Heparanase is an endo-β-glucuronidase that is best known for its pro-cancerous effects but is als... more Heparanase is an endo-β-glucuronidase that is best known for its pro-cancerous effects but is also implicated in the pathogenesis of various viruses. Activation of heparanase is a common strategy to increase viral spread and trigger the subsequent inflammatory cascade. Using a Single Nucleotide Polymorphisms (SNP)-associated approach we identified enhancer and insulator regions that regulate HPSE expression. Although a role for heparanase in viral infection has been noticed, the impact of HPSE functional SNPs has not been determined. We investigated the effect of cytomegalovirus (CMV) serostatus on the involvement of HPSE enhancer and insulator functional SNPs in the risk of acute graft versus host disease (GVHD) and granulocyte-colony stimulating factor related CD34+ mobilization. A significant correlation between the C alleles of insulator rs4364254 and rs4426765 and CMV seropositivity was found in healthy donors and patients with hematological malignancies. The risk of developing...
Conclusions: Baseline quantitative parameters did not correlate with outcome suggesting that seve... more Conclusions: Baseline quantitative parameters did not correlate with outcome suggesting that several others factors could influence the response to CAR T-cells. The variation of metabolic heterogeneity are strictly associated with response suggesting an important role of early evaluation of disease after CAR T-cells infusion. A larger cohort of patients and a validation group are needed in order to verify these observations.
Post-transplant cyclophosphamide (PTCy) effectively prevents alloreactive responses from unmanipu... more Post-transplant cyclophosphamide (PTCy) effectively prevents alloreactive responses from unmanipulated grafts, but its effect on subsequent immune reconstitution remains largely undetermined. A total of 32 patients (pts) (ages > 18) who underwent allogeneic hematopoietic stem cell transplantation (HCT) mainly for acute leukemia and myelodysplastic syndrome were evaluated. Blood samples were collected at 15, 30, 60, 90, 180 and 270 days post-HCT. Cell phenotype was assessed on peripheral blood mononuclear cells (PBMC) using multiparametric flow cytometry. The proliferative and activation T cell capacity in response to mitogenic stimulation was determined. Pts who received graft versus host disease (GVHD) prophylaxis with PTCy/ATG combination (n=8) were compared with pts who received ATG (Grafalon®, Neovii) alone (n=13), and pts undergoing HCT with no ATG (n=11). Five healthy controls served for the functional assays. First, CD4+ and CD8+ T cell differentiation was evaluated assessing CD62L, CD45RA, and CCR7 expression. A smaller percentage of effector cells was detected in the PTCy/ATG group at days 30, 60 and 90 after HCT, compared to the no-ATG and ATG cohorts. Accordingly, the percentage of central memory and effector memory cells was significantly increased in PTCy/ATG pts at the same time points. Next, immuno-modulatory cell populations were assessed. A transient increase in the percentage of CD4+CD25+CD127- Treg cells was detected in both ATG and PTCy/ATG cohorts in the early post-HCT period, reaching maximal frequency of 21% Treg out of the CD4+ subset (range 14-42%) in the ATG group and 19% (range 12-30%) in the PTCy/ATG group on day 21, suggesting that Treg cells are relatively spared by Cy. However, a distinct expression pattern of checkpoint molecules was revealed in pts receiving PTCy/ATG. The percentage of PD1-positive CD4+ and CD8+ cells was comparable in the no-ATG and ATG cohorts (44% vs 45% and 35% vs 41%, respectively). Yet, PD1 expression was significantly increased on both CD4+ (61%, p<0.05) and CD8+ cells (60%, p<0.02) from pts receiving PTCy/ATG. Similarly, an increased frequency of LAG-3+ CD8+ cells was observed in the PTCy /ATG pts, in comparison to the no-ATG and ATG groups. Concomitantly, HLA-DR expression on CD56+ NK subset was decreased in PTCy/ATG pts versus the ATG and non-ATG groups (47% vs 61% vs 67%, respectively, p<0.05), indicating reduced activation of