Emmanuel Olivier

Phosphatase Mg2+/Mn2+ dependent 1D protein (PPM1D) is a serine threonine phosphatase known to regulate oncogenic pathways. PPM1D mutations are enriched in therapy-related MDS and AML, but the role of PPM1D in myeloproliferative neoplasms (MPN) remains largely unexplored. By sequencing 519 MPN patients, we detected activating PPM1D mutations in 3.1% of them. Importantly, PPM1D was overexpressed in hematopoietic stem and progenitor cells (HSPCs) in most PPM1D wild-type (WT) MPN cases. To elucidate the impact of PPM1D mutations on MPN HSPCs, we generated isogenic JAK2V617F+/PPM1D-WT and JAK2V617F+/PPM1D mutant (MUT) induced pluripotent stem cell (iPSC) lines from a MPN patient. Single-cell RNA-sequencing (scRNA-seq) of iPSC derived HSPCs revealed altered TP53, DNA damage response and cell cycle regulation. scRNA-seq analysis of primary PPM1D-MUT MPN patient mononuclear cells (MNCs) further confirmed upregulation of MYC, mTORC1, OXPHOS, JAK/STAT, and NF-κB pathways and corresponding genes in HSCs. The allosteric PPM1D inhibitor (PPM1Di) GSK2830371 preferentially inhibited colony formation in myelofibrosis (MF) samples. Combination treatment of GSK2830371 with low dose inhibition of HDM2, a negative regulator of p53, significantly improved targeting of JAK2V617F+ HSPCs, while sparing normal HSPCs. Single-cell mutational and protein analysis of a clonally complex MF patient sample treated with this combination strategy revealed reduced mutated clones and increased WT clones by >4-fold. Dual PPM1D and HDM2 targeting also significantly decreased leukemic burden in a xenograft MPN model. These findings support PPM1D as a critical MPN HSPCs regulator and support dual PPM1D/HDM2 inhibition as a treatment strategy for MF patients.

Acute myeloid leukemia (AML) that evolves from myeloproliferative neoplasm (MPN) is known as post-MPN AML. Current treatments do not significantly extend survival beyond 12 months. B-cell lymphoma-extra large (BCL-xL) has been found to be overexpressed in leucocytes from patients with MPN, making it a potential therapeutic target. We investigated the role of BCL-xL in post-MPN AML and tested the efficacy of DT2216, a platelet-sparing BCL-xL proteolysis-targeting chimera, in preclinical models of post-MPN AML. We found that BCL2L1, the gene encoding BCL-xL, is expressed at higher levels in patients with post-MPN AML than in those with de novo AML. Single-cell multiomics analysis revealed that leukemia cells harboring both MPN-driver and TP53 mutations exhibited higher BCL2L1 expression and elevated scores for leukemia stem cell, megakaryocyte development, and erythroid progenitor than wild-type cells. BH3 profiling confirmed a strong dependence on BCL-xL in post-MPN AML cells. DT2216 alone, or in combination with standard AML/MPN therapies, effectively degraded BCL-xL, reduced the apoptotic threshold, and induced apoptosis in post-MPN AML cells. DT2216 effectively eliminated viable cells in JAK2-mutant AML cell lines, induced pluripotent stem cell–derived hematopoietic progenitor cells, primary samples, and reduced tumor burden in cell line–derived xenograft model in vivo by degrading BCL-xL. DT2216, either as a single agent or in combination with azacytidine, effectively inhibited the clonogenic potential of CD34+ leukemia cells from patients with post-MPN AML. In summary, our data indicate that the survival of post-MPN AML is BCL-xL dependent, and DT2216 may offer therapeutic advantage in this high-risk leukemia subset with limited treatment options.

Telomere biology disorders (TBDs), caused by pathogenic germ line variants in telomere-related genes, present with multiorgan disease and a predisposition to cancer. Clonal hematopoiesis (CH) as a marker of cancer development and survival in TBDs is poorly understood. Here, we characterized the clonal landscape of a large cohort of 207 patients with TBD with a broad range of age and phenotype. CH occurred predominantly in symptomatic patients and in signature genes typically associated with cancers: PPM1D, POT1, TERT promoter (TERTp), U2AF1S34, and/or TP53. Chromosome 1q gain (Chr1q+) was the commonest karyotypic abnormality. Clinically, multiorgan involvement and CH in TERTp, TP53, and splicing factor genes were associated with poorer overall survival. Chr1q+ and splicing factor or TP53 mutations significantly increased the risk of hematologic malignancies, regardless of clonal burden. Chr1q+ and U2AF1S34 mutated clones were premalignant events associated with the secondary acquisition of mutations in genes related to hematologic malignancies. Similar to the known effects of Chr1q+ and TP53-CH, functional studies demonstrated that U2AF1S34 mutations primarily compensated for aberrant upregulation of TP53 and interferon pathways in telomere-dysfunctional hematopoietic stem cells, highlighting the TP53 pathway as a canonical route of malignancy in TBD. In contrast, somatic POT1/PPM1D/TERTp mutations had distinct trajectories unrelated to cancer development. With implications beyond TBD, our data show that telomere dysfunction is a strong selective pressure for CH. In TBD, CH is a poor prognostic marker associated with worse overall survival. The identification of key regulatory pathways that drive clonal transformation in TBD allows for the identification of patients at a higher risk of cancer development.

