Gene Expression Analyses At Time Of Diagnosis Indicate Biomarkers Predictive Of Therapeutic Response In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2619-2619 ◽  
Author(s):  
Marita Lagergren Lindberg ◽  
Petra Hååg ◽  
Ali Moshfegh ◽  
Lena Kanter ◽  
Magnus Bjorkholm ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Clinical Outcome ◽  
Myeloid Leukemia ◽  
Differentially Expressed ◽  
Training Cohort ◽  
Gene Expression Analyses ◽  
Diagnostic Samples ◽  
Acute Myeloid

Abstract In this study we aimed to identify biomarkers predictive of clinical response in acute myeloid leukemia (AML). For this purpose mRNA was isolated from diagnostic samples from 42 AML patients (“training cohort”) and subjected to Affymetrix® gene expression analysis. All patients entered complete remission (CR) after high-dose induction chemotherapy, reaching a median CR duration of 161 (range 12-3701) days. Samples from patients with “short CR duration” (<6 months, n=24) and “long CR duration” (>6 months n=18), respectively, were pooled and compared. Gene expression analyses revealed 383 genes to be up-regulated and 610 genes down-regulated more than two fold in samples from patients with short, as compared to those with long CR duration. Ten genes were found to be up-regulated >30 times, with the runt-related transcription factor 1; translocated to 1 (cyclin D-related) (RUNX1T1) gene showing the highest differential expression (116-fold), while annexin 1 (ANXA1) was the most down-regulated gene (58-fold). Significantly higher transcript levels of RUNX1T1 were confirmed in the poor outcome group when performing quantitative real time polymerase chain reaction (qRT-PCR) on individual samples (n=20, p<0.002). Subjecting our data to pathway analysis (Ingenuity®) comparing the same groups, we focused on RUNX1T1 and created a network of RUNX1T1 interacting molecules. Utilizing the IPA database to create a network over interacting molecules of RUNX1T1, we identified a majority of these to be transcriptional regulators and among them the transcription factor 3 (TCF3) to be up-regulated 5-fold in patients with short CR duration. Our training cohort data were validated in silico extracting information from an independent study by Metzeler et al, publicly available from Oncomine® (www.oncomine.org) and encompassing diagnostic samples from 162 AML patients. Among genes differentially and similarly regulated in poor responders in both the training and validation cohorts we observed TCF3, chemokine (C-X-C motif) ligand 3 (CXCL3), Zinc finger, MIZ-type containing 1 (ZMIZ1) (up-regulated) and Peroxiredoxin 2 (PRDX2) (down-regulated). Analyses of clinical outcome revealed that AML patients with a high ZMIZ1 expression had a significantly decreased overall survival (OS) as compared to that of patients with a low ZMIZ1 expression (p <0.03). ZMIZ1 has been reported to be involved in tumor growth in general and suggested to interact with activated NOTCH in inducing leukemia, but its more precise role in AML is still unclear. In conclusion, we report clear differences in gene expression in diagnostic samples from AML patients with subsequent poor vs. better long-term clinical outcome to therapy, thus to constitute possible novel predictive biomarkers for response. In our training set RUNX1T1 was the most differentially expressed gene, while ZMIZ1 was upregulated in both the training and validation sets and significantly associated with survival. Further, functional studies of differentially expressed genes in clinical subsets of AML patients appear warranted. Disclosures: No relevant conflicts of interest to declare.

