Bone Marrow Milieu Research Articles

Osteoclasts Promote Immune Suppressive Microenvironment in Multiple Myeloma: Therapeutic Implication

Purpose: The number and activity of osteoclasts (OCs) are strongly enhanced by myeloma cells, leading to significant bone lesion in patients with multiple myeloma (MM). Mechanisms remain elusive whether myeloma-supporting OCs also induce suppressive immune bone marrow (BM) microenvironment.Methods: We examined if OCs could modulate T cell-mediated anti-MM immunity; Inhibitory immune molecules expression during osteoclastogenesis; The impact of APRIL on PDL1 expression in MM and the pathway; The impact of Anti-CD38 mAb on osteoclastogenesis and T cell response;Results: Here, we first show that OCs significantly protect MM cells against T cell-mediated cytotoxicity via direct inhibition of proliferating CD4+and CD8+ T cells. The immune checkpoint molecules PDL1, Galectin-9, HVEM, and CD200, as well as T cell metabolism regulators indoleamine 2, 3-dioxygenase (IDO) and CD38 are significantly up-regulated during osteoclastogenesis. Importantly, levels of these molecules, except CD38, are higher in OCs than MM cells. Anti-PDL1 monoclonal antibody and IDO inhibitor partly overcome OC-inhibited T cell responses against MM cells, confirming their roles in OC-suppressed MM cell lysis by cytotoxic T cells. In addition, Galectin-9 and a proliferation-induced ligand (APRIL), mainly secreted by OCs, are significantly up-regulated during osteoclastogenesis. Galectin-9 specifically induces apoptosis of T cells while sparing monocytes and MM cells. APRIL induces PDL1 expression in MM cells, providing additional immune inhibition by OCs. Moreover, CD38 is significantly up-regulated during osteoclastogenesis. When targeted by an anti-CD38 monoclonal antibody, suppressive T cell function by OCs is alleviated, associated with down-regulation of HVEM and IDO.Conclusion: Taken together, these results define expression of multiple immune proteins and cytokines in OCs essential for suppressive MM BM milieu. These results further support combination of targeting these molecules to improve anti-MM immunity. DisclosuresAnderson:Celgene: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Arginine Methylation Mediated By PRMT5 Has Significant Biological and Clinical Impact in Multiple Myeloma

Arginine-specific methyltransferases critically regulate cellular homeostasis by dictating the biological outcome of target proteins. Among them, Protein Arginine Methyltransferase 5 (PRMT5) has attracted growing interest due to its role as an enzyme mediating epigenetic regulation of anti-cancer target genes, as well as in methylation of non-histone proteins involved in growth-regulating and survival pathways including p53. However, little is known about its biologic function in multiple myeloma (MM).To first evaluate the clinical significance of PRMT5 in MM pathogenesis, we analyzed RNA-seq data from newly-diagnosed MM patients and identified highly upregulated PRMT5 in 320 patients' CD138+ cells compared to 16 samples of normal bone marrow (BM) plasma cells. Additional analysis of PRMT5 expression in two independent datasets also showed further PRMT5 mRNA upregulation during progression of MM. Immunohistochemical staining also confirmed elevated expression of PRMT5 in BM biopsies from MM patients as compared to healthy individuals and monoclonal gammopathy of undetermined significance (MGUS). Moreover, analysis of the prognostic significance of PRMT5 expression in MM patients enrolled on IFM/DFCI 2009 clinical study showed that high PRMT5 expression was associated with poor prognosis in terms of both event free (p= 0.016) and overall survival (p=0.018). Consistently, we also found upregulated PRMT5 expression at both mRNA and protein levels in MM cell lines (N=11) and patients CD138+ MM cells (N=3) as compared to PBMCs from healthy volunteers, associated with a parallel increase of cellular symmetric arginine di-methylation (SDMA) substrates. Interestingly, genetic depletion of PRMT5 in H929 (p53wt) and KMS11 (p53null) MM cell lines by shRNA decreased SDMA levels, associated with cell growth inhibition in a p53-independent manner. Likewise, pharmacological inhibition of PRMT5 with the small molecule inhibitor EPZ015666 triggered decreased SDMA levels, cell growth, survival, and clonogenicity, as well as induction of caspase-dependent apoptosis in MM cell lines. Moreover, although PRMT5 and SDMA levels were increased in MM cells cultured in the presence of BM stromal cell supernatant, cytotoxic activity of EPZ015666 was maintained. Notably, drug treatment significantly impaired cell proliferation of patient MM cells (n=2) even in the presence of BM mononuclear or stromal cells, without toxicity on normal PBMCs. At the level of gene expression modulation, PRMT5 inhibition was associated with downregulation of NF-kB-dependent transcription, evidenced by both gene set enrichment analysis (GSEA) and Ingenuity Upstream Regulator Analysis. Moreover, analysis of protein levels confirmed reduction of both canonical and non-canonical NF-kB pathways, evidenced by significantly decreased NF-kB DNA binding activity by ELISA. Importantly, Mass Spectrometry analysis identified TRIM21 as a new PRMT5 interactor; and EPZ015666-treated cells showed that PRMT5 methylates TRIM21 evidenced by WB analysis. Since TRIM21 mediates monoubiquitination of IKKbeta, thereby triggering its selective autophagy-mediated degradation, we next analyzed EPZ015666 effects on IKKbeta. Treatment increased both monoubiquitination of IKKbeta and the formation of IKKbeta-TRIM21-pBECLIN1-pULK1 autophagic complexes. Conversely, inhibition of autophagosome formation by 3-methyladenine abrogated the anti-MM activity of EPZ015666 and IKKbeta degradation, indicating that selective autophagic degradation of IKKbeta and inhibition of NF-kB signaling mediates EPZ015666-triggered anti-MM activity. Consistent with this view, confocal microscopy analysis also confirmed co-localization of IKKbeta in the autophagosome after EPZ015666 treatment. Finally, stable silencing of TRIM21 in MM cell lines significantly abrogated the anti-proliferative effect of EPZ015666. Collectively, these data delineate arginine methylation as a new control mechanism of MM cell growth, and demonstrate that inhibiting PRMT5 decreases tumor cell survival via blockade of NF-kB signaling, even in the context of the BM milieu. These data demonstrate the biologic and prognostic significance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome in MM. DisclosuresHideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Munshi:Takeda: Consultancy; Celgene Corporation: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Equity Ownership. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Multiple Myeloma Progression: Dependence on Bone Marrow Adipose Tissue

