Targeting Nicotinamide Adenine Dinucleotide (NAD) Glycohydrase Activity of CD38 Exerts Anti-Myeloma Effect Accompanying Intracellular NAD Elevation

Introduction. The development of novel agents has improved the outcomes of multiple myeloma (MM) patients. Especially, daratumumab, an anti-CD38 monoclonal antibody which exerts therapeutic effect against MM cells through direct cell damage, antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), has shown its high efficacy in clinical practice. CD38 is a transmembrane glycoprotein highly expressed in plasma cells. CD38 is also a major nicotinamide adenine dinucleotide (NAD) glycohydrase in mammalian tissues, which regulate cellular levels of NAD. However, the role of CD38 as a NAD glycohydrase (NADase) in survival of MM cells is not well understood. In the present study, we conducted CD38 enzyme activity inhibition on MM cells using a small molecule compound 78c, a specific inhibitor for NADase enzymatic activity of CD38, in order to study the role of CD38 NADase activity in MM cell survival and to examine whether CD38 enzyme inhibition could be a new therapeutic strategy of MM. Materials and methods. MM cell lines (NCI-H929, KMS-12BM, KMS-12PE, U266) were treated with CD38 NADase inhibitor, 78c, in vitro. Viability of MM cell lines and patient-derived MM cells were analyzed by flow cytometry after 7AAD staining. MM cell lines possessing CD38 positive and negative fraction were sorted according to the CD38 expression using CD38 Micro-Beads. CD38 low MM cell lines were treated with All-trans retinoic acid(ATRA)to increase surface CD38 expression. Intracellular NAD and NADH concentrations in MM cells were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Caspase inhibitor, Z-VAD-FMK, was used in combination with 78c to study the mechanism of 78c induced MM cell death. Results. 78c induced cell death in MM cell lines at low concentrations (IC50 10-20 μM). Addition of 78c to patient derived bone marrow cells showed cytotoxicity to MM cells, while toxicity to non-MM cells were limited. CD38 positive fraction of MM cell lines had better sensitivity to 78c compared to CD38 negative fraction. CD38 induction by ATRA in CD38 low MM cell lines showed increased sensitivity to 78c. These results proved that 78c efficacy correlates with surface CD38 expression. Comparison of intracellular NAD and NADH concentrations between CD38 positive and negative fractions of MM cell lines demonstrated a significant increase of NAD in the CD38 negative fraction compared to their positive counterparts, indicating that CD38 is indeed controlling the intracellular NAD concentration. Marked increase of NAD / NADH ratio was observed in 78c treated MM cell lines compared to control, proving that CD38 NADase inhibition affects intracellular NAD concentration in MM cells (Fig. 1). 78c treatment of MM cell lines significantly reduced the number of viable cells in the Annexin- / PI- region, however, addition of Z-VAD-FMK did not lead to recovery of viable cell numbers, indicating non-apototic cell death induction by CD38 NADase inhibition. Conclusions. CD38 is the major NADase in mammalian tissues, and involved in catabolism of NAD. CD38 NADase inhibitor, 78c, inhibited the growth of MM cells at low concentrations. 78c induced cell death was found to be highly specific to MM cells and its cytotoxic effect was associated with surface CD38 expression of MM cells. Increased amount of NAD in MM cells by 78c treatment suggests that NAD elevation is associated with MM cell death induced by CD38 NADase inhibition. Since, daratumumab has limited effect against CD38 NADase activity, modulation of intracellular NAD levels by CD38 NADase inhibition could provide a novel therapeutic strategy for MM (Fig. 2). Disclosures Matsuoka: Kyowa Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

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DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-114206 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5618-5618 ◽

Cited By ~ 1

Author(s):

Priya Choudhry ◽

Margarette C. Mariano ◽

Arun P Wiita

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Research Funding ◽

Plasma Cells ◽

Cpg Island ◽

Ex Vivo ◽

Single Agent ◽

Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

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The Role of CD38 in Multiple Myeloma Cell Biology

Blood ◽

10.1182/blood-2021-150884 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1580-1580

Author(s):

