The team Biology of pediatric leukemia is part of the UMR 1170 Molecular dynamics of hematopoietic transformation and part of the national network CONECT-AML.
Pediatric cancers affect about 1/600 child and represent the second cause of death in western countries. The hematological malignancies account for approximately 45% of the pediatric cancers. Clinical features of these pediatric cancers suggest that they have different bases compared to the corresponding adult cancers. However, the precise molecular mechanisms of transformation and the bases for the exclusive association between several genetic alterations and pediatric cancers are poorly understood.
The overall goal of our studies is to identify the molecular bases of patient with hematological malignancies in order to understand the mechanisms of transformation and develop novel therapeutic strategies. For this purpose, we perform high-throughput sequencing analyses to identify the genetic landscape of leukemia and perform functional analyses through the development of cellular and preclinical in vivo models (e.g. transgenic, patient-derived xenotransplantation, CRISPR/Cas9 approaches).
Functional genetics of acute megakaryoblastic leukemia
Our work on leukemia of the megakaryocyte lineage giving rise to blood platelets, called acute megakaryoblastic leukemia (AML-M7 or AMKL) and associated with poor response to treatment and poor prognosis, has contributed to the identification of several recurrent mutations and fusion oncogenes involving gene expression regulators (e.g. OTT-MAL and ETO2-GLIS2) (Mercher et al, PNAS 2001; Thiollier et al, JEM 2012).
Functional studies showed that the OTT-MAL fusion aberrantly activates the Notch signaling pathway (Mercher et al. JCI 2009), which plays a role in the normal development of the erythro-megakaryocytic lineage (Mercher et al. Cell Stem Cell 2008), but the expression of OTT-MAL alone does not efficiently induce AMKL in mice. Among the other most prevalent mutation in AMKL, we investigated the mutations in signaling proteins, such as MPL, the thrombopoietin receptor, or JAK kinases (~15-20% of AMKL)(Mercher et al. Blood 2006, Walters et al. Cancer Cell 2006, Gu et al. Blood 2007). In mice, co-expression of OTT-MAL with an MPL mutant led to the first model of AMKL with a short latency (Mercher et al. JCI 2009).
De novo pediatric AMKL presenting the ETO2-GLIS2 fusion oncogene is associated with dismal prognosis. Only a few additional mutations have been identified in this subgroup, suggesting that ETO2-GLIS2 may be sufficient for leukemogenesis. We have recently developed models (e.g. CRISPR/Cas9, inducible transgenic) to perform cellular and molecular characterizations (e.g. RNAseq, ATACseq, single cell RNAseq) of the consequences of ETO2-GLIS2 expression in different hematopoietic cell contexts.
Our data indicate that GLIS2 determines the megakaryocyte identity of transformed cells in a DNA-binding dependent manner, while both ETO2 and GLIS2 impose aberrant self-renewal properties. Within the specific transcriptional signature of ETO2-GLIS2, we identified a functional imbalance between the major transcriptional regulators of normal hematopoiesis such as GATA and ETS factors. We also showed that ETO2-GLIS2 binds DNA, together with the ETS factor ERG, at regulatory regions called enhancers or super-enhancers (SE) controlling gene expression. Interference with the transcriptional complexes containing the fusion, through the expression of a small peptide (homologous to the NHR2 domain of ETO2), corrects the ERG/GATA1 imbalance, globally reverses the activity of the enhancers, induces megakaryocyte differentiation and abrogates the in vivo maintenance of human leukemic blasts in xenograft models. Therefore, targeting the integrity of the ETO2-GLIS2 complex may represent a new strategy to target this poor prognostic pediatric leukemia. This work also highlights that the imbalance in HTA/GATA factor activity represents a common mechanism of transformation into genetically distinct AML7 subtypes (e.g. AML7 associated with constitutive Trisomy 21) (Thirant et al. Cancer Cell 2017, Lopez et al. Trends in Cancer 2017).
We have also developed a mouse model of doxycycline-induced expression of ETO2-GLIS2 (Collaboration with J. Schwaller, Basel, Switzerland). We have confirmed that this model develops leukemia upon induction of ETO2-GLIS2 expression. This model allowed us to demonstrate that fetal cells are more permissive to ETO2-GLIS2 fusion transformation than adult cells and that this property is associated with differential activity of several transcription factors, including GATA1 and CEBPA (Lopez et al. Cancer Discovery 2019). We are pursuing the analysis of this model using the single cell approaches and in silico modelling of transcription factor activity. We are also developing models of ETO2-GLIS2 fusion expression in iPSC cells (Bertuccio, Boudia, Cambot et al. HemaSphere 2020) and in primary human cells. On the other hand, we characterize the contributions of ETO2 and GLIS2 factors in hematopoietic cells.
Functional genetics of human erythroleukemia
This axis is motivated by the ontogenic proximity of the erythroid and megakaryocytic lines, by the lack of knowledge of the precise mechanisms of transformation of this lineage and by the description of rare cases of mutations affecting genes in common between megakaryoblastic leukemia (AMKL) and erythroleukaemia (AEL).
For this purpose, we are interacting with numerous french, european (e.g. Switzerland, Germany, United Kingdom, Italy, Spain) and international (e.g. United States, Japan, Australia) collaborators to study these rare patient samples and perform genetic and functional analyses. We were able to classify AEL patients into several molecular subgroups, including patients with mutations in the TP53 gene and patients with alterations in epigenetic regulators, such as TET2 and DNMT3A. We also detected alterations in factors involved in GATA1 transcriptional complexes. We model the expression of these genes in murine erythroid progenitors and perform functional approaches to identify important mediators of the erythroid phenotype in these transformations. The development of xenotransplantation models also allows to carry out preclinical approaches.