The BCR–ABL fusion protein, produced by the Philadelphia chromosome in chronic myeloid leukemia (CML), influences multiple signaling pathways, leading to alterations of apoptosis, cell cycle regulation, and DNA repair. Point mutations of BCR–ABL confer resistance to the oral tyrosine kinase inhibitors (TKIs), rendering well-tolerated therapy ineffective [Citation1–4]. Even with the development of next-generation TKIs, decreased response rates and relapses occur secondary to BCR–ABL point mutations, such as T315I, and genetic instability [Citation5,Citation6]. However, the stage of hematopoiesis at which these mutations are found and the subsequent effect on the mutated and non-mutated leukemic clones have remained unclear.
In this issue of Leukemia and Lymphoma, Chomel and colleagues [Citation7] present data analyzing when the T315I mutation appears in stem cell differentiation and the interaction of three distinct cell populations: the BCR–ABLT315I-containing cells, the BCR–ABLwt-containing cells, and the non-BCR–ABL-containing cell populations in vitro. Specifically, they used blood and bone marrow samples from a patient with an 8-year history of chronic phase CML, treated with interferon/cytarabine, imatinib, and dasatinib, with the eventual development of BCR–ABLT315I and TKI resistance. By performing long-term culture initiating cell (LTC-IC) and colony-forming cell (CFC) assays, Chomel et al. demonstrate the presence of the BCR–ABLT315I in both bone marrow and peripheral blood, indicating the early development of the T315I mutation in the primitive stem cell compartment. Through double gradient-denaturing gradient gel electrophoresis (DG-DGGE) analysis, they describe both T315I and other point mutations in BCR–ABLwt- and BCR–ABLT315I-containing cells as well as in the ABL protein expressed in non-BCR–ABL-containing cells.
Their demonstration that the primitive stem cell contains kinase domain mutations with decreased presence in the LTC-IC indicates that these cells are not affected by TKIs [Citation8]. This finding is supported by previous work from Bhatia et al., which demonstrated the presence of BCR–ABL transcripts in primitive progenitor cells in patients with complete cytogenetic remission and no detectable BCR–ABL by fluorescence in situ hybridization (FISH) [Citation9]. Recently, Sobrinho-Simoes et al. described evidence of patient-specific genomic BCR–ABL (g BCR–ABL) rearrangements in patients treated with imatinib [Citation10]. Treatment with any of the TKIs does not effectively treat the primitive stem cell and therefore does not prevent the genetic instability caused by BCR–ABL, and is unlikely to be curative.
The point mutations and genetic instability seen in the patient analyzed by Chomel et al. [Citation7] are well described, and several pathways likely contribute to this finding [Citation4,Citation11,Citation12]. Stoklosa et al. demonstrated abnormal mismatch repair (MMR) and MMR-dependent apoptosis by exposing BCR–ABL-positive cell lines, CD34+ CML cell lines, and CD34+ cells from normal bone marrow to O6-methyl-N'-nitro-N-nitrosoguanidine (MNNG) [Citation6]. They noted a higher frequency of mutations and decreased MMR efficacy in the BCR–ABL and CD34+ CML cells compared to CD34+ cells from normal bone marrow. Others have noted altered passage through the S-phase checkpoint and increased oxidative damage as effects of the BCR–ABL kinase [Citation13,Citation14].
Chomel et al. provide an important illustration of the T315I mutation occurring in the primitive stem cell compartment further contributing to disease resistance. CML has long been considered a stem cell disease, and this work supports that view [Citation15]. This is notable because the approach to disease treatment changes if the goal becomes eradication of the quiescent stem cell, as it is not effectively targeted by our current therapy. While the presence of point mutations and genetic instability is well established and described in this work, the mechanisms leading to this finding are still being determined. In this study, it is unclear how the patient's previous cytotoxic therapy influenced the point mutations and genetic instability. Further, this work was derived from a single patient, with studies performed in cell culture without the additional interactions and signals of the surrounding stroma. Chomel and colleagues present a thorough analysis of the T315I mutation, but further work is needed to understand the mechanisms of genetic instability and reveal methods to target the CML stem cell.
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