Activity of ARRY-614, an Inhibitor of p38 Map Kinase and Angiogenic Targets, in Hematologic Malignancies.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4798-4798
Author(s):  
Shannon L. Winski ◽  
Stefan D. Gross ◽  
Suzy A. Brown ◽  
Deborah Anderson ◽  
Augusta Garrison ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Ex Vivo ◽  
Single Agent ◽  
Hematologic Malignancies ◽  
P38 Map Kinase ◽  
Matrigel Invasion Assay ◽  
In Vivo Models ◽  
Matrigel Invasion

Abstract Multiple Myeloma (MM) is a malignancy characterized by the clonal expansion of plasma cells within the bone marrow microenvironment. Within this compartment, the proliferative capacity of MM cells is enhanced by pro-inflammatory cytokines, and there is growing recognition that bone marrow stromal cells play an important role in the support of MM growth and chemo-resistance. We have identified a series of compounds, of which ARRY-614 is representative, that are potent inhibitors (EC50<10 nM) of cytokine synthesis through the inhibition of p38 MAPK. This compound is also a potent inhibitor of Abl tyrosine kinases and Tie2/Tek receptor tyrosine kinase. ARRY-614 is active against all of these targets on both the isolated enzymes and in cells. To confirm these activities, ARRY-614 was evaluated in relevant in vitro and in vivo models. ARRY-614 inhibited p38α in ex vivo stimulated human whole blood (EC50=2 nM) and the release of IL-6 and TNFα from SEA- or LPS-challenged mice (ED50<10 mg/kg). Further, ARRY-614 administered as a single agent was efficacious at inhibiting bFGF-driven angiogenesis in an in vivo matrigel invasion assay as well as inhibiting tumor growth in subcutaneous K562 (BCR-Abl dependent) and RPMI 8226 (multiple myeloma) tumor xenografts in mice, at doses ranging from 30 to 100 mg/kg qd, PO. In regulated safety studies, this compound was well-tolerated at doses up to 100 mg/kg qd and 30 mg/kg qd in rats and cynomolgus, respectively. Together, these data support the advancement of this agent into clinical trials for hematologic malignancies.

scholarly journals Systems Biology Analysis Identifies Targetable Vulnerability Networks to Proteasome Inhibitors in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 950-950
Author(s):  
Mark B. Meads ◽  
Rafael Renatino Canevarolo ◽  
Praneeth Reddy Sudalagunta ◽  
Paula S. Oliveira ◽  
Dario M. Magaletti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Single Agent ◽  
Proteasome Inhibitors ◽  
In Vivo Models ◽  
Viability Assay

