Study of the molecular and cellular mechanisms by which the paralogous transcription factors PAX3-FOXO1 and PAX7-FOXO1 exert their oncogenic activity

Le rhabdomyosarcome alvéolaire (RMSA) est un cancer pédiatrique rare des tissus mous causé par des réarrangements chromosomiques majeurs, incluant translocations, duplication du génome et perte ou amplification de pans de chromosomes. Le RMSA reste délétère pour les patients malgré l'utilisation de traitements complexes. Je me suis donc intéressé aux mécanismes moléculaires et cellulaires par lesquels les produits des translocations t(2,13) ou t(1,13) contribuent à la physiopathologie de ce cancer. Il s'agit de protéines chimères constituées des domaines de liaison à l'ADN de PAX3 ou PAX7, à savoir un paired (PD) et un homéodomaine (HD), et du domaine de transactivation de FOXO1. PAX3-FOXO1 et PAX7-FOXO1 sont des facteurs de transcription (FT) avec une capacité de transactivation significativement plus élevée que les PAX sauvages. Je me suis d'abord penchée sur un paradoxe révélé dans de nombreux types cellulaires. Malgré l'exceptionnel pouvoir de transactivation des PAX-FOXO1, leur potentiel de transformation maligne in vivo est faible. C'est également le cas dans le tube neural de l'embryon de poulet, modèle d'étude que nous avons utilisé. Dans ce modèle, les deux PAX-FOXO1 confèrent aux cellules neurales des caractéristiques moléculaires de RMSA, définies au préalable par des analyses transcriptomiques de biopsies et de lignées cellulaires de plusieurs types de RMS. Les PAX-FOXO1 conduisent également les cellules neurales épithéliales à adopter un phénotype mésenchymateux avec des propriétés invasives. En revanche, ils inhibent le cycle cellulaire. Cette inhibition peut être levée par surexpression d'activateurs des complexes CDK-cyclines. Cet impact négatif des PAX-FOXO1 sur la prolifération cellulaire fournit une explication assez simple de leur limitation apparente à induire une transformation maligne in vivo. J'ai ensuite entrepris une analyse structure-fonction de PAX3-FOXO1. Alors que les études génomiques montrent que son recrutement à l'ADN est principalement relayé par le PD, ce domaine n'est pas impliqué dans la délamination des cellules neurales induite par PAX3-FOXO1. Il est néanmoins requis pour l'induction de certains traits moléculaires associés au RMSA. A l'inverse, l'HD contribue à l'ensemble de l'activité de transformation de PAX3-FOXO1. Enfin, la transformation par PAX3-FOXO1 peut aussi être modulée en supprimant d'autres domaines protéiques, comme son extrémité N-terminale. Ainsi, notre étude fournit un faisceau de preuves montrant que la robustesse dans la génération d'un comportement oncogénique par un seul FT repose sur la complexité de sa structure protéique. Enfin, j'ai testé l'hypothèse d'une régulation différentielle de l'expression génique par PAX3-FOXO1 et PAX7-FOXO1, hypothèse qui pourrait expliquer des différences cliniques chez les patients portant la t(2,13) ou la t(1,13). À cette fin, j'ai créé des lignées inductibles de fibroblastes humains exprimant PAX3-FOXO1 ou PAX7-FOXO1. En plus d'une cohorte de gènes communément induits par ces deux FT, il existe des groupes de gènes dont l'expression est spécifiquement modulée par PAX3-FOXO1 ou PAX7-FOXO1, ce dernier induisant plus de gènes et à des niveaux plus forts. Grâce à la technique du 'CUT&Tag', nous avons montré que 30% des régions d'ADN sont spécifiquement liées par PAX3-FOXO1 ou PAX7-FOXO1 en raison d'une utilisation préférentielle respective de leur domaine PD ou HD. De plus, l'analyse du recrutement de la marque d'histone H3K27ac montre que PAX7-FOXO1 a une plus grande capacité à activer les régions enhanceurs que PAX3-FOXO1. Ces résultats à l'échelle du génome corrèlent avec des phénotypes de cycle cellulaire et de morphologie des fibroblastes spécifique à chaque PAX-FOXO1. Dans son ensemble cette étude soutient l'idée que les cellules à l'origine du RMSA interprètent de manière spécifique leur exposition à PAX3-FOXO1 ou à PAX7-FOXO1 ; ce qui pourrait sous-tendre l'hétérogénéité observée chez les patients.Alveolar rhabdomyosarcoma (ARMS) is a rare paediatric soft tissue cancer caused by extensive chromosomal rearrangements, including chromosomal translocations, genome duplication and loss or amplification of chromosome segments. ARMS remains deleterious to patients despite the use of complex treatments. I thus became interested in the molecular and cellular mechanisms by which the products of t(2,13) or t(1,13) chromosomal translocations contribute to the pathophysiology of ARMS, also named fusion positive RMS (FP-RMS). These products are chimeric proteins made of the DNA-binding domains of PAX3 or PAX7, including a paired (PD) and a homeodomain (HD), and the transactivation domain of FOXO1. PAX3-FOXO1 and PAX7-FOXO1 act as transcription factors (TFs) with a significantly higher transactivation capacity than the wild-type PAX. I first looked into a paradox revealed in many cell types. Despite the exceptional transactivation potential of PAX-FOXO1s, their malignant transformation potential in vivo is poor. This is also the case in the neural tube of chick embryos, the model we used to tackle this paradox. We showed that, there, both PAX-FOXO1s confer to neural cells the molecular traits of FP-RMS within 48hours. These traits were defined using the transcriptomes of FP-RMS patient biopsies and refined by profiling patient derived RMS cell lines. The PAX-FOXO1s also force the polarised neural epithelial cells to adopt a mesenchymal phenotype with invasive properties. However, we showed that the PAX-FOXO1s inhibit cell cycle. This inhibition can be lifted by overexpression of cell cycle regulators that promote the activity of CDK-cyclin complexes. This negative impact of the PAX-FOXO1s on cell proliferation provide a fairly simple explanation for their apparent limitation in inducing malignant transformation in vivo. I then undertook a structure-function analysis of PAX3-FOXO1 to identify the molecular basis underlying its potential for transcriptional transactivation and cell transformation. Intriguingly, while genomic studies indicate that the recruitment of this factor to DNA is mainly mediated by its PD, this domain is not required for PAX3-FOXO1 to induce the delamination of neural cells. Yet this domain is necessary for the induction of FP-RMS molecular traits in these cells. In contrast, the HD appeared to be necessary for both PAX3-FOXO1 mediated cell delamination and state transitions. We could also identify other functional domains such as the N-terminal part of PAX3-FOXO1 as key players in establishing robust changes of the neural epithelium morphology and molecular state. Thus, our study provides lines of evidence for the robustness in generating a tumorigenic behaviour by a single TF to stem from several structural domains. Finally, I tested the hypothesis of a differential regulation of gene expression by PAX3-FOXO1 and PAX7-FOXO1. This could explain why there are clinical differences associated with the presence of one or the other factor. For this purpose, I created inducible human fibroblast lines expressing PAX3-FOXO1 or PAX7-FOXO1. First, in addition to a cohort of commonly induced genes, there are sets of genes whose expression is specifically modulated by PAX3-FOXO1 or PAX7-FOXO1, the latter inducing a greater number of genes and at stronger levels. Using CUT&Tag, we showed that 30% of DNA regions are specifically bound by PAX3-FOXO1 or PAX7-FOXO1 due to a respective preferential use of the PD or HD domain. Furthermore, analysis of H3K27ac recruitment shows that PAX7-FOXO1 has a higher ability to activate enhancer regions than PAX3-FOXO1. Moreover, these factors confer distinct phenotypic properties to fibroblasts, such as different cell morphologies and growth potentials. All this argues for discrete interpretations by the cells of origin of FP-RMS of their exposure to PAX3-FOXO1 or PAX7-FOXO1, that in turn could underpin the heterogeneity observed in patients affected by these tumours

