<p>Multiple myeloma (MM) is an incurable B-cell malignancy mainly localized to the bone marrow. Our aim was to examine the growth- and survival-promoting role of the IGF-IR and its downstream signaling components in MM cells to identify potential targets for therapy. </p><p>Octreotide, a somatostatin analog that has been demonstrated to interfere with the actions of IGF-I, induced growth inhibition in both IL-6-dependent and IL-6-independent MM cell lines expressing the somatostatin receptors sst2, sst3 and sst5. Additionally, a slight pro-apoptotic effect could be observed in a few cell lines. In primary MM cells octreotide induced apoptosis, an effect that was abrogated by exogenously added IGF-I, but not by IL-6.</p><p>Inhibition of IGF-I signaling in Karpas 707 cells, using either the anti-IGF-IR antibody αIR3 or the PI 3-K inhibitors LY294002 and wortmannin, increased sensitivity to apoptosis induced by dexamethasone. Exogenously added IGF-I prevented dexamethasone-induced apoptosis, an effect that could partly be mimicked by the pharmacological GSK-3β inhibitors LiCl and SB415286. Thus, we suggest the GSK-3β as an important mediator of the anti-apoptotic effects of IGF-IR signaling in MM.</p><p>Using rapamycin we selectively inhibited mTOR, a phosphoprotein downstream of the IGF-IR. In MM cell lines rapamycin induced G0/G1-arrest, an effect being associated with an increase of the cyclin-dependent kinase inhibitor p27 and a decrease of the cyclins D2, D3 and E. Interestingly, in primary MM cells rapamycin induced apoptosis. Moreover, rapamycin potentiated dexamethasone-induced apoptosis, an effect that was associated with a downregulation of the anti-apoptotic protein survivin. Strikingly, the combinatorial treatment with rapamycin and dexamethasone suppressed the anti-apoptotic effects of exogenously added IGF-I and IL-6, thus suggesting this drug-combination to be active also in vivo. </p><p>Two newly developed, selective IGF-I RTK inhibitors proved to be very effective in MM cell lines and in primary MM cells providing 50-90% growth inhibition within 48 h of incubation. The inhibitors induced massive apoptosis together with a prominent cell cycle arrest in the G2/M-phase. Importantly, the IGF-I RTK inhibitors downregulated the tyrosine phosphorylation of the IGF-IR β-chain but not of the insulin receptor β-chain. </p><p>In conclusion, the IGF-IR potently promotes growth and survival of MM cells. Therefore, interfering with the IGF-IR signaling pathway might be a suitable strategy to improve MM treatment.</p>

<p>Multiple myeloma (MM) is a virtually incurable B cell malignancy of the bone marrow. One important part of tumor progression and an obstacle for successful therapy is resistance to apoptosis. To combat this resistance, the mechanisms of apoptosis and survival in MM must be better defined. </p><p>In this thesis, we identified Fas up-regulation as a mechanism underlying interferon (IFN)-mediated sensitization to Fas-induced apoptosis in the MM cell line U-266-1970. IFN treatment induced activation of signal transducer and activator of transcription (Stat)1 but, intriguingly, also attenuated activation of MM survival factor Stat3. </p><p>Exploring the role of Stat1 further, we established sub-lines of U-266-1970 with a stable over-expression of Stat1 and of its active mutant Stat1C. These sub-lines displayed a decreased expression and activation of Stat3, and an altered expression of apoptosis-related genes Harakiri, Bcl-2 and Mcl-1. In a drug library screening, Stat1 over-expression was associated with an increased sensitivity to Fas-induced apoptosis and, conversely, an increased resistance to several drugs, including the cyclin dependent kinase (cdk)1 inhibitor CGP74514A. We conclude that Stat1 over-expression does not confer a general resistance or sensitivity to apoptosis in MM, but may strongly affect the response to some specific drugs.</p><p>We also explored the effects of picropodophyllin (PPP), an inhibitor of the insulin-like growth factor I (IGF-I) receptor tyrosine kinase (RTK), in MM. PPP selectively inhibited the IGF-I RTK activity without inhibiting the insulin RTK activity. Furthermore, PPP potently induced cell cycle arrest and apoptosis in all MM cell lines and patient samples tested, also in the presence of survival factors IGF-I and IL-6. We conclude that PPP has great therapeutic potential in MM </p><p>Finally, we examined the expression and regulation of the inhibitors of apoptosis proteins (IAPs) in a panel of MM cell lines and patient samples. The glucocorticoid dexamethasone, which is used in MM therapy, induced a transient up-regulation and a subsequent down-regulation of c-IAP2, as well as a down-regulation of XIAP, possibly influencing the sensitivity to apoptosis induced by this drug. Supporting this notion, abrogation of IGF-IR signaling by PPP, which sensitizes MM cells to dexamethasone-induced apoptosis, enhanced the down-regulation of c-IAP2 and XIAP.</p>

