To explore the functional consequences of possible cross-talk between the CXCR4/CXCL12 and the sphingosine-1-phosphate (S1P) pathways in multiple myeloma (MM) cells and to evaluate the effect of S1P ...targeting with the FTY720 modulator as a potential anti-MM therapeutic strategy.
S1P targeting with FTY720 induces MM cell apoptosis. The combination of FTY720 with the SPHK1 inhibitor SKI-II results in synergistic inhibition of MM growth. CXCR4/CXCL12-enhanced expression correlates with reduced MM cell sensitivity to both FTY720 and SKI-II inhibitors, and with SPHK1 coexpression in both cell lines and primary MM bone marrow (BM) samples, suggesting regulative cross-talk between the CXCR4/CXCL12 and SPHK1 pathways in MM cells. FTY720 was found to directly target CXCR4. FTY720 profoundly reduces CXCR4 cell-surface levels and abrogates the CXCR4-mediated functions of migration toward CXCL12 and signaling pathway activation. Moreover, FTY720 cooperates with bortezomib, inducing its cytotoxic activity and abrogating the bortezomib-mediated increase in CXCR4 expression. FTY720 effectively targets bortezomib-resistant cells and increases their sensitivity to bortezomib, promoting DNA damage. Finally, in a recently developed novel xenograft model of CXCR4-dependent systemic MM with BM involvement, FTY720 treatment effectively reduces tumor burden in the BM of MM-bearing mice. FTY720 in combination with bortezomib demonstrates superior tumor growth inhibition and abrogates bortezomib-induced CXCR4 increase on MM cells.
Altogether, our work identifies a cross-talk between the S1P and CXCR4 pathways in MM cells and provides a preclinical rationale for the therapeutic application of FTY720 in combination with bortezomib in patients with MM.
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Purpose To estimate the prevalence, genotype, and clinical spectrum of Best vitelliform macular dystrophy (Best disease). Design Retrospective epidemiologic and clinical and molecular genetic ...observational study. Methods setting: National referral center. participants: Forty-five individuals diagnosed with Best disease. observation procedures: Retrospective review of patients diagnosed according to clinical findings and sequencing of BEST1 . Patients with recently established molecular genetic diagnosis were followed up including multifocal electroretinography (mfERG), spectral-domain optical coherence tomography (SD-OCT), and fundus autofluorescence (FAF) imaging. main outcome measures: BEST1 mutations, SD-OCT and FAF findings, mfERG amplitudes, prevalence estimate of Best disease. Results BEST1 mutations described previously in Danish patients with Best disease are reviewed. In addition, we identified a further 8 families and 1 sporadic case, in whom 6 BEST1 missense mutations were found, 4 of which are novel. The mutation c.904G>T (p.Asp302Asn) was identified in members of 4 unrelated families. Structural alterations ranged from precipitate-like alterations at the level of the photoreceptor outer segments (OS) to choroidal neovascularization. The extent of the former correlated with the reduction of retinal function. A prevalence estimate of Best disease in Denmark based on the number of diagnosed cases was 1.5 per 100 000 individuals. Conclusions Our data expand the mutation spectrum of BEST1 in patients with Best disease. Alterations of the OS overlying lesions with subretinal fluid are similar to those seen in central serous retinopathy and may indicate impaired turnover of OS. Our frequency estimate confirms that Best disease is one of the most common causes of early macular degeneration.
Multiple myeloma (MM) cells specifically attract peripheral-blood monocytes, while interaction of MM with bone marrow stromal cells (BMSCs) significantly increased monocyte recruitment (p<0.01). The ...CXCL12 chemokine, produced by both the MM and BMSCs, was found to be a critical regulator of monocyte migration. CXCL12 production was up-regulated under MM-BMSCs co-culture conditions, whereas blockage with anti-CXCR4 antibodies significantly abrogated monocyte recruitment toward a MM-derived conditioned medium (p<0.01). Furthermore, elevated levels of CXCL12 were detected in MM, but not in normal BM samples, whereas malignant MM cells often represented the source of increased CXCL12 in the BM. Blood-derived macrophages effectively supported MM cells proliferation and protected them from chemotherapy-induced apoptosis. Importantly, MM cells affected macrophage polarization, elevating the expression of M2-related scavenger receptor CD206 in macrophages and blocking LPS-induced TNFα secretion (a hallmark of M1 response). Of note, MM-educated macrophages suppressed T-cell proliferation and IFNγ production in response to activation. Finally, increased numbers of CXCR4-expressing CD163+CD206+ macrophages were detected in the BM of MM patients (n=25) in comparison to MGUS (n=11) and normal specimens (n=8). Taken together, these results identify macrophages as important players in MM tumorogenicity, and recognize the CXCR4/CXCL12 axis as a critical regulator of MM-stroma interactions and microenvironment formation.