Abstract Proteasome inhibitors (PI) such as bortezomib and carfilzomib are critical components of anti-multiple myeloma (MM) therapy, yet all MM patients eventually develop refractory disease. We developed a non-biased method to identify and validate dysregulated pathways associated with PI-resistance in myeloma by combining RNAseq data from 522 MM patient specimens obtained from our Total Cancer Care/M2Gen/ORIEN network at Moffitt Cancer Center with paired ex vivo sensitivity to PIs and kinase inhibitors (KI). Dimensionality reduction analysis (t-SNE) and Fuzzy C-means was used to identify 422 clusters of genes that co-express in individual patients, and Gene Set Enrichment Analysis (GSEA) was used to identify clusters with gene expression patterns that correlated with PI sensitivity. Using publicly curated databases and in silico integrative analyses, we built protein-protein interaction networks to identify putative transcription factors, corresponding master regulators (kinases), and candidate KIs to promote PI sensitization. This systems biology approach identified a Chk1-Cdk1-Plk1 circuit associated with PI-resistance and also found 21 additional kinases (of 501 expressed in our cohort's kinome) that could be targeted to re-sensitize PI-resistant MM, which we confirmed in cell lines, specimens from relapsed patients, and two in vivo models. A panel of paired isogenic PI-resistant and sensitive MM cell lines were differentially screened to find kinases associated with PI-resistance using activity-based protein profiling (ABPP) and KI activity measured by high-throughput viability assay. The MM cell lines 8226 and U266, along with their drug resistant counterparts 8226-B25 and U266-PR, were grown in mono-culture for 24h and lysates were enriched for ATP binding proteins by affinity purification versus a chemical probe. Tryptic peptides were measured using discovery proteomics (nano-UPLC and QExactive Plus mass spectrometer) to identify 85 kinases out of a total of 715 proteins in 8226-B25 MM cells and 35 kinases out of a total of 688 proteins in U266-PR MM cells that were preferentially enriched by 2-fold change compared to parental cell lines. Twenty-four kinases were commonly activated among PI-resistant cell line pairs and were screened in PI-resistant myeloma lines using a label-free, high throughput viability assay that simulates the tumor microenvironment. Three KIs targeting Plk1 (volasertib and GSK461364) and Cdk1/5 (dinaciclib) consistently maintained LD50s in the low-nanomolar range and induced caspase-3 activation in four PI-resistant MM cell lines: 8226-B25, U266-PR, ANBL-6-V10R, and Kas6-V10R. Twenty-four kinases each were identified by RNAseq/ex vivo PI sensitivity of MM specimens and ABPP of PI-resistant/sensitive MM cell line pairs. Of these, 7 kinases were identified by both methods: Cdk1, Chk1, Plk1, ILK, Syk, PKA, and p70S6K. Several KIs targeting Cdk1, Plk1, ILK, DNAPK, Syk, MKK7, Nek2, and mTOR identified in patient specimen or cell-line screens showed single agent activity in MM patient bone marrow specimens purified by a CD138 affinity column. Among these, inhibitors to Cdk1, ILK, mTOR, and Plk1 showed the most activity in patient specimens with an average 96h LD50 of 25 nM (n=56), 2.4 uM (n=42), 2.7 uM (n=57) and 3.8 uM (n=53), respectively. Six KIs targeting Plk1, ILK, Syk, MKK7, Nek2 and MARK3 were synergistic with carfilzomib in 20 patient specimens and maintained or improved ex vivo activity in relapsed refractory MM (RRMM) specimens. Volasertib, which targets Plk1, was the most synergistic with carfilzomib of all KIs tested in patient specimens and was further validated in two in vivo models: a NSG/U266 xenograft model of PI resistance and the syngeneic C57BL/6-KaLwRij/5TGM1 immunocompetent model. Volasertib significantly increased survival and reduced tumor burden in both models as a single agent, and was more effective versus PI-resistant tumors compared to PI-sensitive counterparts. Our pharmaco-proteomic screen, coupled with rich gene expression data from patients identified Plk1 as a target critical to MM survival in the context of acquired PI resistance and represents a unique workflow to find tumor vulnerabilities that arise during therapy. We anticipate that these data will also produce a critical path for the personalized allocation of therapy to maximize efficacy and minimize the use of ineffective therapies in RRMM. Disclosures No relevant conflicts of interest to declare.

scholarly journals A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽  
2016 ◽  
Vol 31 (8) ◽  
pp. 1743-1751 ◽  
Author(s):  
S Hipp ◽  
Y-T Tai ◽  
D Blanset ◽  
P Deegen ◽  
J Wahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cell ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Selective Lysis ◽  
Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

Developing In Vivo Model for Studying Mechanisms of Osteoblast Suppression in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4731-4731
Author(s):  
Chang-Sook Hong ◽  
Alisa Huston ◽  
Flavia Esteve ◽  
Judy Anderson ◽  
Ken Patrene ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Osteoblast Differentiation ◽  
Treated Group ◽  
Bone Lesions ◽  
Neoplastic Disease ◽  
In Vivo Models ◽  
Myeloma Cells ◽  
Ganciclovir Treatment

