High BCR::ABL1 Expression Defines CD34+ Cells with Significant Alterations in Signal Transduction, Short-Proliferative Potential and Self-Renewal Ability

Abstract

Purpose

Chronic Myeloid Leukemia (CML) is a clonal disorder of the hematopoietic stem cell caused by expression of the BCR::ABL1 oncoprotein. High BCR::ABL1 levels have been associated to proliferative advantage of leukemic cells, blast crisis progression and tyrosine kinase inhibitors (TKIs) inefficacy. We have previously shown that high BCR::ABL1/GUS^IS transcripts measured at diagnosis are associated with inferior responses to standard dose Imatinib (IM). However, the mechanisms underlying the higher rates of disease progression and development of TKIs resistance dependent on elevated BCR::ABL1 levels remain unclear.

Methods

Leukemic cells were collected from CML patients showing, at diagnosis, high or low BCR::ABL1/GUS^IS. BCR::ABL1 expression levels were measured using real-time PCR. Short-term culture and long-term culture-initiating cells assays were employed to investigate the role of BCR::ABL1 gene-expression levels on proliferation, clonogenicity, signal transduction, TKIs responsiveness and self-renewal ability. Cell division was performed by carboxyfluorescein-succinimidyl ester (CFSE) assay.

Results

We found that BCR::ABL1 oncogene expression levels correlate in both PMNs and CD34+ cells. Furthermore, high oncogene levels increased both proliferation and anti-apoptotic signaling via ERK and AKT phosphorylation. Moreover, high BCR::ABL1 expression reduced the clonogenicity of leukemic CD34+ cells and increased their sensitivity to high doses IM but not to those of dasatinib. Furthermore, we observed that high BCR::ABL1 levels are associated with a reduced self-renewal of primitive leukemic cells and, also, that these cells showed comparable TKIs responsiveness with cells expressing lower BCR::ABL1 levels. Interestingly, we found a direct correlation between high BCR::ABL1 levels and reduced number of quiescent leukemic cells caused by increasing their cycling.

Conclusion

Higher BCR::ABL1 levels improving the proliferation, anti-apoptotic signaling and reducing self-renewal properties cause an increased expansion of leukemic clone.

Keywords:

• BCR:ABL1
• self-renewal
• CD34
• TKIs
• LTC-IC

Abbreviations

AKT, V-Akt murine thymoma viral oncogene-like protein 1; BFU-E, burst-forming uniterythroid; BM, bone marrow; BOS, Bosutinib; BSA, bovine serum albumin; CFSE, carboxyfluorescein-succinimidyl ester; CFU, colony forming unit; CFU-E, colony forming unit-erythroid; CML, chronic myeloid leukemia; CP, chronic phase; CRKL, CRK like proto-oncogene, adaptor Protein; DAS, Dasatinib; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; FITC, fluorescein-5-isothiocyanate; GUSB, β-glucuronidase; HD, healthy donor; IM, imatinib mesylate; LDA, limiting dilution analysis; LTC-IC, long-term culture-initiating cell; M-MLV, moloney murine leukemia virus; MNC, mononuclear cell; MSC, mesenchymal stem cell; PB, peripheral blood; PE, phycoerythrin; PMN, polymorphonuclear; PON, ponatinib; SCREEN, Sicily and Calabria CML REgional ENterprise; SDS-PAGE, sodium dodecyl sulfate poly-acrylamide gel electrophoresis; SRC, V-Src Avian Sarcoma (Schmidt-Ruppin A-2) Viral Oncogene Homolog; TFR, treatment-free remission; TKI, tyrosine kinase inhibitor.

Acknowledgments

This work was supported by Grant AIRC number 12958 and PRIN 2017 CUP 64I19000710001.

Disclosure

Fabio Stagno reports personal fees from Pfizer, Novartis, and Incyte, outside the submitted work. The authors report no other conflicts of interest in this work.