MYCN-Amplified Neuroblastoma Is Addicted to Iron and Vulnerable to Inhibition of the System Xc-/Glutathione Axis

While evasion of apoptosis has been recognized as a hallmark of cancer for 50 years, the involvement of other recognized modes of programmed cell death remains understudied. Ferroptosis is a cell death program identified by Stockwell and colleagues in 2012 (1) that is characterized by accumulation of iron (Fe)-dependent reactive oxygen species (ROS) causing lipid peroxidation–related cell death. Morphologically, it is defined by smaller mitochondria, increased density of the mitochondrial membrane, vanishing of the mitochondrial cristae, rupture of the outer mitochondrial membrane, and cellular disintegration (2). This form of cell death appears particularly relevant to cancer cells as iron accumulation allows for high activity of iron- and heme-containing enzymes that play roles in diverse processes from cell-cycle progression to cellular metabolism (3). For instance, DNA polymerases require iron (4), as do enzymes involved in mitochondrial respiration and the citric acid cycle (5), and ribonucleotide reductase (6). Indeed, oncogenes like HIF1A can increase iron metabolism (3). Increased iron metabolism, however, is a double-edged sword as it is a major ROS producer through the Fenton reaction, the donation of an electron from ferrous iron to hydrogen peroxide to produce hydroxyl radicals (OH-; ref. 7). Hydroxyl radicals are counteracted by glutathione peroxidase 4 (GPX4), a powerful cellular ROS scavenger that utilizes glutathione (GSH) to directly prevent lipid peroxidation – the oxidative degradation of lipids. Lipid peroxidation results in ferroptotic cell death (8), and, therefore, GPX4 is the central preventive molecule of this process (9, 10).

While RAS was originally connected to the activity of the ferroptosis inducer erastin (11), interactions of oncogenes with the ferroptotic pathway have not been well characterized. High-risk neuroblastoma accounts for upwards of 15% of all pediatric cancer–related deaths, despite comprehensive therapy. Among high-risk cases are about one-fourth with amplification of the oncogenic MYCN. MYCN encodes an E-BOX–binding, basic-helix-leucine zipper (bHLH-LZ) transcription factor (12). Despite its undeniable role in neuroblastomagenesis, MYCN remains undruggable. Here in, we uncover a druggable target in MYCN-amplified neuroblastoma. We find MYCN drives a transcriptional rewiring of the iron import receptor and system Xc-, creating a marked reliance on the GSH pathway. We find that MYCN-amplified neuroblastomas are exquisitely sensitive to targeting this pathway either chemically or genetically and that this pathway is activated by MYCN, at least in part, to detoxify ROS from excess iron import. As such, we find repurposing FDA-approved rheumatoid arthritis drug auranofin and the rheumatoid arthritis and ulcerative colitis drug SAS are effective against MYCN-amplified neuroblastoma, via targeting antioxidant activity.

The cell lines RPE.1, IMR5, SIMA, SMS-SAN, LAN5, NB12, SK-N-BE(2), SK-N-SH, SK-N-DZ, SK-N-AS, and KELLY were from the Molecular Center Therapeutics Laboratory at Massachusetts General Hospital (Boston, MA), which performs routine testing of cell lines by single-nucleotide polymorphism and short tandem repeat analysis. The SK-N-FI cell line was provided by the Children's Hospital of Pennsylvania (Y. Mossé). COG-N-415, COG-N-496, CHLA20, and CHLA172 were kindly provided by the Children's Oncology Group (COG) Cell Culture and Xenograft Repository (C. Pat Reynolds and the Texas Tech Health Sciences Center, Lubbock, TX), powered by Alex's Lemonade Stand Foundation. The SK-N-BE(2), SK-N-AS, SK-N-SH, IMR5, SK-N-DZ and SK-N-FI cell lines were cultured in DMEM/F12 (50:50) supplemented with 10% FBS, 1 μg/mL penicillin and streptomycin. The RPE.1, SIMA, KELLY, NB12, LAN5, and SMS-SAN cell lines were cultured in RPMI1640 supplemented with 10% FBS, 1 μg/mL penicillin and streptomycin. The CHLA20 and CHLA172 cell lines were cultured in DMEM supplemented with 20% FBS, 1× insulin-transferrin-selenium (ITS; 41400045; Thermo Fisher Scientific), and 1 μg/mL penicillin and streptomycin. The COG-N-415 and COG-N-496 cell lines were cultured in Iscove's modified Dulbecco's medium supplemented with 20% FBS, 1× ITS, and 1 μg/mL penicillin and streptomycin. Cell lines were used for less than 40 passages but were not independently authenticated. They were regularly screened for Mycoplasma using a MycoAlert Mycoplasma Detection Kit (LT07–318; Lonza).