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. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A gene expression inflammatory signature specifically predicts multiple myeloma evolution and patients survival.

Multiple myeloma (MM) is closely dependent on cross-talk between malignant plasma cells and cellular components of the inflammatory/immunosuppressive bone marrow milieu, which promotes disease progression, drug resistance, neo-angiogenesis, bone destruction and immune-impairment. We investigated the relevance of inflammatory genes in predicting disease evolution and patient survival. A bioinformatics study by Ingenuity Pathway Analysis on gene expression profiling dataset of monoclonal gammopathy of undetermined significance, smoldering and symptomatic-MM, identified inflammatory and cytokine/chemokine pathways as the most progressively affected during disease evolution. We then selected 20 candidate genes involved in B-cell inflammation and we investigated their role in predicting clinical outcome, through univariate and multivariate analyses (log-rank test, logistic regression and Cox-regression model). We defined an 8-genes signature (IL8, IL10, IL17A, CCL3, CCL5, VEGFA, EBI3 and NOS2) identifying each condition (MGUS/smoldering/symptomatic-MM) with 84% accuracy. Moreover, six genes (IFNG, IL2, LTA, CCL2, VEGFA, CCL3) were found independently correlated with patients' survival. Patients whose MM cells expressed high levels of Th1 cytokines (IFNG/LTA/IL2/CCL2) and low levels of CCL3 and VEGFA, experienced the longest survival. On these six genes, we built a prognostic risk score that was validated in three additional independent datasets. In this study, we provide proof-of-concept that inflammation has a critical role in MM patient progression and survival. The inflammatory-gene prognostic signature validated in different datasets clearly indicates novel opportunities for personalized anti-MM treatment.

NF-Kβ Activation in U266 Cells on Mesenchymal Stem Cells.

Purpose: Mesenchymal Stem Cells (MSCs) are one of the essential members of Bone Marrow (BM) microenvironment and the cells affect normal and malignant cells in BM milieu. One of the most important hematological malignancies is Multiple Myeloma (MM). Numerous studies reported various effects of MSCs on myeloma cells. MSCs initiate various signaling pathways in myeloma cells, particularly NF-kβ. NF-kβ signaling pathway plays pivotal role in the survival, proliferation and resistance of myeloma cells to the anticancer drugs, therefore this pathway can be said to be a vital target for cancer therapy. This study examined the relationship between U266 cells and MSCs. Methods: U266 cells were cultured with Umbilical Cord Blood derived-MSCs (UCB-MSCs) and Conditioned Medium (C.M). Effect of UCB-MSCs and C.M on proliferation rate and CD54 expression of U266 cells were examined with MTT assay and Flowcytometry respectively. Furthermore, expression of CXCL1, PECAM-1, JUNB, CCL2, CD44, CCL4, IL-6, and IL-8 were analyzed by Real Time-PCR (RT-PCR). Moreover, status of p65 protein in NF-kβ pathway assessed by western blotting. Results: Our findings confirm that UCB-MSCs support U266 cells proliferation and they increase CD54 expression. In addition, we demonstrate that UCB-MSCs alter the expression of CCL4, IL-6, IL-8, CXCL1 and the levels of phosphorylated p65 in U266 cells. Conclusion: Our study provides a novel sight to the role of MSCs in the activation of NF-kβ signaling pathway. So, NF-kβ signaling pathway will be targeted in future therapies against MM.