Yawara Kawano ◽

Saki Kushima ◽

Hiroyuki Hata ◽

Masao Matsuoka

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Cell Biology ◽

Proteome Analysis ◽

Cd38 Expression ◽

Mammalian Tissues ◽

Nadh Ratio

Abstract Introduction. Anti-CD38 monoclonal antibodies, such as daratumumab and isatuximab, which exerts therapeutic effect against multiple myeloma (MM) cells through direct cell damage, antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), has shown its high efficacy in clinical practice. However, the role of CD38 in MM cell biology is still unclear. CD38 is known as a major nicotinamide adenine dinucleotide (NAD +) glycohydrase (NADase) in mammalian tissues, which regulate cellular levels of NAD +. In the present study, we compared metabolic and proteomic profile between CD38 positive and negative MM cell lines to analyze the biological significance of CD38 in MM cells. Additionally, we performed CD38 enzyme activity inhibition on MM cells using 78c, a NADase enzyme inhibitor of CD38, in order to study the role of CD38 NADase activity in MM cell survival. Materials and methods. MM cell lines harboring CD38 positive and negative fractions (KMS-12BM, KMS-11) were sorted according to CD38 expression. Intracellular NAD+ and NADH concentrations between CD38 positive and negative cells were analyzed using NAD/NADH Assay kit. CD38 positive and negative MM cells were subjected to metabolome and proteome analysis using Shimadzu TQ8050 GC-MS/MS and TripleTOF 5600 respectively. Metabolites and proteins significantly enriched in CD38 negative MM cells were analyzed using MetaboAnalyst and Metascape. Cell cycle status between CD38 positive and negative cells were determined by flow cytometry after staining by BRDU and 7AAD. CD38 positive MM cell lines (NCI-H929 and KMS-12PE) and patient derived bone marrow cells were treated with 78c, a CD38 NADase inhibitor, in vitro. MM cell viability were determined by flow cytometry post Annexin V and PI staining. Differences in metabolites between 78c treated and control MM cell lines were also analyzed using Shimadzu TQ8050 GC-MS/MS. Results. Higher NAD+ and NAD+/NADH ratio was observed in CD38 negative fraction of MM cell lines compared to their CD38 positive counterparts, demonstrating that cell surface CD38 expression influences intracellular NAD+ concentration. Both metabolome and proteome analysis revealed that CD38 negative cells tend to have higher glycolytic activity compared to CD38 positive cells. Significant suppression of cell cycle, accompanying G0/G1 phase arrest, was observed in CD38 negative MM cells, indicating that metabolic shift in CD38 negative MM cells may lead to change in cell proliferation. Marked increase of NAD+/NADH ratio was observed in 78c treated MM cell lines compared to control, proving that CD38 NADase inhibiton indeed affects intracellular NAD+ concentration in MM cells. 78c was capable of inducing cell death in MM cell lines and patient derived MM cells, accompanying cell cycle arrest. Metabolites significantly upregulated in 78c treated MM cells compared to control were associated with glycolysis, demonstrating that CD38 NADase activity has a significant effect on MM cell metabolism. Conclusions. CD38 is the major NADase in mammalian tissues, involved in catabolism of NAD +. Although CD38 is highly expressed in normal plasma cells and MM cells, its role in MM cell biology has not been studied in detail. By comparing CD38 positive and negative cells and using CD38 NADase inhibitor, we showed for the first time that CD38 on MM cells decrease intracellular NAD+, reduces intracellular glycolysis and as a result, has an influence on cell cycle. The present study sheds light on the significance of CD38 enzyme activity in MM cell biology and may also contribute to understanding the mechanism of resistance to CD38 targeted therapy. Disclosures Kawano: Janssen Pharmaceuticals: Honoraria; Ono pharmaceutical: Honoraria; Sanofi: Honoraria; Bristol Myers Squibb: Honoraria; Takeda Pharmaceuticals: Honoraria.

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JX06, a Novel PDK1 Inhibitor, Induces Myeloma Cell Apoptosis By Metabolic Reprogramming and Works Synergistically with Bortezomib

Blood ◽

10.1182/blood-2019-123619 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1814-1814

Author(s):

