell Signalling Technologies and Becton Dickenson, Inc: Consultancy. Largaespada:Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.

Proliferation Inhibition and Apoptosis Induction Of Imatinib Resistance Chronic Myeloid Leukemia Cells By Down-Regulated PPP2R5C

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder that occurs because of t(9;22)(q34;q11) translocations. The prognosis in CML improved markedly after introduction of abl tyrosine kinase inhibitors (TKI), still a lot of CML patients die due to abl mutation related drug resistance and the blast crisis, moreover, de novo or secondary TKI-resistance is a significant problem in CML. The aim of the study is to down-regulate the PPP2R5C gene expression in imatinib-sensitive or imatinib-resistant chronic myeloid leukemia (CML) cell lines: K562, K562R (imatinib resistance without abl gene mutation), 32D-Bcr-Abl WT (imatinib sensitive, murine CML cell lines with wild type abl gene) and 32D-Bcr-Abl T315I (imatinib resistance, with abl gene T315I mutation) and primary cells from CML patients by RNA interference, thereby inhibit the CML cells proliferation and induce apoptosis. PPP2R5C-siRNAs numbered 799 or 991 were obtained by chemosynthesis. Non-silencing siRNA control (SC)-treated, mock-transfected, untreated cells were used as controls. PPP2R5C expression in mRNA levels from CML cells were analyzed after siRNAs delivered by nucleofection using the real-time quantitative PCR. The PPP2R5C protein levels were analyzed by Western blotting. Cell proliferation in vitro was assayed by the cell count kit-8 method after treatment. The morphology and the percentage of apoptosis were revealed by Hoechst 33258 stain and flow cytometry (FCM). Bone marrow mononuclear cells (BM-MNCs) from healthy individuals were transferred by PPP2R5C-siRNA-991. BFU-E, CFU-Meg and CFU-GM were performed from PPP2R5C-siRNA-991 treated BM-MNCs by methyl cellulose semi-solid culturing method, to estimate the role of differentiation and proliferation in BM-MNCs after PPP2R5C-siRNA transfection. The results showed that both PPP2R5C-siRNA-799 and PPP2R5C-siRNA-991 took best silencing results after nucleofection in all of four cells and primary cells from CML patients. The reduction about 2 to 7 folds in PPP2R5C mRNA level was observed in PPP2R5C-siRNA799 or PPP2R5C-siRNA991 treated cells. And PPP2R5C protein expression inhibition rate reached 38.08%-55.26% at 48 or 72 h after treatment. The proliferation rates of PPP2R5C-siRNA-799 or 991 treated CML cells were significantly decreased at 72 h (P < 0.05). PPP2R5C-siRNA-799 or 991 treated CML cells lines showed a significantly increase in AnnexinV/PI-positive cells (apoptosis) (P < 0.05), similar results in the morphological changes of apoptosis were found by Hoechst 33258 staining test. PPP2R5C gene mRNA expression levels in BM-MNCs from healthy individuals were significantly lower than that in K562 cells (P < 0.05), and the expression level was not significant changed after PPP2R5C-siRNA-991 transfection. The formation of BFU-E, CFU-Meg and CFU-GM from BM-MNCs showed no significant difference between PPP2R5C-siRNA-991 treatment and MOCK control group (P > 0.05). In conclusions, suppression of PPP2R5C by RNA interference could inhibit the proliferation and induce the apoptosis effectively in CML cells either in imatinib sensitive or imatinib resistance cell lines, while no significant effect of PPP2R5C-siRNA on the proliferation and differentiation of BM-MNCs in vitro, suggesting that PPP2R5C-siRNA might specially target on the CML cells. Down-regulating the PPP2R5C gene expression might be considered as a new target therapeutic strategy in CML, especially in imatinib-resistant CML. Disclosures:Li:This work was supported by Grants from National Natural Science Foundation of China (30871091 and 91129720), the Collaborated grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (2012B0506: Research Funding.