Abstract Multiple myeloma (MM) is an incurable neoplastic disease characterized by an accumulation of plasma cells in bone marrow. Osteolytic bone lesions are the major source of morbidity in MM patients and are associated with bone pain and fractures and hypercalcemia. The bone lesions result from increased osteoclastic bone destruction in areas adjacent to the myeloma cells. New bone formation that normally happens at sites of previous bone resorption still occurs in early stages of the disease but is absent in advanced MM. Although the molecular basis for the increased osteoclastic activity has been intensely investigated, the basis for the decreased osteoblast activity is just beginning to be understood. Recently, inhibitors of WNT signaling pathway, Dickkorpf1 (DKK1) and secreted Frizzle-Related Protein-2 (sFRP2) have been identified as factors involved in osteoblast suppression in MM. In addition, IL-3 and IL-7 are increased in plasma of MM patients and suppress osteoblastogenesis in cell culture models. However, the role of those factors in the osteoblastic activity in MM patients is unclear. Studies in patients are confounded by cytotoxic therapy as well as bisphosphonates, which are standard therapy for MM patients. Therefore, preclinical in vivo models are required to delineate the mechanisms responsible for the profound osteoblast suppression in MM. We have developed a mouse model of myeloma bone disease in which genetically modified myeloma cells can be selectively ablated without the confounding effects of cytotoxic therapies and allows us to tract the growth of MM cells. The 5TGM1 cell line which is the most common version of murine MM, was stably transfected with the thymidine kinase (TK) gene from herpes simplex virus, which permits eradication of myeloma cells with ganciclovir, as well as GFP and luciferase genes to detect the presence of MM cells. One ug/ml ganciclovir treatment in culture results in 100% death of the transfected 5TGM1 cells in 4 days. Importantly, ganciclovir treatment of primary marrow cell cultures had no effect on growth and differentiation of osteoblast and hematopoietic progentitors. Co-culturing of primary marrow cells with 5TGM1 expressing TK has no bystander effect on osteoblast differentiation with ganciclovir treatment. Subcutaneously implanted 5TGM1 cells into SCID mice were eradicated by intraperitoneal injection of 20mg/kg ganciclovir/d for 2 weeks. The dose of ganciclovir did not affect osteoblast differentiation of primary marrow culture from the mice treated with ganciclovir. Then we injected the 5TGM1 cells into tibia of SCID nude mice (n=4 per group). After measuring the increase of serum IgG2b level, half of the mice were treated with ganciclovir for 2 weeks and the other with saline. Our preliminary data show that osteogenic cultures of bone marrow from the ganciclovir treated mice had significantly higher alkaline phosphatase activity than cultures derived from the saline treated group (p=0.03). In addition, the ganciclovir treated mice had tendency of higher trabecular bone volume than the saline-treated group (p=0.08). These results demonstrate that this model should be useful for studying mechanisms of osteoblast suppression in MM.

Next Generation XPO1 Inhibitor KPT-8602 for the Treatment of Drug-Resistant Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1818-1818 ◽  
Author(s):  
Joel G Turner ◽  
Jana L Dawson ◽  
Christopher L Cubitt ◽  
Erkan Baluglo ◽  
Steven Grant ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Ex Vivo ◽  
Single Agent ◽  
Resistant Cell ◽  
Drug Resistant ◽  
Newly Diagnosed ◽  
Induced Apoptosis ◽  
Drug Resistant Cell

