Multiple Myeloma Growth Inhibition Research Articles

Mature Osteoblasts Are Critical Bone Marrow Niche Cells Regulating Multiple Myeloma Cell Growth, at Least in Part, By Affecting the Local Immune Microenvironment

Despite high response rates to new therapies, most multiple myeloma (MM) patients relapse and become refractory to treatment. This is likely because MM cells enter dormancy and reactivate only under permissive conditions. Cellular dormancy is ubiquitous in normal tissues as well as in malignant, non-malignant, and metastasizing tumors. Dormant tumor cells (DTC) are protected from therapies which can reactivate later at the time of disease progression, resulting in relapsed refractory MM (RRMM). The tumor microenvironment (TME) is a key determinant of tumor dormancy and reactivation. We have previously shown that inhibition of Activin A in SCID mice transplanted with human MM.1S increased osteoblast numbers and resulted in inhibition of MM growth (Vallet et al., PNAS). We hypothesize that MM-DTCs reside in osteoblastic niche in the BM TME. Our long-term goal is to identify factors regulating DTCs in MM and develop strategies for long term disease control.We have generated mice in which mature osteoblasts can be postnatally deleted in an inducible and reversible manner. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre). This led to the expression of DTR in mature osteoblasts (OC-Cre/iDTR) only. The control mice were littermates lacking the OC-Cre allele (iDTR). Until the treatment with diphtheria toxin (DT), the OC-Cre/iDTR mice were immunocompetent compared to iDTR mice, as they did not show any differences in immune cell populations by flow cytometry, and did not show any skeletal phenotype, as measured by micro-computed CT (micro-CT).We chose to induce mature osteoblast-deficiency at 8-weeks of age to allow skeletal maturation which is assumed to be completed by 6-7weeks of age. To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/kg DT once a week, beginning at 8-weeks of age. Micro-CT analysis showed a significant increase in cortical porosity within 1-week after DT injection. 8-weeks of DT treatment significantly reduced trabecular bone fraction (BV/TV), trabecular numbers (Tb.N), and bone mineral density (BMD) with a significant increase in trabecular spacing (Tb.Sp). Immunohistochemistry for osteocalcin showed rapid loss of mature and endosteal osteoblasts (N.Ob/T.Ar). This was accompanied with a marked decreased in serum sclerostin and serum osteocalcin levels suggesting reduced osteocytes and mature osteoblasts, respectively. Importantly, serum CTX levels or numbers of osteoclasts (N.Oc/T.Ar) were unchanged. To study MM engraftment and progression, 3x10 6 5TGM1 luciferase tdTomato positive (5TGM1-Luc-Tom) MM cells were injected into the tibia of OC-Cre/iDTR and control iDTR mice followed by weekly injection of DT for 8-weeks. Flow cytometry analysis showed a 4-fold increase in the 5TGM1-Tom cells in the BM from OC-Cre/iDTR mice compared to controls. Bioluminescence imaging (BLI) for 5TGM1-Luc-Tom cells at 4-weeks showed dramatic increase in the OC-Cre/iDTR mice compared to controls. Interestingly, by 8-weeks, the BLI imaging showed 5TGM1-Luc-Tom cells in other long-bones of OC-Cre/iDTR mice but not the controls. To identify local BM TME changes, we assessed the levels of 200 cytokines and growth factors in the BM from these mice. The levels of immune regulatory cytokines, such as IL-17F and IL-21, were found to be decreased in the OC-Cre/iDTR mice compared to control iDTR mice, both after 4-weeks from intratibial 5TGM1 cell injection. This was accompanied by significant decrease in the fraction of CD4+ T-helper and Th17+ cells in the OC-Cre/iDTR mice compared to controls, as assessed by flow cytometry.These data show that MM cells engraft and proliferate rapidly within the BM in the absence of mature osteoblasts and have a propensity to migrate to other long bones with time. This may be partially mediated by the altered immune cell profile in the BM in the absence of mature osteoblasts. These data further suggest that expanding the mature osteoblast niche may provide novel therapeutic avenues and reduce disease burden creating an environment for long term tumor control. Importantly, this model will allow us to understand the osteoblastic niche for MM and the mechanisms of activation and dormancy, at least in part, via a role of the immune cells. DisclosuresFulzele: Constellation Pharma: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other.

