The Experts below are selected from a list of 93 Experts worldwide ranked by ideXlab platform
Marco Wachtel - One of the best experts on this subject based on the ideXlab platform.
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PAX3 foxo1 establishes myogenic super enhancers and confers bet bromodomain vulnerability
Cancer Discovery, 2017Co-Authors: Berkley E Gryder, Marco Wachtel, Marielle E Yohe, Hsienchao Chou, Xiaohu Zhang, Joana G Marques, Beat W Schaefer, Nirmalya Sen, Young K Song, Alberto GualtieriAbstract:Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric Transcription Factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the cis-regulatory landscape by inducing de novo super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master Transcription Factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.Significance: PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8); 884-99. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.
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abstract 700 characterization of the mode of action of fenretinide treatment in alveolar rhabdomyosarcoma cells
Cancer Research, 2017Co-Authors: Eva Brack, Marco Wachtel, Beat W SchaeferAbstract:Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by a specific chromosomal translocation encoding the oncogenic Transcription Factor PAX3-FOXO1. As aRMS cells are addicted to the tumor-specific fusion protein, it may serve as an ideal therapeutic target. Previously, we have identified from a large drug library screen the compound Fenretinide (retinoic acid p-hydroxyanilide), which is already in clinical use, to affect both PAX3-FOXO1 expression as well as aRMS cell viability. The aim of this study was therefore to characterize the mode of action of Fenretinide in more detail. First, we were able to show that Fenretinide induced the generation of reactive oxygen species (ROS) in mitochondria. A more detailed characterization revealed that the Fenretinide-induced ROS derived from an interaction of Fenretinide around complex II of the mitochondrial respiratory chain, leading to the production of superoxides. ROS scavenging as well as complexing of iron ions completely abolished cell death. To identify the mode of cell death involved, we then used a range of pharmacological inhibitors of specific cell death pathways including Z-vad (pan -caspase inhibitor), Necrostatin-1 (necroptosis pathway inhibitor (RIP-1 kinase inhibitor)), 3-Methyadenine (3-MA) (autophagy pathway inhibitor (phosphatidylinositol 3-kinase inhibitor)) and Ferrostatin (ferroptosis pathway inhibitor) during Fenretinide treatment. Surprisingly, none of these inhibitors alone was able to prevent cell death and even different combinations were not sufficient to completely inhibit cell death. CRISPR/Cas9 mediated depletion of key players in the apoptotic and necroptotic pathway (Bak, Bax and RIPK1) confirmed the pharmacological data. We therefore conclude that other, less characterized cell death pathways or a combination of several pathways including apoptosis and necroptosis might be crucial. Interestingly, electron microscopic examination of cells pointed towards an excessive accumulation of vacuoles to be characteristic. Taken together, our data show that Fenretinide shows high potential for the treatment of aRMS, inducing several forms of cell death mediated through the production of ROS. These properties open the search for additional compounds acting in a combinatorial manner. Citation Format: Eva Brack, Marco Wachtel, Beat W. Schaefer. Characterization of the mode of action of Fenretinide treatment in alveolar rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2017-700
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abstract pr10 the chromatin remodeler chd4 as a potential specific target for alveolar rhabdomyosarcoma therapy
Cancer Research, 2016Co-Authors: Joana Marques, Marco Wachtel, Maria Boehm, Beat W SchaeferAbstract:Fusion-positive alveolar rhabdomyosarcoma (FP-RMS) is a paediatric tumour driven by an oncogenic fusion Transcription Factor, PAX3-FOXO1. Conventional chemotherapy is only effective for low risk patients which carry no metastasis, achieving a 5-year overall survival of 65%. The unique presence of this fusion protein in FP-RMS as well as the tumour cell survival dependency on PAX3-FOXO1 make this Transcription Factor a promising target for therapy. However, due to the difficulties associated with drug development targeting Transcription Factors, we performed a combined proteomic and genetic screen to identify new druggable co-regulators of PAX3-FOXO1 