MYC Deregulation Sensitizes Cancer Cells to N-myristoyltransferase Inhibition

Long summary of MYC Deregulation Sensitizes Cancer Cells to N-myristoyltransferase Inhibition by Gregor A. Luesg et al.; Audio by Paper2Audio.

We discovered a critical link between MYC deregulation and sensitivity to N-myristoyltransferase inhibitors (NMTis) in cancer cells. Through a large-scale screen of 245 cancer cell lines, we identified that cells with deregulated MYC are significantly more susceptible to NMT inhibition. This increased sensitivity is linked to mitochondrial dysfunction, particularly impacting complex I of the respiratory chain.

Our proteomic analyses revealed that in high-MYC contexts, NMT inhibition leads to the impaired assembly and function of mitochondrial complex I, primarily through the degradation of NDUFAF4, a key assembly factor. In vivo studies in mouse models of MYC-driven cancers demonstrated that NMTis are efficacious and well-tolerated, effectively suppressing tumor growth without causing overt toxicity. These findings highlight the potential of targeting N-myristoylation as a therapeutic strategy in MYC-driven cancers, offering a novel approach to combat these challenging malignancies.

Our initial screening efforts across a diverse panel of cancer cell lines revealed that leukemia cells were particularly sensitive to NMT inhibition. However, we observed no direct correlation between the expression levels of NMT1 or NMT2 and the sensitivity to NMTis, suggesting that the mechanism of action involves more complex factors than simple enzyme abundance. CRISPR-Cas9 knockout experiments further supported this, showing that NMT1 knockout in HeLa cells dramatically increased their sensitivity to NMTis, while NMT2 knockout had minimal impact.

This indicates that NMT1 is the primary catalytic enzyme responsible for N-myristoylation in cancer cells and that inhibiting its activity is crucial for therapeutic efficacy.

To identify the biological pathways associated with NMTi sensitivity, we performed single-sample gene set enrichment analysis (ssGSEA) on cancer cell lines with publicly available transcriptomic data. This analysis revealed that several gene sets were significantly altered in NMTi-sensitive cells, with MYC target genes being particularly enriched. This finding suggested a strong connection between MYC deregulation and sensitivity to NMT inhibition.

To validate this hypothesis, we used inducible cell models of MYC and MYCN, where the expression of these oncogenes can be controlled. In both models, we observed that high levels of MYC or MYCN significantly increased cell death upon treatment with NMTis, confirming that MYC deregulation sensitizes cancer cells to NMT inhibition.

Further investigation into the downstream effects of NMT inhibition in MYC-deregulated cells led us to focus on mitochondrial function. MYC is known to promote mitochondrial biogenesis and metabolic remodeling, making cancer cells highly dependent on mitochondrial respiration. We hypothesized that NMT inhibition disrupts mitochondrial function, particularly affecting the respiratory complex I.

Through proteomic analysis of detergent-enriched membrane fractions, we found that NMT inhibition resulted in the depletion of mitochondrial respiratory complex I proteins specifically in high-MYC cells. This was concurrent with the loss of N-myristoylation and degradation of NDUFAF4, a critical assembly factor for complex I.

We observed a significant reduction in mitochondrial respiration in high-MYC and high-MYCN cells upon NMTi treatment, while low-MYC cells were largely unaffected. This further supported the idea that NMT inhibition selectively impairs mitochondrial function in MYC-deregulated cells. We also found that NMTi treatment decreased mitochondrial potential and increased superoxide generation in high-MYC cells, indicating a disruption of mitochondrial homeostasis.

These effects were consistently observed with different NMTis, confirming the on-target effects of NMT inhibition on mitochondrial function.

To further elucidate the mechanism by which NMT inhibition affects complex I assembly, we performed deep proteomic analysis of the mitochondrial proteome in high- and low-MYC cells treated with NMTi. This analysis revealed a quantitative depletion of specific complex I proteins, including NDUFAF4 and NDUFB7, specifically in high-MYC cells. NDUFAF4 is a direct target of NMT and a transcriptional target of MYC, making it a critical link between NMT inhibition and complex I dysfunction.

We found that NDUFAF4 is degraded via the glycine N-degron pathway when it is not N-myristoylated, leading to impaired complex I assembly.

We also investigated the impact of an NDUFAF4 mutation (Ala3Pro) found in patients with Leigh syndrome, a severe mitochondrial disorder. This mutation disrupts N-myristoylation of NDUFAF4 and leads to complex I assembly defects. We found that expressing the NDUFAF4[Ala3Pro] mutant in cells resulted in reduced expression and proteasomal degradation, similar to the effects of NMT inhibition.

