ALBERT

Description: Abstract 2829 Poster Board II-805 Introduction: We could recently show that the heat shock proteins (HSP) HSP90 and HSP70 are frequently overexpressed in multiple myeloma (MM), stabilize as molecular chaperones various oncogenic proteins and contribute to survival of MM cells. Currently, several clinical Phase I/II studies are under way to evaluate the concept of pharmacological HSP90 blockade in human cancer. Under cellular stress conditions the heat shock transcription factor 1 (HSF1) has a key regulatory role for the up-regulation of HSP. Importantly, it has been observed that treatment with the proteasome inhibitor bortezomib, a clinically effective anti-MM agent, induces up-regulation of HSP90, HSP70 and HSP27. Furthermore, it has recently been demonstrated that HSF1 can protect cells from oncogene-driven malignant transformation. We therefore analyzed the role of HSF1 for the malignant growth of MM cells. Methods: Western analyses were performed to determine HSF1 expression and regulation in different human MM cell lines. To examine the expression of HSF1 and different HSP like HSP90, HSP70 and HSP27 in situ, samples from 60 bone marrow biopsies obtained from MM patients were immunohistochemically stained. To analyze the role of HSF1 for the survival of MM cells, HSF1 was either selectively depleted by siRNA-mediated knockdown using a pSUPER-based siRNA expression vector or targeted by treatment with a novel pharmacological HSF1 inhibitor triptolide. In addition, pharmacological inhibition of HSF1 was combined with concomitant pharmacological inhibition of either HSP90 (with the novel inhibitor NVP-AUY922) or bortezomib. Furthermore, gene expression analyses with an Affymetrix GeneChip were performed to identify HSF1 target genes in MM cells. Results: Here, we show that HSF1 is frequently overexpressed in MM cell lines in vitro and in the majority of the analyzed MM biopsies in situ, but not in MGUS or in normal plasma cells. Blockade of HSF1 either by siRNA-mediated knockdown or treatment with the novel pharamacological HSF1 inhibitor triptolide led to a strong induction of apoptosis in cells of the MM cell lines INA-6 and MM.1s. Importantly, also primary MM cells showed apoptosis induction after triptolide treatment. HSF1 inhibition led to downregulation of HSP70, HSP27 and HSP90. Gene expression analysis revealed a number of additional molecular targets of HSF1 involved in apoptosis regulation. Furthermore, initial experiments indicated that the apoptotic effect of pharmacological HSF1 inhibition is enhanced by the concomitant pharmacological inhibition of either HSP90 or the proteasome. Conclusion: We demonstrate that HSF1 is overexpressed in MM, contributes to the survival of MM cells and controls the activity of oncogenic HSP like HSP90, HSP70 and HSP27. Targeting HSF1 may therefore represent an attractive potential therapeutic strategy in MM, in particular in combination with HSP90 or proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: The PI3K/Akt pathway has been reported to critically contribute to survival and malignant growth of multiple myeloma (MM). Because most of these data are based on pharmacologic inhibition it is not clear if the effects are due to Akt inhibition or off-target effects. Furthermore, the Akt family of kinases consists of three highly homologous isoforms, that may, nonetheless, display individual functional properties. We therefore conducted siRNA experiments to evaluate if any single isoform posesses a special role for the viability of MM cells. This was complemented with extensive analyses into the functional and signaling properties of the Akt pathway in primary MM cells (n = 30). Our knock-down experiments revealed that in MM.1S, an MM cell line with constitutive phospho-Akt signaling, Akt1 and Akt2 both contributed to MM cell survival whereas Akt3 seemed to be of less relevance. Conversely, survival of MM cell line AMO-1 which has no constitutive phospho-Akt signal was completely unaffected. Treatment of these MM cell lines with the Akt1 and Akt2 specific inhibitor Akti-1/2 showed that this drug totally abolished the phospho-Akt signal in MM.1S at a concentration of 10 microM. Again, MM.1S cells underwent apoptosis whereas AMO-1 cells were resistant. Next, we analyzed Akt signaling in a large panel of primary MM samples. Phosphorylated Akt was determined with intracellular staining and flow cytometry analysis in primary tumor samples and could be detected in about 60% of MM cases. This constitutive signal could be blocked with Akti-1/2 in the presence and absence of bone marrow stromal cells. Pharmacologic inhibition of Akt led to strong induction of cell death in 46% of primary MM samples, whereas the rest was largely resistant to Akt inhibition. The samples sensitive to Akt inhibition were mostly identical to those that displayed a constitutive phospho-Akt signal. Of interest, there was no correlation between Akt dependence and mutational inactivation of PTEN. Further inhibition of other signaling cascades implicated in growth and survival of MM cells, such as the MAPK or STAT3 pathways, had only minor additional effects on tumor cell viability of samples resistant to Akt inhibition. Our analysis indicates substantial heterogeneity in MM cells that defines Akt dependent and Akt independent MM subgroups. Akt1 and Akt2 proved relevant for the survival of subsets of MM cell lines and primary samples. Taken together, this is the first comprehensive functional and molecular signaling analysis of primary MM samples which led to the identification of novel functionally defined myeloma subgroups.

