α-Ketoglutarate Supplementation and NAD+ Modulation Enhance Metabolic Rewiring and Radiosensitization in SLC25A1 Inhibited Cancer Cells

doi:10.21203/rs.3.rs-3126927/v1

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α-Ketoglutarate Supplementation and NAD+ Modulation Enhance Metabolic Rewiring and Radiosensitization in SLC25A1 Inhibited Cancer Cells

https://doi.org/10.21203/rs.3.rs-3126927/v1

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published 15 Jan, 2024

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Metabolic rewiring is the result of the growing demands and proliferation of cancer cells, leading to alterations of biological activities and reactions to treatments of cancer cells. The mitochondrial citrate transport protein SLC25A1 is involved in metabolic reprogramming offering a strategy to induce metabolic bottlenecks with relevance for radiosensitization by accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) upon SLC25A1 inhibition (SLC25A1i). Previous studies have uncovered the comparative effects of SLC25A1i or cell permeable D-2HG (octyl-D-2HG) treatments on DNA damage induction and repair as well as on energy metabolism and cellular function crucial for long-term survival of irradiated cells. Here, α-ketoglutarate (αKG), the precursor of D-2HG, potentiated the effects observed upon SLC25A1i on DNA damage repair, cell function and long-term survival in vitro and in vivo, thus making NCI-H460 cancer cells more vulnerable to ionizing radiation. However, αKG treatment alone could hardly exert its effect on the respective phenotypes. Additionally, nicotinamide (NAM) supplementation, as the precursor of NAD (including NAD+ and NADH), was counteracting the effects of SLC25A1i or the combination of SLC25A1i with αKG, emphasizing a potential importance of NAD+/NADH equilibrium on cellular activities relevant for survival of irradiated cancer cells upon SLC25A1i. Furthermore, inhibition of histone lysine demethylases (KDMs), as a major factor affected upon SLC25A1i, by JIB04 treatment alone or in combination with αKG supplementation phenocopied the wide-ranging effects on mitochondrial and cellular function induced upon SLC25A1i. Collectively, αKG-supplementation potentiated the effects on cellular processes observed upon SLC25A1i and increased cellular need for NAD to re-balance the cellular state and ensure survival upon irradiation. Future studies will uncover the underlying metabolic reprogramming induced by SLC25A1i offering novel therapeutic strategies for cancer treatment.

Biological sciences/Cancer/Cancer therapy/Radiotherapy

Biological sciences/Cell biology/Mechanisms of disease

α-ketoglutarate (αKG)

SLC25A1

radiotherapy

DNA repair

nicotinamide (NAM)

JIB-04

As an indispensable part of organism’s biological activity, cellular metabolism is composed of the interaction of a series of metabolites to meet the needs of growth and homeostasis. In malignant cells, cellular metabolism acquires metabolic reprogramming to adapt to the growing demands and alterations of redox homeostasis, which are crucial for cell proliferation, metastasis and survival upon applied treatment regimens [1-4]. During and after the transformation to cancerous states, cells tend to rewire cellular metabolism to satisfy the growing demands of cell growth and proliferation [5]. Due to the diversity of possible metabolic changes and the diverse interconnections of metabolic pathways of cancer cells, it appears to be challenging to describe an accurate static model of altered tumor metabolism predicting the overall state of metabolic changes which support cancerous cell growth [6]. Therefore, concentration on central metabolic processes may represent a strategy to define cancer cell dependent metabolic needs as well as treatment-induced metabolic phenotypes [4]. Here, mitochondria represent important organelle on the one hand providing cells with energy and building blocks, on the other hand, regulating cellular activity through both, modification of redox homeostasis and oncogenic signals [7]. Furthermore, abnormal production of metabolites present in cancer cells but not in normal cells were described to contribute to cancer initiation and progression [8]. It has been well studied that the aberrant production of 2-hydroxyglutarate (2-HG), succinate and fumarate was able to induce cancer initiation and progression thereby linking these oncometabolites to cellular metabolic reprogramming and disturbance of biological processes [8-10]. However, the accumulation of oncometabolites was linked to mutations in respective producing enzymes (e.g. fumarate hydrotase (FH), Succinate dehydrogenase (SDH) or isocitrate dehydrogenase (IDH)) [8]. Interestingly, our previous work revealed a strategy to induce an accumulation of 2-HG as a common phenotype by inhibiting the mitochondrial citrate carrier SLC25A1 in cancer cells without somatic mutation of IDH [11, 12]. SLC25A1 inhibition (SLC25A1i) created a phenotype represented by reduced repair of radiation-induced DNA double strand breaks (DSB) and survival upon radiotherapy (RT) [12]. In more detail, SLC25A1i affected the repair of lethal DNA lesions introduced by ionizing radiation (IR) treatment presumably by inducing accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) and associated restriction of homologous recombination (HR) repair [12]. Furthermore, targeting SLC25A1 indicated vulnerability of cancer cells to inhibition of poly(ADP-ribose)-polymerase (PARP)1 or the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in combination with IR suggesting a window for therapeutic opportunity [12]. Interestingly, SLC25A1i affected not only the DNA repair, but also reduced the abundance of cellular NAD-levels as well as of mitochondrial function [12].

In order to interfere with metabolic alterations induced by SLC25A1i, we used α-ketoglutarate (αKG)- and nicotinamide (NAM)- supplementation as a strategy to reverse the phenotype observed upon SLC25A1i and tested its suitability to affect biological activities of cancer cells alone or in combination with irradiation. Additionally, we used JIB04 as a pan inhibitor of histone lysine demethylases (KDMs) to recapitulate part of the observed phenotype induced by SLC25A1i.

2.1. Cell culture and reagents

Human NSCLC cell line NCI-H460 was cultured in DMEM (+D-Glucose, +L-Glutamine, -Pyruvate) media supplemented with 10% FBS and 1% Penicillin-Streptomycin (Sigma-Aldrich) at the condition of humidified incubator at 37°C and 5% CO₂. This cell line was obtained from ATCC (Bethesda, MD, USA) and was tested for mycoplasma regularly. All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) if not stated otherwise.

