LMO2 Facilitates Synthetic Lethality after PARP Inhibition (PARPi) in Diffuse Large B-Cell Lymphoma (DLBCL)

Abstract Novel therapies for DLBCL are needed to improve patients' outcomes. LIM domain only 2 (LMO2) protein is ubiquitously expressed and plays important roles in endothelial and hematopoietic cell development. LMO2 protein expression is upregulated in germinal center B (GCB) cells, the cell of origin of DLBCL. 73% of GCB and 45% of ABC DLBCLs express LMO2 protein at levels of reactive GCB cells. Although the function of LMO2 in B cells and DLBCL is unknown, expression of LMO2 serves as one of the best prognostic markers of longer survival following R-CHOP therapy. Additionally, LMO2 expression in DLBCL cells results in genomic instability. These observations suggest that LMO2 may decrease DNA repair efficiency. Indeed, here we demonstrate that primary DLBCL tumors and cell lines expressing high levels of LMO2 protein (LMO2HIGH) are defective in DNA double-strand break (DSB) repair via the homologous recombination (HR) pathway. We found that LMO2HIGH DLBCL cells and LMO2LOW DLBCL cells expressing a GFP-LMO2 fusion protein via a doxycycline-inducible system have a defective accumulation of the HR proteins BRCA1 and RAD51 to damaged chromosomes as visualized by immunofluorescence (IF) and Western blot assays. Furthermore, LMO2HIGH DLBCL exposed to ionizing radiation showed decreased levels of phosphorylated (S4 and S8) replication protein A (RPA32 subunit), a marker of DSB end-resection activity and an essential step for HR-dependent DSB repair. Consequently, LMO2HIGH DLBCL showed decreased HR activity as assessed via a DR-GFP reporter system and scoring the frequency of HR-dependent sister chromatid exchanges. Also, in LMO2HIGH vs LMO2LOW DLBCLs, we observed higher levels of ionizing radiation-induced foci (IRIF) for 53BP1 and RIF1 - non-homologous end-joining (NHEJ) core factors playing a critical role in defining DSB repair pathway choice. Similarly, we found a higher accumulation of 53BP1 and RIF1 chromatin-enriched fraction after DNA damage in LMO2HIGH than LMO2LOW DLBCLs. Furthermore, we show that LMO2 forms a complex with 53BP1 but not BRCA1 after DNA damage, as demonstrated by Co-IP, GST pull-down assays and spatial co-localization in IRIFs. This suggests that LMO2 functionally interacts with 53BP1 during DSB repair. Indeed, knockdown of 53BP1 in LMO2HIGH cells increased the levels of BRCA1 and RAD51 IRIF to values similar to LMO2LOW cells without affecting LMO2 levels, revealing that LMO2 depends on 53BP1 to inhibit HR activity. Since LMO2HIGH DLBCL cells exhibit a defective HR-pathway, we next explored the therapeutic potential of PARPi in DLBCL. We found that in LMO2HIGH but not LMO2LOW DLBCL cell lines the PARPi olaparib induced a significant decrease in cell proliferation and colony formation and an increase in cell death via apoptosis. The sensitivity to olaparib directly correlated with LMO2 protein levels. The proliferation defect in LMO2HIGH DLBCL cells was due to the increased DNA damage caused by exposure to PARPi, as observed by an increase in γH2AX foci. Induction of LMO2 expression in LMO2LOW DLBCL cell lines led to sensitivity to olaparib, demonstrating that the proliferation defect induced by olaparib was dependent on LMO2 expression. Silencing of LMO2 via shRNA or CRISP/Cas9 in LMO2HIGH cell lines rescued the proliferation defect induced by olaparib. The proliferation deficiency induced by olaparib was synergistic with doxorubicin in LMO2HIGH but not in LMO2LOW DLBCL cell lines and untreated patient-derived primary DLBCL tumors. We also examined the in vivo efficacy of olaparib in DLBCL mice models. Olaparib treatment prolonged survival of mice harboring LMO2HIGH but not LMO2LOW DLBCL xenograft tumors. Olaparib in combination with RCHOP significantly prolonged survival of mice harboring LMO2HIGH DLBCL tumors compared to cohorts treated with either olaparib or RCHOP alone. Further, analysis of tumors excised from OCI-LY1 bearing animals treated with olaparib for 3 days revealed increased cell death and a higher sensitivity to PARPi in OCI-LY1 LMO2 cells compared to the OCI-LY1 LMO2 negative-GFP control cells, indicating in vivo survival advantage for LMO2LOW cells upon olaparib treatment. In summary, high expression of LMO2 results in HR-dysfunction phenocopying the BRCA1/2 mutations observed in breast and ovarian tumors. In LMO2HIGH DLBCL, PARPi-induced killing is synergistic with doxorubicin, thus providing a clear path for therapeutic development of PARPi in DLBCL. Figure. Figure. Disclosures Lossos: Affimed: Research Funding.

