Genome-wide mapping of histone H3K9me2 in acute myeloid leukemia reveals large chromosomal domains associated with massive gene silencing and sites of genome instability

Drug resistance impedes the long-term effect of targeted therapies in acute myeloid leukemia (AML), necessitating the identification of mechanisms underlying resistance. Approximately 25% of AML patients carry FLT3 mutations and develop post-treatment insensitivity to FLT3 inhibitors, including sorafenib. Using a genome-wide CRISPR screen, we identified LZTR1, NF1, TSC1 or TSC2, negative regulators of the MAPK and MTOR pathways, as mediators of sorafenib resistance. Analyses of ex vivo drug sensitivity assays in FLT3-ITD AML patient samples revealed lower expression of LZTR1, NF1, and TSC2 correlated with sorafenib sensitivity. Importantly, MAPK and/or MTOR complex1 (MTORC1) activity were upregulated in AML cells made resistant to several FLT3 inhibitors, including crenolanib, quizartinib, or sorafenib. These cells were sensitive to MEK inhibitors, and the combination of FLT3 and MEK inhibitors showed enhanced efficacy, suggesting its effectiveness in AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors.

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Genome-wide haplotype association study identify the FGFR2 gene as a risk gene for Acute Myeloid Leukemia

Oncotarget ◽

10.18632/oncotarget.13631 ◽

2016 ◽

Vol 8 (5) ◽

pp. 7891-7899 ◽

Cited By ~ 6

Author(s):

Hongchao Lv ◽

Mingming Zhang ◽

Zhenwei Shang ◽

Jin Li ◽

Shanshan Zhang ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Association Study ◽

Myeloid Leukemia ◽

Haplotype Association ◽

Risk Gene ◽

Genome Wide ◽

Fgfr2 Gene ◽

Acute Myeloid

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Abstract 4763: Genome-wide CRISPR screening identifies TSC1 as a regulator of sorafenib resistance in acute myeloid leukemia

10.1158/1538-7445.am2019-4763 ◽

2019 ◽

Author(s):

Alisa Damnernsawad ◽

Tamilla Nechiporuk ◽

Steve E. Kurtz ◽

Wesley R. Horton ◽

Olga Nikolova ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Genome Wide ◽

Acute Myeloid

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Abstract 4763: Genome-wide CRISPR screening identifies TSC1 as a regulator of sorafenib resistance in acute myeloid leukemia

10.1158/1538-7445.sabcs18-4763 ◽

2019 ◽

Author(s):

Alisa Damnernsawad ◽

Tamilla Nechiporuk ◽

Steve E. Kurtz ◽

Wesley R. Horton ◽

Olga Nikolova ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Genome Wide ◽

Acute Myeloid

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Analysis of genome-wide methylation and gene expression induced by 5-aza-2′-deoxycytidine identifies BCL2L10 as a frequent methylation target in acute myeloid leukemia

Leukemia & Lymphoma ◽

10.3109/10428194.2010.528093 ◽

2010 ◽

Vol 51 (12) ◽

pp. 2275-2284 ◽

Cited By ~ 22

Author(s):

Emiliano Fabiani ◽

Giuseppe Leone ◽

Manuela Giachelia ◽

Francesco D'alo' ◽

Mariangela Greco ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Genome Wide ◽

Acute Myeloid

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Parallel Analyses Disclose Novel Genomic Imbalances in Acute Myeloid Leukemia with Complex Karyotypes.

