Mimicking the Myeloma Niche: A 3D Bone-Derived Co-Culture System to Selectively Assess Bystander BMSCs and to Perform High-Throughput Drug Screening in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1822-1822
Author(s):  
Johannes M. Waldschmidt ◽  
Dagmar Wider ◽  
Andreas R. Thomsen ◽  
Claudius Klein ◽  
Christine Aldrian ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
High Throughput ◽  
Research Funding ◽  
Ex Vivo ◽  
Bm Niche ◽  
Culture Model ◽  
Bystander Cells

Abstract Introduction: Novel substances such as the next generation IMiD pomalidomide or the recently approved next generation proteasome inhibitor carfilzomib (Cfz) have considerably expanded our treatment options in MM, both of them influencing multiple myeloma (MM) interaction with bone marrow stroma cells (BMSCs), that provides an interesting target for anti MM therapy. More compounds directed at this disease critical crosstalk are currently under investigation, however the development of novel drugs remains inefficient, displayed by a substantial drop out rate of the 376 preclinical single agents tested since 1961 (Rongvaux, Annu Rev Immunol. 2013; Schüler, Expert Opin Biol Ther. 2013; Kortüm, CLML 2014). Our focus in the projected presented here was to develop a novel bone-derived in vitro 3D co-culture model specifically adapted to mimic the BM niche to more closely study the role of bone and BM bystander cells and to perform more reliable ex vivo compound screening in MM. Methods: Previous 2D models were compared to a novel 3D co-culture model (agarose matrix interlayer, 100 microwells/cm², 1.5mm in depth, permeable for oxygen+cytokines, but not for BMSCs utilizing U266, RPMI-8226, OPM-2 and primary BM patient (pt) cells, with and without HS-5 vs. M210B4 stroma support (Fig. A + Fig. B.a. for pt characteristics). Analyses covered Trypan Blue, Annexin/PI, MTT, FACS, cell cycle analyses and H2B-mCherry/cytochrome c-GFP assays (Udi, Br J Hematol. 2013). In a next step, primary bone-derived stroma cells were acquired from bones of C57BL/6 J mice. Bones were flushed, digested and FACS sorted in order to acquire single BM and bone bystander cell subtypes (MSPCs [mesenchymal stem and progenitor cells], endothelial cells, osteoblasts, PAS [PDFGRalphaSca1] and CaRs [CXCL12-abundant reticular cells]) which were then compared to HS-5 and M210B4 with regard to growth support, cytokine secretion and protection from anti-MM substances. Results: MMCLs and pt specimens were cultured at different concentrations (10 vs. 100 cells per microwell) with and without M210B4 demonstrating a growth advantage with vs. without M210B4 (Fig. B.b). Liquid overlay technique allowed cluster formation of pt specimens leading to more reliable propagation of pt material for up to 20d of culture. Apoptotic changes were assessed by confocal microscopy of RPMI8226 co-expressing fluorescently labelled histone 2B-mCherry (red) and cytochrome c-GFP (green) as indicators of late and early apoptosis. Comparing BMSCLs with regard to their MM growth support capacities, human HS-5 proved even more beneficent than M210B4 stroma (Fig. B.b). Phenotypic analyses of pt specimens showed decreased CXCR4 expression with vs. without BMSCs suggesting a dynamic regulation of homing molecules. The model was then used as an ex-vivo platform allowing both cytotoxicity and cell cycle analyses for the combination of bortezomib (Btz) vs. Cfz with ARRY-520 (kinesin spindle protein inhibitor). Btz (10nM) and Cfz (20nM) proved significantly cytotoxic compared to the control (U266 and pt specimens, respectively) after 48h of single agent treatment Fig.B.c). Compared to Btz (10nM, B10) and Cfz (20nM, C20) as single agents, the additional combination with ARRY-520 showed stronger additive cytotoxicity for Btz (A5+B10, median: 37.5% vs. 13.1%) than for Cfz (A5+C20, 38.6% vs. 34.1%). To note, the model could also be utilized for more profound analyses as depicted for G2/M cell cycle studies in Fig.B.d. 5nM ARRY-520 (A5) led to significant accumulation of OPM-2 cells in G2/M arrest after 48h of treatment confirming prior analyses (Hernández-Garcia Blood Suppl 4710,2014; Fig. B.d). Co-culture studies with different subsets of BM and bone-derived bystander cells are currently ongoing and will be presented at the meeting (Fig. B.e). Conclusions: More complex, 3D bone-derived high-throughput in vitro models are urgently needed to better predict the potency of preclinically tested agents and to better estimate the likelihood of their later clinical adoption into phase I-II trials. With this work, we provide an innovative model which reflects the BM microenvironment as a crucial predictor for in-vivo sensitivity as shown for ARRY-5200. This ex-vivo approach helps to better incorporate MM growth support by bone and BM-derived bystander cells and thus depicts a valid tool to better characterize the role of the BM niche in myeloma. Figure 1. Figure 1. Disclosures Engelhardt: Deutsch Krebshilfe: Other: grant. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Disruption of MDM2-p53 Interactions by a Small Molecule Inhibitor RG7112 Increases Apoptosis and Impairs Polyploidization of Human Megakaryocytes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1082-1082 ◽  
Author(s):  
Camelia Iancu-Rubin ◽  
Mosoyan Goar ◽  
Ronald Hoffman
Keyword(s):  
Cell Cycle ◽  
Small Molecule ◽  
Research Funding ◽  
Phase I Clinical Trials ◽  
Viable Cells ◽  
Inactive Form ◽  
Cd34 Cells ◽  
P53 Activation