Targeting the GCK pathway: a novel and selective therapeutic strategy against RAS-mutated multiple myeloma

In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus, targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug resistance. We identified germinal center kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown (KD) in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of a short hairpin RNA (shRNA)-resistant GCK, thereby excluding the potential off-target effects of GCK KD. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK KD on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK KD in RASMut cells suggests that targeting GCK is effective in MM, which harbors RAS mutations. In accordance with the effects of GCK KD, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Here, our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome immunomodulatory drug resistance in MM.

A Novel in-Vivo Model to Examine Homing of Multiple Myeloma Cells in Postnatal, Inducible, and Reversible Loss of Mature Osteoblasts

The initiation of Multiple myeloma (MM) coincides with a decrease in osteoblasts and an increase in osteoclasts and adipocytes. However, the contribution of osteoblasts to the initiation and progression of MM remains mostly unknown. In-vitro studies have shown that co-cultures with MC3T3 murine osteoblast cell-line induced quiescence in 5TGM1 murine myeloma cells. In SCID or C57BL/KaLwRijHsd mice, the fluorescently labeled MM cell lines, injected through tail-vein, co-localized with endosteal osteoblasts and were found to be quiescent. We previously demonstrated that increased osteoblast numbers using Activin A inhibition in SCID mice with human-origin MM.1S cells resulted in inhibition of MM growth (Vallet et al., PNAS 2010). Here we aimed to develop a genetic in-vivo model for a better understanding of the interactions between osteoblasts and MM cells. Osteolineage cells develop through the stages of pre-osteoblasts, committed osteoblasts, mature osteoblasts, and osteocytes; each stage governed by a distinct molecular signature and function. To delineate these differences, individual osteoblast populations were FACS-sorted from long bones of Osterix-GFP+ (Osx+) mice (pre-osteoblasts), Collagen 2.3-GFP+ (Col2.3+) mice (committed osteoblasts), and Osteocalcin-YFP+ (OCN+) mice (mature osteoblasts). Co-cultured of these osteoblast populations with 5TGM1 MM cells showed that the OCN+ mature osteoblasts suppressed MM proliferation the most. Therefore, we focused our studies on the role of mature osteoblasts in MM homing and engraftment. We next generated mice in which mature osteoblasts were postnatally deleted in an inducible and reversible manner to better understand the molecular mechanisms of engraftment, proliferation, and migration of 5TGM1 injected intra-tibialy. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre). This led to the expression of DTR in mature osteoblasts (OC-Cre/iDTR) only. The control mice were littermates lacking the OC-Cre allele (iDTR). The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). We chose to induce mature osteoblast-deficiency at 8-weeks to allow skeletal maturation which is assumed to be completed by 6-7weeks of age. To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/Kg DT once a week, beginning at 8-weeks of age. Micro-CT analysis showed a significant increase in cortical porosity within 1-week after DT injection. 8-weeks of DT treatment significantly reduced trabecular bone fraction (BV/TV), trabecular numbers (Tb.N), and bone mineral density (BMD) with a significant increase in trabecular spacing (Tb.Sp). Immunohistochemistry for osteocalcin showed rapid loss of mature and endosteal osteoblasts (N.Ob/T.Ar). This was accompanied with a marked decreased in serum sclerostin and serum osteocalcin levels suggesting reduced osteocytes and mature osteoblasts, respectively. Importantly, serum CTX levels or numbers of osteoclasts (N.Oc/T.Ar) were unchanged. To study MM engraftment and progression, 3x106 5TGM1 luciferase eGFP positive (5TGM1-Luc-GFP) MM cells were injected into the tibia of OC-Cre/iDTR and control iDTR mice followed by weekly injection of DT for 8-weeks. Flow cytometry analysis showed a 4-fold increase in the 5TGM1-GFP cells in the BM from OC-Cre/iDTR mice compared to controls. Bioluminescence imaging (BLI) for 5TGM1-Luc-GFP cells at 4-weeks showed 600-fold increase in the OC-Cre/iDTR mice compared to controls. Interestingly, by 8-weeks, the BLI imaging showed 5TGM1-Luc-GFP cells in other long-bones of OC-Cre/iDTR mice but not the controls. These data show that MM cells engraft and proliferate rapidly in the absence of mature osteoblasts in-vivo. Moreover, in the absence of mature osteoblasts, MM cells have a propensity to migrate to other long bones. These data further suggest that expanding the mature osteoblast niche may provide novel therapeutic avenues and reduce disease burden and create an environment for long term tumor control. Importantly, this model will allow us to follow and analyze the sequential engraftment, dormancy, reactivation, proliferation, and migration of myeloma cells, and evaluate the effects of osteoanabolic and anti-myeloma therapies. Disclosures Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Raje:BMS: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Astrazeneca: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Takeda: Consultancy; Immuneel: Membership on an entity's Board of Directors or advisory committees; Caribou: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy.