Download Full-text

The xCT Antiporter Is a Therapeutic Target in the ABC Subtype of DLBCL

Blood ◽

10.1182/blood.v126.23.3996.3996 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3996-3996

Author(s):

Xiaolei Wei ◽

Yun Mai ◽

Ru Feng ◽

B. Hilda Ye

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

B Cell ◽

Cell Lines ◽

Critical Role ◽

B Cell Lymphoma ◽

Dependent Manner ◽

Ros Accumulation

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in the adult population and can be subdivided into two main subtypes, i.e. GCB-DLBCL and ABC-DLBCL. While both subtypes are derived from normal germinal center (GC) B cells, they differ in B cell maturation stage, transformation pathway, and clinical behavior. When treated with either the combination chemotherapy CHOP or the immuno-chemotherapy R-CHOP, the survival outcome of ABC-DLBCL patients is typically much worse than that of GCB-DLBCL patients. Although the molecular mechanisms underlying this survival disparity remain poorly understood, an attractive hypothesis is that there exist subtype-specific resistance mechanisms directed against the chemo-therapy drugs in the original CHOP formulation. In support of this notion, our previous study has revealed that Doxorubicin (Dox), the main cytotoxic ingredient in CHOP, has subtype-specific mechanisms of cytotoxicity in DLBCLs due to differences in its subcellular distribution pattern. In particular, Dox-induced cytotoxicity in ABC-DLBCLs is largely dependent on oxidative stress rather than DNA damage response. Based on these findings, we hypothesize that agents capable of disturbing the redox balance in ABC-DLBCL cells could potentiate the therapeutic activity of first line lymphoma therapy. As the major route of cystine uptake from extracellular space, the xCT cystine/glutamate antiporter controls the rate-limiting step for glutathione (GSH) synthesis in several types of cancer cells, including CLL. We focused the current study on xCT because its protein stability is known to be positively regulated by a splicing variant of CD44 and we have recently published that expression of CD44 and CD44V6 are poor prognosticators for DLBCL. Indeed, we found that surface CD44 is exclusively expressed in ABC-DLBCL (6/6) but not GCB-DLBCL (0/5) cell lines. In addition, the xCT proteins in two ABC-DLBCL cell lines, Riva and SuDHL2, are extraordinarily stable, with half-lives exceeding 24 hours. As such, transient transfection using siRNA oligos was ineffective in reducing the endogenous xCT protein in ABC-DLBCL cell lines. To circumvent this issue, we turned to a clinically approved anti-inflammatory drug, sulfasalazine (SASP), which is a validated xCT inhibitor in its intact form. When Riva and SuDHL2 cells were treated overnight with the IC50 dose of SASP, the endogenous GSH pool was drastically reduced, leading to significant increase in intracellular ROS, p38 and JNK activation, and progressive apoptosis. Unexpectedly, we found that Dox-treated cells had significantly elevated GSH levels, possibly the result of an antioxidant response to Dox-triggered ROS accumulation. This increase in GSH was completely suppressed when the IC25 dosage of SASP was included in the Dox treatment. As expected, SASP/Dox combination significantly enhanced Dox-triggered ROS accumulation and synergistically promoted cell death in Riva and SuDHL2 cells. Mechanistically, p38 activation and cell death induced by SASP/Dox combination could be markedly attenuated by pretreatment with glutathione monoethyl ester, demonstrating the critical role of oxidative stress. Furthermore, cytotoxicity triggered by SASP/Dox could also be suppressed by the p38 inhibitor, SB203580. We have developed stable cell lines expressing xCT shRNA to confirm the results obtained with SASP. In vivo interactions between SASP and Dox are also being evaluated in xenograft-based ABC-DLBCL models. In summary, we report here for the first time a critical role of xCT in sustaining in vivo GSH production in ABC-DLBCL cells. More importantly, pharmacologic inhibition of xCT function in ABC-DLBCL cells not only prevented Dox-induced endogenous GSH increase, but also potentiated Dox-induced ROS accumulation and cytotoxicity in a p38-dependent manner. With additional evidence from ongoing experiments, our study aims to provide a mechanistic basis for development of novel therapies that target either xCT or redox homeostasis to improve treatment outcomes for ABC-DLBCLs. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Azacitidine/Decitabine Synergism with the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML Models