Distinct association of RUNX family expression with genetic alterations and clinical outcome in acute myeloid leukemia

2020 ◽  
Vol 29 (3) ◽  
pp. 387-397
Author(s):  
Yangli Zhao ◽  
Tingjuan Zhang ◽  
Yangjing Zhao ◽  
Jingdong Zhou
Keyword(s):  
Acute Myeloid Leukemia ◽  
Clinical Outcome ◽  
Myeloid Leukemia ◽  
Disease Free Survival ◽  
Genetic Alterations ◽  
Differentially Expressed ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Frequent Event ◽  
Acute Myeloid

BACKGROUND: The runt-related transcription factor family (RUNXs) including RUNX1, RUNX2, and RUNX3 are key transcriptional regulators in normal hematopoiesis. RUNXs dysregulations caused by aberrant expression or mutation are frequently seen in various human cancers especially in acute myeloid leukemia (AML). OBJECTIVE: We systemically analyzed the expression of RUNXs and their relationship with clinic-pathological features and prognosis in AML patients. METHODS: Expression of RUNXs was analyzed between AML patients and normal controls from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) projects. Correlations between RUNXs expression and clinical features together with survival were further analyzed. RESULTS: All RUNXs expression in AML patients was significantly increased as compared with controls. RUNXs expression was found to be significantly associated with genetic abnormalities such as RUNX1 mutation, t(8;21) and inv(16)/t(16;16). By Kaplan-Meier analysis, only RUNX3 overexpression was associated with shorter overall survival (OS) and disease-free survival (DFS) among non-M3 AML patients. Notably, in high RUNX3 expression groups, patients received hematopoietic stem cell transplantation (HSCT) had markedly better OS and DFS than patients without HSCT among both all AML and non-M3 AML. In low RUNX3 expression groups, there were no significant differences in OS and DFS between HSCT and non-HSCT groups among both all AML and non-M3 AML. In addition, a total of 835 differentially expressed genes and 69 differentially expressed microRNAs were identified to be correlated with RUNX3 expression in AML. CONCLUSION: RUNXs overexpression was a frequent event in AML, and was closely associated with diverse genetic alterations. Moreover, RUNX3 expression may be associated with clinical outcome, and helpful for guiding treatment choice between HSCT and chemotherapy in AML.

Acute Myeloid Leukemia With Biallelic CEBPA Gene Mutations and Normal Karyotype Represents a Distinct Genetic Entity Associated With a Favorable Clinical Outcome

2010 ◽  
Vol 28 (4) ◽  
pp. 570-577 ◽  
Author(s):  
Annika Dufour ◽  
Friederike Schneider ◽  
Klaus H. Metzeler ◽  
Eva Hoster ◽  
Stephanie Schneider ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Clinical Outcome ◽  
Myeloid Leukemia ◽  
Expression Profiles ◽  
Gene Mutations ◽  
Gene Expression Profiles ◽  
Similar Frequency ◽  
Favorable Clinical Outcome ◽  
Acute Myeloid

Purpose CEBPA mutations are found as either biallelic (biCEBPA) or monoallelic (moCEBPA). We set out to explore whether the kind of CEBPA mutation is of prognostic relevance in cytogenetically normal (CN) acute myeloid leukemia (AML). Patients and Methods Four hundred sixty-seven homogeneously treated patients with CN-AML were subdivided into moCEBPA, biCEBPA, and wild-type (wt) CEBPA patients. The subgroups were analyzed for clinical parameters and for additional mutations in the NPM1, FLT3, and MLL genes. Furthermore, we obtained gene expression profiles using oligonucleotide microarrays. Results Only patients with biCEBPA had an improved median overall survival when compared with patients with wtCEBPA (not reached v 20.4 months, respectively; P = .018), whereas patients with moCEBPA (20.9 months) and wtCEBPA had a similar outcome (P = .506). Multivariable analysis confirmed biCEBPA, but not moCEBPA, mutations as an independent favorable prognostic factor. Interestingly, biCEBPA mutations, compared with wtCEBPA, were never associated with mutated NPM1 (0% v 43%, respectively; P < .001) and rarely associated with FLT3 internal tandem duplication (ITD; 5% v 23%, respectively; P = .059), whereas patients with moCEBPA had a similar frequency of mutated NPM1 and a significantly higher association with FLT3-ITD compared with patients with wtCEBPA (44% v 23%, respectively; P = .037). Furthermore, patients with biCEBPA showed a homogeneous gene expression profile that was characterized by downregulation of HOX genes, whereas patients with moCEBPA showed greater heterogeneity in their gene expression profiles. Conclusion Biallelic disruption of the N and C terminus of CEBPA is required for the favorable clinical outcome of CEBPA-mutated patients and represents a distinct molecular subtype of CN-AML with a different frequency of associated gene mutations. These findings are of great significance for risk-adapted therapeutic strategies in AML.

