Getting to the root of the stem cell in mutated chronic myeloid leukemia

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 [1–4]. 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 [5,6]. 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 [7] present data analyzing when the T315I mutation appears in stem cell differentiation and the interaction of three distinct cell populations: the BCR–ABL-containing cells, the BCR– ABL-containing cells, and the non-BCR–ABLcontaining 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–ABL 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–ABL 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–ABLand BCR–ABL-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 [8]. 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) [9]. Recently, Sobrinho-Simoes et al. described evidence of patient-specific genomic BCR–ABL (g BCR–ABL) rearrangements in patients treated with imatinib [10]. 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. [7] are well described, and several pathways likely contribute to this finding [4,11,12]. Stoklosa et al. demonstrated abnormal mismatch repair (MMR) and MMRdependent apoptosis by exposing BCR–ABL-positive cell lines, CD34þ CML cell lines, and CD34þ cells from normal bone marrow to O-methyl-N’-nitro-Nnitrosoguanidine (MNNG) [6]. They noted a higher frequency of mutations and decreased MMR efficacy in the BCR–ABL and CD34þ CML cells compared to CD34þ 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 [13,14].