22 results on '"Asoke K. Mal"'
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2. Supplementary Methods, Figure Legend from Small Molecule Inhibition of PAX3-FOXO1 through AKT Activation Suppresses Malignant Phenotypes of Alveolar Rhabdomyosarcoma
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Asoke K. Mal, Charles Keller, Munmun Mal, and Mathivanan Jothi
- Abstract
PDF file - 115K
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- 2023
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3. Supplementary Figure 2 from Small Molecule Inhibition of PAX3-FOXO1 through AKT Activation Suppresses Malignant Phenotypes of Alveolar Rhabdomyosarcoma
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Asoke K. Mal, Charles Keller, Munmun Mal, and Mathivanan Jothi
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PDF file - 1972K, Supplementary Figure S2. Screen of primary hits for the identification of PAX3-FOXO1 inhibitors.
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- 2023
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4. Data from Small Molecule Inhibition of PAX3-FOXO1 through AKT Activation Suppresses Malignant Phenotypes of Alveolar Rhabdomyosarcoma
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Asoke K. Mal, Charles Keller, Munmun Mal, and Mathivanan Jothi
- Abstract
Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma. Mol Cancer Ther; 12(12); 2663–74. ©2013 AACR.
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- 2023
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5. Supplementary Figure 3 from Small Molecule Inhibition of PAX3-FOXO1 through AKT Activation Suppresses Malignant Phenotypes of Alveolar Rhabdomyosarcoma
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Asoke K. Mal, Charles Keller, Munmun Mal, and Mathivanan Jothi
- Abstract
PDF file - 394K, Supplementary Figure S3. Thapsigargin but not dithiothreitol (DTT) decreases the expression levels of PAX3-FOXO1 protein in ARMS cells.
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- 2023
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6. Supplementary Figure 2 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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Asoke K. Mal, Andrei V. Gudkov, Mathivanan Jothi, and Min-Hyung Lee
- Abstract
Supplementary Figure 2 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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- 2023
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7. Supplementary Figure 6 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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Asoke K. Mal, Andrei V. Gudkov, Mathivanan Jothi, and Min-Hyung Lee
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Supplementary Figure 6 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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- 2023
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8. Supplementary Figure 4 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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Asoke K. Mal, Andrei V. Gudkov, Mathivanan Jothi, and Min-Hyung Lee
- Abstract
Supplementary Figure 4 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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- 2023
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9. Supplementary Figure 5 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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Asoke K. Mal, Andrei V. Gudkov, Mathivanan Jothi, and Min-Hyung Lee
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Supplementary Figure 5 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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- 2023
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10. Supplementary Figure 3 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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Asoke K. Mal, Andrei V. Gudkov, Mathivanan Jothi, and Min-Hyung Lee
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Supplementary Figure 3 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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- 2023
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11. Supplementary Figure Legends 1-6 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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Asoke K. Mal, Andrei V. Gudkov, Mathivanan Jothi, and Min-Hyung Lee
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Supplementary Figure Legends 1-6 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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- 2023
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12. Data from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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Asoke K. Mal, Andrei V. Gudkov, Mathivanan Jothi, and Min-Hyung Lee
- Abstract
Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric muscle cancer, which arrested during the process of skeletal muscle differentiation. In muscle myoblast cells, ectopic expression of the histone H3 lysine 9 (H3K9) methytransferase KMT1A blocks differentiation by repressing a myogenic gene expression program. In this study, we tested the hypothesis that activation of a KMT1A-mediated program of transcriptional repression prevents ARMS cells from differentiating. We investigated whether KMT1A represses the expression of differentiation-associated genes in ARMS cells, thereby blocking muscle differentiation. Our results show that expression of KMT1A is induced in human ARMS cancer cell lines when cultured under differentiation-permissible conditions. shRNA-mediated knockdown of KMT1A decreased anchorage dependent and independent cell proliferation and tumor xenograft growth, increased expression of differentiation-associated genes, and promoted the appearance of a terminally differentiated-like phenotype. Finally, shRNA-directed KMT1A knockdown restored the impaired transcriptional activity of the myogenic regulator MyoD. Together, our results suggested that high levels of KMT1A in ARMS cells under differentiation conditions impairs MyoD function, thereby arresting myogenic differentiation in these tumor cells. Thus, targeting KMT1A may be a novel strategy for the treatment of this disease. Cancer Res; 71(11); 3921–31. ©2011 AACR.