MiR-489 suppresses multiple myeloma cells growth through inhibition of LDHA-mediated aerobic glycolysis.

Dysregulation of miR-489 in human tumors has been widely reported. Lactate dehydrogenase isoform A (LDHA)-mediated aerobic glycolysis participates in proliferation of multiple myeloma (MM) cells. To investigate whether miR-489 induced MM growth inhibition via targeting to LDHA-mediated aerobic glycolysis. Expression of miR-489 in representative MM cell lines was determined via qRT-PCR (quantitative real-time polymerase chain reaction). MTT (3-(4, 5-di methyl thiazol-2-yl)-2, 5-di phenyl tetrazolium bromide) and colony formation assays were utilized to detect cell viability and proliferation. Effect of miR-489 on aerobic glycolysis was detected via glucose uptake, lactate and ATP production. Binding ability between miR-489 and LDHA was conducted via luciferase activity assay. MiR-489 was down-regulated in representative MM cell lines. Gain-of functional assays indicated that over-expression of miR-489 decreased cell viability and inhibited cell proliferation of MM cells. Moreover, miR-489 inhibited aerobic glycolysis via decrease of glucose uptake, lactate and ATP production. LDHA was identified as target of miR-489, suggesting a negative correlation between miR-489 and LDHA in MM cells. Mechanically, the inhibition ability of miR-489 on proliferation of MM cells was through inhibition of LDHA-mediated aerobic glycolysis. miR-489 inhibited MM tumor growth via LDHA-mediated glycolytic metabolism, suggesting potential therapeutic target ability of miR-489/LDHA for MM.

Mature Osteoblasts Promote Multiple Myeloma Survival through Cell-Cell Contact and Immune Modulation Mechanisms

