G6PD promotes cell proliferation and dexamethasone resistance in multiple myeloma via increasing anti-oxidant production and activatingWnt/β-catenin pathway

doi:10.21203/rs.3.rs-1654963/v1

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G6PD promotes cell proliferation and dexamethasone resistance in multiple myeloma via increasing anti-oxidant production and activatingWnt/β-catenin pathway

https://doi.org/10.21203/rs.3.rs-1654963/v1

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Glucose-6-phosphate dehydrogenase (G6PD) has been noticed for its deficiency causing a series of clinical diseases, however, the role of G6PD in cancer remains elusive. In this study, serum metabolic profiling identified that DHEAS level was higher in newly diagnosed multiple myeloma (MM) patients than in normal people. DHEAS can be converted into an endogenously uncompetitive inhibitor of G6PD known as DHEA. Clinical datasets from GEO showed an increasing expression of G6PD during MM progression, especially in those patients resistant to Dexamethasone (Dexa). We further investigated the oncogenic role of G6PD by using two MM cell lines in vitro and xenograft mouse model in vivo. Elevated G6PD promoted MM cell proliferation, in contrast silencing G6PD with RNAi or inhibitor suppressed MM cell growth and improved the survival of MM model mice. Mechanistically, high G6PD expression enhanced enzymatic generation of anti-oxidant NADPH via pentose phosphate pathway (PPP) and decreased the production of reactive oxygen species (ROS) thus inducing proliferation and Dexa resistance in MM cells. Furthermore, canonical Wnt/β-catenin signaling also participated in regulating G6PD-induced drug resistance and cellular redox level of ROS. Intriguingly, DHEA treatment could enhance the sensitivity of MM cells to Dexa mainly through augmenting cellular oxidative stress. In summary, G6PD enhances the enzymatic anti-oxidant NADPH generation and decreases ROS generation, thus encouraging MM cells resistant to Dexa-induced apoptosis mainly via enzymatic PPP pathway and non-enzymatic Wnt/β-catenin signaling pathway in MM. Targeting G6PD to harness cellular redox may afford a promising new strategy to improve therapeutic effect of MM patients.

Multiple myeloma

Glucose-6-phosphate dehydrogenase

Dehydroepiandrosterone

ROS

NADPH

Dexamethasone

Multiple myeloma (MM) is the second most common hematological malignancy worldwide. It is estimated that 34,920 new cases and 14,210 deaths in the United States in 2021[1]. According to statistics collected in China, the incidence of MM in 2016 was 1.60 per 100,000 person-years[2, 3]. The last two decades have seen the development from proteasome inhibitors (PIs, such as Bortezomib/BTZ) and immunomodulatory drugs (IMiDs) to antibody-based[4] or chimeric antigen receptor(CAR)-based[5, 6] targeting immunotherapies, all of which have achieved significant improvement in extending survival of MM patients. However, MM remains incurable for most MM patients inevitably relapsed, which is the most intractable problem to tackle in MM treatment[7]. Currently, the mechanisms driving the pathogenesis and progression of relapsed or refractory MM are poorly understood, and the additional hallmark of metabolic reprogramming in many tumors especially in MM is largely uncovered[8]. There is a growing interest in the pathways associated with reductive biosynthesis and redox homeostasis in cancer[9].

Glucose-6-phosphate dehydrogenase (G6PD), an archetypical housekeeping gene encoding the first and rate-limiting enzyme (EC1.1.1.49) in the pentose phosphate pathway (PPP), is able to catalyze glucose-6-phosphate into 6-phosphogluconolactone[10]. PPP is the major metabolic pathway supplying the major sources of reduced power nicotinamide adenine dinucleotide phosphate (NADPH) in cytoplasm, and it is a route for the dissimilation of carbohydrates besides glycolysis[11]. NADPH regenerates the essential antioxidant glutathione (GSH), which is essential for maintaining cellular redox homeostasis[10, 12]. The critical physiological and pathological roles of G6PD activity in maintaining redox homeostasis of healthy or diseased cells have been extensively discussed[13]. However, the mechanisms of G6PD activity on cell growth, survival and chemo-resistance in MM have not been clearly defined.