Abstract Abstract 1082 Megakaryocyte (MK) development is characterized by polyploidization, cytoplasmic maturation and proplatelet formation, which culminates in the release of platelets into the circulation. The tumor suppressor p53 plays a critical role in the regulation of both cell cycle and apoptosis; its function is tightly controlled by the murine double minute (MDM2) protein which facilitates p53 degradation and inhibits p53 transcriptional activity. MK ploidy results from a disruption of normal cell cycle progression termed endomitosis while platelet release is believed to depend on apoptotic processes. The role of p53-MDM2 in MK in these two processes has not been clearly defined. A small molecule RG7112, which disrupts MDM2-p53 interaction, has shown promising anti-tumor effects in phase I clinical trials. This beneficial outcome has, however, been associated with the development of thrombocytopenia. We, therefore, used RG7112 as pharmacological probe to examine the effects of disruption of the MDM2-p53 regulatory loop on MK. We determined the effects of RG7112 on primary human MK by utilizing an in vitro system in which MK were generated from BM-derived CD34+ cells. We first demonstrated that both p53 and MDM2 transcripts are up-regulated as MK differentiation progresses. The ability of CD34+ cells to proliferate in the absence or presence of various concentrations of RG7112 was then evaluated both in liquid cultures and in CFU-MK colony assays. CD34+ cells exposed to 10 μM RG7112 for 7 days generated 70% fewer viable cells as compared to control cells exposed to the inactive form of the drug (p value = 0.0038). Furthermore, CD34+ cells treated with RG7112 formed up to 40% less CFU-MK colonies as compared to untreated cells. An assessment of apoptosis of MK precursors generated in the presence of RG7112 revealed that 69.5+2.1% were Annexin V positive as compared to 31.5+3.5% present in control cultures. These findings are consistent with the previously reported role of RG7112 in inducing p53 activation and apoptosis. Interestingly, phenotypical characterization of the viable cells generated under identical culture conditions, showed that RG7112 treatment did not interfere with the ability of CD34+ cells to acquire markers of MK differentiation during the first 7 days of culture since similar degrees of CD41 and CD42 expression were observed in the absence and in the presence of the drug. Likewise, exposure of MK precursors to the drug for 7 additional days (i.e. later stages of maturation) did not influence CD41 and CD42 expression. By contrast, cells differentiated in the presence of 5 μM RG7112 generated 50% fewer polyploid MK with greater than 4N DNA content as compared to those treated with the inactive form of the drug. Moreover, the negative effects on ploidy were associated with p53 activation, as assessed by the increased levels of p21 protein, a direct target of p53 which is known to limit polyploidization of primary MK. Finally, platelets generated in vitro were analyzed phenotypically and quantitated by dual labeling with anti-CD41 antibodies and thiazole orange (TO). The number of CD41+/TO+platelets derived from MK generated in the presence of RG7112 was reduced by 22% as compared to control. Based on these findings, we conclude that RG7112 impacts megakaryopoiesis by two potential mechanisms: 1) Impairing the ability of CD34+ cells to generate MK precursors due to increased apoptosis; 2) Limiting polyploidization during the late stages of development due to phamacological activation of p53. A combination of these two effects may provide an explanation for thrombocytopenia observed in patients receiving this drug and suggests that p53 plays an important role in normal human thrombocytopoiesis. Disclosures: Iancu-Rubin: Roche: Research Funding. Hoffman:Roche: Research Funding.