The bone marrow (BM) microenvironment (TME) is a complex mix of cellular and non-cellular components that has a profound effect on hematopoiesis and hematological malignancies. Multiple myeloma (MM), a tumor of plasma cells that originates in the BM is highly influenced by TME components including stromal cells, osteoclasts, and immune cells. Among these cells, osteoblasts are the most versatile regulators of hematopoiesis and also play an integral role in malignant transformations of leukemia and lung cancer. MM is characterized by osteolytic bone lesions partly due to decreased numbers of osteoblasts. Therapies such as denosumab and zoledronic acid aim to restore bone health by inhibiting osteoclastogenesis. However, the contribution of osteoblasts to the initiation and progression of MM is not well understood. We previously demonstrated that increased osteoblast numbers using Activin A inhibition in an in vivo humanized myeloma model resulted in inhibition of MM growth (Vallet et al., 2010). Given that osteoblast numbers are on the decline in a typical MM patient, we hypothesized that the loss of osteoblasts contributes to the initiation and progression of MM. To assess the effects of osteoblasts on MM cells, MC3T3 murine pre-osteoblasts were differentiated into mature osteoblasts by supplementing media with ascorbic acid and β-glycerophosphate. Co-culture of 5TGM1 murine MM cells with the osteoblasts showed a significant decrease in the proliferation of MM cells by 40% (N=4). Osteoblasts go through distinct stages of maturation i.e., pre-osteoblasts, committed osteoblasts, and mature osteoblasts. These individual osteoblast populations were FACS sorted from mice to isolate pre-osteoblasts from the long bones of Osterix-GFP+ (Osx+) mice, committed osteoblasts from Collagen 2.3-GFP+ (Col2.3+) mice, and mature osteoblasts from Osteocalcin-YFP+ (OCN+) mice. Each of the osteoblast populations was co-cultured with 5TGM1 MM cells. Although all the osteoblast populationssignificantly suppressed MM proliferation, the OCN+ mature osteoblasts suppressed MM proliferation the most (N=3). Therefore, we focused our studies on the role of mature osteoblasts in MM progression. We generated mice in which mature osteoblasts could be postnatally deleted in an inducible and reversible manner. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre) to generate OC-Cre/iDTR mice. The control mice were littermates lacking the OC-Cre allele. The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treatedwith 50 µg/Kg DT once a week beginning at 8-weeks of age. To study MM engraftment and progression, 3x1065TGM1-Luciferase MM cells were injected into the tibia of OC-Cre/iDTR and control mice followed by weeklyinjection of DT for 8-weeks. Bioluminescence imaging (BLI) showed that 4-weeks onwards the OC-iDTR mice, but not the control mice, continued to express and increase the BLI signal (N=6). This data suggeststhatunder physiological conditions, mature osteoblasts activelysuppress MM engraftment and progression. We hypothesized that osteoblasts provide niche support to MM cells via direct cell-to-cell contact. To begin to identify the molecular mechanisms, we compared gene expression changes in primary murine osteoblasts between the undifferentiated and 30-day osteogenic differentiation time points. We also examined the BM TME by quantitative protein antibody arrays at 2-weeks following 5TGM1MM intratibial injection into OC-Cre/iDTR and control mice. The mature osteoblasts showed a significant increase in the expression of integrins, including integrin α4, and several immunomodulatory markers. The cytokine array analysis showed altered expressions of cell-cell communication proteins MAdCAM1, BAFF-R, TACI, and immunomodulatory factors IL-33, IL-17F, and IL-13. Taken together, using in vivo and in vitro models, we show that mature osteoblasts may have a negative regulatory impact on MM cells through cell-cell communication or immunomodulatory mechanisms. Expanding the osteoblast niche may provide novel therapeutic avenues to reduce disease burden and create an environment for long term tumor control. Disclosures Raje: Merck: Consultancy; Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy.

Mature Osteoblasts Provide a Protective Niche Against Multiple Myeloma Growth and Survival within the Tumor Microenvironment