Blood ◽

10.1182/blood.v106.11.2357.2357 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2357-2357

Author(s):

Frank G. Rucker ◽

Lars Bullinger ◽

Hans A. Kestler ◽

Peter Lichter ◽

Konstanze Dohner ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

High Resolution ◽

Candidate Genes ◽

Myeloid Leukemia ◽

Parallel Analysis ◽

Genomic Imbalances ◽

Genome Wide ◽

Genomic Regions ◽

Acute Myeloid

Abstract Approximately 10 to 15 % of acute myeloid leukemia (AML) cases exhibit complex karyotypes, i.e., three or more chromosome abnormalities without presence of a specific fusion transcript. To identify novel genomic regions of interest in AML with complex karyotypes we applied comparative genomic hybridization to microarrays (matrix-CGH) allowing high-resolution genome-wide screening of genomic imbalances. We designed a microarray consisting of 2799 different BAC- or PAC-vectors. A set of 1500 of these clones covers the whole human genome with a physical distance of approximately 1.5 Mb. The remaining 1299 clones either contiguously span genomic regions known to be frequently involved in hematologic malignancies (e.g., 1p, 2p, 3q, 7q, 9p, 11q, 12q, 13q, 17p, 18q) (n=600) or contain oncogenes or tumor suppressor genes (n=699). Using this microarray platform, 83 AML cases with complex karyotypes were analyzed. Genomic losses were found more frequently than gains; the most frequent losses were deletions of 5q (71%), 17p (53%), 7q (48%); followed by deletions of 18q (30%), 16q (28%), 3p and 12q (20% each), 12p (18%), 20q (17%), and 11q (12%). The most frequent genomic gains were trisomies of 11q (39%) and 8q (31%); followed by trisomies of 1p (22%), 21q (20%), 9p (14%), 22q (13%), 13q (12%), and 6p (10%). In part, some critical segments were delineated to genomic fragments of 0.8 to a few megabase pairs in size. In lost/gained regions parallel analysis of gene expression using microarray technology detected a gene dosage effect with significant lower/higher average gene expression levels across the genes located in the respective regions. Furthermore, 47 high-level DNA amplifications in 19 different regions were identified; amplifications occurring in at least two cases mapped to (candidate genes in the amplicon) 11q23.3-q24.1 (n=10; ETS, FLI1); 11q23.3 (n=8; MLL, DDX6); 21q22 (n=5; ERG, ETS2); 13q12 (n=3; CDX2, FLT1, FLT3, PAN3); 8q24 (n=3; C8FW, MYC); 9p24 (n=2; JAK2); 12p13 (n=2; FGF6, CCND2); and 20q11 (n=2; ID1, BCL2L1). Parallel analysis displayed overexpressed candidate genes in critical amplified region, e.g., C8FW and MYC in 8q24. In conclusion, using high-resolution genome-wide screening tools such as matrix-CGH allows to unravel the enormous genetic diversity of AML cases with complex karyotypes, and correlation with global gene expression studies facilitates the delineation of disease-related candidate genes located in the critical regions.

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RNAi-Based Functional Genomics Screening of the Human Kinome Identifies Major Growth Regulating Kinases in Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v112.11.2951.2951 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2951-2951

Author(s):

Raoul Tibes ◽

Ashish Choudhary ◽

Amanda Henrichs ◽

Sadia Guled ◽

Irma Monzon ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Acute Myeloid Leukemia ◽