Description: Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Description: The p53 pathway is central to cellular defences against neoplastic transformation, and mutations that impair p53 function are prominent oncogenic events. However, certain malignancies, such as multiple myeloma (MM), rarely present with p53 defects except in late stages. MM might therefore be vulnerable to p53 induction therapy, but it is unknown if the p53 pathway remains actually functional in this disease, and if it can be sufficiently well triggered to induce tumor cell death. We have used nutlin-3, a newly developed small-molecule antagonist against the p53 suppressor murine double minute 2 (MDM2), to analyze the effect on myeloma cell lines and a large panel of patient tumor samples. Nutlin-3 specifically and exclusively induced p53 downstream targets in myeloma cells with wild-type p53, and a large majority of primary medullary myeloma samples was susceptible to nutlin-3-mediated apoptosis. Comparison with the clinically relevant genotoxic drugs melphalan and etoposide showed that nutlin-3 was as effective or better at inducing apoptosis of primary tumor cells, and that combinations of nutlin-3 with genotoxic cytostatics at low doses produced better than additive anti-tumor effects. Importantly, broad anti-tumor activity of nutlin-3 persisted even when primary tumor cells were kept in coculture with bone marrow stromal cells (BMSCs). However, primary tumor isolates often contain a fraction of cells apparently unaffected by drug treatment, and some primary samples were conspicuously less sensitive to nutlin exposure when the cells were kept in coculture with BMSCs, as compared to culture in medium supplemented with interleukin-6. We have begun to analyze to what extent heterogeneity for p53 induction within the tumor cell population may underlie these differences, and if the capacity to activate intracellular growth and survival pathways has an influence on the result of p53 pathway activation. Nutlin-3, as a non-genotoxic agent to specifically induce or strengthen p53-mediated apoptotic responses may be a promising agent to complement standard genotoxic regimens for the treatment of MM.

Description: The interleukin-6 receptor (IL-6R)/signal transducer and activator of transcription 3 (STAT3) pathway contributes to the pathogenesis of multiple myeloma (MM) and protects MM cells from apoptosis. However, MM cells survive the IL-6R blockade if they are cocultured with bone marrow stromal cells (BMSCs), suggesting that the BM microenvironment stimulates IL-6–independent pathways that exert a pro-survival effect. The goal of this study was to investigate the underlying mechanism. Detailed pathway analysis revealed that BMSCs stimulate STAT3 via the IL-6R, and mitogen-activated protein (MAP) kinases via IL-6R–independent mechanisms. Abolition of MEK1,2 activity with PD98059, or ERK1,2 small interfering RNA knockdown, was insufficient to induce apoptosis. However, the combined disruption of the IL-6R/STAT3 and MEK1,2/ERK1,2 pathways led to strong induction of apoptosis even in the presence of BMSCs. This effect was observed with MM cell lines and with primary MM cells, suggesting that the BMSC-induced activation of MEK1,2/ERK1,2 renders MM cells IL-6R/STAT3 independent. Therefore, in the presence of cells from the BM micro-environment, combined targeting of different (and independently activated) pathways is required to efficiently induce apoptosis of MM cells. This might have direct implications for the development of future therapeutic strategies for MM.