Transcriptional activity. Interactor candidates were defined by mass spectrometry analysis of proteins co-purified with the fusion protein and individually validated for their relevance for PAX3-FOXO1 activity through siRNA silencing. The chromodomain-DNA-binding protein 4 (CHD4), a nucleosome remodeler and core member of the NuRD complex (Nucleosome Remodelling and Deacetylase), was identified as an essential positive co-regulator of PAX3-FOXO1 Transcriptional activity. ChIP-qPCR experiments demonstrated that CHD4 not only co-localizes with PAX3-FOXO1 in the FP-RMS genome but also it is necessary for the binding of the fusion protein to cis-regulatory sites for a subset of its target genes. Consequently, CHD4 silencing affected the expression of more than 50% of PAX3-FOXO1 regulated target genes. Additionally, depletion of CHD4 impaired FP-RMS cell proliferation and caused a complete regression of xenograft tumours in mice. Moreover, CHD4 silencing had no effect in cell proliferation of human myoblasts or fibroblasts, suggesting a unique tumour dependency on this chromatin remodeler. In summary, our data propose that CHD4 has a crucial role as a co-regulator of PAX3-FOXO1 driven gene expression whose presence is required for FP-RMS cell viability. To our knowledge, CHD4 is the first identified chromatin remodeler associated with PAX3-FOXO1 Transcriptional activity, thus highlighting the relevance of epigenetic regulation in FP-RMS tumour development. Collectively, our findings suggest CHD4 as a potential novel therapeutic target in this childhood malignancy, and are motivating ongoing work aimed at finding first-in-class small molecules to inhibit CHD4. This abstract is also presented as Poster A20. Citation Format: Joana Marques, Maria Boehm, Marco Wachtel, Beat Schaefer. The chromatin remodeler CHD4 as a potential specific target for alveolar rhabdomyosarcoma therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR10.
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phosphorylation regulates Transcriptional activity of PAX3 fkhr and reveals novel therapeutic possibilities
Cancer Research, 2008Co-Authors: Ralf Amstutz, Marco Wachtel, Felix Niggli, Heinz Troxler, Peter Kleinert, Margret Ebauer, Torsten Haneke, Christoph Oehlerjanne, Doriano Fabbro, Beat W SchaferAbstract:Inhibition of constitutive active signaling pathways, which are a characteristic phenomenon for many tumors, can be an effective therapeutic strategy. In contrast, oncogenic Transcription Factors, often activated by mutational events, are in general less amenable to small-molecule inhibition despite their obvious importance as therapeutic targets. One example of this is alveolar rhabdomyosarcoma (aRMS), in which specific translocations lead to the formation of the chimeric Transcription Factor PAX3/FKHR. Here, we found unexpectedly that the Transcriptional activity of PAX3/FKHR can be inhibited by the kinase inhibitor PKC412. This occurs via specific phosphorylation sites in the PAX3 domain, phosphorylation of which is required for efficient DNA-binding and subsequent Transcriptional activity. Consequently, we show that PKC412 exerts a potent antitumorigenic potential for aRMS treatment both in vitro and in vivo. Our study suggests that postTranscriptional modifications of oncogenic Transcription Factors can be explored as a promising avenue for targeted cancer therapy.
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comparative expression profiling identifies an in vivo target gene signature with tfap2b as a mediator of the survival function of PAX3 fkhr
Oncogene, 2007Co-Authors: M Ebauer, Marco Wachtel, Felix Niggli, Beat W SchaferAbstract:The chromosomal translocation t(2;13), characteristic for the aggressive childhood cancer alveolar rhabdomyosarcoma (aRMS), generates the chimeric Transcription Factor PAX3/FKHR with a well known oncogenic role. However, the molecular mechanisms mediating essential pathophysiological functions remain poorly defined. Here, we used comparative expression profiling of PAX3/FKHR silencing in vitro and PAX3/FKHR-specific gene signatures in vivo to identify physiologically important target genes. Hereby, 51 activated genes, both novel and known, were identified. We also found repression of skeletal muscle-specific genes suggesting that PAX3/FKHR blocks further differentiation of aRMS cells. Importantly, TFAP2B was validated as direct target gene mediating the anti-apoptotic function of PAX3/FKHR. Hence, we developed a pathophysiologically relevant Transcriptional profile of PAX3/FKHR and identified a critical target gene for aRMS development.