Takayuki Sasano ◽

Saki Kushima ◽

Matsushita Yutaka ◽

Masao Matsuoka ◽

Hiroyuki Hata ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Death ◽

Oxidative Phosphorylation ◽

Cell Lines ◽

Mechanism Of Action ◽

Pyruvate Dehydrogenase ◽

Research Funding ◽

Caspase 3 ◽

Low Concentrations

Background: Despite the efficacy of novel agents, multiple myeloma (MM) is still an incurable disease. In order to achieve a cure, it is necessary to develop new therapeutic drugs, which target different pathways from the present anti-MM agents. PDK1 (pyruvate dehydrogenase kinase 1) is a glucose metabolism-related protein often induced by HIF-1. PDK1 inactivates PDH (pyruvate dehydrogenase) through phosphorylation, leading to enhanced glycolysis in the cytoplasm and suppression of oxidative phosphorylation in the mitochondria. PDK1 that is highly expressed in plasma cells is a downstream target of IRF4. We previously reported that PDK1 inhibition is a potent therapeutic strategy in MM (Fujiwara S et al. Br. J. Cancer; 108 (1): 170-178. 2013). However, PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. Materials and methods: MM cell lines (NCI-H929,KMS-12PE,KMS-12BM,U266, KMM1, RPMI-8226) were treated with PDK1 inhibitor, JX06, in vitro. Caspase inhibitor, Z-VAD-FMK, was used in combination with JX06 to study the mechanism of JX06 induced MM cell death. Mitochondrial pyruvate carrier (MPC) inhibitor, UK5099, was utilized to block pyruvate transportation into the mitochondria. Bortezomib was used in combination with or without JX06. Growth inhibition of MM cell lines by JX06 were examined by WST-8 assay. Cytotoxicity of primary MM cells by JX06 was examined using flow cytometry after staining with 7AAD. Caspase 3 activity and PDH phosphorylation of MM cell lines were determined by Western blot. Cell cycle analysis of MM cell lines treated with or without JX06 was performed by flow cytometry using BrdU. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Results: JX06 suppressed cell growth of various MM cell lines and primary myeloma cells at low concentrations (0.5-1.0 µM). MM cell death by JX06 accompanied caspase 3 activation and this cell death was suppressed under addition of Z-VAD-FMK, indicating that JX06 induced apoptosis in MM cells. Moreover, phosphorylation of PDH, known as a target of PDK1, was significantly suppressed under JX06 treatment, demonstrating that indeed JX06 exerts anti-MM effect by inhibiting PDK1-PDH pathway. Addition of UK5099 to JX06 suppressed JX06-induced MM cell death, demonstrating that the efficacy of JX06 depends on pyruvate transported into the mitochondria through MPC. There was no significant difference in cell cycle distribution between JX06 treated MM cells compared to control, suggesting that JX06 exerts cytotoxicity independent of cell cycle phase. Moreover, significant increase of cell death was observed in NCI-H929 cell line treated in combination with 0.25 µM JX06 and 2.5 nM bortezomib, although bortezomib alone at concentration of 2.5 nM didn't induce cell death. Conclusion: We demonstrated that JX06 could induce apoptosis of MM cell lines and primary MM cells by inhibiting PDK1. JX06-induced MM cell death is mediated by metabolic shift from glycolysis in the cytoplasm to oxidative phosphorylation in the mitochondria (Fig. 1). Considering its efficacy and the distinct mechanism of action from the current anti-MM agents, JX06 can be a promising anti-MM agent. Furthermore, JX06 not only works as single agent, but can also enhance the efficacy of current anti-MM drugs, suggesting this combination lead to better treatment response and less toxicity. Disclosures Matsuoka: Kyowa Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

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MDM2 Inhibitor Nutlin-3a Triggers Autophagic Cell Death In Addition to Apoptosis In Leukemia Cell Lines with Wild-Type p53

Blood ◽

10.1182/blood.v116.21.3300.3300 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3300-3300

Author(s):

Seshagiri Duvvuri ◽

Vivian Ruvolo ◽

Duncan H. Mak ◽

Kensuke Kojima ◽

Marina Konopleva ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Western Blot ◽