Multiple myeloma (MM), a tumor of B-lymphocyte lineage cells, originates in the bone marrow (BM) and is highly influenced by the BM tumor microenvironment (TME). Among cells of the TME, osteoblasts are the most versatile regulators of many hematopoietic lineage cells through either direct cell-cell communication or secreted factors. Specifically relevant to MM, G-protein coupled receptor signaling in pre-osteoblasts is essential for the differentiation, maturation, and egress of B-cells (Panaroni et al., 2015). Despite these key roles, the contribution of osteoblasts to the initiation and progression of MM is not well understood. MM is characterized by osteolytic bone lesions partly due to decreased numbers of osteoblasts. Here, we hypothesize that osteoblasts provide niche support to maintain myeloma cells in a quiescent stage and that the loss of the osteoblastic niche leads to the progression of MM. As a proof of concept, we previously showed that increasing osteoblastogenesis by inhibiting Activin A led to inhibition of MM growth in an in vivo humanized myeloma model (Vallet et al., 2010).We generated mice in which mature osteoblasts could be postnatally deleted in an inducible and reversible manner. Diphtheria toxin receptor floxed mice were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter to generate OC-Cre/iDTR mice. Littermates heterozygous for DTR but lacking the OC-Cre expression were used as controls. The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/Kg DT beginning at 8-weeks of age. Osteocalcin immunohistochemistry of trabecular bone showed that the DT treated OC-Cre/iDTR mice were completely devoid of endosteal osteoblasts and young osteocytes. Consequently, serum levels of sclerostin were also significantly reduced in OC-Cre/iDTR mice compared to the controls.To study MM engraftment and progression, 3x106 5TGM1-Luciferase MM cells were inject into tibia of OC-Cre/iDTR and control mice followed by a weekly injection of DT for 8-weeks. Bioluminescence imaging (BLI) was used to assess tumor progression. Both the control and OC-Cre/iDTR mice started with similar BLI signal at 1-week. Interestingly, 4-weeks onwards only the OC-iDTR mice continued to express and increase the BLI signal indicating that the MM cells engrafted and continued to proliferate only in the OC-Cre/iDTR mice. This data suggests that under physiological conditions mature osteoblasts actively suppress MM engraftment and progression.To determine the direct effects of osteoblasts on MM cells, we established co-cultures of osteoblasts with MM cells. FACS sorting was used to isolate pre-osteoblasts from the long bones of Osterix-GFP+ (Osx+) mice, committed osteoblasts from Collagen 2.3-GFP+ (Col2.3+) mice, and mature osteoblasts from Osteocalcin-YFP+ (OCN+) mice. 5TGM1 MM cells were co-cultured along with 10,000 cells from each of the osteoblast populations. Although all three populations of osteoblasts significantly suppressed MM proliferation, the OCN+ mature osteoblasts suppressed MM proliferation the most.The mature osteoblastic niche may regulate MM cells via a) direct cell-to-cell contact, b) secreted factors, and/or c) other intermediary cells. To identify these possibilities, the BM TME was examined at 2-weeks following the 5TGM1 intratibia injection into the OC-Cre/iDTR and control mice. Quantitative protein antibody arrays analysis on the BM supernatant identified numerous key factors involved in cell-cell communication and immunomodulation in MM. These factors included BAFF-R, TACI, IL-33, IL-3, IL-21, and IL-17F. Gene expression analysis of the sorted 5TGM1 cells from the injected tibia indicated increased expression of genes involved in mitochondrial metabolism.Taken together, using in vivo and in vitro models, we show that mature osteoblasts offer specialized niches for MM cells where tumor cells are maintained in quiescence. The loss of the niche support allows the reactivation and progression of MM through the loss of cell-cell communication or through activation of immunomodulatory intermediary cells. Normalizing osteoblasts, such as through Activin A treatment, could provide novel avenues to reduce disease burden and long term tumor control. DisclosuresNo relevant conflicts of interest to declare.

The Sphingosine-1-Phosphate Modulator FTY720 Targets Multiple Myeloma via the CXCR4/CXCL12 Pathway.

Purpose: To explore the functional consequences of possible cross-talk between the CXCR4/CXCL12 and the sphingosine-1-phosphate (S1P) pathways in multiple myeloma (MM) cells and to evaluate the effect of S1P targeting with the FTY720 modulator as a potential anti-MM therapeutic strategy.Experimental Design and Results: S1P targeting with FTY720 induces MM cell apoptosis. The combination of FTY720 with the SPHK1 inhibitor SKI-II results in synergistic inhibition of MM growth. CXCR4/CXCL12-enhanced expression correlates with reduced MM cell sensitivity to both FTY720 and SKI-II inhibitors, and with SPHK1 coexpression in both cell lines and primary MM bone marrow (BM) samples, suggesting regulative cross-talk between the CXCR4/CXCL12 and SPHK1 pathways in MM cells. FTY720 was found to directly target CXCR4. FTY720 profoundly reduces CXCR4 cell-surface levels and abrogates the CXCR4-mediated functions of migration toward CXCL12 and signaling pathway activation. Moreover, FTY720 cooperates with bortezomib, inducing its cytotoxic activity and abrogating the bortezomib-mediated increase in CXCR4 expression. FTY720 effectively targets bortezomib-resistant cells and increases their sensitivity to bortezomib, promoting DNA damage. Finally, in a recently developed novel xenograft model of CXCR4-dependent systemic MM with BM involvement, FTY720 treatment effectively reduces tumor burden in the BM of MM-bearing mice. FTY720 in combination with bortezomib demonstrates superior tumor growth inhibition and abrogates bortezomib-induced CXCR4 increase on MM cells.Conclusions: Altogether, our work identifies a cross-talk between the S1P and CXCR4 pathways in MM cells and provides a preclinical rationale for the therapeutic application of FTY720 in combination with bortezomib in patients with MM. Clin Cancer Res; 23(7); 1733-47. ©2016 AACR.