Blood ◽

10.1182/blood.v118.21.578.578 ◽

2011 ◽

Vol 118 (21) ◽

pp. 578-578 ◽

Cited By ~ 11

Author(s):

Peter G Smith ◽

Tary Traore ◽

Steve Grossman ◽

Usha Narayanan ◽

Jennifer S Carew ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Death ◽

Synthetic Lethality ◽

Single Agent ◽

Xenograft Model ◽

S Phase ◽

Myelogenous Leukemia ◽

Phase Accumulation

Abstract Abstract 578 MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). To identify potential combination partners of MLN4924 we performed a high-throughput viability screen in AML cells with 40 approved and investigational agents. In vitro characterization of AML cell lines revealed two distinct cell cycle phenotypes suggesting alternate mechanism of action following MLN4924 inhibition of NAE. One group demonstrated moderate S-phase accumulation with greater than 4N DNA content consistent with DNA-rereplication as a result of CDT1 dysregulation. The second group demonstrated distinct and rapid accumulation of subG1 cells without S-phase accumulation or DNA re-replication suggesting induction of apoptosis and cell death. These observations led us to choose two cells lines representative of each mechanism to understand potential for synergy in AML cells. Two hypomethylating agents were included in the screen (decitabine and azacitidine) and were found to be synergistic with MLN4924 by Combination Index and Blending Synergy Analysis. These data were confirmed with a second NAE inhibitor that is structurally dissimilar to MLN4924. The combination of azacitidine and MLN4924 were shown to result in significantly increased DNA-damage and cell death compared to single agent alone as measured by Western Blotting and FACS analysis of cell cycle distributions. In vivo studies were performed in HL-60 and THP-1 xenografts using MLN4924 on a clinically relevant dosing schedule twice weekly. Single agent azacitidine at its Maximum Tolerated Dose (MTD) had minimal activity in the HL-60 model and was combined with a sub-optimal dose of MLN4924 that when combined induced complete and sustained tumor regressions. The mechanism for the apparent synthetic lethality in this in vivo model is currently under evaluation; however it is supported by a dramatic elevation in DNA damage and cleaved caspase-3 in vivo in the combination arm. A second xenograft model (THP-1) that was also insensitive to single agent azacitidine treatment underwent complete and sustained tumor regressions when combined with MLN4924. Thus MLN4924 and azacitidine can combine to produce synergistic antitumor activity in pre-clinical models of AML. Coupled with their non-overlapping clinical toxicities these data suggest the potential for future combination studies in clinical trials. Disclosures: Smith: Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Grossman:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Carew:Millennium Pharmaceuticals: Research Funding. Lublinksky:Millennium Pharmaceuticals: Employment. Kuranda:Millennium Pharmaceuticals: Employment. Milhollen:Millennium Pharmaceuticals: Employment.