Cell Lines ◽

Research Funding ◽

Leukemia Cell ◽

Annexin V ◽

Dependent Manner ◽

Autophagic Vacuoles ◽

Leukemia Cell Lines

Abstract Abstract 3300 Background: Nutlin-3a is a small molecule inhibitor of MDM2 and has been shown to induce apoptosis and cell cycle arrest in various cancer models in a p53 dependent manner. Autophagy is a programmed cell death that can occur concurrently with apoptosis or in its absence. There is significant debate whether autophagy is a protective mechanism or a bona fide mechanism of cell death. While autophagy can function as tumor cell defense mechanism against cellular stress induced death, mutation/loss of alleles of certain genes regulating autophagy have been associated with development of cancer (e.g. Beclin-1 in breast cancer [Nature, 1999, 402: 672–676]). Multiple proteins involved in autophagy are transcriptional targets of p53 but Nutlin-3a has not been evaluated for its role in inducing autophagy. Here we present data suggesting that low dose Nutlin-3a induces autophagy in addition to apoptosis in leukemia cell lines in a p53 dependent manner. Methods and results: OCI-AML-3 cells (p53-WT) treated with Nutlin-3a (2.5 and 5.0μM for 48, 72 and 96 hrs) were stained with mono-dansyl-cadaverine (MDC), a dye that accumulates in acidic autophagic vacuoles. OCI-AML-3 cells showed increasing staining with MDC in a dose and time dependent fashion by both flow cytometry (54%, 57% and 51% MDC positive after treatment with Nutlin-3a 5.0μM for 48, 72 and 96 hrs) and by confocal microscopy. Nutlin-3a treated cells also were positive for Annexin-V (flow cytometry 22%, 26% and 36% at 48, 72 and 96 hrs time points), and some of the cells were double-positive for Annexin-V and MDC (9.2%, 5% and 7% at 48, 72 and 96 hrs) suggesting that both apoptosis and autophagy can occur simultaneously. Autophagy induction was confirmed by Transmission Electron Microscopy (TEM). Large, multiple autophagic vacuoles were observed in OCI-AML-3 cells treated with Nutlin-3a. OCI-AML-3 cells with stable p53 knockdown by shRNA or HL-60 cells (p53-null) did not show increased MDC staining by flow cytometry (both cell lines) or autophagic vacuoles by TEM (HL-60) after similar treatment. Western blot analysis showed increases in LC3-II and in conjugation of Atg5/12, early and late autophagy markers respectively, in OCI-AML-3 cells after treatment with Nutlin-3a. Increased expression of the autophagy markers (LC3-II and Atg 5/12 conjugate) were also seen by Western blot analysis in the ALL cell lines REH and NALM-6 (both p53-WT) after treatment with Nutlin-3a. Western blot and/or RT-PCR analysis showed upregulation of other p53 related proteins involved in autophagy e.g. DRAM, AMPK-β, LKB1, pLKB1 in OCI-AML-3 cells treated with Nutlin-3a. As mTOR/Akt pathway inhibits autophagy, analysis of mTOR targets showed downregulation of the total and phospho-ribosomal-S6-protein levels, whereas there was no change in total or phospho-4-EBP-1 levels. Knockdown of Beclin-1 (ATG6), one of the proteins required for initiation of the formation of autophagic vacuoles, caused reduction in autophagic vacuoles (MDC staining by confocal microscopy) in OCI-AML-3 and REH cells without affecting apoptosis induction (Annexin V by flow cytometry). Pharmacologic inhibition of late autophagy by Bafilomycin (10nM for 2 hours) reduced MDC staining in OCI-AML-3 cells treated with Nutlin-3a for 48 hrs (32% without and 9% with Bafilomycin) while having limited inhibition of apoptosis (Annexin V positive 42% without and 33% with Bafilomycin). Conclusion: Nutlin-3a induces autophagy in leukemia cells by a p53 dependent manner. We also demonstrate that autophagy could go hand-in-hand with apoptosis and in a fraction of cells both processes may occur concomitantly. Inhibition of autophagy does not necessarily enhance apoptosis. Disclosures: Andreeff: Roche: Research Funding. Borthakur:ASCO: Research Funding.

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Role Of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) and Its Ligands In The Regulation Of Functional OCT-1 Activity In CML Cells

Blood ◽

10.1182/blood.v122.21.1470.1470 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1470-1470

Author(s):

Jueqiong Wang ◽

Chung Hoow Kok ◽

Richard J. D'Andrea ◽

Timothy P. Hughes ◽

Deborah L. White

Keyword(s):

