Abstract
Despite significant progress obtained in CML therapy by using tyrosine kinase inhibitors (TKIs), progression towards accelerated phase and blast crisis (AP/BC) occurs, especially in patients developing resistance towards several lines of TKIs. AP/BC is characterized by an increased genetic instability, which is one of the major characteristics of CML and facilitated by the expression of the BCR-ABL fusion protein in leukemic cells. Although the major genomic events leading to BC have been described over several years of research (such as TP53 alterations, abnormalities of DNA repair genes) there is no model of reproducing these events in the context of stem cells in vitro. To this purpose we have designed a CML patient-derived, iPSC-based in vitro mutagenesis strategy to uncover novel genomic events associated with progression of CML. To mimic AP/BC in CML patients and to generate a "blast crisis in a dish" model, we have designed a strategy based on patient-derived iPSCs. For this purpose, we have generated iPSC from leukemic cells of three CML patients. At the time of cell reprogramming, two patients (UPN PB27 and UPN PB32) were in CP and the other (UPN PB34) in AP. The latter patient also had JAK2V617F mutation in the same Ph1+ stem cell clone. iPSC were treated using the mutagenic agent N- ethyl-N-nitrosourea (ENU) by daily addition of the drug in cultures. They were characterized for their pluripotency (expression of pluripotency genes, teratoma assays in NSG mice) as well as for their hematopoietic potential using StemDiff technology, before and after mutagenesis. Using this technology, iPSCs gave rise to hematopoietic cells from day +5 up to day 13, with a major expansion of hematopoietic cells and progenitors. As expected, cytological patterns differed between 3 pts. Amongst the three pts, UPN-PB32-ENU as compared to non-mutagenized cells, generated myeloid blasts exhibiting monoblastic patterns (AML5) and expression of MPO, CD34, CD33, CD11b, and CD43. To analyze the molecular events occurring during CML iPSC-based mutagenesis, we have performed single cell RNAseq analyses in hematopoiesis-induction cultures, in mutagenized versus non-mutagenized iPSC. These experiments have been performed at D+5, D+9 and D+13 of differentiation using 104 cells in each condition. They revealed 13 clusters, with evidence of delay of hematopoietic differentiation in ENU-treated iPSC and accumulation of Msx1-expressing cells at the mesoderm stage. Hematopoietic cells generated from the three mutagenized iPSCs (PB27, 32, and 34) and collected at day +13 were then compared to their non-mutagenized counterparts using transcriptomic analyses. As can be seen in Figure 1, several up-and down-regulated genes were identified in all three ENU-mutagenized hematopoietic cells. In UPN PB32-ENU (harboring the most potent BC phenotype), 1439 up- and 1641-down-regulated genes were identified. These results were analyzed along with the GSE4170 GEO dataset, in which Radich et al (PNAS 2006) have compared gene expression between CP, AP and BC by analyzing 91 cases of CML using normal immature CD34+ cells as a reference. A significant overlap was found between primary BC cells (GSE4170) and the signature induced in UPN PB32-ENU compared to its non-mutagenized counterparts. Among the overexpressed genes, the IL2RA (CD25) was found to be of interest as it has been shown to be overexpressed in AML. We have therefore analyzed its relevance using a cohort of CML patients in CP (n=22) AP/BP (n=14) and compared the CD25 mRNA expression to that observed in a cohort of AML (n=15), ALL (n=17), and normal controls (n=15). The mean percentage of CD25/ABL1 was highly increased in AP/BP CML as compared to other groups and, in particular, to CP-CML (Mean % 75 vs. 19.3). In addition, CD25 mRNA expression was evaluated in seven CML patients who have progressed to BC, in both CP and BP samples. CD25 up-regulation was clearly observed in all patients during AP/BC, with a 5- to 10-fold increase in BP of 4 of these 7 patients as compared to CP. Overall, our results provide a novel iPSC-based experimental tool to study CML progression, allowing to reveal genetic abnormalities that correlate with those observed in primary BC. They also suggest that iPSC technology could pave the way for discovering new therapeutic targets, not only for CML progression, but also for several other hematological malignancies progressing towards aggressive phases.
Disclosures
Cayssials:BMS: Honoraria; Pfizer: Honoraria; Incyte Biosciences: Honoraria; Novartis: Honoraria. Turhan:Abbvie: Consultancy, Honoraria; Incyte Biosciences: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.
