Haematologica 2003 - Supplements

myeloma patients. Phase I data indicate the combination of ATO
and ascorbic acid is well tolerated and may be a promising
therapeutic agent for myeloma. However, the mechanism (s) of
ATO action in myeloma remains elusive. We have begun to
address this question in two ways. First, we asked whether
caspase activity is required for ATO-mediated cell death.
Second, we developed a panel of ATO-resistant lines as tools to
identify determinants of ATO sensitivity in myeloma.
Here we report that ATO exerts cytotoxic effects in myeloma cell
lines via both caspase-dependent and caspase-independent
pathways. We monitored ATO-induced changes in cell viability,
caspase activity, superoxide production, and m in the
presence or absence of pharmacological caspase inhibitors and
the anti-oxidant N-acetylcysteine. Consistent with GSH levels
dictating ATO action, NAC abrogated ATO-induced changes and
protected cells from ATO-induced cell death up to 7 days.
Surprisingly, experiments with caspase inhibitors suggested at
least two models for ATO death signaling in myeloma. In 8226/S
cells, blockade of caspases had no effect on loss of cell viability,
increase in ROS production, and minimal effects on the loss of
m. In contrast, caspase inhibition conferred significant
protection from ATO effects in U266, MM1.S, KMS11, and
KMS18 cells. Studies with myeloma cells freshly isolated from
bone marrow biopsies indicated that both the caspase-dependent
and caspase-independent pathways might be clinically relevant.
In an effort to further characterize the ATO death pathways, we
developed myeloma lines that are resistant to ATO. The ATO
resistant lines 8226/Ar, DoxAr, U266/Ar, and MM1.S/Ar were
generated from the 8226/S, 8226/Dox40, U266, and MM1.S cell
lines, respectively. The 8226/Ar, U266/Ar, and MM1.S/Ar lines
were selected by continuous culture in 200 nM of clinical grade
ATO. The DoxAr line arose spontaneously. Consistent with
GSH levels being a key determinant of ATO sensitivity in other
cell types, all the resistant myeloma lines display significantly
higher GSH levels than their respective parental lines. BSO
restored ATO sensitivity in all the resistant lines, while ascorbic
acid restored sensitivity in the DoxAr, U266/Ar and MM1.S/Ar
resistant lines. We also analyzed the ATO resistant variants for
the development of cross-resistance to other chemotherapeutic
agents. All the resistant lines displayed cross-resistance to the
kinase inhibitor staurosporine. The DoxAr and MM1.S/Ar cells
were resistant to melphalan, and either BSO or ascorbic acid
restored melphalan sensitivity. Cisplatin resistance was detected
in 8226/Ar, U266/Ar and MM1.S/Ar cells and etoposide
resistance was found in both the 8226/Ar and DoxAr cells.
Interestingly, the MM1.S/Ar cells also developed cross-resistance
to dexamethasone, despite the presence of a full-length
glucocorticoid receptor. These studies may have implications for
the rational design of effective ATO-based regimes for the
treatment of refractory myeloma.
376
Melphalan, Arsenic Trioxide (ATO) and Ascorbic Acid
Combination Therapy (MAC) in Refractory and
Relapsing Multiple Myeloma (MM)
Berenson J.R., Borad M.J., Vescio R.A., Yang H.H., Swift
R.
Cedars-Sinai Medical Center
Pre-clinical studies in our laboratory have shown that the addition
of arsenic trioxide to chemotherapy can greatly reduce the
concentration of chemotherapy required to kill myeloma tumor
cells compared to chemotherapy alone. As a result on this, we
evaluated a combination of oral melphalan (0.05-0.1 mg/kg qd
for four days every six weeks), ATO (0.25 mg/kg IV qd for the
first four days followed by twice weekly infusions) and ascorbic
acid (1 g IV qd for the first four days followed by twice weekly
infusions) to treat eight patients with refractory MM (ages 47-63;
two males and six females; IgG subtype (n=3), IgA subtype (n=3)
and light chain disease (n=2) associated renal failure (n=5; serum
creatinine 2.1, 3.1, 5.1, 5.2, and 6.1 mg/dL)) for a duration
ranging from 14-37 weeks. The patients had failed multiple prior
therapeutic regimens including high dose glucocorticoids (n=6),
VAD (n=6), melphalan (n=3), autologous peripheral stem cell
transplantation (n=3), thalidomide (n=6) and PS-341 (n=3) prior
to MAC administration. At the time of initiation of MAC
therapy, all the patients were on oral glucocorticoids (oral
methylprednisolone, prednisone or dexamethasone) which was
maintained during MAC therapy. Seven of the eight patients
responded with a reduction of serum paraprotein from 25-45%
and 26-58% reduction in the urine M-protein. Importantly, in four
of the five patients with renal failure, renal functions improved
with a reduction of serum creatinine from 39-56% reduction in
the serum creatinine (from 2.3 to 1.4 mg/dL, 5.1 to 2.4 mg/dL,
5.1 mg/dL to 3.0 mg/dL and 6.2 to 2.7 mg/dL). One patient with
profound hypercalcemia despite treatment with zoledronic acid
normalized her serum calcium after one cycle of MAC therapy.
Two of the eight patients progressed after responding for 21 and
26 weeks. MAC was generally well tolerated. One patient with
light chain disease with associated renal failure failed to respond
to MAC treatment. The most common adverse events noted with
this combination were marrow suppression (anemia n=8,
leukopenia n=6 and thrombocytopenia n=4), prolongation of the
QT interval (n=3), gastrointestinal symptomatology (n=3),
reactivation of zoster (n=2), headache (n=2), pulmonary edema
(n=1), hyperpigmentation (n=1) and neuropathy (n=1). The
combination of arsenic trioxide, ascorbic acid and low-dose oral
melphalan appears to be effective and well tolerated treatment for
patients with refractory and relapsing myeloma, including
patients with renal failure. Because of these encouraging results,
a large Phase II trial is beginning.
377
A Peptide Trivalent Arsenical Induces Apoptosis in
Myeloma Cell Lines
William W Xu1,2, Malcolm A King3, Philip Hogg4,
Christopher M Ward1,2
1Northern Blood Research Centre, University of Sydney;
2Department of Haematology and Transfusion Medicine, Royal
North Shore Hospital; 3Department of Clinical Immunology, Pacific
Laboratory Medical Services; 4Center of Vascular Research,
University of New South Wales
High-dose chemotherapy and autologous transplantation are
unable to prevent disease progression in the great majority of
patients with multiple myeloma. Novel therapies which target the
interactions between malignant plasma cells and the bone marrow
microenvironment, such as thalidomide, proteasome inhibitors
and arsenic trioxide have the potential to achieve durable
remissions, even in patients whose disease has become refractory
to standard chemotherapy. In previous reports, arsenic trioxide
(As2O3) has been shown to induce apoptosis in a range of
myeloma cell lines via caspase-9 activation. This effect can be
augmented in vitro by the concurrent addition of ascorbic acid,
which depletes intracellular glutathione. Clinical trials of As2O3
are underway in myeloma, but as with thalidomide, drug toxicity
may limit its utility in older patients. “Second-generation” agents
with reduced toxicity are urgently needed to improve patient
outcomes.
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