Haematologica 2003 - Supplements

P12.2.4
NFKB. A NEW THERAPEUTIC TARGET FOR
OVERCOMING DRUG RESISTANCE.
James R.Berenson, MD
Director of Multiple Myeloma and Bone Metastasis Programs at
Cedars-Sinai Medical Center, Los Angeles, CA
Activation of apoptosis in cancer cells resulting from NF-kB
inhibition suggests that NF-kB inhibition could be used as a
mechanism to treat cancers. To inhibit the activity of NF-kB,
several genetic studies were carried out by homologous
recombination to either directly destroy NF-kB/p65 function or
indirectly suppress NF-kB activity through destroying IKKb (IkB
kinase) function and thereby upregulating IkB activity. To
establish that inhibition of NF-kB activity induces apoptosis in
MM cells, we recently carried out viral transduction experiments
in which dominant negative IkBa was introduced into both
melphalan-sensitive and -resistant MM cells. The cellular
apoptosis was noted to greatly increase in melphalan-sensitive
and resistant MM cells compared to viral vector alone. These
studies confirm the notion that inhibition of NF-kB activity can
precipitate cell death in MM cells through induction of apoptosis.
The proteasome inhibitor, PS-341 (Bortezomib or Velcade), is a
novel drug that was designed to specifically block the signal
transduction pathways mediated by NF-kB. The destruction of
IkBa proteins following their phosphorylation by IKK and
subsequent ubiquitination is primarily mediated by the
proteasome degradation that can be inhibited by PS-341.
Blocking the proteasome degradation of IkBa by PS-341
significantly inhibits NF-kB activity, resulting in the stimulation
of apoptosis of myeloma cells.
PS-341 has been shown to be quite effective in inhibiting human
myeloma cell growth both in vitro and in vivo. Specifically, the
growth of both chemo-sensitive and -resistant MM cell-lines was
substantially inhibited by PS-341 treatment. Interestingly, there is a
"right shift" in the dose-response curves for chemoresistant cell
lines, suggesting that the chemoresistant cell-lines appear to be
more sensitive to the treatment of PS-341 than the chemosensitive
lines. The alteration in NF-kB activity appears to be one of the
major mechanisms of anti-myeloma activity of PS-34.
In support of this, the nuclear translocation of NF-kB and its
subsequent DNA binding are decreased in MM cell lines that
were treated with PS-341.
Many tumor cells especially MM tumor cells display
constitutively high levels of NF-kB activity. In response to
chemotherapy, the activity of NF-kB is further enhanced resulting
in chemoresistance. Thus, the inhibition of its activity can be
used to reverse the chemoresistant phenotype of a variety of
cancer cells. Using NF-kB inhibition together with cytotoxic
agents has also been tried in MM treatment, and this combination
strategy has been evaluated using in vitro studies since the
availability of PS-341. We demonstrated a marked synergistic
effect exists between PS-341 and various chemotherapeutic
agents in inhibiting MM cell growth. We treated several chemosensitive
MM cell-lines along with chemo-resistant lines with
several chemotherapeutic agents, including doxorubicin,
mitoxantrone and melphalan, that were used either alone or in
combination with a low, non-cytotoxic dose of PS-341 (5 ng/ml).
We saw no significant growth inhibition of chemo-resistant lines
when they are treated with chemotherapeutic agents alone until
high concentrations of chemotherapy were applied. However,
when the cells were treated with PS-341 together with
chemotherapeutic agents, these chemo-resistant cell-lines became
extremely sensitive to chemotherapeutic agents. For example, the
cytotoxic dose of melphalan when used together with PS-341 was
1,000,000-fold lower than the concentration necessary for
melphalan alone to induce cytotoxicity in a highly melphalanresistant
MM cell lines. Similar effects were observed between
PS-341 and doxorubicin or mitoxantrone as the combination
markedly increased the sensitivity of both doxorubicin-resistant
and mitoxantrone-resistant MM cell-lines by approximately
100,000-fold. Parallel with the increase in chemosensitivity, there
also was a marked increase in apoptosis of chemoresistant MM
cell lines induced by this combined approach.
The synergy observed between PS-341 and chemotherapeutic
agents appear to be cell-type specific. Synergistic effects between
PS-341 and chemotherapeutic agents were not found when they
were used together to treat other types of tumor cell lines. Similar
experiments were also performed on normal unstimulated and
mitogen stimulated peripheral blood mononuclear cells (PBMCs)
and CD34-selected BMMCs obtained from healthy individuals.
Suppression of proliferation in these non-MM cell-lines or
normal hematopoietic cells was not found with PS-341 treatment
except at higher concentrations (IC50 50 - 75 ng/ml). Moreover,
the addition of PS-341 to chemotherapy had minimal synergistic
inhibitory effects on cell growth in these same samples. This
observation is interesting because the extent of synergy between
PS-341 and cytotoxic agents also correlates with the baseline
levels of NF-kB activity identified in each cell type evaluated.
This finding is also important and clinically relevant since the
difference in cell response to the combined treatments between
myeloma cells and normal cells could provide an excellent
therapeutic/toxicity ratio for this approach for treating MM
patients. As a result of these encouraging in vitro results, we
began a Phase I clinical trial to study the efficacy lower doses of
both PS-341 (using 40% of the dose/month in the previous large
Phase II SUMMIT trial) and oral melphalan as combination
therapy in treating refractory and relapsed MM patients. Even
among all three patients receiving the lowest melphalan dose
(only 0.025 mg/kg daily X 4), dramatic decreases in paraprotein
levels were observed, and, in fact, responses have been observed
in all five 3-patient cohorts (see H Yang et al, IXth International
Workshop on Multiple Myeloma for details). Importantly, this
combination has been associated with minimal neurotoxicity.
In addition to the proteasome inhibitors, other
pharmacotherapeutic agents also can block NF-kB signaling.
One example is arsenic trioxide. Arsenic trioxide has been shown
to be a potent NF-kB inhibitor. It binds to the cysteine residue
179 in the activation loop of IKK catalytic subunits and thereby
blocks the IKK activity. This results in a lack of IkB
phosphorylation and inability for the IkB to be ubiquitinated and
proteasome degraded. Indeed, exposure of MM cells to arsenic
results in accumulation of IkB and reduced nuclear accumulation
of NF-kB and DNA-binding of this transcription factor. Similar
to PS-341, arsenic trioxide also sensitizes myeloma cells to
chemotherapy in vitro and in vivo. Based on these preclinical
findings, we treated eight relapsing myeloma patients with a
combination of low-dose oral melphalan, arsenic trioxide and
ascorbic acid.
The latter agent was used because previous studies showed that
this vitamin was able to reduce glutathione levels and increase the
anti-myeloma effects of arsenic trioxide. Seven of the eight
patients showed reduction in paraprotein (25-58%), and four of
the patients with renal failure showed marked improvement in
renal function on this regimen which was well tolerated. These
studies suggest that inhibition of NF-kB activity may allow use of
reduced doses of both chemotherapy and the NF-kB blocking
agents resulting in enhanced anti-myeloma effects with reduced
toxicity.
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