Modeling Antileukemic Adoptive Immunotherapy In Mouse-Humans Chimeras To Identify Novel Mechanisms Of Cancer Immunoediting

IntroductionIn spite of the documented efficacy of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) in the cure of Acute Myeloid Leukemia (AML), post-transplantation relapse remains an unmet clinical need. According to the “leukemia immunoediting” hypothesis relapse may be due to the outgrowth of immune-resistant leukemic variants upon the selective pressure of the transplanted immune system. We provided a clinically relevant proof-of-principle of this model in transplanted patients (Vago et al, N Engl JMed, 2009), but ex vivo studies often lack mechanistic insights, due to high inter-individual variability and lack of suitable controls. Conversely, “mouse-in-mouse” models of cancer immunoediting can provide precious and reproducible insights into molecular mechanisms, but those results are often difficult to translate into clinical practice due to species-specific factors. To overcome these limitations, here we set up a novel mouse-human chimeric model of antileukemic adoptive immunotherapy, to dissect how T cell immune pressure can sculpt leukemia gene expression profile. MethodsPurified primary human AML blasts were infused into non-irradiated immunodeficient NOD/SCID γ-chain null (NSG) mice. Upon documentation of leukemia engraftment, mice received serial infusions of human T cells, either autologous or allogeneic (HLA-identical, HLA-haploidentical or HLA-disparate) to the leukemic cells to mimic immune pressure. Absolute counts of human leukemic and T cells were monitored weekly in mice peripheral blood. At sacrifice, leukemic cells were FACS-purified, total RNA was extracted and gene expression profile was analyzed using Illumina microarray. Deregulated genes and signatures were identified by pairwise LIMMA analysis. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) curated databases were interrogated to identify deregulated processes. ResultsInfused leukemic cells stably and reproducibly engrafted into the murine bone marrow, and could be detected circulating in the peripheral blood of treated mice from three weeks after infusion. Leukemic cells exponentially expanded, could be transferred to secondary and tertiary recipients, and, in the absence of immune pressure, displayed a stable gene expression profile amongst littermates and upon serial transfer. HLA-disparate and HLA-haploidentical T cells eradicated AML from 10/10 treated mice, and complete eradication was confirmed by no disease recurrence in second transplant recipients. HLA-identical T cells granted only temporary control in 6/6 mice, while autologous T cells were completely inefficacious in 6/6 mice. Leukemic blasts subjected to T cell-mediated immune pressure showed a specific and reproducible gene signature. GO and GSEA demonstrated the selective deregulation of genes involved in immune processes. Among the top-ranked upregulated genes we identified genes related to response to interferons, comprising proteasome and immunoproteasome subunits, as well as molecules and receptors involved in antigen processing and presentation, comprising classical and non-classical HLA Class I and II molecules (HLA-DMA, HLA-DRA, CD74, B2M, TAPBP, TAPBPL). ConclusionsOur findings provide further proof of the leukemia immunoediting hypothesis, demonstrating that leukemic cells modify their expression profile, and their immunogenicity, in response to T cell-mediated immune pressure, and that antigen presentation pathways represent key targets in these processes. The model we set up provides a novel and valuable tool to investigate these mechanisms in detail. Experiments with T cells genetically modified to express a suicide gene are currently ongoing, for a time-wise control of alloreactivity aimed at modeling a longer phase of equilibrium between adoptively transferred immune cells and leukemia. Disclosures:Bordignon:MolMed SpA: Employment. Bonini:MolMed SpA: Consultancy.

Detection of SALL4 mRNA expression in leukemia by real-time fluorescent quantitative PCR

This study was purposed to establish a real-time fluorescent quantitative PCR (FQ-PCR) for quantifying SALL4 mRNA and to investigate its expression in different types of leukemia patients. SALL4 mRNA expression were measured in 60 leukemia patients of different periods and 10 normal controls sequentially by FQ-PCR. The results showed that the expression of SALL4 mRNA in de novo leukemia patients and relapsed patients was higher than that in controls (P < 0.05), which was significantly decreased at complete remission (CR). In relapsed patients, the expression of SALL4 mRNA increased slightly higher than that in de novo leukemia group, but the difference was not statistically significant (P > 0.05). However, the expression of SALL4 mRNA was low in CLL, T-ALL and AML-M3. The expression pattern of BMI-1 was same as SALL4, and the expression of BMI-1 positively correlated with that of SALL4 in leukemia (r = 0.825, P < 0.01). It is concluded that the detection of SALL4 gene expression in acute and chronic leukemia by real-time gTR-PCR displays high sensitivity and specificity. SALL4 gene may be one of indicators for monitoring the therapeutic outcome of partial leukemia and minimal residual disease.