Download Full-text

The Novel HDAC Inhibitor Chidamide Synergizes with Rituximab to Inhibit DLBCL Tumor Growth in Vitro and In Vivo By up-Regulating CD20 Expression

Blood ◽

10.1182/blood-2018-99-114598 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2947-2947

Author(s):

Xu-Wen Guan ◽

Wang Hua-Qing ◽

Li Jia ◽

Feng-Ting Liu

Keyword(s):

Cell Death ◽

Tumor Growth ◽

Cell Lines ◽

Cell Lymphoma ◽

Combined Treatment ◽

B Cell Lymphoma ◽

Surface Expression ◽

Cd20 Expression

Abstract Background: Histone deacetylases (HDACs) are crucial proteins for supporting tumorigenesis. HDACs reverse chromatin acetylation and alter transcription of oncogenes and tumor suppressor genes by removing acetyl groups from histones. HDAC inhibitors are considered as promising anti-cancer drugs, particularly in combination with other standard treatment regimens. Chidamide is the world first oral HDAC inhibitor which selectively inhibits class I HDAC1, HDAC2, and HDAC3 as well as class IIb HDAC10. Chidamide has been approved by China FDA in 2015 for the treatment of relapsed or refractory peripheral T-cell lymphoma. Diffuse large B-cell lymphoma (DLBCL) is the most aggressive form of B-cell lymphoma. Treatment with R-CHOP i.e. Rituximab (the anti-CD20 monoclonal antibody) plus CHOP (Cyclophosphamide, doxorubicin, vincristine, and prednisone) has significantly improved clinical outcome for DLBCL patients. However, treatment-induced deacetylation of CD20 gene and consequently down-regulation of CD20 protein expression causes an acquired resistance to further treatment with R-CHOP. We hypothesize that inhibition of HDACs by Chidamide could overcome Rituximab-mediated down-regulation of CD20 and facilitate Rituximab-induced DLBCL tumor growth inhibition. The aim of this study is to determine the synergistic effect of Chidamide and Rituximab in the treatment of DLBCL in vitro and in vivo. Methods: The levels of CD20 (MS4A1) mRNA expression and clinical outcomes in patients with DLBCL treated either with R-CHOP or CHOP were obtained from the Gene Expression Omnibus (GEO) repository (NCBI GSE 10846). The association of CD20 expression with overall survival (OS) was analyzed by Cox regression analysis and the cut-off point was calculated by the X-tile software. CD20 protein surface expression and Rituximab-induced cell death were analyzed by flow cytometry. The IC50s of Chidamide and the synergisms with Rituximab (10 µg/ml) on five DLBCB cell lines (OCI-LY3, OCI-LY7, Su-DHL6, Su-DHL8, and Su-DLH10) were determined by MTT test after cells were treated with a range of concentrations of Chidamide with or without Rituximab for 24 hours. The synergism was calculated using ComboSyn software to obtain the combination index (CI). For in vivo experiments, the human DLBCL cell line OCI-LY7 were injected to 6 weeks BALB/C nude mice to develop xenograft DLBCL mice models. After tumors were palpable, mice were divided into four groups and injected with NaCl (control), Rituximab, Chidamide and Rituximab plus Chidamide daily for three weeks. The tumor volumes were monitored frequently during the treatment. Results: In R-CHOP treated cohort (n=233), higher expression of CD20 expression (n=137) is significantly associated with superior clinical outcomes compared with lower CD20 expression (n=96) with P=0.0038, HR=0.4753, 95% CI=0.274-0.779. However, the levels of CD20 have no effect on clinical outcome in DLBCL patients treated with CHOP (n=183). The levels of CD20 protein surface expression on five DLBCL cell lines were significantly and positively correlated with the sensitivities of cells to Rituximab-induced cell death (P=0.0018, R=0.88). HDAC1, HDCA2 and HDCA3 proteins were detected in these DLBCL cell lines. Treatment with Rituximab significantly reduced CD20 surface expression but treatment with Chidamide significantly increased CD20 surface expression in DLBCL cells. The CI numbers for combined treatment with Chidamide and Rituximab were either <0.01 (very strong synergism) or <0.3 (strong synergism), indicating that Chidamide significantly synergized Rituximab-induced cell death. For in vivo assay, treatment with either Rituximab or Chidamide alone slightly but not significantly reduced tumor volume. Combination with Chidamide and Rituximab significantly inhibited tumor growth in DLBCL xenograft mice (P<0.0001). Mice with combined treatment showed significantly prolonged survival compared with other groups. Conclusions: our data demonstrate for the first time that inhibition of HDACs by Chidamide significantly synergized Rituximab-induced tumor growth inhibition in vitro and in vivo. We propose that CD20 surface expression should be used clinically to evaluate treatment response in patients with DLBCL. Chidamide is a promising sensitizer for the treatment of DLBCL with R-CHOP. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine

Blood ◽

10.1182/blood.v122.21.377.377 ◽

2013 ◽

Vol 122 (21) ◽

pp. 377-377 ◽

Cited By ~ 3

Author(s):

Shruti Bhatt ◽

Daxing Zhu ◽

Xiaoyu Jiang ◽

Seung-uon Shin ◽

John M Timmerman ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cd20 Antibody ◽

Anti Cd20

Abstract The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome. Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013). Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components. Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting. These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures: Rosenblatt: Seattle Genetics, Inc.: Research Funding.

Download Full-text

PRL2 serves as a negative regulator in cell adaptation to oxidative stress

Cell & Bioscience ◽

10.1186/s13578-019-0358-z ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Xinyue Du ◽

Yang Zhang ◽

Xiao Li ◽

Qi Li ◽

Chenyun Wu ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Ionizing Radiation ◽

Bactericidal Activity ◽

Critical Role ◽

Negative Regulator ◽

Effector Protein ◽

Cell Adaptation ◽

In Vivo Experiments

AbstractHigh levels of ROS cause oxidative stress, which plays a critical role in cell death. As a ROS effector protein, PRL2 senses ROS and controls phagocyte bactericidal activity during infection. Here we report PRL2 regulates oxidative stress induced cell death. PRL2 senses oxidative stress via highly reactive cysteine residues at 46 and 101. The oxidation of PRL2 causes protein degradation and supports pro-survival PDK1/AKT signal which in turn to protect cells against oxidative stress. As a result, PRL2 levels have a high correlation with oxidative stress induced cell death. In vivo experiments showed PRL2 deficient cells survive better in inflammatory oxidative environment and resist to ionizing radiation. Our finding suggests PRL2 serves as a negative regulator in cell adaptation to oxidative stress. Therefore, PRL2 could be targeted to modulate cell viability in inflammation or irradiation associated therapy.

Download Full-text

Antineoplastic Effect of a Combined Mitotane Treatment/Ionizing Radiation in Adrenocortical Carcinoma: A Preclinical Study

Cancers ◽

10.3390/cancers11111768 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1768

Author(s):

Cerquetti ◽

Bucci ◽

Carpinelli ◽

Lardo ◽

Proietti ◽

...

Keyword(s):