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- 2023
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13. Supplementary Figure 1 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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Asoke K. Mal, Andrei V. Gudkov, Mathivanan Jothi, and Min-Hyung Lee
- Abstract
Supplementary Figure 1 from Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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- 2023
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14. Camptothecin exhibits topoisomerase1-independent KMT1A suppression and myogenic differentiation in alveolar rhabdomyosarcoma cells
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Min-Hyung Lee, Fengzhi Li, David W. Wolff, Asoke K. Mal, Munmun Mal, and Mathivanan Jothi
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0301 basic medicine ,Chemistry ,Myogenesis ,camptothecin ,MyoD ,medicine.disease ,3. Good health ,03 medical and health sciences ,030104 developmental biology ,Oncology ,Downregulation and upregulation ,Histone methyltransferase ,Cancer research ,medicine ,Alveolar rhabdomyosarcoma ,Myocyte ,methyltransferase ,rhabdomyosarcoma ,myogenesis ,Rhabdomyosarcoma ,neoplasms ,Camptothecin ,Research Paper ,medicine.drug - Abstract
Alveolar rhabdomyosarcoma (aRMS) is an aggressive subtype of the most common soft tissue cancer in children. A hallmark of aRMS tumors is incomplete myogenic differentiation despite expression of master myogenic regulators such as MyoD. We previously reported that histone methyltransferase KMT1A suppresses MyoD function to maintain an undifferentiated state in aRMS cells, and that loss of KMT1A is sufficient to induce differentiation and suppress malignant phenotypes in these cells. Here, we develop a chemical compound screening approach using MyoD-responsive luciferase reporter myoblast cells to identify compounds that alleviate suppression of MyoD-mediated differentiation by KMT1A. A screen of pharmacological compounds yielded the topoisomerase I (TOP1) poison camptothecin (CPT) as the strongest hit in our assay system. Furthermore, treatment of aRMS cells with clinically relevant CPT derivative irinotecan restores MyoD function, and myogenic differentiation in vitro and in a xenograft model. This differentiated phenotype was associated with downregulation of the KMT1A protein. Remarkably, loss of KMT1A in CPT-treated cells occurs independently of its well-known anti-TOP1 mechanism. We further demonstrate that CPT can directly inhibit KMT1A activity in vitro. Collectively, these findings uncover a novel function of CPT that downregulates KMT1A independently of CPT-mediated TOP1 inhibition and permits differentiation of aRMS cells.
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- 2018
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15. Small Molecule Inhibition of PAX3-FOXO1 through AKT Activation Suppresses Malignant Phenotypes of Alveolar Rhabdomyosarcoma
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Charles Keller, Mathivanan Jothi, Munmun Mal, and Asoke K. Mal
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Cancer Research ,SERCA ,Thapsigargin ,Oncogene Proteins, Fusion ,Transcription, Genetic ,Antineoplastic Agents ,Apoptosis ,Biology ,Article ,Calcium in biology ,Cell Line ,Small Molecule Libraries ,Mice ,chemistry.chemical_compound ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Paired Box Transcription Factors ,Phosphorylation ,Protein kinase B ,Rhabdomyosarcoma, Alveolar ,Endoplasmic reticulum ,musculoskeletal system ,medicine.disease ,Xenograft Model Antitumor Assays ,Fusion protein ,Tumor Burden ,Enzyme Activation ,Gene Expression Regulation, Neoplastic ,Cell Transformation, Neoplastic ,Phenotype ,Oncology ,chemistry ,Proteolysis ,embryonic structures ,Cancer research ,Alveolar rhabdomyosarcoma ,Calcium ,Drug Screening Assays, Antitumor ,Proto-Oncogene Proteins c-akt ,Protein Binding - Abstract
Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma. Mol Cancer Ther; 12(12); 2663–74. ©2013 AACR.
