A Single Chronic Myeloid Leukemia (CML) Stem Cell Harboring Both BCR-ABL and JAK2-V617F Mutation Modeled by Induced Pluripotency
Authors: Sloma Ivan , Mitjavila-Garcia Maria Teresa , Feraud Olivier , Tosca Lucie , El Marsafy Sanaa , Gobbo Emilie , Divers Dominique , Proust Alexis , Bonnet Marie Laure , Griscelli Frank , Tachdjian Gerard , Bennaceur-Griscelli Annelise , Turhan Ali G 
Published: October 2019

Abstract
Abstract 3728
The occurrence of JAK2-V617F mutation has been reported during the evolution of several patients with Ph1-positive CML. In all cases where a clonal analysis has been performed, the involvement of two different hematopoietic stem cells (HSC) has been demonstrated, with the presence of two different myeloproliferative disorders (MPDs). The occurrence of the V617F mutation in a leukemic stem cell (LSC) harboring the BCR-ABL rearrangement has not been reported so far. In a 63- year old patient with a diagnosis of high Sokal score Ph1+ CML, the initial therapy with Imatinib led to efficient cytoreduction and complete cytogenetic response but with persistent splenomegaly. Upon disease progression, he was treated successively with IFN-alpha, ARA-C, Dasatinib and Nilotinib with transient responses and persistent splenomegaly. The progressive increase of platelet counts led to the discovery of JAK2-V617F mutation, which appeared to be present from the diagnosis of CML with a progressive increase (40 % at diagnosis up to 70% at the time of the discovery of JAK2 mutation). To determine if BCR-ABL and JAK2 mutation were present in the same cells, a clonogenic assay was performed and single BFU-E's (n=11) were analyzed individually by q-RT-PCR. All 11 BFU-E expressed BCR-ABL at high levels with a mean BCR-ABL/ABL ratio of 157% (Range 91–207 %). The same individual BFU-E also revealed the presence of a heterozygous JAK2-V617F mutation. To generate a HSC model harboring simultaneously these two major molecular events, we have generated induced pluripotent stem cells (iPSC) from leukemic cells using Sendai-virus mediated Oct4, Sox2, Klf4 and c-Myc gene transfer. We have also generated iPSC from non-leukemic (BCR-ABL-negative, JAK2-V617F-negative) endothelial progenitor cells (EPC) obtained from peripheral blood. iPSC generated from both sources expressed pluripotency markers (Tra-1–60, SSEA-3, SSEA-4) and generated teratoma in NOD/SCID mice. In 24 individual leukemic iPSC clones which were amplified, Ph1-chromosome was found to be present in all mitoses and BCR-ABL was highly expressed (Mean ratio 140%). Each iPSC exhibited the same V617F mutation which co-existed in the primary leukemic BFU-E. None of the non-leukemic iPSC expressed BCR-ABL neither V617F. To determine the hematopoietic potential of both iPSC, we have generated day-16 embryoid bodies (EB) followed by induction of hematopoiesis. Day 16 EB's generated variable numbers of CD34+ cells (12–40 %, n=4) and clonogenic potential (25–340 CFC/105 D16-EB) with evidence of growth-factor-independent CFC's. Interestingly, IM and Dasatinib had partial inhibitory effects on CFC activity, but a combination of Pimozide (STAT5 inhibitor) and IM had a major synergistic effect. As neither normal iPSC nor human embryonic stem cells (hESC) are able to generate definitive, long-term hematopoiesis, we have tested the long-term HSC potential of these iPSC in long-term culture initiating cell (LTC-IC) assays as compared to human ESC line H1. In these conditions, leukemic iPSC generated hematopoietic cells with terminal granulocytic and erythroblastic differentiation and gave rise for the first time to an LTC-IC- derived progeny at 6 weeks (200 CFU-C/105 Day-16 EB) whereas in the same time H1-derived cells did not generate any significant LTC-IC potential (3 CFU-C/105 D-16 EB). Interestingly, non-leukemic iPSC generated also hematopoietic cells but exhibited a major genetic instability in culture with appearance after several passages, of a t (5;7) translocation and major deletions/duplications in CGH arrays. To determine a potential upstream molecular event which might be involved at the origin of the occurrence of BCR-ABL and JAK2 mutation in the same HSC, a whole genome sequencing strategy is underway. In conclusion, we report for the first time the occurrence of two major molecular events involved in CML and non-CML MPDs in the same LSC and demonstrate the utility of iPSC modeling to study the earliest HSC involved. These results suggest strongly that the co-existence of BCR-ABL and V617F enables the specification of iPSC towards hematopoieis which could lead to the identification of specific hematopoietic signaling pathways. Finally, clonal analysis of hematopoietic cells in CML patients with demonstration of Jak2V617 mutations is worthwhile to determine the prevalence of the simultaneous occurrence of these two molecular events in the same cell.

Disclosures:
Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.