NK cells. Furthermore, a significant increase in the percentage of CD28-CD127- cells out of CD8+ cells was detected in PTCy/ATG pts compared to the ATG and no-ATG groups on day 60 post-HCT (82% vs 56% vs 38%, respectively, p<0.02). CD57 up-regulation combined with CD28 down-regulation on T cells reflected an exhausted/senescent phenotype; therefore, we further analyzed the frequency of CD28-CD57+ T cells. Significantly elevated expression of CD57 on CD8+CD28- cells was detected in the PTCy/ATG cohort, in comparison to the ATG and no-ATG groups (65% vs 43% vs 21%, respectively, p<0.01). Exhausted/senescent T cells showed functional impairment, which manifested as reduced potential for cytokine production and poor proliferative capacity. Notably, both CD4+ and CD8+ cells from ATG and PTCy/ATG cohorts demonstrated diminished proliferation in response to ex-vivo αCD3/αCD28 stimulation, when compared to the no-ATG cohort and healthy controls (p<0.01). Moreover, significantly reduced production of IFNγ in response to stimulation was detected in PTCy/ATG and ATG pts (p<0.01). PBMC from the no-ATG cohort displayed inducible IFNγ production levels comparable with healthy controls. Finally, PTCy/ATG-treated patients demonstrated altered phenotypic and functional recovery of the CD8+ cells. While CD4+ cells gradually downregulated PD1 expression and up-regulated co-stimulatory molecules CD28 and CD127 on days 180 and 270, notably CD8+ cells retained exhausted/tolerogenic phenotype as late as 270 days post-HCT. Our results demonstrate that the acquisition of senescent/exhausted phenotype by cytotoxic CD8+ cells is a distinctive feature of HCT with ATG conditioning. PTCy further compromises recovery of CD8+ cells and impairs NK activation, resulting in a sustained immunosuppression. Hopefully, these results will not only lead to a better understanding of the tolerance mechanisms behind PTCy/ATG anti GVHD prophylaxis but will also assist in the development of novel clinical treatments. Disclosures No relevant conflicts of interest to declare.
Background: Chemoresistance remains a major treatment obstacle in multiple myeloma (MM). Novel ne... more Background: Chemoresistance remains a major treatment obstacle in multiple myeloma (MM). Novel new therapies are thus in need. Transient Receptor Potential Vanilloid type 1 (TRPV1) is a calcium-permeable ion channel that has been demonstrated to be expressed in solid tumors. Calcium channels have been shown to be involved in the regulation of cell proliferation, chemoresistance, migration and invasion. The aim of the current study was to evaluate its possible role in MM. Methods: Pharmacological inhibitor was used to evaluate the role of TRPV1 in MM cell lines and primary MM cells. Flow cytometry, molecular analysis, fluorescent microscopy, proteomic analysis and xenograft in vivo model of MM with BM involvement were employed to assess the effect of TRPV1 inhibition and decipher its unique mechanism of action in MM. Results: TRPV1 was found to be expressed by MM cell lines and primary MM cells. TRPV1 inhibition using the antagonist AMG9810-induced MM cell apoptosis and synergized with bortezomib, overcoming both CXCR4-dependent stroma-mediated and acquired resistance. In accordance, AMG9810 suppressed the expression and activation of CXCR4 in MM cells. TRPV1 inhibition increased mitochondrial calcium levels with subsequent mitochondrial ROS accumulation and depolarization. These effects were reversed by calcium chelation, suggesting the role of calcium perturbations in oxidative stress and mitochondrial destabilization. Furthermore, AMG9810 abolished bortezomib-induced accumulation of mitochondrial HSP70 and suppressed protective mitochondrial unfolded protein response. Proteomics revealed unique molecular signature related to the modification of ubiquitin signaling pathway. Consequently, 38 proteins related to the ubiquitylation machinery were downregulated upon combined bortezomib/AMG9810
Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents rem... more 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. Disclosures Peled: Cellect Biotherapeutics Ltd: Consultancy.