2.2. Quantification of D-2HG

D-2-Hydroxyglutarate (D-2HG) Assay Kit (Colorimetric) (BioVision, Milpitas, CA, USA) was applied to quantify the intracellular D-2HG levels as previously described [11, 12]. In details, 10 million cells were homogenized, lysed and spun down. Supernatant was collected and transferred to 96-well plate followed by measuring the enzymatic conversion of D-2HG to αKG which could interact with the probe and produce a detectable colored product. Absorbance at 450nm was measured with a BioTek Synergy H1 Microplate reader (BioTek Instruments, Inc., Winooski, VT, USA)

2.3. Irradiation

Irradiation was performed as previously described [2, 11, 13, 14]. In brief, cells were irradiated at room temperature with an X-ray machine (Precision X-ray Inc., North Branford, CT, USA) operated at 320kV, 12.5mA with a 1.65mm Al filter, at a distance of 50cm and a dose rate of 3.71Gy/min. Cells were returned to the incubator immediately after exposure to ionizing radiation (IR).

2.4. Alkaline comet assay

To quantify the DNA damage levels, alkaline comet assay was conducted as described previously [12] . Cells were plated in 12 well plates in a triplicate manner at a cell density of 200.000 cells per well. Treatments were administrated 24h after plating at different concentrations. Slides were covered with 1% low melting point (LMP) agarose to form the first layer for gel retention. The second or cell-containing layer was a mixture of 30% cell containing media and 70% 1% LMP agarose. Slides were placed in a lysis solution (Content:1.2M NaCl,100mM Na₂EDTA, 0.1% sodium lauryl sarcosinate and 0.26M NaOH, pH>13) for 1h at 4°C after the agarose gel became solid. Then the slides were put into freshly prepared alkaline electrophoresis solution (Content: 2mM Na₂EDTA and 0.03M NaOH, pH=12.3) for 10 minutes before electrophoresis. Electrophoresis was conducted for 1 hour at 20V. After that, slides were immersed in water and 100% ethanol to drain excess electrophoresis solution. Propidium iodide (PI) was used to detect the DNA under fluorescence microscopy.

2.5. Colony formation assay

The effect of different treatments on long-term survival was analyzed by clonogenic survival analysis as described previously [2, 12]. Different densities (200, 400, 800, 1600, 3200 cells per well) were plated into the 6-well plates and treated with indicated concentrations of drugs 24 hours later. Radiation was initiated 2 hours after drug treatment at 2Gy, 5Gy, 8Gy separately. Non-irradiated cells were sham irradiated at room temperature for the same period of time as their irradiated counterparts. 8-10 days later, colonies were stained with methanol containing 0.1% (w/v) Coomassie Blue dye and counted manually.

2.6. Flow cytometry analysis

30.000 cells were plated in 6 well plates 24 hours before treatment. The supernatant of cells was collected in flow cytometry tubes before trypsinizing the cells with accutase (PAN Biotech, Germany). Detached cells were equally aliquoted into 3 flow cytometry tubes before spun down (1.500 rpm, 5min) and the supernatant was discarded. Then the cells were stained with following staining solutions separately for different purpose: a) Cytoplasmic ROS levels: 0.5nM DHE diluted in PBS; b) Mitochondrial ROS levels: 5μM MitoSOX-Staining Solution (Invitrogen, USA) diluted in DMEM (+D-Glucose, +L-Glutamine, −Pyruvate) media; c) Apoptosis: 5µg/ml PI diluted in hypotonic buffer (0,05% Triton X-100+0,1% Natriumcitrat in PBS); d) Cell death: 1µg/ml PI diluted in PBS; e) γH2AX antibody was diluted at the ratio of 1:100 in permeable buffer before staining the cells, as previously described [12]. CytoFLEX Flow Cytometer (Beckman Coulter, Inc. U.S.A) was employed to evaluate samples.

2.7. Crystal violet assay

To quantify the alteration of cell proliferation/viability induced by treatments, crystal violet assay was applied as previously described [12]. In brief, 5.000 cells per well were seeded into a 96 well plate and incubated 24h at 37°C before treatment. Media was discarded before fixation with 1% glutaraldehyde which followed by adding 0.1% crystal violet staining solution. 0.2% Triton-X 100 was applied to lyse the cells. At last, absorbance at 540nm was measured by using the BioTek Synergy H1 Microplate reader (BioTek Instruments, Inc., Winooski, VT, USA)

2.8. Cell redox state determination

NAD⁺, NADP⁺, NADH and NADPH levels, as well as NAD⁺/NADH and NADP⁺/NADPH ratios were determined using the NAD/NADH-Glo™ and NADP/NADPH-Glo™ Assays kits (Promega, USA) according to manufacturer`s protocol. Briefly, 10.000 cells were plated in a 96 well plate 24h before treatment. Measurement procedure started with cell lysis followed by analyzing NAD(P)⁺ or NAD(P)H separately. The ratio of NAD⁺/NADH and NADP⁺/NADPH were calculated based on the instruction of the kits, as previously described [12].

2.9. Mitochondrial function analysis (Seahorse technology)

In accordance with the previously described steps, we seeded 10.000-15.000 cells in each well, except the four corners for background correction, of a seahorse XF 96 well plate and incubated the plate at 37°C in 5% CO₂overnight. The cell culture medium was replaced with 180 μl of Seahorse XF DMEM Media (Seahorse XF DMEM Media (with HEPE) + 1mM Pyruvate, 2mM Glutamine, 10mM Glucose) and cells were incubated at 37°C in a CO₂ free incubator for 45 min before measurement. Oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) were measured by Seahorse XF96 Analyzer (Agilent, Santa Clara, USA). OCR was determined at four Consecutive steps: (1) without any treatment, (2) oligomycin (1μM), (3) carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 2μM), (4) Rotenone and Antimycin A (0.5μM). For individual well normalization of cell number, DNA content fluorescence was measured after cells were stained with 10μg/mL Hoechst 33342 (Sigma-Aldrich) solution after each assay. Data were analyzed using Wave 2.6.1 software (Agilent Technologies). All metabolic parameters were normalized to Hoechst intensity (relative fluorescence units, RFU) in each well as previously described [4, 11-13].