Effects of ω-3 polyunsaturated fatty acids on multiple myeloma growth inhibition and enhanced sensitivity of dexamethasone

Effects of ω-3 polyunsaturated fatty acids on multiple myeloma growth inhibition and enhanced sensitivity of dexamethasone

Mesenchymal stem cells inhibit multiple myeloma cells via the Fas/Fas ligand pathway

IntroductionCell-based therapy represents a new frontier in the treatment of a wide variety of human diseases traditionally associated with morbidity outcomes, including those involving inflammation, autoimmunity, tissue damage, and cancer. However, the use of mesenchymal stem cells (MSCs) to treat multiple myeloma (MM) bone disease has raised concerns. Specifically, evidence has shown that infused MSCs might support tumor growth and metastasis.MethodsIn this study, we used a standard disseminated MM model in mice to identify the in vivo effects of intravenous MSC infusion. In addition, a series of in vitro co-culture assays were preformed to explore whether Fas/Fas ligand (Fas-L) is involved in the inhibitory effects of MSCs on MM cells.ResultsIn the MM mouse model, treatment of MSCs with highly expressed Fas ligand (Fas-Lhigh MSCs) showed remarkable inhibitory effects on MM indenization in terms of extending the mouse survival rate and inhibiting tumor growth, bone resorption in the lumbus and collum femoris, and MM cell metastasis in the lungs and kidneys. In addition, reduced proliferation and increased apoptosis of MM cells was observed when co-cultured with Fas-Lhigh MSCs in vitro. Furthermore, mechanistically, the binding between Fas and Fas-L significantly induced apoptosis in MM cells, as evidenced through an increase in the expression of apoptosis marker and Fas in MM cells. In contrast, Fas-Lnull MSCs promote MM growth.ConclusionsThese data suggest that Fas/Fas-L-induced MM apoptosis plays a crucial role in the MSC-based inhibition of MM growth. Although whether MSCs inhibit or promote cancer growth remains controversial, the levels of Fas-L expression in MSCs determine, at least partially, the effects of MSCs on MM cell growth.

Effects of short-hairpin RNA-inhibited β-catenin expression on the growth of human multiple myeloma cells in vitro and in vivo

Multiple myeloma (MM) is thrombogenic as a consequence of multiple hemostatic effects. Overexpression of β-catenin has been observed in several types of malignant tumors, including MM. However, the relationship between β-catenin expression and MM remains unclear. In the present study, RNA interference was used to inhibit β-catenin expression in RPMI8226 cells. RT-PCR and Western blotting analyses showed that β-catenin mRNA and protein expression were markedly down-regulated by CTNNB1 shRNA. Western blotting showed that the protein levels of cyclin D1 and glutamine synthetase were downregulated and supported the transcriptional regulatory function of β-catenin. The MTT assay showed that CTNNB1 shRNA could have significant inhibitory effects on the proliferation of RPMI8226 cells. The TOPflash reporter assay demonstrated significant downregulation after CTNNB1 shRNA transfection in RPMI8226 cells. Flow cytometric analyses also showed significantly profound apoptosis in CTNNB1 shRNA cells. We found CTNNB1 silence led to growth inhibition of MM growth in vivo. Immunohistochemical analyses showed that c-myc and β-catenin were reduced in CTNNB1 shRNA tumor tissues, but that expression of cleaved caspase-3 was increased. These results show that β-catenin could be a new therapeutic agent that targets the biology of MM cells.