Cell Death ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

K562 Cells ◽

Vehicle Control ◽

Peroxisome Proliferator ◽

Advisory Committees ◽

Peroxisome Proliferator Activated Receptor

Abstract Introduction The human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL positive cells. The functional activity of the OCT-1 protein (OCT-1 activity, OA) is predictive of molecular response in de-novo chronic phase chronic myeloid leukemia (CP-CML) patients. We have previously demonstrated that diclofenac, a competitive peroxisome proliferator-activated receptor-γ (PPARγ) antagonist, can significantly increase OA in CML cells 1. However, the role of PPARγ and its ligands in OA regulation remain unknown. Thus, the link between OA and PPARγ in CML cells has been investigated in this study. Methods OA was determined by intracellular uptake and retention assay (IUR) in the presence and absence of the OCT-1 inhibitor, prazosin 2. To assess the effect of PPARγ ligands on OA, BCR-ABL positive cell lines (KU812, K562) were incubated with PPARγ antagonist (GW9662, T0070907) or agonists (GW1929, rosiglitazone) respectively for 1 hour immediately prior to the IUR assays. The OA was also assessed in the mononuclear cells (MNCs) of 77 CP-CML patients enrolled to the TIDEL II trial. PPARγ activity in CML MNC nuclear extracts was determined through the use of a PPARγ Transcription Factor Assay Kits according to the manufacturer's instructions. To assess the effect of PPARγ ligands on cell death, KU812 or K562 cells were stained with AnnexinV and 7-AAD for detection of apoptosis after the co-administration of imatinib and PPARγ ligands for 72 hours. Results A significant increase in OA was observed in KU812 and K562 cells treated with PPARγ antagonists. In contrast, PPARγ agonists significantly decreased the OA in both cell lines (Table 1). A negative link between OA and PPARγ activity was observed in CML MNC samples (R=-0.585, p<0.001). PPARγ activity was significantly elevated in CML patients who had a low OA at diagnosis (less than 4 ng/200,000 cells) compared with those who had higher OA (p<0.001). After 72 hours co-administration with 0.1µM imatinib, KU812 cells treated with PPARγ antagonists (GW9662 and T0070907) showed a significantly lower cell viability (40% and 18% respectively) compared with vehicle control (70%, p<0.001). Similar results were also observed in K562 cells after co-administration with 1.0µM imatinib for 72 hours. K562 cells treated with PPARγ antagonists (GW9662: 51% and T0070907: 47%) showed a significantly lower cell viability (51% and 47% respectively) compared with vehicle control (61%, p<0.05). Conclusion Ligand-activation or inhibition of PPARγ is a regulator of OA in CML cell lines, and the low MNC OCT-1 activity in CML patients is consistent with the high level of PPARγ activity in these cells. Low PPARγ activity may be the key driver for low OA and poor imatinib response observed in a subset of CML patients. Importantly, the enhanced OA as a result of PPARγ antagonist treatment resulted in increased cell death following co-administration with imatinib. Ongoing studies relating to the upstream pathways involved in PPARγ activation aim to reveal the possible mechanism of OA modulation by PPARγ. Enhancement of OA by PPARg antagonists is likely to provide an important axis for clinical application to improve the clinical efficacy of imatinib. This would be particularly important in patients with low OA who currently have inferior outcomes with imatinib therapy. 1. Wang J, Hughes TP, Kok CH, et al. Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells. British Journal of Cancer. 2012;106(11):1772-1778. 2. White DL, Saunders VA, Dang P, et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: Higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood. 2007;110(12):4064-4072. Disclosures: Hughes: Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

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Targeting CD99 in T-Cell Neoplasms with Monoclonal Antibodies

Blood ◽

10.1182/blood.v126.23.2749.2749 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2749-2749

Author(s):

Montreh Tavakkoli ◽

Dong H. Lee ◽

Benjamin Durham ◽

Stephen S. Chung ◽

Christopher Y. Park

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Death ◽

T Cell ◽

Cell Lines ◽

Functional Role ◽

Normal Peripheral Blood ◽

T Cell Lymphomas ◽

Cd34 Cells