2.10. Tumor growth assessment using an in vivo CAM-model

The chick embryo chorioallantoic membrane (CAM) assay was utilized as an in vivo model to explore the effect of indicated treatments on the tumor growth of NCI-H460 cells. Chicken eggs were incubated in the environment of relative air humidity of 65% and a temperature of 37°C with and automatically turned 4 times a day for 10 days before grafting to ensure proper Embryo development. On the grafting day, large vessel area was marked by candling the eggshell before opening a “window”. A hole to lower the CAM was created at the bottom of the eggs with scissor and widened with tweezers. The chosen window was opened by using a drill. Two million cells were resolved in 50μL PBS and pipetted onto the CAM of the chicken eggs and the window was sealed with tape. Seven days after grafting, the tumor was dissected and its diameters were measured, as previously described [12, 15, 16].

Statistical analysis was performed by using GraphPad Prism 7.0. Calculations of various formulas was performed by using Microsoft Excel 2019. Experiments were repeated 3 times. Assuming a normal distribution, statistical significances were calculated. For this, either the unpaired Students t-test or the two-way ANOVA (analysis of variance, means comparison test) with a post-hoc test according to Bonferroni was applied. The confidence interval was set to 95%. The significance level was set at α = 0.05 (equivalent to 5%), i.e., the difference between two data sets was considered significant if the p value was ≤0.05. Significances were marked with asterisks (*) in the Figures. Here, *p<0.05 stands for significant, **p<0.01 for highly significant, ***p<0.001 for extremely significant and ****p<0.0001 for most significant, ns for non-significant.

-ketoglutarate (αKG) -supplementation potentiated DNA damage and tumor growth delay of CTPI2-treated NCI-H460 cells.

Since D-2HG acts as competitive inhibitor of α-ketoglutarate dependent dioxygenases (αKGDD) by replacing αKG as a substrate, we hypothesized that αKG-supplementation upon SLC25A1i or octyl-D-2HG-treatment could reverse or save observed effects on DNA damage response and cell function alteration induced by respective treatments. In our previous study, we have found that the 3^rd generation small molecule inhibitor of SLC25A1, CTPI2, induced D-2HG accumulation, thereby affecting homologous recombination repair (HRR) [12]. In this study, we first supplemented αKG to NCI-H460 lung cancer cell line treated with CTPI2 in an attempt to modulate the D-2HG production. Here, αKG-supplementation alone had no significant influence on D-2HG production of NCI-H460 cell line (Fig. 1a). Surprisingly, D-2HG production induced by CTPI2 treatment was significantly amplified by additional αKG-supplementation (Fig. 1a). Consistent with the increased accumulation of D-2HG, additional αKG-supplementation in combination with CTPI2-treatment significantly potentiated the induction of radiation-induced DNA damage 6h after irradiation as determined by the alkaline comet assay (Fig. 1b). Again, αKG-supplementation alone exerted no significant effect on the radiation-induced DNA damage (Fig. 1b). Testing, whether the observed DNA damage induction by αKG-supplementation is a consequence of D-2HG accumulation, we applied cell permeable octyl-D-2HG treatment alone as previously described [12] and in combination with additional αKG-supplementation. Here, αKG-supplementation potentiated radiation-induced DNA damage in NCI-H460 cell line upon octyl-D-2HG-treatment (Fig. 1b) hinting to D-2HG related mechanisms potentiated by αKG-treatment.

Next, we compared the ability of NCI-H460 cells to repair radiation-indued DSBs upon CTPI2 or octyl-D-2HG treatment alone or in combination with αKG-supplementation by quantifying radiation-induced γ-H2AX foci using flow cytometry as previously described [12]. Here, αKG-supplementation in combination with CTPI2-treatment further enhanced the γ-H2AX signal induced by CTPI2 treatment alone at 6h timepoint after irradiation with a dose of 5Gy (Fig. 1c). Nevertheless, increased levels of radiation-induced γ-H2AX signal induced by octyl-D-2HG treatment at 6h time point post-irradiation with a dose of 5Gy were not increased by additional αKG-supplementation, hinting to more complex metabolic reprogramming induced upon CTPI2 treatment compared to octyl-D-2HG treatment (Fig. 1c). Next, we employed the CAM model as a proof-of-concept platform for validation of αKG-induced phenotype potentiation observed upon CTPI2-inhibition in vivo.

In order to explore the ability of αKG-supplementation to enhance the tumor growth reduction of NCI-H460 cancer cells induced by CTPI2-treatment in vivo, we used the well-described chick embryo chorioallantoic membrane (CAM) model as previously reported [12, 15, 17, 18]. Here, additional αKG-supplementation further reduced tumor volume of CTPI2-treated NCI-H460 cells (Fig. 1d, e). Noteworthy, additional application of IR further potentiated the reduction of tumor volume in NCI-H460 cells treated with both, CTPI2 and αKG (Fig. 1e). Interestingly, in case of octyl-D-2HG treated tumors, αKG-supplementation had no additional effect on tumor growth of NCI-H460 cells without IR and displayed tendencies towards increased tumor volume in combination with IR (Fig. 1e). Taken together our results hint to a complex metabolic reprogramming induced upon CTPI2 treatment compared to octyl-D-2HG treatment.

Disturbance of cellular and mitochondrial function induced by CTPI2 treatment is enhanced in combination with α-ketoglutarate (αKG) - supplementation in NCI-H460 cells.

