scholarly journals BT7480, a novel fully synthetic Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA™) induces tumor localized CD137 agonism

2021 ◽  
Vol 9 (11) ◽  
pp. e002883
Author(s):  
Kristen Hurov ◽  
Johanna Lahdenranta ◽  
Punit Upadhyaya ◽  
Eric Haines ◽  
Heather Cohen ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Drug Exposure ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Therapeutic Potential ◽  
Human Cancer ◽  
Human Peripheral Blood ◽  
Unmet Need ◽  
Mouse Tumor ◽  
Human Cancers

BackgroundCD137 (4-1BB) is an immune costimulatory receptor with high therapeutic potential in cancer. We are creating tumor target-dependent CD137 agonists using a novel chemical approach based on fully synthetic constrained bicyclic peptide (Bicycle®) technology. Nectin-4 is overexpressed in multiple human cancers that may benefit from CD137 agonism. To this end, we have developed BT7480, a novel, first-in-class, Nectin-4/CD137 Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA™).MethodsNectin-4 and CD137 co-expression analyses in primary human cancer samples was performed. Chemical conjugation of two CD137 Bicycles to a Nectin-4 Bicycle led to BT7480, which was then evaluated using a suite of in vitro and in vivo assays to characterize its pharmacology and mechanism of action.ResultsTranscriptional profiling revealed that Nectin-4 and CD137 were co-expressed in a variety of human cancers with high unmet need and spatial proteomic imaging found CD137-expressing immune cells were deeply penetrant within the tumor near Nectin-4-expressing cancer cells. BT7480 binds potently, specifically, and simultaneously to Nectin-4 and CD137. In co-cultures of human peripheral blood mononuclear cells and tumor cells, this co-ligation causes robust Nectin-4-dependent CD137 agonism that is more potent than an anti-CD137 antibody agonist. Treatment of immunocompetent mice bearing Nectin-4-expressing tumors with BT7480 elicited a profound reprogramming of the tumor immune microenvironment including an early and rapid myeloid cell activation that precedes T cell infiltration and upregulation of cytotoxicity-related genes. BT7480 induces complete tumor regressions and resistance to tumor re-challenge. Importantly, antitumor activity is not dependent on continuous high drug levels in the plasma since a once weekly dosing cycle provides maximum antitumor activity despite minimal drug remaining in the plasma after day 2. BT7480 appears well tolerated in both rats and non-human primates at doses far greater than those expected to be clinically relevant, including absence of the hepatic toxicity observed with non-targeted CD137 agonists.ConclusionBT7480 is a highly potent Nectin-4-dependent CD137 agonist that produces complete regressions and antitumor immunity with only intermittent drug exposure in syngeneic mouse tumor models and is well tolerated in preclinical safety species. This work supports the clinical investigation of BT7480 for the treatment of cancer in humans.

scholarly journals MET Receptor Tyrosine Kinase Regulates the Expression of Co-Stimulatory and Co-Inhibitory Molecules in Tumor Cells and Contributes to PD-L1-Mediated Suppression of Immune Cell Function

2019 ◽  
Vol 20 (17) ◽  
pp. 4287 ◽  
Author(s):  
Hyun Kyung Ahn ◽  
Sehui Kim ◽  
Dohee Kwon ◽  
Jaemoon Koh ◽  
Young A. Kim ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Cell Function ◽  
Immune Cell ◽  
Immune Escape ◽  
Human Cancer ◽  
Human Peripheral Blood ◽  
The Cancer Genome Atlas ◽  
Peripheral Blood Mononuclear ◽  
Human Cancer Cell Lines ◽  
Inhibitory Molecules

