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

Abstract

Recent evidence suggests chronic myeloid leukemia (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, after retransplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off were able to persist in vivo and reinitiate leukemia in secondary recipients on Bcr-Abl reexpression. Bcr-Abl knockdown in human CD34(+) CML cells cultured for 12 days in physiologic 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 a long-term culture-initiating cell assay and expansion on 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.

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

“…The primary tissue is isolated by flow cytometry using leukapheresis samples, followed by culture in growth factors and TKIs as previously described 42 . A low-ionic-strength buffer wash (20mM Bicine, 250mM Sucrose; pH 7.5) is used prior to lysis with zwitterionic detergent (20mM Bicine; pH 7.6, 0.6% CHAPS).…”

“…The quantity of clinical starting material needed for this approach is significantly smaller than those required in previous studies using a cIEF platform 1,2 , and this technique also enables a more detailed analysis of protein phosphorylation than was possible with earlier technologies. 42 • Anti-human CD38-FITC (Becton Dickinson, cat. no.…”

“…no. 555821) 42 • Dimethyl sulfoxide (DMSO), ACS spectrophotometric grade, e99.9% (Sigma-Aldrich, cat. no.…”

“…Sort cells into CD34 + CD38 -and CD34 + CD38 + subpopulations using a BD FACSAria Cell-Sorting System. 42 ii. Culture sorted sells overnight in SFM plus growth factors.…”

Stem cell persistence in chronic myeloid leukemia

“…Recent reports from several groups including ours strongly suggest that primitive CML cells are not oncogene addicted and do not require BCR-ABL kinase activity for their survival. 12,13 This suggests that eradication of all CML LSCs by BCR-ABL TKIs may be biologically impossible. Moreover, evidence is accumulating that the signals affording survival of BCR-ABL LSCs despite BCR-ABL inhibition are extrinsic, derived from their microenvironment.…”