Whole Genome Sequencing Of Chronic Myeloid Leukemia (CML)-Derived Induced Pluripotent Stem Cells (iPSC) Reveals Faithful Genocopying Of Highly Mutated Primary Leukemic Cells

Genetic instability is a hallmark of chronic myeloid leukemia (CML). Recently, several major abnormalities in DNA repair mechanisms have been identified in primitive CML cells that likely explain the additional mutations these cells develop leading to their selective growth under tyrosine kinase inhibitor (TKI) therapies. It seems likely that such mechanisms also underlie disease progression in CML. However, an understanding of the specific somatic mutations involved and investigations of their resulting effects on the biological behavior of primary sources of primitive chronic phase (CP) CML cells is extremely challenging. As an alternative approach, we have now explored the possibility of applying whole genome sequencing (WGS) to induced pluripotent stem cells (iPSCs) derived from primitive CML cells to determine if such iPSCs, genocopy the mutations present in the diagnostic sample from which they were generated and whether primitive hematopoietic cells derived from these iPSCs might be useful for future drug screening experiments. To this end, we chosen a CML patient whose CP clonogenic cells contained both the Ph1 chromosome and the JAK2 V617F mutation and whose disease progressed into an accelerated phase (AP) during TKI therapy. iPSC were generated from leukemic cells obtained at the time of AP using Oct4, Sox2, Klf4 and c-Myc gene transfer. The presence of both BCR-ABL and JAK2 V617F was confirmed in 24/24 iPSC colonies. A control iPSC line negative for both genes was similarly established from the patient’s CD34+CD31+ endothelial progenitors purified from peripheral blood. We then performed WGS on DNA prepared from the leukemic cells obtained at diagnosis of CP (CML 006), the AP cell-derived iPSCs (PB34), and the control non-leukemic iPSCs (PB13), using a HighSeq Illumina platform. WGS revealed 845,175 somatic SNVs and 68,817 somatic short Indels in the CP leukemic cells at diagnosis that were not present in the non-leukemic iPSCs (PB13). 49,225 of these SNVs and 11,665 of the short Indels were novel (absent in the dbSNP database), and 419 were found in the COSMIC database. We identified 274 novel SNVs (3 missense, 161 nonsense, 108 synonymous and 2 splice site mutations) and 46 short Indels (19 insertions and 27 deletions). Most of the novel coding SNVs and Indels were heterozygous and an estimation of the variant allele frequency indicated these were present in virtually all leukemic cells. In addition to the JAK2 V617F mutation that was present at diagnosis, we found a novel frame shift mutation in exon 12 of ASXL1 gene (p.S871YfsX5) leading to protein truncation, a genetic event that has also been associated with myeloproliferative neoplasms (MPNs) and AML. We also identified several novel SNVs predicted by SIFT, Provean and PolyPhen-2 algorithms to be deleterious for protein structure. These novel mutations were found in genes relevant for the pathophysiology of MPNs, including the catenin (CTNNA1 R204C, and AIDA K235T), RAS (RREB1 P789T), autophagy (ULK1 R553C) cellular antioxidant defense (GSR S293C), RNA nuclear transport (NUP160 start loss) pathways. Individual sequencing confirmed the presence of these mutations in PB34 and their absence in PB13 (non-leukemic iPSC). We next compared the sequence data from the AP leukemic cell-derived iPSCs (PB34) with the diagnostic data (CML006). This analysis showed only 799 additional somatic SNVs and 96 new short Indels compared with those already evident in the cells present at diagnosis. Only 4 (3 non synonymous and 1 synonymous) SNVs and no Indels were found in exons. These mutations could have appeared during the application of the reprogramming process to the AP leukemic cell-derived iPSCs; none was an obvious contributor to MPN pathophysiology. Finally, we showed that day16 embryoid bodies derived from the PB34 iPSCs contained expected numbers of CD34+ cells (18±11%, n=6) and BCR-ABL-expressing hematopoietic colony-forming cells (CFCs, 143±64 / 10⁵ cells, n= 6). These CFCs showed a slight inhibitory response to imatinib (54±15% colonies obtained in 1 µM IM, n=4) whereas a combination of IM and Pimozide (a STAT5 phosphorylation inhibitor), reduced survival another ∼10-fold. In conclusion, we have provided proof-of-principle results illustrating the potential of iPSC technology in combination with WGS to dissect the clonal evolution of disease progression in CML and develop patient specific drug screens that could build on this data.