The MET tyrosine receptor kinase is essential for embryonic development and tissue regeneration by promoting cell survival, proliferation, migration, and angiogenesis. It also contributes to tumor development and progression through diverse mechanisms. Using human cancer cell lines, including Hs746T (MET-mutated/amplified), H596 (MET-mutated), and H1993 (MET-amplified) cells, as well as BEAS-2B bronchial epithelial cells, we investigated whether MET is involved in the regulation of immune checkpoint pathways. In a microarray analysis, MET suppression using a MET inhibitor or siRNAs up-regulated co-stimulatory molecules, including 4-1BBL, OX40L, and CD70, and down-regulated co-inhibitory molecules, especially PD-L1, as validated by measuring total/surface protein levels in Hs746T and H1993 cells. MET activation by HGF consistently increased PD-L1 expression in H596 and BEAS-2B cells. Co-culture of human peripheral blood mononuclear cells with Hs746T cells suppressed interferon-γ production by the immune cells, which was restored by MET inhibition or PD-L1 blockade. A significant positive correlation between MET and PD-L1 expression in lung cancer was determined in an analysis based on The Cancer Genome Atlas (TCGA) and in an immunohistochemistry study. The former also showed an association of MET overexpression in a PD-L1high tumor with the decreased expressions of T-cell effector molecules. In summary, our results point to a role for MET overexpression/activation in the immune escape of tumors by PD-L1 up-regulation. MET-targeted-therapy combined with immunotherapy may therefore be an effective treatment strategy in patients with MET-dependent cancer.

scholarly journals NF-κB and Human Cancer: What Have We Learned over the Past 35 Years?

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 889
Author(s):  
Thomas D. Gilmore
Keyword(s):  
Cell Cycle Progression ◽  
Cell Function ◽  
Immune Cell ◽  
Human Cancer ◽  
Cancer Prognosis ◽  
Cycle Progression ◽  
The Past ◽  
Human Cancers ◽  
Limited Success ◽  
Upstream Regulators

Transcription factor NF-κB has been extensively studied for its varied roles in cancer development since its initial characterization as a potent retroviral oncogene. It is now clear that NF-κB also plays a major role in a large variety of human cancers, including especially ones of immune cell origin. NF-κB is generally constitutively or aberrantly activated in human cancers where it is involved. These activations can occur due to mutations in the NF-κB transcription factors themselves, in upstream regulators of NF-κB, or in pathways that impact NF-κB. In addition, NF-κB can be activated by tumor-assisting processes such as inflammation, stromal effects, and genetic or epigenetic changes in chromatin. Aberrant NF-κB activity can affect many tumor-associated processes, including cell survival, cell cycle progression, inflammation, metastasis, angiogenesis, and regulatory T cell function. As such, inhibition of NF-κB has often been investigated as an anticancer strategy. Nevertheless, with a few exceptions, NF-κB inhibition has had limited success in human cancer treatment. This review covers general themes that have emerged regarding the biological roles and mechanisms by which NF-κB contributes to human cancers and new thoughts on how NF-κB may be targeted for cancer prognosis or therapy.

scholarly journals Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist

2021 ◽  
Vol 9 (1) ◽  
pp. e001762
Author(s):  
Punit Upadhyaya ◽  
Johanna Lahdenranta ◽  
Kristen Hurov ◽  
Sailaja Battula ◽  
Rachel Dods ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Tumor Antigens ◽  
Immune Cell ◽  
Checkpoint Inhibitors ◽  
Human Peripheral Blood ◽  
Interleukin 2 ◽  
Cancer Therapeutics ◽  
Peripheral Blood Mononuclear ◽  
Tnf Superfamily