Description: Mutation of p53 is a rare event in multiple myeloma, but it is unknown if p53 signaling is functional in myeloma cells, and if targeted nongenotoxic activation of the p53 pathway is sufficient to kill tumor cells. Here, we demonstrate that treatment of primary tumor samples with a small-molecule inhibitor of the p53–murine double minute 2 (MDM2) interaction increases the level of p53 and induces p53 targets and apoptotic cell death. Significantly, given the importance of the bone marrow microenvironment for the support and drug resistance of myeloma cells, tumor cells undergo effective apoptosis also in the presence of stromal cells, which themselves appear to tolerate exposure to nutlin-3. The in vitro toxicity of nutlin-3 was similar to that of the genotoxic drug melphalan. Because nutlin-mediated p53 activation is not dependent on DNA damage, MDM2 antagonists may help to avoid or reduce the severe genotoxic side effects of chemotherapeutic agents currently used to treat multiple myeloma. Therefore, MDM2 antagonists may offer a new treatment option for this disease.

Description: The combined blockade of the IL-6R/STAT3 and the MAPK signaling pathways has been shown to inhibit bone marrow microenvironment (BMM)–mediated survival of multiple myeloma (MM) cells. Here, we identify the molecular chaperones heat shock proteins (Hsp) 90α and β as target genes of both pathways. The siRNA-mediated knockdown of Hsp90 or treatment with the novel Hsp90 inhibitor 17-DMAG attenuated the levels of STAT3 and phospho-ERK and decreased the viability of MM cells. Although knockdown of Hsp90β—unlike knockdown of Hsp90α—was sufficient to induce apoptosis, this effect was strongly increased when both Hsp90s were targeted, indicating a cooperation of both. Given the importance of the BMM for drug resistance and MM-cell survival, apoptosis induced by Hsp90 inhibition was not mitigated in the presence of bone marrow stromal cells, osteoclasts, or endothelial cells. These observations suggest that a positive feedback loop consisting of Hsp90α/β and major signaling pathways supports the survival of MM cells. Finally, in situ overexpression of both Hsp90 proteins was observed in most MMs but not in monoclonal gammopathy of undetermined significance (MGUS) or in normal plasma cells. Our results underpin a role for Hsp90α and β in MM pathogenesis.

Description: Introduction: Oncogenic activation of signaling cascades, such as the IL-6R/STAT3-, Ras/MAPK- and PI3K/Akt-pathways, is implied to contribute to proliferation and survival of multiple myeloma (MM) cells. However, investigations have mostly focused on selected MM cell lines because of the numerous limitations that apply to working with primary tumor material. Analysis of the activation status and roles of these pathways in primary MM cells is necessary, though, since immortalized MM cell lines are in many ways unrepresentative of the intramedullary cancer that is diagnosed in the large majority of patients. Experimental model: We have therefore established intracellular phosphoepitope staining for pathway components in MM cells, which permits their FACS-based analysis with relatively small cell numbers. This assay was used to conduct a detailed examination of the activation status of ERK, STAT3 and Akt, and of the effect of selective pathway inhibitors, on a large panel (n=15) of primary MM samples. CD138-purified primary MM cells were either kept in medium alone or in coculture with BMSCs or osteoclasts. After two days in culture the phosphorylation levels of the respective proteins were measured in cells that served as controls and in cells that had been treated with selective pathway inhibitors (Sant7 for the IL-6R/STAT3-, PD98059 for the Ras/MAPK-, and triciribine for the PI3K/Akt-pathway). In addition, survival rates were determined for cells in the different settings and under different drug regimens. Results: 60% of MM samples displayed some basal STAT3 phosphorylation. The signal was distinctly higher when MM cells were cocultured with BMSCs or osteoclasts, and was abrogated through treatment with Sant7. 70% of MM samples showed basal phosphorylation of ERK, which was generally enhanced in coculture and inhibited by treatment with PD98059. Low phosphorylation levels of Akt were observed in 80% of samples, with only slight increases in coculture and blockade through triciribine. Inhibition of any single pathway alone had at best slight effects on the survival of MM cells when they were cocultured with BMSCs. However, the simultaneous blockade of the Ras/MAPK and PI3K/Akt pathway reduced the number of viable cells to about 40% of controls. Interestingly, additional blockade of the IL-6R/STAT3 pathway did not significantly enhance this anti-myeloma effect. We are currently extending these studies in primary MM cells and in MM cell lines to include reagents more specific for the different Akt-isoforms. Conclusions: These experiments represent a detailed functional analysis of three important survival pathways in primary MM tumor cells. They also establish the suitability of intracellular phosphoepitope staining for their FACS-based analysis. The results indicate, that under coculture conditions inhibition of any single pathway alone will not lead to appreciable levels of apoptosis. The combined targeting of survival supporting pathways leads to better proapoptotic effects but combination with drugs against other classes of target might be required for truly effective clinical applications.

