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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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THP1 cell characteristics influenced by 10 μM of the dopamine receptor antagonist SCH39166 compared to DMSO control. (A) Proliferation in serum reduced conditions. (B) Colony formation in methyl cellulose. Images show representative wells of DMSO control (left) and 10 μM SCH39166 treatment (right). (C) Cell cycle distribution in serum reduced conditions. (D) Cell migration in 5 μm transwells. (E) DNA new synthesis rate measured via EdU incorporation and SYTOX AADvanced nuclear stain. Ratio of SCH39166 compared to DMSO control treatment is shown. Mean over three (A-C) or two (D-E) independent experiments are shown. Bars indicate standard error of the mean; *p < 0.05. † Non significant differences because of interexperimental fluorescence intensity standard deviation, although the ratio between the two treatments remains similar.
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Figure 7: THP1 cell characteristics influenced by 10 μM of the dopamine receptor antagonist SCH39166 compared to DMSO control. (A) Proliferation in serum reduced conditions. (B) Colony formation in methyl cellulose. Images show representative wells of DMSO control (left) and 10 μM SCH39166 treatment (right). (C) Cell cycle distribution in serum reduced conditions. (D) Cell migration in 5 μm transwells. (E) DNA new synthesis rate measured via EdU incorporation and SYTOX AADvanced nuclear stain. Ratio of SCH39166 compared to DMSO control treatment is shown. Mean over three (A-C) or two (D-E) independent experiments are shown. Bars indicate standard error of the mean; *p < 0.05. † Non significant differences because of interexperimental fluorescence intensity standard deviation, although the ratio between the two treatments remains similar.

Mentions: Treatment of THP1 cells with 10 μM SCH39166 led to a number of significant changes in cell characteristics: (1) proliferation and (2) colony forming capacity were reduced, (3) cell cycle analysis revealed alternating changes in G1 and S phase distributions which may suggest a slowdown of cell cycle progression, (4) cells were slower to reach G0/G1 phase after DNA new synthesis which was reduced and (5) the cell migration rate was decreased (Figure 7). No increase in the rate of apoptotic THP1 cells was observed (Additional file 3: Figure S3).


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

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

THP1 cell characteristics influenced by 10 μM of the dopamine receptor antagonist SCH39166 compared to DMSO control. (A) Proliferation in serum reduced conditions. (B) Colony formation in methyl cellulose. Images show representative wells of DMSO control (left) and 10 μM SCH39166 treatment (right). (C) Cell cycle distribution in serum reduced conditions. (D) Cell migration in 5 μm transwells. (E) DNA new synthesis rate measured via EdU incorporation and SYTOX AADvanced nuclear stain. Ratio of SCH39166 compared to DMSO control treatment is shown. Mean over three (A-C) or two (D-E) independent experiments are shown. Bars indicate standard error of the mean; *p < 0.05. † Non significant differences because of interexperimental fluorescence intensity standard deviation, although the ratio between the two treatments remains similar.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: THP1 cell characteristics influenced by 10 μM of the dopamine receptor antagonist SCH39166 compared to DMSO control. (A) Proliferation in serum reduced conditions. (B) Colony formation in methyl cellulose. Images show representative wells of DMSO control (left) and 10 μM SCH39166 treatment (right). (C) Cell cycle distribution in serum reduced conditions. (D) Cell migration in 5 μm transwells. (E) DNA new synthesis rate measured via EdU incorporation and SYTOX AADvanced nuclear stain. Ratio of SCH39166 compared to DMSO control treatment is shown. Mean over three (A-C) or two (D-E) independent experiments are shown. Bars indicate standard error of the mean; *p < 0.05. † Non significant differences because of interexperimental fluorescence intensity standard deviation, although the ratio between the two treatments remains similar.
Mentions: Treatment of THP1 cells with 10 μM SCH39166 led to a number of significant changes in cell characteristics: (1) proliferation and (2) colony forming capacity were reduced, (3) cell cycle analysis revealed alternating changes in G1 and S phase distributions which may suggest a slowdown of cell cycle progression, (4) cells were slower to reach G0/G1 phase after DNA new synthesis which was reduced and (5) the cell migration rate was decreased (Figure 7). No increase in the rate of apoptotic THP1 cells was observed (Additional file 3: Figure S3).

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