BackgroundIn contrast to immune checkpoint inhibitors, the use of antibodies as agonists of immune costimulatory receptors as cancer therapeutics has largely failed. We sought to address this problem using a new class of modular synthetic drugs, termed tumor-targeted immune cell agonists (TICAs), based on constrained bicyclic peptides (Bicycles).MethodsPhage libraries displaying Bicycles were panned for binders against tumor necrosis factor (TNF) superfamily receptors CD137 and OX40, and tumor antigens EphA2, Nectin-4 and programmed death ligand 1. The CD137 and OX40 Bicycles were chemically conjugated to tumor antigen Bicycles with different linkers and stoichiometric ratios of binders to obtain a library of low molecular weight TICAs (MW <8 kDa). The TICAs were evaluated in a suite of in vitro and in vivo assays to characterize their pharmacology and mechanism of action.ResultsLinking Bicycles against costimulatory receptors (e.g., CD137) to Bicycles against tumor antigens (e.g., EphA2) created potent agonists that activated the receptors selectively in the presence of tumor cells expressing these antigens. An EphA2/CD137 TICA (BCY12491) efficiently costimulated human peripheral blood mononuclear cells in vitro in the presence of EphA2 expressing tumor cell lines as measured by the increased secretion of interferon γ and interleukin-2. Treatment of C57/Bl6 mice transgenic for the human CD137 extracellular domain (huCD137) bearing EphA2-expressing MC38 tumors with BCY12491 resulted in the infiltration of CD8+ T cells, elimination of tumors and generation of immunological memory. BCY12491 was cleared quickly from the circulation (plasma t1/2 in mice of 1–2 hr), yet intermittent dosing proved effective.ConclusionTumor target-dependent CD137 agonism using a novel chemical approach (TICAs) afforded elimination of tumors with only intermittent dosing suggesting potential for a wide therapeutic index in humans. This work unlocks a new path to effective cancer immunotherapy via agonism of TNF superfamily receptors.

scholarly journals In Vitro Antitumor Activity of Endophytic and Rhizosphere Gram-Positive Bacteria from Ibervillea sonorae (S. Watson) Greene against L5178Y-R Lymphoma Cells

2022 ◽  
Vol 19 (2) ◽  
pp. 894
Author(s):  
Ricardo Romero-Arguelles ◽  
César Iván Romo-Sáenz ◽  
Karla Morán-Santibáñez ◽  
Patricia Tamez-Guerra ◽  
Ramiro Quintanilla-Licea ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Growth Inhibition ◽  
Mononuclear Cells ◽  
Colorimetric Assay ◽  
Human Peripheral Blood ◽  
Antitumor Effects ◽  
Lymphoma Cells ◽  
Gram Positive ◽  
Peripheral Blood Mononuclear ◽  
Gram Positive Bacteria

Plant-associated microorganisms represent a potential source of new antitumor compounds. The aim of the present study was to isolate endophytic and rhizosphere Gram-positive bacteria from Ibervillea sonorae and produce extracts with antitumor activity. Methanol and ethyl acetate extracts were obtained from 28 d bacterial fermentation, after which murine L5178Y-R lymphoma cells growth inhibition was evaluated at concentrations ranging from 15.62 µg/mL to 500 µg/mL by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide reduction colorimetric assay. IC50 and the selectivity index (SI) were calculated and compared with healthy control human peripheral blood mononuclear cells (PBMC). Identification of the isolated strains was performed using the 16S ribosomal gene and by MALDI-TOF MS mass spectrometry. The endophytic and rhizosphere bacterial extracts from strains ISE-B22, ISE-B26, ISE-B27, ISS-A01, ISS-A06, and ISS-A16 showed significant (p < 0.05) L5178Y-R cell growth inhibition, compared with an untreated control. The rhizosphere Micromonospora echinospora isolate ISS-A16 showed the highest (90.48%) percentage of lymphoma cells growth inhibition and SI (19.1) for PBMC, whereas the Bacillus subtilis ISE-B26 isolate caused significant (p < 0.01) growth inhibition (84.32%) and a SI of 5.2. Taken together, results of the present study evidenced antitumor effects by I. sonorae endophytic and rhizosphere bacteria culture extracts. Further research will involve the elucidation of the compounds that exert the antitumor activity and their evaluation in pre-clinical studies.

scholarly journals Liposome/gold hybrid nanoparticle encoded with CoQ10 (LGNP-CoQ10) suppressed rheumatoid arthritis via STAT3/Th17 targeting

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241080
Author(s):  
Jooyeon Jhun ◽  
Jeonghyeon Moon ◽  
Jaeyoon Ryu ◽  
Yonghee Shin ◽  
Seangyoun Lee ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Mononuclear Cells ◽  
Therapeutic Potential ◽  
Human Peripheral Blood ◽  
Treated Group ◽  
Hybrid Nanoparticles ◽  
Peripheral Blood Mononuclear ◽  
Hematoxylin And Eosin ◽  
Anti Inflammatory ◽  
Safranin O