Knockdown Of Matrix Metalloproteinase 13 (MMP13) In 5TGM1 Multiple Myeloma Cells Inhibits Development Of Lytic Bone Lesions In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 879-879
Author(s):  
Jing Fu ◽  
Shirong Li ◽  
Huihui Ma ◽  
G. David Roodman ◽  
Markus Y. Mapara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Trabecular Bone ◽  
Research Funding ◽  
Bone Lesions ◽  
Mineral Density ◽  
Lytic Bone Lesions

Abstract Background Multiple myeloma (MM) cells secrete osteoclastogenic factors that activate osteoclasts (OCL) and contribute to development of pure lytic bone lesions in MM patients. We have recently shown that i) MMP13 is highly expressed by MM cells and ii) exogenous MMP13 increases OCL fusion and bone resorption (Feng et al, 2009). Further, MMP13 mediates these effects by upregulating dendritic cell-specific transmembrane protein (DC-STAMP), which is critical for OCL fusion and activation (Fu et al, 2012). Here, we investigated the role of MMP13 in MM-related bone disease (MMBD) in vivo and the underlying osteoclastogenic mechanisms. Methods and Results The role of MMP13 in MMBD was examined in vivo by the intratibial 5TGM1-GFP mouse MMBD model. Mouse MM cell line 5TGM1-GFP cells were transduced by pLKO.1-puro empty vector (EV) or sh-MMP13 (MMP13-KD) lentivirus followed by puromycin selection for 2 weeks. MMP13 knockdown in 5TGM1-MMP13-KD cells were confirmed by quantitative RT-PCR. 1×105 5TGM1-GFP-EV and 5TGM1-GFP-MMP13-KD cells were bilaterally intratibially injected into Recombination Activating Gene 2 (Rag2) knockout mice (n=9). After 4 weeks of tumor growth, tibiae were separated for micro quantitative computed tomography (micro-QCT) followed by immunohistochemistry (IHC) analysis. Following 5TGM1-GFP-EV injection, micro-QCT analysis of the tibiae and adjacent femurs indicated severe bone erosions, especially within trabecular bone. By contrast MMP13 KD inhibited the development of MM-induced bone lesions. Bone histomorphologic analysis showed that compared to 5TGM1-GFP-EV, MMP13-KD significantly reduced the MM induced trabecular bone loss with increased relative bone volume (0.069 ± 0.018 vs 0.0499 ± 0.016%; P=0.001), connective density (54.94 ± 33.03 vs 27.33 ± 18.97mm3; P=0.002), trabecular bone numbers (3.26 ± 0.29 vs 3.06 ± 0.33mm-1; P=0.032) and bone mineral density (159.1 ± 20.7 vs 134.2 ± 18.6mg/cm3; P=6E-04); as well as decreased triangulation bone surface to volume ratio (66.12 ± 6.67 vs 73.28 ± 10.07; P=0.017) and triangulation structure model index (3.05 ± 0.36 vs 3.42 ± 0.35 mm-1; P=0.002). In accordance with our finding that MMP13 induced OCL fusion, IHC results confirmed the presence of smaller TRAP+OCLs adjacent to the tumor in mice injected with 5TGM1-GFP-MMP13-KD cells compared with 5TGM1-GFP-EV cells. Although MMP13 knockdown showed no effects on 5TGM1-GFP cell growth in vitro, in vivo tumor progression represented by fluorescence imaging and sera immunoglobin 2G level (0.96 ± 0.12 vs 1.10 ± 0.11 mg/ml) was significantly inhibited (P=0.009 and 0.03 respectively), indicating MMP13 depletion in MM cells impaired OCL activation which, in turn, failed to support MM cell growth in bone marrow microenvironment as effectively in EV control group. In vitro studies demonstrated that MMP13 directly induced ERK1/2 phosphorylation in pre-osteoclasts. Consistent with a critical role for ERK1/2 phosphorylation in regulating OCL formation, U0126 (ERK1/2 inhibitor) blocked MMP13-induced ERK1/2 phosphorylation, ERK1/2-dependent DC-STAMP upregulation and MMP13-induced OCL fusion (P<0.01). Conclusion Our results demonstrate that silencing MMP13 expression in MM cells inhibits MM cell-induced OCL fusion and development of lytic bone lesions in vivo, indicating that MMP13 is essential for MM-induced bone diseases. Further, MMP13 upregulates DC-STAMP expression and OCL fusion via the activation of ERK1/2 signaling. Our data suggest that targeting MMP13 may represent a novel therapeutic approach for the treatment of MMBD. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Lilly: Research Funding. Lentzsch:Celgene: Research Funding.

scholarly journals Integrative Analysis of FISH, Transcriptomics and Mutational Status Predicts Responsiveness to Novel Agents in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 574-574
Author(s):  
Cecilia Bonolo De Campos ◽  
Caleb K Stein ◽  
Nathalie Meurice ◽  
Laura Ann Bruins ◽  
Joachim L Petit ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Throughput ◽  
Predictive Models ◽  
Research Funding ◽  
Ex Vivo ◽  
Integrative Analysis ◽  
Drug Screen ◽  
Mrna Sequencing ◽  
Bet Inhibitors ◽  
Selection Of

