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RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes.

Fleischmann KK, Pagel P, Schmid I, Roscher AA - Mol. Cancer (2014)

Bottom Line: The differential gene expression profile was validated for leukemia-association by gene set enrichment analysis of published gene sets from patient studies and MLL-AF9 overexpression studies and revealed 425 differentially expressed genes.Gene ontology analysis was consistent with a more differentiated state of MLL-AF9 depleted cells, with involvement of a wide range of downstream transcriptional regulators and with defined functional processes such as ribosomal biogenesis, chaperone binding, calcium homeostasis and estrogen response.Applying the antagonist SCH39166 against the dopamine receptor DRD5 resulted in reduced leukemic cell characteristics of THP1 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Children's Research Center, Division of Pediatric Hematology and Oncology, Dr, von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, Lindwurmstrasse 2a, München 80337, Germany. katrin.fleischmann@med.uni-muenchen.de.

ABSTRACT

Background: The translocation t(9;11)(p22;q23) leading to the leukemogenic fusion gene MLL-AF9 is a frequent translocation in infant acute myeloid leukemia (AML). This study aimed to identify genes and molecular processes downstream of MLL-AF9 (alias MLL-MLLT3) which could assist to develop new targeted therapies for such leukemia with unfavorable prognosis.

Methods: In the AML cell line THP1 which harbors this t(9;11) translocation, endogenous MLL-AF9 was silenced via siRNA while ensuring specificity of the knockdown and its efficiency on functional protein level.

Results: The differential gene expression profile was validated for leukemia-association by gene set enrichment analysis of published gene sets from patient studies and MLL-AF9 overexpression studies and revealed 425 differentially expressed genes. Gene ontology analysis was consistent with a more differentiated state of MLL-AF9 depleted cells, with involvement of a wide range of downstream transcriptional regulators and with defined functional processes such as ribosomal biogenesis, chaperone binding, calcium homeostasis and estrogen response. We prioritized 41 gene products as candidate targets including several novel and potentially druggable effectors of MLL-AF9 (AHR, ATP2B2, DRD5, HIPK2, PARP8, ROR2 and TAS1R3). Applying the antagonist SCH39166 against the dopamine receptor DRD5 resulted in reduced leukemic cell characteristics of THP1 cells.

Conclusion: Besides potential new therapeutic targets, the described transcription profile shaped by MLL-AF9 provides an information source into the molecular processes altered in MLL aberrant leukemia.

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Related in: MedlinePlus

SiRNA knockdown of MLL-AF9 in THP1 cells. (A) Alignments of siRNAs to MLL-AF9 mRNA. Bold sequence represents the MLL part of the fusion transcript. (B) Confirmation of MLL-AF9 knockdown via RT-qPCR. Mean relative MLL-AF9 and HOXA9 transcript levels of knockdown (siRNA-A, siRNA-B) as compared to control treatments (control siRNA-1, -2) during the time course of experiments. Graph represents data from five independent experiments. Bars indicate standard deviation. (C) Immunoblot of MLL-AF9 from total protein extracted from day 8 experimental samples. Normalized MLL-AF9 levels are indicated at the bottom.
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Figure 1: SiRNA knockdown of MLL-AF9 in THP1 cells. (A) Alignments of siRNAs to MLL-AF9 mRNA. Bold sequence represents the MLL part of the fusion transcript. (B) Confirmation of MLL-AF9 knockdown via RT-qPCR. Mean relative MLL-AF9 and HOXA9 transcript levels of knockdown (siRNA-A, siRNA-B) as compared to control treatments (control siRNA-1, -2) during the time course of experiments. Graph represents data from five independent experiments. Bars indicate standard deviation. (C) Immunoblot of MLL-AF9 from total protein extracted from day 8 experimental samples. Normalized MLL-AF9 levels are indicated at the bottom.

Mentions: To specifically target MLL-AF9 without disturbing MLL and AF9 wildtype expression levels, siRNAs were designed to target the THP1 specific fusion point of MLL-AF9 transcript (Figure 1A). Off-target effects were controlled by utilizing two different MLL-AF9 specific as well as two different non-targeting control siRNAs. An experimental setup with prolonged knockdown of MLL-AF9 (over 8 days) was chosen, because (1) the half-life of MLL-AF9 protein is unknown and (2) MLL-AF9 is believed to lead to changes in the expression of target genes via epigenetic mechanisms [7,8] whose reversal may take a long time [15] thus leading to a delayed effect on transcriptional level.


RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes.