Graft-versus-Host disease (GVHD) is the most significant treatment-related complication after all... more Graft-versus-Host disease (GVHD) is the most significant treatment-related complication after allogeneic stem cell transplantation (alloSCT). HLA, KIR and cytokine gene polymorphisms were previously reported to be involved in determining the risk of GVHD after alloSCT. The current study was designed to explore the role of polymorphism in the multi-drug resistance 1 (MDR1) gene in predicting GVHD. Single nucleotide polymorphisms at C3435T and G2677T were determined in patients (pts) and donors using RFLP- based assay and correlated with the occurrence of acute and chronic GVHD after alloSCT. The study included 115 pts with various hematological malignancies after alloSCT from related (n=73) or unrelated (n=42) donors. Fifty pts had myeloablative and 65 had reduced-intensity conditioning. C3435T polymorphism included CC, CT and TT genotypes. CC genotype was detected in 36% and 34% of pts and donors, respectively. CT and TT genotypes were detected in 47%/47%, and 17%/19% respectively. G2677T polymorphism included GG, GT and TT genotypes that were detected in 29%/34%, 55%/49%, and 16%/17% of pts and donor pairs, respectively. The incidence of acute and chronic GVHD were found to be increased after alloSCT in pt-donor pairs when the donor had CC in the C3435T site. The cumulative incidence of acute GVHD grade II–IV after alloSCT was 53% (95%CI, 37–74), 40% (28–57) and 42% (25–71) when the donors had CC, CT and TT in the C3435T site, respectively (p=NS), while the cumulative incidence of acute GVHD grade III–IV was 38% (24–62), 18% (9–35) and 18% (6–50), respectively (p=0.02). Multivariable analysis determined donor CC (HR 2.4, p=0.05) and alloSCT from female to male (HR 5.1, p=0.03) as the most significant factors predicting for severe acute GVHD while donor, disease and conditioning types were not significant. Pt C3435T polymorphism and pt or donor G2677T polymorphisms had no correlation with GVHD. Similarly the cumulative incidence of chronic GVHD was 84% (95%CI, 68–100), 53% (38–75) and 38% (39–63) when the donors had CC, CT and TT in the C3435T site, respectively (p=0.02). Multivariable analysis determined donor CC (HR 2.2, p=0.01), alloSCT from female to male (HR 2.5, p=0.03) and alloSCT from unrelated donor (HR 1.8, p=0.05) as the most significant factors predicting for chronic GVHD. The same three factors predicted for extensive chronic GVHD. The CC genotype in C3435T is known to be associated with higher expression level of P-glycoprotein (Pgp). We speculate that increased Pgp may result in lower intracellular levels of cyclosporine in donor T-cells leading to higher incidence of GVHD. Furthermore, Pgp may be involved in transporting Th1 and Th2 cytokines that are critical to the pathogenesis of acute and chronic GVHD, respectively. Further studies will be required to determine the mechanism of the association of C3435T polymorphism, Pgp expression and GVHD. In conclusion, C3435T polymorphism in the MDR1 gene may be an important factor in predicting GVHD and may be considered when selecting the most suitable donor.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Background We assessed the mechanism by which multiple myeloma (MM) shapes the bone marrow (BM) m... more Background We assessed the mechanism by which multiple myeloma (MM) shapes the bone marrow (BM) microenvironment and affects MΦ polarization. Methods In vivo xenograft model of BM-disseminated human myeloma, as well as analysis of MM cell lines, stromal components, and primary samples from patients with MM, was utilized. Results Analysis of the BM from MM-bearing mice inoculated with human CXCR4-expressing RPMI8226 cells revealed a significant increase in M2 MΦ cell numbers (p < 0.01). CXCL13 was one of the most profoundly increased factors upon MM growth with increased levels in the blood of MM-bearing animals. Myeloid cells were the main source of the increased murine CXCL13 detected in MM-infiltrated BM. MM cell lines induced CXCL13 and concurrent expression of M2 markers (MERTK, CD206, CD163) in co-cultured human MΦ in vitro. Interaction with MΦ reciprocally induced CXCL13 expression in MM cell lines. Mechanistically, TGFβ signaling was involved in CXCL13 induction in MM cell...
Despite the high rates of complete remission following chimeric antigen receptor (CAR) T cell the... more Despite the high rates of complete remission following chimeric antigen receptor (CAR) T cell therapy, its full capacity is currently limited by the generation of dysfunctional CAR T cells. Senescent or exhausted CAR T cells possess poor targeting and effector functions, as well as impaired cell proliferation and persistence in vivo. Strategies to detect, prevent or reverse T cell exhaustion are therefore required in order to enhance the effectiveness of CAR T immunotherapy. Here we report that CD19 CAR T cells from non-responding patients with B cell malignancies show enrichment of CD8+ cells with exhausted/senescent phenotype and display a distinct transcriptional signature with dysregulation of genes associated with terminal exhaustion. Furthermore, CAR T cells from non-responding patients exhibit reduced proliferative capacity and decreased IL-2 production in vitro, indicating functional impairment. Overall, our work reveals potential mediators of resistance, paving the way to s...
Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, under... more Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic modalities that bypass these resistance mechanisms. FTY720, also known as fingolimod, is an S1P modulator approved by the FDA to treat the relapsing form of multiple sclerosis. Previously we reported that FTY720 exhibits potent anti-myeloma effect in vitro and in vivo in disseminated xenograft model of MM (Beider et al., Clin Cancer Res 2017). Cytotoxic activity of FTY720 was associated with down-regulation of anti-apoptotic protein MCL-1 while not affecting BCL-2 levels. It is therefore conceivable that BCL-2 inhibition using BH3-mimetic venetoclax may improve responses to FTY720. Incubation of human MM cell lines (n=8) and primary MM cells (n=3) with venetoclax and FTY720 combination synergistically potentiated cell death (CI<0.02), regardless of the MM cells t (11; 14) status. The robust apoptosis induced by venetoclax /FTY720 treatment was acco...
Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents rem... more 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 resi...
Graft-versus-Host disease (GVHD) is the most significant treatment-related complication after all... more Graft-versus-Host disease (GVHD) is the most significant treatment-related complication after allogeneic stem cell transplantation (alloSCT). HLA, KIR and cytokine gene polymorphisms were previously reported to be involved in determining the risk of GVHD after alloSCT. The current study was designed to explore the role of polymorphism in the multi-drug resistance 1 (MDR1) gene in predicting GVHD. Single nucleotide polymorphisms at C3435T and G2677T were determined in patients (pts) and donors using RFLP- based assay and correlated with the occurrence of acute and chronic GVHD after alloSCT. The study included 115 pts with various hematological malignancies after alloSCT from related (n=73) or unrelated (n=42) donors. Fifty pts had myeloablative and 65 had reduced-intensity conditioning. C3435T polymorphism included CC, CT and TT genotypes. CC genotype was detected in 36% and 34% of pts and donors, respectively. CT and TT genotypes were detected in 47%/47%, and 17%/19% respectively. ...
Heparanase is an endo-β-glucuronidase that is best known for its pro-cancerous effects but is als... more Heparanase is an endo-β-glucuronidase that is best known for its pro-cancerous effects but is also implicated in the pathogenesis of various viruses. Activation of heparanase is a common strategy to increase viral spread and trigger the subsequent inflammatory cascade. Using a Single Nucleotide Polymorphisms (SNP)-associated approach we identified enhancer and insulator regions that regulate HPSE expression. Although a role for heparanase in viral infection has been noticed, the impact of HPSE functional SNPs has not been determined. We investigated the effect of cytomegalovirus (CMV) serostatus on the involvement of HPSE enhancer and insulator functional SNPs in the risk of acute graft versus host disease (GVHD) and granulocyte-colony stimulating factor related CD34+ mobilization. A significant correlation between the C alleles of insulator rs4364254 and rs4426765 and CMV seropositivity was found in healthy donors and patients with hematological malignancies. The risk of developing...
Conclusions: Baseline quantitative parameters did not correlate with outcome suggesting that seve... more Conclusions: Baseline quantitative parameters did not correlate with outcome suggesting that several others factors could influence the response to CAR T-cells. The variation of metabolic heterogeneity are strictly associated with response suggesting an important role of early evaluation of disease after CAR T-cells infusion. A larger cohort of patients and a validation group are needed in order to verify these observations.