Abstract Purpose Human multiple myeloma (MM) remains an incurable disease despite relatively effective treatments, including proteasome inhibitors, immunomodulator-based therapies, and high-dose chemotherapy with autologous stem cell rescue. New agents are needed to further improve treatment outcomes. In previous studies, we have shown that inhibitors of the nuclear export receptor XPO1, in combination with bortezomib, carfilzomib, doxorubicin, or melphalan, synergistically induced apoptosis in MM cells in vitro, in vivo and ex vivo without affecting non-myeloma cells. In early clinical trials, the oral, brain penetrating XPO1 inhibitor selinexor showed clear anti-myeloma activity however adverse events have been recorded, including nausea and anorexia. Our purpose was to investigate the use of oral KPT-8602, a novel small-molecule inhibitor of XPO1 with minimal brain penetration, which has been shown to have reduced toxicities in rodents and primates while maintaining potent anti-tumor effects. Experimental Procedures To test the efficacy of KPT-8602, we treated human MM cell lines (both parental and drug-resistant) with KPT-8602 ± currently used MM drugs, including bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, and VP-16. Human MM cell lines assayed included RPMI-8226 (8226), NCI-H929 (H929), U266, and MM1.S, PI-resistant 8226-B25 and U266-PSR, doxorubicin-resistant 8226-Dox6 and 8226-Dox40, and melphalan-resistant 8226-LR5 and U266-LR6 cell lines. MM cells (2-4x106 cells/mL) were treated for 24 hours with KPT-8602 (300 nM), followed by treatment with one of the listed anti-MM agents for an additional 24 hours. MM cells were then assayed for cell viability (CellTiter-Blue, Promega). In addition, cells were treated with KPT-8602 ± anti-MM agents concurrently for 20 hours and assayed for apoptosis by flow cytometry. In vivo testing was done in NOD/SCID-g mice by intradermal injection of U266 MM cells. Treatment started 2 weeks after tumor challenge with KPT-8602 (10 mg/kg) ± melphalan (1 or 3 mg/kg) 2X/week (Tuesday, Friday) or with KPT-8602 alone 5X weekly (10 mg/kg) (Monday-Friday). A parallel experiment was run using the clinical XPO1 inhibitor KPT-330 (selinexor). Ex vivo testing was performed on MM cells from newly diagnosed/relapsed patient bone marrow aspirates with KPT-8602 ± bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, or VP16. CD138+/light-chain+ cells were assayed for apoptosis by flow cytometry. Results Viability assay showed that KPT-8602 had low IC50values (~140 nM) as a single agent and functioned synergistically with bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16. (CI values < 1.0). This synergistic effect was less pronounced in myeloma cells when KPT-8602 was used in combination with dexamethasone or lenalidomide. KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 combination therapy also induced apoptosis in all MM cell lines tested, including drug-resistant cell lines, as shown by caspase 3 cleavage and flow cytometric analyses. NOD/SCID-gamma mouse tumor growth was reduced and survival increased in KPT-8602/melphalan-treated mice when compared to single-agent controls. In addition, mice treated with KPT-8602 5X weekly had significantly reduced tumor growth and increased survival when compared to 2X weekly drug administration. No toxicity was observed in KPT-8602-treated mice as determined by weight loss in both the 2X and 5X groups. In patient bone marrow biopsies, the combination of KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 was more effective than single agents at inducing apoptosis in CD138+/LC+ MM cells in both newly diagnosed and relapsed/refractory patient samples. Conclusions We found that the novel XPO1 inhibitor KPT-8602 sensitizes MM cells to bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 as shown by apoptosis in parental and drug-resistant cell lines and by cell viability assays. Sensitization was found to be synergistic. In addition, KPT-8602 was effective in treatment of human MM tumors in mice as a single agent or in combination with melphalan and was effective when combined with several MM drugs in MM cell lines and MM patient bone marrow aspirates. KPT-8602 may be a potential candidate for future clinical trials. Disclosures Shacham: Karyopharm: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties.

scholarly journals A pilot study of 3D tissue-engineered bone marrow culture as a tool to predict patient response to therapy in multiple myeloma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kinan Alhallak ◽  
Amanda Jeske ◽  
Pilar de la Puente ◽  
Jennifer Sun ◽  
Mark Fiala ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Clinical Response ◽  
Ex Vivo ◽  
Drug Efficacy ◽  
Improve Patient Care ◽  
Hematologic Malignancies ◽  
Time Frame ◽  
Response To Therapy

AbstractCancer patients undergo detrimental toxicities and ineffective treatments especially in the relapsed setting, due to failed treatment attempts. The development of a tool that predicts the clinical response of individual patients to therapy is greatly desired. We have developed a novel patient-derived 3D tissue engineered bone marrow (3DTEBM) technology that closely recapitulate the pathophysiological conditions in the bone marrow and allows ex vivo proliferation of tumor cells of hematologic malignancies. In this study, we used the 3DTEBM to predict the clinical response of individual multiple myeloma (MM) patients to different therapeutic regimens. We found that while no correlation was observed between in vitro efficacy in classic 2D culture systems of drugs used for MM with their clinical efficacious concentration, the efficacious concentration in the 3DTEBM were directly correlated. Furthermore, the 3DTEBM model retrospectively predicted the clinical response to different treatment regimens in 89% of the MM patient cohort. These results demonstrated that the 3DTEBM is a feasible platform which can predict MM clinical responses with high accuracy and within a clinically actionable time frame. Utilization of this technology to predict drug efficacy and the likelihood of treatment failure could significantly improve patient care and treatment in many ways, particularly in the relapsed and refractory setting. Future studies are needed to validate the 3DTEBM model as a tool for predicting clinical efficacy.