Beat W Schaefer - One of the best experts on this subject based on the ideXlab platform.
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PAX3 foxo1 establishes myogenic super enhancers and confers bet bromodomain vulnerability
Cancer Discovery, 2017Co-Authors: Berkley E Gryder, Marco Wachtel, Marielle E Yohe, Hsienchao Chou, Xiaohu Zhang, Joana G Marques, Beat W Schaefer, Nirmalya Sen, Young K Song, Alberto GualtieriAbstract:Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric Transcription Factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the cis-regulatory landscape by inducing de novo super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master Transcription Factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.Significance: PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8); 884-99. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.
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abstract 700 characterization of the mode of action of fenretinide treatment in alveolar rhabdomyosarcoma cells
Cancer Research, 2017Co-Authors: Eva Brack, Marco Wachtel, Beat W SchaeferAbstract:Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by a specific chromosomal translocation encoding the oncogenic Transcription Factor PAX3-FOXO1. As aRMS cells are addicted to the tumor-specific fusion protein, it may serve as an ideal therapeutic target. Previously, we have identified from a large drug library screen the compound Fenretinide (retinoic acid p-hydroxyanilide), which is already in clinical use, to affect both PAX3-FOXO1 expression as well as aRMS cell viability. The aim of this study was therefore to characterize the mode of action of Fenretinide in more detail. First, we were able to show that Fenretinide induced the generation of reactive oxygen species (ROS) in mitochondria. A more detailed characterization revealed that the Fenretinide-induced ROS derived from an interaction of Fenretinide around complex II of the mitochondrial respiratory chain, leading to the production of superoxides. ROS scavenging as well as complexing of iron ions completely abolished cell death. To identify the mode of cell death involved, we then used a range of pharmacological inhibitors of specific cell death pathways including Z-vad (pan -caspase inhibitor), Necrostatin-1 (necroptosis pathway inhibitor (RIP-1 kinase inhibitor)), 3-Methyadenine (3-MA) (autophagy pathway inhibitor (phosphatidylinositol 3-kinase inhibitor)) and Ferrostatin (ferroptosis pathway inhibitor) during Fenretinide treatment. Surprisingly, none of these inhibitors alone was able to prevent cell death and even different combinations were not sufficient to completely inhibit cell death. CRISPR/Cas9 mediated depletion of key players in the apoptotic and necroptotic pathway (Bak, Bax and RIPK1) confirmed the pharmacological data. We therefore conclude that other, less characterized cell death pathways or a combination of several pathways including apoptosis and necroptosis might be crucial. Interestingly, electron microscopic examination of cells pointed towards an excessive accumulation of vacuoles to be characteristic. Taken together, our data show that Fenretinide shows high potential for the treatment of aRMS, inducing several forms of cell death mediated through the production of ROS. These properties open the search for additional compounds acting in a combinatorial manner. Citation Format: Eva Brack, Marco Wachtel, Beat W. Schaefer. Characterization of the mode of action of Fenretinide treatment in alveolar rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2017-700
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abstract pr10 the chromatin remodeler chd4 as a potential specific target for alveolar rhabdomyosarcoma therapy
Cancer Research, 2016Co-Authors: Joana Marques, Marco Wachtel, Maria Boehm, Beat W SchaeferAbstract:Fusion-positive alveolar rhabdomyosarcoma (FP-RMS) is a paediatric tumour driven by an oncogenic fusion Transcription Factor, PAX3-FOXO1. Conventional chemotherapy is only effective for low risk patients which carry no metastasis, achieving a 5-year overall survival of 65%. The unique presence of this fusion protein in FP-RMS as well as the tumour cell survival dependency on PAX3-FOXO1 make this Transcription Factor a promising target for therapy. However, due to the difficulties associated with drug development targeting Transcription Factors, we performed a combined proteomic and genetic screen to identify new druggable co-regulators of PAX3-FOXO1 Transcriptional activity. Interactor candidates were