Divergent transcriptional and transforming properties of PAX3-FOXO1 and PAX7-FOXO1 paralogs

Abstract The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Both PAX-FOXO1s result in related cell transformation in animal models, but both mutations are associated with distinct pathological manifestations in patients. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 causes stronger de novo transactivation of its bound regions than PAX3-FOXO1, resulting in greater transcriptomic dynamics involving genes regulating cell shape and cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into M phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into S phase, and causes more genomic instabilities. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the transcriptional activities of PAX3-FOXO1 and PAX7-FOXO1. Furthermore, the identified pronounced deleterious effects of PAX7-FOXO1 provide an explanation for the low frequency of the translocation generating this factor in patients with rhabdomyosarcoma

Divergent transcriptional and transforming properties of PAX3-FOXO1 and PAX7-FOXO1 paralogs.

The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Overexpression of either PAX-FOXO1s results in related cell transformation in animal models. Yet, in patients the two structural genetic aberrations they derived from are associated with distinct pathological manifestations. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 binding results in greater recruitment of the H3K27ac activation mark than PAX3-FOXO1 binding and is accompanied by greater transcriptional activation of neighbouring genes. These effects are associated with a PAX-FOXO1-specific alteration in the expression of genes regulating cell shape and the cell cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into S phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into M phase, and causes increased DNA damage. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the genomic occupancy and transcriptional activity of PAX3-FOXO1 and PAX7-FOXO1

The Drosophila Fab-7 boundary modulates Abd-B gene activity by guiding an inversion of collinear chromatin organization and alternate promoter use

Summary: Hox genes encode transcription factors that specify segmental identities along the anteroposterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, a feature known as collinearity. In Drosophila, the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive changes in histone modifications, chromatin architecture, and use of boundary elements and cis-regulatory regions. To dissect functional hierarchies, we compare chromatin organization in cell lines and larvae, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary for insulation between 3D domains carrying different histone modifications. Interestingly, we detect a non-canonical inversion of collinear chromatin dynamics at Abd-B, with the domain of active histone modifications progressively decreasing in size. This dynamic chromatin organization differentially activates the alternative promoters of the Abd-B gene, thereby expanding the possibilities for fine-tuning of transcriptional output

The Drosophila Fab-7 boundary element modulates Abd-B gene activity in the genital disc by guiding an inversion of collinear chromatin organization and alternative promoter use

Hox genes encode transcription factors that specify segmental identities along the Antero-Posterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, an evolutionary conserved feature known as collinearity. In Drosophila , the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive replacement of histone modifications, reorganization of 3D chromatin architecture and sequential activation of boundary elements and cis -regulatory regions. To dissect functional hierarchies, we compared chromatin organization in larvae and in cell lines, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary element for insulation between 3D domains marked by different histone modifications. Interestingly, we detected a non-canonical inversion of collinear chromatin dynamics at the Abd-B gene, with the active histone domain decreasing in size. This chromatin organization differentially instructed alternative Abd-B promoter use, thereby expanding the possibilities to regulate transcriptional output

The PAX-FOXO1s trigger fast trans-differentiation of chick embryonic neural cells into alveolar rhabdomyosarcoma with tissue invasive properties limited by S phase entry inhibition

International audienceThe chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS

BMP2 and BMP7 cooperate with H3.3K27M to promote quiescence and invasiveness in pediatric diffuse midline gliomas

Pediatric diffuse midline gliomas (pDMG) are an aggressive type of childhood cancer with a fatal outcome. Their major epigenetic determinism has become clear, notably with the identification of K27M mutations in histone H3. However, the synergistic oncogenic mechanisms that induce and maintain tumor cell phenotype have yet to be deciphered. In 20 to 30% of cases, these tumors have an altered BMP signaling pathway with an oncogenic mutation on the BMP type I receptor ALK2, encoded by ACVR1. However, the potential impact of the BMP pathway in tumors non-mutated for ACVR1 is less clear. By integrating bulk, single-cell and spatial transcriptomic data, we show here that the BMP signaling pathway is activated at similar levels between ACVR1 wild type and mutant tumors and identify BMP2 and BMP7 as putative activators of the pathway in a specific subpopulation of cells. By using both pediatric isogenic glioma lines genetically modified to overexpress H3.3K27M and patients-derived DIPG cell lines, we demonstrate that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. These data suggest a generic oncogenic role for the BMP pathway in gliomagenesis of pDMG and pave the way for specific targeting of downstream effectors mediating the BMP/K27M crosstalk