scholarly journals Relationship of differential gene expression profiles in CD34+ myelodysplastic syndrome marrow cells to disease subtype and progression

Blood ◽  
2009 ◽  
Vol 114 (23) ◽  
pp. 4847-4858 ◽  
Author(s):  
Kunju Sridhar ◽  
Douglas T. Ross ◽  
Robert Tibshirani ◽  
Atul J. Butte ◽  
Peter L. Greenberg
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Differential Gene Expression ◽  
Myeloid Leukemia ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Differentially Expressed ◽  
Differential Gene ◽  
Acute Myeloid

AbstractMicroarray analysis with 40 000 cDNA gene chip arrays determined differential gene expression profiles (GEPs) in CD34+ marrow cells from myelodysplastic syndrome (MDS) patients compared with healthy persons. Using focused bioinformatics analyses, we found 1175 genes significantly differentially expressed by MDS versus normal, requiring a minimum of 39 genes to separately classify these patients. Major GEP differences were demonstrated between healthy and MDS patients and between several MDS subgroups: (1) those whose disease remained stable and those who subsequently transformed (tMDS) to acute myeloid leukemia; (2) between del(5q) and other MDS patients. A 6-gene “poor risk” signature was defined, which was associated with acute myeloid leukemia transformation and provided additive prognostic information for International Prognostic Scoring System Intermediate-1 patients. Overexpression of genes generating ribosomal proteins and for other signaling pathways was demonstrated in the tMDS patients. Comparison of del(5q) with the remaining MDS patients showed 1924 differentially expressed genes, with underexpression of 1014 genes, 11 of which were within the 5q31-32 commonly deleted region. These data demonstrated (1) GEPs distinguishing MDS patients from healthy and between those with differing clinical outcomes (tMDS vs those whose disease remained stable) and cytogenetics [eg, del(5q)]; and (2) molecular criteria refining prognostic categorization and associated biologic processes in MDS.

TET2 Mutations in Acute Myeloid Leukemia (AML): Results From a Comprehensive Genetic and Clinical Analysis of the AML Study Group

2012 ◽  
Vol 30 (12) ◽  
pp. 1350-1357 ◽  
Author(s):  
Verena I. Gaidzik ◽  
Peter Paschka ◽  
Daniela Späth ◽  
Marianne Habdank ◽  
Claus-Henning Köhne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Clinical Outcome ◽  
Expression Pattern ◽  
Myeloid Leukemia ◽  
Gene Expression Pattern ◽  
Genetic Alterations ◽  
Response To Therapy ◽  
Adult Patients ◽  
Acute Myeloid

Purpose The tet oncogene family member 2 (TET2) gene was recently identified to be mutated in myeloid disorders including acute myeloid leukemia (AML). To date, there is increasing evidence for a functional role of TET2 mutations (TET2mut) in AML. Thus, we explored the frequency, gene-expression pattern, and clinical impact of TET2mut in a large cohort of patients with AML in the context of other AML-associated aberrations. Patients and Methods Samples from 783 younger adult patients with AML were analyzed for the presence of TET2mut (coding exons 3 to 11), and results were correlated with data from molecular genetic analyses, gene-expression profiling, and clinical outcome. Results In total, 66 TET2mut were found in 60 patients (60 of 783 patients; 7.6%), including missense (n = 37), frameshift (n = 16), and nonsense (n = 13) mutations, which, with one exception, were all heterozygous. TET2mut were not correlated with distinct clinical features or genetic alterations, except for isocitrate dehydrogenase mutations (IDHmut) that were almost mutually exclusive with TET2mut (P < .001). TET2mut were characterized by only a weak gene-expression pattern, which, nevertheless, reflected TET2mut-associated biology. TET2mut did not impact the response to induction therapy and clinical outcome; the combination of patients who exhibited TET2mut and/or IDHmut revealed shorter overall survival (P = .03), although this association was not independent from known risk factors. Conclusion TET2mut were identified in 7.6% of younger adult patients with AML and did not impact the response to therapy and survival. Mutations were mutually exclusive with IDHmut, which supported recent data on a common mechanism of action that might obscure the impact of TET2mut if compared against all other patients with AML.

