BCR and BCR/ABL Regulation during Myeloid Differentiation in Healthy Donors and in Chronic Phase/Blast Crisis CML Patients
Authors: Marega Manuela , Piazza Rocco , Meneghetti Iris , Redaelli Sara , Mogavero Angela , Gambacorti Carlo 
Published: April 2020

Abstract
The fusion protein BCR/ABL leads to chronic myeloid leukaemia (CML). The corresponding fusion gene is under the transcriptional control of BCR promoter. It is known that in CML progenitors the ability to block myeloid differentiation is directly related to BCR/ABL levels. These observations open new questions about BCR/ABL and BCR expression control. However, up to date only few studies have been focused on the characterization of the BCR promoter, so little is known about the transcriptional regulation of this gene. We studied BCR expression in sorted myeloid precursors in healthy donors (HD) and in CML patients in Chronic Phase (CP) and Blast Crisis (BC). CML samples were also analyzed for BCR/ABL. The expression level was analyzed by Real Time PCR normalized against GUS as housekeeping gene, in haematopoietic stem cells (HSCs defined as CD34+/CD38−/Thy+/−), common myeloid and granulocyte-monocyte progenitors (CMPs and GMPs, respectively defined as CD34+/CD38+/IL−3Ralo/CD45RA− and CD34+/CD38+/IL−3Ralo/CD45RA+,) sorted by Fluorescence Activated Cell Sorting (FACS). Preliminarly, we analyzed the BCR and BCR/ABL mRNA halflife. We performed a RNA stability assay using Actinomycin D in the K562 cell line and in CP and BC samples: the halflife of both genes were comparable thus excluding major differences in RNA stability. Then, we analyzed BCR levels in healthy donors. A statistically significant BCR downregulation was noted during myeloid maturation in HD (HSCs [0.815±0.27SD] vs CMPs [0.176±0.1] and vs GMPs [0.167±0.17], p=0.0079 in both cases). The same analysis performed in CP patients showed that both BCR and BCR/ABL were downregulated upon committement to differentiation (BCR: HSCs [0.326±0.24] vs CMPs [0.0899±0.054], p=0.0078, and vs GMPs [0.0277±0.07], p=0.0008; BCR/ABL: HSCs [2.99±1.76] vs CMPs [0.781±0.41], p=0.0005 and vs GMPs [0.304±0.46], p=0.028). However, while BCR levels were lower in CML samples compared to HD (HSC samples [0.815±0.27 vs 0.326±0.24]: p=0.0120), probably due to haploinsufficiency, BCR/ABL values were higher than BCR ones, when compared to CML (HSCs: p=0.0020) and even to HD samples (HSCs: p=0.0074). These data indicate that in CML a potent upregulation of BCR/ABL compared to BCR is present, probably caused by a selective mechanism acting on BCR/ABL. In a limited set of BC samples (4 patients), the decrease of BCR and BCR/ABL was less evident and did not reach statistical significance (BCR: HSCs 0.339±0.235; CMPs: 0.135±0.12; GMPs: 0.126±0.06; BCR/ABL: HSCs: 8.58±5.78; CMPs: 5.55±7.39; GMPs: 3.36±2.61); thus as already indicated by Jamieson and colleagues (NEJM, 351:657, 2004), in BC the downregulation of BCR/ABL during maturation is impaired. Moreover, we detected a tendency for higher expression of BCR/ABL in BC than CP in all the subpopulations under study, which reached statistical significance only in GMPs (HSCs [8.58±5.78 BC vs 2.99±1.76 CP]: p=0.1483; CMPs [5.55±7.39 BC vs 0.781±0.41 CP]: p=0.1483; GMPs [3.36±2.61 BC vs 0.304±0.46 CP]: p=0.028); BCR was upregulated only in GMPs (HSCs [BC 0.339±0.235 vs 0.326±0.24 CP]: p=0.9399; CMPs [0.135±0.12 vs 0.0899±0.054]: p=0.4818; GMPs [0.126±0.06 vs 0.0277±0.07]: p=0.0755). In conclusion, BCR is physiologically downregulated during myeloid maturation, and this regulation is conserved in CP-CML, not only for BCR but also for BCR/ABL. In BC-CML, this mechanism of regulation seems to be impaired for BCR/ABL and possibly for BCR too. In addition, a potent upregulation of BCR/ABL compared to BCR is present in all stages of CML, probably caused by a selective mechanism acting on BCR/ABL.