Miguel Lemaire

Bone marrow stromal cell–derived exosomes as communicators in drug resistance in multiple myeloma cells

The interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells performs a crucial role in MM pathogenesis by secreting growth factors, cytokines, and extracellular vesicles. Exosomes are membranous vesicles 40 to 100 nm in diameter constitutively released by almost all cell types, and they mediate local cell-to-cell communication by transferring mRNAs, miRNAs, and proteins. Although BMSC-induced growth and drug resistance of MM cells has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here we investigate the effect of BMSC-derived exosomes on the viability, proliferation, survival, migration, and drug resistance of MM cells, using the murine 5T33MM model and human MM samples. BMSCs and MM cells could mutually exchange exosomes carrying certain cytokines. Both naive and 5T33 BMSC-derived exosomes increased MM cell growth and induced drug resistance to bortezomib. BMSC-derived exosomes also influenced the activation of several survival relevant pathways, including c-Jun N-terminal kinase, p38, p53, and Akt. Exosomes obtained from normal donor and MM patient BMSCs also induced survival and drug resistance of human MM cells. Taken together, our results demonstrate the involvement of exosome-mediated communication in BMSC-induced proliferation, migration, survival, and drug resistance of MM cells.

Polycomb target genes are silenced in multiple myeloma.

Multiple myeloma (MM) is a genetically heterogeneous disease, which to date remains fatal. Finding a common mechanism for initiation and progression of MM continues to be challenging. By means of integrative genomics, we identified an underexpressed gene signature in MM patient cells compared to normal counterpart plasma cells. This profile was enriched for previously defined H3K27-tri-methylated genes, targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts. Additionally, the silenced gene signature was more pronounced in ISS stage III MM compared to stage I and II. Using chromatin immunoprecipitation (ChIP) assay on purified CD138+ cells from four MM patients and on two MM cell lines, we found enrichment of H3K27me3 at genes selected from the profile. As the data implied that the Polycomb-targeted gene profile would be highly relevant for pharmacological treatment of MM, we used two compounds to chemically revert the H3K27-tri-methylation mediated gene silencing. The S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobinostat), reactivated the expression of genes repressed by H3K27me3, depleted cells from the PRC2 component EZH2 and induced apoptosis in human MM cell lines. In the immunocompetent 5T33MM in vivo model for MM, treatment with LBH589 resulted in gene upregulation, reduced tumor load and increased overall survival. Taken together, our results reveal a common gene signature in MM, mediated by gene silencing via the Polycomb repressor complex. The importance of the underexpressed gene profile in MM tumor initiation and progression should be subjected to further studies.

Bortezomib Alone or in Combination with the Histone Deacetylase Inhibitor JNJ-26481585: Effect on Myeloma Bone Disease in the 5T2MM Murine Model of Myeloma

The proteasome inhibitor bortezomib (Velcade) is currently approved as second-line treatment of multiple myeloma (MM). MM-related bone disease is one of the most debilitating complications of MM. Besides supportive care with biphosphonates, which have proven efficacy in reducing and delaying skeletal-related events, there is no specific treatment of lytic bone lesions. The present study investigated the effect of bortezomib alone or in combination with a hydroxamate-based histone deacetylase inhibitor, JNJ-26481585 on tumor burden, and MM bone disease in the 5T2MM model. Injection of 5T2MM cells into C57Bl/KaLwRij mice resulted in MM bone disease, characterized by an increase in the percentage osteoclasts, a decrease in osteoblasts, trabecular bone volume, trabecular number, and the development of bone lesions. Treatment of 5T2MM-bearing mice with bortezomib significantly reduced tumor burden, angiogenesis, and MM bone disease. More importantly, the combination of bortezomib with JNJ-26481585 resulted in a more pronounced reduction of osteoclasts and increase of osteoblasts, trabecular bone volume, and trabecular number compared with bortezomib as single agent. These data suggest that bortezomib has bone remodeling properties that can be improved in combination with low dose JNJ-26481585. The study indicates that this combination therapy could be a useful strategy for the treatment of MM patients, especially in those patients with skeletal complications.

The microenvironment and molecular biology of the multiple myeloma tumor.

Multiple myeloma (MM) is a deadly plasma cell cancer that resides in the bone marrow (BM). Numerous studies have demonstrated the involvement of the BM microenvironment supporting tumor growth, angiogenesis, bone disease and drug resistance. Reciprocal interactions between the different components of the BM microenvironment and the MM cells are necessary to regulate migration, differentiation, proliferation and survival of the malignant plasma cells. In this review we focus on the interactions and molecular mechanisms by which the BM microenvironment exert these effects. Better understanding of these interactions and the study of the epigenetic changes that tumor cells undergo are necessary in order to improve current treatments and for the discovery of new therapies that may eventually lead to a potential cure.