Osteoclasts promote immune suppressive microenvironment in multiple myeloma: therapeutic implication

AbstractThe number and activity of osteoclasts (OCs) are strongly enhanced by myeloma cells, leading to significant bone lesions in patients with multiple myeloma (MM). Mechanisms remain elusive as to whether myeloma-supporting OCs also induce suppressive immune bone marrow (BM) microenvironment. Here, we first show that OCs significantly protect MM cells against T-cell–mediated cytotoxicity via direct inhibition of proliferating CD4+ and CD8+ T cells. The immune checkpoint molecules programmed death ligand 1 (PD-L1), Galectin-9, herpesvirus entry mediator (HVEM), and CD200, as well as T-cell metabolism regulators indoleamine 2, 3-dioxygenase (IDO), and CD38 are significantly upregulated during osteoclastogenesis. Importantly, the levels of these molecules, except CD38, are higher in OCs than in MM cells. Anti–PD-L1 monoclonal antibody (mAb) and IDO inhibitor partly overcome OC-inhibited T-cell responses against MM cells, confirming their roles in OC-suppressed MM cell lysis by cytotoxic T cells. In addition, Galectin-9 and a proliferation-induced ligand (APRIL), secreted by OCs, are significantly upregulated during osteoclastogenesis. Galectin-9 specifically induces apoptosis of T cells while sparing monocytes and MM cells. APRIL induces PD-L1 expression in MM cells, providing additional immune inhibition by OCs. Moreover, CD38 is significantly upregulated during osteoclastogenesis. When targeted by an anti-CD38 mAb, suppressive T-cell function by OCs is alleviated, associated with downregulation of HVEM and IDO. Taken together, these results define the expression of multiple immune proteins and cytokines in OCs essential for suppressive MM BM milieu. These results further support the combination of targeting these molecules to improve anti-MM immunity.

MicroRNAs: novel crossroads of multiple myeloma treatment

Multiple myeloma (MM) is a hematologic malignancy of differentiated plasma cells that accumulate in the bone marrow, where a complex microenvironment made by different cell types supports proliferation, survival, and drug resistance of tumor cells. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at post-transcriptional level. Emerging evidence indicates that miRNAs are aberrantly expressed or functionally deregulated in MM cells as the result of multiple genetic or epigenetic mechanisms and that also the tumor microenvironment regulates MM cell functions by miRNAs. Consistently, modulation of miRNA levels in MM cells has been demonstrated to impair their functional interaction with the bone marrow microenvironment and to produce significant antitumor activity to overcome the protective bone marrowmilieu. This review will describe the most recent findings on miRNA function in the context of MM bone marrow microenvironment, focusing on the therapeutic potential of miRNA-based approaches. Key words: MicroRNAs; Multiple myeloma/TH; Review

A novel hypoxia-selective epigenetic agent RRx-001 triggers apoptosis and overcomes drug resistance in multiple myeloma cells.

The hypoxic bone-marrow (BM) microenvironment confers growth/survival and drug-resistance in multiple myeloma (MM) cells. Novel therapies targeting the MM cell in its hypoxic-BM milieu may overcome drug resistance. Recent studies led to the development of a novel molecule RRx-001 with hypoxia-selective epigenetic and Nitric Oxide-donating properties. Here we demonstrate that RRx-001 decreases the viability of MM cell lines and primary patient cells, as well as overcomes drug-resistance. RRx-001 inhibits MM cell growth in the presence of BM stromal cells. RRx-001 induced apoptosis is associated with: 1) activation of caspases; 2) release of ROS and nitrogen-species; 3) induction of DNA damage via ATM/γ-H2AX; and 4) decrease in DNA methytransferase (DNMT) and global methylation. RNA interference study shows a predominant role of DNMT1 in MM cell survival versus DNMT3a or DNMT3b. Deubiquitylating enzyme USP7 stimulates DNMT1 activity; and conversely, USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. RRx-001 plus USP7 inhibitor P5091 triggered synergistic anti-MM activity. MM xenograft studies show that RRx-001 is well tolerated, inhibits tumor growth, and enhances survival. Combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity. Our results provide the rationale for translation of RRx-001, either alone or in combination, to clinical evaluation in MM.

The KDM3A-KLF2-IRF4 axis maintains myeloma cell survival.