Fleischmann KK, Pagel P, Schmid I, Roscher AA - Mol. Cancer (2014)

SiRNA knockdown of MLL-AF9 in THP1 cells. (A) Alignments of siRNAs to MLL-AF9 mRNA. Bold sequence represents the MLL part of the fusion transcript. (B) Confirmation of MLL-AF9 knockdown via RT-qPCR. Mean relative MLL-AF9 and HOXA9 transcript levels of knockdown (siRNA-A, siRNA-B) as compared to control treatments (control siRNA-1, -2) during the time course of experiments. Graph represents data from five independent experiments. Bars indicate standard deviation. (C) Immunoblot of MLL-AF9 from total protein extracted from day 8 experimental samples. Normalized MLL-AF9 levels are indicated at the bottom.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924703&req=5

Figure 1: SiRNA knockdown of MLL-AF9 in THP1 cells. (A) Alignments of siRNAs to MLL-AF9 mRNA. Bold sequence represents the MLL part of the fusion transcript. (B) Confirmation of MLL-AF9 knockdown via RT-qPCR. Mean relative MLL-AF9 and HOXA9 transcript levels of knockdown (siRNA-A, siRNA-B) as compared to control treatments (control siRNA-1, -2) during the time course of experiments. Graph represents data from five independent experiments. Bars indicate standard deviation. (C) Immunoblot of MLL-AF9 from total protein extracted from day 8 experimental samples. Normalized MLL-AF9 levels are indicated at the bottom.
Mentions: To specifically target MLL-AF9 without disturbing MLL and AF9 wildtype expression levels, siRNAs were designed to target the THP1 specific fusion point of MLL-AF9 transcript (Figure 1A). Off-target effects were controlled by utilizing two different MLL-AF9 specific as well as two different non-targeting control siRNAs. An experimental setup with prolonged knockdown of MLL-AF9 (over 8 days) was chosen, because (1) the half-life of MLL-AF9 protein is unknown and (2) MLL-AF9 is believed to lead to changes in the expression of target genes via epigenetic mechanisms [7,8] whose reversal may take a long time [15] thus leading to a delayed effect on transcriptional level.

Bottom Line: The differential gene expression profile was validated for leukemia-association by gene set enrichment analysis of published gene sets from patient studies and MLL-AF9 overexpression studies and revealed 425 differentially expressed genes.Gene ontology analysis was consistent with a more differentiated state of MLL-AF9 depleted cells, with involvement of a wide range of downstream transcriptional regulators and with defined functional processes such as ribosomal biogenesis, chaperone binding, calcium homeostasis and estrogen response.Applying the antagonist SCH39166 against the dopamine receptor DRD5 resulted in reduced leukemic cell characteristics of THP1 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Children's Research Center, Division of Pediatric Hematology and Oncology, Dr, von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, Lindwurmstrasse 2a, München 80337, Germany. katrin.fleischmann@med.uni-muenchen.de.

ABSTRACT

Background: The translocation t(9;11)(p22;q23) leading to the leukemogenic fusion gene MLL-AF9 is a frequent translocation in infant acute myeloid leukemia (AML). This study aimed to identify genes and molecular processes downstream of MLL-AF9 (alias MLL-MLLT3) which could assist to develop new targeted therapies for such leukemia with unfavorable prognosis.

Methods: In the AML cell line THP1 which harbors this t(9;11) translocation, endogenous MLL-AF9 was silenced via siRNA while ensuring specificity of the knockdown and its efficiency on functional protein level.

Results: The differential gene expression profile was validated for leukemia-association by gene set enrichment analysis of published gene sets from patient studies and MLL-AF9 overexpression studies and revealed 425 differentially expressed genes. Gene ontology analysis was consistent with a more differentiated state of MLL-AF9 depleted cells, with involvement of a wide range of downstream transcriptional regulators and with defined functional processes such as ribosomal biogenesis, chaperone binding, calcium homeostasis and estrogen response. We prioritized 41 gene products as candidate targets including several novel and potentially druggable effectors of MLL-AF9 (AHR, ATP2B2, DRD5, HIPK2, PARP8, ROR2 and TAS1R3). Applying the antagonist SCH39166 against the dopamine receptor DRD5 resulted in reduced leukemic cell characteristics of THP1 cells.

Conclusion: Besides potential new therapeutic targets, the described transcription profile shaped by MLL-AF9 provides an information source into the molecular processes altered in MLL aberrant leukemia.

Show MeSH
Related in: MedlinePlus