Dna Damage ◽

Cell Death ◽

Ionizing Radiation ◽

Tumor Growth ◽

Growth Inhibition ◽

Cell Lines ◽

Adrenocortical Carcinoma ◽

Repair System ◽

Msh2 Gene ◽

Sw13 Cell

Mitotane (MTT) is an adrenolytic drug used in adjuvant and advanced treatments of adrenocortical carcinoma (ACC). Ionizing radiation (IR) is also used in adrenal cancer treatment, even though its biological action remains unknown. To provide a reliable in vivo preclinical model of ACC, we used mouse xenografts bearing human ACC to test the effects of MTT and IR alone and in combination. We evaluated tumor growth inhibition by the RECIST criteria and analyzed the cell cycle by flow cytometry (FCM). In the xenograft ACC model treated with MTT/IR in combination, we observed a marked inhibition of tumor growth, with strong tumor regression (p < 0.0001) compared to MTT and IR given alone (p < 0.05). The MTT results confirm its antisteroidogenic activity (p < 0.05) in the xenograft ACC model, revealing its ability to render cancer cells more prone to radiotherapy treatment. In addition, to explain the biological effect of these treatments on the Mismatch Repair System (MMR), we interfered with the MSH2 gene expression in untreated and MTT/IR-treated H295R and SW13 cell lines. Moreover, we observed that upon treatment with MTT/IR to induce DNA damage, MSH2 gene inhibition in both the H295R and SW13 cell lines did not allow DNA damage repair, thus inducing cell death. In conclusion, MTT seems to have a radiosensitizing property and, when given in combination with IR, is able to promote neoplastic growth inhibition, leading to a significant reduction in tumor size due to cell death.

Download Full-text

Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis

Cancers ◽

10.3390/cancers13143579 ◽

2021 ◽

Vol 13 (14) ◽

pp. 3579

Author(s):

Mimoune Berehab ◽

Redouane Rouas ◽

Haidar Akl ◽

Hugues Duvillier ◽

Fabrice Journe ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Apoptotic Cell ◽

Critical Role ◽

B Cell Lymphoma ◽

Cytosolic Calcium ◽

Store Operated Calcium Entry ◽

Safe Mechanism ◽

Apoptotic Pathways

Targeting non-apoptotic modalities might be therapeutically promising in diffuse large B cell lymphoma (DLBCL) patients with compromised apoptotic pathways. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells, but little is known about its effect on non-apoptotic pathways. This work investigates TQ selectivity against DLBCL cell lines and the cell death mechanisms. TQ reduces cell viability and kills cell lines with minimal toxicity on normal hematological cells. Mechanistically, TQ promotes the mitochondrial caspase pathway and increases genotoxicity. However, insensitivity of most cell lines to caspase inhibition by z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) pointed to a critical role of non-apoptotic signaling. In cells dying through non-apoptotic death, TQ increases endoplasmic reticulum (ER) stress markers and substantially increases cytosolic calcium ([Ca2+]c) through ER calcium depletion and activation of store-operated calcium entry (SOCE). Chelation of [Ca2+]c, but not SOCE inhibitors, reduces TQ-induced non-apoptotic cell death, highlighting the critical role of calcium in a non-apoptotic effect of TQ. Investigations showed that TQ-induced [Ca2+]c signaling is primarily initiated by necroptosis upstream to SOCE, and inhibition necroptosis by necrostatin-1 alone or with z-VAD-fmk blocks the cell death. Finally, TQ exhibits an improved selectivity profile over standard chemotherapy agents, suggesting a therapeutic relevance of the pro-necroptotic effect of TQ as a fail-safe mechanism for DLBCL therapies targeting apoptosis.

Download Full-text

Bio-Inspired Functional Lipoprotein-like Nanoparticles (Flip-NPs) Cause Cholesterol Starvation and Ferroptosis in B-Cell Lymphomas: Studies in Cell Lines, Xenograft Models and Primary Patient Samples

Blood ◽

10.1182/blood-2019-127805 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2857-2857

Author(s):

Jonathan Rink ◽

Adam Yuh Lin ◽

Shuo Yang ◽

Amir Behdad ◽

Reem Karmali ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cholesterol Reduction ◽