Post-transplant cyclophosphamide (PTCy) effectively prevents alloreactive responses from unmanipu... more Post-transplant cyclophosphamide (PTCy) effectively prevents alloreactive responses from unmanipulated grafts, but its effect on subsequent immune reconstitution remains largely undetermined. A total of 32 patients (pts) (ages &amp;amp;gt; 18) who underwent allogeneic hematopoietic stem cell transplantation (HCT) mainly for acute leukemia and myelodysplastic syndrome were evaluated. Blood samples were collected at 15, 30, 60, 90, 180 and 270 days post-HCT. Cell phenotype was assessed on peripheral blood mononuclear cells (PBMC) using multiparametric flow cytometry. The proliferative and activation T cell capacity in response to mitogenic stimulation was determined. Pts who received graft versus host disease (GVHD) prophylaxis with PTCy/ATG combination (n=8) were compared with pts who received ATG (Grafalon®, Neovii) alone (n=13), and pts undergoing HCT with no ATG (n=11). Five healthy controls served for the functional assays. First, CD4+ and CD8+ T cell differentiation was evaluated assessing CD62L, CD45RA, and CCR7 expression. A smaller percentage of effector cells was detected in the PTCy/ATG group at days 30, 60 and 90 after HCT, compared to the no-ATG and ATG cohorts. Accordingly, the percentage of central memory and effector memory cells was significantly increased in PTCy/ATG pts at the same time points. Next, immuno-modulatory cell populations were assessed. A transient increase in the percentage of CD4+CD25+CD127- Treg cells was detected in both ATG and PTCy/ATG cohorts in the early post-HCT period, reaching maximal frequency of 21% Treg out of the CD4+ subset (range 14-42%) in the ATG group and 19% (range 12-30%) in the PTCy/ATG group on day 21, suggesting that Treg cells are relatively spared by Cy. However, a distinct expression pattern of checkpoint molecules was revealed in pts receiving PTCy/ATG. The percentage of PD1-positive CD4+ and CD8+ cells was comparable in the no-ATG and ATG cohorts (44% vs 45% and 35% vs 41%, respectively). Yet, PD1 expression was significantly increased on both CD4+ (61%, p&amp;amp;lt;0.05) and CD8+ cells (60%, p&amp;amp;lt;0.02) from pts receiving PTCy/ATG. Similarly, an increased frequency of LAG-3+ CD8+ cells was observed in the PTCy /ATG pts, in comparison to the no-ATG and ATG groups. Concomitantly, HLA-DR expression on CD56+ NK subset was decreased in PTCy/ATG pts versus the ATG and non-ATG groups (47% vs 61% vs 67%, respectively, p&amp;amp;lt;0.05), indicating reduced activation of NK cells. Furthermore, a significant increase in the percentage of CD28-CD127- cells out of CD8+ cells was detected in PTCy/ATG pts compared to the ATG and no-ATG groups on day 60 post-HCT (82% vs 56% vs 38%, respectively, p&amp;amp;lt;0.02). CD57 up-regulation combined with CD28 down-regulation on T cells reflected an exhausted/senescent phenotype; therefore, we further analyzed the frequency of CD28-CD57+ T cells. Significantly elevated expression of CD57 on CD8+CD28- cells was detected in the PTCy/ATG cohort, in comparison to the ATG and no-ATG groups (65% vs 43% vs 21%, respectively, p&amp;amp;lt;0.01). Exhausted/senescent T cells showed functional impairment, which manifested as reduced potential for cytokine production and poor proliferative capacity. Notably, both CD4+ and CD8+ cells from ATG and PTCy/ATG cohorts demonstrated diminished proliferation in response to ex-vivo αCD3/αCD28 stimulation, when compared to the no-ATG cohort and healthy controls (p&amp;amp;lt;0.01). Moreover, significantly reduced production of IFNγ in response to stimulation was detected in PTCy/ATG and ATG pts (p&amp;amp;lt;0.01). PBMC from the no-ATG cohort displayed inducible IFNγ production levels comparable with healthy controls. Finally, PTCy/ATG-treated patients demonstrated altered phenotypic and functional recovery of the CD8+ cells. While CD4+ cells gradually downregulated PD1 expression and up-regulated co-stimulatory molecules CD28 and CD127 on days 180 and 270, notably CD8+ cells retained exhausted/tolerogenic phenotype as late as 270 days post-HCT. Our results demonstrate that the acquisition of senescent/exhausted phenotype by cytotoxic CD8+ cells is a distinctive feature of HCT with ATG conditioning. PTCy further compromises recovery of CD8+ cells and impairs NK activation, resulting in a sustained immunosuppression. Hopefully, these results will not only lead to a better understanding of the tolerance mechanisms behind PTCy/ATG anti GVHD prophylaxis but will also assist in the development of novel clinical treatments. Disclosures No relevant conflicts of interest to declare.