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- 2013
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16. p38α MAPK disables KMT1A-mediated repression of myogenic differentiation program
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David W. Wolff, Biswanath Chatterjee, Munmun Mal, Mathivanan Jothi, and Asoke K. Mal
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p38α ,0301 basic medicine ,Cellular differentiation ,KMT1A ,Biology ,MyoD ,Cell Line ,Mitogen-Activated Protein Kinase 14 ,Myoblasts ,Mice ,03 medical and health sciences ,MyoD Protein ,Animals ,Humans ,Orthopedics and Sports Medicine ,Phosphorylation ,Promoter Regions, Genetic ,Molecular Biology ,Skeletal muscle differentiation ,Myogenin ,PITX2 ,Myogenesis ,Research ,Cell Differentiation ,Methyltransferases ,Cell Biology ,musculoskeletal system ,Molecular biology ,Repressor Proteins ,030104 developmental biology ,tissues ,Chromatin immunoprecipitation ,C2C12 ,Signal Transduction - Abstract
Background Master transcription factor MyoD can initiate the entire myogenic gene expression program which differentiates proliferating myoblasts into multinucleated myotubes. We previously demonstrated that histone methyltransferase KMT1A associates with and inhibits MyoD in proliferating myoblasts, and must be removed to allow differentiation to proceed. It is known that pro-myogenic signaling pathways such as PI3K/AKT and p38α MAPK play critical roles in enforcing associations between MyoD and transcriptional activators, while removing repressors. However, the mechanism which displaces KMT1A from MyoD, and the signals responsible, remain unknown. Methods To investigate the role of p38α on MyoD-mediated differentiation, we utilized C2C12 myoblast cells as an in vitro model. p38α activity was either augmented via overexpression of a constitutively active upstream kinase or blocked via lentiviral delivery of a specific p38α shRNA or treatment with p38α/β inhibitor SB203580. Overexpression of KMT1A in these cells via lentiviral delivery was also used as a system wherein terminal differentiation is impeded by high levels of KMT1A. Results The association of KMT1A and MyoD persisted, and differentiation was blocked in C2C12 myoblasts specifically after pharmacologic or genetic blockade of p38α. Conversely, forced activation of p38α was sufficient to activate MyoD and overcome the differentiation blockade in KMT1A-overexpressing C2C12 cells. Consistent with this finding, KMT1A phosphorylation during C2C12 differentiation correlated strongly with the activation of p38α. This phosphorylation was prevented by the inhibition of p38α. Biochemical studies further revealed that KMT1A can be a direct substrate for p38α. Importantly, chromatin immunoprecipitation (ChIP) studies show that the removal of KMT1A-mediated transcription repressive histone tri-methylation (H3K9me3) from the promoter of the Myogenin gene, a critical regulator of muscle differentiation, is dependent on p38α activity in C2C12 cells. Elevated p38α activity was also sufficient to remove this repressive H3K9me3 mark. Moreover, ChIP studies from C2C12 cells show that p38α activity is necessary and sufficient to establish active H3K9 acetylation on the Myogenin promoter. Conclusions Activation of p38α displaces KMT1A from MyoD to initiate myogenic gene expression upon induction of myoblasts differentiation. Electronic supplementary material The online version of this article (doi:10.1186/s13395-016-0100-z) contains supplementary material, which is available to authorized users.
