Human chronic myelogenous leukemia K562 cells are relatively resistant to the anti-metabolite cytosine arabinoside (Ara-C) and, when treated with Ara-C, they differentiate into erythrocytes without undergoing apoptosis. In this study we investigated the mechanism by which Ara-C induces K562 cells to differentiate. We first observed that Ara-C-induced differentiation of these cells is completely inhibited by the radiosensitizing agent caffeine, an inhibitor of ATM and ATR protein kinases. We next found that Ara-C activates Chk1 and Chk2 in the cells, and that the activation of Chk1, but not of Chk2, was almost completely inhibited by caffeine. Proteasome-mediated degradation of Cdc25A and phosphorylation of Cdc25C were induced by Ara-C treatment, presumably due to the activation of Chk2 and Chk1, respectively. To directly observe the effects of checkpoint kinase activation in Ara-C-induced differentiation, we suppressed Chk1 or Chk2 with the Chk1-specific inhibitor Go6976, by generating cell lines stably over-expressing dominant-negative forms of Chk2, or by siRNA-mediated knock-down of the Chk1 or the Chk2 gene. The results suggest that Ara-C-induced erythroid differentiation of K562 cells depends on both Chk1 and Chk2 pathways.

We performed chromosome analysis on the bone marrow of a patient with BCR/ABL negative chronic myelogenous leukemia (CML). By interphase fluorescence in situ hybridization (FISH), an extra ABL signal was present in interphase nuclei and appeared to be located at 17p in the metaphase cells. Chromosome analysis showed a subtle abnormality at 17p13 and 12p13 but no visible rearrangement at 9q34 (ABL). Additional FISH experiments disclosed a rearrangement between the short arms of chromosomes 12 and 17 at approximately bands 12p13 and 17p13, respectively. In addition, subtelomeric FISH analysis confirmed the presence of terminal 12p at 17p13 and showed terminal 9q34 to be intact on each chromosome 9. Taken together, these results indicated a rearrangement involving chromosomes 9, 12, and 17 that suggested the possibility of juxtaposition of part of the ETV6 (also known as TEL) locus (12p13) with a portion of ABL (9q34) together at 17p13. The ETV6/ABL fusion was confirmed by RT-PCR, which showed that the first 5 exons of ETV6 were fused in frame with ABL at exon 2. Wild-type ETV6 and ABL were also expressed, in accordance with the FISH results that showed no loss of the second ETV6 or ABL allele.

Myeloid cells (granulocytes and monocytes) are derived from multipotent hematopoietic stem cells. Gene transcription plays a critical role in hematopoietic differentiation. However, there is no single transcription factor that is expressed exclusively by myeloid cells and that, alone, acts as a “master” regulator of myeloid fate choice. Rather, myeloid gene expression is controlled by the combinatorial effects of several key transcription factors. Hematopoiesis has traditionally been viewed as linear and hierarchical, but there is increasing evidence of plasticity during blood cell development. Transcription factors strongly influence cellular lineage during hematopoiesis and expression of some transcription factors can alter the fate of developing hematopoietic progenitor cells. PU.1 and CCAAT/enhancer-binding protein alpha (C/EBPalpha) regulate expression of numerous myeloid genes, and gene disruption studies have shown that they play essential, nonredundant roles in myeloid cell development. They function in cooperation with other transcription factors, co-activators, and co-repressors to regulate genes in the context of chromatin. Because of their essential roles in regulating myeloid genes and in myeloid cell development, it has been hypothesized that abnormal expression of PU.1 and C/EBPalpha would contribute to aberrant myeloid differentiation, i.e. acute leukemia. Such a direct link has been elusive until recently. However, there is now persuasive evidence that mutations in both PU.1 and C/EBPalpha contribute directly to development of acute myelogenous leukemia. Thus, normal myeloid development and acute leukemia are now understood to represent opposite sides of the same hematopoietic coin.

Imatinib, which is an inhibitor of the BCR-ABL tyrosine kinase, has been a remarkable success for the treatment of Philadelphia chromosome-positive (Ph(+)) chronic myelogenous leukemias (CMLs). However, a significant proportion of patients chronically treated with imatinib develop resistance because of the acquisition of mutations in the kinase domain of BCR-ABL. Mutations occur at residues directly implicated in imatinib binding or, more commonly, at residues important for the ability of the kinase to adopt the specific closed (inactive) conformation to which imatinib binds. In our quest to develop new BCR-ABL inhibitors, we chose to target regions outside the ATP-binding site of this enzyme because these compounds offer the potential to be unaffected by mutations that make CML cells resistant to imatinib. Here we describe the activity of one compound, ON012380, that can specifically inhibit BCR-ABL and induce cell death of Ph(+) CML cells at a concentration of <10 nM. Kinetic studies demonstrate that this compound is not ATP-competitive but is substrate-competitive and works synergistically with imatinib in wild-type BCR-ABL inhibition. More importantly, ON012380 was found to induce apoptosis of all of the known imatinib-resistant mutants at concentrations of <10 nM concentration in vitro and cause regression of leukemias induced by i.v. injection of 32Dcl3 cells expressing the imatinib-resistant BCR-ABL isoform T315I. Daily i.v. dosing for up to 3 weeks with a >100 mg/kg concentration of this agent is well tolerated in rodents, without any hematotoxicity.