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Although having promising anti-myeloma properties, the pan-histone deacetylase inhibitor (HDACi) panobinostat lacks therapeutic activity as a single agent. The aim of the current ...study was to elucidate the mechanisms underlying multiple myeloma (MM) resistance to panobinostat monotherapy and to define strategies to overcome it. Sensitivity of MM cell lines and primary CD138+ cells from MM patients to panobinostat correlated with reduced expression of the chemokine receptor CXCR4, whereas overexpression of CXCR4 in MM cell lines increased their resistance to panobinostat. Decreased sensitivity to HDACi was associated with reversible G0/G1 cell growth arrest while response was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators revealed the pro-survival mTOR pathway to be regulated by CXCR4 overexpression. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance to HDACi and induced synergistic cell death. The combination of panobinostat/everolimus resulted in sustained DNA damage and irreversible suppression of proliferation accompanied by robust apoptosis. Gene expression analysis revealed distinct genetic profiles of single versus combined agent exposure. Whereas panobinostat increased the expression of the cell cycle inhibitor p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated the expression of DNA repair genes and mitotic checkpoint regulators. Importantly, the combination of panobinostat with everolimus effectively targeted CXCR4-expressing resistant MM cells in vivo in the BM niche. In summary, our results uncover the mechanism responsible for the strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel potential therapeutic approach to treat MM.
Best disease is a monogenic macular degeneration caused mainly by heterozygous mutations in the BEST1 gene. The objective was to characterize the molecular and clinical features of patients with the ...classical form of Best disease that is inherited in an autosomal recessive mode.
Clinical evaluation included detailed family history, a full ophthalmologic examination, electro-oculography (EOG), electroretinography, color vision testing, and ocular imaging. Mutation analysis was performed by direct sequencing of PCR products.
Two young siblings affected by Best disease, as confirmed by funduscopy, retinal imaging, and electrophysiologic assessment, were recruited for the study. Molecular analysis revealed a novel homozygous deletion (c.1415delT) in the BEST1 gene leading to a frameshift followed by a premature stop codon, which cosegregated with the disease in a recessive mode. The heterozygous parents had normal visual acuity, retinal appearance, and function. The two heterozygous grandmothers, ages 61 and 62, also had normal Arden ratios on EOG, but one of them manifested moderate-to-severe dry non-neovascular age-related macular degeneration.
We show here that the typical vitelliform phenotype of Best disease, usually transmitted in an autosomal dominant fashion, can be inherited as an autosomal recessive disease due to homozygosity for a frameshift mutation.
Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents remains a major treatment obstacle, therefore novel therapies are in need. Wild-type p53-induced ...phosphatase 1 (WIP1) is an oncogenic serine/threonine phosphatase implicated in silencing of cellular responses to genotoxic stress. WIP1 overexpression was documented in various solid cancers in correlation with aggressive features and poor prognosis. Thus, we studied WIP1 in MM addressing its potential role in mediating resistance and aggressive phenotype.
Increased expression of WIP1 was detected in MM cell lines (n=8) and primary samples (n=18) at both mRNA and protein level as compared with normal PBMCs (n=5). Furthermore, a positive correlation between WIP1 and CXCR4 levels (p<0.02, R2=0.5) was revealed. The latter is a well-known oncogenic receptor in MM. WIP1 expression levels were significantly up-regulated following bortezomib (Bort) treatment. Using MM cell lines with acquired resistance to Bort (RPMI8226BortRes and CAGBortRes), a higher induction of WIP1 upon Bort exposure could be demonstrated, suggesting a possible role for WIP1 in the acquisition of MM drug resistance to proteasome inhibitors. WIP1 was also upregulated in MM cells cultured on human BM stroma (BMSC) known to protect the tumor cells from Bort-induced apoptosis, further supporting its function in mediating resistance.
GSK2830371 (GSK), a novel allosteric inhibitor of WIP1, significantly suppressed MM cells proliferation (p<0.01) and induced apoptosis, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization (ψm), caspase 3 and PARP cleavage, and DNA fragmentation. Moreover, combined treatment with GSK and Bort synergistically potentiated cell death in both Bort-sensitive and resistant MM cells and overcame BMSC protection (CI<0.5). The robust apoptosis induced by Bort/GSK treatment was accompanied by increased mitochondrial ROS accumulation, subsequent mitochondrial destabilization and extensive DNA damage. GSK treatment resulted in a reduction of WIP1 basal expression and abrogated WIP1 induction upon Bort treatment. Thus, we defined that GSK can regulate WIP1 expression in MM cells.