Mounting evidences demonstrate that G6PD is upregulated and promotes tumor progression in many cancers[14]. Inhibition of G6PD has emerged as a potential therapeutic strategy for cancer treatment, for example, the applications of a small molecule G6PD inhibitor RRX-001 in colorectal cancer & hepatoma cells[15–17] and endogenous G6PD inhibitor dehydroepiandrosterone (DHEA: 5-androsten-3beta-ol-17-one) in cervical & breast cancer cells[18, 19]. Interestingly, our previous non-targeted UHPLC-MS metabolomic study identified unique metabolites changes in serum samples from MM patients and normal people, especially the reduction of dehydroepiandrosterone sulfate (DHEAS). DHEAS is the more stable sulfate ester form of DHEA in the blood, and both of them can be interconverted into each other via de-sulfation/sulfation by hydroxysteroid sulfur transferase (HST) or steroid sulfatase (STS), respectively [20, 21]. DHEA as a metabolic precursor for the synthesis of steroid androgen and estrogen[22] possesses an uncompetitive inhibition of G6PD activity thus targeting PPP[23, 24].

The present study aimed to investigate the oncogenic role of G6PD in MM progression. We constructed G6PD knockdown (G6PD-KD) & overexpression (G6PD-OE) MM cell lines and established MM cell line-derived xenograft (CDX) mouse model to explore the functions and potential mechanism of G6PD. The mechanism studies demonstrated that G6PD was involved in the regulation of anti-oxidative activity and Wnt/β-catenin signaling pathway. Our data provide a novel insight of G6PD acting as promising therapeutic target for MM treatment, especially in relapsed MM patients.

Metabolomics profiling identifies a significant decrease of DHEAS in MM patients

Untargeted metabolomics analyses were performed on the serum samples from 70 normal people (Ctrl group) and 70 newly diagnosed MM patients (MM group). The resulting metabolic profiles were clearly segregated between Ctrl and MM groups based on Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) analysis (Figure 1A). Glucose-6-phosphate and DHEAS were considered as significantly differential metabolites with P value < 0.05 and variable importance in the projection (VIP) of >1.0 (Table 1). In addition, ELISA assay showed that serum DHEAS concentrations were statistically lower in MM group than in Ctrl group (Figure 1B).

High expression of G6PD is related to poor prognosis of MM patients

We followed to explore the relationship between G6PD and the prognosis of MM patients since DHEA and its ester sulfate DHEAS interconvert freely[20, 21] and DHEA is the endogenous inhibitor of G6PD[23, 24] (Figure 1C). Based on public available datasets from NCBI Gene Expression Omnibus (GSE2658 and GSE5900), the data showed that G6PD mRNA was significantly increased during MM progression from normal bone marrow plasma cells (NP, n = 22), “premalignant” individuals with monoclonal gammopathy of undetermined significance (MGUS, n = 44) to malignant plasma cells of newly diagnosed multiple myeloma (MM, n = 351) (Figure 1D). Furthermore, the Kaplan-Meier analysis indicated a statistically significant inverse correlation between G6PD levels and overall survival (OS) of MM patients from the clinical datasets of both University of Arkansas Total Therapy 2 (TT2) and the dutch-belgian cooperative trial group for hematology oncology group-65 (HOVON65) cohorts (Figure 1E & 1F). The above results suggest that high G6PD expression is correlated with inferior clinical outcomes in MM.

Elevated G6PD promotes cellular proliferation in MM cells and xenograft mouse model

In order to identify the functions of G6PD in MM cells, we constructed two G6PD-OE ARP1 and H929 MM cell lines using lentivirus expression system. WB analysis validated the G6PD-OE MM cells were constructed successfully (Figure 2A). MTT assay demonstrated obviously enhanced cellular viability in G6PD-OE MM cells relative to wildtype (WT) MM cells (P < 0.05) (Figure 2B). Consistently, flow cytometry analysis showed that elevated G6PD induced a cell cycle arrest at the G0/G1 phase while the proportion of S phase was increased in both ARP1 and H929 cells (Figure 2C & 2D), suggesting that G6PD facilitated MM cell proliferation. Subsequently, we established MM cell line-derived xenograft (CDX) mouse model to investigate the role of G6PD in vivo. Two weeks after MM cell injection, the tumors in G6PD-OE group developed faster than WT group (Figure 2E & 2F). Accordingly, the tumor weight was remarkably increased in G6PD-OE group compared with WT group (Figure 2G).

Deceased G6PD suppresses MM cell viability and improves the survival of MM mice.