The effects of JNJ-26481585, a novel hydroxamate-based histone deacetylase inhibitor, on the development of multiple myeloma in the 5T2MM and 5T33MM murine models

Multiple myeloma (MM) is a B-cell malignancy, which often remains incurable because of the development of drug resistance governed by the bone marrow (BM) microenvironment. Novel treatment strategies are therefore urgently needed. In this study, we evaluated the anti-MM activity of JNJ-26481585, a novel ‘second-generation’ pyrimidyl-hydroxamic acid-based histone deacetylase inhibitor, using the syngeneic murine 5TMM model of MM. In vitro, JNJ-26481585 induced caspase cascade activation and upregulation of p21, resulting in apoptosis and cell cycle arrest in the myeloma cells at low nanomolar concentrations. Similar results could be observed in BM endothelial cells using higher concentrations, indicating the selectivity of JNJ-26481585 toward cancer cells. In a prophylactic and therapeutic setting, treatment with JNJ-26481585 resulted in an almost complete reduction of the tumor load and a significant decrease in angiogenesis. 5T2MM-bearing mice also developed a MM-related bone disease, characterized by increased osteoclast number, development of osteolytic lesions and a reduction in cancellous bone. Treatment of these mice with JNJ-264815 significantly reduced the development of bone disease. These data suggest that JNJ-26481585 has a potent anti-MM activity that can overcome the stimulatory effect of the BM microenvironment in vivo making this drug a promising new anti-MM agent.

Imaging and radioimmunotherapy of multiple myeloma with anti-idiotypic Nanobodies

Multiple myeloma (MM) is characterized by the monoclonal expansion of malignant plasma cells in the bone marrow (BM) and the production of monoclonal protein (M-protein). With the implementation of autologous stem cell transplantation and highdose chemotherapy using dexamethasone, bortezomib, thalidomide and lenalidomide, the survival rate has improved but MM patients still relapse, even if they achieve complete remission (CR). Therefore, new therapeutic strategies are needed to target residual malignant cells and eliminate minimal residual disease (MRD) in order to improve patient outcome. Nanobodies are the smallest (15 kDa) antigen-binding fragments derived from camelid heavy-chain-only antibodies. Because of their biochemical characteristics including high stability, solubility and target affinity, they are ideal therapeutic and diagnostic tools. Nanobodies can also recognize epitopes that remain undetected by conventional antibodies. Previous work has already demonstrated that Nanobody conjugates are able to reach, bind and kill cancer cells. Moreover, Nanobodies conjugated with radionuclides have been successfully used with the single-positron emission tomography (SPECT) technology combined with micro-computed tomography (micro-CT) for imaging purposes. Despite the extensive Nanobody-based research, there is so far only little evidence about their potential for diagnostic and/or therapeutic applications in MM. Here, we take advantage of the M-protein present in the murine 5T2MM model to prove the potential use of Nanobodies in MM. The 5TMM models are syngeneic, immunocompetent models that resemble human MM clinically and biologically. The best characterized are the 5T33MM and the 5T2MM models. The former represents an aggressive tumor, which develops in a short period of time (4 weeks), whereas the latter represents a more moderate tumor that develops over a period of 3 months. Both express different idiotypes (5T33MMid and 5T2MMid, respectively) on the cell membrane surface. By immunization of a dromedary with purified 5T2MM M-protein and a simple selection method (see Supplementary Materials and Methods), we were able to select, produce and purify a panel of very specific anti-5T2MM-idiotype Nanobodies (a5T2MMid-Nbs) that recognize nearby epitopes on the idiotype (Supplementary Figure 1). After in vitro characterization of these Nanobodies, R3B23 came up as the better binder (see Supplementary Figures 2, 3 and 4) and was therefore selected for in vivo testing. R3B23 was labeled with radionuclides Technetium (Tc) and Lutetium (Lu) using previous established protocols. Tc (half-life: 6 h) is used in SPECT for nuclear medicine imaging techniques, whereas Lu (half-life: 6.7 days) is mainly used for therapeutic applications due to the emission of low-energy b-minus particles. Several radiotherapeutic monoclonal antibodies (MoAbs) are currently under (pre)clinical evaluation in cancers, including B-cell disorders. In MM MRD, which is characterized by a small tumor load in BM, radiotherapeutic MoAbs might not be ideal as radiotoxicity levels to healthy tissues are high, providing a rationale for the use of highly specific and rapidly cleared radiotherapeutic Nanobodies instead. First, we studied the specificity of R3B23 in vivo. At 1 h post injection (p.i.), anesthetized mice were imaged using pinhole SPECT and micro-CT, as described previously. At 30 min after imaging, the mice were killed, different organs were removed, weighed and the radioactivity was measured. Fused SPECT/microCT images obtained from naive mice with nontargeting control Nanobody Tc-cAbBCII10 showed tracer uptake only in the bladder and kidneys (Figure 1a). Biodistribution experiments (Figure 1d) confirmed a high tracer uptake in both kidneys (4200%IA/g) and only marginal levels of uptake in other organs (ranging from 0.20±0.04%IA/g in muscle tissue to 1.02±0.26%IA/g in lungs) as expected for unbound tracers that are eliminated from the body through renal filtration. Importantly, similar results were observed in naive mice injected with Tc-R3B23 (Figures 1e and h) indicating that R3B23 does not bind to circulating immunoglobulins or other in vivo targets. SPECT/micro-CT scan images and biodistribution studies of terminally diseased 5T33MM mice injected with either Tc-cAbBCII10 (Figures 1b and d) or Tc-R3B23 (Figures 1f and h) and 5T2MM mice injected with Tc-cAbBcII10 (Figures 1c and d) showed analogous patterns with a high tracer uptake in kidneys and bladder and a low uptake in all other organs, demonstrating that MM disease does not influence Nanobody uptake. SPECT/micro-CT scan images of 5T2MM mice injected with Tc-R3B23 (Figure 1g) revealed a systemic tracer uptake, which was confirmed by biodistribution studies (Figure 1h). The up to 100-fold-elevated tracer levels in blood (44.56±2.54%IA/g) can be attributed to binding of the antiidiotypic Nanobody to the high levels of circulating M-protein in this late-stage disease model. The elevated tracer blood-pool activity accounts for the decreased uptake observed in the kidneys (circa 8%IA/g) and is responsible for the elevated uptake in other organs (ranging from 0.91±0.04%IA/g in the muscle to 11.63±2.35%IA/g in the lungs). It is noteworthy that the in vivo increase in circulating M-protein can be monitored over time using SPECT/micro-CT scans with Tc-R3B23 (Supplementary Figure 5). In conclusion, the biodistribution studies demonstrate that R3B23 does not bind to any target in healthy mice or to M-protein with a different idiotype, and it is therefore truly antiidiotypic. Finally, we evaluated the effect of Nanobody R3B23 conjugated with Lutetium (Lu-R3B23) on tumor growth. One week after inoculation of 5T2MM cells into naive mice, we started weekly treatments with intravenously administrated Lu-R3B23 or negative control Lu-cAbBCII10. After 5 weeks of treatment, animals were imaged using SPECT/micro-CT scans with TcR3B23 (Figure 2). On the basis of micro-CT images, an ellipsoid region of interest was drawn around the heart. Tracer uptake in heart, as a measurement of blood-pool activity, is expressed as the counts in the tissue divided by the injected activity/cubic centimeter (%IA/cm). The %IA/cm detected in the heart of mice treated with Lu-R3B23 (5.55±1.42) was significantly lower than the values measured in untreated mice (10.03±0.27; Po0.005) and mice treated with control Nanobody Lu-cAbBcII10 (9.19±0.84; Po0.05). The lower blood value of Tc-R3B23 uptake in mice treated with Lu-R3B23 is not due to in vivo competition with the therapeutic Lu-labeled Nanobody, as the latter was already systemically cleared. Indeed, the 10 mg Tc-R3B23 injection and subsequent SPECT/micro-CT scanning were performed 5 days after injection with 10 mg Lu-Nanobody.