To determine the molecular mechanism of Bort/GSK synergism we performed gene and protein expression analysis. Combination of both agents significantly reduced expression of anti-apoptotic proteins such as cIAP1, cIAP2, XIAP and Survivin. Previous studies indicate that maintaining IAPs expression is part of an adaptive unfolded protein response that promotes MM survival upon Bort-induced endoplasmic reticulum (ER) stress. Therefore, it is conceivable that targeting IAPs upon WIP1 inhibition may overcome protective responses, inducing unresolved ER stress and MM cell death.
Indeed, we found that combination of Bort and GSK significantly enhanced ER stress, as indicated by increase in the pro-apoptotic factors ATF4, CHOP and GADD34. Concomitantly, mitosis-inducing factors Cyclin B1, CDK1 and PLK1 were prominently reduced upon Bort/GSK treatment.
To assess the potential role of p53 activation in GSK-mediated effects, p53-stabilizing agents nutlin3a and PRIMA1 were applied in combination with WIP1 inhibition. We observed a significant (p<0.01) increase in the responsiveness of both p53WT and p53mut MM cells to GSK-mediated apoptosis. Consistently, combined GSK/Bort treatment upregulated p53 targets, including PUMA, NOXA, GADD45A and p21 genes. These data suggest that p53 may potentiate the WIP1 inhibition mediated stress induction.
Finally, we assessed the signaling pathways that may be involved in WIP1 mediated cessation of stress response. GSK profoundly augmented Bort-induced phosphorylation of JNK and c-Jun, without affecting p38 phosphorylation. Accordingly, JNK inhibitor SP600125 successfully reverted both the apoptosis and the downregulation of IAPs induced by Bort/GSK treatment. Altogether, these results identify pro-apoptotic JNK/c-Jun signaling being preferential target of WIP1 in the process of dampening Bort-induced stress response.
To conclude, we disclose the role of WIP1 in blunting stress response and promoting resistance to bortezomib. Collectively, WIP1 suppression prevents MM cell adaptation and recovery upon ER stress. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy.
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Peled:Cellect Biotherapeutics Ltd: Consultancy.
Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Acquired or de novo resistance to current anti-MM ...therapy remains a major treatment obstacle. Novel new therapies are thus in need. Recent data have highlighted the contribution of Ca2+channels in the regulation of cell proliferation, chemo-resistance, migration and invasion. Transient Receptor Potential Vanilloid type-1 (TRPV1) is a calcium-permeable ion channel that has been demonstrated to be expressed in solid tumors. As no data is available evaluating TRPV1 in MM, the aim of the current study was to evaluate its possible role in MM.
Results: Elevated levels of TRPV1 transcript was detected in MM cell lines (n=8) and BM aspirates from MM patients (n=24) in comparison to normal BM (n=5). AMG9810 a specific antagonist of TRPV1, significantly reduced the viability of MM cell lines (n=8) and primary CD138+ cells (n=6),in a time- and dose-dependent manner (p<0.01) and induced apoptosis manifested by phosphatidylserine externalization, loss of mitochondrial membrane potential (ψm), caspase 3 cleavage and DNA fragmentation. AMG9810-triggered apoptosis could be partially blocked by inhibition of calpains and cathepsins, indicating the role of lysosomal rapture in AMG9810-mediated cell death. Indeed, treatment with TRPV1 antagonist induced rapid lysosomal acidification and increased the number of acidic vesicles (detected by acridine orange stain). The acidic vesicles appeared as early as 1 hour post exposure to AMG9810 preceding the mitochondrial destabilization and apoptosis, thus suggesting that TRPV1 blockade induces lysosomal-induced cell death in MM. Furthermore, TRPV1 inhibition with AMG9810 completely suppressed the pro-survival AKT/mTOR pathway and significantly reduced the levels of anti-apoptotic factors BCL-2 and BCL-XL. Combining AMG9810 with the proteasome inhibitor bortezomib (Bort) induced synergistic cell death in both native and Bort-resistant cells (CI<0.4). Moreover, TRPV1 inhibition successfully overcame the CXCR4-mediated protection from Bort provided by BM stromal cells. This finding suggests that the TRPV1 channel may regulate the activity of CXCR4 chemokine receptor in MM cells affecting the MM-microenvironment interactions. In accordance, the TRPV1 antagonist AMG8910 prevented the responsiveness of CXCR4-expressing MM cells to CXCL12 stimulation, decreased the phosphorylation of signaling mediators like Erk1/2 and AKT and suppressed cell migration, while TRPV1 activator capsaicin promoted the CXCR4-mediated signaling and migration. Gene and protein expression analysis were next performed to delineate the molecular mechanisms underlying the observed synergism between Bort and AMG9810. Bort treatment resulted in robust induction of endoplasmic reticulum (ER) stress genes including the increase in pro-apoptotic factors ATF4, CHOP and