NOD CRISPR Prkdc Il2r Gamma (NCG) male mice were injected with approximately 5 × 10⁶ IMR5 or SK-N-SH cells per 200 μL of 1:1 [cells: Matrigel; (354248; Corning)]. Cells were injected on both flanks of the mice and monitored for tumor growth. When tumors reached an average size of 150–200 mm³, the tumor-bearing mice were randomized (IMR5: no treatment = 4 tumors, auranofin = 6 tumors, sulfasalazine = 3 tumors; SK-N-SH: no treatment = 4 tumors, auranofin = 4 tumors). The tumors were measured every other day by electronic caliper, in two dimensions (length and width), with the formula v = l × (w)2 (π/6), where v is the tumor volume, l is the length, and w is the width (the smaller of the two measurements). Mice were treated with auranofin (10 mg/kg/qd, i.p.); 20 μL; DMSO) 6 days/week (Monday–Saturday) or sulfasalazine (250 mg/kg/qd, i.p.; 100 μL; PBS) 6 days/week (Monday–Saturday) for 13 days (IMR5) or 10 days (SK-N-SH) total. Mice bearing IMR5 or SK-N-SH cell xenografts were euthanized after the 13th and 10th day of the treatment, respectively, because some of the tumors of the “control” (no treatment) or auranofin cohort reached or exceeded the size of 1,000 mm³. All mouse experiments were approved and performed in accordance with the Institutional Animal Care and Use Committee at VCU (Richmond, VA).

The COG-N-561, COG-N-452, COG-N-415, and COG-N-496 patient-derived xenograft (PDX) models were kindly provided by the Children's Oncology Group (COG) Cell Culture and Xenograft Repository (C. Pat Reynolds and the Texas Tech Health Sciences Center, Lubbock, TX), powered by Alex's Lemonade Stand Foundation, and were injected into NOD CRISPR Prkdc Il2r Gamma (NCG) male mice at 2 × 10⁶ cells per flank on both flanks of the mouse using a 1:1 ratio of cells/Matrigel. Mice were randomized when tumors reached an average size of 150–250 mm³, (COG-N-561: no treatment = 6 tumors, auranofin = 6 tumors; COG-N-452: no treatment = 6 tumors, auranofin = 4 tumors; COG-N-415: no treatment = 7 tumors, sulfasalazine = 7 tumors, COG-N-496: no treatment = 6 tumors, auranofin = 3 tumors). Mice were treated with auranofin (10 mg/kg/qd, i.p.; 20 μL; DMSO) 6 days/week (Monday–Saturday) for 30 days (COG-N-561) for 16 days (COG-N-452), for 24 days (COG-N-415), or for 11 days (COG-N-496). Mice bearing COG-N-452 as well as COG-N-496 tumors were euthanized after the 16th and 11th day of the treatment, respectively, because some of the tumors of the “control” (no treatment) cohort reached (COG-N-452 model) or exceeded (COG-N-496 model) the size of 1,000 mm³. All animal experiments were approved by the Virginia Commonwealth University (VCU) Institutional Animal Care and Use Committee.

The phospholipid hydroperoxidase GPX4 serves as the primary protection of cells from ferroptotic cell death and lipid peroxidation (13), and its genetic inhibition results in ferroptosis (13–15). GPX4 depletion/ferroptosis induction has recently been reported to be a vulnerability of cells that undergo EMT (8, 15). Our investigations began when upon examining the Broad Institute depository of three genome-wide siRNA screens (Depmap) covering more than 700 cancer cell lines, we noted the highest toxicity observed following GPX4 knockdown was in neuroblastoma (Fig. 1A; Supplementary Fig. S1A); interestingly, most of the neuroblastoma cell lines had amplification of MYCN (Supplementary Fig. S1B).