To understand the mechanism behind the effect of αKG-supplementation on the DNA damage response and even tumor growth reduction when combined with octyl-D-2HG or CTPI2, short-term effects of respective treatments on the cell function were investigated. Analysis of cytoplasmic ROS levels by flow cytometry 6h after respective treatments alone or in combination with IR using a dose of 5Gy revealed highest and significant increase in cytoplasmic ROS-levels upon combinatory treatment of αKG and octyl-D-2HG without IR in NCI-H460 cells (Fig. 2a). Similar tendency to increased cytoplasmic ROS levels was also observed upon combinatory treatment with CTPI2 and αKG, though with lower absolute levels of cytoplasmic ROS-positive cells without IR (Fig. 2a). When adding IR with a single radiation dose of 5Gy in combination with CTPI2 and αKG treated NCI-H460 cancer cells, cytoplasmic ROS levels were increased, whereas no significant effect was observed with combination of octyl-D-2HG and αKG-supplementation (Fig. 2a). Next, the mitochondrial ROS levels were assessed by MitoSOX staining 24h after treatments. Again, αKG-supplementation alone exerted no significant effect on mitochondrial ROS levels compared to the non-treated control group (Fig. 2b). In line with cytoplasmic ROS, αKG-supplementation strongly potentiated mitochondrial ROS production in NCI-H460 cells pre-treated with CTPI2 (Fig. 2b). Contrary to the potentiating effects on the cytoplasmatic ROS production, αKG-supplementation in combination with octyl-D-2HG even reduced mitochondrial ROS levels either with or without IR (Fig. 2b). Significant increase in cytoplasmatic or mitochondrial ROS levels might result in apoptosis and cell death induction [12]. Here, αKG-supplementation only potentiated the apoptosis (Fig. S1a) and cell death-levels (Fig. S1b) induced by CTPI2-treatment in both, irradiated and non-irradiated NCI-H460 cells. No additional effect on apoptosis or cell death levels was observed, when supplementing octyl-D-2HG treated NCI-H460 cells with αKG (Fig. S1a, b). It was surprising to observe that treatment with αKG influenced cell death levels in NCI-H460 cells when combined with IR, whereas αKG treatment was not cytotoxic without IR (Fig. S1b).

Since CTPI2 exerts its function on the mitochondrial citrate carrier, the mitochondrial function was measured by using extracellular flux analyser (Seahorse-Analyzer). Here, treating the NCI-H460 cancer cells with αKG alone exerted no significant effect on the mitochondrial basal respiration (Fig. 2c). Again, CTPI2 or octyl-D-2HG treatment alone or in combination with IR, reduced measured basal respiration (Fig. 2c). Again, supplementation of αKG reduced mitochondrial function only in CTPI2 pre-treated NCI-H460 cells, whereas αKG- supplementation exerted no effect on mitochondrial function in combination with octyl-D-2HG (Fig. 2c). Consistent results upon respective treatments were observed on mitochondrial maximal respiration (Fig. S1c) and mitochondrial ATP production (Fig. S1d). Reduction of mitochondrial respiration was associated with observed increase in ROS-production and induction of cell death in NCI-H460 cells [4, 12].

To explain the differences observed on mitochondrial function induced by additional αKG-supplementation, the balance of NAD⁺/NADH and NADP⁺/NADPH ratios was investigated upon the respective treatments. Additional αKG-supplementation in CTPI2-treated NCI-H460 cells significantly increased the ratios of NAD⁺/NADH towards oxidative state, whereas no significant effect was observed in octyl-D-2HG-treated cells (Fig. 2d). Additionally, no significant change in NADP⁺/NADPH ratios was determined upon additional αKG-supplementation in CTPI2- or octyl-D-2HG-treated NCI-H460 cells (Fig. 2e). This was in consistent with the potential of αKG-supplementation to enhance mitochondrial ROS production in CTPI2-treated NCI-H460 cells (Fig. 2b). However, αKG-supplementation in octyl-D-2HG-treated cells displayed a tendency of NAD⁺/NADH or NADP⁺/NADPH ratios toward the oxidative state, which was observed in only octyl-D-2HG treated NCI-H460 cells (Fig. 2d, e). Remarkably, the applied treatments and their combinations decreased the relative amounts of of NAD⁺, NADH, NADP⁺ and NADPH with a higher tendency on the respective reductive form (Fig. S1e, f). Reduction of redox or energy carrier molecules has already been linked to affect cellular proliferation [19, 20]. Here, αKG-supplementation in combination with CTPI2 further reduced cell viability/proliferation of non-irradiated or irradiated NCI-H460 cells already 24h after respective treatments (Fig. 2f). Again, αKG-supplementation in the context of octyl-D-2HG treated NCI-H460 cells exerted no additional effects on cell viability/proliferation reduction in NCI-H460 cells line (Fig. 2f).

Nicotinamide (NAM)-supplementation rescued mitochondrial function and reduced DNA damage in CTPI2-treated NCI-H460 cancer cells.

As demonstrated in the current study, CTPI2 treatment alone, or in combination with αKG-supplementation increased the ratio of NAD⁺/NADH by decreasing the relative amount of NAD⁺ and NADH (Fig. 2d, S1e). Since nicotinamide (NAM) is the precursor of NAD⁺ [21], we hypothesized that NAM-supplementation could shift the ratio of NAD⁺/NADH to reductive state, restore the decline in NAD⁺ levels observed upon CTPI2 or octyl-D-2HG-treatments, restore the mitochondrial function and thus rescue radiation-induced DNA damage.

Assessing the DNA damage by flow cytometric measurement of γ-H2AX signal revealed that supplementation of NAM was able to overcome DNA damage induction upon single or combined treatment approaches using CTPI2 or octyl-D2-HG in combination with αKG treatment (Fig. 3a). However, NAM- supplementation did not exert any effects on only αKG-treated NCI-H460 cell line (Fig. 3a). In addition to DNA damage, NAM-supplementation also counteracted the effect of CTPI2 or octyl-D-2HG treatment, as well as its combination with αKG, on cytoplasmic ROS levels (Fig. 3b).

