A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation

Abstract

IntroductionChronic myeloid leukemia (CML) is a clonal disorder of pluripotent hematopoietic cells that is characterized by the presence of the Philadelphia chromosome (Ph ϩ ), the result of a reciprocal translocation between chromosomes 9 and 22. The translocation encodes a chimeric protein, BCR-ABL, which is a constitutively activated protein tyrosine kinase (PTK) 1 and which has an essential role in the molecular pathology of CML. CML is a progressive disease with an initial chronic phase in which there is a marked expansion in the late myeloid cell population (reviewed by Clarkson and Strife 2 ). CML progenitor cells possess only a subtle defect in maturation, while retaining their requirements for bone marrow stromal cells or cytokines to survive and proliferate. 3 This chronic phase, which is of variable duration, is usually followed by an accelerated phase leading to blast crisis. The blast crisis is the most severe manifestation of the disease in which differentiation is apparently blocked. Imatinib (Gleevec or STI571), an Abl tyrosine kinase inhibitor, is the first line and gold standard for treating CML. Nonetheless, there is clear evidence of resistance to this drug. The development of other approaches to treat the disease with smallmolecule inhibitors to new targets has been, and will be, a focus for future research into treatment of imatinib-resistant patients and those in accelerated phase or blast crisis. [4][5][6][7] Thus, understanding the process of leukemogenesis driven by this oncogene is still an important research objective.Most cell-line models expressing BCR-ABL PTK are already differentiation blocked, and assume a growth factor-independent phenotype. [8][9][10][11] In this respect they mirror the properties of cells from patients with CML in blast crisis. However, we have established a cell-line model for CML that has properties of chronic-phase progenitor cells that progressively change to those resembling accelerated phase by using an inducible temperaturesensitive BCR-ABL PTK (ts-BCR-ABL FDCP-Mix). 12-14 DNA microarray technology has been used to identify differential expression as a result of the initial activation of BCR-ABL. Activation of BCR-ABL in FDCP-Mix cells has identified transcriptional and secretory events regulated by BCR-ABL. These effects are observed to be due to BCR-ABL in primary CML cells. Materials and methods Cell linesFDCP-Mix cells transfected with the retroviral vector pM5-neo carrying a p210 ts-BCR-ABL cDNA were used as a model system for CML. 12 The L.M. carried out the majority of the practical work, data analysis, and interpretation, and contributed to preparation of the manuscript; S.P. played a major part in experimental design and execution; N.P. and B.P. provided essential biological material and made a major contribution to the interpretation of data; A.P. and A.D.W. provided essential biological reagents and contributed to experimental design and writing of the manuscript; A.E.I. was responsible for the overall conception and design of the project, in...

CCN family of proteins: critical modulators of the tumor cell microenvironment

“…Again, and from another type of cancer perspective, studies from the Irvine lab (McCallum et al 2006(McCallum et al , 2009Suresh et al 2013) investigating the role of CCN3 in myelopoiesis and leukemia, provided evidence that exogenous CCN3 decreases NOTCH1 signaling and BCR-ABL causes downregulation of CCN3 allowing the NOTCH1 drive to continue suggesting a form of combinatorial therapy including CCN3. Whether future studies will identify similar therapeutic permutations involving CCN proteins have yet to be realized.…”

CCN3 suppresses mitogenic signalling and reinstates growth control mechanisms in Chronic Myeloid Leukaemia

“…Primer and probe sets for CCN3, Integrins alpha 6, beta 1 and beta 4 were designed against Genbank published sequences in association with Primer Express (Applied Biosystems). CCN3 primers and probe set were used as previously described (McCallum et al 2006) having the 5′ reporter 6-carboxyfluorescein (FAM) and the 3′ quencher 6-carboxy-tetramethylrhodamine (TAMRA). The amplification reactions (12.5 μL) contained 50 ng cDNA equivalents (or control), 1×Taqman universal PCR master mix, final concentrations of 5 mM MgCl 2, 0.2 mM deoxyadenosine deoxycytosine deoxyguanosine triphosphate (dATP/ dCTP/ dGTP), 0.4 mM deoxyuridine triphosphate (dUTP), 0.125 U AmpliTaq Gold, 2 μM primers (forward and reverse) and 200 nM TaqMan probe.…”

“…Our group were the first to identify a role for CCN3 in haematological malignancy showing CCN3 was downregulated as a result of the BCR-ABL oncogene in CML (McCallum et al 2006). CML is a clonal disorder of pluripotent haematopoietic stem cells and CML progenitor cells possess a subtle defect in cell maturation (Wong and Witte 2001).…”

“…We have previously shown, using a transient expression model, that increasing CCN3 expression results in reduced cell growth and enhanced apoptosis of CML cells (McCallum et al 2006). We now report on the establishment of a CCN3 stable expression system in K562 CML cells and characterization of this model.…”

MicroRNAs 130a/b are regulated by BCR-ABL and downregulate expression of CCN3 in CML

“…Bone marrow cells from CML patients had reduced Ccn3 expression and the expression was restored upon treatment with imatinib. Increased secretion of Ccn3 is also observed in progenitor CD34 + CML cells, indicating loss of CCN3 as an early event in CML (McCallum et al 2006). In vitro studies using CML cell lines overexpressing Ccn3 or treated with recombinant Ccn3 showed reduced cell proliferation and increased apoptosis (McCallum et al 2009).…”