The team Biology of pediatric leukemia is part of the UMR 1170 Molecular dynamics of hematopoietic transformation. It is part of the national network on childhood leukemia CONECT-AML and coordinates the PEDIAC consortium.
Pediatric cancers affect about 1 in 600 children and represent the second leading cause of death in children in France. Hematological malignancies represent 45% of pediatric cancers. The clinical characteristics of pediatric cancers suggest that they have a different molecular basis compared to similar cancers in adults. However, the mechanisms of leukemic transformation and the basis for the exclusive association between several mutations and some pediatric cancers are poorly understood.
The objective of our studies is to identify the molecular basis of childhood leukemia and to characterize the mechanisms of transformation in order to allow the development of new clinical perspectives. Following genetic analyses to identify chromosomal and gene alterations observed in leukemia, we develop models using human cells (xenografts of primary patient cells, CRISPR/Cas9 approach) or transgenic models. Molecular and functional characterizations (e.g. gene expression, chromatin status, functional screening through inactivation) are analyzed by computational approaches performed within the team and allow the identification of new therapeutic targets.
Functional genetics of acute megakaryoblastic leukemia
Our work on leukemia of the megakaryocytic lineage (acute megakaryoblastic leukemia or AMKL) associated with poor response to treatments and unfavorable prognosis has led to the identification of several recurrent fusion mutations and oncogenes involving regulators of gene expression (e.g. OTT-MAL and ETO2-GLIS2) (Mercher et al, PNAS 2001; Thiollier et al, JEM 2012).
Our functional studies have shown that the OTT-MAL fusion alters Notch signaling (Mercher et al. JCI 2009), which plays a role in normal erythro megakaryocyte lineage development (Mercher et al, Cell Stem Cell 2008). However, expression of the OTT-MAL fusion alone does not induce leukemic development in mice. Other recurrent genetic alterations in AML7 include Trisomy 21 and proteins with tyrosine kinase activity.
De novo AMKL with ETO2-GLIS2 is associated with the worst prognosis of pediatric AMKL. Only a few additional mutations have been identified in this subgroup suggesting that ETO2-GLIS2 may be sufficient for leukemogenesis. We are developing modeling approaches (e.g. CRISPR/Cas9, inducible transgenic, PDX, IPSC) to perform cellular and molecular characterizations (e.g. transcriptome, chromatin accessibility, protein interactors, single cell approaches) aimed at characterizing the consequences of the ETO2-GLIS2 fusion in hematopoietic cells.
Using primary leukemia cells from patients and models, we have shown that the ETO2-GLIS2 fusion binds to DNA through both its ETO2 and GLIS2 parts, in particular at regulatory regions of gene expression called "enhancers". It controls the expression of major transcription factors. It activates ERG which is essential for the expression of a stem cell-associated transcriptional program, including the up-regulation of KIT growth factor receptor expression. It represses GATA1, which is a master regulator of erythro/megakaryocyte differentiation. From a mechanistic point of view, we showed that the transcriptional program imposed by ETO2-GLIS2 depends on the functional interaction between ETO2-GLIS2 and ETO2 via the NHR2 domain allowing ETO2 oligomerization. Indeed, ectopic expression of a peptide interfering with the NHR2 domain inhibits the expression of enhancer-associated genes, restores the expression balance of ERG and GATA1 factors, and abrogates the proliferation of ETO2-GLIS2 leukemia cells in in vivo models. These results established that functional interference with the activity of transcriptional complexes involving ETO2-GLIS2 inhibits the proliferation/survival of these leukemia cells (Thirant et al. Cancer Cell 2017, Lopez et al. Trends in Cancer 2017).
We have developed a mouse model of doxycycline-inducible ETO2-GLIS2 expression (coll. with J. Schwaller, Basel, Switzerland). We confirmed that this model develops leukemia upon induction of expression by fusion. This model allowed us to show that fetal cells are more permissive to transformation by ETO2-GLIS2 fusion 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 analyses to better understand the activity of transcription factors as well as the influence of extrinsic factors on tumor development.
We are developing expression models of the ETO2-GLIS2 fusion from human cells. To do so, we follow two approaches. First, we use iPSC cells to obtain in vitro embryonic hematopoiesis. In this system, we have shown that fusion alters megakaryocyte differentiation, increases progenitor self-renewal, and recapitulates the transcriptional deregulations observed in patients (Bertuccio et al Hemasphere 2020). This model represents the first ETO2-GLIS2 model using human cells at an early stage of development. On the other hand, we collaborate with the team of Dr. F. Pflumio (CEA, Fontenay-aux-roses) for the use of primary human cells at different stages of development.
The molecular data obtained in our analyses allow for the development of new targeted preclinical approaches in interaction with experts in pharmacology (coll. Dr. G. Barratt and Dr. F.X. Legrand, Paris Saclay) and pharmaceutical companies.
Functional genetics of human erythroleukemia
Acute erythroid leukemia (AEL) is a rare disease. In order to study AEL, we have initiated with the group of Dr. J. Schwaller (Basel, Switzerland) a large collaborative work with teams from the Gustave Roussy Institute (Dr. S. DeBotton and Dr. J.B. Micol), French clinical centers (Dr. E. Delabesse: Toulouse, Dr. D. Birnbaum: Marseille, Dr. L. Garcon: Amiens), European centers (Dr. P. Vyas: England, Dr. E. Anguita: Spain, Dr. C. Dierks: Germany, Dr. A. Rambaldi: Italy, Dr. P. Valent: Austria) and international centers (Dr. M. Caroll: USA, Dr. J. Maciejewski: USA, Dr. S. Kazuya: Japan, Dr. C. Carmichael: Australia) to obtain samples from AEL patients. We identified genetic and transcriptional alterations and classified 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 of factors involved in GATA1 transcriptional complexes such as ERG, ETO2 or SKI, in 18% of the patients of our cohort, some of them described for the first time in human AEL. We then showed that the expression of these genes in erythroid progenitors allows their transformation in vitro and induces erythroid leukemia when injected into mice. We have also demonstrated that inhibition of ETO2 and SKI in erythroid cell lines leads to a decrease in cell proliferation (Fagnan et al Blood 2020). This work is currently being pursued with molecular studies of the mechanisms controlled by ETO2 using PDX and cellular models of inactivation of ETO2 function (Fagnan et al, submitted).