KDM3A is implicated in tumorigenesis; however, its biological role in multiple myeloma (MM) has not been elucidated. Here we identify KDM3A–KLF2–IRF4 axis dependence in MM. Knockdown of KDM3A is toxic to MM cells in vitro and in vivo. KDM3A maintains expression of KLF2 and IRF4 through H3K9 demethylation, and knockdown of KLF2 triggers apoptosis. Moreover, KLF2 directly activates IRF4 and IRF4 reciprocally upregulates KLF2, forming a positive autoregulatory circuit. The interaction of MM cells with bone marrow milieu mediates survival of MM cells. Importantly, silencing of KDM3A, KLF2 or IRF4 both decreases MM cell adhesion to bone marrow stromal cells and reduces MM cell homing to the bone marrow, in association with decreased ITGB7 expression in MAF-translocated MM cell lines. Our results indicate that the KDM3A–KLF2–IRF4 pathway plays an essential role in MM cell survival and homing to the bone marrow, and therefore represents a therapeutic target.

The Role of Intracellular Signaling Pathways in the Pathogenesis of Multiple Myeloma and Novel Therapeutic Approaches.

The introduction of novel agents, such as bortezomib, thalidomide, and lenalidomide, into daily practice has dramatically improved clinical outcomes and prolonged survival of patients with multiple myeloma (MM). However, despite these advanced clinical benefits, MM remains an incurable hematological malignancy. Therefore, development of new agents and novel therapeutic strategies is urgently needed. Recent advances toward understanding the mechanism of myeloma cell growth and drug resistance in the bone marrow milieu have provided clues for the development of next-generation agents aimed at improving patient outcomes. In this review article, we discuss new possible agents for the treatment of MM based on recent advances in the understanding of signaling pathways in myeloma cells.

Bone Disease in Multiple Myeloma.

Bone involvement represented by osteolytic bone disease (OBD) or osteopenia is one of the pathognomonic and defining characteristics of multiple myeloma (MM). Nearly 90% of patients with MM develop osteolytic bone lesions, frequently complicated by skeletal-related events (SRE) such as severe bone pain, pathological fractures, vertebral collapse, hypercalcemia, and spinal cord compression. All of these not only result in a negative impact on quality of life but also adversely impact overall survival. OBD is a consequence of increased osteoclast (OC) activation along with osteoblast (OB) inhibition, resulting in altered bone remodeling. OC number and activity are increased in MM via cytokine deregulation within the bone marrow (BM) milieu, whereas negative regulators of OB differentiation suppress bone formation. Inhibition of osteolysis and stimulation of OB differentiation leads to reduced tumor growth in vivo. Therefore, novel agents targeting OBD are promising therapeutic strategies not only for the treatment of MM OBD but also for the treatment of MM. Several novel agents in addition to bisphosphonates are currently under investigation for their positive effect on bone remodeling via OC inhibition or OB stimulation. Future studies will look to combine or sequence all of these agents with the goal of not only alleviating morbidity from MM OBD but also capitalizing on the resultant antitumor activity.

MiR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth.

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3′UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

MicroRNAs: Novel Crossroads between Myeloma Cells and the Bone Marrow Microenvironment.

Multiple myeloma (MM) is a hematologic malignancy of differentiated plasma cells that accumulate in the bone marrow, where a complex microenvironment made by different cell types supports proliferation, survival, and drug resistance of tumor cells. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at posttranscriptional level. Emerging evidence indicates that miRNAs are aberrantly expressed or functionally deregulated in MM cells as the result of multiple genetic or epigenetic mechanisms and that also the tumor microenvironment regulates MM cell functions by miRNAs. Consistently, modulation of miRNA levels in MM cells has been demonstrated to impair their functional interaction with the bone marrow microenvironment and to produce significant antitumor activity even able to overcome the protective bone marrow milieu. This review will describe the most recent findings on miRNA function in the context of MM bone marrow microenvironment, focusing on the therapeutic potential of miRNA-based approaches.

A Novel Agent SL-401 Triggers Anti-Myeloma Activity By Targeting Plasmacytoid Dendritic Cells: Implications for a Novel Immune-Associated Mechanism

Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. Our studies have identified an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. The functional significance of increased numbers of pDCs in MM BM is evident from our observations that pDCs: are relatively resistant to novel and conventional therapies; protect tumor cells from therapy-induced cytotoxicity; promote tumor growth and survival; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323). Aberrant pDC function is evidenced in their interactions not only with MM cells, but also with other immune effector T cells and NK cells in the MM BM milieu (Ray et al, Leukemia 2015, 29:1441-1444). Directly targeting pDC interactions with MM and immune effector cells in the MM BM milieu will be required to enhance both anti-tumor immunity and cytotoxicity. However, therapies targeting pDCs are lacking. We found that IL-3R is highly expressed on pDCs, and that pDC-MM interactions trigger secretion of IL-3, which in turn, promotes both pDC survival and osteolytic bone disease. Recent efforts have led to the development of a novel therapeutic agent SL-401, which specifically targets IL-3R-expressing pDCs. Here we examined the effect of SL-401 on pDC-induced MM cell growth both in vitro and in vivo, as well as on IL-3R-expressing osteoclasts.Methods Patient MM cells, pDCs, and MNCs were obtained from normal donors or MM patients. Cell growth/viability was analyzed using MTT/WST assays. OCL function and bone resorption were measured using the OsteoAssays and TRAP staining. The RPMI-8226 cell line was used to isolate MM-SPs by flow-cytometry based Hoechst 33342 staining. SL-401 is a recombinant protein expressed in E. coli. The hybrid gene is comprised of human IL-3 fused to truncated diphtheria toxin (DT). The IL-3 domain of SL-401, which replaces the native binding domain of DT, targets SL-401 to cells that overexpress IL-3R. SL-401 was obtained from Stemline Therapeutics, NY; bortezomib, lenalidomide, pomalidomide, and melphalan were purchased from Selleck Chemicals. For animal model studies, SL-401 (16.5 μg/kg) was administered intravenously daily for 2 weeks.Results SL-401 triggered significant apoptosis in pDCs (>95%) at low picomolar concentrations that are well within clinically achievable doses.Higher concentrations of SL-401 trigger a modest apoptosis (30%± 1.3% apoptosis at 83 ng/ml or 1.3 nM) in MM cells due to lower IL-3R expression versus pDCs. Moreover, SL-401 did not significantly induce apoptosis of normal PBMCs (8% ± 0.5% apoptosis at 83 ng/ml), suggesting a favorable therapeutic index for SL-401. SL-401 inhibited pDC-induced growth of MM cell lines and patient MM cells in a dose-dependent manner. Moreover, 6 of 9 MM samples were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Combinations of SL-401 with melphalan, bortezomib, lenalidomide, or pomalidomide induced synergistic anti-MM activity (Combination index < 1). SL-401 blocked monocyte-derived osteoclast formation in a dose-dependent fashion, as well as restored MM patient BM-derived osteoblast formation. Having defined the efficacy of SL-401 in targeting pDCs and pDC-triggered MM cell growth in vitro, we validated these findings in vivo using our murine xenograft model of human MM, under auspices of protocols approved by our institutional animal protection committee. SL-401 inhibited pDC-induced MM cell growth in vivo and prolonged survival in a murine xenograft model of human MM. We also evaluated the efficacy of SL-401 in vivo using our SCID-human (SCID-hu) mouse model, which reflects the human BM milieu with human cytokines and extracellular matrix proteins. SL-401 significantly abrogated pDC-triggered MM cell growth in vivo in SCID-hu model.Conclusions Our data provide the basis for using SL-401 to directly target pDCs and inhibit the pDC-MM interaction as well as target osteolytic bone disease in novel therapeutic strategies in order to enhance MM cytotoxicity, overcome drug resistance, and improve patient outcome. The interactions of immune effector cells in the MM tumor microenvironment also provide a rationale for combining SL-401 with checkpoint inhibitors.Correspondence: Dharminder Chauhan DisclosuresMacri:Stemline Therapeutics, Inc., New York, NY USA: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership. Richardson:Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: 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; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Chauhan:Stemline Therapeutics: Consultancy.

The Dual PIM/PI3-K Inhibitor Ibl-202 Overcomes Microenvironmental Mediated Resistance in Multiple Myeloma and Prevents PIM1 Induced CXCR4 Upregulation