Prognostic Impact and Concurrent Genetic Alterations of CEBPA Double Mutations in Adults with Cytogenetically Intermediate-Risk Acute Myeloid Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2538-2538
Author(s):  
Shunichiro Yamaguchi ◽  
Kenji Tokunaga ◽  
Eisaku Iwanaga ◽  
Tomoko Nanri ◽  
Taizo Shimomura ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Clinical Outcome ◽  
Myeloid Leukemia ◽  
Gene Mutations ◽  
Prognostic Impact ◽  
Intermediate Risk ◽  
Disease Entity ◽  
Cebpa Mutations ◽  
Acute Myeloid

Abstract Abstract 2538 Aims: Among acute myeloid leukemia (AML) patients with intermediate-risk cytogenetics, C/EBPa mutations represent a distinct disease entity with a favorable clinical outcome and is adopted in the current WHO classification of AML as a provisional disease entity in the category AML with recurrent genetic abnormalities. CEBPA encodes a transcription factor that is essential for neutrophil development. AML patients with CEBPA mutations can be separated into two subgroups with a single mutation in the CEBPA (CEBPA sm) and double mutations (CEBPA dm). Biallelic mutations consisted of an N-terminal frameshift mutation and a C-terminal inframe bZIP mutation were detected in the majority of CEBPA dm, whereas CEBPA sm occurs in either N-terminal or C-terminal regions. More recent data indicate that favorable outcome is mainly observed in AML patients with CEBPA dm but not with CEBPA sm. In addition, concurrent gene mutations may occur more frequently in AML with CEBPA sm than in CEBPA dm. In contrast, transcription factor GATA2 mutations are frequently identified in AML with CEBPA dm. In this study, we examined incidence, concurrent gene mutations and clinical significance of CEBPA dm and CEBPA sm in Japanese adults with cytogenetically intermediate-risk AML. Methods: To identify the prevalence and prognostic impact of CEBPA dm and CEBPA sm, we examined 111 patients with intermediate-risk AML who were mainly treated with the JALSG protocols. Age ranged from 16 to 86 years, with a median of 58.5 years. DNA was extracted from bone marrow or peripheral blood mononuclear cells at diagnosis and subjected to PCR amplification and direct sequencing of the CEBPA, FLT3, NPM1, IDH1, IDH2, DNMT3A and GATA2 genes. This study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the revised Declaration of Helsinki. Results: Of 111 cytogenetically intermediate-risk AML, we found 12 (10.8%) CEBPA dm and 7 (6.3%) CEBPA sm. In 7 CEBPA sm, one NPM1 mutation and one FLT3-ITD were detected. Two FLT 3-ITD and no concurrent mutation of NPM1 were found in CEBPA dm. No mutation in the IDH1, IDH2, DNMT3A exon 23 was identified in both patients with CEBPA sm and CEBPA dm. On the other hand, mutations in the GATA2 zinc finger domains were detected in 3 of 12 (25%) patients with CEBPA dm. No GATA2 mutations were found in 7 CEBPA sm. One of 21 patients with wild-type CEBPA (CEBPA wt) had a GATA2 mutation. Patients with CEBPA double or single mutations showed a better 5-year overall survival (OS) compared to CEBPA wt (51.3% vs 16.0%, P=0.0048). CEBPA dm AML was associated with a significant superior clinical outcome compared with CEBPA wt (5-year OS, 55.6% vs 16.0%, P=0.0025). However, no significant difference was identified between CEBPA dm and CEBPA sm AML (5-years OS, 55.6% vs 42.9%, P=0.1375) or between CEBPA sm and CEBPA wt AML (5-year OS, 42.9% vs 16.0%, P=0.4827). In addition, the presence of additional GATA2 mutations did not significantly influence the clinical outcome of AML patients with CEBPA dm. Conclusions: A total of 19 (17.1%) patients with cytogenetically intermediate-risk AML harbored CEBPA mutations. Our study indicates that the presence of the CEBPA dm but not CEBPA sm is associated with favorable outcome in Japanese patients with cytogenetically intermediate-risk AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeting MTHFD2 in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 443-443
Author(s):  
Yana Pikman ◽  
Alexandre Puissant ◽  
Gabriela Alexe ◽  
Andrew Furman ◽  
Stacey Frumm ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Carbon Metabolism ◽  
Myeloid Leukemia ◽  
Differentially Expressed ◽  
Bet Inhibitor ◽  
Folate Pathway ◽  
One Carbon Metabolism ◽  
Acute Myeloid