scholarly journals ATR addiction in multiple myeloma: synthetic lethal approaches exploiting established therapies

Haematologica ◽  
2019 ◽  
Vol 105 (10) ◽  
pp. 2440-2447 ◽  
Author(s):  
Oronza A. Botrugno ◽  
Silvia Bianchessi ◽  
Desirée Zambroni ◽  
Michela Frenquelli ◽  
Daniela Belloni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Synthetic Lethality ◽  
Ex Vivo ◽  
Myeloma Cell ◽  
Tumor Burden ◽  
In Vivo Models ◽  
Cancer Multiple

Therapeutic strategies designed to tinker with cancer cell DNA damage response have led to the widespread use of PARP inhibitors for BRCA1/2-mutated cancers. In the haematological cancer multiple myeloma, we sought to identify analogous synthetic lethality mechanisms that could be leveraged upon established cancer treatments. The combination of ATR inhibition using the compound VX-970 with a drug eliciting interstrand cross-links, melphalan, was tested in in vitro, ex vivo, and most notably in vivo models. Cell proliferation, induction of apoptosis, tumor growth and animal survival were assessed. The combination of ATM inhibition with a drug triggering double strand breaks, doxorucibin, was also probed. We found that ATR inhibition is strongly synergistic with melphalan, even in resistant cells. The combination was dramatically effective in targeting myeloma primary patient cells and cell lines reducing cell proliferation and inducing apoptosis. The combination therapy significantly reduced tumor burden and prolonged survival in animal models. Conversely, ATM inhibition only marginally impacted on myeloma cell survival, even in combination with doxorucibin at high doses. These results indicate that myeloma cells extensively rely on ATR, but not on ATM, for DNA repair. Our findings posit that adding an ATR inhibitor such as VX-970 to established therapeutic regimens may provide a remarkably broad benefit to myeloma patients.

scholarly journals Multiple Myeloma Progression: Dependence on Bone Marrow Adipose Tissue

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3262-3262 ◽  
Author(s):  
Michaela Reagan ◽  
Carolyne Falank ◽  
Heather Fairfield ◽  
Michelle McDonald ◽  
Peter Croucher ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Adipose Tissue ◽  
Cell Lines ◽  
Expression Profiles ◽  
Artificial Culture ◽  
In Vivo Models ◽  
Bone Marrow Adipose Tissue ◽  
Marrow Adipose Tissue