To corroborate these data chemically and to begin to assess a possible role of MYCN in sensitivity, we analyzed within the Depmap consortium, screening data with the GPX4 chemical inhibitor, ML210 (9). Here, we found MYCN-amplified neuroblastoma cells were hypersensitive to pharmaceutical GPX4 inhibition (Fig. 1B), consistent with the genetics data. These data evidenced a sensitivity of MYCN-amplified neuroblastoma to GPX4 inhibition.

Upstream of GPX4 is the antioxidant GSH, which enables GPX4 by donating an electron. In turn, GSH synthesis is controlled primarily by the system Xc- receptor pathway. As MYCN-amplified neuroblastoma were highly represented among the neuroblastoma cancer cell lines in the GPX4 siRNA assay, we hypothesized that MYCN may create a particular vulnerability along the system Xc- pathway/GSH axis. To do so, we interrogated a panel of MYCN-amplified neuroblastoma cell lines and MYCN-wild-type neuroblastoma cell lines with buthionine-(S,R)-sulfoximine (BSO), a potent inhibitor of the rate-limiting GSH-synthesizing enzyme, gamma-glutamylcysteine synthetase (13, 16, 17). Of significance, we found enhanced sensitivity to BSO in the MYCN-amplified neuroblastoma cell line panel versus the wild-type panel (Fig. 1C). To directly assess whether MYCN sensitizes neuroblastoma cells to BSO, we expressed exogenous MYCN in MYCN-wild-type CHLA20 and SK-N-SH neuroblastoma cells. The presence of MYCN conferred synthetic lethality to BSO (Fig. 1D). We next knocked down MYCN with siRNA in two MYCN-amplified neuroblastoma cell lines, IMR5 and SK-N-BE(2). In both cases, we found a marked reduction in sensitivity to BSO when MYCN was reduced (Fig. 1E; Supplementary Fig. S1C). Consistent with on-target activity, BSO depleted GSH (Fig. 1F), induced ROS (Fig. 1G), and was protected by cotreatment with the antioxidant N-acetyl-l-cysteine (NAC) in MYCN-amplified neuroblastoma cell lines (Fig. 1H; Supplementary Fig. S1D). Moreover, BSO-mediated toxicity was protected by the ferroptosis and lipid peroxidation inhibitor a-tocopherol in MYCN-amplified neuroblastoma cell lines (Fig. 1I; Supplementary Fig. S1E), demonstrating on-target activity of ferroptotic cell death by BSO. These data indicated a vulnerability to GSH inhibition by MYCN in neuroblastoma, resulting in ferroptosis.

The import of cystine serves as the limiting step to synthesize GSH (cystine is reduced to cysteine in the cell; ref. 18). Our data above demonstrate MYCN confers sensitivity to GSH targeting in neuroblastoma. On the basis of these data, we hypothesized there is a likeliness that there is a significant influx of cystine that creates high activity of the GSH catabolic pathway. We looked at the two genes that encode the subunits of the system Xc- antiporter, as expression of the receptor is the key hub for controlling cystine import and subsequent activity of this pathway (2). Indeed, we found both genes encoding the heterodimeric system Xc- (SLC7A11 and SLC3A2) were markedly increased in MYCN-amplified neuroblastoma tumors versus MYCN-wild-type neuroblastoma tumors (Fig. 2A; ref. 19). As an oncogenic transcriptional factor, MYCN drives expression of target genes that participate in establishing and maintaining a cancerous phenotype. We reasoned because of the increase in expression of the system Xc- receptor subunits, that MYCN may directly bind to and promote transcription of the system Xc- receptor. For this, we parsed data from a study utilizing an elegant system of a MYCN-wild-type cell line (SHEP21; ref. 20). This system includes the SHEP21 parental cells, a MYCN-negative cell line, and the SHEP21N cells, modified to express exogenous MYCN, and which expression is lost with the addition of doxycycline (20). Indeed, there was marked direct binding of MYCN to SLC3A2 at canonical E-boxes in the SLC3A2 promoter in both systems (Fig. 2B), indicating direct transcriptional upregulation by MYCN. Binding of MYCN to the promoter of SLC7A11 was also present, although not as pronounced (Supplementary Fig. S2A). Consistent with the upregulation of the system Xc- receptor in MYCN-amplified neuroblastoma tumors and a direct upregulation of SLC3A2 by MYCN, exogenous MYCN expression in MYCN wild-type neuroblastoma cells and the RPE.1 neural crest cell line led to increased protein levels of both SLC7A11 and SLC3A2 (Fig. 2C, left). Conversely, silencing MYCN in the MYCN-amplified KELLY and SK-N-DZ cells results in a significant loss of SLC7A11 and SLC3A2 (Fig. 2C, right). While cystine uptake is rate-limiting to GSH synthesis (21), when intracellular cysteine is abundant, the glutamate–cysteine ligase, made up of the GCL catalytic subunit, GCLC, and a modifier subunit, becomes the rate-limiting step toward the biosynthesis of GSH (22). Again, consistent with increased flux through this pathway, we found MYCN-amplified neuroblastoma tumors had higher levels of GCLC compared with wild-type tumors (Supplementary Fig. S2B; refs. 19, 23), which coincided with increased binding of MYCN to the GCLC promoter (Supplementary Fig. S2C; ref. 20). Exogenous expression of MYCN was sufficient to increase GCLC expression, while knockdown of endogenous MYCN in the MYCN-amplified neuroblastoma cell lines was sufficient to reduce GCLC (Supplementary Fig. S2D).