To further confirm the role of G6PD in cellular proliferation, we utilized RNAi technology to knockdown G6PD in MM cell lines, which would transiently express shRNA-G6PD induced by doxycycline (DOX). As Figure 3A shown, G6PD-KD MM cells were constructed successfully. MTT analysis showed that the cell viability was significantly decreased in G6PD-KD cells compared with control cells (Figure 3B). In addition, we took advantage of RRX-001 (1-bromoacety l-3,3-dinitroazetidine) to suppress G6PD activity. RRX-001 is a G6PD inhibitor and multifunctional anticancer drug in phase III clinical trials[15-17]. As evidenced in Figure 3C and D, overexpression of G6PD caused MM cells more sensitive to RRX-001 than WT cells (IC₅₀/μΜ of RRX-001 in ARP1 WT: 1.75 vs OE: 0.997; H929 WT: 2.50 vs OE: 1.38). Then, we used 5TMM3VT MM mouse model to test the protective effect of RRX-001 on survival of MM mice in vivo (Figure 3E). The conditions of control mice rapidly deteriorated from day 20 until all mice in control group were dead on day 38. In comparison, RRX-001 treatment led to prolonged survival up to ~50 days in MM mice (p = 0.0088).

Increased G6PD enhances MM cell resistance to Dexa, but not BTZ.

As to better understand the association between high expression of G6PD and poor prognosis in the relapsed MM patients, we examined G6PD expression in the recurrent MM patients from APEX (Assessment of Proteasome Inhibition for Extending Remissions) cohort. Based on the data of Affymetrix U133 Plug 2.0 gene chip, the level of G6PD signal in relapsed MM patients was significantly higher than the baseline and positively correlated with inferior outcomes compared with normal people (Figure 4A & 4B). Both of bortezomib (BTZ) and dexamethosane (Dexa) are front-line drugs. The expression of G6PD in MM patients not responding to Dexa treatment was significantly higher than patients responding to Dexa treatment (P < 0.05) (Figure 4C). In contrast, there was no differential expression of G6PD in patients responding to BTZ or not (Figure 4D). It indicates that G6PD may be involved in specific response to Dexa in MM patients.

In agreement with above findings, MTT assays indicated that G6PD-OE cells were able to endure higher dosages of Dexa with 4~5 folds of IC₅₀ values higher than WT ARP1 (OE/WT: 4629/81.76) and H929 (OE/WT: 901.7/265.8) cells, respectively (Figure 4E & 4F). The flow cytometry analysis demonstrated that Dexa treatment caused significant apoptotic events in Dexa-treated WT cells compared with untreated cells (p = 0.0335), however Dexa did not trigger similar level of apoptosis in G6PD-OE cells (Figure 4G & 4H). Additionally, the combination treatment of Dexa and RRX-001 on Dexa-resistant MM1.R cells significantly increased the proportion of apoptotic cells compared to single Dexa or RRX-001 treatment (Figure 4I & 4J).

G6PD-mediated Dexa resistance in MM cells is related to cellular redox levels of NADPH and ROS

Considering the central role of G6PD in PPP to produce ribose and reducing equivalent NADPH, we measured NADPH content in MM cells. The NADP⁺/NADPH ratio in both ARP1 and H929 cells was significantly lower than in G6PD-OE cells, indicating more NADPH contents were generated upon G6PD overexpression (Figure 5A). Then we enforced the production of ROS by adding 1 mM H₂O₂into G6PD-OE cells to explore the association between anti-oxidative NADPH and ROS. The dihydroethidium (DHE) fluorescent probe exhibits blue fluorescence in the cytosol until it is oxidized upon DNA intercalation to emit bright red fluorescence. We observed that supplement of extra H₂O₂ significantly triggered ROS generation of 4 folds in ARP1 G6PD-OE cells and 8 folds in H929 G6PD-OE cells compared with counterpart cells (Figure 5B & 5C). Then we followed to treat MM cells with 10 μM DHEA or 15 μM DHEAS for 24 h, respectively. WB analysis showed that DHEA or DHEAS significantly inhibited G6PD expression at protein level (Figure 5D & 5F). Higher NADP⁺/NADPH ratios were observed in G6PD-OE cells treated with DHEA or DHEAS compared with non-treated cells (Figure 5E & 5G), supporting the critical role of G6PD in producing NADPH. We further found that DHEA or DHEAS significantly triggered more ROS generation of ～1.5 folds in ARP1 G6PD-OE cells than non-treated cells (Figure 5H & 5I) . Similar tendency was observed in H929 G6PD-OE cells (Figure 5J & 5K), supporting that high level of G6PD with relatively low oxidative stress confers MM cell resistant to Dexa.