In case of mitochondrial ROS production induced upon both CTPI2 and CTPI2+αKG treatments, NAM-supplementation eliminated the mitochondrial ROS levels induced by the respective treatments (Fig. 3c). Unexpectedly, NAM-treatment potentiated the mitochondrial ROS-production of both octyl-D-2HG and octyl-D-2HG+αKG treated NCI-H460 cells (Fig. 3c). In line with the elimination of mitochondrial ROS, NAM-treatment was able to reduce apoptosis levels in CTPI2-treated and in CTPI2+αKG-treated NCI-H460 cells, whereas no effect was observed in octyl-D-2HG-, octyl-D-2HG+αKG-, αKG- or non-treated groups (Fig. 3d). Interestingly, NAM-treatment revealed a trend to reducing cell death levels in only CTPI2-treated group (p = 0.53) but only reached statistically significant differences in cells treated with CTPI2+αKG or octyl-D-2HG+αKG (Fig. 3e). In addition, we tested potential beneficial effects of NAM- supplementation upon CTPI2 or octyl-D-2HG-treatments on the basal mitochondrial function by using extracellular flux analyser. As illustrated in Fig. 3f, NAM-treatment for 24h recuperated the basal mitochondrial respiration almost to the level of untreated control group, which was inhibited in CTPI2 or CTPI2+αKG-treated NCI-H460 cells but had no effect on octyl-D-2HG-treated NCI-H460 cells alone or in combination with αKG- treatment (fig. 3f).

Furthermore, cell proliferation/viability analysis assessed by crystal violet assay further validated differences of NAM-supplementation in CTPI2- and octyl-D-2HG-treated NCI-H460 cells (Fig. 3g). In our study, reduced cell viability/proliferation induced by CTPI2 or CTPI+αKG-treatment was rescued by NAM-supplementation (Fig. 3g). However, no significant rescue effect by NAM-supplementation was observed in octyl-D-2HG or octyl-D-2HG+αKG treated groups underlining different alterations induced upon CTPI2 or octyl-D-2HG treatments (Fig. 3g). To further explore the mechanism behind the saving effects observed upon NAM-supplementation in CTPI2-treated NCI-H460 cells, relative amounts and ratio of NAD⁺/NADH were assessed. In line with our previous observations on cellular and mitochondrial function, NAM-supplementation only rescued the NAD⁺/NADH ratio in NCI-H460 cells treated with CTPI2 hinting to increased demand for NAD in CTPI2 treated NCI-H460 cells (Fig. 3h).

Inhibition of Histon-lysin-demethylases (KDMs) recapitulated the effects observed upon SLC25A1 inhibition by CTPI2.

Recent studies identified D-2HG accumulation as a result of mutation in isocitrate dehydrogenase (IDH) to affect the function of Histone-lysin demethylases 4B (KDM4B), a subgroup of αKG dependent dioxygenases (αKGDDs), and thereby disrupting local chromatin signalling and supressing DNA repair by HR [22]. In our recent study, we proposed a strategy for metabolic induction of a phenotype mimicking a defect of HR repair pathway (HRness) by targeting of SLC25A1 and respective inhibition of KDM4 due to induced accumulation of D-2HG [12]. SLC25A1i allowed to create context-dependent lethality in combination with inhibitors of end-joining (EJ) repair pathways like PARP in vitro and in vivo [12].

Based on this observation, we wondered whether direct KDM inhibition could recapitulate the functional phenotype induced by CTPI2 treatment. In order to mimic the inhibiting effect of CTPI2 on KDM, NCI-H460 cells were treated with JIB-04, a pan-inhibitor of KDMs. Similar to the effect induced by CTPI2-treatement, JIB-04-treatment stimulated radiation-induced γ-H2AX formation, which was further enhanced by additional αKG-supplementation (Fig. 4a). Accordingly, JIB-04-treatment induced cytoplasmic and mitochondrial ROS-levels, apoptosis levels and cell death levels of NCI-H460 cells without IR 24h after treatment, respectively (Fig. 4b-e). These effects were significantly enhanced when adding αKG (Fig. 4b-e). When the cells were treated with IR in addition to JIB-04 treatment alone or in combination with αKG, similar augmentation on ROS and cell death levels upon αKG+JIB-04 treatment was observed (Fig. 4b-e). As an exception, the apoptosis levels were not significantly altered upon the described treatments (Fig. 4d). Interestingly, inhibition of JIB-04 reduced basal mitochondrial respiration which was not significantly enhanced by αKG-supplementation (Fig. 4f). However, cell viability/proliferation was remarkably inhibited after 24h treatment with JIB-04, and the effect was more pronounced when combined with αKG treatment, no matter with or without IR (Fig. 4g). Taken together, inhibition of KDMs was able to recapitulate the effects on DNA repair, mitochondrial and cellular function induced by CTPI2 treatment hinting to KDM inhibition as one important factor contributing to cellular response observed upon SLC25A1i.

α-ketoglutarate (αKG) further radiosensitized NCI-H460 cancer cells treated with CTPI2.