Increased flux through the system Xc- pathway and increased GCLC suggested MYCN also increased GSH levels. Therefore, in our MYCN syngeneic models, we determined whether there was enhanced GSH production. Indeed, we found GSH levels were higher in the syngeneic models expressing exogenous MYCN and lower in the si MYCN-treated models, when compared with their respective controls (Fig. 2C–E; Supplementary Fig. S2D). Altogether, these data demonstrate that MYCN orchestrates increased system Xc- expression and subsequent flux through the system Xc-/GSH pathway.

We next sought to determine whether this translated to enhanced sensitivity of targeting the system Xc- directly. To do so, we first limited available cystine for the cells in culture, and measured viability in the syngeneic models. As demonstrated in Fig. 2F, cystine deprivation was substantially more toxic to neuroblastoma cells in the presence of MYCN. Consistent with an enhanced reliance on this pathway for survival, genetic inhibition of SLC3A2 and SLC7A11 was sufficient to induce toxicity in MYCN-amplified neuroblastoma lines (Supplementary Fig. S2E). To assay whether these data together would translate to a pharmaceutical vulnerability, we treated our panel of MYCN-amplified and MYCN-wild-type cells with SAS, a system Xc- receptor inhibitor (24). Similar to the results from BSO (Fig. 1C), we found MYCN-amplified neuroblastoma cells were considerably more sensitive than the MYCN-wild-type neuroblastoma cell lines following therapy with SAS (Fig. 3A), and SAS potently inhibited GSH levels in the MYCN-amplified neuroblastoma cells (Fig. 3B). Consistent with SAS depletion of GSH resulting in ferroptotic cell death, treatment with the ferroptosis inhibitors ferrostatin-1 or liproxstatin-1 resulted in mitigated toxicity (Fig. 3C). Consistent with the notion MYCN was driving sensitivity, we found enhanced sensitivity in the syngeneic models when MYCN was present (Fig. 3D), whereas silencing of MYCN resulted in profound resistance (Fig. 3E; Supplementary Fig. S2D); again, ferrostatin-1 prevented SAS toxicity in the si Control-treated MYCN-amplified neuroblastoma cells (Supplementary Fig. S2F–S2H). As SAS is FDA-approved, we evaluated its efficacy in vivo. SAS (250 mg/kg, every day) was sufficient to block tumor growth in two MYCN-amplified neuroblastoma models (Fig. 3F). In tumor tissue, SAS induced transferrin receptor 1 (TfR1), as well as malondialdehyde (MDA; Fig. 3G; Supplementary Fig. S3A), a combination of antibodies that are specific ferroptosis markers in vivo (25). In addition, no significant alterations were observed in the expression of SLC3A2 and SLC7A11 (Supplementary Fig. S3B). Altogether, MYCN increases system Xc- expression and pathway activation, resulting in a hypersensitivity of MYCN-amplified neuroblastoma to system Xc- receptor targeting.

These data indicated a new therapeutic vulnerability in MYCN-amplified neuroblastoma. We sought to understand the basis for enhanced system Xc- pathway activation and toxicity from genetic or pharmaceutical targeting in MYCN-amplified neuroblastoma. Increased expression of system Xc- suggests a particularly large need for MYCN-amplified neuroblastoma to detoxify ROS. Iron is a major contributor to increased cancer proliferation and survival (26); however, it is also detrimental to the cell as it is a major ROS producer through the Fenton reaction, the donation of an electron from ferrous iron to hydrogen peroxide to produce hydroxyl radicals (OH-). Indeed iron-induced ROS at lipids causes ferroptotic cell death (1).