This further confirmed that N-myristoylation of NDUFAF4 is essential for its stability and function in complex I assembly. Our data suggest that failure to N-myristoylate NDUFAF4 is sufficient to impair physiological complex I assembly, as seen in patients with Leigh syndrome.

To assess the in vivo efficacy of NMT inhibition, we used mouse models of MYC-driven cancers. In a double-hit DLBCL model, we found that NMTi treatment resulted in significant tumor regression with minimal residual tumor. We also observed that NMTi treatment was efficacious in an immune-competent neuroblastoma mouse model, leading to strong tumor regression without obvious toxicity.

Proteomic analyses of tumors from these mice revealed a significant reduction of mitochondrial respiratory complex I proteins, further supporting the on-target effects of NMT inhibition in vivo.

Our findings provide a comprehensive understanding of the link between MYC deregulation and sensitivity to NMT inhibition in cancer cells. We have identified that NMT inhibition impairs mitochondrial function, particularly affecting complex I assembly through the degradation of NDUFAF4. These findings highlight the potential of targeting N-myristoylation as a therapeutic strategy in MYC-driven cancers and offer a novel approach to combat these challenging malignancies.

Our investigation began with a broad screening approach to identify cancer subtypes that exhibit heightened sensitivity to NMT inhibition. We utilized a panel of 245 diverse cancer cell lines, exposing them to varying concentrations of IMP-1320, a potent NMT inhibitor. The results indicated a selective sensitivity, with leukemia cell lines showing particularly strong responses.

Surprisingly, we found no significant correlation between the expression levels of NMT1 or NMT2 and the IC50 values across the cell line panel. This suggested that the mechanism underlying NMTi sensitivity is not directly related to the abundance of these enzymes.

To delve deeper into the role of NMT1 and NMT2, we performed CRISPR-Cas9 knockout experiments in HeLa cells. Knocking out NMT1 resulted in a dramatic increase in sensitivity to IMP-1088, shifting the EC50 value from 10 nM to 10 pM. In contrast, NMT2 knockout had a negligible impact on NMTi sensitivity.

This stark difference underscores the predominant role of NMT1 in cellular N-myristoylation, at least in the context of HeLa cells. Given that all potent human NMTis reported to date are dual NMT1 and NMT2 inhibitors, our findings suggest that pharmacological inhibition primarily targets NMT1, regardless of NMT2 expression levels.

We next sought to identify the biological pathways that predict sensitivity to NMTi. We used single-sample gene set enrichment analysis (ssGSEA) to obtain enrichment scores for the Hallmark gene sets in 211 cell lines with publicly available transcriptomic annotation. By comparing sensitive and less-sensitive cell lines, we identified 11 gene sets that correlated with sensitivity to NMT inhibition.

Notably, transcription of MYC target genes was significantly enriched in sensitive lines, consistent with previous screens using other NMT inhibitors. Furthermore, cancer cell lines with greater dependence on NMT1 expression were overrepresented in high-MYC expression and/or structural alterations in MYC or MYCN.

Based on these findings, we hypothesized that deregulated MYC increases the sensitivity of cancer cells to NMTi. To test this hypothesis, we used P493-6 immortalized B cells, a model of Burkitt's lymphoma known to be sensitive to NMT inhibition and highly expressing MYC. By regulating MYC expression using doxycycline and β-estradiol, we could induce high-MYC or low-MYC states.

High-MYC P493-6 cells showed a pronounced cell death response upon treatment with IMP-1088, whereas low-MYC P493-6 cells exhibited minimal death. This confirmed that MYC deregulation sensitizes cancer cells to NMTi.

We observed similar sensitivity in MYCN tet-off SHEP21N cells, a model of aggressive neuroblastoma driven by MYCN amplification. High-MYCN SHEP21N cells treated with NMTi experienced high levels of cytotoxicity relative to DMSO-treated controls, while low-MYCN SHEP21N cells were less sensitive. Notably, NMTi did not deplete MYC or MYCN levels in either model, suggesting a sensitizing mechanism downstream of MYC/MYCN expression.

We confirmed the dependency of NMTi efficacy on MYC or MYCN using cell quantification assays in P493-6 cells and MYCN-ER-SHEP cells, as well as using a chemically distinct NMTi, DDD86481. These data collectively support MYC deregulation as a sensitizing factor for NMTi in cancer cells.