Colony formation assay (CFA) was used to determine the long-term survival of tumor cells after treatment with ionizing radiation [23]. In the present study, the respective treatments were applied in combination with IR to assess the long-term radiosensitization effect in NCI-H460 cell line. Compared with the irradiated control group, the survival fraction was significantly decreased when the NCI-H460 cells were treated with CTPI2 in combination with αKG- supplementation (Fig. 5a, b, Fig. S2a-c). Furthermore, inhibition of SLC25A1 by CTPI2, of the KDMs by JIB-04 or octyl-D2-HG treatment significantly decreased the survival fraction (SF) of NCI-H460 cells irradiated with a dose of 5 or 8Gy (Fig. 5b, Fig. S2b, c). Again, the reduction of the survival fraction was potentiated by adding αKG (Fig. 5b, Fig. S2b, c). Interestingly, treatment with octyl-D-2HG in combination with αKG-supplementation rescued the survival fraction of irradiated NCI-H460 cancer cells compared to octyl-D-2HG treatment alone at irradiation doses of 5Gy and 8Gy (Fig. 5b, Fig. S2b, c). It was surprising to observe, that NAM-supplementation was able to increase the survival fraction of all indicated treatments, except for the octyl-D-2HG treatment alone or in combination with αKG-supplementation (Fig. 5b, Fig. S2b, c) hinting to increased cellular requirement for NAD for survival upon CTPI2 treatment in combination with IR. However, the pronounced saving effect of NAM-supplementation on the survival of IR-treated NCI-H460 cells was observed upon CTPI2+αKG treatment, compared to CTPI2 treatment alone (Fig. 5b). Our results strongly hint to global metabolic reprogramming induced by SLC25A1 inhibition alone and in combination with αKG-supplementation leading to increased cellular demand for NAD for survival upon IR (Fig. 5c). Therefore, metabolic reprogramming induced by CTPI2 treatment may offer an opportunity for radiosensitization in combination with NAD-producing pathways.

Aerobic glycolysis was reported by Warburg a century ago as a metabolic pathway used in tumor cells, drawing attention to the alterations in cancer metabolism [24, 25]. In the last two decades, the relationship between cancer metabolism and oncogenes was discovered and more extensively investigated [5, 26]. However, the development of targeted therapies related to metabolic alterations in cancers was limited within the past decade [27]. The combination of therapies which could represent one vulnerability for another has attracted considerable attention in recent years [28, 29]. The development of strategies combining radiotherapy (RT) with drugs targeting phenotype-specific metabolic vulnerabilities to increase the lethality of cancer cells to RT and overcome the radioresistance associated with metabolic deregulation has been proposed as a new area of research [30, 31].

Our group has reported that genetic (siRNA) or pharmacologic targeting of a mitochondrial citrate carrier SLC25A1 by CTPI2 results in accumulation of the oncometabolite D-2HG [11, 12]. Both CTPI2 and cell permeable D-2HG (octyl-D-2HG) altered cancer cell metabolism, thus increasing the sensitivity to RT [12]. As mentioned above, D-2HG is a competitive inhibitor of αKGDD, which uses O₂ and αKG as cofactors to perform a range of oxidation reactions, e.g. modification of chromatin or regulation of protein stability [8]. It has been reported that αKG is involved in numerous biological processes including anti-oxidative defence, energy production, signalling modules, and genetic modification [32]. Additionally, αKG has been used as a nutritional supplement and therapeutic agent, which means it has been examined and approved for clinical use [32].

Here, in the purpose of counterbalancing the metabolic effects induced by 2-HG-accumulation, αKG-supplementation was applied in our study. Surprisingly, supplementation of αKG promoted CTPI2-induced D-2HG production whereas αKG-treatment alone was unable to increase the concentration of D-2HG. The primary source for 2-HG production was described to be the abundance of αKG, which is used as a substrate to produce the 2-HG [33]. However, αKG-treatment alone was not capable of stimulating the production of D-2HG, implying the reaction conditions didn’t meet the requirements for D-2HG generation. Nevertheless, the combination of CTPI2-treatment in combination with αKG-supplementation potentiated the production of D-2HG induced upon CTPI2 treatment alone. Our surprising finding indicate that αKG-supplementation enhanced necessary conditions for 2-HG production induced by CTPI2 treatment. To further explore the effect of αKG-supplementation in combination with CTPI2-treatment on cell biological activities, the combinatory treatment was tested to investigate their ability to potentiate radiation-induced DNA damage, short-term cell function and cell proliferation as well as long-term survival.

Exploring the radiation-induced DNA damage by using the alkaline comet assay, αKG-treatment alone was unable to potentiate radiation-induced DNA damage compared to non-treated control group. However, αKG-supplementation significantly enhanced radiation-induced DNA damage in combination with CTPI2-treatment compared to CTPI2-treatment alone. Similar results were also observed upon octyl-D-2HG treatment alone and in combination with αKG-supplementation, implying that CTPI2-induced DNA damage in combination with ionizing irradiation (IR) was based on the effect of metabolically induced D-2HG production upon CTPI2 treatment. Slightly different results were detected by monitoring the removal of radiation-induced γ-H2AX signal, as a marker for DNA-DSBs [34]. Here, αKG-treatment alone stimulated the formation of IR-induced γ-H2AX signal and potentiated the amount of IR-induced γ-H2AX in combination with CTPI2-treatment. On the contrary, αKG-treatment in combination with IR did not enhance γ-H2AX formation induced by octyl-D-2HG-treatment, which indicated that CTPI2 and octyl-D-2HG acted differently on the repair of IR-induced DSBs [34]. In line with the observation on IR-induced DNA damage potentiated by αKG-supplementation in combination with CTPI2 treatment, the long-term effect on the survival of IR-treated NCI-H460 cancer cells measured by colony formation assay revealed that αKG-supplementation in combination with CTPI2 treatment potentiated radiosensitization of NCI-H460 lung cancer cells.