Introduction Despite continuous improvement of clinical outcome in multiple myeloma (MM), disease relapse remains a major challenge, leading to progressively shorter remissions and fewer treatment options. Strategies attempting to counteract this challenge include recent efforts resulting in an increase in the availability of novel promising anti-MM agents and targeting specific genetic profiles of the disease. In this context, we aim to develop predictive models of sensitivity and resistance to novel compounds by connecting an ex vivo high-throughput drug screen with genetic, transcriptomics, FISH, and clinical features. Methods Twenty compounds (afatinib, afuresertib, belinostat, buparlisib, cobimetinib, CPI-0610, crenolanib, dinaciclib, dovitinib, JQ1, LGH447, osimertinib, OTX015, panobinostat, romidepsin, selinexor, sunitinib, trametinib, venetoclax, and vorinostat) were selected based on overall promising anti-MM activity from an ex vivo high throughput drug screen with a panel of 79 single agents incubated for 24 hours. The area under the curve (AUC) was used to rank order the ex vivo responses for each compound and the lowest and highest quartile samples were identified for further analysis. Clinical data and FISH data, including t(11;14), t(4;14), t(14;16), del(17p), +1q, monosomy 13, and MYC rearrangement, were collected. Targeted DNA sequencing was performed using a 2.3 Mb custom capture panel covering 139 MM-relevant genes. mRNA-sequencing was performed and differential gene expression analysis in the highest and lowest quartile identified subsets of markers positively and negatively associated with the AUC response for a given compound. An additional unbiased selection of markers using lasso techniques was performed, resulting in predictive generalized linear models (GLM) for each agent. Responses from the remaining intermediate samples were estimated with the predictive models, with overall predictive ability assessed by correlating predicted AUCs with their actual counterparts. Results Our integrative analysis was performed on 50 primary patient samples (36% untreated and 64% relapsed MM). Venetoclax, dinaciclib, romidepsin, panobinostat, osimertinib, belinostat and selinexor were the most active compounds in the cohort. Interestingly, LGH447, dovitinib, selinexor, JQ1, OTX-015, cobimetinib, and trametinib showed increased activity in relapsed MM when compared to untreated samples (Wilcoxon Test; p&lt;0.05). We generated GLMs using an average of 92 markers (range 64-107) per compound, combining mRNA-sequencing expression with FISH and mutation data. The analysis proposed in the present study was validated through the unbiased selection of BCL2 among the subset of markers included in the GLM predicting sensitivity to venetoclax, a first-in-class orally bioavailable selective BCL2 inhibitor. Expression level of critical NF-kB and cell cycle genes, such as BIRC3, CKS1B, PAX5, NFKB2, and CCND2, were included in 60% of our predictive models. Mutations of DNA repair genes (ATM,TP53) were included in the GLMs of three epigenetic therapies, one histone deacetylase inhibitor and two BET inhibitors, associated to ex vivo resistance to the drugs. The presence of monosomy 13 was also a marker for ex vivo resistance for five epigenetic therapies, four HDAC inhibitors and one BET inhibitor. The three BET inhibitors, JQ1, CPI-0610, and OTX015, were among the compounds most accurately predicted by our integrative approach, with Spearman correlation values between 0.773-0.858. Overall, our models accurately predicted the ex vivo response for 16 (80%) of the compounds (r&gt;0.7). Five (25%) of these compounds displayed a remarkably accurate prediction model in both training (highest and lowest quartiles) and validation (intermediate quartiles) samples (r&gt;0.8). Conclusions The GLM data integration approach enabled the establishment of effective predictive models, identifying FISH, transcriptomics, and mutations of putative driver genes important in anti-MM agent responsiveness. In addition, the resulting dataset is promising for future research focusing on the discovery of novel mechanisms of action and establishing markers of sensitivity and resistance to novel compounds. We are currently increasing our dataset and seek to create an omnibus approach that predicts responses to multiple anti-MM agents simultaneously. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Stewart:Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy; Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy.

scholarly journals Multiple Myeloma Pathogenesis: The Role of Junb in Bone Marrow Angiogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4341-4341
Author(s):  
Fengjuan Fan ◽  
Stefano Malvestiti ◽  
Yujia Shen ◽  
Eugenio Morelli ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Angiogenic Factors ◽  
University Research ◽  
Advisory Committees ◽  
Cell Clones ◽  
In Vitro Angiogenesis