As sensitivity to GPX4 inhibition is linked to ferroptosis (1, 15), this further suggested to us the possibility of aberrant iron accumulation in MYCN-amplified neuroblastoma to drive its proliferative program. To begin to test whether MYCN increased iron metabolism in neuroblastoma, we assessed the reliance of MYCN-overexpressing cells on iron accumulation. Indeed, while the MYCN wild-type cells showed only modest sensitivity (∼25%) to the higher concentrations of the iron scavenger deferoxamine (DFO) used in the dose–response assay, the presence of exogenous MYCN conferred susceptibility (Fig. 4A). Consistent with this, the MYCN-amplified neuroblastoma cell lines were more sensitive than the MYCN-wild-type neuroblastoma cell lines (Fig. 4B). Iron is controlled at the receptor level: Tfr1 is the receptor responsible for iron import, and ferroportin is responsible for iron export (3). We first evaluated the expression of the Tfr1 (encoded by the TFRC gene). In fact, TFRC levels were increased in neuroblastoma tumors with MYCN amplification (Fig. 4C; ref. 27). Similar to MYCN binding directly to system Xc- receptors to upregulate its expression, we found, in the SHEP21 system, that MYCN directly binds to E-box sites in the promoter of TFRC (Fig. 4D). Interrogation of the syngeneic pairs RPE.1, CHLA20, and CHLA172 by Western blotting verified the increase of the transferrin receptor in the MYCN-overexpressing cells (Fig. 4E). We next tested whether iron levels were higher when MYCN is present. In line with our hypothesis, there was an increased labile Fe (II) pool in the syngeneic RPE.1 and MYCN-wild-type neuroblastoma cells when in the presence of exogenous MYCN, as demonstrated by a Fe (II) selective probe (Fig. 4F). Although TFRC was higher, cellular iron levels are also controlled by the iron export receptor, ferroportin. Indeed, we found expression of ferroportin, (encoded by the SLC40A1 gene), was lower in MYCN-amplified neuroblastomas across the tumor datasets (Fig. 4G). These data were also corroborated at the protein level in the syngeneic models (Fig. 4E). As DNA methylation plays a large role in expression patters in neuroblastoma (28), we interrogated the status of ferroportin methylation across 105 neuroblastoma tumors (28). Interestingly, we discovered the SLC40A1 gene had increased methylation along the promoter and first exon in the MYCN neuroblastomas (Supplementary Fig. S4), likely contributing to lower expression in MYCN-amplified neuroblastoma. Therefore, both MYCN-directed TFRC expression and a decreased level of the SLC40A1 gene, encoding the ferroportin iron export receptor, contribute to enhanced iron capture in MYCN-amplified neuroblastoma.

Amplified MYCN drives increased iron accumulation through receptor modulation and enhanced sensitivity to cystine deprivation and system Xc- receptor inhibition. As our goal is for clinical translation, while SAS is FDA-approved, we sought to find an additional clinical drug that could be immediately translated to neuroblastoma clinical trials. Of note, the antioxidant thioredoxin reductase is a powerful ROS detoxifier, which, along with GSH, is responsible for controlling the redox state of the cell.

We reasoned that MYCN-amplified neuroblastoma would also need this pathway to detoxify the ROS from the increased demands from enhanced iron metabolism. We therefore studied auranofin, an FDA-approved drug for rheumatoid arthritis (29) that is a potent thioredoxin reductase inhibitor and has recently been repurposed in preclinical studies as an anticancer agent by increasing ROS (30). Consistent with this hypothesis and known mode of activity as a thioredoxin reductase inhibitor, we found that auranofin had preferentially activity in MYCN-amplified neuroblastoma (Fig. 5A). The sensitivity auranofin conferred to the MYCN-amplified NB cell lines was reversed by the addition of the ferroptosis inhibitor, ferrostatin-1 (Fig. 5B), implementing ferroptosis in the toxicity.