Wnt/β-catenin signaling pathway is involved in G6PD-induced Dexa resistance in MM

Transcriptomic RNA sequencing (RNA-seq) technology was performed to screen the downstream genes of G6PD in ARP1 and H929 WT and G6PD-OE cells. We found 19 differentially expressed genes (DEGs, 12 upregulated & 7 downregulated) in ARP1 cells and 31 DEGs (16 upregulated & 15 downregulated) in H929 cells (Figure 6A). The gene ontology (GO) and KEGG enrichment analyses revealed that eight signaling pathways were identified as the top enriched pathways, including Wnt and mTOR (mammalian target of rapamycin) signaling pathways (Figure 6B). We detected the components of the enriched pathways by WB assay and found obviously altered AKT protein of mTOR signaling in H929 cells but not in ARP1 cells (Figure S1A & S1B). Therefore, we focused on Wnt/β-catenin signaling pathway to verify whether it would be deregulated by G6PD at protein level. As a core component of Wnt/β-catenin pathway, β-catenin expression was increased in G6PD-OE cells and decreased in G6PD-KD cells (Figure 6C & 6D). Furthermore, suppressed β-catenin in G6PD-KD cells was increased by supplying extra Wnt3a into the culture medium to activate canonical Wnt pathway (Figure 6E & 6F). To further validate the link between β-catenin and G6PD mediated anti-ROS generation under the condition of Dexa resistance, we examined the changes of ROS level by adding Wnt3a into MM cells with or without RRX-001 treatment. The results showed that Wnt3a treatment effectively increased the ROS levels, whereas the elevation of ROS levels was significantly decreased by using RRX-001 in both MM cell lines (Figure 6G & 6H). The critical contribution of Wnt/β-catenin to G6PD-mediated anti-ROS generation may be in a G6PD dependent manner in MM (Figure S2). Importantly, the high expression of β-catenin in G6PD-OE cells was significantly decreased by Dexa and H₂O₂ individually or collectively (Figure 6I). Collectively, Wnt/β-catenin signaling pathway was involved in G6PD-induced Dexa resistance of MM cells.

DHEA relieves G6PD-mediated Dexa resistance in MM cells

We assessed the efficacy of DHEA in combination with Dexa for treating G6PD-mediated Dexa resistant in MM. MTT assay demonstrated that DHEA enhanced the sensitivity of MM.1S, ARP1 and H929 G6PD-OE cells to Dexa (Figure 7A-C). The protein levels of G6PD were significantly decreased by a combination treatment of DHEA and Dexa compared with Dexa treatment alone (Figure 7D & 7E). Furthermore, the intracellular oxidative activities of G6PD-OE cells before and after Dexa treatment were detected, which showed that Dexa treatment did not trigger more ROS generation upon G6PD overexpression in MM cells (Figure 7F & 7G). Intriguingly, the combination treatment of DHEA and Dexa significantly increased the ROS levels in G6PD-OE cells (Figure 7H & 7I), suggesting that DHEA treatment may reverse Dexa resistance induced by elevated G6PD.

G6PD is profoundly known for its significant role in preventing hemolysis of mammal red blood cells (erythrocytes), which lack both nucleus and mitochondria. G6PD deficiency is a well-known inherited chromosome X-linked enzymopathy prevalent in Africa, Mediterranean and southeast Asia, which predisposes G6PD-deficient individuals to favism, neonatal jaundice and infection/drug-induced hemolysis mainly due to oxidative stress[25]. One recent preclinical study using G6PD deficient (G6PDd) cells infected with human coronavirus (hCoV) ascribed a possible biological role of G6PDd in SARS-CoV2 viral proliferation, which may cause disparity in COVID19 outcomes[26]. It has been extensively studied with respect to G6PD deficiency for more than a half-century[10, 25], however an increasing evidence in the last decades documented the aberrantly high expression of G6PD in many cancers[10, 12, 14, 27]. Our present study aimed to reveal the oncogenic role of G6PD in promoting cellular proliferation, survival and chemotherapeutic drug resistance in MM.