In contrast, αKG-supplementation saved the survival fraction of NCI-H460 cancer cells treated with octyl-D-2HG. This phenomenon may suggest that induction of DSBs upon IR, rather than overall DNA damage, correlate to the long-term survival of cancer cells upon IR and thus to radiosensitization as previously described by others [35, 36]. Although αKG-treatment enhanced the generation of cytoplasmic and mitochondrial ROS induced by CTPI2-treatment with or without IR, αKG-treatment alone did not have any significant effect on cytoplasmatic or mitochondrial ROS formation in NCI-H460 cancer cells. However, αKG-supplementation was able to potentiate the effects of CTPI2 treatment on short-term cell function (mitochondrial respiration, cell death, proliferation) and in vivo tumor growth (CAM-Assay), whereas αKG-supplementation alone had no effect. On the other hand, treating the NCI-H460 cancer cells with the oncometabolite octyl-D-2HG were not consistently modulating measured cellular function compared to CTPI2-treatment. This result underlined the broad multifactorial metabolic reprogramming induced by CTPI2-mediated inhibition of SLC25A1, among those accumulation of D-2HG seems to be one major mechanism affecting cellular function and DNA damage repair upon IR (Fig. 5c). Thus, treatment of cancer cells with octyl-D-2HG, might still allow the cancer cell to exchange citrate between the mitochondria and the cytosol, which seems to be less lethal for the cancer cell and thus less suitable for radiosensitization.

Analysing cellular viability/proliferation, αKG-treatment tended to counteract the proliferation suppression induced by octyl-D-2HG treatment alone, suggesting, that αKG-supplementation may recapture the binding ability to αKG-dependent dioxygenases (αKGDD) thus restoring their enzymatic function. However, again, αKG-supplementation was not able to abrogate the inhibition of proliferation induced by CTPI2 treatment, hinting to distinct effects of octyl-D-2HG application in the presence of functional SLC25A1 and the complex metabolic reprogramming induced upon inhibition of SLC25A1 by CTPI2 treatment.

Generally, SLC25A1 mediates trafficking of citrate between the mitochondria and cytosol, thereby supporting redox homeostasis and lipid metabolism [11, 37-39]. So far others reported that accumulation of the 2HG enantiomers L-2HG or D-2HG can occur under certain conditions as pathologic metabolites in hypoxic cancer cells produced by lactate dehydrogenase (LDH) or malate dehydrogenase (MDH) [33, 40, 41] or as so-called “oncometabolites” as a result of gain-of-function mutations in the genes coding for isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) [42, 43]. We described previously that inhibition of the citrate export into the cytosol by blocking SLC25A1 using CTPI2 treatment, lead to downregulation of cellular mitochondrial oxidation, accompanied ROS production and inhibited DNA repair by HR pathway through accumulation of D2-HG and accompanied inhibition of KDMs [12].

Others have linked the metabolic reprogramming of cancer cell to cellular metabolic phenotype and anabolic state by influencing epigenetic and genetic processes, thus activating oncogenic cascades [44]. Here, cellular energy metabolism, mitochondrial function as well as the cellular antioxidant systems are fundamentally regulated/affected by nicotinamide adenine dinucleotide, NAD (including NAD⁺ and NADH) and nicotinamide adenine dinucleotide phosphate, NADP (including NADP⁺ and NADPH) [45]. In particular, NAD⁺/NADH redox balance not only fuels oxidative phosphorylation (OXPHOS), but also triggers biosynthesis, particularly in glycolysis pathway and the tricarboxylic acid (TCA) cycle, where NAD⁺ is required as an electron receptor to maintain pathway flux of glycolysis [5]. Elevated NAD⁺ levels enhance glycolysis via glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which requires NAD⁺ as co-enzyme [46, 47]. Based on the multi-facetted and pathway connecting role of NAD within the cell, the rate limiting enzyme for NAD synthesis, nicotinamide phosphoribosyl transferase (NAMPT), has been identified as one target for tumor therapy [48, 49]. NAMPT inhibitors, like FK866, reduced NAD levels and abrogated cancer cell proliferation by interfering with energy production pathways [49]. Cells with active mitochondrial oxidation require NADH to drive ATP synthesis by electron transport chain (ETC) [50]. The ETC is the main utilizer of NADH so that dysfunction of the ETC leads to accumulation of mitochondrial and cytosolic NADH [33, 41, 51]. Under these conditions, activity of the malate dehydrogenase MDH, which is stimulated by αKG accumulation, might help the cells to avoid accumulation of excess NADH in the cytosol, by MDH-dependent reduction of aKG to 2-HG that is associated with NADH-oxidation [33, 40, 52]. In our study, aKG-supplementation potentiated the accumulation of D2-HG in CTPI2-treated cells, possibly by activation the MDH-dependent reduction of aKG to 2-HG to increase the oxidation of NADH.

Both treatments with CTPI2 or octyl-D2-HG, as well as in combination with aKG- supplementation to CTPI2, shifted the NAD⁺/NADH as well as the NADP⁺/NADPH ratios towards the oxidized form and additionally reduced the amount of NAD⁺, NADH, NADP⁺ and NADPH within the NCI-H460 cancer cells. The high demand of NAD⁺ as the electron acceptors is the common feature of proliferating cancer cells [5]. In an effort to save the decreasing level of NAD⁺ and other NAD-related species their precursor Nicotinamide (NAM) was added in our study. It was intriguing for us to further examine whether NAM-supplementation would save the effect of CTPI2 and octyl-D-2HG on remodelled biological activities of NCI-H460 cancer cells. NAM is an amide form of vitamin B3 and the precursor of NAD⁺, an essential co-enzyme of redox reactions for adenosine triphosphate (ATP) production and for several other metabolic processes [53]. In our study NAM-supplementation alleviated the amount of γ-H2AX signal thereby reducing the damaging effect induced by CTPI2- or CTPI2+αKG-treatments on the formation of radiation-induced DSBs. In addition, NAM-supplementation was able to rescue the radiosensitization of NCI-H460 cancer cells induced by treatment with CTPI2, octyl-D-2HG, CTPI2+αKG or octyl-D-2HG+αKG, to a different extent. These results are in accordance with documented effect of NAM-supplementation on genomic stability supported by the function of ATP-dependent DNA repair enzymes [53]. Furthermore, NAM-supplementation rescued the effects of CTPI2 alone or in combination with αKG-supplementation relieving induced cytoplasmic/mitochondrial ROS production, apoptosis-, cell death-levels, mitochondrial dysfunction and cell proliferation (Fig 5c). Our observations imply that decreasing amount of NAD⁺, NADH, NADP⁺ and NADPH may become critical for cells survival upon inhibition of SLC25A1 in NCI-H460 cancer cells.

