What implications are there linking metformin to inhibition of CD19-CAR T cells?

Question

What implications are there linking metformin to inhibition of CD19-CAR T cells? If you were a oncologist, would you recommend to do co-treatment with CAR T to upregulate immune function to fight tumor cells and metformin to target glucose metabolism in cancer? Thank you.

Introduction

Cancer immunotherapy has emerged as a hopeful approach in cancer treatment for the weak side effect and favorable applicability.1 Recently, chimeric antigen receptor (CAR)- modified T cells has been proven to be a promising strategy in cancer therapy. Among them, T cells genetically modified with CD19 CARs which specifically bind to normal and malignant B lymphocytes but not to other normal myeloid cells, have been well documented to possess potent antitumor property against CD19-expressing leukemia cells.3,4 Recent studies in clinical trials have reported that the objective tumor response in patients with acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM) after infusion with CD19-CAR T cells.5–7 However, various outcomes were found in different clinical trials. These various outcomes might be due to various procedures such as the design of CAR structure, transfect methods, T-cell source and culture conditions, and the dosage of CAR T cells. Until now, how to improve the persistence and efficiency is still a challenge for the application of CD19-CAR T cells. However, the crucial factor in regulating the efficiency as well as its potential mechanism still remains unclear. In recent years, glucose metabolism is gaining increased attention in type II diabetes mellitus and cardiovascular disease, especially in cancers.9–11 Moreover, a study has demonstrated that glucose uptake and metabolism is a key cellular transcription factor in T-cell function, such as activation, proliferation, and differentiation.12 As a traditional medicine, metformin has been widely used in type II diabetes mellitus for over 50 years. It has long been demonstrated in inhibiting glucose production, increasing insulin sensitivity, and reducing insulin secretion by ?-pancreatic cells.13,14 Intriguingly, recent reports have focused on the relationship between metformin and cancer. For example, some studies have established the direct suppressive effect of metformin on breast and pancreatic cancer cells.15,16 Furthermore, studies have provided evidences that diabetic patients receiving metformin have a reduced risk of cancer progress and mortality.17,18 Meanwhile, metformin has been shown to affect the glycolytic metabolism of immunocytes.19,20 A study further showed the regulatory role of glycolytic metabolism on T-cell proliferation and effective function. These findings implied the close relationship between glycolytic metabolism and biological functions of CAR T cells. However, no evidences have evaluated whether the regulatory role of glycolytic metabolism by metformin on T cells could be affected when genetically modified with CD19-CARs, as well as its underlying mechanisms. It is well established that AMP-activated protein kinase (AMPK) played a key role in maintaining multifunctionality of immunocytes under low nutrient conditions by regulating glycolytic metabolism.20,22 A study also found that mTOR, the downstream kinase of AMPK, functions as a main regulator of glucose metabolism in activated lymphocytes.23 Furthermore, mTOR induction by glycolysis is modulated through the activation of HIF1?. 24 Therefore, the AMPK/mTOR/HIF1? pathway is an overt signal pathway to reflect T-cell glycometabolism. Thus, the aims of the current study were to evaluate the effect of metformin on the biological functions of CD19-CAR T cells and explore the mechanisms involved.

Results

Metformin inhibits CD19-CAR T cell proliferation

The lentiviral expression vectors encoding the CD19-targeted CARs include CD19 scFv, human CD8a molecule transmembrane region, and intracellular signaling domains of CD28, 4-1BB, and CD3zeta and enhanced green fluorescent protein (eGFP) using the “self-cleaving” F2A peptide (Figure 1A). By using flow cytometry, we detected that the anti-CD19- CAR can be expressed efficiently on the T-cell surface. The transduction efficiency was about 63.1%±8.2% (Figure 1B). Based on these results, the CD19-CAR T cells can be used for the following experiments. To evaluate the effect of metformin on CD19-CAR T cells, the CCK-8 assay was used to detect CD19-CAR T-cell proliferation. As shown in Figure 1C, the proliferative ability of CD19-CAR T cells was significantly suppressed by metformin (1, 10, and 20 mM) in a dose-dependent manner. To assess the changes in the actual cell numbers, the effect of metformin on CD19-CAR T-cell proliferation was detected by direct cell counting. As shown in Figure 1D, we found that 1 mM metformin restrained CD19-CAR T-cell proliferation and 10 and 20 mM metformin had greater inhibitive effects. These results indicated that metformin inhibited CD19-CAR T-cell proliferation.