A significant increase in bone marrow (BM) angiogenesis represents a key event in early, microenvironment-dependent, multiple myeloma (MM). Angiogenic growth factor- and cytokine- production and secretion is a complex process regulated by a plethora of transcription factors (TFs). Over the past years, members of the AP-1 family of TFs have emerged as potential new therapeutic targets. Our recent work demonstrated for the first time a pivotal role for the AP-1 family member JunB in MM pathogenesis (Fan et al., 2017). Whether JunB also contributes to MM BM angiogenesis is currently unknown. In silico and immunohistochemical analyses revealed a correlative increase of JunB and angiogenic growth factors in samples isolated from healthy donors to MGUS and MM patients; and a decrease in samples isolated from patients with plasma cell leukemia. These data were supported by the utilization of an innovative in vivo MM model of clonal evolution. Specifically, JunB as well as selected angiogenic factors were significantly increased in tumor cell clones at primary sites (bone chips) versus tumor cell clones at metastatic (distant BM) sites, as evidenced by whole exome and RNA sequencing. Functionally, doxycyclin- induced inhibition of stroma cell: MM cell co-culture- as well as of IL-6- mediated JunB upregulation in TetR-shJunB/ MM.1S cells significantly reduced production and secretion of angiogenic factors; and consequently inhibited in vitro angiogenesis. Conversely, 4-hydroxytamoxifen (4-OHT)-mediated upregulation of JUNB activity in JUNB-ER/MM cells strongly increased the expression and secretion of angiogenic factors and in vitro angiogenesis. The interaction of JunB with angiogenic factor- encoding DNA in MM cells was further confirmed utilizing chromatin immunoprecipitation (ChIP)- sequencing. Finally, treatment with doxycycline effectively inhibited JunB levels and consistently reduced microvessel density in immunodeficient NSG mice inoculated with TetR-shJUNB/ MM.1S, but not TetR-SCR/ MM.1S. In conclusion, our findings demonstrate a pivotal role of JUNB in MM BM angiogenesis; they thereby provide further evidence that JUNB is a promising therapeutic target particularly in early MM. Disclosures Vallet: Pfizer: Honoraria; Roche Pharmaceuticals: Consultancy; MSD: Honoraria. Roccaro:Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; European Hematology Association: Research Funding. Goldschmidt:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Molecular Partners: Research Funding. Podar:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Amgen Inc.: Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding.

scholarly journals DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5618-5618 ◽  
Author(s):  
Priya Choudhry ◽  
Margarette C. Mariano ◽  
Arun P Wiita
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Ex Vivo ◽  
Single Agent ◽  
Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

scholarly journals TBK1/Ikkε Inhibitor Amlx Blocks Multiple Myeloma Cell Growth in Vitro and In Vivo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4504-4504
Author(s):  
Quanhong Sun ◽  
Peng Zhang ◽  
Juraj Adamik ◽  
Konstantinos Lontos ◽  
Valentina Marchica ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Growth ◽  
Cell Lines ◽  
Bone Disease ◽  
Research Funding ◽  
Ex Vivo ◽  
Dominant Negative ◽  
Induction Of Apoptosis

Abstract Multiple myeloma (MM) is the most frequent cancer to involve the skeleton and remains incurable for most patients, thus novel therapies are needed. MM bone disease is characterized by osteolytic lesions that contribute significantly to patient morbidity and mortality. We showed that TBK1 signaling is a novel pathway that increases osteoclast (OCL) formation in Paget's disease, an inflammatory bone disease. Therefore, we hypothesized that TBK1 plays a similar role in MM induction of OCL. We found that MM conditioned media (MM-CM) dose-dependently increased bone marrow monocyte (BMM) expression of activated TBK1 protein and enhanced RANKL-driven OCL formation. TBK1 knockdown by shRNA transduction into BMM significantly attenuated the ability of MM-CM to increase OCL differentiation without altering OCL differentiation in control media. We found that the TBK1/IKKε inhibitor Amlexanox (Amlx) blocked normal and MM-enhanced OCL formation. Importantly, TBK1 mRNA expression in CD138+ plasma cells (PC) isolated from MM or PC leukemia patients is significantly higher as compared to PC from Monoclonal Gammopathy of Undetermined Significance (MGUS) patients. Therefore, we tested whether targeting the TBK1/ IKKε signaling pathways would also affect MM cells. We found that Amlx strongly decreased the viability of several MM cell lines and primary MM cells via induction of apoptosis. Amlx treatment of MM cell lines also induced a G1/S blockade, decreased activated ERK1/2, and increased translation of the dominant-negative C/EBPb-LIP isoform in several MM cell lines. The positive-acting C/EBPb-LAP isoform was previously shown to be a critical transcription factor for MM viability. Importantly, Amlx also enhanced the effectiveness of the proteasome inhibitors bortezomib and carfilzomib to kill MM cells in culture. Further, Amlx sensitized MM1.S cells to the induction of apoptosis by the autophagic inhibitor Bafilomycin A. Amlx dose-dependently inhibited tumor growth in a syngeneic MM mouse model in which 5TGM1 MM cells expressing secreted GLuc were injected subcutaneously into immunocompetent C57Bl/KaLwRij. Tumor growth was assessed by measuring tumor volumes and by the levels of secreted GLuc in the blood. Further, OCL formation ex vivo from bone marrow monocytes obtained from AMLX-treated mice versus controls was decreased. Amlx did not affect the viability of primary BMM, bone marrow stromal cells (BMSC), or splenocytes. Further, Amlx treatment of primary BMSC from MM patients or normal donors decreased expression of TNFα, IL-6 and RANKL, thereby decreasing BMSC support of MM survival and OCL differentiation. Amlx pretreatment of BMSC and murine pre-osteoblast MC4 cells also decreased VCAM1 expression and reduced MM cell adhesion, another mechanism for Amlx reduction of bone microenvironmental MM support. These data suggest that targeting TBK1/IKKε signaling may decrease MM bone disease by slowing MM growth, directly and indirectly, and preventing MM-induced osteolysis. Disclosures Giuliani: Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding. Roodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Towards CD38-Targeted Immunochemotherapy of Multiple Myeloma: Significant Improvement of Anti-Myeloma Effects of Lenalidomide, Bortezomib and Dexamethason by CD38-Specific Antibody Daratumumab, Even in Chemotherapy Resistant/Intolerant Patients

