Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival

Recent evidence suggests CML stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, following re-transplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off, were able to persist in vivo and re-initiate leukemia in secondary recipients upon Bcr-Abl re-expression. Bcr-Abl knockdown in human CD34+ CML cells cultured for 12 days in physiological growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%, however the surviving fraction was enriched for primitive leukemic cells capable of growth in long-term culture initiating cell assay and expansion upon removal of dasatinib and addition of growth factors. Together these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance

Optimization of methods for the detection of BCR-ABL activity in Philadelphia-positive cells

<b>Objective:</b> The recent success in treating chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKI), such as imatinib mesylate (IM), has created a demand for reproducible methods to accurately assess inhibition of BCR-ABL activity within CML cells, including rare stein and progenitor cells, either in vitro or in vivo. The purpose of this study was to develop an enzyme-linked immunosorbent (ELISA) method to measure total tyrosine phosphorylation (P-Tyr) in small samples of cells that express BCR-ABL and to compare to more established methods.<p></p> <b>Materials and Methods:</b> The assay was first validated in BCR-ABL wild-type and mutant vs BCR-ABL-negative cell lines. P-Tyr levels were then measured by ELISA in primary CD34(+) CML cells treated with IM.<p></p> <b>Results:</b> In vitro exposure to TKI resulted in decreases in the level of P-Tyr, in both BCR-ABL-positive cell lines and primary CD34(+) CML samples, which were comparable to the reduction in P-Tyr by flow cytometry and phosphorylation of CrkL by either Western blot or flow cytometry.<p></p> <b>Conclusion:</b> We have developed an accurate ELISA method to measure BCR-ABL, activity within Ph+ cells, which is comparable to other in vitro BCR-ABL, assessment techniques in terms of sensitivity and could be adapted for high throughput.<p></p&gt

Elevated Expression of LGR5 and WNT Signaling Factors in Neuroblastoma Cells With Acquired Drug Resistance

Neuroblastoma (NB) is a pediatric solid cancer with high fatality, relapses, and acquired resistance to chemotherapy, that requires new therapeutic approaches to improve survival. LGR5 is a receptor that potentiates WNT/signaling pathway and has been reported to promote development and survival in several adult cancers. In this study we investigated LGR5 expression in a panel of NB cell lines with acquired resistance to vincristine or doxorubicin. We show LGR5-LRP6 cooperation with enhanced expression in drug resistant NB cell lines compared to parental cells, suggesting a role for LGR5 in the emergence of drug resistance, warranting further investigation

BCR-ABL activity and its response to drugs can be determined in CD34⁺ CML stem cells by CrkL phosphorylation status using flow cytometry

In chronic myeloid leukaemia, CD34<sup>+</sup> stem/progenitor cells appear resistant to imatinib mesylate (IM) <i>in vitro</i> and <i>in vivo</i>. To investigate the underlying mechanism(s) of IM resistance, it is essential to quantify Bcr-AbI kinase status at the stem cell level. We developed a flow cytometry method to measure CrkL phosphorylation(P-CrkL) in samples with < 10<sup>4</sup> cells. The method was first validated in wild-type (K562) and mutant (BAF3) BCR-ABL<sup>+</sup> as well as BCR-ABL<sup>-</sup> (HL60) cell lines. In response to increasing IM concentration, there was a linear reduction in P-CrkL, which was Bcr-AbI specific and correlated with known resistance. The results were comparable to those from Western blotting. The method also proved to be reproducible with small samples of normal and Ph<sup>+</sup> CD34<sup>+</sup> cells and was able to discriminate between Ph<sup>-</sup>, sensitive and resistant Ph<sup>+</sup> cells. This assay should now enable investigators to unravel the mechanism(s) of IM resistance in stem cells