defined by mass spectrometry analysis of proteins co-purified with the fusion protein and individually validated for their relevance for PAX3-FOXO1 activity through siRNA silencing. The chromodomain-DNA-binding protein 4 (CHD4), a nucleosome remodeler and core member of the NuRD complex (Nucleosome Remodelling and Deacetylase), was identified as an essential positive co-regulator of PAX3-FOXO1 Transcriptional activity. ChIP-qPCR experiments demonstrated that CHD4 not only co-localizes with PAX3-FOXO1 in the FP-RMS genome but also it is necessary for the binding of the fusion protein to cis-regulatory sites for a subset of its target genes. Consequently, CHD4 silencing affected the expression of more than 50% of PAX3-FOXO1 regulated target genes. Additionally, depletion of CHD4 impaired FP-RMS cell proliferation and caused a complete regression of xenograft tumours in mice. Moreover, CHD4 silencing had no effect in cell proliferation of human myoblasts or fibroblasts, suggesting a unique tumour dependency on this chromatin remodeler. In summary, our data propose that CHD4 has a crucial role as a co-regulator of PAX3-FOXO1 driven gene expression whose presence is required for FP-RMS cell viability. To our knowledge, CHD4 is the first identified chromatin remodeler associated with PAX3-FOXO1 Transcriptional activity, thus highlighting the relevance of epigenetic regulation in FP-RMS tumour development. Collectively, our findings suggest CHD4 as a potential novel therapeutic target in this childhood malignancy, and are motivating ongoing work aimed at finding first-in-class small molecules to inhibit CHD4. This abstract is also presented as Poster A20. Citation Format: Joana Marques, Maria Boehm, Marco Wachtel, Beat Schaefer. The chromatin remodeler CHD4 as a potential specific target for alveolar rhabdomyosarcoma therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR10.
Beat W Schafer - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation regulates Transcriptional activity of PAX3 fkhr and reveals novel therapeutic possibilities
Cancer Research, 2008Co-Authors: Ralf Amstutz, Marco Wachtel, Felix Niggli, Heinz Troxler, Peter Kleinert, Margret Ebauer, Torsten Haneke, Christoph Oehlerjanne, Doriano Fabbro, Beat W SchaferAbstract:Inhibition of constitutive active signaling pathways, which are a characteristic phenomenon for many tumors, can be an effective therapeutic strategy. In contrast, oncogenic Transcription Factors, often activated by mutational events, are in general less amenable to small-molecule inhibition despite their obvious importance as therapeutic targets. One example of this is alveolar rhabdomyosarcoma (aRMS), in which specific translocations lead to the formation of the chimeric Transcription Factor PAX3/FKHR. Here, we found unexpectedly that the Transcriptional activity of PAX3/FKHR can be inhibited by the kinase inhibitor PKC412. This occurs via specific phosphorylation sites in the PAX3 domain, phosphorylation of which is required for efficient DNA-binding and subsequent Transcriptional activity. Consequently, we show that PKC412 exerts a potent antitumorigenic potential for aRMS treatment both in vitro and in vivo. Our study suggests that postTranscriptional modifications of oncogenic Transcription Factors can be explored as a promising avenue for targeted cancer therapy.
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comparative expression profiling identifies an in vivo target gene signature with tfap2b as a mediator of the survival function of PAX3 fkhr
Oncogene, 2007Co-Authors: M Ebauer, Marco Wachtel, Felix Niggli, Beat W SchaferAbstract:The chromosomal translocation t(2;13), characteristic for the aggressive childhood cancer alveolar rhabdomyosarcoma (aRMS), generates the chimeric Transcription Factor PAX3/FKHR with a well known oncogenic role. However, the molecular mechanisms mediating essential pathophysiological functions remain poorly defined. Here, we used comparative expression profiling of PAX3/FKHR silencing in vitro and PAX3/FKHR-specific gene signatures in vivo to identify physiologically important target genes. Hereby, 51 activated genes, both novel and known, were identified. We also found repression of skeletal muscle-specific genes suggesting that PAX3/FKHR blocks further differentiation of aRMS cells. Importantly, TFAP2B was validated as direct target gene mediating the anti-apoptotic function of PAX3/FKHR. Hence, we developed a pathophysiologically relevant Transcriptional profile of PAX3/FKHR and identified a critical target gene for aRMS development.