Growth Inhibition of Human Multiple Myeloma Cells by an Oncolytic Adenovirus Carrying the CD40 Ligand Transgene

The growth-inhibitory activity of recombinant CD40 ligand (CD40L) is well documented in human multiple myeloma (MM). We examined MM-targeted delivery of CD40L by a conditional replicative oncolytic adenovirus, AdEHCD40L. The growth-regulatory activity of AdEHCD40L was determined in vitro and in vivo. Differential analysis with AdEHCD40L and parental virus (AdEHNull)-infected cultures allowed the identification of cellular and molecular pathways modulated by the CD40L transgene. Conditional expression of viral E1A and CD40L transgene was shown in human MM lines RPMI 8226 [interleukin (IL)-6 independent] and Kas-6/1 (IL-6 dependent) under hypoxic conditions commonly found in MM in situ. AdEHCD40L inhibited MM cell growth more effectively than AdEHNull. This enhanced growth-inhibitory activity was abrogated by cotreatment with a CD40L antibody. Chemoresistant MM lines (MR20 and LR5) were similarly susceptible to AdEHCD40L treatment. AdEHCD40L induced apoptosis and S-phase cell cycle blockade while uniquely up-regulating the previously described proapoptotic elements tumor necrosis factor-related apoptosis-inducing ligand, Fas, and IL-8. Intratumoral injections of AdEHCD40L reduced the growth of severe combined immunodeficient/hu RPMI 8226 xenografts by >50% compared with 28% reduction by AdEHNull. Adenoviral hexon and CD40L were detected in AdEHCD40L-treated tumors at day 35 after infection primarily in necrotic areas, suggesting viral replicative activity. These findings show that CD40L acts in concert with viral oncolysis to produce MM growth inhibition through activation of cellular apoptosis. The direct growth-inhibitory activity of AdEHCD40L, together with the well-known immune-potentiating features of CD40L, may be clinically applicable for the experimental treatment of MM or plasma cell leukemia.

Combination of the Proteasome Inhibitor Bortezomib and a Histone Deacetylase Inhibitor PXD101 Results in Synergistic Inhibition of Osteoclastogenesis and Significantly Stronger Inhibition of Multiple Myeloma Growth In Vitro and In Vivo.

Bortezomib, a proteasome inhibitor, has been known to have in vitro and in vivo activity against multiple myeloma (MM). PXD101, a novel, low molecular weight histone deacetylase (HDAC) inhibitor recently has been recognized to induce growth arrest and apoptosis in MM cells. We hypothesized that these two agents, with different molecular targets, can be combined with a high probability of additive or synergistic effects without exerting cross-resistance.In this study, we investigated the activity of the combination of PXD101 and bortezomib against osteoclast generation and MM cell growth. To evaluate the effects of bortezomib and PXD101 on the formation of osteoclasts from mononuclear hematopoietic precursors, we generated osteoclasts cultured from human bone marrow cells from healthy donors and MM patients using in vitro RANKL/M-CSF stimulation. Proliferation of MM cell lines treated with PXD101, bortezomib, or their combination was determined by measuring 3H-Thymidine incorporation. Annexin V-FITC/PI staining was used to determine the degree of apoptosis, and trypan blue was used to assess cell viability. We utilized a xenograft murine model to test the in vivo synergistic activity of the two agents against MM tumor formation.We found that even though either drug alone significantly inhibited osteoclast formation at concentrations of PXD101 at 25 nM or bortezomib at 1 nM, the combination resulted in synergistic inhibition in osteoclast assays. Treatment of MM cells lines (MM.1S, RPMI-8226, OPM2) with serial dilutions (10–100 nM) of the combination also led to significantly stronger inhibition of proliferation, apoptosis and cell death than single drug treatment. In addition, the combination demonstrated synergistic induction of cell death in primary human CD138+ myeloma cells isolated from patient samples. In vivo, the combination of PXD101 and bortezomib resulted in significantly higher xenograft tumor inhibition compared to either drug treatment alone and was not more toxic.The current study provides the rationale for clinical evaluation of this novel PXD101-bortezomib combination for patients with MM. The data generated here support the hypothesis that simultaneous targeting of different proliferative and anti-apoptotic pathways both in tumor cells and activated osteoclasts avoids cross-resistance and constitutes a dynamic approach to treat MM.