Background: Chemoresistance remains a major treatment obstacle in multiple myeloma (MM). Novel ne... more Background: Chemoresistance remains a major treatment obstacle in multiple myeloma (MM). Novel new therapies are thus in need. Transient Receptor Potential Vanilloid type 1 (TRPV1) is a calcium-permeable ion channel that has been demonstrated to be expressed in solid tumors. Calcium channels have been shown to be involved in the regulation of cell proliferation, chemoresistance, migration and invasion. The aim of the current study was to evaluate its possible role in MM. Methods: Pharmacological inhibitor was used to evaluate the role of TRPV1 in MM cell lines and primary MM cells. Flow cytometry, molecular analysis, fluorescent microscopy, proteomic analysis and xenograft in vivo model of MM with BM involvement were employed to assess the effect of TRPV1 inhibition and decipher its unique mechanism of action in MM. Results: TRPV1 was found to be expressed by MM cell lines and primary MM cells. TRPV1 inhibition using the antagonist AMG9810-induced MM cell apoptosis and synergized with bortezomib, overcoming both CXCR4-dependent stroma-mediated and acquired resistance. In accordance, AMG9810 suppressed the expression and activation of CXCR4 in MM cells. TRPV1 inhibition increased mitochondrial calcium levels with subsequent mitochondrial ROS accumulation and depolarization. These effects were reversed by calcium chelation, suggesting the role of calcium perturbations in oxidative stress and mitochondrial destabilization. Furthermore, AMG9810 abolished bortezomib-induced accumulation of mitochondrial HSP70 and suppressed protective mitochondrial unfolded protein response. Proteomics revealed unique molecular signature related to the modification of ubiquitin signaling pathway. Consequently, 38 proteins related to the ubiquitylation machinery were downregulated upon combined bortezomib/AMG9810
Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents rem... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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. Disclosures Peled: Cellect Biotherapeutics Ltd: Consultancy.
Graft-versus-Host disease (GVHD) is the most significant treatment-related complication after all... more Graft-versus-Host disease (GVHD) is the most significant treatment-related complication after allogeneic stem cell transplantation (alloSCT). HLA, KIR and cytokine gene polymorphisms were previously reported to be involved in determining the risk of GVHD after alloSCT. The current study was designed to explore the role of polymorphism in the multi-drug resistance 1 (MDR1) gene in predicting GVHD. Single nucleotide polymorphisms at C3435T and G2677T were determined in patients (pts) and donors using RFLP- based assay and correlated with the occurrence of acute and chronic GVHD after alloSCT. The study included 115 pts with various hematological malignancies after alloSCT from related (n=73) or unrelated (n=42) donors. Fifty pts had myeloablative and 65 had reduced-intensity conditioning. C3435T polymorphism included CC, CT and TT genotypes. CC genotype was detected in 36% and 34% of pts and donors, respectively. CT and TT genotypes were detected in 47%/47%, and 17%/19% respectively. G2677T polymorphism included GG, GT and TT genotypes that were detected in 29%/34%, 55%/49%, and 16%/17% of pts and donor pairs, respectively. The incidence of acute and chronic GVHD were found to be increased after alloSCT in pt-donor pairs when the donor had CC in the C3435T site. The cumulative incidence of acute GVHD grade II–IV after alloSCT was 53% (95%CI, 37–74), 40% (28–57) and 42% (25–71) when the donors had CC, CT and TT in the C3435T site, respectively (p=NS), while the cumulative incidence of acute GVHD grade III–IV was 38% (24–62), 18% (9–35) and 18% (6–50), respectively (p=0.02). Multivariable analysis determined donor CC (HR 2.4, p=0.05) and alloSCT from female to male (HR 5.1, p=0.03) as the most significant factors predicting for severe acute GVHD while donor, disease and conditioning types were not significant. Pt C3435T polymorphism and pt or donor G2677T polymorphisms had no correlation with GVHD. Similarly the cumulative incidence of chronic GVHD was 84% (95%CI, 68–100), 53% (38–75) and 38% (39–63) when the donors had CC, CT and TT in the C3435T site, respectively (p=0.02). Multivariable analysis determined donor CC (HR 2.2, p=0.01), alloSCT from female to male (HR 2.5, p=0.03) and alloSCT from unrelated donor (HR 1.8, p=0.05) as the most significant factors predicting for chronic GVHD. The same three factors predicted for extensive chronic GVHD. The CC genotype in C3435T is known to be associated with higher expression level of P-glycoprotein (Pgp). We speculate that increased Pgp may result in lower intracellular levels of cyclosporine in donor T-cells leading to higher incidence of GVHD. Furthermore, Pgp may be involved in transporting Th1 and Th2 cytokines that are critical to the pathogenesis of acute and chronic GVHD, respectively. Further studies will be required to determine the mechanism of the association of C3435T polymorphism, Pgp expression and GVHD. In conclusion, C3435T polymorphism in the MDR1 gene may be an important factor in predicting GVHD and may be considered when selecting the most suitable donor.