The HDAC Inhibitor LBH589 Enhances the Antimyeloma Effects of the IGF-1RTK Inhibitor Picropodophyllin

Purpose: We have previously shown the use of the insulin-like growth factor type 1 receptor tyrosine kinase (IGF-1RTK) inhibitor picropodophyllin (PPP) as an attractive strategy to combat multiple myeloma (MM) in vitro and in vivo. After a combinatorial drug screening, the histone deacetylase inhibitor LBH589 was shown to act in synergy with PPP reducing survival of MM cells. In this study, we tried to elucidate the molecular mechanisms underlying this combinatorial effect. Experimental Design: The in vitro anti-MM effects of PPP and LBH589 alone and in combination were evaluated by studying apoptosis, cell cycle distribution, and downstream transcriptome using both human MM cell lines and cells from the murine 5T3MM model. In vivo the effect on survival of 5T33MM-inoculated mice was evaluated. Results: In the human MM cell line RPMI8226, treatment with PPP and LBH589 in combination resulted in a five-fold increase of apoptosis, and an additive effect on the cleavage of the active forms of caspase-8 was observed as compared with the single drug treatments. Cell cycle analysis revealed an accumulation of cells in the G2–M phase and subsequent downregulation of cell cycle regulating proteins. These data were also confirmed in the 5T33MM cells in vitro. Also, the transcriptome was analyzed by Affymetrix arrays showing gene expression alterations mainly in categories of genes regulating apoptosis and cell adhesion. Combined treatment in vivo resulted in a significantly prolonged survival of 5T33MM-inoculated mice. Conclusions: The results indicate an improved MM treatment opportunity in using a combination of PPP and LBH589. Clin Cancer Res; 18(8); 2230–9. ©2012 AACR.