Cancer driver mutations often show distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in the course of acute myeloid leukaemia, upon progression or relapsed/refractory disease^1,2,3,4,5,6. Here, by using human leukaemogenesis models, we first show that RAS mutations are obligatory late events that need to succeed earlier cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed progenitors of the myelomonocytic lineage (granulocyte–monocyte progenitors) harbouring previously acquired driver mutations, showing that advanced leukaemic clones can originate from a different cell type in the haematopoietic hierarchy than ancestral clones. Furthermore, we demonstrate that RAS-mutant leukaemia stem cells (LSCs) give rise to monocytic disease, as observed frequently in patients with poor responses to treatment with the BCL2 inhibitor venetoclax. We show that this is because RAS-mutant LSCs, in contrast to RAS-wild-type LSCs, have altered BCL2 family gene expression and are resistant to venetoclax, driving clinical resistance and relapse with monocytic features. Our findings demonstrate that a specific genetic driver shapes the non-genetic cellular hierarchy of acute myeloid leukaemia by imposing a specific LSC target cell restriction and critically affects therapeutic outcomes in patients.

Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.

Development of advanced gene and cell therapies for the treatment of genetic diseases requires confident animal and cellular models to test their efficacy and is crucial in the cases where no primary samples from patients are available. CRISPR/Cas9 technology, has become one of the most spread endonuclease tools for editing the genome at will. Moreover, it is known that the use of two guides tends to produce the precise deletion between the guides via NHEJ. Different distances between guides were tested (from 8 to 500 base pairs). We found that distances between the two cutting sites and orientation of Cas9 protein-DNA interaction are important for the efficiency within the optimal range (30-60 bp), we could obtain new genetically reproducible models for two rare disease, a Pyruvate Kinase Deficiency model, using human primary cells, and a (for in vivo primary hyperoxaluria therapeutic mouse model. We demonstrate that the use of a 2-guideRNAs at the optimal distance and orientation is a powerful strategy for disease modelling in those diseases where the availability of primary cells is limited.

As cell culture methods and stem cell biology have progressed, the in vitro production of cultured RBCs (cRBCs) has emerged as a viable option to produce cells for transfusion or to carry therapeutic cargoes. RBCs produced in culture can be quality-tested either by xeno-transfusion of human cells into immuno-deficient animals, or by transfusion of autologous cells in immuno-competent models. Although murine xeno-transfusion methods have improved, they must be complemented by studies in immuno-competent models. Non-human primates (NHPs) are important pre-clinical, large animal models due to their high biological and developmental similarities with humans, including their comparable hematopoietic and immune systems. Among NHPs, baboons are particularly attractive to validate cRBCs because of the wealth of data available on the characteristics of RBCs in this species that have been generated by past blood transfusion studies. We report here that we have developed a method to produce enucleated cRBCs by differentiation of baboon induced pluripotent stem cells (iPSCs). This method will enable the use of baboons to evaluate therapeutic cRBCs and generate essential pre-clinical data in an immuno-competent, large animal model. Production of the enucleated baboon cRBCs was achieved by adapting the PSC-RED protocol that we previously developed for human cells. Baboon-PSC-RED is an efficient chemically-defined method to differentiate iPSCs into cRBCs that are about 40% to 50% enucleated. PSC-RED is relatively low cost because it requires no albumin and only small amounts of recombinant transferrin.

This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.8% ± 5.4% express CD34 at day 7, 98.6% ± 1.3% express CD43 at day 10, and 99.1% ± 0.95% of cells are CD235a positive by day 31 of the differentiation process. Moreover, this differentiation protocol supports extensive expansion, with a single hPSC producing up to 150 hematopoietic progenitor cells by day 10 and 50,000–200,000 erythroid cells by day 31. The erythroid cells produced exhibit a definitive fetal hematopoietic type, with 90%–95% fetal globin and variable proportion of embryonic and adult globin at the protein level. The presence of small molecules during the differentiation protocol has quantitative and qualitative effects; it increases the proportion of adult globin and decreases the proportion of embryonic globin. Given its level of definition, this system provides a powerful tool for investigation of the mechanisms governing early hematopoiesis and erythropoiesis, including globin switching and enucleation. The early stages of the differentiation protocol could also serve as a starting point for the production of endothelial cells and other hematopoietic cells, or to investigate the production of long-term reconstituting hematopoietic stem cells from hPSCs. Significance: This differentiation protocol allows the production of a large amount of erythroid cells from pluripotent stem cells. Its efficiency is compatible with that of in vitro red blood cell production, and it can be a considerable asset for studying developmental erythropoiesis and red blood cell enucleation, thereby aiding both basic and translational research. In addition to red cells, the early stages of the protocol could also be used as a starting point for the large-scale production of other hematopoietic cell types, including the ultimate goal of generating long-term reconstituting hematopoietic stem cells.