To understand the downstream consequences of NMT inhibition, we hypothesized that it leads to the mislocalization or depletion of biologically relevant protein complexes from the membrane. We isolated detergent-enriched membrane fractions using Triton X-114 (TX-114) phase separation and applied LC-MS/MS-based analysis to determine changes upon NMTi treatment. In high-MYC P493-6 cells, we identified 3,532 proteins, of which 72% had UniProt membrane annotations and 87 are known to be co-translationally N-myristoylated.

As expected, NMT inhibition predominantly depleted N-myristoylated proteins in detergent fractions.

Protein-protein interaction analysis of proteins significantly depleted in the detergent fraction by NMTi revealed several distinct protein clusters, including mTOR signaling through the Ragulator-Rag complex. However, the cluster with the most affected proteins was related to mitochondrial respiratory complex I, consistent with previous reports on complex I impacts upon NMT inhibition or NMT1 knockout. An analogous experiment in high-MYCN SHEP21N cells produced similar results in terms of affected biological functions, implying a conserved mechanism of action across MYC paralogues.

Given that MYC upregulates mitochondrial biogenesis, we hypothesized that NMTi-induced disruption of complex I causes mitochondrial dysfunction in high-MYC cancer models, contributing to cell death.

We confirmed increased mitochondrial respiration in high-MYC P493-6 or high-MYCN SHEP21N cells compared to low-MYC P493-6 or low-MYCN SHEP21N cells. Exposure of high-MYC or high-MYCN cells to NMTi significantly reduced respiratory parameters, whereas low-MYC or low-MYCN cells were unaffected by NMTi treatment. These effects were observable after only 12 hours of NMTi treatment in high-MYC P493-6 cells.

Additionally, IMP-1088 treatment decreased mitochondrial potential and increased superoxide generation in high-MYC, but not low-MYC, P493-6 cells. Both IMP-1088 and DDD86481 induced similar impacts on mitochondrial function in patient-derived LY11212 DLBCL cancer cells, derived from a patient with multi-chemotherapy-resistant lymphoma carrying MYC and BCL2 translocations. Taken together, NMTi drives mitochondrial respiratory complex I defects and subsequent mitochondrial dysfunction in MYC-deregulated cancer cells.

Deep proteomic analysis of the fractionated mitochondrial proteome in high- and low-MYC P493-6 cells with or without NMTi treatment revealed quantitative depletion of specific complex I proteins, with NMTi only in high- but not in low-MYC cells. NDUFAF4 and NDUFB7, previously shown to be human NMT substrates, were particularly affected. We focused on NDUFAF4, as it is specifically depleted in the mitochondrial proteome in high-MYC cells treated with NMTi and in total protein extracts in both our MYC- and MYCN-inducible cell systems.

NDUFAF4 is a complex I assembly factor important for complex I expression and activity and is transcriptionally regulated by MYC and MYCN. Non-N-myristoylated NDUFAF4 is subject to degradation via the glycine N-degron pathway.

We hypothesized that an Ala3Pro mutation in NDUFAF4, found in patients with Leigh syndrome, phenocopies the impact of NMTi by abolishing NDUFAF4 N-myristoylation, leading to its proteasomal degradation. We expressed wild-type NDUFAF4 or NDUFAF4[Ala3Pro] with a C-terminal FLAG tag in HEK293 cells and found that NDUFAF4[Ala3Pro] expression was significantly reduced relative to wild type, which could be rescued by proteasome inhibition. NDUFAF4[Ala3Pro] N-terminal peptide is not a substrate for recombinant human NMT, in contrast to efficient N-myristoylation of wild-type NDUFAF4 peptide.

NDUFAF4, but not NDUFAF4[Ala3Pro], protein could be metabolically labeled with myristate analogue YnMyr in HEK293 cells.

The impact of NMTi on mitochondrial localization of complex I components in high-MYC P493-6 cells is clustered around the Q module, which is dependent on NDUFAF4 for incorporation into complex I, supporting the hypothesis that impaired NDUFAF4 N-myristoylation upon NMTi leads to specific complex I assembly defects in high MYC cells. These data suggest that failure to N-myristoylated NDUFAF4 is sufficient to impair physiological complex I assembly in humans, as seen in patients with Leigh syndrome bearing the NDUFAF4[Ala3Pro] mutation.

We examined the in vivo impact of NMTi in a double-hit DLBCL model. DoHH2 cells were engrafted subcutaneously into CB17/SCID mice to establish tumors to a volume of 100 to 150 mm3. Mice were treated with vehicle or IMP-1320 at 25 milligrams/kg/day delivered intraperitoneally at 12.5 milligrams/kg BID using a 3 days on/3 days off dosing schedule.