Abstract Multiple myeloma (MM) is characterized by clonal proliferation of transformed plasma cells1 and is extremely dependent on bone marrow (BM) niche molecules and cells, such as osteoclasts. Unlike osteoclasts, the roles of BM adipocytes (BMAs) in MM are poorly understood, despite their great therapeutic potential. This year we published a study of body composition PET/CT parameters that serve as predictors of monoclonal gammopathy of undetermined significance (MGUS) progression to MM(Veld J, O'Donnell EK, Reagan MR, et al. Abdominal adipose tissue in MGUS and multiple myeloma. Skeletal Radiol.). We found that recently diagnosed MM patients had higher abdominal white adipose tissue (WAT) than MGUS patients, even after correction for BMI. Bone Marrow Adipose Tissue (BMAT), a newly appreciated adipose depot with endocrine and paracrine signaling functions, resides near MM cells and has unique expression profiles and phenotypic responses compared to WAT. Because obesity and aging, risk factors for MM, correlate with increased BMAT, and BMAs and MM cells are closely physically associated, we hypothesized that BMAs contribute to an optimal microenvironment for MM cell proliferation and/or drug resistance. We performed direct and indirect co-culture experiments to study the effects of BMAT and BMAT-derived cytokines and lipids on MM proliferation and chemoresistance. MM cells were cultured on, or with conditioned media (CM) from, human and mouse BM-derived mesenchymal stem cells (MSCs) differentiated into adipocytes. MM proliferation, assessed by bioluminescence imaging, was dependent on MM cell line, MSC donor, and adipogenic stage. IL6 is a highly potent MM-supportive cytokine elevated in MM patient BM and thought to be derived mainly from MSCs. MM cells (OPM2 and MM1R) grown in CM from MSCs differentiated for 21 days into adipocytes (Fat CM) treated with IL6 neutralizing antibodies had significantly decreased proliferation vs MM cells treated with Fat CM alone. MM1S cells also showed this trend. These data identified BMAs as a novel BM IL6 source. MM cells typically proliferated in response to donor "lipid fractions", the oil layer on top of human hip surgery BM samples, after 24, 48 and 72 hours, although donor variability was again observed. Lipid droplet content (Oil Red O quantification) of these BMAs also significantly decreased upon culture with MM cells, suggesting that MM cells induce lipolysis or uptake BMAT lipids to fuel their proliferation. In contrast to the literature, we found that adiponectin can be either MM-supportive or MM-inhibitory, depending on the MM line tested and on the presence of dex. Certain MM cell lines (MM1S) became dexamethasone (dex) resistant when treated with Fat CM. Strikingly, all 3 cell lines tested (MM1S, MM1R and OPM2) showed significant decreases in cell number at 24, 48 and 72 hours after treatment with a neutralizing adiponectin antibody vs IgG control, when grown in the presence of 0.1μM dex + Fat CM (which contained high levels of adiponectin from ELISA analysis) (Fig 1A). These data suggest that adiponectin can induce dex resistance, indicating that adiponectin inhibitors + dex may be a novel MM therapy. Lastly, we developed a physiologically relevant 3D in vitro tissue engineered BMAT model utilizing biocompatible, porous silk fibroin scaffolds to more accurately define BMA-MM interactions. Our 3D models provide the correct mechanical robustness and biomaterial properties to mimic trabecular bone and unilocular BMAT (Fig 1 B-D). We generated long-term cultures of BMAT from MSCs and cultured MM cells (GFP+ MM1S) on these for up to 1 week, demonstrating the development of the first 3D BMAT artificial culture system, with or without MM cells. We are now using this novel platform to more deeply explore the relationship between BMAT and MM cells. In conclusion, BMAT likely plays a role in MM progression. 3D tissue engineered models of the BM milieu are a crucial link between 2D and in vivo models, maintaining the high-throughput capacity of 2D studies and the translational relevancy of in vivo models. Our data demonstrate important interactions between BMAT and MM cells, highlighting our need for further research into the roles of BM adipokines and adipocytes in MM pathogenesis and chemoresistance. Disclosures No relevant conflicts of interest to declare.

SCIO-469, a Potent and Selective Inhibitor of p38a MAPK, Normalizes the Bone Marrow Microenvironment and Inhibits Multiple Myeloma Cell Proliferation in In Vitro and In Vivo Models.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1501-1501 ◽  
Author(s):  
Aaron N. Nguyen ◽  
Mamatha Reddy ◽  
Margaret Henson ◽  
Elizabeth G. Stebbins ◽  
Gilbert O’Young ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Critical Role ◽  
Bone Destruction ◽  
Great Promise ◽  
In Vivo Models

