Haematologica 2003 - Supplements

quiescent counterpart, the human umbilical vein endothelial cells
(HUVEC). MM endothelial cells express highly: a) vascular
endothelial growth factor receptor-2 (VEGFR-2) and tyrosin
kinase with Ig and EGF homology-2 (Tie2/Tek), suggesting their
engagement in vessel sprouting, i.e. in angiogenesis; b) CD34 and
CD133 (AC133), suggesting recruitment of endothelial
progenitor cells into an ancillary vascular network, i.e. into
embryonic vasculogenesis; c) basic fibroblast growth factor
receptor-2 (bFGFR-2) and bFGFR-3, suggesting that they are
prone to this growth factor secreted by plasma cells and stromal
cells; d) endoglin, a marker of tumor vessels; d) E-selectin and β3
molecules, suggesting more opportunities of interactions with
plasma cells; e) aquaporin 1, suggesting hyperpermeability. On
the contrary, they poorly express vascular-endothelial (VE)-
cadherin, as angiosarcoma cells. Fluorescent activated cell sorting
(FACS) analysis of some antigens shows their heterogeneous
expression, suggesting well defined subpopulations of cells. The
main genetic markers are Tie-2/Tek, VEGFs, bFGFs and the
corresponding receptors. MM endothelial cells rapidly form a
close capillary network in vitro (matrigel assay), and generate on
their turn numerous new vessels in vivo (chick embryo
chorioallantoic membrane [CAM] assay). They secrete VEGF,
bFGF, metalloproteinase-2 (MMP-2) and MMP-9, that are
growth and invasive factors both for themselves and plasma cells.
Ultrastructurally, they show vescicles, fenestrae, and hyperplasia
of endoplasmic reticulum that are absent in HUVEC.
Thalidomide interferes with their proliferative activity and
capillarogenesis on matrigel. Our data suggest that both
embryonic vasculogenesis and angiogenesis concur to the
formation of vascular tree of MM bone marrow and disease
progression. Because of the heterogeneous antigenic phenotype, a
mixture (or sequence) of antiangiogenic agents coupled with
thalidomide is envisaged as a possibler biologic therapy (5) of
MM.
Holmgren L, O'Reilly MS, Folkman J. Dormancy of micrometastases:
balanced proliferation and apoptosis in the presence of angiogenesis
suppression. Nat Med 1995;1:149-153.
Vacca A, Ribatti D, Roncali L, Ranieri G, Serio G, Silvestris F,
Dammacco F. Bone marrow angiogenesis and progression in multiple
myeloma. Br J Haematol 1994;87:503-508.
Rajkumar SV, Mesa RA, Fonseca R, Schroeder G, Plevak MF,
Dispenzieri A, Lacy MQ, Lust JA, Witzig TE, Gertz MA, Kyle RA,
Russel SJ, Greipp PR. Bone marrow angiogenesis in 400 patients with
monoclonal gammopathy of undetermined significance, multiple
myeloma, and primary amyloidosis. Clin Cancer Res 2002;8:2210-2216.
Bellamy WT, Richter L, Frutiger Y, Grogan TM. Expression of vascular
endothelial growth factor and its receptors in hematopoietic malignancies.
Cancer Res 1999;59:728-733.
Hideshima T and Anderson KC. Molecular mechanisms of novel
therapeutic approaches for multiple myeloma. Nat Rev Cancer
2002;2:927-937.
P6.5
GENOMIC AND PROTEOMIC CHANGES FOLLOWING
MM CELL-MICROENVIRONMENTAL INTERACTION
Constantine S. Mitsiades1,2, Nicholas S. Mitsiades1,2,
Ciaran McMullan1,2, Galinos Fanourakis1,2, Reshma
Shringarpure1,2, Nikhil C. Munshi1,2, Towia Liberman3,
Kenneth C. Anderson1,2.
1. Jerome Lipper Multiple Myeloma Center, Department of Medical
Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts,
USA; 2. Department of Medicine, Harvard Medical School, Boston,
Massachusetts, USA; 3. Harvard Institutes of Medicine, Beth Israel
Deaconess Medical Center, Boston, Massachusetts, USA.