Abstract CD99 is a 32-kDa glycoprotein involved in leukocyte migration and homotypic cell aggregation. Since its initial discovery as a marker on acute lymphoblastic leukemia (ALL), few studies have investigated its potential targeting and biological role in this disease. We have shown that CD99 is up-regulated in malignant stem cells in acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS), and that monoclonal antibodies (mAbs) targeting CD99 induce cell death. Given that targeting CD99 holds promise in AML/MDS, we sought to determine whether it is an effective target in other hematologic malignancies. We began by screening 15 T-, B-, and plasma cell lines as well as normal peripheral blood and umbilical cord CD34+ cells for CD99 expression by flow cytometry. CD99 expression was 7- and 10-fold higher on 1/1 T-ALL and 1/2 anaplastic large cell lymphoma (ALCL) cell lines compared with CD34+ cells, and 2- and 3-fold higher relative to normal peripheral blood T cells, respectively. However, it was minimally expressed in 11/12 B cell lymphomas, plasma cell dyscrasias, and peripheral T cell neoplasms. CD99 expression (degree, localization) was also assessed on 264 lymphoma patient samples by immunohistochemistry (IHC) using the CD99 mAb, 12E7. We found that 11/20 (55%) T-lymphoblastic lymphomas, 7/16 (44%) angioimmunoblastic T-cell lymphomas, 4/13 (31%) ALCLs, 10/63 (16%) peripheral T-cell lymphomas, and 0/3 (0%) of NK/T cell lymphomas express CD99 by IHC, while only 1/70 (1.4%) diffuse large B cell lymphomas, 2/24 (8%) mantle cell lymphomas, 2/17 (12%) follicular lymphomas, 4/22 (18%) chronic lymphocytic leukemias, and 3/16 (19%) marginal zone lymphomas express CD99. Staining was predominately moderate and cytoplasmic. Using a BioGPS dataset from T-ALL patient bone marrow samples, CD99 transcript was found to be up-regulated in T-ALL bone marrow (n=117) relative to normal bone marrow (n=7) (p<0.0001), and was expressed at similar levels at diagnosis (n=14) and relapse (n=14), suggesting it is stably expressed and may be a candidate therapeutic target. To test whether CD99 mAbs are cytotoxic to T-ALL and ALCL cell lines, cells were incubated with 5µg/ml CD99 mAb in the presence of 7µg/ml anti-IgG antibody, and cell survival was assessed by flow cytometry following 72-hours relative to IgG isotype control. 4/5 T-ALL cell lines (KOPTK1, Loucy, CCRF HSB-2, PF283) were sensitive to the cytotoxicity of CD99 mAb, mediating 30-96% cell death (p≤0.003), with 2/4 cell lines displaying 90-96% cytotoxicity. Remarkably, incubating CD99 mAb with a primary T-ALL patient sample induced 100% cell death within 48 hours of treatment (p<0.0001). 1/2 ALCL cell lines (Karpas-299) were sensitive to cytotoxic CD99 mAb (46% cell death, p=0.02). Furthermore, CD99 mAb treatment induced Annexin V positivity, and cell death occurred independent of complement and within 3 hours of treatment. To determine whether CD99 mAb cytotoxicity depends on the level of CD99 expression, we stably transduced KOPTK1 cells with an optimized CD99 shRNA (199-fold reduction in CD99 mean fluorescence intensity [MFI]), stably transduced CD99-low Mac2A (ALCL) cells with TetOn CD99 (17-fold increase in CD99 MFI), and analyzed the cells for cytotoxicity following 24-hour incubations with CD99 mAbs. CD99 mAb-induced cell death increased from 4.4% to 88% upon overexpressing CD99, and decreased from 89% to 20% upon knocking down CD99, suggesting that cell death is dependent on the level of CD99 expression. To elucidate the functional role of CD99 in T-cell neoplasms, we xenografted KOPTK1 cells expressing CD99 shRNA into sublethally irradiated NOD/SCID/IL-2Rgc-null (NSG) mice. Animals transplanted with CD99 knockdown showed no improved survival compared to controls (n=4 and 5 in each group, respectively). We further evaluated the potential oncogenic role of CD99 in vitro, and observed no effect of CD99 knock down in KOPTK1 or overexpression in Mac2A on cell cycle status or proliferation by PI staining and cell counting. Our data indicate that CD99 is expressed in a subset of T-lineage neoplasms. While there is no evidence for a functional role of CD99 in the growth or survival of T-ALL and ALCL, CD99 can be targeted by CD99 mAbs to induce apoptosis with rapid kinetics and in a manner that is dependent on levels of CD99 expression and independent of complement. Thus, CD99 is a promising target in the treatment of a subset of T-cell neoplasms. Disclosures No relevant conflicts of interest to declare.