Abstract Despite recent advances in the treatment of multiple myeloma (MM), this disease remains incurable. Accumulating evidence suggest that the bone marrow (BM) microenvironment of MM plays a critical role in tumor growth, survival, and drug resistance. A key aspect of this tumor-supportive environment is elevated levels of cytokines and other soluble factors. Most prominent among these is IL-6, which acts as a survival factor for MM cells and promotes their proliferation, migration, and drug resistance. Other mediators also implicated in the disease are VEGF and TNFa. The p38 MAPK is activated by a multitude of signals, including pro-inflammatory cytokines (e.g., TNFa and IL-1ß) and environmental stress. Furthermore, p38 activation has been shown to be important for the synthesis and secretion of IL-6, VEGF, and TNFa. Consequently, inhibition of p38 is postulated to reduce the production of these factors implicated in MM and to have therapeutic benefit by suppressing the tumor-supportive state of the BM microenvironment. Here, we demonstrate that SCIO-469, a specific and potent inhibitor of p38a MAPK, strongly inhibits MM cell proliferation by affecting MM cells directly as well as the BM microenvironment. SCIO-469 directly inhibits MM cell proliferation in long term culture. Importantly, SCIO-469 potently inhibits IL-6 and VEGF secretion from BM stromal cells (BMSC). To examine the effect of inhibiting BMSC-derived factors important in MM, we measured MM cell proliferation using transwell plates that separate BMSC from MM cells via a porous membrane. In transwell plates containing only MM cells, MM cell proliferation was modest and was inhibited by SCIO-469. In contrast, the presence of BMSC in transwell inserts dramatically increased the proliferation of MM cells over the course of the study. This result suggests that factors (e.g., IL-6) secreted by BMSC greatly stimulate MM cell proliferation. When SCIO-469 was added to these transwell cultures containing BMSC, MM cell proliferation was inhibited significantly. Consistent with these results, we show that levels of IL-6 under these conditions mirror exactly the proliferation of MM cells; IL-6 level is high in vehicle-treated cultures and is suppressed in SCIO-469-treated cultures. Finally, in a mouse xenograft plasmacytoma model of MM, we show that p38 inhibition significantly inhibited the increase in MM tumor volume. Collectively, our data indicate that SCIO-469 is a suppressor of the BM microenvironment and an effective inhibitor of MM cell proliferation in vitro and in vivo. Since SCIO-469 also inhibits secretion of osteoclast-stimulating factors (RANKL, IL-11, and MIP1a) in the microenvironment, SCIO-469 may not only inhibit MM cell survival but may also alleviate bone-related pathologies (bone destruction and osteolytic lesions) commonly associated with MM. Therefore, SCIO-469 may offer great promise for an improved outcome for patients with MM.

The Lack of Clinical Efficacy of Flavopiridol in Patients with Relapsed Refractory Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3461-3461
Author(s):  
Angela Dispenzieri ◽  
Keith C. Bible ◽  
Martha Q. Lacy ◽  
Tom R. Fitch ◽  
Susan M. Geyer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Efficacy ◽  
Ex Vivo ◽  
Single Agent ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Myeloma Cells ◽  
Refractory Multiple Myeloma

Abstract Purpose: Flavopiridol is a cyclin-dependent kinase inhibitor with preclinical activity against multiple myeloma cells in vitro and ex vivo. It can induce apoptosis in non-cycling human cell lines, including RPMI-8226, a myeloma cell line. Suggested mechanisms of cytotoxicity include down regulation of anti-apoptotic regulators, direct binding to duplex DNA, disruption of transcription factor/DNA binding, upregulation of p53, inhibition of transcription, and inhibition of angiogenesis. A Phase II multicenter trial was conducted to determine the activity of flavopiridol in patients with relapsed or refractory multiple myeloma. Experimental Design: Eighteen patients were treated with 1 hour flavopiridol infusions (50 mg/m(2)/day) for 3 consecutive days every 21 days. Responses were assessed according to IBMTR criteria each cycle. Serial bone marrow aspirates were collected before and immediately after flavopiridol treatment to measure in vivo and ex vivo effects of flavopiridol on patient plasma cells. Immunoblotting for Mcl-1, Bcl-2, p53, cyclin D, phosphoRNA polymerase II and phosphoSTAT 3 was conducted on total cellular proteins isolated from sorted plasma/myeloma cells. Ex vivo assessment of flavopiridol sensitivity of freshly collected myeloma cells was performed for 5 patients pre-therapy. Results: Median age of patients ranged from 49 to 81 years, with a median of 65 years. Patients had received a median of 3 (range, 1–5) treatment regimens prior to enrollment. Sixty-one percent of patients were refractory to prior therapy and fifty-five percent had received prior high dose therapy with hematopoietic stem cell support. The immunoglobulin isotypes of the patients were as follows: IgG (10), IgA (4), light chain (3), and non-secretory (1). At enrollment, 13 patients had a beta-2 microglobulin greater than 3.5. Seven had a plasma cell labeling index greater than or equal to 1%. Four had an elevated LDH. The trial was stopped at time of interim analysis due to a lack of clinical efficacy. Of eighteen treated patients, 15 progressed (including 1 death on study) and 3 discontinued due to toxicity. All patients have ended the active treatment phase, and the mean number of cycles administered for all enrolled patients was 1.6 cycles (range 1–3). No objective responses were observed. The most frequent adverse events were leukopenia and diarrhea (83% each), followed by thrombocytopenia, nausea, and fatigue (61% each). Ex vivo flavopiridol treatment of pre-therapy patient plasma/myeloma cells led to cytotoxicity, but only after longer exposure times at higher flavopiridol concentrations than were anticipated to be achieved in vivo. Further, immunoblotting demonstrated no indication that known in vitro cellular effects of flavopiridol were recapitulated in vivo. Conclusions: Flavopiridol has little single-agent activity in relapsed or refractory multiple myeloma when administered at this dose and schedule, which is likely due to the inability to achieve biologically relevant concentrations in patients. .