Description: We have recently shown that Hsp90 is overexpressed in multiple myeloma (MM) and critically contributes to tumor cell survival. Pharmacologic blockade of Hsp90 has consistently been shown to induce MM cell death. However, most data have been obtained with MM cell lines whereas knowledge about the molecular effects of pharmacologic Hsp90 blockade in primary tumor cells is lacking. Furthermore, these investigations have so far exclusively relied on geldanamycin derivatives. Here, we analyzed the anti-tumor effects of a novel diarylisoxazole-based Hsp90 inhibitor (NVP-AUY922) in a large set of primary MM samples and in MM cell lines. The majority of cell lines (n = 8), as well as most primary samples (n = 20), displayed profound apoptotic responses and steep dose-effect curves with EC50 values in the range of 5–15 nM and EC90 values between 8 and 25 nM. This effect was not attenuated in coculture with cells from the bone marrow microenvironment. Some cell lines and about a quarter of primary MM samples displayed greater resilience to drug treatment, with EC50 values but not EC90 values reached at concentrations up to 50 nM. Sensitivity of MM cells to the Hsp90 inhibitor was not correlated with TP53 mutation or Hsp70 induction levels. Western analyses of MM cell lines and flow cytometric analyses of antibody-stained Hsp90 client proteins in primary tumor cells showed that NVP-AUY922-treatment entailed molecular effects and pharmacodynamic properties consistent with abrogation of Hsp90 function. Consequently, downregulation of multiple signaling and survival pathways was detectable through, for example, decreases in the phosphorylated (activated) forms of extracellular signal-regulated kinase (ERK) 1 and 2, signal transducer and activator of transcription (STAT) 3 and glycogen synthase kinase-3beta. All samples treated displayed strong upregulation of Hsp70. Importantly, peripheral blood mononuclear cells as well as primary bone marrow stromal cells were much less affected by high (50–100 nM) concentrations of NVP-AUY922, showing that a therapeutic window might be established for the treatment of multiple myeloma. Taken together, NVP-AUY922 could be a promising new drug for the treatment of a majority of myeloma patients.

Description: Multiple myeloma (MM) is a fatal plasma cell tumor that accounts for about 1% of cancers. A hallmark of the disease is its location in the bone marrow where the tumor cells receive prosurvival support from the microenvironment and cause extensive osteolytic damage. Novel drugs are currently being developed into a range of new treatment options. However, because the problems of cancer relapse and eventual selection of therapy-resistant offspring remain, additional therapeutic targets should still be investigated. ILK is a multifunctional protein that, as an adaptor and/or as a kinase, may relay adhesion- and growth factor receptor-mediated signals to downstream signaling cascades that promote growth and survival. We have analysed the expression of ILK in MM cells and have tested the effects of a novel small molecule ILK-inhibitor (QLT0267; QLT Inc., Vancouver, Canada) in MM cell lines, primary MM tumor cells and healthy cells, respectively. ILK expression at either cDNA or protein level was detectable in virtually every MM sample tested. Treatment with QLT0267 for up to 3 days resulted in extensive apoptotic death in MM cell lines (EC50 values below 10 microM in 8/9 MM cell lines tested) and in a majority of primary (anti-CD138-purified) MM samples (EC50 values below 10 microM in 8/14 primary MM samples tested). Drug treatment led to rapid decreases in the levels of phospho-STAT3, phospho-GSK3beta and total Akt protein, whereas levels of ILK and of phospho-ERK were unaffected or, in the latter case, showed a slight increase. Similar to other current pharmacologic approaches, targeting ILK may have several detrimental impacts on the signaling network that sustains MM cells. Such pleiotropic effects could prove valuable for combination treatments. The survival of peripheral blood mononuclear cells and of bone marrow stromal cells (BMSCs) at 20 microM QLT0267 was just slightly affected, indicating that the scope for establishment of a therapeutic window in MM might exist. High (20 microM) concentrations of QLT0267 gradually (and reversibly) promoted detachment of BMSCs from the culture dish, indicating that the drug might be useful to temporarily impair their effectiveness to support myeloma cells. Taken together, these experiments provide a rationale to further explore the utility of ILK-inhibition for the treatment of MM.