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Identification of Deubiquitinase OTUD1 As a Novel Player in Resistance of Multiple Myeloma to Bortezomib

Blood ◽

10.1182/blood-2019-127930 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5526-5526

Author(s):

Alexander Vdovin ◽

Tomas Jelinek ◽

Matous Hrdinka ◽

Juli R. Bago ◽

Tereza Sevcikova ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Death ◽

Research Funding ◽

Molecular Mechanisms ◽

Plasma Cells ◽

Hek293 Cells

Introduction Multiple myeloma (MM) being one of the most widely spread haematological malignancies remains an incurable disease. As malignant plasma cells produce abnormally large amounts of immunoglobulins they particularly rely on the ubiquitin-proteasome system (UPS) to avoid aberrant protein overload. This unique feature is targeted by proteasome inhibitors (PI) that induce MM cell death especially by increasing levels of reactive oxygen species (ROS). Despite the high efficacy of PI most of the MM patients eventually relapse and expansion of drug resistant clones makes the treatment ineffective. Therefore, uncovering new molecular mechanisms of drug resistance is a crucial task. UPS is a very complex system that involves hundreds of proteins. While the roles of the proteasome and E3 ligases in PI resistance are well established, the third UPS component, deubiquitinating enzymes (DUBs), is much less explored. In this work, we performed a comprehensive search for DUBs with impact on MM pathogenesis and PI resistance, and further investigated the underlying molecular mechanisms. Methods Gene expression and survival For analysis of DUB genes (n = 101) expression in blood cells, expression dataset Gds3997, DICE database and data from (Jourdan et al., J Immunol. 2011 Oct 15;187(8):3931-41., Jourdan et al., Blood. 2009 Dec 10;114(25):5173-81.) submitted to http://www.genomicscape.com were used. For survival analysis MM patients were divided into two groups by median of gene expression for each DUB (datasets GSE2658, GSE4581 and GSE9782). Cell line models RPMI8226 and HEK293 cells were used as model cell lines. Cells with OTUD1 knockdowns and overexpression were generated by lentiviral infection using vectors containing doxycycline-inducible shRNA's and different versions of OTUD1 gene, respectively. Proliferation and cell death MTT assay was used for the analysis of cell proliferation and viability. Cell death was also evaluated by flow cytometry by staining with Annexin V and 7-AAD. Cell migration Cells were labelled with calcein-AM and placed into the upper chamber of transwell insert with 8-µM pores. After 16 hours of migration towards SDF-1α gradient, cells were counted by fluorescence detection. ROS analysis ROS was detected by labelling cells with 2′,7′-dichlorodihydrofluorescein diacetate and measuring fluorescence intensity using flow cytometry. Immunoprecipitation HEK293 cells were co-transfected with HA-OTUD1 and FLAG-KEAP1, and reciprocal co-immunoprecipitation and western blot analyses were performed. Results We analysed the expression of all human DUBs in different blood cell types and identified OTUD1 as the most differentially expressed DUB between B-cell lineage and other haematopoietic cells. During B-cell maturation OTUD1 expression reaches the maximum in the bone marrow plasma cells. MM patients with low OTUD1 expression had significantly worse prognosis in OS based on three large datasets (p value= 0,035; 0,008; 2.4e−06. HR=0,55; 0,19; 0,41). Expression of shRNA targeting OTUD1 in MM cell line RPMI8226 did not affect cell proliferation and migration but dramatically increased survival under oxidative stress (high ROS) conditions induced by bortezomib. Treatment with bortezomib promoted expression of OTUD1 in the wild type MM cells in a ROS-dependent manner. Additionally, we identified oxidative stress regulator, the E3 ligase KEAP1 as a novel direct interaction partner of OTUD1 that regulates OTUD1 stability under high ROS conditions. Conclusion Based on the gene expression analysis, OTUD1 was identified as a novel, potentially important player in MM pathogenesis. Low levels of OTUD1 expression in MM patients correlate with significantly worse OS. Knocking down OTUD1 in MM cells causes resistance to bortezomib. Mechanistically, bortezomib-induced ROS promotes transcription of OTUD1 mRNA and further induces stabilization of OTUD1 on protein level via disruption of OTUD1-KEAP1 complex. Our collective data suggest on a crucial role of OTUD1 in bortezomib-mediated MM cytotoxity. Further mechanistic studies delineating the role of OTUD1 in MM pathogenesis and PI resistance are ongoing. Disclosures Hajek: Janssen: Honoraria, Other: Consultant or advisory relationship, Research Funding; Amgen: Honoraria, Other: Consultant or advisory relationship, Research Funding; Celgene: Honoraria, Other: Consultant or advisory relationship, Research Funding; AbbVie: Other: Consultant or advisory relationship; Bristol-Myers Squibb: Honoraria, Other: Consultant or advisory relationship, Research Funding; Novartis: Other: Consultant or advisory relationship, Research Funding; PharmaMar: Honoraria, Other: Consultant or advisory relationship; Takeda: Honoraria, Other: Consultant or advisory relationship, Research Funding.

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Cytotoxicity of Environmentally Relevant Concentrations of Aluminum in Murine Thymocytes and Lymphocytes

Journal of Toxicology ◽

10.1155/2011/796719 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Jamal Kamalov ◽

David O. Carpenter ◽

Irina Birman

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Immune System ◽

Dna Binding ◽

Aluminum Chloride ◽

Cell Swelling ◽

Cytotoxic Effects ◽

Minute Exposure ◽

Low Concentrations ◽

Dose Dependent

The effects of low concentrations of aluminum chloride on thymocytes and lymphocytes acutely dissociated from young mice were studied using flow cytometry with a DNA-binding dye. We demonstrate a rapid and dose-dependent injury in murine thymocytes and lymphocytes resulting from exposure to aluminum, as indicated by an increase in the entry into the cell of the DNA-binding dye, propidium iodine. A 60-minute exposure to 10 μM AlCl3caused damage of about 5% of thymocytes, while 50% were injured after 10 minutes at 20 μM. Nearly all thymocytes showed evidence of damage at 30 μM AlCl3after only 5 minutes of incubation. In lymphocytes, injury was observed at 15 μM AlCl3and less than 50% of cells were injured after a 60-minute exposure to 20 μM. Injury only rarely proceeded to rapid cell death and was associated with cell swelling. These results suggest that aluminum has cytotoxic effects on cells of the immune system.