Coenzyme Q10 (CoQ10), also known as ubiquinone, is a fat-soluble antioxidant. Although CoQ10 has not been approved as medication by the Food and Drug Administration, it is widely used in dietary supplements. Some studies have shown that CoQ10 has anti-inflammatory effects on various autoimmune disorders. In this study, we investigated the anti-inflammatory effects of liposome/gold hybrid nanoparticles encoded with CoQ10 (LGNP-CoQ10). Both CoQ10 and LGNP-CoQ10 were administered orally to mice with collagen-induced arthritis (CIA) for 10 weeks. The inflammation pathology of joint tissues of CIA mice was then analyzed using hematoxylin and eosin and Safranin O staining, as well as immunohistochemistry analysis. We obtained immunofluorescence staining images of spleen tissues using confocal microscopy. We found that pro-inflammatory cytokines were significantly decreased in LGNP-CoQ10 injected mice. Th17 cell and phosphorylated STAT3-expressed cell populations were also decreased in LGNP-CoQ10 injected mice. When human peripheral blood mononuclear cells (PBMCs) were treated with CoQ10 and LGNP-CoQ10, the IL-17 expression of PBMCs in the LGNP-CoQ10-treated group was significantly reduced. Together, these results suggest that LGNP-CoQ10 has therapeutic potential for the treatment of rheumatoid arthritis.

scholarly journals Therapeutic Potential of a Novel Bifidobacterium Identified Through Microbiome Profiling of RA Patients With Different RF Levels

2021 ◽  
Vol 12 ◽  
Author(s):  
Yunju Jeong ◽  
JooYeon Jhun ◽  
Seon-Yeong Lee ◽  
Hyun Sik Na ◽  
JeongWon Choi ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Relative Abundance ◽  
Th17 Cells ◽  
Mononuclear Cells ◽  
Therapeutic Potential ◽  
Human Peripheral Blood ◽  
Cartilage Damage ◽  
Therapeutic Effects ◽  
Bioinformatics Analyses ◽  
Proinflammatory Mediators

The potential therapeutic effects of probiotic bacteria in rheumatoid arthritis (RA) remain controversial. Thus, this study aimed to discover potential therapeutic bacteria based on the relationship between the gut microbiome and rheumatoid factor (RF) in RA. Bacterial genomic DNA was extracted from the fecal samples of 93 RA patients and 16 healthy subjects. Microbiota profiling was conducted through 16S rRNA sequencing and bioinformatics analyses. The effects of Bifidobacterium strains on human peripheral blood mononuclear cells and collagen-induced arthritis (CIA) mice were assessed. Significant differences in gut microbiota composition were observed in patients with different RF levels. The relative abundance of Bifidobacterium and Collinsella was lower in RF-high than in RF-low and RF-negative RA patients, while the relative abundance of Clostridium of Ruminococcaceae family was higher in RF-high than in RF-low and RF-negative patients. Among 10 differentially abundant Bifidobacterium, B. longum RAPO exhibited the strongest ability to inhibit IL-17 secretion. Oral administration of B. longum RAPO in CIA mice, obese CIA, and humanized avatar model significantly reduced RA incidence, arthritis score, inflammation, bone damage, cartilage damage, Th17 cells, and inflammatory cytokine secretion. Additionally, B. longum RAPO significantly inhibited Th17 cells and Th17-related genes—IL-17A, IRF4, RORC, IL-21, and IL-23R—in the PBMCs of rheumatoid arthritis patients. Our findings suggest that B. longum RAPO may alleviate RA by inhibiting the production of IL-17 and other proinflammatory mediators. The safety and efficacy of B. longum RAPO in patients with RA and other autoimmune disorders merit further investigation.

scholarly journals Identification of human immune cell subtypes most responsive to IL-1β–induced inflammatory signaling using mass cytometry