The role of MYCN was confirmed in the syngeneic models expressing MYCN compared with those expressing GFP (Fig. 5C). When MYCN was knocked down by short hairpin (sh)RNA (Fig. 5D), or siRNA (Supplementary Fig. S5A), auranofin sensitivity was mitigated. The cell viability data were further verified by crystal violet assays demonstrating increased sensitivity in the presence of exogenous MYCN (Supplementary Fig. S5B) or when MYCN was knocked down by shRNA (Supplementary Fig. S5C). Similar to ferrostatin-1, treatment with NAC mitigated auranofin toxicity in the MYCN-amplified IMR5 cells (Supplementary Fig. S5D, left), and did so in a MYCN-dependent manner (Supplementary Fig. S5D, right).

To further demonstrate auranofin was preferentially active in MYCN-amplified neuroblastoma by interfering with ROS neutralization, we measured both soluble ROS levels and ROS formed in lipids. Consistent with auranofin-mediated inhibition of thioredoxin reductase as central to its activity, ROS levels tracked with the antigrowth effects of auranofin: in the SK-N-DZ cells, ROS levels only substantially increased at 3,330 nmol/L, where activity was first seen, but in the KELLY and SIMA cells, ROS levels substantially increased in response to much lower doses (330 nmol/L), where auranofin was effective (Fig. 5E; Supplementary Fig. S5E). The amount of ROS increased by auranofin was higher in the presence of MYCN, and, importantly, the iron scavenger DFO markedly hindered ROS production by auranofin and to a greater degree in the presence of MYCN (Fig. 5F). Furthermore, the increase of the GSH levels (Supplementary Fig. S5G) is likely the outcome of a cellular feedback mechanism when thioredoxin activity is inhibited by auranofin, that has also been reported (31). In line with these observations, an increase of SLC7A11 was also displayed at 1,000 nmol/L of auranofin (Supplementary Fig. S5H).

To further probe the relationship between auranofin and iron accumulation in MYCN-amplified neuroblastoma, we cotreated the ex vivo MYCN-amplified PDX COG-N-496 cells with auranofin and the iron scavenger DFO. Strikingly, DFO blocked auranofin toxicity in a dose-dependent manner, consistent with ROS generation from iron accumulation driving sensitivity to auranofin (Fig. 5G, left). We further tested this relationship in the CHLA20 neuroblastoma cells with GFP and exogenous MYCN expression. Similar to the enhanced sensitivity in the MYCN-amplified neuroblastoma, MYCN was sufficient to induce sensitivity to DFO, and, strikingly, auranofin efficacy was mitigated by DFO and only in the cells expressing MYCN (Fig. 5H). Although MYCN was insufficient to induce sensitivity to DFO in the CHLA172 syngeneic line, once again, DFO robustly mitigated auranofin sensitivity, and once again, only in the presence of MYCN (Supplementary Fig. S5I). These data strongly infer that auranofin is selectively active in MYCN-amplified neuroblastoma only in the presence of increased iron influx, consistent with our hypothesis.

High c-MYC is a driver of a distinct subset of high-risk neuroblastomas (32), and c-MYC and MYCN share, to an extent, an overlapping transcriptome (33). Thus, we hypothesized that c-MYC should also serve as an activator for the GSH antioxidant pathway and cystine import in neuroblastomas. Indeed, consistent with an inverse correlation between MYCN and c-MYC in neuroblastoma (34), we found the highest c-MYC–expressing neuroblastoma lines in our panel were MYCN-wild-type (Supplementary Fig. S6A), where SLC3A2 levels were also higher than in the neuroblastomas without either MYCN amplification or high c-MYC levels (Supplementary Fig. S6A). To directly assess whether c-MYC affects the iron pathway and system Xc- receptor/GSH pathway, we exogenously expressed c-MYC in the RPE.1 and the MYCN-wt, c-MYC low, CHLA172 cell line, and investigated potential alterations in the expression of SLC3A2, SLC7A11, and Tfr1. In line with this hypothesis, there was significant upregulation in the presence of c-MYC in both cell lines of these proteins, except for SLC7A11 protein in the CHLA172 cells (Supplementary Fig. S6B and S6C). Treatment of both syngeneic cell line models with auranofin revealed significant sensitization of c-MYC–overexpressing cells (Supplementary Fig. S6B and S6C), and, consistent with upregulation of all three proteins in the RPE.1 cells, this effect was greater in the RPE.1 cells than the CHLA172 cells (75% more toxicity in the c-MYC–expressing RPE.1 cells compared with the control cells, with 25% more toxicity in the c-MYC–expressing CHLA172 cells compared with control cells). These data demonstrate c-MYC has a similar effect as MYCN on regulating iron uptake and system Xc-/GSH pathway activation and rendering c-MYC–high neuroblastomas sensitive to ferroptosis-inducing drugs like auranofin.