Abstract Alterations in differentiation pathways contribute to the development of acute myeloid leukemia (AML). Differentiation therapy with all-trans retinoic acid (ATRA) has dramatically altered the treatment of acute promyelocytic leukemia, transforming it from a nearly fatal disease to a curable one. We set out to identify cellular pathways that contribute to AML differentiation, with the goal of identifying new therapeutic targets. We analyzed gene expression data from AML cell lines treated with phorbol 12-myristate 13-acetate (PMA), ATRA, Vitamin D, the BET inhibitor JQ1 and the DOT1L inhibitor EPZ00477, treatments known to induce AML differentiation and impair growth. Folate-mediated one-carbon metabolism was one of only three metabolic pathways altered by these compounds, with expression of MTHFD2 consistently downregulated with each compound. MTHFD2 is an NAD-dependent, bi-functional mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase. It is differentially expressed in embryonic and transformed tissues and is upregulated in myeloid progenitors. MTHFD2 is the most differentially expressed metabolic enzyme in cancer cells versus normal cells, including normal proliferating cells. We thus investigated the role of MTHFD2 in myeloid malignancy. First, we demonstrated using ChIP-qPCR, MYC knockdown and MYC inhibition with a BET inhibitor, that MYC directly regulates MTHFD2 expression in AML. Knockdown of MTHFD2 with two shRNAs confirmed to have on-target activity, induced myeloid differentiation in AML cell lines, as measured by Cd11b expression, morphologic changes and induction of a previously validated AML differentiation gene expression signature. MTHFD2 knockdown decreased cell growth in AML cell lines, as well as decreased colony formation in methylcellulose in both AML cell lines and primary patient blasts. AML cells transduced with these two MTHFD2-directed shRNAs demonstrated attenuated growth in an orthotopic mouse model of AML. Furthermore, three MTHFD2-directed shRNAs prolonged survival in an MLL-AF9 mouse leukemia model. Additionally, using a doxycycline inducible shRNA system, we demonstrated that two miR30-shRNAs directed against MTHFD2 decreased AML burden in mice with established disease and prolonged survival. To identify biomarkers of response to MTHFD2 suppression, we used single sample Gene Set Enrichment Analysis (ssGSEA) to identify primary patient AML samples enriched for gene expression signatures of folate-mediated one-carbon metabolism and MTHFD2. We found in both independent data sets that the cluster of patients enriched for expression of the one-carbon folate pathway gene signatures was also enriched for patients with FLT3-ITD mutations, a subset of AML with a particularly poor prognosis. In addition, in an shRNA screen targeting 11,194 genes and performed in 216 cancer cell lines, including 17 AML lines, FLT3-ITD was a biomarker of response to MTHFD2 knockdown. We next validated that while MTHFD2 suppression induced measureable differentiation in all six AML cell lines examined, it induced the most robust induction of apoptosis in FLT3-ITD mutant AML. The mitochondrial one-carbon folate pathway is thought to contribute to cellular oxidative balance by providing reducing power in the form of NAD(P)H, and suppression of MTHFD2 was previously shown to increase ROS levels. Indeed, suppression of MTHFD2 led to a marked increase in ROS in the FLT3-ITD positive AML cell lines in which apoptosis was induced. In summary, a decrement in MTHFD2 expression was found at the center of multiple AML perturbations that impair AML growth and induce differentiation. Our data support MTHFD2 as an AML dependency and FLT3-ITD as a potential biomarker of response. We thus provide critical preclinical evidence for targeting of MTHFD2 as a therapeutic strategy in AML. Disclosures Stone: Celgene: Consultancy; Merck: Consultancy. DeAngelo:Celgene: Consultancy; Pfizer: Consultancy; Incyte: Consultancy; Agios: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Amgen: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.