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Background We assessed the mechanism by which multiple myeloma (MM) shapes the bone marrow (BM) m... more Background We assessed the mechanism by which multiple myeloma (MM) shapes the bone marrow (BM) microenvironment and affects MΦ polarization. Methods In vivo xenograft model of BM-disseminated human myeloma, as well as analysis of MM cell lines, stromal components, and primary samples from patients with MM, was utilized. Results Analysis of the BM from MM-bearing mice inoculated with human CXCR4-expressing RPMI8226 cells revealed a significant increase in M2 MΦ cell numbers (p < 0.01). CXCL13 was one of the most profoundly increased factors upon MM growth with increased levels in the blood of MM-bearing animals. Myeloid cells were the main source of the increased murine CXCL13 detected in MM-infiltrated BM. MM cell lines induced CXCL13 and concurrent expression of M2 markers (MERTK, CD206, CD163) in co-cultured human MΦ in vitro. Interaction with MΦ reciprocally induced CXCL13 expression in MM cell lines. Mechanistically, TGFβ signaling was involved in CXCL13 induction in MM cell...
Despite the high rates of complete remission following chimeric antigen receptor (CAR) T cell the... more Despite the high rates of complete remission following chimeric antigen receptor (CAR) T cell therapy, its full capacity is currently limited by the generation of dysfunctional CAR T cells. Senescent or exhausted CAR T cells possess poor targeting and effector functions, as well as impaired cell proliferation and persistence in vivo. Strategies to detect, prevent or reverse T cell exhaustion are therefore required in order to enhance the effectiveness of CAR T immunotherapy. Here we report that CD19 CAR T cells from non-responding patients with B cell malignancies show enrichment of CD8+ cells with exhausted/senescent phenotype and display a distinct transcriptional signature with dysregulation of genes associated with terminal exhaustion. Furthermore, CAR T cells from non-responding patients exhibit reduced proliferative capacity and decreased IL-2 production in vitro, indicating functional impairment. Overall, our work reveals potential mediators of resistance, paving the way to s...
Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, under... more Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic modalities that bypass these resistance mechanisms. FTY720, also known as fingolimod, is an S1P modulator approved by the FDA to treat the relapsing form of multiple sclerosis. Previously we reported that FTY720 exhibits potent anti-myeloma effect in vitro and in vivo in disseminated xenograft model of MM (Beider et al., Clin Cancer Res 2017). Cytotoxic activity of FTY720 was associated with down-regulation of anti-apoptotic protein MCL-1 while not affecting BCL-2 levels. It is therefore conceivable that BCL-2 inhibition using BH3-mimetic venetoclax may improve responses to FTY720. Incubation of human MM cell lines (n=8) and primary MM cells (n=3) with venetoclax and FTY720 combination synergistically potentiated cell death (CI<0.02), regardless of the MM cells t (11; 14) status. The robust apoptosis induced by venetoclax /FTY720 treatment was acco...
Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents rem... more 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 resi...
Graft-versus-Host disease (GVHD) is the most significant treatment-related complication after all... more Graft-versus-Host disease (GVHD) is the most significant treatment-related complication after allogeneic stem cell transplantation (alloSCT). HLA, KIR and cytokine gene polymorphisms were previously reported to be involved in determining the risk of GVHD after alloSCT. The current study was designed to explore the role of polymorphism in the multi-drug resistance 1 (MDR1) gene in predicting GVHD. Single nucleotide polymorphisms at C3435T and G2677T were determined in patients (pts) and donors using RFLP- based assay and correlated with the occurrence of acute and chronic GVHD after alloSCT. The study included 115 pts with various hematological malignancies after alloSCT from related (n=73) or unrelated (n=42) donors. Fifty pts had myeloablative and 65 had reduced-intensity conditioning. C3435T polymorphism included CC, CT and TT genotypes. CC genotype was detected in 36% and 34% of pts and donors, respectively. CT and TT genotypes were detected in 47%/47%, and 17%/19% respectively. ...
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