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4988-4988
Author(s):  
Inger S. Nijhof ◽  
Jeroen Lammerts van Bueren ◽  
Berris van Kessel ◽  
Michel de Weers ◽  
Joost M Bakker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Research Funding ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Human Antibody ◽  
Synergistic Activity ◽  
Effector Cells ◽  
Complement Dependent Cytotoxicity

Abstract Abstract 4988 To date, multiple myeloma (MM) remains an incurable malignancy of antibody-producing clonal plasma cells. The introduction of a new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT), used alone or in combination with steroids (dexamethasone; DEX or prednisone; PRED) has significantly improved the overall survival of MM patients. Nonetheless, all chemotherapy strategies are eventually hampered by the development of drug-resistance. Towards a novel and effective targeted immunotherapy for MM, we have developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity. In vitro, DARA induces substantial anti-MM effects mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In ex vivo assays, which allowed us to address killing of MM cells in bone marrow aspirates isolated from MM patients, enhanced or even synergistic MM cell killing was observed when DARA was combined with LEN, or with cocktails of LEN/BORT/DEX and melphalan/BORT/DEX. We now extended these ex vivo analyses to evaluate whether DARA in combination with LEN, BORT and DEX could improve the lysis of MM cells in bone marrow aspirates derived from 22 patients of whom 9 became refractory for LEN and 6 for LEN and BORT. DARA significantly enhanced the lysis of MM cells when combined with LEN or BORT in virtually all patients, including the LEN- and LEN/BORT-refractory patients. The combination of DARA+BORT and DARA+DEX induced additive killing, suggestive of lysis by independent mechanisms. When combined with LEN, DARA improved the lysis of MM cells in a synergistic manner in both non-refractory and LEN-refractory patients. This is suggestive of killing by at least partly complementary mechanisms. Synergistic activity of LEN and DARA was attributable to LEN-induced activation of effector cells that were involved in DARA-mediated ADCC. In addition, enhanced/synergistic direct killing of MM cells was observed. Experiments are under way to further investigate the mechanism underlying synergistic activity of DARA and LEN. In conclusion, our results provide a rationale for clinical evaluation of DARA in combination with LEN, BORT and DEX including in patients refractory to these drugs. Disclosures: van Bueren: genmab: Employment. de Weers:genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Lokhorst:genmab: Consultancy, Research Funding. Mutis:genmab: Research Funding.

scholarly journals 3D-Tissue Engineered Bone Marrow (3DTEBM) Culture Retrospectively Predicts Treatment Clinical Outcomes of Multiple Myeloma Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1987-1987
Author(s):  
Amanda Jeske ◽  
Feda Azab ◽  
Pilar De La Puente ◽  
Barbara Muz ◽  
Justin King ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Ex Vivo ◽  
In Vitro Models ◽  
Advisory Committees ◽  
Treatment Plans ◽  
Equity Ownership