Our work found that G6PD played critical role in assuring an essential life of MM cells as well healthy cells mainly via anti-oxidative processes. Untargeted metabolomics and GEO analyses suggested high expression of G6PD leading to poor clinical outcomes during MM progression (Fig. 1). To some extent different from the report that the modest increase of G6PD protected cells from oxidative damage and extended a healthy lifespan in mice[27], our CDX model mice with G6PD-OE cell injection had a higher tumor burden (Fig. 2E-G) while the 5TMM3VT mice treated with G6PD inhibitor RRX-001 presented prolonged survival (Fig. 3E). It may dependent on the spatiotemporal expression pattern and activity of G6PD in normal/diseased tissues or local microenvironment. The contributions of G6PD on proliferation and pro-survival of cancer cells occur at several levels. Firstly, as the first and rate-limiting enzyme in PPP signaling, G6PD facilitates the production of ribose-5-phosphate as well as cytoplasmic NADPH, both of them are required for the massive biosynthesis of essential nucleic acids and fatty acids during the accelerated replication and progression of cancer cells. Secondly, it is well known that NADPH is extremely important cellular antioxidant in the maintenance of redox homeostasis. ROS serving as one class of the main highly-bioactive and easily-diffusible small signaling molecules, such as superoxide anion O₂⁻, hydrogen peroxide H₂O₂ and hydroxyl radical OH•, have a dual role of either facilitating cancer cell proliferation, increasing metabolism and adaptation to hypoxia or triggering oxidative damage-induced cell death[28]. As shown in Fig. 5, the antioxidant capacity of G6PD-OE MM cells was increased to counteract hyperactive ROS generation during cellular transformation and tumorigenesis. Thirdly, the unique hallmark of cancer cells with relatively high turnover of ROS production and scavenging makes them more sensitive to the alterations of ROS level or redox environment, which explains why MM cells are partially resistant to Dexa that is a commonly used immunosuppressive anti-tumor drug and able to enhance ROS producing capacity and apoptosis of myeloid cells as well as osteoblasts[29, 30]. As a consequence, knockdown or inhibition of G6PD improved the susceptibility of MM cells to Dexa and aggravated cell death (Fig. 3). Therefore, targeting G6PD might be a promising treatment for MM therapy strategy.

From a broader perspective of metabolic reprogramming in cancer, it may enable cancer cells to evolve to utilize a variety of unconventional nutrient sources for survival and growth[31], in addition to the aerobic glycolysis (named Warburg Effect) in the cytoplasm[13]. Elevated G6PD impels cancer cells to hijack and reprogram the intracellular metabolic pathway, which influence DNA replication, cell division, redox equilibrium and proliferation to obtain an advantageous condition especially during chemical therapy. Since cancer cells generally exhibit high ROS level or exposure, the rewired metabolic network of PPP may enable cancer cells to counteract persistent oxidative stress by NADPH reduction, as NADPH is the direct or indirect donor of many reductive powers including thioredoxins, glutathione, glutaredoxins, peroxiredoxins and glutathione peroxidases[32]. Interestingly, one recent study using G6PD deleted (G6PD-KO) cells demonstrated that G6PD neutralized oxidative stresses by supporting reductive glutamine metabolism and activating AMPK pathway to balance NADH/NAD⁺ ratio[33]. Therefore, the critical role of enzymatic G6PD in balancing cellular oxidative redox of cancer was supported by our group and other research groups. We also provided evidence for that Wnt/β-catenin pathway was involved in G6PD-induced proliferation and Dexa resistance of MM cells in a non-enzymatic manner (Fig. 6). This finding is consistent with mounting reports that oncogenic Wnt/β-catenin signaling promotes cancer cell growth, migration and/or invasion in many solid and blood cancers in vitro and in vivo[34, 35], despite an opposite relationship in the case of melanoma[36]. However, we could not exclude other signaling pathways potentially involved in G6PD-induced MM cell growth and drug resistance, such as mTOR signaling (Figure S1). It is in accordance with one work in acute myeloid leukemia (AML) of interplay between G6PD inhibition and mTORC1 activity[37], since mTOR is a conserved serine/threonine kinase controlling cell growth and metabolism in response to nutrients, growth factors, cellular energy and stress[38]. In addition, G6PD transcription and PPP switching on in turn can be mediated by Wnt/β-catenin signal activation including c-MYC, consequently conferring oxaliplatin chemoresistance in HCC cells[39]. There is an intricate interplay between Wnt/β-catenin and oxidative ROS[40], and Wnt/β-catenin signaling also plays key roles in metabolic reprogramming and drug resistance[41]. Thus, it is plausible to speculate that G6PD may well coordinate the enzymatic/non-enzymatic supply of necessary cellular energy and support of anti-oxidant defense during tumorigenesis, which can be translated into MM therapy in future.