scholarly journals Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sumiko Takao ◽  
Lauren Forbes ◽  
Masahiro Uni ◽  
Shuyuan Cheng ◽  
Jose Mario Bello Pineda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Myeloid Leukemia ◽  
Human Cancers ◽  
Co Activation ◽  
Alternative Transcription ◽  
Transcription Factor Complexes ◽  
Oncogenic Gene ◽  
Acute Myeloid

Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2, and SATB1. They are organized convergently in genetically diverse subtypes of AML and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control.

Functional miRNA Expression Library Screen Identifies miRNAs That Alter Proliferation and Differentiation in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3541-3541 ◽  
Author(s):  
Valerie A. Morris ◽  
Carrie Cummings ◽  
Soheil Meshinchi ◽  
Vivian Oehler
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Mirna Expression ◽  
Myeloid Leukemia ◽  
Differentially Expressed ◽  
Nb4 Cells ◽  
Library Screen ◽  
Differentiation And Proliferation ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by increased self-renewal of leukemia stem/progenitor cells and failure of differentiation to mature myeloid cells. MicroRNAs (miRNAs, miRs) are short non-coding RNAs whose power stems from the ability to regulate hundreds of targets by interfering with gene expression by degrading messenger RNA or blocking protein translation. It has become evident that miRNAs are important regulators of normal cell processes that are altered in cancer, and restoring or inhibiting miRNAs may be a therapeutic strategy in cancer. MiRNAs play a significant role in normal hematopoiesis and differentiation; however, our understanding of how miRNAs contribute to impaired differentiation and proliferation in acute leukemia remains limited. We have used a combined approach to prioritize miRNAs for investigation for functional roles in leukemogenesis. To identify miRNAs that may play a role in altered cell proliferation or differentiation of AML cells, we used a functional miRNA library screen. We transduced a high-throughput lentiviral library containing 613 precursor miRNAs (Systems Biology) into the AML cell line THP-1 harboring a MLL-AF9 translocation. These cells were then treated with 12-O-tetra-decanoylphorbol-13-acetate (TPA), an agent that induces monocytic differentiation and cell adhesion. After recovery of proliferating cells we compared miRNA expression before and after TPA induced differentiation using quantitative PCR to identify enriched miRNAs (may block differentiation) and miRNAs that drop out (may promote differentiation). To prioritize miRNAs with phenotypic effects that may be relevant to AML, we focused on miRNAs that are differentially expressed in various AML subtypes, using both miRNA-sequencing data from primary pediatric patient AML samples (N=117) and mature miRNA expression of primary adult patient AML samples (N=20). Selected miRNAs were cloned into a lentiviral expression vector and retested in THP-1 cells to assess whether proliferation or differentiation was altered by expression. In line with previously published data we found enrichment of miR-9-1 and members of the miR-17-92 cluster, and its paralogs miR-106a-363 and miR-106b-25, as inhibitors of myeloid differentiation. In addition, we have validated three miRNAs (miR-590, miR-550A1 and miR-550A2) with previously uncharacterized roles that block differentiation and increase proliferation of THP-1 cells. These phenotypes were confirmed in other AML cell lines, including NB4 cells (PML-RARA translocation) and U937 cells (MLL-AF9 translocation). Increased expression of either miR-590 or miR-550A2 enhances MAPK signaling, as detected by increased phospho-ERK1/2, which influences cell proliferation. We have identified that PPP2R2A, the regulatory B55 alpha subunit of the tumor suppressor protein phosphatase 2A (PP2A), is a miR-590 target. Regulation of this complex by miR-590 could contribute to alterations in MAPK/ERK signaling. To identify additional miR-590 and miR-550A putative targets, we profiled gene expression changes by microarray (Illumina Human HT-12v.4) in THP-1 and NB4 cells expressing miR-590 or miR-550A2 versus control cells. Validation of identified putative targets, as well as functional investigations assessing the impact of the identified miRNAs and targets on AML cell proliferation or differentiation, are underway. In conclusion, functional miRNA screening is an important tool to prioritize differentially expressed miRNAs identified in primary AML patient samples that alter differentiation and proliferation. Using this approach we have identified new roles for miR-590 and miR-550A2 in AML. Our goal is to determine mechanistically how these miRNAs block differentiation in AML cells and to establish how to target these pathways therapeutically to promote differentiation and AML cell death. Disclosures No relevant conflicts of interest to declare.

