Methods: We used a patient-specific induced pluripotent stem cell (iPSC) line treated with the mutagenic agent N-ethyl-N-nitrosourea (ENU). Genomic instability was validated using γ-H2AX and micronuclei assays and CGH array for genomic events. Results: An increased number of progenitors (x5-Fold), which proliferated in liquid cultures with a blast cell morphology, was observed in the mutagenized condition as compared to the unmutagenized one. CGH array performed for both conditions in two different time points reveals several cancer genes in the ENU-treated condition, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Transcriptome GEO-dataset GSE4170 allowed us to associate 125 of 249 of the aberrations that we detected in CML-iPSC with the CML progression genes already described during progression from chronic and AP to BC. Among these candidates, eleven of them have been described in CML and related to tyrosine kinase inhibitor resistance and genomic instability. Conclusions: These results demonstrated that we have generated, for the first time to our knowledge, an in vitro genetic instability model, reproducing genomic events described in patients with BC.

REarranged during Transfection (RET) oncogenic rearrangements can occur in 1–2% of lung adenocarcinomas. While RET-driven NSCLC models have been developed using various approaches, no model based on patient-derived induced pluripotent stem cells (iPSCs) has yet been described. Patient-derived iPSCs hold great promise for disease modeling and drug screening. However, generating iPSCs with specific oncogenic drivers, like RET rearrangements, presents challenges due to reprogramming efficiency and genotypic variability within tumors. To address this issue, we aimed to generate lung progenitor cells (LPCs) from patient-derived iPSCs carrying the mutation RET^C634Y, commonly associated with medullary thyroid carcinoma. Additionally, we established a RET^C634Y knock-in iPSC model to validate the effect of this oncogenic mutation during LPC differentiation. We successfully generated LPCs from RET^C634Y iPSCs using a 16-day protocol and detected an overexpression of cancer-associated markers as compared to control iPSCs. Transcriptomic analysis revealed a distinct signature of NSCLC tumor repression, suggesting a lung multilineage lung dedifferentiation, along with an upregulated signature associated with RET^C634Y mutation, potentially linked to poor NSCLC prognosis. These findings were validated using the RET^C634Y knock-in iPSC model, highlighting key cancerous targets such as PROM2 and C1QTNF6, known to be associated with poor prognostic outcomes. Furthermore, the LPCs derived from RET^C634Y iPSCs exhibited a positive response to the RET inhibitor pralsetinib, evidenced by the downregulation of the cancer markers. This study provides a novel patient-derived off-the-shelf iPSC model of RET-driven NSCLC, paving the way for exploring the molecular mechanisms involved in RET-driven NSCLC to study disease progression and to uncover potential therapeutic targets.

Purpose: To model CML progression in vitro and generate a blast crisis (BC-CML) model in vitro in order to identify new targets. Methods: Three different CML-derived iPSC lines were mutagenized with the alkylating agent ENU on a daily basis for 60 days. Cells were analyzed at D12 of hematopoietic differentiation for their phenotype, clonogenicity, and transcriptomic profile. Single-cell RNA-Seq analysis has been performed at three different time points during hematopoietic differentiation in ENU-treated and untreated cells. Results: One of the CML-iPSCs, compared to its non-mutagenized counterpart, generated myeloid blasts after hematopoietic differentiation, exhibiting monoblastic patterns and expression of cMPO, CD45, CD34, CD33, and CD13. Single-cell transcriptomics revealed a delay of differentiation in the mutated condition as compared to the control with increased levels of MSX1 (mesodermal marker) and a decrease in CD45 and CD41. Bulk transcriptomics analyzed along with the GSE4170 GEO dataset reveal a significant overlap between ENU-treated cells and primary BC cells. Among overexpressed genes, CD25 was identified, and its relevance was confirmed in a cohort of CML patients. Conclusions: iPSCs are a valuable tool to model CML progression and to identify new targets. Here, we show the relevance of CD25 identified in the iPSC model as a marker of CML progression.

Introduction: Previous studies have suggested that the tyrosine kinase receptor RET plays a sig-nificant role in the hematopoietic potential in mice and could also be used to expand cord-blood derived hematopoietic stem cells (HSCs). The role of RET in the human iPSC-derived hematopoi-esis has not been tested so far. Methods: To test the implication of RET on the hematopoietic potential of iPSCs, we activated its pathway with the lentiviral overexpression of RETWT or RETC634Y mutation in normal iPSCs. An iPSC derived from a patient harboring the RETC634Y mutation (iRETC634Y) and its CRISPR-corrected isogenic control iPSC (iRETCTRL) were also used. Hematopoietic potential was tested using 2D cul-tures and evaluated regarding the phenotype and the clonogenic potential of generated cells. Results: Hematopoietic differentiation from iPSCs with RET overexpression (WT or C634Y) led to a significant reduction in the number and in the clonogenic potential of HSCs (CD34+/CD38-/CD49f+) as compared to control iPSCs. Similarly, the hematopoietic potential of iRETC634Y was reduced as compared to iRETCTRL. Transcriptomic analyses revealed a specific ac-tivated expression profile for iRETC634Y compared to its control with evidence of overexpression of genes which are part of the MAPK network with negative hematopoietic regulator activities. Conclusion: RET activation in iPSCs is associated with an inhibitory activity in iPSC-derived hematopoiesis, potentially related with MAPK activation.

Les cellules souches induites à la pluripotence (CSIP) dans la Leucémie Myéloïde Chronique (LMC) : Modélisation de la crise blastique et design d'un modèle de thérapie à base de cellules CAR: L'histoire naturelle de la leucémie myéloïde chronique (LMC) qui était une affection constamment mortelle il y a 20 ans en raison d'une évolution vers une transformation en leucémie aiguë (ou crise blastique, CB) a été modifiée par l'utilisation des inhibiteurs tyrosine kinase (ITK). Malgré le succès de ces traitements, la résistance aux ITK et la progression clonale vers une crise blastique (CB) restent des problèmes majeurs, dus à l'instabilité génétique inhérente à l'oncogène BCR::ABL. Pour modéliser les évènements impliqués dans cette progression, nous avons utilisé des cellules souches pluripotentes induites (CSPI) dérivées des cellules leucémiques Ph1+ des patients atteints de LMC. Nous avons montré la possibilité de générer une instabilité globale in vitro par une stratégie de mutagenèse (Article 1). Les expériences de différenciation hématopoïétique ont montré la possibilité de générer en 12 jours, des cellules ayant des caractéristiques de leucémie aiguë, mimant la CB. Les analyses de transcriptome ont permis de mettre en évidence la surexpression du gène CD25 (IL2RA) dans ce cellules leucémiques, confirmée dans les cellules primaires de patients en BP (Article 2). La possibilité d'utiliser une stratégie de CAR-CD25 basée sur les cellules NK a ensuite été développée. Nous avons utilisé dans ce but, la lignée NK92 que nous avons génétiquement modifiée par des lentivirus CAR-CD25. Nous avons montré la possibilité de cibler les cellules leucémiques surexprimant CD25 dans les modèles in vitro et in vivo. Nous avons ensuite transduit les cellules iPSC par les lentivirus CAR et montré l'expression de CAR-CD25 via les promoteurs constitutif ou inductibles à la surface des iPSC (Article 3). Ces expériences de preuve de concept représentent une étape importante pouvant conduire aux thérapies CAR-NK-CD25 dérivées des CSPI dans le futur.