Margaret Buckingham - One of the best experts on this subject based on the ideXlab platform.
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Skeletal Muscle Differentiation of Embryonic Mesoangioblasts Requires PAX3 Activity
Stem cells (Dayton Ohio), 2009Co-Authors: Graziella Messina, Silvia Brunelli, Frederic Relaix, Beatriz G. Gálvez, Dario Sirabella, Stefania Monteverde, Rossana Tonlorenzi, Esther Schnapp, Luciana De Angelis, Margaret BuckinghamAbstract:Mesoangioblasts have been characterized as a population of vessel-associated stem cells able to differentiate into several mesodermal cell types, including skeletal muscle. Here, we report that the paired box Transcription Factor PAX3 plays a crucial role in directing mouse mesoangioblasts toward skeletal myogenesis in vitro and in vivo. Mesoangioblasts isolated from the aorta of PAX3 null embryos are severely impaired in skeletal muscle differentiation, whereas most other differentiation programs are not affected by the absence of PAX3. Moreover, PAX3(-/-) null mesoangioblasts failed to rescue the myopathic phenotype of the alpha-sarcoglycan mutant mouse. In contrast, mesoangioblasts from PAX3 gain of function, PAX3(PAX3-FKHR/+), mice display enhanced myogenesis in vitro and are more efficient in regenerating new muscle fibers in this model of muscular dystrophy. These data demonstrate that PAX3 is required for the differentiation of mesoangioblast stem cells into skeletal muscle, in keeping with its role in orchestrating entry into the myogenic program.
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Skeletal muscle differentiation of embryonic mesoangioblasts requires PAX3 activity
'Alphamed Press', 2009Co-Authors: Graziella Messina, Silvia Brunelli, Frederic Relaix, Beatriz G. Gálvez, Dario Sirabella, Stefania Monteverde, Rossana Tonlorenzi, Esther Schnapp, Luciana De Angelis, Margaret BuckinghamAbstract:Mesoangioblasts have been characterized as a population of vessel-associated stem cells able to differentiate into several mesodermal cell types, including skeletal muscle. Here, we report that the paired box Transcription Factor PAX3 plays a crucial role in directing mouse mesoangioblasts toward skeletal myogenesis in vitro and in vivo. Mesoangioblasts isolated from the aorta of PAX3 null embryos are severely impaired in skeletal muscle differentiation, whereas most other differentiation programs are not affected by the absence of PAX3. Moreover, PAX3-/- null mesoangioblasts failed to rescue the myopathic phenotype of the α-sarcoglycan mutant mouse. In contrast, mesoangioblasts from PAX3 gain of function, PAX3PAX3-FKHR/+, mice display enhanced myogenesis in vitro and are more efficient in regenerating new muscle fibers in this model of muscular dystrophy. These data demonstrate that PAX3 is required for the differentiation of mesoangioblast stem cells into skeletal muscle, in keeping with its role in orchestrating entry into the myogenic program. ©AlphaMed Press
Berkley E Gryder - One of the best experts on this subject based on the ideXlab platform.
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PAX3 foxo1 establishes myogenic super enhancers and confers bet bromodomain vulnerability
Cancer Discovery, 2017Co-Authors: Berkley E Gryder, Marco Wachtel, Marielle E Yohe, Hsienchao Chou, Xiaohu Zhang, Joana G Marques, Beat W Schaefer, Nirmalya Sen, Young K Song, Alberto GualtieriAbstract:Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric Transcription Factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the cis-regulatory landscape by inducing de novo super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master Transcription Factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.Significance: PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8); 884-99. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.
