Renate Burger

The bisphosphonate zoledronic acid has antimyeloma activity in vivo by inhibition of protein prenylation

Nitrogen‐containing bisphosphonates (N‐BPs) are effective antiosteolytic agents in patients with multiple myeloma. Preclinical studies have also demonstrated that these agents have direct antitumor effects in vitro and can reduce tumor burden in a variety of animal models, although it is not clear whether such effects are caused by direct actions on tumor cells or by inhibition of bone resorption. N‐BPs prevent bone destruction in myeloma by inhibiting the enzyme farnesyl pyrophosphate synthase in osteoclasts, thereby preventing the prenylation of small GTPase signaling proteins. In this study, utilizing a plasmacytoma xenograft model without complicating skeletal lesions, treatment with zoledronic acid (ZOL) led to significant prolongation of survival in severe combined immunodeficiency mice inoculated with human INA‐6 plasma cells. Following treatment with a clinically relevant dose of ZOL, histological analysis of INA‐6 tumors from the peritoneal cavity revealed extensive areas of apoptosis associated with poly (ADP‐ribose) polymerase cleavage. Furthermore, Western blot analysis of tumor homogenates demonstrated the accumulation of unprenylated Rap1A, indicative of the uptake of ZOL by nonskeletal tumors and inhibition of farnesyl pyrophosphate synthase. These studies provide, for the first time, clear evidence that N‐BPs have direct antitumor effects in plasma cell tumors in vivo and this is executed by a molecular mechanism similar to that observed in osteoclasts.

Impact of Interleukin-6 in Hematological Malignancies

Almost 3 decades have passed since the discovery and cloning of IL-6, and a tremendous amount of work has contributed to the current knowledge of the biological functions of this cytokine, its receptor, and the signaling pathways that are activated. The understanding of the role of IL-6 in human disease has led to the development of novel therapeutic strategies that block the biological functions of IL-6. In clinical studies, IL-6 and IL-6 receptor antibodies have proven efficacy in rheumatoid arthritis, systemic juvenile idiopathic arthritis, and Castlemans disease, conditions that are known to be driven by IL-6. The focus of this overview is the role of IL-6 in the pathophysiology of hematological malignancies.

Activity of everolimus (RAD001) in relapsed and/or refractory multiple myeloma: a phase I study.

The mammalian target of rapamycin plays an important role in multiple myeloma. The allosteric mammalian target of rapamycin inhibitor everolimus has long been approved for immunosuppression and has shown activity in certain cancers. This investigator-initiated phase I trial explored the use of everolimus in relapsed and/or refractory multiple myeloma patients who had received two or more lines of prior treatment. Following a dose-escalation design, it called for a fixed dose of oral everolimus. Blood drug levels were monitored and the biological activity of everolimus was evaluated in bone marrow. Seventeen patients were enrolled (age range, 52 to 76 years). All had been previously treated with stem cell transplantation and proteasome inhibitors and almost all with immunomodulatory drugs. No dose-limiting toxicity was observed and the intended final daily dose of 10 mg was reached. Only one severe adverse event was assessed as possibly related to the study drug, namely atypical pneumonia. Remarkably few infections were observed. Although the trial was mainly designed to evaluate feasibility, anti-myeloma activity, defined as clinical benefit, was documented in ten of 15 evaluable patients at every dose level including eight patients with stable disease, one patient with minor remission and one with partial remission. However, the median time to progression was 90 days (range, 13 to 278 days). The biomarker study documented on-target activity of everolimus in malignant plasma cells as well as the microenvironment. The observed responses are promising and allow further studies to be considered, including those testing combination strategies addressing escape pathways. This trial is registered with EudraCT number 2006-002675-41.

CD20-Specific Immunoligands Engaging NKG2D Enhance γδ T Cell-Mediated Lysis of Lymphoma Cells

Human γδ T cells are innate‐like T cells which are able to kill a broad range of tumour cells and thus may have potential for cancer immunotherapy. The activating receptor natural killer group 2 member D (NKG2D) plays a key role in regulating immune responses driven by γδ T cells. Here, we explored whether recombinant immunoligands consisting of a CD20 single‐chain fragment variable (scFv) linked to a NKG2D ligand, either MHC class I chain‐related protein A (MICA) or UL16 binding protein 2 (ULBP2), could be employed to engage γδ T cells for tumour cell killing. The two immunoligands, designated MICA:7D8 and ULBP2:7D8, respectively, enhanced cytotoxicity of ex vivo‐expanded γδ T cells against CD20‐positive lymphoma cells. Both Vδ1 and Vδ2 γδ T cells were triggered by MICA:7D8 or ULBP2:7D8. Killing of CD20‐negative tumour cells was not induced by the immunoligands, indicating their antigen specificity. MICA:7D8 and ULBP2:7D8 acted in a dose‐dependent manner and induced cytotoxicity at nanomolar concentrations. Importantly, chronic lymphocytic leukaemia (CLL) cells isolated from patients were sensitized by the two immunoligands for γδ T cell cytotoxicity. In a combination approach, the immunoligands were combined with bromohydrin pyrophosphate (BrHPP), an agonist for Vδ2 γδ T cells, which further enhanced the efficacy in target cell killing. Thus, employing tumour‐directed recombinant immunoligands which engage NKG2D may represent an attractive strategy to enhance antitumour cytotoxicity of γδ T cells.