scholarly journals Lycorine Downregulates HMGB1 to Inhibit Autophagy and Enhances Bortezomib Activity in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3265-3265
Author(s):  
Mridul Roy ◽  
Long Liang ◽  
Xiaojuan Xiao ◽  
Yuanliang Peng ◽  
Yuhao Luo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Xenograft Model ◽  
Hmgb1 Expression ◽  
Resistant Cells

Abstract Multiple myeloma (MM) is the second most prevalent hematologic malignancy, characterized by the infiltration of malignant plasma cells into bone marrow. In spite of current efficient therapeutic regimens, which have significantly increased patients overall survival, the major features inevitably present in MM are the intrinsic and acquired resistance with nearly universal relapse. In addition, the diverse heterogeneous characteristics of this largely incurable disease emphasize the importance of innovative therapies and identification of more effective drugs. Autophagy removes defective cellular organelles, protein aggregates, and intracellular microbes and is associated with cell survival and tumor maintenance. Inhibition of autophagy enhances sensitivity of a number of anticancer agents and induces cell death in MM. High-mobility group box-1 (HMGB1) protein plays an important subcellular localization-dependent role during autophagy. The importance of HMGB1 for induction of autophagy and tumor development has made this protein as a novel target for cancer therapy. Lycorine is a natural alkaloid with significant anti-cancer activity. While previous studies mainly showed lycorine as a potential apoptosis inducer, recent studies stated that apoptosis is not the primary underlying anti-proliferative mechanism of this compound. This led the interest to investigate the role of lycorine on other cell maintenance systems, such as autophagy. In addition single-agent efficacy of lycorine or in combination with other anti-MM agents has not been evaluated in vivo. Herein we investigated the anti-MM effect of lycorine and the role of this natural agent on regulation of autophagy in vitro and in vivo. We found that lycorine inhibits proliferation and induces apoptosis in MM cells with less sensitivity to the normal B-cell at the same concentrations. We also found that lycorine promisingly inhibits autophagy, the mechanism that MM cells use to survive and defeat treatment. We identified HMGB1, an important regulator of autophagy, as the most aberrantly expressed protein after lycorine treatment. Furthermore, we characterized HMGB1 as a critical mediator of lycorine activity against MM. Gene expression profiling (GEP) analysis showed that higher expression of HMGB1 is linked with the poor prognosis of MM. We further confirmed this correlation in human bone marrow CD138+ primary myeloma cells and MM cell lines. Mechanistically, by activating the proteasomal degradation of HMGB1, lycorine induces a rapid turnover of HMGB1. This led to decreased Bcl-2 phosphorylation by MEK-ERK pathway and increased association of Bcl-2 with Beclin-1 resulting in autophagy inhibition and growth attenuation. In addition, we observed higher HMGB1 expression in bortezomib resistant cells. The combination of bortezomib plus lycorine was highly efficient against MM cells and MM cells grown in bone marrow micro-environment. Lycorine showed the capability of inhibiting bortezomib induced autophagy as well as re-sensitizing resistant cells to bortezomib. In agreement with our in vitro observations, in vivo study using human MM xenograft model showed that lycorine is well tolerated, inhibits HMGB1 expression and thereby autophagy and induces enhanced bortezomib activity. These observations indicated lycorine as an effective autophagy inhibitor and revealed that lycorine alone or in combination with bortezomib is a potential therapeutic strategy. Our study supports the feasibility of lycorine in anti-MM treatment and provides a preclinical framework for combining lycorine with bortezomib in clinical setting. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document