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- 2016
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17. AKT and PAX3-FKHR cooperation enforces myogenic differentiation blockade in alveolar rhabdomyosarcoma cell
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Kochi Nishijo, Charles Keller, Asoke K. Mal, and Mathivanan Jothi
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Oncogene Proteins, Fusion ,Transcription, Genetic ,Cellular differentiation ,Biology ,MyoD ,Mice ,MyoD Protein ,Report ,Cell Line, Tumor ,medicine ,Animals ,Gene Knock-In Techniques ,Phosphorylation ,RNA, Small Interfering ,Molecular Biology ,Protein kinase B ,Rhabdomyosarcoma, Alveolar ,PI3K/AKT/mTOR pathway ,Cell Nucleus ,Regulation of gene expression ,Skeletal muscle ,Cell Differentiation ,Cell Biology ,musculoskeletal system ,Disease Models, Animal ,medicine.anatomical_structure ,embryonic structures ,Cancer research ,RNA Interference ,Proto-Oncogene Proteins c-akt ,Developmental Biology - Abstract
The chimeric PAX3-FKHR transcription factor is present in a majority of alveolar rhabdomyosarcoma (ARMS), an aggressive skeletal muscle cancer of childhood. PAX3-FKHR-mediated aberrant myogenic gene expression resulting in escape from terminal differentiation program is believed to contribute in ARMS development. In skeletal muscle differentiation, activation of AKT pathway leads to myogenic gene activation and terminal differentiation. Here, we report that AKT acts, in part, by modulating PAX3-FKHR transcriptional activity via phosphorylation in the maintenance of the myogenic differentiation blockade in established mouse models of ARMS cells. We observed that low levels of AKT activity are associated with elevated levels of PAX3-FKHR transcriptional activity, and AKT hyperactivation results in PAX3-FKHR phosphorylation coupled with decreased activity once cells are under differentiation-permissible conditions. Subsequent data shows that attenuated AKT activity-associated PAX3-FKHR activity is required to suppress the function of MyoD, a key myogenic regulator of muscle differentiation. Conversely, decreased PAX3-FKHR activity results in the eradication of MyoD expression and subsequent suppression of the myogenic differentiation. Thus, AKT regulation of the PAX3- FKHR suppresses myogenic gene expression in ARMS cells, causing a failure in differentiation. Evidence is presented that provides a novel molecular link between AKT and PAX3-FKHR in maintaining myogenic differentiation blockade in ARMS.
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- 2012
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18. Histone Methyltransferase KMT1A Restrains Entry of Alveolar Rhabdomyosarcoma Cells into a Myogenic Differentiated State
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Asoke K. Mal, Min-Hyung Lee, Andrei V. Gudkov, and Mathivanan Jothi
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Cancer Research ,Cellular differentiation ,Transplantation, Heterologous ,Cell Growth Processes ,Biology ,MyoD ,Article ,Mice ,MyoD Protein ,Transduction, Genetic ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Myocyte ,RNA, Small Interfering ,Child ,Promoter Regions, Genetic ,Rhabdomyosarcoma, Alveolar ,Myogenin ,Regulation of gene expression ,Cell Differentiation ,Histone-Lysine N-Methyltransferase ,medicine.disease ,Gene Expression Regulation, Neoplastic ,Oncology ,Gene Knockdown Techniques ,Histone Methyltransferases ,Cancer research ,Alveolar rhabdomyosarcoma ,Ectopic expression - Abstract
Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric muscle cancer, which arrested during the process of skeletal muscle differentiation. In muscle myoblast cells, ectopic expression of the histone H3 lysine 9 (H3K9) methytransferase KMT1A blocks differentiation by repressing a myogenic gene expression program. In this study, we tested the hypothesis that activation of a KMT1A-mediated program of transcriptional repression prevents ARMS cells from differentiating. We investigated whether KMT1A represses the expression of differentiation-associated genes in ARMS cells, thereby blocking muscle differentiation. Our results show that expression of KMT1A is induced in human ARMS cancer cell lines when cultured under differentiation-permissible conditions. shRNA-mediated knockdown of KMT1A decreased anchorage dependent and independent cell proliferation and tumor xenograft growth, increased expression of differentiation-associated genes, and promoted the appearance of a terminally differentiated-like phenotype. Finally, shRNA-directed KMT1A knockdown restored the impaired transcriptional activity of the myogenic regulator MyoD. Together, our results suggested that high levels of KMT1A in ARMS cells under differentiation conditions impairs MyoD function, thereby arresting myogenic differentiation in these tumor cells. Thus, targeting KMT1A may be a novel strategy for the treatment of this disease. Cancer Res; 71(11); 3921–31. ©2011 AACR.