The response of multiple myeloma (MM) patients to conventional
therapies is significantly affected by interactions of MM tumor
cells with their local bone marrow (BM) microenvironment,
including biologic sequelae induced by BM-derived cytokines,
and adhesion to extracellular matrix proteins and BM stromal
cells (BMSCs). Indeed, these interactions can confer protection to
MM cells against pro-apoptotic therapies (e.g. dexamethasone or
cytotoxic chemotherapy), with adverse implications for patient
outcome. The need to develop rational strategies to target and
abrogate this microenvironment-derived drug-resistance of MM
cells has provided the impetus for comprehensive profiling of the
molecular sequelae triggered by exposure of MM cells to
microenvironmental stimuli, such as BM-derived cytokines (such
as IL-6 and insulin-like growth factors (IGFs)) or co-culture with
BMSCs. IL-6 is known for its role as a growth/survival factor for
MM cells and an important regulator of osteoclastogenesis, while
the MM-BMSCs interaction is known to trigger NF-êB-mediated
IL-6 secretion by BMSCs. The major emphasis on IGFs is
warranted by our recent studies showing that IGFs not only
stimulate MM cell proliferation, survival and attenuated response
to apoptosis-inducing agents (e.g. Dex or Apo2L/TRAIL), but are
also expressed at high levels in serum of MM patients (endocrine
IGF), as well as locally in the BM microenvironment by
autocrine (MM cells) and paracrine (including BMSCs and
osteoblsts) sources (CS Mitsiades et al. Blood 2002; 100, 170a).
Importantly, we have recently shown that IGF-1 receptor (IGF-
1R/CD221) is expressed on all MM cell lines and patients cells
tested and that its inhibition by several different strategies
(including neutralizing antibodies, inhibitory peptides or small
molecule Tyr kinase inhibitors) significantly suppresses MM cell
proliferation, survival and resistance to other drugs, both in vitro
and in vivo (CS Mitsiades et al. Blood 2002; 100, 170a).
To characterize the molecular sequelae triggered by these
microenvironemtal interactions of MM cells with their BM
milieu, we performed gene expression profiling, using U133A
Affymetrix oligonucleotide microarrays, and proteomic analyses
of the signaling state of MM cells, using multiplex
immunoblotting arrays, as recently described (N. Mitsiades et al.
Blood 2003;101(6):2377 and CS Mitsiades et al. Semin Oncol, in
press). These studies involved ex vivo stimulation of MM cells
with pathophysiologically-relevant concentrations of IGF-1, IGF-
2 and IL-6, as well as incubation of MM cells in an ex vivo
model of co-culture with BMSCs. In this model, MM-1S cells
stably transfected with a construct for Green fluorescent protein
(GFP) were co-cultured with BMSCs: the 2 cellular
compartments were subsequently sorted by fluorescence activated
cell sorting (FACS) on the basis of the GFP+ status of MM cells
vs GFP- of BMSCs (thereby minimizing any potential
background signaling and transcriptional changes that may be
induced during mAb-based positive selection and maximizing the
post-sort yield of tumor cells).
Molecular profiles of co-cultured cells were compared with their
respective profiles in isolated cultures, as well as with profiles
generated by co-culture in the setting of treatment with novel
anti-MM agents such as proteasome inhibitor PS-341, hsp90
inhibitor 17-AAG, histone deacetylase inhibitor SAHA, and anti-
IGF-1R inhibitor.
Analyses of these gene expression and proteomic data (using
hierarchical clustering, functional clustering and relevenace
networks algorithms, as well as subsequent confirmatory and
mechanistic assays) showed that the distinct molecular signatures
of MM cells treated with cytokine or co-cultured with BMSCs
also feature overlapping patterns of activation of proliferative
/anti-apoptotic signaling events. Indeed, BM-derived cytokines
and co-culture with BMSCs triggered activation of PI-3K/Akt
and Raf/MAPK signaling pathways in MM cells; upregulated the
S41