Identification, characterisation and regulation by CD40 activation of novel CD95 splice variants in CD95-apoptosis-resistant, human, B-cell non-Hodgkin's lymphoma.

CD95 gene and splicing aberrations have been detected in B-cell non-Hodgkin lymphoma (B-NHL) where they are thought to contribute to CD95 apoptosis resistance. To further investigate this, we have performed extensive CD95 transcript sequencing and functional analysis in B-NHL with demonstrated resistance to CD95-induced apoptosis (B-NHLr). Strikingly, instead of showing CD95 mutations per se, B cells from B-NHLr co-expressed wild-type and multiple, normal (CD95nv) and novel alternatively spliced variant CD95 transcripts (CD95av). CD95av were predicted, by sequencing, to encode soluble, potentially apoptosis inhibitory proteins. However, their overexpression, by transfection, in Jurkat cells did not interfere with endogenous CD95 death signalling. Furthermore, CD95av-expressing B-NHLr did not show mutations in CD95 splice-regulatory elements and CD95av expression was 'reversible' by CD40 activation. This, in conjunction with treatment by the protein synthesis inhibitor, cycloheximide, could sensitise a subset of B-NHLr to CD95 apoptosis. In normal and lymphoma B cells, this correlated to increased CD95 membrane expression, enhanced DISC activity and engagement of the mitochondrial death pathway via Bid cleavage, although the latter occurred less efficiently in B-NHLr. Thus, immune modulation of CD95 transcription and alternative splicing combined with enhanced engagement of mitochondrial death signalling offer potential for restoration of CD95 apoptosis sensitivity in B-NHLr.

1q12 chromosome translocations form aberrant heterochromatic foci associated with changes in nuclear architecture and gene expression in B cell lymphoma.

Epigenetic perturbations are increasingly described in cancer cells where they are thought to contribute to deregulated gene expression and genome instability. Here, we report the first evidence that a distinct category of chromosomal translocations observed in human tumours--those targeting 1q12 satellite DNA--can directly mediate such perturbations by promoting the formation of aberrant heterochromatic foci (aHCF). By detailed investigations of a 1q12 translocation to chromosome 2p, in a case of human B cell lymphoma, aberrant aHCF were shown to be localized to the nuclear periphery and to arise as a consequence of long range 'pairing' between the translocated 1q12 and chromosome 2 centromeric regions. Remarkably, adjacent 2p sequences showed increased levels of repressive histone modifications, including H4K20me3 and H3K9me3, and were bound by HP1. aHCF were associated to aberrant spatial localization and deregulated expression of a novel 2p gene (GMCL1) that was found to have prognostic impact in diffuse large B cell lymphoma. Thus constitutive heterochromatin rearrangements can contribute to tumourigenesis by perturbing gene expression via long range epigenetic mechanisms.