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- 2011
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19. p300- and Myc-mediated regulation of glioblastoma multiforme cell differentiation
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Sreejith P. Panicker, Baisakhi Raychaudhuri, Pankaj Sharma, Russell Tipps, Tapati Mazumdar, Asoke K. Mal, Juan M. Palomo, Michael A. Vogelbaum, and S. Jaharul Haque
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Transcription, Genetic ,GBM stem cell ,Blotting, Western ,Genes, myc ,Fluorescent Antibody Technique ,Mice, Nude ,p300 ,Nerve Tissue Proteins ,Myc ,urologic and male genital diseases ,glioblastoma multiforme (GBM) ,Nestin ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Intermediate Filament Proteins ,Neural Stem Cells ,Cell Line, Tumor ,Glial Fibrillary Acidic Protein ,Animals ,Humans ,Neoplasm Invasiveness ,Cell Proliferation ,030304 developmental biology ,0303 health sciences ,urogenital system ,Cell Differentiation ,differentiation ,Cell Dedifferentiation ,invasion ,Research Papers ,female genital diseases and pregnancy complications ,nervous system diseases ,Gene Expression Regulation, Neoplastic ,Oncology ,030220 oncology & carcinogenesis ,Neoplastic Stem Cells ,RNA Interference ,Glioblastoma ,E1A-Associated p300 Protein - Abstract
Tumorigenic potential of glioblastoma multiforme (GBM) cells is, in part, attributable to their undifferentiated (neural stem cell-like) phenotype. Astrocytic differentiation of GBM cells is associated with transcriptional induction of Glial Fibrillary Acidic Protein (GFAP) and repression of Nestin, whereas the reciprocal transcription program operates in undifferentiated GBM cells. The molecular mechanisms underlying the regulation of these transcription programs remain elusive. Here, we show that the transcriptional co-activator p300 was expressed in GBM tumors and cell lines and acted as an activator of the GFAP gene and a repressor of the Nestin gene. On the other hand, Myc (formerly known as c-Myc overrode these p300 functions by repressing the GFAP gene and inducing the Nestin gene in GBM cells. Moreover, RNAi-mediated inhibition of p300 expression significantly enhanced the invasion potential of GBM cells in vitro. Taken together, these data suggest that dedifferentiated/undifferentiated GBM cells are more invasive than differentiated GBM cells. Because invasion is a major cause of GBM morbidity, differentiation therapy may improve the clinical outcome.
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- 2010
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20. Too much AKT turns PAX3-FKHR dead: A prospect of novel therapeutic strategy for alveolar rhabdomyosarcoma
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Asoke K. Mal and Mathivanan Jothi
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medicine.medical_specialty ,Oncogene Proteins, Fusion ,Cellular differentiation ,AKT1 ,Antineoplastic Agents ,Cell Differentiation ,Biology ,medicine.disease_cause ,Disease Models, Animal ,Editorial ,Endocrinology ,Oncology ,Differentiation therapy ,Internal medicine ,Cancer cell ,Cancer research ,medicine ,Animals ,Humans ,Phosphorylation ,Carcinogenesis ,Proto-Oncogene Proteins c-akt ,Protein kinase B ,Rhabdomyosarcoma, Alveolar ,PI3K/AKT/mTOR pathway - Abstract
Alveolar rhabdomyosarcoma (ARMS) is the aggressive subtype of muscle cancer in the pediatric population and children with ARMS have significantly poor prognosis and frequently present metastasis [1]. More than 80% of ARMS have chromosomal translocation t(2;13) generated novel fusion transcription factor PAX3-FKHR in which the DNA binding domain of PAX3 is fused to the carboxyl terminus of FKHR [1]. PAX3-FKHR protein acts as a potent transcriptional activator and influences expression of genes that ultimately contribute to its oncogenic behavior by modifying cell growth, migration, apoptosis, and muscle differentiation [1]. Fusion-positive PAX3-FKHR status in patients correlates with worse outcome and displays a more aggressive clinical phenotype that is consistent with cell lines and mouse models of ARMS [1]. Therefore, inhibiting PAX3-FKHR activity, a potential Achilles' heel, is a sound therapeutic strategy to eliminate ARMS cells that have benefit to children with this disease. ARMS exhibit undifferentiated muscle lineage phenotype and resistance to terminal differentiation [1]. PAX3-FKHR activity sustains the undifferentiated state by