Abstract

Background

   Members of the inhibitor of DNA-binding (ID) family of helix-loop-helix
   proteins have been causally implicated in the pathogenesis of several
   types of B-cell lineage malignancy, either on the basis of mutation or
   by altered expression. B-cell chronic lymphocytic leukemia encompasses
   a heterogeneous group of disorders and is the commonest leukaemia type
   in the Western world. In this study, we have investigated the
   pathobiological functions of the ID2 and ID3 proteins in this disease
   with an emphasis on their role in regulating leukemic cell
   death/survival.

Methods

   Bioinformatics analysis of microarray gene expression data was used to
   investigate expression of ID2/ID3 in leukemic versus normal B cells,
   their association with clinical course of disease and molecular
   sub-type and to reconstruct a gene regulatory network using the
   ‘maximum information coefficient’ (MIC) for target gene inference. In
   vitro cultured primary leukemia cells, either in isolation or
   co-cultured with accessory vascular endothelial cells, were used to
   investigate ID2/ID3 protein expression by western blotting and to
   assess the cytotoxic response of different drugs (fludarabine,
   chlorambucil, ethacrynic acid) by
   3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
   ID2/ID3 protein levels in primary leukemia cells and in MEC1 cells were
   manipulated by transduction with siRNA reagents.

Results

   Datamining showed that the expression profiles of ID2 and ID3 are
   associated with distinct pathobiological features of disease and
   implicated both genes in regulating cell death/survival by targeting
   multiple non-overlapping sets of apoptosis effecter genes. Consistent
   with microarray data, the overall pattern of ID2/ID3 protein expression
   in relation to cell death/survival responses of primary leukemia cells
   was suggestive of a pro-survival function for both ID proteins. This
   was confirmed by siRNA knock-down experiments in MEC1 cells and in
   primary leukemia cells, but with variability in the dependence of
   leukemic cells from different patients on ID protein expression for
   cell survival. Vascular endothelial cells rescued leukemia cells from
   spontaneous and cytotoxic drug-induced cell death at least in part, via
   an ID protein-coupled redox-dependent mechanism.

Conclusions

   Our study provides evidence for a pro-survival function of the ID2/ID3
   proteins in chronic lymphocytic leukemia cells and also highlights
   these proteins as potential determinants of the pathobiology of this
   disorder.

Electronic supplementary material

   The online version of this article (doi:10.1186/s12943-014-0286-9)
   contains supplementary material, which is available to authorized
   users.

   Keywords: Chronic lymphocytic leukemia, ID helix-loop-helix proteins,
   Cell survival/cell death, Drug resistance

Introduction

   The ‘Inhibitor of DNA-binding’ (ID) family of helix-loop-helix proteins
   function as key regulators of lineage specification and cell fate
   determination in Metazoa [[29]1-[30]3]. In mammals, there are four ID
   family members (ID1-ID4) that function by heterodimerising with and
   antagonising the activities of several classes of transcription factor.
   The E-protein family of basic helix-loop-helix transcription factors
   (E2A/TCF3, E2-2/TCF4 and HEB/TCF12) are the best characterised ID
   protein targets [[31]1-[32]4]. In hematopoietic cells, individual ID
   proteins perform distinct, but overlapping functions in a lineage- and
   differentiation-stage-specific manner [[33]4-[34]7]. ID proteins have
   also been causally implicated in the pathogenesis of leukemias and
   lymphomas; as in many solid tumour types, ID-mediated tumourigenesis is
   coupled to various oncogene/tumour suppressor pathways in hematopoietic
   cells [[35]6]. Compelling evidence from loss- and gain-of-function
   studies in transgenic mice and cell line models supports a role for ID
   proteins in hematopoietic malignancies. Individual ID proteins have
   been ascribed either an ‘oncogene’ or ‘tumour suppressor’ function in
   primary human hematopoietic malignancies on the basis of expression
   level, mutational pattern and functional properties. For example, ID1
   is a common downstream target of oncogenic tyrosine kinases,
   exemplified by BCR-ABL in chronic myeloid leukaemia, driving cell
   proliferation, survival and invasiveness [[36]6]. High ID1 expression
   is also associated with a poor-prognosis subgroup of acute myeloid
   leukaemia [[37]8]. Deregulated expression of ID2 is a consistent
   feature of Hodgkin’s lymphoma and appears to function in concert with
   ABF-1 in sequestering E2A and probably also PAX5 to augment the
   B-cell-specific gene regulatory programme in Hodgkin’s-Reed/Sternberg
   cells [[38]9,[39]10]. In Burkitt lymphoma by contrast, the function of
   the ID3 protein is recurrently inactivated through the acquisition of
   missense mutations in the ID3 gene, predominantly affecting the
   helix-loop-helix dimerisation domain [[40]11-[41]13]. The ID4 gene
   similarly behaves as a tumour suppressor through epigenetic silencing
   in most cases of acute myeloid leukemia [[42]14], while in a sub-group
   of B-cell precursor acute lymphoblastic leukemia, expression of the ID4
   gene is deregulated by the recurrent t(6;14)(p22;q32) chromosomal
   translocation [[43]15,[44]16].

   B-cell chronic lymphocytic leukemia (CLL) is the most prevalent type of
   leukemia in the Western world and it manifests as a clonal expansion of
   CD5^+, CD19^+, CD23^+ B cells [[45]17,[46]18]. In this leukemia type,
   the status of only the ID4 family member has been evaluated in detail.
   In the Eμ-TCL1 mouse model of CLL, loss of an ID4 allele leads to more
   aggressive disease while hemizygous loss of ID4 in nontransformed
   TCL-1-positive B cells enhances cell proliferation [[47]19]. These
   findings, together with the observation that ID4 mRNA and protein
   expression is universally silenced in primary human CLL [[48]14],
   strongly implicate ID4 as a tumour suppressor in this disease [[49]19].
   For the ID3 family member, microarray gene expression profiling data
   has shown that the expression of this gene is deregulated in CLL. An
   analysis of published microarray datasets of Zheng and colleagues
   [[50]20] reveals a four-fold upregulation of ID3 gene expression in CLL
   compared to normal CD5^+ B-cells. An independent study [[51]21] showed
   that ID3 is among the most significantly overexpressed genes in a
   multivariate gene expression analysis comparing CLL with normal CD19^+
   B-cells, consistent with a potential role in CLL pathogenesis.

   In addition to the various roles ascribed to individual ID proteins in
   regulating cell cycle/cell growth, differentiation, invasiveness,
   angiogenesis and metastasis in tumours of diverse histological origin,
   these proteins have also been widely documented to play a key role in
   regulating cell survival [[52]1-[53]4]. However, the behavior of
   individual ID proteins in functioning as either positive or negative
   regulators of cell viability is highly cell type-dependent, as
   illustrated by their contrasting functions in mediating cell survival
   or cell death in different solid tumour types in response to cytotoxic
   drugs [[54]22-[55]24] (and references therein). Since the primary