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abstract pr16 targeting the chromatin architecture established by PAX3 foxo1 in rhabdomyosarcoma
Cancer Research, 2016Co-Authors: Berkley E Gryder, Marielle E Yohe, Hsienchao Chou, Jack F Shern, Young Song, Rajesh Patidar, Sivasish Sindiri, Abigail Cleveland, Hongling Liao, Xinyu WenAbstract:Master Transcription Factors establish enhancers to regulate cell identity genes by recruiting epigenetic machinery, and are sequentially exchanged during changes in cell identity (ie, differentiation). Commonly, the fusion of Transcription Factors profoundly alters proper progression of cell identity, serving as the signature oncogenic event in many malignancies. The most common soft tissue cancer of childhood, rhabdomyosarcoma (RMS), is characterized by an inability to exit the proliferative myoblast-like state, presumably by blocking myogenic Transcription Factors from advancing the active enhancer landscape. This is achieved by either chromosomal translocation resulting in the oncogenic fusion Transcription Factor PAX3/7-FOXO1 (Fusion-Positive alveolar subtype, FP-RMS) or mutations in the tyrosine kinase/RAS/PIK3C axis (Fusion-Negative embryonal subtype, FN-RMS). Patients who harbor a PAX-fusion typically relapse despite aggressive therapy and have very poor survival. Here we hypothesized that the PAX3-FOXO1 fusion gene causes epigenetic reprogramming resulting in increased proliferation and a failure to terminally differentiate. Furthermore we hypothesized that disrupting the epigenetic machinery recruited by this fusion gene would provide a tractable target for therapy. We mapped the landscape of epigenetic alterations caused by the PAX3-FOXO1 fusion gene using a combination of RNA-seq, DNase hypersensitivity, and ChIP-seq against histone marks and Transcription Factors in cell lines and models of FP-RMS. We found high expression of several master Transcription Factors (including MYOD1, MYOG, MYCN, and SOX8) in FP-RMS primary tumors and cell lines, resembling human skeletal muscle myoblasts. ChIP-seq revealed that PAX3-FOXO1 is exclusively bound to distal, active enhancers and the histone modification most enriched surrounding PAX3-FOXO1 was acetylated H3K27. Furthermore we found that the introduction of the fusion gene into fibroblast cells opened up the chromatin at these same sites, completely rewiring the active enhancer landscape, recapitulating a transcriptome locked in a myoblast-like state. Genome-wide profiling of MYOD1, MYOG and MYCN reveals that all three master regulators collaborative bind at nearly every PAX3-FOXO1 driven super enhancer (SE), while typical enhancers (TEs) rarely have more than two of these four. PAX3-FOXO1 has a 7-fold preference for SEs over TEs. We also find that PAX3-FOXO1 bound, myogenic enhancers are decommissioned throughout normal skeletal muscle differentiation. To identify small molecules that would inhibit the PAX3-FOXO1 induced epigenetic machinery we treated a panel of FP-RMS cell lines with 1912 targeted agents and chemical probes at multiple concentrations and measured cell viability. Classes of molecules selectively potent for PAX3-FOXO1 driven cells (as compared to normal fibroblasts) hit connected biologically relevant targets including SE controlled receptor tyrosine kinases (including FGFR4, IGF1R, ALK), and Transcriptional coFactors involved in SE complexes (including HDACs and BRD). In an expanded panel of RMS cell lines we confirmed that FP-RMS is selectively sensitive to the BET bromodomain inhibitors with the most potent being JQ1. These inhibitors selectively suppress PAX3-FOXO1 dependent Transcription as measured by reporter assays and RNA-seq analysis. Indeed, coactivators of looped chromatin p300, MED1 and BRD4 excessively co-localize with PAX3-FOXO1 genome wide. In vivo , JQ1 selectively ablated PAX3-FOXO1 dependent SE driven Transcription, and significantly delayed tumor progression in xenografts of PAX3-FOXO1 driven cell lines. In conclusion we found that PAX3-FOXO1 establishes myogenic super enhancers that are sensitive to BET bromodomain inhibition which constitutes a novel therapeutic strategy for children with PAX-fusion driven rhabdomyosarcoma. This abstract is also presented as Poster A16. Citation Format: Berkley E. Gryder, Marielle E. Yohe, Jack Shern, Hsien-Chao Chou, Young Song, Rajesh Patidar, Sam Li, Sivasish Sindiri, Abigail Cleveland, Hongling Liao, Xinyu Wen, Xiaohu Zhang, Lesley Mathews-Griner, Rajarshi Guha, Paul Shinn, Marc Ferrer, Scott Martin, Madhu Lal, Craig Thomas, Javed Khan. Targeting the chromatin architecture established by PAX3-FOXO1 in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR16.