Project description:To identify potential target genes of leukemia-related miRNAs such as miR-196b and miR-150 in human MLL-associated leukemia, we performed Affymetrix Human Exon 1.0 ST array assay of 15 human MLL-associated samples and 9 human normal bone marrow (including 3 each of CD34+, CD33+, and MNC) cell samples.
Project description:Chronic lymphocytic leukemia shows a variable clinical course which is associated with distinct alterations such as chromosomal aberrations, gene mutations or IGHV mutation status. Refining biologic categories may help to improve clinical management with available and future treatment approaches. We used Affymetrix Exon 1.0 ST microarrays to investigate differences in CLL biology.
Project description:Chronic lymphocytic leukemia shows a variable clinical course which is associated with distinct alterations such as chromosomal aberrations, gene mutations or IGHV mutation status. Refining biologic categories may help to improve clinical management with available and future treatment, lymphoma/leukemia celllines and non-tumour cells of the microenvironment can be used for models to better categorize biologic CLL entities. We used Affymetrix Exon 1.0 ST microarrays to investigate differences in CLL biology.
Project description:The recently released Affymetrix Human Gene 1.0 ST array has two major differences compared with standard 3'™ based arrays: (1) it interrogates the entire mRNA transcript, and (2) it uses cDNA targets. To assess the impact of these differences on array performance, we performed series of comparative hybridizations between the Human Gene 1.0 ST and the Affymetrix HG-U133 Plus 2.0 and the Illumina HumanRef-8 BeadChip arrays. Additionally, both cRNA and cDNA targets were probed on the HG-U133 Plus 2.0 array. The results show that the overall reproducibility is best using the Gene 1.0 ST array. When looking only at the high intensity probes, the reproducibility of the Gene 1.0 ST array and the Illumina BeadChip array is equally good. Concordance of array results was assessed using different inter-platform mappings. The Gene 1.0 ST is most concordant with the HG-U133 array hybridized with cDNA targets, thus showing the impact of the target type. Agreements are better between platforms with designs which choose probes from the 3' end of the gene. Overall, the high degree of correspondence provides strong evidence for the reliability of the Gene 1.0 ST array. Keywords: Cross-platform comparison
Project description:To identify potential target genes of leukemia-related miRNAs such as miR-196b and miR-150 in human MLL-associated leukemia, we performed Affymetrix Human Exon 1.0 ST array assay of 15 human MLL-associated samples and 9 human normal bone marrow (including 3 each of CD34+, CD33+, and MNC) cell samples. A total of 15 MLL-associated (9 primary untreated leukemia and 6 leukemia cell line) samples and 9 human normal bone marrow samples (including 3 each of CD34+ hematopoietic stem/progenitor, CD33+ myeloid, and mononuclear cell (MNC) samples) were analyzed by use of Affymetrix Human Exon 1.0 ST arrays (Affymetirx, Santa Clara, CA).
Project description:Background: The oncogenic protein HOXA9 plays a critical role in leukemia transformation and maintenance, and its aberrant expression is a hallmark of most aggressive acute leukemia. Although inhibiting the upstream regulators of HOXA9 has been proven as a significant therapeutic intervention, the comprehensive regulation network controlling HOXA9 expression in leukemia has not been systematically investigated. Results: Here, we perform genome-wide CRISPR/Cas9 screening in the HOXA9-driven reporter acute leukemia cells. We identify a poorly characterized RNA-binding protein, RBM5, as the top candidate gene required to maintain leukemia cell fitness. RBM5 is highly overexpressed in acute myeloid leukemia (AML) patients compared to healthy individuals. RBM5 loss triggered by CRISPR knockout and shRNA knockdown significantly impairs leukemia maintenance in vitro and in vivo. Through domain CRISPR screening, we reveal that RBM5 functions through a noncanonical transcriptional regulation circuitry rather than RNA splicing, such an effect depending on DNA-binding domains. By integrative analysis and functional assays, we identify HOXA9 as the downstream target of RBM5. Ectopic expression of HOXA9 rescues impaired leukemia cell proliferation upon RBM5 loss. Importantly, acute protein degradation of RBM5 through auxin-inducible degron system immediately reduces HOXA9 transcription. Conclusions: We identify RBM5 as a new upstream regulator of HOXA9 and reveal its essential role in controlling the survival of AML. These functional and molecular mechanisms further support RBM5 as a promising therapeutic target for myeloid leukemia treatment. Keywords: Acute myeloid leukemia; CRISPR screen; Genome editing; HOXA9; RBM5.
Project description:Acute myeloid leukemia (AML) is a type of heterogeneous and fatal hematopoietic malignancy. The ten-eleven translocation (TET) mediated DNA demethylation is known to be critically associated with AML pathogenesis. Through chemical compound screening, we found that the opioid receptor agonist, loperamide hydrochloride (OPA1), significantly suppresses AML cell viability. The potential therapeutic effects of opioid receptor agonists, especially OPA1, were then verified in AML cells in vitro, and t(11q23) and t(8;21) AML mouse models in vivo. OPA1-induced activation of OPRM1 enhanced the transcription of TET2, increased DNA 5-hydroxymethylcytosine (5hmC) modification, and in turn, activated NFκB signaling. Notably, AML with TET2 mutations or chemotherapy resistance were highly sensitive to OPA1. Our results reveal a previously unknown OPRM1-TET2-5hmC-TRAF2 regulatory axis in AML, and suggest that opioid agonists, particularly OPA1, an FDA-approved antidiarrheal drug, have therapeutic potential in AML, especially in TET2 mutated and chemotherapy-resistant AML, which have a poor prognosis.