Background:The interaction of multiple myeloma (MM) cells with bone marrow (BM) cells along with factors in the BM milieu such as chemokines and cytokines play a crucial role in both progression of MM and drug resistance. Activation of the PI3-K/Akt survival pathway is a characteristic of both human MM cell lines and patient samples. This activation can be linked to BM microenvironmental signalling and use of proteasome inhibitors in treatment, suggesting this as a crucial point of therapeutic intervention to abrogate growth and survival signals in MM. However, the efficacy of such therapeutics has been modest and is likely to be compromised by the stimulation of compensatory signalling pathways, such as the PIM kinases, which like the PI3-K/Akt pathway are also induced by BM microenvironmental influences and share similar downstream targets. These proto-oncogenic kinases are constitutively active and play an important role in proliferation and survival in MM. The influence of these kinases on homing and migration has been observed in other malignancies, this has yet to be reported in MM. Here we report the effects of a dual inhibitor of PIM/PI3-K, IBL-202, and provide novel insights into effects on cell survival, signaling and migration.Methods:We investigated the effect of IBL-202 against a panel of MM cell lines (MM.IS, NCI-H929s, KMS11 and RPMI-8226) and primary MM patient samples. The in vitro efficacy of IBL-202 was compared to that of single pan-PIM inhibitors pPIMi and AZD1208 and also the pan-PI3-K inhibitor GDC-0941. Apoptosis was measured with AnnexinV staining and cell cycle analysed with Edu/DAPI staining.To mimic BM microenvironmental conditions MM cells were cultured under hypoxic conditions (1% O2) and in co-culture with the human stromal cell line HS5.Surface expression of CXCR4 was assessed in MM cell lines by flow cytometry. PIM kinases, pCXCR4 and downstream targets of PIM/PI3-K were examined by western blot. Transwell migration assays were carried out in the presence of 50ng SDF-1α for 4h @ 37o C.Results:Simultaneous inhibition of PIM and PI3-K using IBL-202 in vitro was significantly more potent at inducing apoptosis than GDC-0941, pPIMi or AZD1208 in all MM cell lines tested. IC50 values were under 1μM for IBL-202 at 48h whilst in comparison the pan PIM inhibitors pPIMi and AZD1208 scored IC50 values between 5 and 10μM. The IC50 for GDC-0941 was on average 5μM (Figure 1).At the molecular level there was a notable decrease in phosphorylation of known PIM/PI3-K targets Akt (Ser473), Bad (Ser112) and the translational targets S6 (Ser235/236) and 4EBP1 (Thr37/46). The levels of total proteins were unchanged.Treatment with increasing doses of IBL-202 led to a marked reduction in cells in S phase of the cell cycle. These changes were paralled by down regulation of the cell cycle promoting proteins cyclin D1 and c-myc.IBL-202 was also effective in inducing apoptosis in primary MM patient samples (n=4) after just 24h as assessed by Annexin-V staining (Figure 2).To explore the role of the BM microenvironment we co-cultured MM cell lines with HS5s. This led to strong induction of PIM2 in MM cells. While MM cells in this setting were protected from Bortezomib-induced cell death, the apoptotic effect of IBL-202 was enhanced. In a further effort to mimic the tumour microenvironment we cultured MM cell lines in hypoxia. This may be of particular relevance as Pim-1 has been reported to be a pivotal regulator involved in hypoxia-induced chemoresistance. MM cells were further sensitised to IBL-202 in hypoxia. In addition, hypoxia increased the surface expression of CXCR4, a chemokine receptor critical for homing of MM cells to the bone marrow, with a concomitant increase in PIM1. Treatment of MM cell lines with IBL-202 reduced the level of PIM1 and CXCR4 Ser339 phosphorylation, along with down regulation of CXCR4 surface expression resulting in reduced migration of MM cells along an SDF-1 gradient.Conclusion:Together these data provide direct evidence of the potency of IBL-202 in MM in conditions that mimic the BM microenvironment. Moreover, they indicate a potential role for PIM kinases in facilitating dissemination and invasiveness of MM by CXCR4 and provide an added rationale for targeting PIM kinases in MM. [Display omitted] [Display omitted] DisclosuresO'Neill:Inflection Biosciences: Employment.

Macrophage Migration Inhibitory Factor Drives Multiple Myeloma IL-6/8 Pro-Survival Signals in the Tumor Microenvironment

BackgroundMultiple myeloma (MM) accounts for 10-15% of all newly diagnosed hematological malignancies in the western world and despite recent significant progress in the treatment of MM, it presently remains an incurable disease. Average survival following diagnosis is currently approximately 5 years. Current treatments are effective at reducing malignant cell number (and therefore alleviating symptoms), however a small sub-population of MM cells will survive within the bone marrow milieu and relapse is inevitable. Interactions between the MM and bone marrow mesenchymal stromal cells (BM-MSCs) have been shown to be highly beneficial to the MM cells, protecting them from chemotherapeutic agents and upregulating the expression of genes associated with increased survival and proliferation. We hypothesize that the myeloma cells initiate the re-programming of the BM-MSCs for this very purpose, providing themselves with a more favorable environment.The cytokine MIF (Macrophage Migratory Inhibitory Factor) has been shown to be present in abnormally high levels in many cancer types. Here we show that MIF is aberrantly produced by primary myeloma cells and induces transcriptional changes and cytokine release from the BM-MSC. We also show these signals contribute to MM cell survival and drug resistance.MethodsPrimary MM and BM-MSC were obtained from patient's bone marrow as previously described. Primary MM cells at 1x106 were co-cultured on confluent autologous BM-MSC. Conditioned media from MM alone, MM/BM-MSC and BM-MSC cultures were collected and analyzed using Proteome Profiler Human XL Cytokine Array and cytokine specific ELISAs. Real-time PCR was used to analyze the changes in BM-MSC transcriptional levels in response to specific cytokine stimulation or inhibition. Recombinant MIF and the MIF inhibitor ISO-1 were used to verify the MM-BMSC findings.ResultsHere we report that bone marrow, aspirated from MM patients, had a higher level of MIF expression when compared to samples obtained from healthy donors. We examined the cytokine profile in human primary MM monoculture compared to MM cultured with autologous BM-MSC or BM-MSC alone and found that MIF was highly expressed by primary MM and that IL-6 and IL-8 were also increased in MM/BM-MSC co-cultures. RT-PCR confirmed that the majority of MIF transcription within the bone marrow microenvironment occurs within the myeloma cells themselves, with relatively minimal levels found within the bone marrow stromal cells.Next BM-MSCs from MM patients (n=5) were stimulated with recombinant MIF and cytokine release was measured using the XL cytokine array. The results showed that that recombinant human MIF stimulated the release of IL-6 and IL-8 to the media from BM-MSC monocultures. These observed changes were confirmed with the use of cytokine specific ELISA assays. RT-PCR also showed that IL-6 and IL-8 RNA expression were induced in BM-MSCs in response to recombinant MIF. These results confirm that MM derived MIF is instrumental in the transcriptional regulation of IL-6 and IL-8 in BM-MSCs.Next we found that the MIF inhibitor ISO-1 inhibited MM and MIF induced BM-MSC IL-6 and IL-8 RNA expression and cytokine release. Finally we found that MIF inhibition in MM/BM-MSC co-cultures reduced MM survival as well as enhancing the therapeutic potential of proteasome inhibition.SummaryTogether, these data suggest that MM signals via MIF to initiate the re-programming of the BM-MSCs, which creates a pro-tumoral environment in which MM can thrive and be protected from anti-MM therapeutics. DisclosuresNo relevant conflicts of interest to declare.