Metformin induces CD19-CAR T-cell apoptosis

To determine whether metformin has a direct effect on CD19-CAR T-cell apoptosis, the cells were incubated with metformin (1, 10, and 20 mM) for 24 hours. As shown in flow cytometry results, metformin (1, 10, and 20 mM) effectively induced CD19-CAR T-cell apoptosis (Figure 2A and B). These data demonstrated the pro-apoptotic role of metformin on CD19-CAR T cells.

Cytotoxicity of CD19-CAR T cells was inhibited by metformin

To verify the effect of metformin on CD19-CAR T-cell cytotoxicity, CD19-CAR T cells were cocultured with CD19+ Raji cells or CD19? K562 cells for 24 hours. The effector to target cell ratio (E:T) was from 1:1 to 40:1. As shown in Figure 3A, compared to CD19? K562 cells, the cytotoxic effect of CD19-CAR T cells was significantly activated when cocultured with CD19+ Raji cells at various E:T. Then, metformin was introduced in the cocultured CD19-CAR T cells and CD19+ Raji cells. As shown in Figure 3B, we found that metformin significantly inhibited the cytotoxicity of CD19- CAR T cells at various E:T. Furthermore, the IL-2 and INF-? release were detected by using ELISA assay. Our data showed that the secretion of IL-2 and INF-? at various E:T was significantly reduced when treated with metformin (Figure 3C and D). Collectively, these data indicated that metformin inhibited the cytotoxic activity of CD19-CAR T cells.

Metformin promotes AMPK phosphorylation and suppresses mTOR and HIF1? expression in CD19-CAR T cells

It is well documented that AMPK/mTOR/HIF1? signal pathway play a critical role in metformin-modulated lymphocytes energetic metabolism.20,24 To further explore the latent mechanism about metformin on CD19-CAR T cells, the AMPK, mTOR, and HIF1? signaling pathway was examined. As shown in Figure 4A, the AMPK phosphorylation was upregulated when treated with 10 mM metformin, whereas mTOR phosphorylation was downregulated. Meanwhile, metformin also downregulated the expression of HIF1?. The regulation was statistically significant, as quantified by densitometry (Figure 4B). These results indicated that AMPK/mTOR/HIF1? signal pathway may take part in the metformin-modulated glucose metabolism of CD19-CAR T cells.

AMPK pathway is involved in metforminregulated CD19-CAR T-cell apoptosis and cytotoxicity

To further delineate whether the role of metformin on CD19-CAR T cells was AMPK-dependent, compound C, a potent AMPK inhibitor, was applied to block AMPK phosphorylation (Figure 5A). As shown in Figure 5B–D, inhibition of AMPK phosphorylation with compound C reversed the metformin-modulated cytotoxicity and apoptosis in CD19-CAR T cells, whereas the metforminsuppressed mTOR and HIF1? expression was not affected by compound C (Figure 5E and F). Taken together, these results strengthen the opinion that AMPK pathway is involved in metformin-modulated biological functions of CD19-CAR T cells.

Metformin suppresses the cytotoxicity effect of CD19-CAR T cells in vivo

Having shown the effect of metformin on CD19-CAR T cells in vitro, next we were interested in determining whether metformin had the same role on suppressing the cytotoxicity of CD19-CAR T cells in vivo. NSG mice were engrafted with Raji-ffluc cells intravenously 3 days prior to CD19-CAR T-cell injection (Figure 6A). The data show that metformin treatment exhibits negative effect on the cytotoxicity of CD19-CAR T cells. Three of 5 mice in metformin treatment group showed tumor progression leading to their sacrifice at days 10, 15, and 17, respectively. The other two mice had large clusters of tumor cells but not sacrificed. However, tumor cells in the control group mice were mostly eliminated, and all mice survived at day 18 (Figure 6B). The survival rate of mice was reduced by the treatment of metformin (Figure 6C). Strikingly, the data demonstrated that metformin plays suppressant role on CD19-CAR T-cell cytotoxicity in vivo.