Xenograft Models

Introduction: Hematologic malignancies, including B cell lymphomas such as diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), have increased demands for cholesterol and cholesteryl esters to maintain membrane anchored pro-proliferative and pro-survival signaling pathways, including B cell receptor signaling. Recent evidence suggests that certain cancer cell lines, including several anaplastic large T cell lymphoma (ALCL) cell lines, are auxotrophic for cholesterol and are sensitive to cholesterol reduction-induced ferroptosis (Garcia-Bermudez, Nature 2019), an iron dependent form of programmed cell death characterized by accumulation of lipid peroxides. We have developed a cholesterol depleting functional lipoprotein-like nanoparticle (Flip-NP) that specifically targets the high-affinity HDL receptor, scavenger receptor type B1 (SCARB1), which maintains cellular and cell membrane cholesterol homeostasis. Our prior data demonstrated that Flip-NPs induce B cell lymphoma cell death in vitro and in in vivo xenograft models. Accordingly, we hypothesized that the mechanism of cell death by Flip-NPs in B cell lymphomas is ferroptosis, and that Flip-NPs would be potent therapy for an expanded number of cholesterol-addicted malignancies, including ALCL. Methods: After informed consent, primary B cell lymphoma cells were isolated from excisional biopsies from patients with FL or DLBCL. The SUDHL4 [germinal center (GC) DLBCL], Ramos [Burkitt's lymphoma], SUDHL1 [ALCL] and SR-786 [ALCL] cell lines were used for in vitro experiments. SCARB1 expression was quantified using flow cytometry and western blot analysis. Cell viability was quantified using the MTS assay and flow cytometry. Ferroptosis was measured using the lipophilic antioxidant ferrostatin-1 or the iron chelator deferoxamine. Gene expression changes were quantified using RT-qPCR. Lipid peroxidation was measured using C11-BODIPY and flow cytometry. SUDHL1 and SUDHL4 flank tumor xenografts were initiated in SCID-beige mice, with Flip-NPs administered 3 times per week IV. Results: Primary B cell lymphoma cells were isolated from patients with FL (n=4) or DLBCL (n=2), and all samples expressed some level of SCARB1 by flow cytometry. Flip-NPs increased cell death in 3 of the 4 FL samples and 1 of 2 DLBCL samples. In Ramos and SUDHL4 cells, RT-qPCR data showed that Flip-NP-mediated cholesterol reduction led to up-regulation of cholesterol biosynthesis genes and down-regulation of glutathione peroxidase-4 (GPX4), a critical protein responsible for degradation of lipid peroxides. Correspondingly, as shown with C11-BODIPY, Flip-NP treatment increased lipid peroxide accumulation in Ramos and SUDHL4 cells. Addition of ferrostatin-1 or deferoxamine reduced Flip-NP induced cell death, demonstrating that the mechanism-of-action of Flip-NPs involves, at least in part, ferroptosis. Given the sensitivity of cholesterol auxotrophic cell lines to cholesterol reduction-induced ferroptosis, we tested the efficacy of the Flip-NPs against cholesterol auxotrophic ALK+ ALCL cell lines SUDHL1 and SR-786. SCARB1 was expressed in both cell lines. Flip-NPs potently induced cell death in both SUDHL1 and SR-786 cells in vitro. In vivo, systemic administration of Flip-NPs reduced tumor volumes in both SUDHL4 and SUDHL1 tumor xenograft models. Conclusions: Our data show that Flip-NPs reduce GPX4 expression and increase lipid peroxide accumulation in B cell lymphoma cell lines, resulting in ferroptosis. Expanding on these results, Flip-NP efficacy was also demonstrated in cholesterol auxotrophic ALK+ ALCL cell lines and primary patient-derived B cell lymphoma cells. These in vitro results translated to in vivo murine models, as systemic administration of Flip-NPs potently reduced DLBCL and ALK+ ALCL tumor xenograft burden. Flip-NPs are a molecularly targeted, first-in-class therapy that may be effective for malignancies reliant upon cellular cholesterol. Disclosures Behdad: Pfizer: Other: Speaker; Thermo Fisher: Membership on an entity's Board of Directors or advisory committees; Loxo-Bayer: Membership on an entity's Board of Directors or advisory committees. Karmali:Astrazeneca: Speakers Bureau; Takeda, BMS: Other: Research Funding to Institution; Gilead/Kite; Juno/Celgene: Consultancy, Speakers Bureau. Thaxton:Zylem: Other: Co-founder of the biotech company Zylem. Gordon:Juno/Celgene: Other: Advisory Board, Research Funding; Gilead: Other: Advisory Board; Bayer: Other: Advisory Board; Zylem LLC: Other: co-founder; research in nanoparticles in cancer.