Clinically, the inhibition of G6PD has been proved to be a useful strategy in reversing chemotherapy resistance, such as competitive G6PD inhibitor 6-aminonicotinamide (6-AN)[42] and uncompetitive G6PD inhibitor DHEA[18, 19]. Our untargeted metabolomic and ELISA assays revealed that DHEAS was significantly decreased in the serum of MM group compared with Ctrl group (Fig. 1A-B). Furthermore, after G6PD-OE cells were treated by DHEA, G6PD protein and NADPH levels were significantly downregulated (Fig. 5D-G) while the ROS levels were significantly upregulated (Fig. 5I & 5K). It has been reported that DHEA, as the active form of DHEAS, evidently reduces Dexa-induced oxidative stress in renal cortex[43], possibly via inhibiting glucocorticoid receptor (GR) translocation into the nucleus[44, 45]. DHEA could enhance oxidative stress upon G6PD overexpression in MM cells and increase β-catenin expression (Fig. 7D-I). Although DHEA possesses anti-cancer, anti-obesity and anti-inflammatory effects on animal models[46, 47], the clinical trials with DHEA were unsuccessful due to a requirement of high oral doses and the ready conversion of endogenous DHEA into the active androgens[48]. More studies are still needed to investigate the relationships among G6PD, Wnt/β-catenin and ROS-mediated drug resistance in MM.

In conclusion, our findings demonstrate that high expression of G6PD enhances the intracellular anti-oxidative activity via generating more NADPH and less ROS, which encourages MM cells to be antagonistic to Dexa-induced apoptosis via enzymatic PPP pathway and non-enzymatic Wnt/β-catenin signaling (Fig. 8). Targeting G6PD to regulate cell redox balance may provide a promising new strategy to improve therapeutic effect of MM patients and sensitize relapsed MM patients to treatment.

Untargeted metabolomics

70 normal people were recruited at Affiliated Hospital of Nanjing University of Chinese Medicine. 70 newly diagnosed MM patients were enrolled from Shanghai Changzheng Hospital. The serum samples from all subjects were collected and stored in a digital-alarm-controlled freezer at -80°C before analysis. Sample preparation for untargeted metabolomic was detected as described in previous study[49]. The Thermo Scientific™ Vanquish™ Horizon UHPLC system coupled to an Thermo Scientific™ Q Exactive hybrid quadrupole-Orbitrap mass spectrometer and operated in full scan mode was used for untargeted analysis of plasma samples. For the analyses carried out in the negative ESI mode, mobile phase A consisted of water containing 0.1% (vol/vol) formic acid (#F809712, Macklin, Biochemical Co., Ltd, Shanghai, China) and mobile phase B was acetonitrile (#1.00030.4008, Merck, Carrigtwohill, Ireland). The gradient profile was presented as described in previous study[50]. Data processing and statistics were analyzed by SIMCA 14.1 software and MetaboAnalyst 5.0. The metabolites with P value < 0.05 and VIP of >1.0 were considered as statistically significant metabolites.

Measurement of serum DHEAS level

After thawed at 4°C, the serum samples were used for measuring the concentrations of DHEAS by utilizing a human DHEAS ELISA kit (YFXEH00908, YIFEIXUE BIO TECH, Jiangsu, China). 20 serum samples were selected randomly from normal people and MM groups respectively in duplicated wells for each assay. According to the manufacturer’s instructions, two identical treatments were conducted. All the experimental operations were carried out by the same operator.

Myeloma cell lines and cell culture

Human MM cell lines ARP1 and H929 were kind gifts from Prof. Zhiqiang Liu (Department of Physiology and Pathophysiology, School of Basic Medical Science, Tianjin Medical University). MM.1S and MM.1R cells were purchased from ATCC (CRL-2974 and CRL-2975, respectively). Mouse 5TMM3VT cells were donated by Dr. Wen Zhou (Xiangya School of Medicine, Central South University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education; Key Laboratory of Carcinogenesis, National Health and Family Planning Commission, Changsha, China). The cells were maintained at 37 °C with 5 % CO₂ in RPMI 1640 (#05-065-1A, Biological Industries, Beit Haemek, Israel) supplemented with 10% heat-inactivated fetal bovine serum (FBS; #04-002-1A, Biological Industries, Israel) and 1% penicillin/streptomycin.