Minimal Reduction of PU.1 Is Sufficient to Induce a Preleukemic State and Promote Development of Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 305-305
Author(s):  
Britta Will ◽  
Thomas O. Vogler ◽  
Swathi-rao Narayanagari ◽  
Boris Bartholdy ◽  
Tihomira I. Todorova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Malignant Transformation ◽  
Myeloid Leukemia ◽  
Complete Loss ◽  
Hematopoietic Stem ◽  
Minimal Reduction ◽  
Acute Myeloid

Abstract Genomic studies have shown that human cancer is rarely associated with a complete loss of transcripts; instead, acquired DNA alterations often occur within the non-coding part of the genome, are enriched in gene-regulatory regions, and cause only moderate transcriptional changes. It is currently not well understood how such moderate gene expression changes impact normal tissue function and how they contribute to malignant transformation. Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) develop through a multi-step transformation process originating in hematopoietic stem cells (HSCs) and mainly present in the elderly (median age of >65 years at diagnosis). Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces AML in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet moderate PU.1 inhibition is common in AML patients. At the example of the Ets-family transcription factor PU.1, which is indispensable for HSC function and the differentiation of cells within the myeloid as well as lymphoid lineages, we tested the hypothesis that even moderate gene expression alterations of key regulators can drive malignant transformation. We assessed the effects of minimal PU.1 inhibition on hematopoiesis in a novel mouse model that co-models the genomic context found in aging human individuals and patients with MDS/AML. Mice lacking Msh2, the key component of the MutSα and MutSβ complexes mediating DNA mismatch repair, accumulate elevated numbers of point mutations, in particular C/G>T/A transitions and small insertions/deletions resembling the mutation spectrum acquired in HSCs in aging human individuals and patients with MDS and AML. We crossed Msh2-/- mice with animals carrying a heterozygous deletion of an upstream regulatory element of PU.1 (UREΔ/+). UREΔ/+Msh2-/- mice exhibited a significant, but very modest reduction of PU.1 expression on average by 26-37% in fractionated hematopoietic multipotent stem and myeloid progenitor cells. Strikingly, this minimal reduction of PU.1 led to the emergence of an aggressive, transplantable AML in more than two thirds of UREΔ/+Msh2-/- mice which was never observed in URE+/+Msh2-/- mice. Overt leukemia was preceded by a preleukemic phase hallmarked by an expanded population of multipotent murine hematopoietic stem cell enriched cells (HSPCs) that was myeloid-biased and less quiescent than their wild type counterpart. Longitudinal monitoring of preleukemic UREΔ/+Msh2-/- mice revealed a progressive increase in immature myeloid cells along with a gradual decrease in mature myeloid cells, as well as expansion of phenotypic HSPC compartments and multi-lineage dysplasia resembling human MDS. AML progression was accompanied by additional inhibition of a PU.1-cooperating factor, interferon responsive factor 8 (Irf8). Irf8 expression restoration rescued impaired expression of genes harboring