Abstract Background: Multiple Myeloma (MM) is the second most common hematological malignancy, and continues to be a fatal disease even with the development of novel therapies. Despite promising preclinical data in standard tissue culture models, most drugs fail in clinical trials and show lower efficacy in patients. This highlights the discrepancy between the current in vitro models, the pathophysiology of the disease in the patients, and the urgent need for better in vitro models for drug development and improved prediction of efficacy in patients. We have previously developed a patient-derived 3D-Tissue Engineered Bone Marrow (3DTEBM) culture model, which showed superior properties for proliferation of primary MM cells ex vivo, and better recapitulated drug resistance. The long-term goal of this study is to use the 3DTEBM model as a tool to perform drug screens on BM aspirates of MM patients and prospectively predict the efficacy of different therapies in individual patients, and help treatment providers develop personalized treatment plans for each individual patient. In the current study, we used the 3DTEBM model to, retrospectively, predict clinical responses of MM patients to therapy, as a proof of concept. Methods: We used whole-BM, viably frozen tissue banked samples from 20 MM patients with clear clinical response patterns of complete remission, and either very good partial response (sensitive) or progressive disease (non-sensitive). The BM aspirates were used to develop a 3DTEBM that represents each individual patient. The patient-derived 3DTEBM cultures were treated ex vivo with the same therapeutic regimen that the patient received in the clinic for 3 days. The treatment ex vivo was based on combinations at different concentrations which mimic the steady state concentrations (Css) of each drug. The efficacy of the treatment ex vivo was evaluated by digestion of the 3DTEBM matrix, extraction of the cells, and analysis for prevalence of MM cells in the treatment groups compared to the non-treated controls. Patients were defined "sensitive" if the effect reached 50% killing in the range of 10xCss. The ex vivo sensitivity data was then correlated with the clinical response outcomes. Results: We found that the 3DTEBM was predictive in approximately 80% of the cases (in about 85% of the combination therapy cases, and in about 70% of the single therapy cases). Broken down by individual drug, it was predictive in 80% of the cases treated with Bortezomib, 78% Lenalidomide, 84% Dexamethasone, 100% Daratumumab, 50% Carfilzomib, 50% Pomalidomide, and 100% Doxorubicin. Conclusions: The 3DTEBM is a more pathophysiologically relevant model which predicts clinical efficacy of drugs in multiple myeloma patients, retrospectively. This data provides the bases for future studies which will examine the ability of the 3DTEBM model to predict treatment efficacy, prospectively, for development of personalized treatment plans in individual multiple myeloma patients. Disclosures Jeske: Cellatrix LLC: Employment. Azab:Cellatrix LLC: Employment. De La Puente:Cellatrix LLC: Other: Co-founder. Vij:Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Azab:Ach Oncology: Research Funding; Cellatrix LLC: Equity Ownership, Other: Founder and owner; Glycomimetics: Research Funding; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner.

scholarly journals The Role of PHF19 As a Promoter of Tumorigenicity and Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 508-508
Author(s):  
Carolina D. Schinke ◽  
Pingping Qu ◽  
Shmuel Yaccoby ◽  
Valeriy V Lyzogubov ◽  
Veronica MacLeod ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Candidate Gene ◽  
Cell Lines ◽  
Crucial Role ◽  
Plasma Cells ◽  
Total Therapy

Introduction - Multiple Myeloma (MM) is a hematologic malignancy characterized by clonal growth of differentiated plasma cells (PCs). Despite improvement in MM therapy, the disease remains mostly incurable and is characterized by recurrent relapses with development of resistant clones that eventually lead to patient death. The pathways that lead to resistant and aggressive MM are not fully understood highlighting the need to improve our understanding of MM biology to identify potential new pathways and therapeutical targets. PHD Finger Protein 19 (PHF19) is a regulator of Polycomb Repressive Complex 2 (PRC2), the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. PRC2 employs enhancer of zeste homolog 1 and 2 (EZH1/EZH2) as enzymatic subunits to hypermethylate H3K27. While overexpression and gain of function mutations of EZH1/2 have been observed in many cancers the role of this particular pathway in MM remains poorly understood. In the present study, we report on PHF19 as a new candidate gene to play a potential crucial role in MM oncogenesis. Methods- Gene expression profiling (GEP; Affymetrix U133 Plus 2.0) was performed on 739 MM patients (from total therapy trials [TT] 3-5; low risk MM n=636, high risk MM n=103), 42 patients with monoclonal gammopathy of undetermined significance (MGUS), 73 smoldering MM patients, 42 patients with primary plasma cell leukemia and 34 healthy donors. Myeloma risk was determined by the GEP 70 signature as previously defined. To test the implications of functional PHF19 knock down (KD) we used TRIPZ inducible PHF19 shRNA vs. scrambled control (Dharmacon) in two MM cell lines (JJN3 and ARP1). Real time PCR as well as western blotting was used to confirm PHF19 KD as well as to elucidate the effect on H3K27me3 (Cell Signaling). Functional in vitro studies included proliferation (Promega), clonogenic assays (StemCell), cell cycle and apoptosis assays (both Invitrogen). In vivo studies were performed using SCID mice that were subjected to tail vain injection with PHF19 KD JJN3 cells (n=10) or scrambled shRNA control (n=10). Weekly ELISA (Bethyl) and in vivo imaging (Xenogen) were performed and survival was recorded. Results- GEP of the previously mentioned patient populations and healthy controls identified PHF19 (chr9q33.2) as a candidate gene that was consistently dysregulated in MM patients. Mean expression levels at different MM stages correlated with disease aggressiveness (ANOVA, p&lt;0.0001), Figure 1. High expression of PHF19 (log2&gt;10.46) at diagnosis correlated significantly with adverse clinical parameters, including ISS III, anemia and elevated LDH, as well as worse overall survival (5 yr OS = 29% for patients with high PHF19 expression vs 77% for patients with low PHF19 expression [log2&lt;10.46], p&lt; 0.0001). These results led us to test the implications of functional PHF19 KD using TRIPZ inducible PHF19 shRNA vs. scrambled control in the JJN3 and ARP1 MM cell lines. PHF19 KD led to a drastic reduction of H3K27me3 thereby resulting in significantly reduced proliferation via cell cycle arrest, while apoptosis was not substantially altered. Clonogenic assays showed a significant reduction in colony numbers and size of MM cells with PHF19 KD compared to the control (&gt;75% reduction in both cell lines, p&lt;0.05). Xenograft studies showed consistently less tumor burden in the mice injected with PHF19 KD cells compared to scrambled control, evident through ELISA testing for IgG Kappa (Median =180 mg/ml for scrambled control vs 80 mg/ml for PHF19 KD at week 8, p=0.07) and bioimaging (Median bioilumisence 2.1x108 p/s for scrambled control vs. 0.8x108 p/s for PHF19 KD at week 8, non-significant). Median OS in mice injected with PHF19 KD cell was substantially longer (66 days) compared to mice subjected to scrambled control cells (54 days), p=0.052. Conclusion- In summary we show that PHF19 is upregulated in malignant plasma cells of MM patients and that PHF19 expression levels increase with advanced MM stages. High PHF19 expression was a marker of adverse prognosis in our total therapy (TT 3-5) cohort. Most importantly, in-vitro and in-vivo functional studies showed that PHF19 has important biological functions in MM. These results suggest that epigenetic regulation through histone methylation, in particular, H3K27 trimethylation, plays a crucial role in MM and the affected downstream pathways should be further elucidated. Disclosures Boyle: Janssen: Honoraria, Other: Travel; Abbvie: Honoraria; Amgen: Honoraria, Other: travel; Takeda: Honoraria, Other: travel; Celgene Corporation: Honoraria, Other: Travel. van Rhee:Kite Pharma: Consultancy; Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy. Walker:Celgene: Research Funding.