Download Full-text

Kinase Inhibitors of DNA-PK, ATM and ATR in Combination with Ionizing Radiation Can Increase Tumor Cell Death in HNSCC Cells While Sparing Normal Tissue Cells

Genes ◽

10.3390/genes12060925 ◽

2021 ◽

Vol 12 (6) ◽

pp. 925

Author(s):

Eva-Maria Faulhaber ◽

Tina Jost ◽

Julia Symank ◽

Julian Scheper ◽

Felix Bürkel ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Side Effects ◽

Ionizing Radiation ◽

Cell Lines ◽

Normal Tissue ◽

Kinase Inhibitors ◽

Induced Response ◽

Tumor Control ◽

Tumor Cell Death

(1) Kinase inhibitors (KI) targeting components of the DNA damage repair pathway are a promising new type of drug. Combining them with ionizing radiation therapy (IR), which is commonly used for treatment of head and neck tumors, could improve tumor control, but could also increase negative side effects on surrounding normal tissue. (2) The effect of KI of the DDR (ATMi: AZD0156; ATRi: VE-822, dual DNA-PKi/mTORi: CC-115) in combination with IR on HPV-positive and HPV-negative HNSCC and healthy skin cells was analyzed. Cell death and cell cycle arrest were determined using flow cytometry. Additionally, clonogenic survival and migration were analyzed. (3) Studied HNSCC cell lines reacted differently to DDRi. An increase in cell death for all of the malignant cells could be observed when combining IR and KI. Healthy fibroblasts were not affected by simultaneous treatment. Migration was partially impaired. Influence on the cell cycle varied between the cell lines and inhibitors; (4) In conclusion, a combination of DDRi with IR could be feasible for patients with HNSCC. Side effects on healthy cells are expected to be limited to normal radiation-induced response. Formation of metastases could be decreased because cell migration is impaired partially. The treatment outcome for HPV-negative tumors tends to be improved by combined treatment.

Download Full-text

A Critical Role for Fas Ligand in the Active Suppression of Systemic Immune Responses by Ultraviolet Radiation

Journal of Experimental Medicine ◽

10.1084/jem.189.8.1285 ◽

1999 ◽

Vol 189 (8) ◽

pp. 1285-1294 ◽

Cited By ~ 57

Author(s):

Laurie L. Hill ◽

Vijay K. Shreedhar ◽

Margaret L. Kripke ◽

Laurie B. Owen-Schaub

Keyword(s):

Dna Damage ◽

Immune Responses ◽

Immune Suppression ◽

Fas Ligand ◽

Critical Role ◽

Cell Activity ◽

Contact Hypersensitivity ◽

Delayed Type Hypersensitivity ◽

Intact Cells

Induction of antigen-specific suppression elicited by environmental insults, such as ultraviolet (UV)-B radiation in sunlight, can inhibit an effective immune response in vivo and may contribute to the outgrowth of UV-induced skin cancer. Although UV-induced DNA damage is known to be an initiating event in the immune suppression of most antigen responses, the underlying mechanism(s) of such suppression remain undefined. In this report, we document that Fas ligand (FasL) is critical for UV-induced systemic immune suppression. Normal mice acutely exposed to UV exhibit a profound suppression of both contact hypersensitivity and delayed type hypersensitivity (DTH) reactions and the development of transferable antigen-specific suppressor cells. FasL-deficient mice exposed to UV lack both transferable suppressor cell activity and primary suppression to all antigens tested, with the exception of the DTH response to allogeneic spleen cells. Interestingly, suppression of this response is also known to occur independently of UV-induced DNA damage. Delivery of alloantigen as protein, rather than intact cells, restored the requirement for FasL in UV-induced immune suppression of this response. These results substantiate that FasL/Fas interactions are essential for systemic UV-induced suppression of immune responses that involve host antigen presentation and suggest an interrelationship between UV-induced DNA damage and FasL in this phenomenon. Collectively, our results suggest a model whereby UV-induced DNA damage disarms the immune system in a manner similar to that observed in immunologically privileged sites.

Download Full-text