Hypermethylation of two CpG sites upstream of CASP8AP2 promoter influences gene expression and treatment outcome in childhood acute lymphoblastic leukemia

DNA hypermethylation of Caspase 8 associated protein 2 (CASP8AP2) and its role in childhood acute lymphoblastic leukemia (ALL) is unclear. We analyzed methylation status of CpG sites upstream of CASP8AP2 gene in 86 children with ALL by bisulfite sequencing and quantitative PCR. Methylation percentage of two CpG sites at positions of -1189 and -1176 was inversely correlated with mRNA expression (Spearman correlation: -0.333, P=0.002). High methylation was associated with the existence of minimal residual disease (MRD) at day 78 (P=0.035), The patients in high methylation group had a poor treatment outcome. The combination of methylation level and MRD at day 33 might improve current risk stratification.

ANGPT2 promoter methylation is strongly associated with gene expression and prognosis in chronic lymphocytic leukemia

Increasing evidence suggests a key role for angiopoietin-2 (ANGPT2) in influencing the aggressiveness of chronic lymphocytic leukemia (CLL). In the presence of vascular endothelial growth factor (VEGF), ANGPT2 causes vessel destabilization leading to neoangiogenesis. Accordingly, high expression levels of ANGPT2 and high degree of angiogenesis have consistently been associated with poor prognosis in CLL; however, the molecular mechanisms behind the variability in ANGPT2 expression are still to be discovered. Here, for the first time, we investigated the DNA methylation status of the ANGPT2 promoter in a large CLL cohort (n = 88) using pyrosequencing and correlated methylation data with ANGPT2 expression levels, prognostic factors and outcome. Importantly, methylation levels of the ANGPT2 gene correlated inversely with its mRNA expression levels (p < 0.001). Moreover, low ANGPT2 methylation status was highly associated with adverse prognostic markers, shorter time to first treatment and overall survival. Finally, treatment with methyl inhibitors induced re-expression of ANGPT2 in two B-cell lymphoma cell lines, underscoring the importance of DNA methylation in regulating transcriptional silencing of this gene. In conclusion, we believe that the known variability in ANGPT2 expression among CLL patients could be explained by differential promoter DNA methylation and that low methylation levels of the ANGPT2 promoter have an adverse prognostic impact in CLL.

Abstract 1518: Microenvironment regulation of acute lymphoblastic leukemia miRNA profiles.

Abstract It is well established that the bone marrow microenvironment provides cues to hematopoietic tumors that initiate and progress in this unique anatomical site that likely mimics, to some degree, cell signaling that directs steady state hematopoiesis. We have previously documented diverse changes in gene and protein expression in acute lymphoblastic leukemia (ALL) cells in contact with either primary bone marrow stromal cells (BMSC obtained from consenting donors) or human derived osteoblasts (HOB); two components of the marrow stem cell niche (Wang 2007). Importantly, many of these changes are coincident with chemoresistance. In the current study we have expanded our investigation to include investigation of miRNA profile comparisons of the human Ph+ ALL cell line Sup-B15 (ATCC # CRL-1929) grown in media versus tumor cells grown in long term co-culture (greater than 3 months) with adherent BMSC or HOB. We have previously described the stem cell-like characteristics of Sup-B15, making it an intriguing cell line for continued study (Wang 2007). In addition, comparisons were completed for the cell lines Nalm-27 and Nalm-30 (Fujisaki Cancer Center, Okayama, Japan) for evaluation of tumors at diagnosis versus relapse of disease. Total RNA was isolated using Ambion miRVana RNA extraction kit and a microarray assay was performed using a service provider (LC Sciences, Houston TX) and μParafloTM MicroRNA Microarray Assays as described (Wei 2009). Preliminary observations identified over 15 different microRNAs expressed at baseline in all three lines evaluated which met the medium to high relative threshold according to the analysis. Profiles of Nalm-27 and Nalm-30 were highly conserved with the exception of miR-7-1-3p and miR-99b-5p which were upregulated and miR-142-3p, miR-1260b, and miR-19a-3p which were down regulated when the cell line established at relapse (Nalm-30) was compared to that from diagnosis (Nalm-27). Consistent with microRNAs as critical regulators of diverse cellular processes including proliferation, survival, and differentiation, several targets were noted to be significantly influenced by LTCC of Sup-B15 ALL cells with BMSC or HOB. Preliminary evaluation suggests miR-4254 expression was increased by BMSC and miR-4484, miR-34b-3p and miR-1237-5p were increased when tumor cells were provided continual access to HOB. In contrast, miR-221-3p, miR-222-3p, miR-4521, levels were decreased during BMSC co-culture as well as miR-221-3p, miR-222-3p, miR-199b-5p, miR-185-5p, miR-4521 during co-culture with HOB. Collectively these changes highlight the dynamic expression of microRNAs and emphasize their responsiveness to microenvironment derived signals. These observations will contribute to identification of novel pathways that include miRNAs that warrant consideration as therapeutic targets in efforts to eradicate residual disease in the setting of ALL. Citation Format: Blake Moses, Sriganesh G. Sharma, J. Michael Ruppert, Laura F. Gibson. Microenvironment regulation of acute lymphoblastic leukemia miRNA profiles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1518. doi:10.1158/1538-7445.AM2013-1518