Multiple myeloma (MM) is an incurable B-cell malignancy mainly localized to the bone marrow. Our aim was to examine the growth- and survival-promoting role of the IGF-IR and its downstream signaling components in MM cells to identify potential targets for therapy. Octreotide, a somatostatin analog that has been demonstrated to interfere with the actions of IGF-I, induced growth inhibition in both IL-6-dependent and IL-6-independent MM cell lines expressing the somatostatin receptors sst2, sst3 and sst5. Additionally, a slight pro-apoptotic effect could be observed in a few cell lines. In primary MM cells octreotide induced apoptosis, an effect that was abrogated by exogenously added IGF-I, but not by IL-6. Inhibition of IGF-I signaling in Karpas 707 cells, using either the anti-IGF-IR antibody αIR3 or the PI 3-K inhibitors LY294002 and wortmannin, increased sensitivity to apoptosis induced by dexamethasone. Exogenously added IGF-I prevented dexamethasone-induced apoptosis, an effect that could partly be mimicked by the pharmacological GSK-3β inhibitors LiCl and SB415286. Thus, we suggest the GSK-3β as an important mediator of the anti-apoptotic effects of IGF-IR signaling in MM. Using rapamycin we selectively inhibited mTOR, a phosphoprotein downstream of the IGF-IR. In MM cell lines rapamycin induced G0/G1-arrest, an effect being associated with an increase of the cyclin-dependent kinase inhibitor p27 and a decrease of the cyclins D2, D3 and E. Interestingly, in primary MM cells rapamycin induced apoptosis. Moreover, rapamycin potentiated dexamethasone-induced apoptosis, an effect that was associated with a downregulation of the anti-apoptotic protein survivin. Strikingly, the combinatorial treatment with rapamycin and dexamethasone suppressed the anti-apoptotic effects of exogenously added IGF-I and IL-6, thus suggesting this drug-combination to be active also in vivo. Two newly developed, selective IGF-I RTK inhibitors proved to be very effective in MM cell lines and in primary MM cells providing 50-90% growth inhibition within 48 h of incubation. The inhibitors induced massive apoptosis together with a prominent cell cycle arrest in the G2/M-phase. Importantly, the IGF-I RTK inhibitors downregulated the tyrosine phosphorylation of the IGF-IR β-chain but not of the insulin receptor β-chain. In conclusion, the IGF-IR potently promotes growth and survival of MM cells. Therefore, interfering with the IGF-IR signaling pathway might be a suitable strategy to improve MM treatment.

Multiple myeloma (MM) is a virtually incurable B cell malignancy of the bone marrow. One important part of tumor progression and an obstacle for successful therapy is resistance to apoptosis. To combat this resistance, the mechanisms of apoptosis and survival in MM must be better defined. In this thesis, we identified Fas up-regulation as a mechanism underlying interferon (IFN)-mediated sensitization to Fas-induced apoptosis in the MM cell line U-266-1970. IFN treatment induced activation of signal transducer and activator of transcription (Stat)1 but, intriguingly, also attenuated activation of MM survival factor Stat3. Exploring the role of Stat1 further, we established sub-lines of U-266-1970 with a stable over-expression of Stat1 and of its active mutant Stat1C. These sub-lines displayed a decreased expression and activation of Stat3, and an altered expression of apoptosis-related genes Harakiri, Bcl-2 and Mcl-1. In a drug library screening, Stat1 over-expression was associated with an increased sensitivity to Fas-induced apoptosis and, conversely, an increased resistance to several drugs, including the cyclin dependent kinase (cdk)1 inhibitor CGP74514A. We conclude that Stat1 over-expression does not confer a general resistance or sensitivity to apoptosis in MM, but may strongly affect the response to some specific drugs. We also explored the effects of picropodophyllin (PPP), an inhibitor of the insulin-like growth factor I (IGF-I) receptor tyrosine kinase (RTK), in MM. PPP selectively inhibited the IGF-I RTK activity without inhibiting the insulin RTK activity. Furthermore, PPP potently induced cell cycle arrest and apoptosis in all MM cell lines and patient samples tested, also in the presence of survival factors IGF-I and IL-6. We conclude that PPP has great therapeutic potential in MM Finally, we examined the expression and regulation of the inhibitors of apoptosis proteins (IAPs) in a panel of MM cell lines and patient samples. The glucocorticoid dexamethasone, which is used in MM therapy, induced a transient up-regulation and a subsequent down-regulation of c-IAP2, as well as a down-regulation of XIAP, possibly influencing the sensitivity to apoptosis induced by this drug. Supporting this notion, abrogation of IGF-IR signaling by PPP, which sensitizes MM cells to dexamethasone-induced apoptosis, enhanced the down-regulation of c-IAP2 and XIAP.

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