PU.1/IRF consensus binding sites, led to the loss of aberrant self-renewal, promoted myeloid differentiation, and induced apoptosis in leukemic UREhetMsh2-/- cells demonstrating that Irf8 impairment functionally cooperates with minimally reduced PU.1 expression in our model. We also found evidence of disease-relevant joint PU.1/IRF8 inhibition in human myeloid leukemogenesis: (1) patients with MDS with a higher risk for the progression to AML had lower IRF8 levels; (2) lower IRF8 expression was detected specifically in AML patients with reduced PU.1 levels; (3) restoration of IRF8 expression induced differentiation in IRF8 low expressing AML cells, and (4) a positive correlation of PU.1 and IRF8 expression was found in human leukemia stem cells, but not in healthy HSCs. Strikingly, comparative pathway analysis revealed a genome-wide molecular resemblance of preleukemic and leukemic UREΔ/+Msh2-/- mice with gene expression profiles from human MDS and AML patients, respectively. Our study demonstrates that minimal reduction of a key lineage-specific transcription factor that commonly occurs in human disease is sufficient to initiate cancer development and provides mechanistic insight into the formation and progression of preleukemic stem cells in MDS and AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Epigenetic down-regulation of the HIST1 locus predicts better prognosis in acute myeloid leukemia with NPM1 mutation

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Sylvain Garciaz ◽  
Lia N’guyen Dasi ◽  
Pascal Finetti ◽  
Christine Chevalier ◽  
Julien Vernerey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Expression Profiling ◽  
Myeloid Leukemia ◽  
Histone Genes ◽  
Npm1 Mutation ◽  
Epigenetic Machinery ◽  
Gene Expression Analyses ◽  
Lower Expression ◽  
Acute Myeloid

Abstract Background The epigenetic machinery is frequently altered in acute myeloid leukemia. Focusing on cytogenetically normal (CN) AML, we previously described an abnormal H3K27me3 enrichment covering 70 kb on the HIST1 cluster (6.p22) in CN-AML patient blasts. Here, we further investigate the molecular, functional, and prognosis significance of this epigenetic alteration named H3K27me3 HIST1 in NPM1-mutated (NPM1mut) CN-AML. Results We found that three quarter of the NPM1mut CN-AML patients were H3K27me3 HIST1high. H3K27me3 HIST1high group of patients was associated with a favorable outcome independently of known molecular risk factors. In gene expression profiling, the H3K27me3 HIST1high mark was associated with lower expression of the histone genes HIST1H1D, HIST1H2BG, HIST1H2AE, and HIST1H3F and an upregulation of genes involved in myelomonocytic differentiation. Mass spectrometry analyses confirmed that the linker histone protein H1d, but not the other histone H1 subtypes, was downregulated in the H3K27me3 HIST1high group of patients. H1d knockdown primed ATRA-mediated differentiation of OCI-AML3 and U937 AML cell lines, as assessed on CD11b/CD11c markers, morphological and gene expression analyses. Conclusions Our data suggest that NPM1mut AML prognosis depends on the epigenetic silencing of the HIST1 cluster and that, among the H3K27me3 silenced histone genes, HIST1H1D plays a role in AML blast differentiation. Graphical abstract

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