Discussion

The present study documented several new findings about metformin. Metformin can inhibit the proliferation and cytotoxicity of CD19-CAR T cells. Metformin can also induce apoptosis of CD19-CAR T cells. Metformin acts on CD19-CAR T cells through AMPK signaling pathway. In vivo, cytotoxicity of CD19-CAR T cells was restrained by metformin. In recent years, CD19-CAR T cells have shown valid outcomes in B-cell malignancies.3,4 However, the response rate of CD19-CAR T cells varied in different B-cell malignancies.5–7 Thus, seeking effective drugs to regulate the persistence and efficiency of CD19-CAR T cells and exploring its mechanisms are crucial issues in cancer treatment. Metformin is frequently used in type II diabetes mellitus treatment for over 50 years. Recent studies have focused on the glucose metabolism in cancers.9–11 Many preclinical and clinical studies have been reported that metformin treatment reduced cancer incidence in type II diabetes patients. For example, Evans et al reported the relationship between metformin usage and cancer incidence in patients with type II diabetes mellitus.27 Also, a meta-analysis revealed that diabetic patients with metformin treatment tend to have a lower incidence of cancer.28 Further studies demonstrated that metformin reduced the risk of various cancers such as colorectal, breast, ovarian, prostate, and endometrial cancer.29–32 Moreover, a study has demonstrated that glucose uptake and metabolism is a key cellular transcription factor in T-cell functions, such as activation, proliferation, and differentiation.12 Although previous studies reported the regulatory effect of metformin, the role of metformin on the biological functions of CD19-CAR T cells as well as its detailed molecular mechanisms has not been clearly elucidated. In the current study, we explored the role of metformin on the proliferation, apoptosis, and cytotoxicity in CD19-CAR T cells. Our research revealed that metformin could significantly suppress the proliferation and cytotoxicity and induce its apoptosis in CD19-CAR T cells. For the first time, our research revealed the negative effect of metformin usage in CD19-CAR T-cell treatment. However, Eikawa et al pointed out the positive effect of metformin in CD8+ TIL.20 This study verified that the physiological dose of metformin could induce tumor rejection in vivo. Our study indicated that metformin in physiological dose plays a negative role in diminishing CD19+ Raji cells by CD19-CAR T cells in vivo. The differences between these two studies brought interesting thoughts. Maybe metformin can be affected by tumor microenvironment. Perhaps metformin treatment differentially affects CAR and TCR signaling. Further research will be conducted on these details. It is well documented that metformin was the agonist of AMP-activated protein kinase.33 Studies further revealed that the crucial role of AMPK in regulating the glycolytic metabolism of immunocytes.20,22 Also, studies demonstrated that mTOR promoted CD8+ T-cell differentiation by modulating the transcription factor hypoxia inducible factor 1? (HIF1?).23 Moreover, studies found that HIF1? was caused T-cell activation in mTOR-dependent manner.34–36 In our study, the expression of AMPK, mTOR, and HIF1? in CD19-CAR T cells was examined. We found that the expression of phosphor-AMPK was upregulated, while expressions of phosphor-mTOR and HIF1? expression were downregulated when treated with metformin. To testify that AMPK, mTOR, and HIF1? were involved in metformin-regulated biological function in CD19- CAR T cells, we blocked AMPK activity by compound C, the AMPK inhibitor. Our research revealed that inhibition of AMPK activity by compound C significantly reversed the apoptosis and cytotoxicity of CD19-CAR T cells when treated with metformin. Meanwhile, the expression of mTOR and HIF1? was not affected when AMPK inhibitor was added. These results indicated that AMPK is crucial for metformin-modulated apoptosis and cytotoxicity in CD19-CAR T cells. Also, these results demonstrated that metformin-inhibited mTOR and HIF1? expression might be AMPK-independent. In conclusion, our study for the first time indicated that metformin could affect proliferation, apoptosis, and cytotoxicity of CD19-CAR T cells and validated in vivo. Moreover, the regulatory effect of metformin in CD19-CAR T cells was AMPK-dependent. The results in our study will guide medications on patients with B-cell malignancies and type II diabetes.

Explanation / Answer

Metformin inhibits proliferation and cytoxicity of CD19 - CAR T cells . It also induces apoptosis of the cell. Metformin at physiological doses have anti tumor activity. This is supported by studies on diabetic patients who is on metformin for several years. These patients have low incidence of tumor . Metformin exerts its activity through AMP activated protein kinase. It activates protein kinase and inactivates mTOR which is necesary for cell proliferation . If the concentration of metformin is reduced in diabetic patients , then the effect of metformin is not visible. Yes , as metformin at higher concentration produces anti tumor activity , it can be used as anti cancer drug . More over this drug is safe in patients with very less side effects and it is known to be in use for over 50 years . Hence , it can be used in cancer therapy also.

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