Haematologica 2003 - Supplements
Haematologica 2003 - Supplements Haematologica 2003 - Supplements
121 The role of membrane raft in initiating CD40 signaling and ERK activation in modulating CD40-induced cytokine production in human multiple myeloma cells Yu-Tzu Tai,1 Leurence P. Catley,1 Klaus Podar,1 Stine- Kathrein Kraeft,2 Renate Burger,1 Reshma Shringarpure,1 Masaharu Akiyama,1 Teru Hideshima,1 Dharminder Chauhan,1 Nicholas Mitsiades,1 Constantine S. Mitsiades,1 Nikhil C. Munshi,1 Paul Richardson,1 Lan Bo Chen,2 and Kenneth C. Anderson1 1The Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; 2Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, MA, USA. CD40 activation of human multiple myeloma (MM) induces PI3K/AKT/NF-κB activation, which mediates MM cell transmigration. CD40 activation in human MM also activates the MAP kinase (MEK) pathway, evidenced by phosphorylation of ERK, but not JNK or p38. In the present study, we examined the role of membrane raft in initiating CD40 signaling and the role of ERK activation in CD40- dependent regulation of cytokine/chemokine production in MM.1S cells, as well as patient plasma cell leukemia cells. Following crosslinking by sCD40L or anti-CD40 mAb G28.5, CD40 receptors on human MM cells move to membrane rafts, cholesterol-rich plasma membrane microdomains, as illustrated by dual immunofluorescence staining with FITC-labeled cholera toxin B against membrane raft ganglioside GM1 and Alexa 568-conjugated anti-CD40 mAb. Since Src is a membrane raft associated-signaling protein, we determined whether Src and intact membrane rafts modulate tyrosine phosphorylation (pTyr) following CD40 activation. Using methyl-βcyclodextrin to disrupt cholesterol structure on cell membrane or the Src-family specific inhibitor PP2, we demonstrated that CD40-induced pTyr events are initiated in membrane rafts and are dependent on Src family kinase activation. Since CD40 activation of MM.1S and plasma cell leukemia cells, even at concentrations as high as 20 µg/ml, did not significantly alter DNA synthesis (p = 0.15 and 0.19, respectively), we next studied effects of CD40-induced ERK phosphorylation on cytokine/chemokine mRNA expression in MM.1S and plasma cell leukemia cells. Following CD40 activation, ERK activation was induced and persisted for an hour following treatment of sCD40L (2 µg/ml) and phosphorylation of c-Myc was also observed. We found that chemokine RANTES and IL-8 mRNAs were up-regulated in MM.1S and plasma cell leukemia cells by CD40 activation for 6-24 hr. Macrophage chemoattractant protein-1 (MCP-1) mRNA was also significantly induced. These results are in contrast to published reports in tonsillar B cells where only p38 is induced (J Immunol. 163, 5786- 95, 1999). Importantly, conditioned medium collected from CD40- activated and unstimulated MM.1S cells significantly increased normal PBMC cell transmigration (6.3% + 1.3 migrating cells vs 2.8% + 1.2, p
123 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 S142
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121<br />
The role of membrane raft in initiating CD40 signaling<br />
and ERK activation in modulating CD40-induced<br />
cytokine production in human multiple myeloma cells<br />
Yu-Tzu Tai,1 Leurence P. Catley,1 Klaus Podar,1 Stine-<br />
Kathrein Kraeft,2 Renate Burger,1 Reshma Shringarpure,1<br />
Masaharu Akiyama,1 Teru Hideshima,1 Dharminder<br />
Chauhan,1 Nicholas Mitsiades,1 Constantine S.<br />
Mitsiades,1 Nikhil C. Munshi,1 Paul Richardson,1 Lan Bo<br />
Chen,2 and Kenneth C. Anderson1<br />
1The Jerome Lipper Multiple Myeloma Center, Department of Medical<br />
Oncology, Dana-Farber Cancer Institute, Boston, MA, USA;<br />
2Department of Cancer Biology, Dana-Farber Cancer Institute, and<br />
Department of Medicine, Harvard Medical School, Boston, MA, USA.<br />
CD40 activation of human multiple myeloma (MM) induces<br />
PI3K/AKT/NF-κB activation, which mediates MM cell transmigration.<br />
CD40 activation in human MM also activates the MAP kinase (MEK)<br />
pathway, evidenced by phosphorylation of ERK, but not JNK or p38.<br />
In the present study, we examined the role of membrane raft in<br />
initiating CD40 signaling and the role of ERK activation in CD40-<br />
dependent regulation of cytokine/chemokine production in MM.1S<br />
cells, as well as patient plasma cell leukemia cells. Following crosslinking<br />
by sCD40L or anti-CD40 mAb G28.5, CD40 receptors on<br />
human MM cells move to membrane rafts, cholesterol-rich plasma<br />
membrane microdomains, as illustrated by dual immunofluorescence<br />
staining with FITC-labeled cholera toxin B against membrane raft<br />
ganglioside GM1 and Alexa 568-conjugated anti-CD40 mAb. Since<br />
Src is a membrane raft associated-signaling protein, we determined<br />
whether Src and intact membrane rafts modulate tyrosine<br />
phosphorylation (pTyr) following CD40 activation. Using methyl-βcyclodextrin<br />
to disrupt cholesterol structure on cell membrane or the<br />
Src-family specific inhibitor PP2, we demonstrated that CD40-induced<br />
pTyr events are initiated in membrane rafts and are dependent on Src<br />
family kinase activation. Since CD40 activation of MM.1S and plasma<br />
cell leukemia cells, even at concentrations as high as 20 µg/ml, did not<br />
significantly alter DNA synthesis (p = 0.15 and 0.19, respectively), we<br />
next studied effects of CD40-induced ERK phosphorylation on<br />
cytokine/chemokine mRNA expression in MM.1S and plasma cell<br />
leukemia cells. Following CD40 activation, ERK activation was<br />
induced and persisted for an hour following treatment of sCD40L (2<br />
µg/ml) and phosphorylation of c-Myc was also observed. We found<br />
that chemokine RANTES and IL-8 mRNAs were up-regulated in<br />
MM.1S and plasma cell leukemia cells by CD40 activation for 6-24 hr.<br />
Macrophage chemoattractant protein-1 (MCP-1) mRNA was also<br />
significantly induced. These results are in contrast to published reports<br />
in tonsillar B cells where only p38 is induced (J Immunol. 163, 5786-<br />
95, 1999). Importantly, conditioned medium collected from CD40-<br />
activated and unstimulated MM.1S cells significantly increased normal<br />
PBMC cell transmigration (6.3% + 1.3 migrating cells vs 2.8% + 1.2,<br />
p