Haematologica 2003 - Supplements

immunoglobulin heavy chain (IgH) locus. Overexpression of
receptor for hyaluronan mediated motility (RHAMM), and its
splice variants (RHAMM-exon4 and RHAMM-exon13), also
characterizes the myeloma clone. Recently, we reported that
RHAMM, an itinerant protein that functions outside and within
the cell, is a centrosomal/spindle pole protein that maintains
mitotic stability. GFP-RHAMMFL and GFP-RHAMM-exon13,
but not GFP-RHAMM-exon4, interact with microtubules.
RHAMM, like nuclear-mitotic apparatus protein (NuMA),
depends upon direct microtubule contact, mediated by exon 4,
and indirect microtubule contact through the dynein motor
complex to crosslink spindle microtubules. As RHAMM
overexpression characterizes MM, we investigated centrosomal
structure within CD138+ plasma cells from archived bone
marrow cores taken from MM (n=41), MGUS (n=8) and control
(n=4) patients. Centrosomal number and qualitative structural
abnormalities were analysed visually. Immunofluorescence
analysis, in combination with confocal microscopy and 3-
dimensional reconstruction, allowed quantitative assessment of
structural abnormalities. While visual inspection of centrosomal
numbers outlined a difference between MM and control (p=0.03)
but not MGUS (p=0.09), quantitation of centrosomal structure
demonstrated significant differences between MM and control
(p=0.002) as well as MGUS (p=0.01) samples. These data
illustrate the necessity for quantitative analysis of centrosomal
structure and the pervasive centrosomal abnormalities in MM.
Other centrosomal/spindle pole gene products that are intimately
associated with RHAMM mitotic function(s) (TACC3, NuMA,
dynein light chain 2B) map telomeric to recurrent IgH
translocation sites (4p16.3, 11q13, 16q23.3) and may also be
disregulated in MM. IgH translocations may elevate the
expression of gene products by positioning them next to strong
enhancer elements; moreover, the occurrence of unbalanced IgH
translocations, and occasional loss of derivative chromosomes,
may induce haploinsufficiency of translocated gene products.
Using RHAMM as a model of spindle pole proteins, we show
that both overexpression and inhibition of function affects mitotic
integrity. Overexpression of GFP-RHAMMFL induces ectopic
nucleation of microtubules and, in the absence of centrosomal
replication defects, multipolar spindles. Inhibition of RHAMM
function, through the microinjection of purified RHAMM
antibodies, disrupts mitotic integrity and induces tripolar (11+/-
3.4% of injected cells) and tetrapolar (14+/- 4.3%) spindles.
Isoform balance may be another important determinant of
RHAMM function. Fluorescent recovery after photobleaching
examination of GFP-RHAMMFL and GFP-RHAMM-exon4
dynamics illustrates differences in isoform mobility within the
cytoplasm and at the centrosome. Within the cytoplasm, GFP
alone was highly mobile (99.24% mobility) which translated as
an extremely low time for 50% recovery (t1/2=0.218s). The
dynamics of GFP-RHAMMFL(t1/2=8.42s, 70.6% mobile)
differed from GFP-RHAMM-exon4(t1/2=2.96s, 88.9% mobile)
within the cytoplasm but not at the centrosome. Although not yet
examined at spindle poles, relative overexpression of RHAMMexon4,
shown by us to significantly correlate with poor survival,
may inhibit microtubule crosslinking, adversely affect the ability
of RHAMM complexes to maintain spindle poles and lead to
CIN. Thus, MM is characterised by centrosomal disregulation
that can result from elevated, inhibited and/or differential isoform
expression of RHAMM, perhaps in concert with other
centrosomal proteins that may be disregulated by recurrent
translocations in MM.
031
Altered RHAMM splicing is an adverse prognostic
factor, and upregulated RHAMM correlates with lytic
bone disease.
Tony Reiman MD, Christopher A. Maxwell, John
Shaughnessy PhD, Andrew R. Belch MD, and Linda M.
Pilarski PhD.
Oncology, Cross Cancer Institute, University of Alberta AND
Lambert Laboratory of Myeloma Genetics, University of Arkansas
for Medical Sciences.
RHAMM is a centrosomal protein which is overexpressed in
myeloma bone marrow plasma cells (BMPC) relative to normal B
cells. Aberrant RHAMM expression in vitro leads to mitotic
errors. In myeloma we have observed variable deletion of
RHAMM exon 4, a region critical to binding of microtubules.
These observations imply a model in which aberrant RHAMM
expression and/or splicing leads to compromised mitotic spindle
integrity, contributing to the chromosome instability that drives
myeloma progression.
Our model is supported by microarray-based global gene
expression profiles (GEP) of BMPC from 112 myeloma patients
with lytic bone disease, in comparison to 43 patients lacking bone
lesions. RHAMM was found to be more highly expressed in
patients with lytic bone lesions (p