Cell proliferation

MM cell growth was detected by performing a colorimetric MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. The cells were seeded in a 96-well plate at a density of 5×10³ cells per well containing 180 μL complete medium. After 48 h, 20 μL MTT (5 mg/mL) was added into each well. After culturing for 4 h, the supernatant was removed by centrifuge and dimethyl sulfoxide was added to each well. Then a microplate reader (Thermo Fisher Scientific, Inc., USA) was used to measure the absorbance at 570 nm.

Cell cycle analysis

The cells were washed twice with ice-cold PBS and fixed with 75% ice ethanol overnight. Next day, after the cells were washed with PBS, RNase A (200 μg/mL) was added to treat the cells on ice for 1 h. Then the cells were added 50 μg/mL PI (#25535-16-4, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) to incubate for 5 minutes at room temperature, and detected by a flow cytometer (Guava Technologies, Hayward, CA, USA). Data were analyzed by Modifit software.

Apoptosis assay

MM cells were seeded in a 6-well plate with 1.5×10⁶ cells per well. The administration group was treated with Dexa (#D4902, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), RRX-001 (#925206-65-1, Shanghai yuanye Bio-Technology, Shanghai, China) in an incubator at 37°C with 5% CO₂ for 48 h. The cells were collected in a centrifuge tube, centrifuged at 1200 rpm for 5 min, discarded the supernatant, washed twice with pre-cooled PBS and finally discarded the supernatant. Subsequently, cell pellets were resuspended with 100 μL of 1×Binding buffer, added 5μL Annexin V-APC (#640920, Biolegend, Beijing, China) and 5μL PI (50 μg/mL, #25535-16-4, Solarbio Life Science, Beijing, China) and incubated at room temperature for 5 min, then added 400 μL 1×Binding buffer. At last, the samples were loaded on a flow cytometer for detection.

Animal experiments

ARP1 WT and G6PD-OE cells (1 × 10⁶) were subcutaneously injected into the abdominal area of 6∼8-week old NOD-SCID mice (n = 6 per group) from Beijing Vital River Laboratory Animal Technology, Co., Ltd (Beijing, China). The tumor volumes were measured using calipers at the indicated time points. When the tumor diameters reached 20mm, the mice would be sacrificed. Tumor volume (mm³) was calculated as: (length × width²)/2. The 8-week old male/female C57BL/KaLwRij mice (n = 10 per group) were injected intravenously with 5TMM3VT (1×10⁶ cells per mouse). Treatments with RRX-001 started from Day 2 (2 times/week), and continued until the mice were dead. All animal procedures were conducted in accordance with government-published recommendations for the Care and Use of Laboratory Animals and approved by the Institutional Ethics Review Boards of Nanjing University of Chinese Medicine (No. ACU170501).

ROS determination

MM cells were cultured with different concentrations of DHEA (#D806984, Macklin Biochemical Co., Shanghai, China), DHEAS (#HY-B0765, MedChemExpress, New Jersey, USA) or Dexa for 24 h. The intracellular ROS levels were determined by flow cytometry (Guava Technologies, Hayward, CA, USA). The cells were incubated with 100 μM Dihydroethidium (#104821-25-2, Beyotime Institute of Biotechnology, Shanghai, China) at 37°C in the dark for 30 min, then washed twice and resuspended in PBS. ROS levels were determined by a flow cytometer.

Western Blot

MM cells were harvested, washed and lysed with assistance of RIPA lysis buffer. Total protein samples (20-40 μg) were heated in SDS/b-mercaptoethanol buffer and loaded on 10%-15% SDS-PAGE gels. The proteins were separated by electrophoresis in the gels, and then transferred onto PVDF membrane. The membrane was blocked with 5% non-fat milk and incubated with primary antibodies against G6PD (1:1,000 dilution, #ab210702, Abcam, Shanghai, China), β-catenin (1:1 000 dilution, #51067-2-AP, Proteintech Group, Wuhan, China) and β-actin (1:1,000 dilution, #4970S, Cell Signaling Technology, Mass, USA) overnight at 4°C. Blots were incubated with secondary antibodies using horseradish peroxidase conjugated rabbit anti-mouse (1:10,000 dilution, #S0002, Affinity, OH, USA) or goat anti-rabbit IgG (1:10,000 dilution, #FMS-Rb01, Fcmacs, Nanjing, China) at room temperature for 1h. Finally, blots were developed by chemiluminescence using ECL kit (#180-5001, Tanon, Shanghai, China).