2021 ◽  
Vol 14 (673) ◽  
pp. eabc5763 ◽  
Author(s):  
Hema Kothari ◽  
Corey M. Williams ◽  
Chantel McSkimming ◽  
Fabrizio Drago ◽  
Melissa A. Marshall ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Human Peripheral Blood ◽  
Effector Memory ◽  
High Morbidity ◽  
Cytokine Storm ◽  
Mass Cytometry ◽  
Myeloid Dendritic Cells

IL-1β is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1β blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4+ T cells and CD4−CD8low/−CD161+ T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1β. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c+ myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16−CD56brightCD161− and CD16−CD56dimCD161+), and lineage− (Lin−) cells expressing CD161 and CD25. IL-1β also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1β stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1β–induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1β in CCR6+ T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1β–neutralizing therapies.

scholarly journals Chloroquine and Rapamycin Augment Interleukin-37 Expression via the LC3, ERK, and AP-1 Axis in the Presence of Lipopolysaccharides

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Xiaoyi Shi ◽  
Chunhui Lai ◽  
Lianyu Zhao ◽  
Mingying Zhang ◽  
Xi Liu ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Potential ◽  
Inflammatory Diseases ◽  
Human Peripheral Blood ◽  
Signal Pathways ◽  
U937 Cells ◽  
Healthy Human ◽  
Peripheral Blood Mononuclear ◽  
Phosphorylated Proteins

IL-37 is a cytokine that plays critical protective roles in many metabolic inflammatory diseases, and its therapeutic potential has been confirmed by exogenous IL-37 administration. However, its regulatory mechanisms remain unclear. U937 cells were treated with autophagy-modifying reagents (3-MA, chloroquine, and rapamycin) with or without LPS stimulation. Thereafter, IL-37 expression and autophagic markers (Beclin1, P62/SQSTM1, and LC3) were determined. For regulatory signal pathways, phosphorylated proteins of NF-κB (p65 and IκBα), AP-1 (c-Fos/c-Jun), and MAPK signal pathways (Erk1/2 and p38 MAPK) were quantified, and the agonists and antagonists of MAPK and NF-κB pathways were also used. Healthy human peripheral blood mononuclear cells were treated similarly to confirm our results. Four rhesus monkeys were also administered chloroquine to evaluate IL-37 induction in vivo and its bioactivity on CD4 proliferation and activation. IL-37 was upregulated by rapamycin and chloroquine in both U937 cells and human PBMCs in the presence of LPS. IL-37 was preferentially induced in autophagic cells associated with LC3 conversion. AP-1 and p65 binding motifs could be deduced in the sequence of the IL-37 promoter. Inductive IL-37 expression was accompanied with increased phosphorylated Erk1/2 and AP-1 and could be completely abolished by an Erk1/2 inhibitor or augmented by Erk1/2 agonists. In monkeys, chloroquine increased IL-37 expression, which was inversely correlated with CD4 proliferation and phosphorylated STAT3. IL-37 levels were induced by rapamycin and chloroquine through the LC3, Erk1/2, and NF-κB/AP-1 pathways. Functional IL-37 could also be induced in vivo.

scholarly journals Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2396
Author(s):  
Katarzyna Zielniok ◽  
Anna Burdzinska ◽  
Beata Kaleta ◽  
Radoslaw Zagozdzon ◽  
Leszek Paczek
Keyword(s):  
Real Time ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Real Time Pcr ◽  
Mononuclear Cells ◽  
Therapeutic Potential ◽  
Human Peripheral Blood ◽  
Immunomodulatory Activity ◽  
Peripheral Blood Mononuclear ◽  
Immunomodulatory Properties