scholarly journals Glaucocalyxin A suppresses multiple myeloma progression in vitro and in vivo through inhibiting the activation of STAT3 signaling pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mei Li ◽  
Cailong Chen ◽  
Qian Wang ◽  
Xiaolu Jiang ◽  
Lanlan Tan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Xenograft Model ◽  
Mice Model ◽  
Cycle Arrest ◽  
Nude Mice Model

Abstract Background Multiple myeloma (MM) is the most common malignant hematological disease in the people worldwide. Glaucocalyxin A (GLA) is a bioactive ent-kauranoid diterpenoid, that is derived from Rabdosia japonica var. GLA has been demonstrated that it had various pharmacological activities, such as anti-coagulation, anti-bacterial, anti-tumor, anti-inflammation, antioxidant activities. Although GLA has effective anti-tumor properties, its effects on multiple myeloma remain unclear. The aim of this study was to examine the possible anti-cancer effects of GLA and their molecular mechanisms on MM cells in vitro and in vivo. Methods To evaluate the role of GLA on the proliferation of MM cells in vitro and in vivo, we used MTT method to detect the role of GLA on the proliferation of MM cells. Cell apoptosis and cell cycle assay were evaluated by flow cytometry. Protein expressions in GLA-treated and untreated MM cells were evaluated by western blot analyses. MM xenograft nude mice model was used to investigate the role of GLA on the proliferation of MM cells in vivo. IHC assay was used to examine the role of GLA on the MM xenograft model in vivo. Results In the present study, we firstly reported the potent anti-myeloma activity of GLA on MM cells. We found that GLA could induce apoptosis in vitro and in vivo. GLA could inhibit the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and downregulate interleukin IL-6 induced STAT3 phosphorylation in MM. Overexpression of STAT3 could significantly prevent apoptosis induced by GLA; while knockdown of STAT3 enhanced it. Moreover, GLA could inhibit cell proliferation by inducing the cell cycle arrest. GLA reduced the expression of cell cycle-related proteins CCNB1, CCND1, CCND2, and CCND3 and increased the expression of p21 in MM cell lines. In addition, in the MM xenograft nude mice model, GLA exhibited very good anti-myeloma activity. Administration of GLA almost completely inhibited tumor growth within 19 days without physical toxicity. And the IHC results showed GLA significantly inhibited cell proliferation and interfered STAT3 pathway on MM xenograft model tumor tissues. Conclusions Taken together, our present research indicated that GLA inhibits the MM cell proliferation, induces MM cell apoptosis and cell cycle arrest through blocking the activation of STAT3 pathway. Thus, GLA may be a potential therapeutic candidate for MM patients in the future.

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