suppressing differentiation, thus contributing ARMS tumorigenesis [1]. In this scenario, PAX3-FKHR exerts a negative regulation on key myogenic regulator MyoD-driven terminal differentiation in ARMS cells [1]. We investigated the molecular regulation of PAX3-FKHR activity to evade terminal differentiation in ARMS cells. Intriguingly, we discover that activated AKT status in ARMS cells balance the transcriptional activity of PAX3-FKHR via phosphorylation. In particular, we find that attenuated AKT activity sustains PAX3-FKHR activity, however, acute AKT activation blocks PAX3-FKHR in gene activation including its bona fide target MyoD expression, preventing ARMS cell from escaping differentiation. However, acute AKT activation along with ectopic MyoD expression permits ARMS cells to undergo terminal differentiation. We recently published this finding in Cell Cycle Journal [2]. AKT downstream effector GSK mediated phosphorylation of PAX3-FKHR increases its transcriptional activity and is inhibited by GSK specific inhibitors [3], raising the possibility that AKT mediated inhibition of PAX3-FKHR activity either directly or via GSK. PAX3-FKHR retains two consensus AKT phosphorylation sites in the FKHR domain [2]. Mutation of the AKT sites in PAX3-FKHR results in increased transcriptional activity (personal communication), strongly suggesting that AKT directly regulating PAX3-FKHR activity. Activation of AKT acts as a promyogenic signal for terminal differentiation (Fig. (Fig.1).1). Conversely, AKT activation has been linked to both oncogenesis and poor prognosis and consequently AKT inhibitors that limit AKT signals are being developed as cancer therapeutics [4]. However, inhibiting AKT increases cancer cell motility and invasion abilities that are important for tumor metastasis. Indeed, expression of activated AKT1 in ErbB2 background enhances tumorigenesis, but shows fewer metastatic lesions compared with control mice [5], indicating that AKT1 functions as a suppressor of tumor metastasis. The anti-metastatic function of AKT was also reported in breast cancer cells [4]. Moreover, AKT allosteric inhibitor MK-2206 showed limited in vivo activity against solid tumor and ALL xenografts [6]. Thus, there are pressing concerns of inhibiting AKT for therapeutic benefit in some cancers. Figure 1 This model represents the action of AKT on PAX3-FKHR activity in ARMS and terminal muscle differentiation Our study revealed the unexpected finding that elevating AKT activity can inactivate PAX3-FKHR in ARMS cells [2]. This is important because it shows that acute activation of AKT inactivates PAX3-FKHR activity known to be involved in development and metastasis of ARMS. This new discovery poses a host of new questions and challenges that activator-induced AKT activation may hinder PAX3-FKHR activity, thus offer a therapeutic benefit to ARMS. Dilling et al. showed that cell lines derived from childhood ARMS are very sensitive to the growth-inhibitory effects of the rapamycin [7]. Rapamycin induced feedback activation of AKT is well documented [8]. Therefore, sensitivity of ARMS cells to rapamycin may result in AKT activation induced inhibition of PAX3-FKHR activity. In a similar way, activation of AKT may provide a therapeutic benefit in other cancers. In this case, we may envision the potential benefit of AKT activation by TNF-α, which is used in the regional treatment of locally advanced soft tissue sarcomas, metastatic melanomas and other unresectable tumors [9]. Constitutive PI3K/AKT activation in AML patients showed a better prognosis [10], indicating a potential function of AKT activation in clinical success of cancer treatment. A characteristic abnormality of ARMS cells is a blockade of terminal differentiation. A strategy of inducing ARMS cells to differentiate, termed ‘differentiation therapy’ is an elegant alternative that could limit the current toxic therapeutic approach. The bi-transgenic AKT1 and ErbB-2 developed mammary tumors exhibit more differentiated phenotypes than ErbB2 alone [5]. This study suggests that AKT could promote tumor differentiation in certain setting. PAX3-FKHR abolition in ARMS cells led to terminal differentiation [1], therefore, it is worth considering the potential role of AKT activation-dependent inactivation of PAX3-FKHR in this effort (Fig. (Fig.1).1). Taken together, we venture that acute AKT activation should be evaluated for therapeutic benefit in ARMS and also in certain types of tumor.