NADP⁺/NADPH Assay

NADP⁺/NADPH Assay Kit with WST-8 (#S0179) was purchased from Beyotime Institute (Shanghai, China). The experiment was conducted according to the accompanying instructions.

Statistical analysis

Data were expressed as mean ± SD. Two independent experimental groups were assessed by Student’s t-test, while multiple groups were compared with one-way ANOVA. The difference between groups was set at P < 0.05 (∗), P < 0.01 (∗∗), and P < 0.001 (∗∗∗). All data were analyzed by GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA).

G6PD: Glucose-6-phosphate dehydrogenase; BTZ: Bortezomib; CAR: Chimeric antigen receptor; CDX: Cell line-derived xenograft; Dexa: Dexamethasone; DHEAS: Dehydroepiandrosterone sulfate; DOX: Doxycycline; GEO: Gene expression omnibus; GO: Gene ontology; GR: Glucocorticoid receptor; GSH: Glutathione; HST: Hydroxysteroid sulfur transferase; IMiDs: Immunomodulatory drugs; MGUS: Monoclonal gammopathy of undetermined significance; MM: Multiple Myeloma; NADPH: Nicotinamide adenine dinucleotide phosphate; NP: Normal bone marrow plasma cells; OPLS-DA: Orthogonal Projections to Latent Structures Discriminant Analysis; OS: Overall survival; PIs: Proteasome inhibitors; PPP: Pentose phosphate pathway; ROS: Reactive oxygen species; STS: Steroid sulfatase; VIP: Variable importance in the projection.

Funding

This work was supported by National Natural Science Foundation of China 82173849 (to CG); Natural Science Foundation of Jiangsu Province BK20200097 (to CG); Jiangsu Postgraduate Research and Practice Innovation Program KYCX20_1451 (to MK).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics declarations

All animal procedures were conducted in accordance with government-published recommendations for the Care and Use of Laboratory Animals and were approved by the Animal Ethical and Experimental Committee of Nanjing University of Chinese Medicine (No. ACU170501). The studies involving human participants were reviewed and approved by Institutional Review Board and the Ethics Committee of the First Affiliated Hospital of Nanjing University of Traditional Chinese Medicine. The patients/participants provided their written informed consent to participate in this study.

Author contributions

YY and CG designed and conceived this project. RL, MK, MQ, ZH, YC and ZD performed the experiments. RL, MK and JQ analyzed the data. JQ drafted the manuscript. YY and CG revised the manuscript. All authors read and approved the final version of the manuscript.

Data availability statement

Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2658. The RNA-seq datasets generated for this study were also deposited in NCBI GEO database (accession number GSE172318).

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Table 1 is available in the Supplemental Files section.

Additional File 2 is not available with this version.

No competing interests reported.

FiguresS1S2.pdf
Table1.xlsx
Table 1 Top10 differential metabolites between the serums of Ctrl group and MM group.

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You are reading this latest preprint version

G6PD promotes cell proliferation and dexamethasone resistance in multiple myeloma via increasing anti-oxidant production and activatingWnt/β-catenin pathway

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Version 1

Abstract

Figures

Introduction

Results

Metabolomics profiling identifies a significant decrease of DHEAS in MM patients

High expression of G6PD is related to poor prognosis of MM patients

Elevated G6PD promotes cellular proliferation in MM cells and xenograft mouse model

Deceased G6PD suppresses MM cell viability and improves the survival of MM mice.

Increased G6PD enhances MM cell resistance to Dexa, but not BTZ.

DHEA relieves G6PD-mediated Dexa resistance in MM cells

Discussion

Materials And Methods

Untargeted metabolomics

Measurement of serum DHEAS level

Myeloma cell lines and cell culture

Cell proliferation

Cell cycle analysis

Apoptosis assay

Animal experiments

ROS determination

Western Blot

Statistical analysis

Abbreviations

Declarations

Funding

Conflict of interest

Ethics declarations

Author contributions

Data availability statement

References

Tables

Additional File

Additional Declarations

Supplementary Files

Status:

Version 1