HOXA9 Gene Expression in Acute Myeloid Leukemia

Homeobox genes encode the class of transcription factors in vertebrates and are found in clusters called A, B, C, and D on four separate chromosomes. HOXA9 gene is part of the cluster A on chromosome 7 and encodes a DNA-binding transcription factor which may regulate gene expression, morphogenesis, and differentiation. The objective of this study was to determine the HOXA9 gene expression in acute myeloid leukemia (AML). For this purpose, semi-quantitative reverse transcriptase-polymerase chain reaction was used to measure HOXA9 gene expression in human erythroleukemia (HEL) cell line and bone marrow mononuclear cells from 54 AML patients and 20 healthy individuals. The data show that HOXA9 mRNA expression was negative in 20 healthy individuals but was positive in HEL cells and in 22 out of 54 (40.74%) AML patients. The complete remission rate (45.45%) of the patients who expressed the gene was significantly lower than that (71.86%) in patients who did not express the gene after chemotherapy. Therefore, it was concluded that HOXA9 gene might be involved in the pathogenesis of AML and played as a worse prognostic factor in AML.

Curative effect of decitabine combined with cytokine-induced killer cells in two elderly patients with acute myeloid leukemia

This study was aimed to evaluate the effectiveness and safety of low methylation drug decitabine combined with autologous cytokine induced killer cells (CIK) to treat the elderly patients with acute myeloid leukemia (AML). Two AML patients aged over 80 years old were diagnosed and treated in our department from 2006 to 2012; both company with MDS history, and one case was M4-type, another case was M6-type according to FAB classification. The changes in lymphocyte subsets, hematologic response, transfusion frequency, leukemic gene expression, obtaining CR or PR, quality of life and survival time of the patients with different treatment regimen (decitabine alone; CIK alone; decitabine combined with CIK) were systematically observed. The results showed that therapy of decitabine combined with CIK cells could reduce bone marrow suppression extent, decrease the frequency and volume of blood transfusion, and prolong the duration of partial remission, compared with the single use of CIK cell infusion and single use of decitabine treatment. Meanwhile, the kinds of expressed genes associated with leukemia decreased and the survival time was prolonged obviously. The patients' life quality significantly improved. It is concluded that decitabine combined with CIK for treatment of elderly patients with AML is safe and effective.

Prognostic Implication of BAALC Gene Expression in Adult Acute Myeloid Leukemia

Recently various molecular markers and global gene expression profiling have been investigated to improve risk profile characterization of AML with normal cytogenetics. Our main objective is to investigate the prognostic impact of brain and acute leukemia, cytoplasmic (BAALC) expression in AML with normal karyotype. BAALC expression was analysed using quantitative real time (QRT) PCR. High expression was detected in 22 of 45 patients (48.9%) and its expression did not correlate with the clinical parameters of patients. High BAALC expressers had significantly lower incidence of CR (22.7% vs. 73.9%; p = 0.001), higher mortality rate (72.1% vs. 39.1%; p = 0.023), showed significantly shorter DFS (mean 4.5 vs. 13.21 months, p < 0.001), and inferior overall survival (7.02 vs. 15.02 months, p < 0.001). Multivariable analysis confirmed high BAALC expression as an independent risk factor for DFS and OS. BAALC expression is an important prognostic factor in AML patients with normal karyotype and its incorporation into novel risk-adapted therapeutic strategies will improve the currently disappointing cure rate of this group of patients.