We next tested auranofin in vivo, as dosed previously (35), in three MYCN-amplified PDXs and the MYCN-amplified IMR5 xenograft model. These tumors were grown in NOD CRISPR Prkdc Il2r Gamma (NCG) mice, and upon reaching 150–250 mm³, were randomized, followed by treatment with 10 mg/kg every day (i.p.) auranofin or with no drug. Auranofin was sufficient to slow or block tumor growth in all three MYCN-amplified PDX models, including the MYCN-amplified COG-N-561 PDX model, taken from a patient following chemo-relapse (Fig. 6A; ref. 36). Additional experiment testing auranofin in mice bearing SK-N-SH (MYCN-wt) tumors demonstrated that these tumors were resistant to auranofin (Fig. 6A, right), consistent with the differential sensitivity of MYCN-amplified neuroblastoma tumors to auranofin. Similar to SAS, evaluation of tumor tissues treated with auranofin demonstrated a robust ferroptotic response (Fig. 6B; Supplementary Figs. S3A and S7). These data demonstrate that a well-tolerated FDA-approved drug may be repurposed to induce ferroptosis in MYCN-amplified neuroblastomas, adding to SAS as tailored therapies for MYCN-amplified neuroblastoma. Our model is depicted in Fig. 6C.

Recently, the therapeutic opportunities to kill cancer cells vulnerable to ferroptosis has been highlighted by two published studies (8, 15), which indicated cancer cells that underwent epithelial-to-mesenchymal transition (EMT) were dependent on GPX4 and disruption of GPX4 or treatment with ferroptosis inducers were selectively toxic to this subpopulation of cells. As the field of ferroptosis is still in its infant stages, potential opportunities have not yet been significantly explored.

In this study, we demonstrate that: (i) MYCN primes neuroblastoma cells for sensitivity to ferroptosis induction through increased iron import; (ii) an oncogenic transcription factor (i.e., MYCN) directs in concert the expression of several receptors to create both an iron dependency and subsequent vulnerability to system Xc- receptor/GSH targeting; (iii) a subset of oncogene-driven cancer (i.e., neuroblastoma characterized by amplified MYCN) is hypersensitive to system Xc-/GSH disruption; (iv) there exists evidence of an opportunity to exploit these findings with two FDA-approved drugs, SAS and auranofin, both of which induce ferroptosis in PDX and xenograft models of MYCN-amplified neuroblastoma; and (v) sensitivity to ferroptosis induction can also be seen in c-MYC–high neuroblastoma cell lines, which makes up an additional subset of high-risk neuroblastoma (34), and this potentially through an identical mechanism.

MYCN continues to be one of the most important targets in cancer therapeutics but remains difficult to drug directly. As an oncogenic transcription factor with high levels of expression in neuroblastoma, MYCN binds throughout the genome to confer oncogenesis (33). Our study highlights a new and, from a therapeutic standpoint, important way MYCN does this, through activation of the iron import and subsequent system Xc-/GSH axis, by increasing the expression of key receptors governing these processes. The system Xc-/GSH axis is a vital mechanism for cells to detoxify ROS from iron metabolism. Indeed, increase of Tfr1 is a marker for ferroptosis (25), and GPX4 is the primary defense against ferroptosis (10, 13).

The enhanced sensitivity to ROS targeting is conceptually consistent with the study from Wang and colleagues (37) demonstrating MYCN drives glutaminolysis and enhances ROS production. Indeed, MYCN drives glutamine catabolism (38). Therefore, enhanced glutaminolysis in MYCN-amplified neuroblastoma likely also contributes to the high need of detoxification from ROS that is present in MYCN-amplified neuroblastoma.

The important role of iron to drive growth and proliferation in cancer has come under increasing interest. The diverse pathways that contain enzymes require iron to function, including those found in the TCA cycle, helicases in DNA synthesis, and signaling molecules like nitric oxide synthase (3, 26). In breast cancer, low levels of ferroportin (which increases intracellular iron levels) correlated with metastatic relapse in patients with breast cancer (39). Glioblastoma stem cells require transferrin to survive (40). In ovarian cancer, targeting iron is sufficient to slow the growth of a genetic mouse model of the disease (41).