The therapeutic potential of mesenchymal stromal cells (MSCs) is largely attributed to their immunomodulatory properties, which can be further improved by hypoxia priming. In this study, we investigated the immunomodulatory properties of MSCs preconditioned with hypoxia-mimetic Vadadustat (AKB-6548, Akebia). Gene expression analysis of immunomodulatory factors was performed by real-time polymerase chain reaction (real-time PCR) on RNA isolated from six human bone-marrow derived MSCs populations preconditioned for 6 h with 40 μM Vadadustat compared to control MSCs. The effect of Vadadustat preconditioning on MSCs secretome was determined using Proteome Profiler and Luminex, while their immunomodulatory activity was assessed by mixed lymphocyte reaction (MLR) and Culturex transwell migration assays. Real-time PCR revealed that Vadadustat downregulated genes related to immune system: IL24, IL1B, CXCL8, PDCD1LG1, PDCD1LG2, HIF1A, CCL2 and IL6, and upregulated IL17RD, CCL28 and LEP. Vadadustat caused a marked decrease in the secretion of IL6 (by 51%), HGF (by 47%), CCL7 (MCP3) (by 42%) and CXCL8 (by 40%). Vadadustat potentiated the inhibitory effect of MSCs on the proliferation of alloactivated human peripheral blood mononuclear cells (PBMCs), and reduced monocytes-enriched PBMCs chemotaxis towards the MSCs secretome. Preconditioning with Vadadustat may constitute a valuable approach to improve the therapeutic properties of MSCs.

Anti-Tumor Activity of a Novel Immunosuppressant FTY720 in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3456-3456
Author(s):  
Hiroshi Yasui ◽  
Teru Hideshima ◽  
Aldo M. Roccaro ◽  
Norihiko Shiraishi ◽  
Makoto Hamasaki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Human Cancer ◽  
Human Peripheral Blood ◽  
Parp Cleavage ◽  
Igf I ◽  
Sphingosine 1 Phosphate

Abstract Sphingosine and its metabolites are bioactive sphingolipids involved in lipid biosynthesis, signal transduction and apoptosis. FTY720, a synthetic sphingosine analogue of myriocine derived from culture filtrates of Isaria sinclairii, has been reported to interact with the sphingosine-1-phosphate specific G protein-linked receptors (S1P1, 3, 4 and 5) (Mandala S et al. Science, 2002) and alter the migration and homing of lymphocytes, thereby inhibiting the immune response (Matloubian M et al. Nature, 2004). Recent studies have also shown that FTY720 induces growth inhibition and/or apoptosis in human cancer cells in vitro as well as in vivo murine model (Azuma H et al. Cancer Research, 2002). To date, however, the biologic sequelae of inhibiting sphingosine-1-phosphate activity on multiple myeloma (MM) cells have not been demonstrated. In the present study, we examined whether FTY720 triggers anti MM activity. FTY720 induced potent cytotoxicity against MM cell lines including MM.1S, U266, RPMI8226, with IC50 at 24 h of 3.0 – 7.0 mM, assessed by trypan-blue exclusion and MTT assays. FTY720 also inhibited growth of doxorubicin (Dox)-resistant RPMI8226-Dox40 and dexamethasone (Dex)-resistant MM.1R cell lines, with IC50 values similar to the parental drug-sensitive cell lines. In contrast, no cytotoxicity of FTY720 was recognized against human peripheral blood mononuclear cells from normal healthy donors. The combination of Dex with FTY720 demonstrated enhanced cytotoxicity compared to either agent alone. Importantly, neither interleukin-6 (IL-6) nor insulin like growth factor-I (IGF-I), which induces MM cell growth and protection against Dex-induced apoptosis, protected against FTY720-induced growth inhibition. The anti-MM mechanisms of action of FTY720 were next studied, and FTY720 induced caspase-dependent apoptosis in MM cell lines: FTY720 triggers caspase−8, −9 and −3 cleavage, followed by PARP cleavage and DNA fragmentation, as confirmed by Western blotting and agarose gel electrophoresis, respectively. Moreover, FTY720 abrogated both IL-6 mediated phosphorylation of Akt-1, STAT3 and p42/44MAPK, and IGF-I mediated Akt-1 phosphorylation. Importantly, paracrine MM cell growth with bone marrow stromal cells was strongly inhibited by FTY720. These results suggest that FTY720 overcomes drug resistance in MM cells and, providing the rationale for its clinical evaluation to improve patient outcome in MM.

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