STAT3-induced long noncoding RNAs in multiple myeloma cells display different properties in cancer

Interleukin-6 (IL-6)-activated Signal Transducer and Activator of Transcription 3 (STAT3) facilitates survival in the multiple myeloma cell line INA-6 and therefore represents an oncogenic key player. However, the biological mechanisms are still not fully understood. In previous studies we identified microRNA-21 as a STAT3 target gene with strong anti-apoptotic potential, suggesting that noncoding RNAs have an impact on the pathogenesis of human multiple myeloma. Here, we describe five long noncoding RNAs (lncRNAs) induced by IL-6-activated STAT3, which we named STAiRs. While STAiRs 1, 2 and 6 remain unprocessed in the nucleus and show myeloma-specific expression, STAiRs 15 and 18 are spliced and broadly expressed. Especially STAiR2 and STAiR18 are promising candidates. STAiR2 originates from the first intron of a tumor suppressor gene. Our data support a mutually exclusive expression of either STAiR2 or the functional tumor suppressor in INA-6 cells and thus a contribution of STAiR2 to tumorigenesis. Furthermore, STAiR18 was shown to be overexpressed in every tested tumor entity, indicating its global role in tumor pathogenesis. Taken together, our study reveals a number of STAT3-induced lncRNAs suggesting that the interplay between the coding and noncoding worlds represents a fundamental principle of STAT3-driven cancer development in multiple myeloma and beyond.

Due to interleukin-6 type cytokine redundancy only glycoprotein 130 receptor blockade efficiently inhibits myeloma growth

Interleukin-6 has an important role in the pathophysiology of multiple myeloma where it supports the growth and survival of the malignant plasma cells in the bone marrow. It belongs to a family of cytokines which use the glycoprotein 130 chain for signal transduction, such as oncostatin M or leukemia inhibitory factor. Targeting interleukin-6 in plasma cell diseases is currently evaluated in clinical trials with monoclonal antibodies. Here, efforts were made to elucidate the contribution of interleukin-6 and glycoprotein 130 signaling in malignant plasma cell growth in vivo. In the xenograft severe combined immune deficiency model employing our interleukin-6-dependent plasma cell line INA-6, the lack of human interleukin-6 induced autocrine interleukin-6 production and a proliferative response to other cytokines of the glycoprotein 130 family. Herein, mice were treated with monoclonal antibodies against human interleukin-6 (elsilimomab/B-E8), the interleukin-6 receptor (B-R6), and with an antibody blocking glycoprotein 130 (B-R3). While treatment of mice with interleukin-6 and interleukin-6 receptor antibodies resulted in a modest delay in tumor growth, the development of plasmacytomas was completely prevented with the anti-glycoprotein 130 antibody. Importantly, complete inhibition was also achieved using F(ab’)2-fragments of monoclonal antibody B-R3. Tumors harbor activated signal transducer and activator of transcription 3, and in vitro, the antibody inhibited leukemia inhibitory factor stimulated signal transducer and activator of transcription 3 phosphorylation and cell growth, while being less effective against interleukin-6. In conclusion, the growth of INA-6 plasmacytomas in vivo under interleukin-6 withdrawal remains strictly dependent on glycoprotein 130, and other glycoprotein 130 cytokines may substitute for interleukin-6. Antibodies against glycoprotein 130 are able to overcome this redundancy and should be explored for a possible therapeutic window.

B289 Farnesyltransferase Inhibitors are Synergistic with Bisphosphonates in Leading to Myeloma Cell Death

Introduction: Activation of the Ras-dependent MAPK pathway–induced by cytokines like IL-6 and IGF-1 or by Ras mutations is essential for the gro wth of malignant plasma cells. Proteins of the Ras family are active only in their membranebound form depending on posttranscriptional prenylation by farnesyltransferase (FTase). Farnesyltransferase inhibitors (FTIs), designed to block prenylation of Ras and related small G proteins, have already shown activity in multiple myeloma (MM). However, geranylgeranyl transferase might substitute for blocked FTase. Nitrogen-containing bisphosphonates (N-BP) inhibit farnesyl pyrophoshate synthase, an essential enzyme for the geranylgeranylation of small G proteins and were recently shown to block myeloma growth in vitro and in vivo. Therefore, a combination of both classes of inhibitors, FTI and N-BP, should be able to effectively block Ras as well as other G proteins by inhibiting both farnesylation and geranylgeranylation. Materials and Methods: The potential of such an approach was ev aluated in 6 human myeloma cell lines, measuring growth by MT S or [3H] thymidine assay upon treatment with 3 different FTI alone or in combination with the N-BP zoledronate (ZOL). Apoptosis was determined by propidium iodide or 7-AAD/Annexin V staining. Ras and p53 mutation status was determined by sequencing. Western blot analysis was performed to look for phosphorylated MAPK. Results and Discussion: Both classes of inhibitors, FTI as well as N-BP, were able to induce apoptosis. All FTI inhibited gr owth of plasma cell lines in a dose-dependent manner. The activity of FTI and/or ZOL was not dependent on N-/K-ras or p53 mutation status. The combination of FTI inhibitor L744.832 and ZOL showed the strongest synergistic activity in the plasmacytoma cell lines INA-6 and JK-6L (q 0.5 μM of L744.832 and q 50 μM of ZOL). Because the decrease of phosphorylation of p 44/42 MAPK required high drug concentrations, essential prenylated proteins are more likely to be important targets than those of the Ras pathway. These findings could have important clinical implications, and combinations of FTI and N-BP might provide a less toxic innovative approach in the treatment of plasma cell tumors.