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GATA1 Target Gene CSF2RB Is Functionally Coupled to MPL and Marks a TPO-Dependent Preleukemic Cell Population Sustained by the Fetal Liver, but Not Bone Marrow
David Cruz Hernandez1 †, Nicolas Papadopoulos2-4†, Elise Sepulchre2,3, Marlen Metzner1, Batchimeg Usukhbayar1, Mirian Angulo Salazar1, Leila Varghese2-4, Gabriel Levy2-4, Irene Roberts1, Stefan N. Constantinescu2-5†, Paresh Vyas1†.1MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford United Kingdom.
2Université catholique de Louvain and de Duve Institute, Brussels, Belgium,
3Ludwig Cancer Research Laboratories, Brussels, Belgium,
4Wel Research Institute, WelBio Department,
5Ludwig Institute of Cancer Research, Nuffield Department of Medicine, Oxford University. †These authors contributed equally.
Many paediatric cancers exhibit a strikingly different genetic basis and biology compared to adult cancers. Understanding the mechanistic basis for these differences will shed light on factors important in oncogenesis during development. One exemplar are the genetically linked myeloid pre-leukemic and leukemic disorders in newborns and children under 4 years old with Trisomy 21 (T21) Down syndrome (DS). Children with DS have a 400-fold increased risk of developing myeloid leukaemia Down Syndrome (ML-DS) in early life[1, 2]. ML-DS is preceded by a fetal/neonatal clonal, pre-leukemic phase, transient abnormal myelopoiesis (TAM) when fetal T21 hematopoietic cells acquire somatic mutations in the X-linked GATA1 gene[3, 4]. These mutations produce an N-terminal truncated GATA1, GATA1-short (GATA1s). Remarkably, the pre-leukemic proliferative effect exerted by germline T21 and GATA1s are exquisitely developmentally restricted. As hemopoiesis shifts from the fetal liver to newborn bone marrow, TAM usually spontaneously regresses. The biological basis for the requirement of a fetal liver environment for TAM cells and their failure to thrive in the bone marrow is unknown. However, a possible clue is that TAM cells can proliferate in bone marrow if they acquire additional somatic oncogenic activating mutations, typically in cytokine signalling or JAK-STAT pathway genes. These acquired mutations transform the TAM clone to a leukemic ML-DS clone[5].
To address the question of why TAM cells, expressing only GATA1s, survive only in a fetal liver environment, we initially performed single cell RNA sequencing, coupled with genotyping from 3 primary TAM samples and as controls, 4 T21 and 3 normal disomic cord blood (CB) samples. Using the whole transcriptome to identify cell states, we identified a marked expansion of mutant GATA1s TAM cells, compared to controls, at the earliest GATA1-expressing progenitors and through erythropoiesis and megakaryopoiesis (Fig 1A). In these cell compartments, TAM cells displayed transcriptional upregulation of the PI3K/AKT/mTOR, TNFA/NFκB and JAK/STAT pathways. Using the program SCENIC[6], we identified enhanced activity of the STAT3 regulon in GATA1s cells compared to T21 and disomic CB cells (Fig 1B).
From this, we hypothesized that aberrant cytokine receptor activation leading to increased STAT3 transcriptional activity was essential for uncontrolled mutant GATA1s cell myeloproliferation. To identify the cytokine receptor(s) leading to enhanced STAT3 activity, we examined the most highly differentially expressed genes in TAM cells, compared to T21 and disomic CB cells. This pinpointed to CSF2RB, a cytokine receptor subunit that directly activates the JAK-STAT signalling pathway[7] . Next, we demonstrated that CSF2RB is also overexpressed at the protein level in TAM and marks an impressive 81% of TAM CD34+ cells compared to 5% of disomic, and 10% of T21 CB CD34+ cells (Fig 1C). Next, we showed CSF2RB was a direct GATA transcriptional target gene. Using CUT&TAG we showed GATA1s and GATA2 bind two GATA sites in the CSF2RB locus (12kb upstream and 68kb downstream of the CSF2RB promoter)[8] in an ML-DS cell line (Fig 1D). CRISPR-Cas9 disruption of either GATA2, or the GATA motif within the -12kb site led to downregulation of CSF2RB protein. We are testing if GATA1 represses CSF2RB and whether GATA1s fails to repress CSF2RB, allowing sustained abnormal CSF2RB expression in TAM. Finally, CSF2RB is an essential gene for ML-DS; CRISPR knockout in a ML-DS cell line model leads to cell death. Taken together, CSF2RB is a direct GATA target gene and abnormal sustained, CSF2RB expression in TAM cells is required for mutant GATA1s cell survival.
CSF2RB functions as a heterodimer. Classically, CSF2RB partners with the a chain of either IL-3, or IL5 or GM-CSF receptors. Most interestingly, none of the a chains of IL-3 or IL5 or GM-CSF are expressed neither at the protein nor RNA level on TAM CSF2RB+ cells, suggesting that CSF2RB might partner with another, unexpected cytokine receptor. We nominated MPL, the thrombopoietin (TPO) receptor as a new candidate binding partner of CSF2RB for the following reasons: (i) we documented co-expression of MPL with CSF2RB at the RNA and protein level in TAM cells. (ii) we confirmed functional coupling between CSF2RB and MPL in Ba/F3 and TF-1 cells, where at equivalent MPL levels, Tpo signaling via the JAK-STAT pathway is significantly enhanced by CSF2RB. Importantly, this effect is stronger at high Tpo levels. (iii) A mutantion in CSFR2RB (A455D) that we described in ML-DS[9] requires specific interaction with human MPL to induce autonomous growth of cytokine-dependent hematopoietic cells[10]. Notably, direct interaction between MPL and CSF2RB A455D is detected by Bioluminescence Resonance Energy Transfer[10].
Finally, we hypothesize that high levels of fetal liver produced TPO sustain TAM myeloproliferation because of the functional coupling between MPL and CSF2RB. This required survival/proliferation signal is lost when cells migrate to the low TPO bone marrow environment (Fig 1E). Further work will precisely assess TPO concentration in DS fetal liver environment and whether TAM cells display increased proliferation and activation of the JAK-STAT signaling in response to varying doses of TPO.
In summary, we propose a model whereby the reduced TPO levels experienced by TPO addicted TAM cells during their migration from fetal liver to bone marrow creates selective pressure for the acquisition of somatic mutations (including the A455D CSF2RB mutation that we previously identified)[9], that permit GATA1s mutant cells to proliferate independently of the high TPO environment of the fetal liver.
References
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Speakers
Paresh Vyas