Gene Silencing ◽

Functional Genomics ◽

Cell Lines ◽

Myeloid Leukemia ◽

Large Scale ◽

Cell Cycle Checkpoint ◽

Acute Myeloid

Abstract In order to improve treatment strategies for Acute Myeloid Leukemia (AML), we adapted a functional genomics approach using RNAi screening to identify molecular targets that are vital to the growth of AML. Herein we report the first large-scale kinome gene silencing screen in AML. A high throughput RNAi screen was developed for the efficient siRNA transfection of AML cell lines. Eight commercially available cationic lipid-based transfection reagents were tested for their ability to transfect several AML cell lines with siRNA. These extensive transfection optimization experiments identified two AML cells lines TF-1 and ML4 with up to 95–100 and 70–75% transfection efficiency respectively. Two independent replicate kinome screens were performed on both cell lines using a siRNA library targeting 572 kinase genes with 2 siRNA/gene. At 96 hours post transfection, cell proliferation was assessed and the B-score method was used to background correct and analyze the screening data. Several siRNA to specific kinases were identified that significantly inhibit cell proliferation of up to ~40–88%. Hits were defined at two thresholds: siRNA having a B-score of <−2 providing a statistically significance of p<0.05 (confidence of > 95%) and a cutoff B-score of <−1.5 providing greater than 87% confidence for each siRNA hit. Two different kinases (2 siRNA/gene/screen) were identified as major growth regulating kinases in TF1 cells with all 4 siRNA/gene having a B-score <−2. For two additional kinases, 3/4 siRNA for each gene had a Bscore <−2. Expanding the cutoff to a B-score <−1.5 three further kinases were targeted by at least 3/4 siRNA/gene. Similar analysis using the same criteria for ML4 cells identified one kinase targeted by 3/4 siRNA at a B-score <−2, seven kinases with 2/4 siRNA <−2 and two kinases with 3/4 siRNA/gene at a B-score of <−1.5. Common hits for both cell lines with at least 6/8 siRNA per gene from 4 screens performing at a B-score <−2 identified two kinases, one of them PLK1. Applying a B-score threshold of <−1.5, we identified five kinases for which at least 5/8 siRNA/gene from 4 screens met these criteria. Kinases/genes will be presented at the meeting.Confirmation of gene silencing and validation of growth response is currently underway for a subset of genes. Among the strongest hits are siRNA targeting PLK1, as well as siRNA targeting three other kinase-genes involved in regulating cell cycle progression and checkpoints and gene ontology (GO) analysis showed enrichment in cell cycle and cell cycle-checkpoint processes. Inhibitors against PLK1 and other kinase hits identified in the screen are in (pre)-clinical development and if confirmed, our experiments provide a strong rational to test these in AML. The application of RNAi based screening is useful in the identification of genes important in AML proliferation, which could serve as targets for therapeutic intervention and guide AML drug development. Furthermore, results from these types of functional genomics approaches hold promise to be rapidly translated into clinical application.

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Selective and Titratable Effects On AML Genome Wide Methylation Patterning, Transcription and Clonogenicity Using Very Low Concentrations of 5-Aza-2'deoxycytidine.

Blood ◽

10.1182/blood.v114.22.2385.2385 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2385-2385

Author(s):

Elisabeth Heuston ◽

Jason E. Farrar ◽

Timothy Triche ◽

Jonathan Buckley ◽

Poul Sorensen ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Cellular Reprogramming ◽

Transcription Profiles ◽

Genome Wide ◽

Low Concentrations ◽

Xenograft Models ◽

Methylation Patterns ◽

Acute Myeloid

Abstract Abstract 2385 Poster Board II-362 5-Aza-2'deoxycytidine (5AzadC) has significantly contributed to the treatment of myelodysplatic syndromes (MDS) and acute myeloid leukemia (AML). But while the cytotoxic effects of 5AzadC have been well characterized, its influence on methylation-induced cellular reprogramming remains poorly understood. We have treated several AML cell lines at extremely low concentrations of 5AzadC (0 nM to 1.0 nM) over the course of three days, followed by the determination of genome wide methylation changes, alterations in transcription profiles as well as cell viability, proliferation, apoptosis and changes in clonogenicity. The results demonstrate titratable responses on both genomic methylation and transcriptional patterns as well as a selective effect on clonogenicity compared to cytotoxicity. An alternative chemotherapeutic cytosine analog, cytosine arabinofuranoside (AraC), does not show the same selective depletion of clonogenic cells, suggesting that 5AzadC's effects are likely due to altered epigenetic changes associated with cellular reprogramming rather than a direct cytotoxic effect. We are currently evaluating 5AzadC and AraC effects on this population using immunophenotyping methods as well as xenograft models of tumorigenicity. These findings describe a potential role for very low concentrations of 5AzadC in treating acute myeloid leukemia through a selective affect on genome wide methylation patterns leading to altered transcription that differentially effects the clonogenic, leukemic stem cell compartment. Disclosures: No relevant conflicts of interest to declare.

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