In this study, we demonstrated MYCN-amplified neuroblastoma have a reliance on system Xc-, presumably to counteract increased ROS from iron metabolism. Indeed, targeting the GSH pathway results in ferroptotic cell death (42). We identified within the Depmap portal (Broad Institute; ref. 43), that GPX4 knockdown was, across hundreds of cancer cell lines and dozens of subtypes of cancer, most effective in neuroblastoma, which was attributed to the MYCN-amplified subset. Because GSH has a short half-life, the pool of cysteine (produced by the import and subsequent reduction of cystine) is critical for the antioxidant activity of this pathway (18). Indeed, the uptake of cystine is thought to be the rate-limiting step for GSH production in the cell (21), and we found limiting cystine in cell culture medium is particularly toxic in the presence of MYCN in neuroblastoma. The Xc- antiporter shuttles in cystine and shuttles out glutamate, and in some cells at least, is the only known transporter that can bring cystine into the cell (44). Targeting this pathway, at the receptor level (with SAS) or the level of GSH biosynthesis (with BSO), has proven effective in several cancer models (24). Indeed, in our study, MYCN demonstrated synthetic lethality with both drugs. These data are consistent with a recent finding in TH-MYCN mice (45), an elegant mouse model of MYCN-amplified neuroblastoma (46), demonstrating enhanced GSH biosynthesis in these mice.

We also demonstrated that auranofin, a thioredoxin reductase inhibitor that is approved for rheumatoid arthritis, was an effective drug against MYCN-amplified neuroblastoma both in vitro and in neuroblastoma PDX models. Thioredoxin reductase is part of the thioredoxin antioxidant system, a key cellular antioxidant system (47), that works in parallel with the GSH pathway (47). Consistently, auranofin has recently been shown to increase ROS in vitro in neuroblastoma cells (48) and is a bona fide ferroptosis inducer (49). Auranofin can radiosensitize diverse cancer cells through increased ROS, which was negated by treatment with the ROS scavenger NAC (48). In addition, we found by cotargeting SAS directly in vivo was sufficient to block the grown of neuroblastoma tumors. These data provide two FDA-approved drugs that specifically target MYCN-amplified neuroblastoma.

Overall, we demonstrate that overexpressed MYCN increases iron influx and system Xc-/GSH pathway activation directly through upregulation of the key receptors governing these pathways. These data provide novel insights into how MYCN alters the transcriptome in neuroblastoma to confer growth and survival advantages. These changes create a vulnerability to ferroptosis inducers and outline a new strategy to treat these cancers with repurposed FDA-approved drugs.

B.R. Belvin reports grants from NIH during the conduct of the study. J.E. Koblinski reports grants from NIH/NCI during the conduct of the study. A.C. Faber reports personal fees from AbbVie and grants from IDP Pharma outside the submitted work. No disclosures were reported by the other authors.

K.V. Floros: Conceptualization, data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. J.Y. Cai: Data curation, methodology. S. Jacob: Data curation, methodology. R. Kurupi: Data curation. C.K. Fairchild: Data curation. M. Shende: Data curation. C.M. Coon: Methodology. K.M. Powell: Data curation. B.R. Belvin: Data curation, methodology. B. Hu: Data curation, methodology. M. Puchalapalli: Data curation, methodology. S. Ramamoorthy: Data curation, formal analysis, methodology. K. Swift: Data curation, formal analysis, methodology. J.P. Lewis: Formal analysis, methodology. M.G. Dozmorov: Data curation, formal analysis, methodology. J. Glod: Formal analysis, investigation, methodology. J.E. Koblinski: Formal analysis, methodology. S.A. Boikos: Investigation, methodology. A.C. Faber: Conceptualization, data curation, formal analysis, supervision, funding acquisition, investigation, methodology, writing–original draft, writing–review and editing.

This work was supported by a National Cancer Institute award (R01CA215610), to A.C. Faber, an American Cancer Society Mission boost grant (MBG-20-133-01-MBG) to A.C. Faber and an Andrew McDonough B+ Foundation Childhood Cancer Research Grant to A.C. Faber. Services and products in support of the research project were generated by the Virginia Commonwealth University Cancer Mouse Models Core Laboratory, supported, in part, with funding to the Massey Cancer Center from NIH-NCI Cancer Center Support Grant P30 CA016059.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.