GADD34. Compensatory unfolded protein response (UPR) was activated as well, with increase in chaperons HSP27, HSP70, HSP90, and lysosomal chaperon LAMP3 known to stabilize lysosome, protecting cells against lysosomal membrane permeabilization (LMP) and subsequent cell death. AMG9810 further increased ER stress, elevating CHOP and GADD34 expression, while significantly reducing both basal and Bort-increased levels of HSP70 and LAMP3, thus overcoming the protective response to Bort treatment and prompting lethal LMP. Finally, combining Bort with AMG9810 resulted in significantly reduced ROS that was correlated with impaired mitochondria and increased MM apoptosis, suggesting that dissipation of intracellular ROS may be involved in AMG9810-promoted cytotoxicity.
Conclusions: Altogether, our data indicate that TRPV1 is implicated in MM cell survival, proliferation, migration, microenvironment interactions and stress response. TRPV1 inhibition by AMG9810 inhibits CXCR4-mediated migration and stromal protection, synergizes with Bort, amplifies ER stress, targets cytoprotective HSP70 and LAMP3, destabilizes lysosome, impairs mitochondria and promotes MM cell death. These results unravel the mechanism mediating the strong synergistic anti-MM activity of Bort in combination with TRPV1 inhibition which may be translated into the clinic.
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Peled:Biokine: Consultancy; Biosight: Consultancy.
Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Despite the initial efficacious treatment, MM ...patients often become refractory to common anti-MM drugs, therefore novel therapies are in need. Pan-histone deacetylase (HDAC) inhibitor panobinostat exerts multiple cytotoxic actions in MM cells in vitro, and was approved for the treatment of relapsed/refractory MM in combination with bortezomib and dexamethasone. Although having promising anti-MM properties, panobinostat lacks therapeutic activity as monotherapy. The aim of the current study was to elucidate the mechanisms underlying MM resistance to panobinostat and to define strategies to overcome it.
Results: Panobinostat at the low concentrations (IC50 5-30 nM) suppressed the viability in MM cell lines (n=7) and primary CD138+ cells from MM patients (n=8) in vitro. Sensitivity to panobinostat correlated with reduced expression of chemokine receptor CXCR4, while overexpression of CXCR4 or its ligand CXCL12 in RPMI8226 and CAG MM cell lines significantly (p<0.001) increased their resistance to panobinostat, pointing to the role of the CXCR4 axis in HDACi response. Notably, similar expression levels of class I HDACs (HDAC1-3) were detected in MM cells with either low or high CXCR4. Interaction with BM stromal cells that represent the source of CXCL12 also protected MM cells from panobinostat-induced apoptosis, further strengthening a role for CXCR4 downstream pathway. Decreased sensitivity to cytotoxic effect was concomitant with reduced histone (H3K9 and H4K8) acetylation in response to panobinostat treatment. In addition, resistance to HDACi was associated with the reversible G0/G1 cell growth arrest, whereas sensitivity was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators involved in CXCR4-mediated HDACi resistance revealed the pro-survival AKT/mTOR pathway to be regulated by both CXCR4 over-expression and interaction with BMSCs. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance and induced synergistic cell death of MM cell lines and primary MM cells, but not of normal mononuclear cells (CI<0.4). This effect was concurrent with the increase in DNA double strand breaks, histone H2AX phosphorylation, loss of Dψm, cytochrome c release, caspase 3 activation and PARP cleavage. The increase in DNA damage upon combinational treatment was not secondary to the apoptotic DNA fragmentation, as it occurred similarly when apoptosis onset was blocked by caspase inhibitor z-VAD-fmk. Kinetics studies also confirmed that panobinostat-induced DNA damage preceded apoptosis induction. Strikingly, combined panobinostat/everolimus treatment resulted in sustained DNA damage and irreversible suppression of MM cell proliferation accompanied by robust apoptosis, in contrast to the modest effects induced by single agent. Gene expression analysis revealed distinct genetic profiles of single versus combined exposures. Whereas panobinostat increased the expression of cell cycle inhibitors GADD45G and p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated DNA repair genes, including RAD21, Ku70, Ku80 and DNA-PKcs. Furthermore, combined treatment markedly decreased both mRNA and protein expression of anti-apoptotic factors survivin and BCL-XL, checkpoint regulator CHK1, and G2/M-specific factors PLK1, CDK1 and cyclin B1, therefore suppressing the DNA damage repair and inhibiting mitotic progression. Given the anti-apoptotic role of p21, the synergistic lethal effect of everolimus could be attributed to its ability to suppress p21 induction by panobinostat ensuing the shift in the DNA damage response toward apoptosis.
