Haematologica 2003 - Supplements

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THE IGF/IGF-1R SYSTEM IS A MAJOR THERAPEUTIC
TARGET FOR MULTIPLE MYELOMA AND OTHER
MALIGNANCIES
Constantine S. Mitsiades1,2, Nicholas Mitsiades1,2,
Andrew L. Kung3, Ciaran J. McMullan1,2, Carlos Garcia-
Echeverria4, Mark A. Pearson4, Francesco Hofmann4,
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. Department of Cell Biology and Pediatric
Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts,
USA; 4. Oncology Research, Novartis Pharma AG, Basel,
Switzerland
We and others have shown that insulin-like growth factors (IGFs)
stimulate proliferation of
multiple myeloma (MM) cells and protect them from
Dexamethasone (Dex)- or Apo2L/TRAILinduced apoptosis. We
now show that the IGF/IGF-1 receptor (IGF-1R/CD221) pathway
represents a major therapeutic target for MM cells and other
neoplasias. We studied a panel of 25 MM cell lines, including
cells resistant to Dex, melphalan, anthracyclines, Apo2L/TRAIL,
thalidomide and its novel immunomodulatory derivatives; 30
tumor samples from MM patients, including patients resistant to
IMiDs or PS-341; as well as 30 cell lines from several
hematologic malignancies, including B- and T-ALL, AML,
CML, various non-Hodgkin's lymphoma (NHL) subtypes, and
solid tumors (e.g. breast, prostate, lung, colon, thyroid, ovarian,
renal Ca, retinoblastoma). Cell surface IGF-1R expression was
confirmed by flow cytometry in all cell lines and 30/30 MM
patient samples tested. No correlation was noted between the
degree of IGF-1R expression and the drug sensitivity pattern of
MM cells. Because IGFs are present in high levels in peripheral
blood serum (fetal bovine, from healthy donors or autologous
MM patients’ sera) and can stimulate proliferation/survival of
tumor cells throughout the body, we specifically evaluated the
impact of inhibiting the IGF/IGF-1R pathway in serum-cultured
tumor cells, using anti-IGF-1R neutralizing monoclonal
antibodies (mAb); an IGF-1-like peptide that binds IGF-1R but
competitively inhibits activation of its Tyr kinase; and a specific
IGF-1R Tyr kinase small molecule inhibitor (Novartis AG, Basel,
Switzerland). All 3 IGF-1R-inihibitory molecules had
comparable effect in profoundly suppressing serum-induced
proliferation/survival of all MM cells (median % inhibition of
>70%) and cell lines from both solid tumors and hematologic
malignancies (with less activity, though, against some NHL
cells), while specific anti-IL-6R neutralizing mAb’s had minimal,
if any, effect on serum-cultured MM cells. We also found that
IGFs are not only present in circulation, but are also produced in
the BM microenvironment by autocrine (MM cells) and paracrine
(BMSCs, osteoblasts) sources. Through gene expression and
proteomic profiling of IGF-1R-inhibitor-treated MM cells, we
found that IGF-1R inhibition blocks key growth/survival
pathways (e.g. PI-3K/Akt, Ras/Raf/MAPK, IKK-á/NF-êB);
blocks expression of several inhibitors of apoptosis (e.g. FLIP,
XIAP, cIAP-2, survivin); neutralizes pro-apoptotic Forkhead
transcription factors; suppresses both constitutive and serum- or
IGF-1-induced upregulation of proteasome activity and
telomerase activity; increases sensitivity of MM cells to Dex,
chemotherapy and PS-341; and overcomes the protection
conferred to MM cells by their co-culture with BMSCs.
Importantly, our in vivo studies in SCID/NOD mice model of
diffuse MM bone lesions showed that IGF-1R kinase inhibitor
significantly suppresses MM tumor growth, improves overall
survival of mice and enhances the anti-MM effect of cytotoxic
chemotherapy, without major toxicities. Our studies indicate that
IGFs (present in serum or from autocrine/paracrine sources) and
IGF-1R signaling in tumor cells play critical roles in stimulation
of proliferation, survival and drug-resistance of MM, as well as
other malignancies. Most importantly, our studies provide the
first proof-of-principle that inhibition of IGF/IGF-1R pathway
can be achieved with favorable therapeutic window and that
small molecule IGF-1R kinase inhibitors constitute promising
therapeutic agents for MM, and other neoplasias.
5.2 Apoptosis and survival signalling
124
In myeloma cells apoptosis induced by inhibition of
Janus kinase 2 is independent of IL-6- or BMSCmediated
STAT3 activation
Manik Chatterjee, Thorsten Stühmer, Kurt Bommert, Bernd
Dörken, and Ralf C. Bargou
Department of Hematology, Oncology and Tumorimmunology,
Robert-Rössle Cancer Center at the Max-Delbrück-Center for
Molecular Medicine, Humboldt University of Berlin, Germany
Introduction: It has recently been reported that constitutive
activation of STAT3 signaling confers resistance to apoptosis in
human multiple myeloma (MM) cell lines. Accordingly, it has
been demonstrated that the Janus kinase 2 (Jak2) inhibitor
tyrphostin AG490 inhibits STAT3 phosphorylation and induces
apoptosis in certain MM cell lines. Therefore, tyrphostins are
thought to be useful drugs for the treatment of MM. However,
data concerning the role of tyrphostins in human MM are limited
to cell lines and do not generally consider contributions of the
bone marrow microenvironment, which has been found to confer
resistance to drug treatment in MM. Data on primary MM cells
are missing and data describing the specificity and potential side
effects of typhostins are scant.
Experimental model: To determine the role of the STAT3
activation status for the induction of apoptosis by AG490, we
analyzed two different human MM cell lines: INA-6, which is
strictly dependent on IL-6 and consequently maintains an
activated STAT3 pathway, and MM.1S which is independent on
IL-6 and lacks constitutive activation of STAT3. We also
employed a co-culture model with MM cells (INA-6 or primary
MM cells) and bone marrow stromal cells (BMSCs) to analyze
AG490-mediated effects. In order to assess effects of Jak2
inhibition on hematopoietic stem cells CD34+ enriched cells were
treated with AG490.
Results: Here we show that treatment with AG490 induces
apoptosis of human MM cells in the absence or presence of
BMSCs to a similar extent. Interestingly, this effect was observed
not only with INA-6 cells, where strict dependence on IL-6 keeps
the STAT3 pathway activated, but also with MM.1S cells, where
STAT3 activity is absent. Primary MM cells were susceptible to
AG490-induced apoptosis as well. Of interest, these experiments
revealed a strong inhibitory effect of AG490 on the growth of
hematopoietic stem cells in vitro. Signaling analysis revealed that
treatment with AG490 led to a significant inhibition of
phosphorylation of STAT3 only in the absence of BMSCs and
only at very high concentrations. In the presence of BMSCs and
at lower concentrations, that were nonetheless sufficient to induce
apoptosis, no inhibition of STAT3 could be observed.
Additionally, treatment with AG490 did not result in significant
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