In contrast, no consistent conclusion can be drawn from NAM-supplementation experiments in combination with octyl-D-2HG treatment. These observations corroborate the conclusion on distinct mechanisms induced by octyl-D-2HG treatment compared to CTPI2-treatment. Furthermore, NAM-supplementation was used in addition to CTPI2 or octyl-D-2HG to determine the ability of NAM to save the oxidative state induced by the latter two treatments. Here, NAM-supplementation only rescued the NAD-depletion induced by CTPI2 treatment. NAM-supplementation was able to restore cellular dysfunction regarding DNA repair, ROS production and cell death induction which was induced by CTPI2 treatment. These observations implied that shifting back the ratio between NAD⁺/NADH to a more reductive state upon CTPI2 treatment is important for the survival of NCI-H460 cancer cells upon irradiation.

The salvage pathway, whose rate-limiting enzyme is nicotinamide phosphoribosyl transferase (NAMPT), is the main route of NAD⁺ synthesis from NAM, which is first step of downstream NADH, NADP⁺, NADPH production [53]. Recent study revealed that the inhibition of NAMPT disturbed cell proliferation, mitochondrial function and DNA damage response, which are in accordance with the effects induced by CTPI2 in our study [54]. This phenomenon further emphasizes the role of NAMPT on the disturbance of NAD⁺/NADH ratio induced by CTPI2 and requires further investigations.

A potential involvement of KDM inhibition in the proposed mechanism of action on the disturbance of DNA repair by D-2HG accumulation was examined by direct inhibition of KDMs by using a pan KDM-inhibitor JIB-04 alone or in combination with αKG-supplementation in order to mimic the part of the effects observed upon CTPI2 treatment (Fig 5c). JIB-04 inhibits the demethylase activity of Jumonji enzymes, one of the major KDM sub-families in the cell, without affecting αKG-dependent prolyl hydroxylases and TET enzymes or other chromatin-modifying enzymes such as histone deacetylases [55, 56]. Treatment with JIB-04 increased radiation-induced γ-H2AX accumulation at 6h after IR. These results further consolidate the ability of the JIB-04-treatment to impair the removal of radiation-induced γ-H2AX and thus potentially impair the repair of radiation-induced DNA damage [57].

Furthermore, the present study provides first evidence on a combinatorial effect of KDM-inhibition by JIB-04 and additional αKG-supplementation for radiosensitization as well as the ability of NAM-supplementation to rescue the survival of irradiated NCI-H460 cancer cells upon those treatments. JIB-04-treatment was suggested for application in clinical trial since it altered transcriptional growth programs in cancer cells but not in normal cells, resulting in cancer-specific cell death induction [56, 57]. In accordance with the reported results, JIB-04 treatment increased the level of cell death and reduced cell proliferation, with or without IR in the present study. Of note, the antineoplastic effects of JIB-04-treatment recapitulated the phenotype induced by CTPI2-treatment and was significantly enhanced by αKG-supplementation which is consistent with the findings obtained upon CTPI2 treatment.

In conclusion, the present study describes the synergistic effect of αKG-supplementation in combination with SLC25A1 inhibition on cellular and mitochondrial function creating the cellular need for NAD to balance cellular activities important for the survival of cancer cells and radiosensitization. Furthermore, our results provide a new angle for understanding the novel context-dependent role αKG in the cancer progression and the treatment of cancer.

AUTHOR CONTRIBUTIONS

J.M. designed and conceptualized the research; KX., MK., SL., JM. performed experiments, analyzed, validated, and visualized the results; M.B. and N.D. provided technology and equipment; KX., J.M. wrote the original manuscript draft. J.M. and V.J. supervised the work; V.J. and J.M. acquired the funding. All authors critically revised, edited, and approved the final version of the manuscript.

ACKNOWLEDGMENTS

We would like to thank Prof. Dr. George Iliakis (Institute of Medical Radiation Biology, Essen, Germany) for the opportunity to use the X-RAD 320 X-Ray Biological Irradiator. Furthermore, we thank the Dünker lab for providing their CAM facility equipment and technical support. Additionally, we thank Brigitte und Dr. Konstanze Wegener-Stiftung for funding of the Seahorse XFe 96 analyzer. We thank Angelika Warda for support with the STR analysis. The work was supported by grants of the German Research Foundation DFG GRK1739/2 to V.J., and MA 8970/1-1 to J.M., the Federal Ministry of Education and Research (BMBF, 02NUK061B) to J.M., the European Union’s Framework Program for Research and Innovation Horizon 2020 (2014-2020) under Marie Skłodowska-Curie (Grant Agreements No. 860245 (ITN THERADNET) to J.M. and V.J.), the China Cooperation Office of the Medical Faculty of the University of Duisburg-Essen within the frame of a research scholarship to K.X. and the Internal Discipline Development Grant - Department of Gastroenterology of Chongqing University Cancer Hospital (Y133) to K.X.

DECLARATION OF INTRESTS

The authors declare no competing interests.

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α-Ketoglutarate Supplementation and NAD+ Modulation Enhance Metabolic Rewiring and Radiosensitization in SLC25A1 Inhibited Cancer Cells

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Abstract

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1. Introduction

2 Materials And Methods

2.1. Cell culture and reagents

2.2. Quantification of D-2HG

2.3. Irradiation

2.4. Alkaline comet assay

2.5. Colony formation assay

2.6. Flow cytometry analysis

2.7. Crystal violet assay

2.8. Cell redox state determination

2.9. Mitochondrial function analysis (Seahorse technology)

2.10. Tumor growth assessment using an in vivo CAM-model

3. Results

4. Discussion

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