Haematologica 2003 - Supplements

ligation or dexamethasone (Georgii-Hemming et al 1996, Nilsson
et al 1998). However, the biological significance of individual
anti-apoptotic elements downstream the IGF-IR are not fully
elucidated.
Upon ligand interaction with the IGF-R -subunit, tyrosine
residues in the intracellular, membrane-bound ß-subunit are
autophosphorylated. This enables phosphorylation and docking of
the insulin receptor substrates (IRS), thereby activating two
important pathways mediating proliferation and survival, i.e. the
phosphatidylinositol 3-kinase (PI 3-K)/Akt and the mitogenactivated
protein kinase (MAPK) pathways. Thus, interfering
with signaling at the level of the receptor tyrosine kinase (RTK)
might represent an attractive strategy to sensitize MM cells to
apoptosis.
To characterize the effects of the IGF-I RTK inhibitors in MM,
we used a panel of authentic MM cell lines as well as freshly
purified CD138+ tumor cells from MM patients. The IGF-I RTK
inhibitors proved to be effective in the MM cell lines and in the
primary MM cells providing 50-90% cell death within 48 h
incubation during standard, serum-containing culture conditions.
The two drug resistant subclones of the MM cell line RPMI8226,
RPMI8226/Dox 40 (doxorubicin) and RPMI8226/LR5
(melphalan), were also sensitive to the RTK inhibitors showing
similar IC50s as the parental cell line. Additionally, the IGF-I
RTK inhibitors were studied in combination with conventional
cytotoxic drugs, e.g. dexamethasone.
Analysis of the IGF-I RTK activity using immunoprecipitation of
the IGF-IR -subunit and Western blotting shows that the IGF-I
RTK inhibitors downregulate both basal and ligand-induced RTK
activity. Furthermore, the downstream consequences of the
perturbed IGF-IR signaling were investigated using
phosphorylation site-specific antibodies directed against signaling
molecules and substrates of the PI 3-kinase/Akt and the MAPK
pathways. To identify target genes crucial for the IGF-IR
mediated survival, gene expression of candidate regulators of
apoptosis, e.g. the Bcl-2 family of genes, were analysed by
ribonuclease-protection assay and Western blotting.
133
Inhibition of Protein Kinase C delta negatively
regulates the Akt signaling pathway and induces
apoptosis of myeloma cells
Nizar J Bahlis, Christopher Maisel, Stanton Gerson
Case Western Reserve University, Ireland Comprehensive Cancer
Center, Cleveland, OH 44106
Multiple myeloma (MM) is an invariably fatal disease of
terminally differentiated B-lineage or plasma cells. Several
growth factors (IL-6, IGF-I, VEGF) provide proliferative and
oncogenic signals to these cells, contributing to their resistant
phenotype. These signals are often transduced by a family of
phospholipid-regulated, serine / threonine kinases known as
Protein Kinase C. The expression of these PKC isoenzymes is
cell specific and each isoenzyme is reported to regulate a specific
cellular function. We first determined that several myeloma cell
lines (8226s, U266, MM1S and MM1R) expressed classical PKC
isoforms (α,β,γ) as well as the novel isoform, PKC δ. Treatment
of myeloma cells with the classical and novel PKC inhibitor, N-
benzoyl-staurosporine (PKC412, Novartis) (500-1000nM)
induced apoptosis in dexamethasone sensitive (MM1S) as well as
dexamethasone (MM1R) and chemo-resistant (U266) cells. Cotreatment
with IL-6 (100ng/ml) did not inhibit PKC412 induced
cell death while the caspase inhibitor Z-VAD-fmk (100µM)
significantly abrogated its effect. PKC412 induced activation of
caspase 9, caspase 3 and cleavage of PARP. Most importantly
PKC412 reduced ser 473 phosphorylation of Akt and its
downstream phosphorylated substrates GSK3-β, FKHR and Bad.
Similarly to PKC412, the specific PKC δ inhibitor, Rottlerin
(3µM), reduced Akt phosphorylation, induced caspase 3 and
PARP cleavage and apoptosis of treated myeloma cells as well.
However the classical PKC (α, β, γ) inhibitor GÖ6976 did not
significantly affect their viability. Finally PKC412 (1µM) and
Rottlerin (3µM) induced apoptosis in primary myeloma cells
(CD138 high) while they had no effect on CD138 low bone
marrow mononuclear cells.
Our studies demonstrate that inhibition of PKCδ negatively
regulates Akt in myeloma cells and activates the downstream
apoptotic machinery providing a rationale for targeting PKC
isoforms, especially PKCδ in multiple myeloma.
134
MOLECULAR PROFILE OF THE ANTI-MYELOMA
ACTIVITY OF HISTONE DEACETYLASE (HDAC)
INHIBITORS: BIOLOGICAL AND THERAPEUTIC
IMPLICATIONS.
Constantine S. Mitsiades1,2, Nicholas S. Mitsiades1,2,
Ciaran J. McMullan1,2, Vassiliki Poulaki3, Reshma
Shringarpure1,2, Teru Hideshima1,2, Masaharu
Akiyama1,2, Dharminder Chauhan1,2, Nikhil Munshi1,2,
Xuesong Gu4, Charles Bailey4, Marie Joseph4, Towia A.
Libermann4, Victoria M. Richon5, Paul A. Marks5,6,
Kenneth C. Anderson1,2.
1. Jerome Lipper Multiple Myeloma Center, Department of Medical
Oncology, Dana-Farber Cancer Institute, Boston, MA 02115; 2.
Department of Medicine, Harvard Medical School, Boston, MA
02115; 3. Massachusetts Eye and Ear Infirmary, Harvard Medical
School, 234 Charles Street, Boston, MA 02114; 4. Genomics
Center, Beth Israel Deaconess Medical Center, Harvard Institutes
of Medicine, Boston, MA 02115; 5. Aton Pharma, Inc. Tarrytown,
NY; 6. Memorial Sloan–Kettering Cancer Center, 1275 York
Avenue, New York, NY 10021, USA.
Histone deacetylases (HDACs) affect cell differentiation and
survival at the transcriptional level, by regulating the acetylation
status of nucleosomal histones and the function of transcription
factor complexes. HDAC inhibition induces differentiation and/or
apoptosis in transformed cells.
We recently showed (Blood, in press) that HDAC inhibitors, such
as the prototypic hydroxamic acid-based HDAC inhibitor
suberoylanilide hydroxamic acid (SAHA), potently induce cell
death (through caspase-independent/calpain-dependent
mechanism) of human multiple myeloma (MM) cells, including
cell lines and MM patient-derived tumor cells, either sensitive or
resistant to conventional or novel anti-tumor agents. SAHA also
sensitized MM cells to death receptor (e.g. Fas or TRAIL
receptor)-mediated apoptosis and inhibited IL-6 secretion in cocultures
of bone marrow stromal cells (BMSCs) with MM cells.
These comprehensive effects of SAHA, both on MM cells
directly and on their microenvironmental interactions, prompted
further investigation of the molecular sequelae of this class of
agents, with particular focus on the transcriptional profile of
SAHA-treatment, since HDAC inhibition exerts its anti-tumor
activity by targeting predominantly the regulation of gene
expression. HDAC inhibition was originally pursued with intent
to induce differentiation of malignant (e.g. leukemic) cells, by derepressing
transcriptional programs of cellular differentiation.
Interestingly, however, our gene expression profiling (using
U133A Affymetrix oligonucleotide microarrays) and subsequent
confirmatory mechanistic and functional assays, indicate that
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