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- 2012
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21. Abstract LB-20: Transcriptionally inactive PAX3-FKHR fails to induce ARMS cells differentiation
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Charles Keller, Asoke K. Mal, and Mathivanan Jothi
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Regulation of gene expression ,Cancer Research ,PAX3 ,Skeletal muscle ,Biology ,musculoskeletal system ,medicine.disease_cause ,Cell biology ,Transactivation ,medicine.anatomical_structure ,Oncology ,embryonic structures ,Immunology ,medicine ,Progenitor cell ,Carcinogenesis ,Protein kinase B ,PI3K/AKT/mTOR pathway - Abstract
Alveolar rhabdomyosarcoma (ARMS) is an aggressive muscle cancer of childhood and believed to arise from committed muscle progenitor cells that have arrested during the process of skeletal muscle differentiation. One of the most established genetic signatures in ARMS is the presence of chimeric aberrant transcription factor PAX3-FKHR generated by fusion of PAX3 and FKHR genes. The stronger transcriptional activity of this fusion PAX3-FKHR protein has previously been attributed to the deregulation of genes, which are known to be associated with tumorigenesis and suppression of skeletal muscle differentiation. Currently, we are investigating the relative contribution of PAX3-FKHR's transcriptional activity in abrogating ARMS cells differentiation using mouse ARMS cells as an in vitro model differentiation readout system. Here, we show that transactivation potential of PAX3-FKHR is declined in mouse ARMS cells that have been induced to differentiate by growing these cells in mitogens-deficient in vitro differentiation-permissible condition. Moreover, this diminished activity of PAX3-FKHR is not due to the alteration of its sub-cellular localization or changes in the level of either mRNA or protein in these ARMS cells. Results also show the lack of muscle-specific gene activation in these ARMS cells despite the reduced transcriptional activity of PAX3-FKHR under condition of differentiation. We also observed that decrease in transcriptional activity of PAX3-FKHR is coincided with the activation of PI3/AKT pathway. The results further show that blockade of PI3/AKT pathway sustains the transcriptional activity of PAX3-FKHR whereas forced activation of AKT inhibits this activity. Together, our results signify that transcriptionally inactive PAX3-FKHR by AKT is incompetent to induce mouse ARMS cells differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-20.
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- 2010
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22. Small molecule inhibition of PAX3-FOXO1 through AKT activation suppresses malignant phenotypes of alveolar rhabdomyosarcoma.
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Jothi M, Mal M, Keller C, and Mal AK
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- Animals, Antineoplastic Agents pharmacology, Apoptosis drug effects, Calcium metabolism, Cell Line, Cell Line, Tumor, Cell Transformation, Neoplastic drug effects, Drug Screening Assays, Antitumor, Enzyme Activation drug effects, Gene Expression Regulation, Neoplastic drug effects, Humans, Mice, Oncogene Proteins, Fusion metabolism, Paired Box Transcription Factors metabolism, Phenotype, Phosphorylation drug effects, Protein Binding, Proteolysis, Small Molecule Libraries, Thapsigargin pharmacology, Transcription, Genetic, Tumor Burden drug effects, Xenograft Model Antitumor Assays, Oncogene Proteins, Fusion antagonists & inhibitors, Paired Box Transcription Factors antagonists & inhibitors, Proto-Oncogene Proteins c-akt metabolism, Rhabdomyosarcoma, Alveolar metabolism, Rhabdomyosarcoma, Alveolar pathology
- Abstract
Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma., (©2013 AACR.)
- Published
- 2013
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