Conclusions: Collectively, our findings indicate that CXCR4/CXCL12 activity promotes the resistance of MM cells to HDACi with panobinostat through mTOR activation. Inhibition of mTOR by everolimus synergizes with panobinostat by simultaneous suppression of p21, G2/M mitotic factors and DNA repair machinery, rendering MM cells incapable of repairing accumulated DNA damage and promoting their apoptosis.
Our results unravel the mechanism responsible for strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel therapeutic strategy to eradicate MM.
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No relevant conflicts of interest to declare.
Polyclonal anti-human thymocyte globulins (ATG) have been recently shown to significantly reduce the incidence of graft versus host disease (GVHD) post allogeneic stem cell transplantation (HSCT) ...from both sibling and unrelated donors. Induction of regulatory T cells has been suggested as one of the possible mechanisms. The aim of current study was to further characterize the T cell populations induced by ATG treatment and to delineate the mechanisms involved in ATG-induced tolerance. Phenotypic characterization revealed a significant increase in the expression of FoxP3, GITR, CD95, PD-1 and ICOS as well as the complement inhibitory molecules CD55, CD58 and CD59 on CD4+CD25+ T cells upon ATG treatment. Addition of ATG-treated cells to autologous and allogeneic peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/anti-CD28 antibodies resulted in significant inhibition of proliferation. Moreover, T-cell activation and IFNγ secretion were reduced in the presence of ATG-induced Treg cells. The CD4+CD25+CD127-low Treg fraction sorted from ATG-treated culture demonstrated greater suppressive potency than negative fraction. Conditioned medium produced by ATG-treated but not IgG-treated cells contained TGFβ and suppressed T cell proliferation and activation in a TGFβ receptor-dependent manner. TGFβ receptor kinase inhibitor SB431542 interfered with the suppressive activity of ATG-primed cells, enabling partial rescue of proliferation and IFNγ secretion. Moreover, SB431542 prevented Treg phenotype induction upon ATG treatment. Altogether, our data reveal the role of TGFβ signaling in ATG-mediated immunosuppression and further support the use of ATG, a potent inducer of regulatory T cells, for prevention of GVHD post HSCT and potentially other therapeutic applications.
To analyze retinal structure and function in vitelliform macular dystrophy (VMD) due to mutations in BEST1.
Patients from five Swedish and four Danish families were examined with electrooculography ...(EOG), full-field electroretinography (ffERG), multifocal ERG (mfERG), optical coherence tomography (OCT), and fundus autofluorescence photography (FAF). Genetic analysis of the BEST1 gene was performed by direct sequencing.
Mutations in BEST1 have been reported previously in the Swedish families. In the Danish families, four disease-causing missense mutations were found, one of which is novel: c.936C>A (p.Asp312Glu). The mutation was homozygous in a 9-year-old boy and heterozygous in his father in a consanguineous family. ffERG rod response was reduced in the homozygous boy, but normal in the heterozygous father. EOG was reduced in all but two patients and did not correlate with the ffERG results. OCT ranged from normal to cystoid edema and thickening of the outer retina-choroid complex. Decreased mfERG amplitudes, increased mfERG latencies, and loss of integrity of the foveal photoreceptor inner/outer segment junction, correlated with decreased vision. FAF demonstrated hyperautofluorescence beyond the ophthalmoscopic changes in several patients.
The finding of a homozygous dominant mutation in a patient with VMD and evidence of widespread retinal degeneration may imply that the pathogenesis of the generalized retinal degeneration differs from that of the macular degeneration. A relative agreement between hyperautofluorescence by FAF, reduced retinal function, and VMD implies that the hyperautofluorescence emanates from lipofuscin and A2E. A potential therapy for VMD, involving the inhibition of the retinoid cycle, is suggested.
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