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PRMT5 Inhibition Drives Therapeutic Vulnerability to BCL-2 Inhibition with Venetoclax and Provides Rationale for Combination Therapy in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2019-130797 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 302-302 ◽

Cited By ~ 1

Author(s):

Fiona Brown ◽

Yang Zhang ◽

Claire Hinterschied ◽

Alexander Prouty ◽

Shelby Sloan ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Combination Treatment ◽

Cell Lymphoma ◽

Targeted Agents ◽

Mantle Cell ◽

Anti Tumor Activity

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, defined by the t(11;14) translocation and comprises 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 5 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life. Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting the cell cycle, PRC2 activity, and signaling via the BCR and PI3K/AKT pathways. We have developed first-in-class selective inhibitors of PRMT5 and, in collaboration with Prelude Therapeutics, we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models of human MCL. Selective inhibition of PRMT5 in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to chromatin. We identified 136 newly emerged FOXO1-bound genomic loci following 48 hours of PRMT5 inhibition in the CCMCL1 MCL line by performing chromatin immunoprecipitation-seq analysis. These genes were markedly upregulated in CCMCL1 cells treated with the PRMT5 inhibitor PRT382 as determined by RNA-seq analysis. Among those genes, we identified and confirmed FOXO1 recruitment to the promoter of BAX, a pro-apoptotic member of the BCL2 family of proteins. Treatment of MCL cell lines (Granta-519, CCMCL1, Z-138, and SEFA) with the selective PRMT5 inhibitor PRT382 (10, 100nM) led to upregulation of BAX protein levels and induction of programmed cell death as measured by annexin V/PI staining and flow cytometry. We hypothesized that induction of BAX would trigger a therapeutic vulnerability to the BCL2 inhibitor venetoclax, and that combination PRMT5/BCL2 inhibitor therapy would drive synergistic cell death in MCL. Single agent and combination treatment with venetoclax and PRT382 was performed in eight MCL lines including a new cell line generated from our ibrutinib-refractory PDX model (SEFA) and IC50 and synergy scores were calculated. The Z-138 line was most sensitive to venetoclax (IC50<10nM) while CCMCL-1, SP53, JeKo-1, and Granta-519 demonstrated relative resistance (IC50>1uM). All lines reached an IC50 <1uM when co-treated with PRT382, with IC50 values ranging from 20 - 500nM. Combination treatments showed high levels of synergy (scores > 20) in 4 lines and moderate synergy (scores 10-20) in 2 lines. The two lines with the highest levels of synergy, Z-138 and SEFA, express high levels of BCL-2 and are Ibrutinib resistant. Overall there was a strong positive correlation between BCL2 expression and synergy score (r=0.707), and no correlation between PRMT5 expression and synergy score (r=0.084). In vivo evaluation in two preclinical MCL models (Granta-519 NSG mouse flank and an ibrutinib-resistant MCL PDX) showed therapeutic synergy with combination venetoclax/PRT382 treatment. In both models, mice were treated with sub-therapeutic doses of venetoclax and/or PRT543 (Granta) or PRT382 (IR-MCL PDX) and tumor burden assessed weekly via flank mass measurement (Granta) or flow cytometry (IR-MCL-PDX). Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in both MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. PRT543, a selective PRMT5 inhibitor, has been advanced into clinical studies for the treatment of patients with solid tumors and hematologic malignancies, including MCL (NCT03886831). Disclosures Zhang: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

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The emerging role of ferroptosis in non-cancer liver diseases: hype or increasing hope?

Cell Death and Disease ◽

10.1038/s41419-020-2732-5 ◽

2020 ◽

Vol 11 (7) ◽

Cited By ~ 5

Author(s):

Lihong Mao ◽

Tianming Zhao ◽

Yan Song ◽

Lin Lin ◽

Xiaofei Fan ◽

...

Keyword(s):

Cell Death ◽

Liver Diseases ◽

Therapeutic Strategy ◽

Kidney Injury ◽

Hepatic Disease ◽

Therapeutic Concept ◽

Regulated Cell Death ◽

Dependent Form ◽

Molecular Machinery

Abstract Ferroptosis is an iron- and lipotoxicity-dependent form of regulated cell death (RCD). It is morphologically and biochemically distinct from characteristics of other cell death. This modality has been intensively investigated in recent years due to its involvement in a wide array of pathologies, including cancer, neurodegenerative diseases, and acute kidney injury. Dysregulation of ferroptosis has also been linked to various liver diseases and its modification may provide a hopeful and attractive therapeutic concept. Indeed, targeting ferroptosis may prevent the pathophysiological progression of several liver diseases, such as hemochromatosis, nonalcoholic steatohepatitis, and ethanol-induced liver injury. On the contrary, enhancing ferroptosis may promote sorafenib-induced ferroptosis and pave the way for combination therapy in hepatocellular carcinoma. Glutathione peroxidase 4 (GPx4) and system xc− have been identified as key players to mediate ferroptosis pathway. More recently diverse signaling pathways have also been observed. The connection between ferroptosis and other forms of RCD is intricate and compelling, where discoveries in this field advance our understanding of cell survival and fate. In this review, we summarize the central molecular machinery of ferroptosis, describe the role of ferroptosis in non-cancer hepatic disease conditions and discuss the potential to manipulate ferroptosis as a therapeutic strategy.

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