The Hypoxia-Selective Epigenetic Agent, Rrx-001, Triggers Apoptosis and Overcomes Drug-Resistance in Multiple Myeloma Cells

Introduction The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in multiple myeloma (MM) cells (Chauhan et al, Cancer Cell 2009, 16:309-323) BM hypoxia (low oxygenation) plays a role in promoting MM cell survival, drug resistance, migration, and metastasis. Novel therapies that selectively target the MM cell in its hypoxic BM milieu may therefore overcome conventional drug resistance. Recent studies led to the development of a novel aerospace industry-derived Phase 2 molecule RRx-001 with hypoxia-selective epigenetic and NO-donating properties. A Phase I clinical trial demonstrated promising evidence of anti-tumor activity in a heavily pretreated population with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578, Reid et al, Lancet Oncology, in press). RRx-001 is currently under investigation in multiple Phase II clinical trials. Here we examined both the mechanism of action and anti-MM activity of RRx-001 using in vitro and in vivo models of MM.Methods Cell viability, apoptosis, and migration assays were performed using MTT, Annexin V staining, and transwell Inserts, respectively. ROS and NO generation was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). Synergistic anti-MM activity was assessed by isobologram analysisusing "CalcuSyn" software program. In vitro angiogenesis was assessed using matrigel capillary-like tube formation assays. DNMT1 activity was measured using DNMT1 assay kit. USP7 siRNA was purchased from Dharmacon. CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Statistical significance of data was determined using a Student's t test. RRx-001 was obtained from EpicentRx, CA, USA; USP7 inhibitor P5091, bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA.Results Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 48h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells, even in the presence of BM stromal cells. Washout experiments showed that a short time (3h) exposure of MM cells to RRx-001 triggered irreversible cell death. RRx-001-triggered apoptosis is associated with: 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNMT1 activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. Deubiqyitylating enzyme USP7 stimulates DNMT1 enzymatic activity. USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. Importantly, the combination of USP7 inhibitor P5091 and RRx-001 triggered synergistic anti-MM activity associated with a robust decrease in DNMT1 activity, as well as increased degradation of USP7 substrate MDM2 and induction of downstream p21/p53 signaling axis. In vivo studies using a subcutaneous human MM xenograft model shows that RRx-001 is well tolerated, inhibits tumor growth, and enhances survival. Finally, combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity in p53-WT and p53-null MM cells, and overcomes drug resistance.Conclusion Our preclinical studies demonstrate that RRx-001, a ROS-mediated epigenetic inhibitor with anti-angiogenic properties selectively targets MM cells in vivo and synergizes with existing anti-MM agents to overcome therapeutic resistance. Our data also suggest a potential mechanism of action for RRx-001-induced epigenetic changes via USP7-DNMT1 complex and downstream p53/p21 signaling cascade. Collectively, these results provide a rationale for rapid translation of RRx-001, either alone or in combination, in a clinical trial of relapsed refractory MM. DisclosuresOronsky:epicentrx: Employment. Scicinski:epicentrx: Employment. Chauhan:Stemline Therapeutics: Consultancy.

Lenalidomide Enhances Immune Checkpoint Blockade-Induced Immune Response in Multiple Myeloma.

PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell-mediated antitumor immune responses. Here, we assessed the impact of single and dual blockade of PD-1/PD-L1, alone or in combination with lenalidomide, on accessory and immune cell function as well as multiple myeloma cell growth in the bone marrow (BM) milieu. Surface expression of PD-1 on immune effector cells, and PD-L1 expression on CD138(+) multiple myeloma cells and myeloid-derived suppressor cells (MDSC) were determined in BM from newly diagnosed (ND) multiple myeloma and relapsed/refractory (RR) multiple myeloma versus healthy donor (HD). We defined the impact of single and dual blockade of PD-1/PD-L1, alone and with lenalidomide, on autologous anti-multiple myeloma immune response and tumor cell growth. Both ND and RR patient multiple myeloma cells have increased PD-L1 mRNA and surface expression compared with HD. There is also a significant increase in PD-1 expression on effector cells in multiple myeloma. Importantly, PD-1/PD-L1 blockade abrogates BM stromal cell (BMSC)-induced multiple myeloma growth, and combined blockade of PD-1/PD-L1 with lenalidomide further inhibits BMSC-induced tumor growth. These effects are associated with induction of intracellular expression of IFNγ and granzyme B in effector cells. Importantly, PD-L1 expression in multiple myeloma is higher on MDSC than on antigen-presenting cells, and PD-1/PD-L1 blockade inhibits MDSC-mediated multiple myeloma growth. Finally, lenalidomide with PD-1/PD-L1 blockade inhibits MDSC-mediated immune suppression. Our data therefore demonstrate that checkpoint signaling plays an important role in providing the tumor-promoting, immune-suppressive microenvironment in multiple myeloma, and that PD-1/PD-L1 blockade induces anti-multiple myeloma immune response that can be enhanced by lenalidomide, providing the framework for clinical evaluation of combination therapy.

Notch signaling deregulation in multiple myeloma: A rational molecular target.

Despite recent therapeutic advances, multiple myeloma (MM) is still an incurable neoplasia due to intrinsic or acquired resistance to therapy. Myeloma cell localization in the bone marrow milieu allows direct interactions between tumor cells and non-tumor bone marrow cells which promote neoplastic cell growth, survival, bone disease, acquisition of drug resistance and consequent relapse. Twenty percent of MM patients are at high-risk of treatment failure as defined by tumor markers or presentation as plasma cell leukemia. Cumulative evidences indicate a key role of Notch signaling in multiple myeloma onset and progression. Unlike other Notch-related malignancies, where the majority of patients carry gain-of-function mutations in Notch pathway members, in MM cell Notch signaling is aberrantly activated due to an increased expression of Notch receptors and ligands; notably, this also results in the activation of Notch signaling in surrounding stromal cells which contributes to myeloma cell proliferation, survival and migration, as well as to bone disease and intrinsic and acquired pharmacological resistance. Here we review the last findings on the mechanisms and the effects of Notch signaling dysregulation in MM and provide a rationale for a therapeutic strategy aiming at inhibiting Notch signaling, along with a complete overview on the currently available Notch-directed approaches.

Adiponectin as a biomarker linking obesity and adiposopathy to hematologic malignancies

Higher body mass index and adiposopathy have been associated with increased risk of hematologic malignancies such as leukemia, multiple myeloma, myeloproliferative disorders, Hodgkin's and non-Hodgkin's lymphoma, and myelodysplastic syndromes. Adiponectin is a multimeric protein of the white adipose tissue presenting anti-inflammatory, insulin-sensitizing, anti-atherogenic, cardioprotective, and anti-neoplastic properties. Its anti-neoplastic actions are manifested via two mechanisms: (i) direct action on tumor cells by enhancing receptor-mediated signaling pathways and (ii) indirect action by regulating inflammatory responses, influencing cancer angiogenesis, and modulating insulin sensitivity at the target tissue site. In the bone marrow milieu, adiponectin and its main receptors are expressed by the majority of bone marrow stromal cell populations influencing hematopoietic stem cells function. Adiponectin may represent a molecular mediator relating adiposopathy with leukemogenesis and myelomagenesis. Several epidemiological studies conducted to date relate hypoadiponectinemia to the risk of myeloid-derived hematopoietic cancer and multiple myeloma. Adiponectin may be a promising biomarker with potential diagnostic and prognostic utility in determining the likelihood of myeloma and leukemia progression in certain cohorts of monoclonal gammopathy of undetermined significance patients and in myeloid hematologic malignancies, respectively. This review summarizes experimental and epidemiologic data regarding the role of adiponectin in hematologic malignancies in the context of adiposopathy. Enhancement of endogenous adiponectin, adiponectin replacement, or manipulation of adiponectin receptor sensitivity may be an attractive goal for prevention and an effective therapeutic strategy against hematopoietic cancer, specifically in overweight/obese individuals. Further studies are required to elucidate the role of the bone marrow microenvironment adiponectin in complex interactions involved in preleukemic and leukemic states.