NMTi treatment resulted in significant tumor regression, with minimal residual tumor present at day 22 of the experiment, while tumors grew in all vehicle-treated controls. No significant effect on body weight was observed, suggesting that IMP-1320 was well tolerated under this dosing schedule.

IMP-1320 was also efficacious in an immune-competent neuroblastoma mouse model. The TH-MYCN genetically engineered mouse (GEM) model spontaneously develops tumors and models MYCN-amplified neuroblastoma. TH-MYCN tumor cells were engrafted into 129SvJ mice to establish a syngeneic model, and tumors were allowed to grow to ca.

5 millimeters in diameter, after which mice were treated with IMP-1320 at 25 milligrams/kg QD (i.p.) or vehicle on a 3 days on/4 days off schedule. IMP-1320 treatment resulted in strong tumor regression without obvious toxicity. Proteomic analyses of tumors following 3-day initial NMTi treatment revealed a significant reduction of mitochondrial respiratory complex I proteins compared to vehicle controls, in both DoHH2 xenograft and TH-MYCN GEM mice.

Meta-analysis further confirmed that this was the most significantly downregulated protein complex in both mouse models.

DDD86481 profoundly inhibited tumor growth in NOD scid gamma (IL2R-NSG) mice subcutaneously injected with PD LY11212 cells, which are highly sensitive to both IMP-1088 and DDD86481 in vitro. Taken together, these data are consistent with the hypothesis that NMTi treatment could be efficacious in a range of MYC- or MYCN-driven cancers.

In this study, we identified that MYC-deregulated cancers are particularly sensitive to NMTi in a 245-cell-line screen. Although expression of NMT enzymes does not predict sensitivity, consistent with screens against other NMTi, the effects of NMTi in cells are predominantly attributed to inhibition of NMT1, given that NMT1, but not NMT2, KO in HeLa cells greatly increased sensitivity to NMTis. The impact of MYC or MYCN deregulation on NMTi-induced cell death was verified in two distinct isogenic cell models, consistent with the sensitivity of MYC deregulation across cancer types.

N-myristoylation directs substrates to the membrane to regulate multiple signaling pathways, and we describe a systems-level analysis of the impact of NMTi in detergent-enriched membrane fractions. Our proteomics data are consistent with previous studies in NMT biology that connect NMT substrates such as LAMTOR1 and Src to NMT inhibition, while also highlighting respiratory complex I as a highly affected node. Differences in affected complexes upon NMTi were seen between each cell line investigated, such as the proteasome.

These differences may arise due to the different cell lineages of the two models or through differences between the cellular states induced by MYC and MYCN, respectively.

Mitochondrial dysfunction is both a hallmark and a liability of MYC deregulation, and we show that the impact of NMTi on mitochondria is both robust and MYC-dependent, as measured by mitochondrial respiration and mitochondrial proteomics analyses. NDUFAF4 is a direct target of NMT and a direct transcriptional target of MYC, and we hypothesized that its N-myristoylation is important for NDUFAF4 expression and subsequent complex I assembly. Indeed, we found that NMTi treatment phenocopied a pathogenic A3P NDUFAF4 mutant, which is not N-myristoylated and sufficient to drive physiological complex I defects in human patients with Leigh syndrome.

Moreover, NMTi-induced mitochondrial dysfunction and loss of complex I has also been reported in lymphoma models, including acute myeloid leukemia (AML) cell lines and HAP1 cells, although we show here the significant role of MYC deregulation on this phenotype.

The differential responses driven by MYC modulation revealed in our study may arise from lower NDUFAF4 expression and turnover in low-MYC contexts. However, it is clear that the mechanisms by which NMTi induces cancer cell death are complex and perturbations in multiple cellular pathways occur simultaneously. It is likely that contributions from several affected pathways combine to drive enhanced anticancer activity.

In addition to its effects on mitochondria and complex I, a major function of MYC is to drive general protein synthesis. As N-myristoylation is an irreversible and predominantly co-translational modification, the response of a cell to an NMTi is intrinsically linked to NMT substrate turnover. It is possible that MYC deregulation also sensitizes cancer cells to NMTi through its effects on proteome dynamics.

Our data are consistent with NMTi being highly effective in cancers in which MYC or MYCN is a driver oncogene, and indeed NMT1, but not NMT2, was recently identified in a genetic screen as a potential synthetic lethal knockout in combination with MYC overexpression. The promising in vitro results translated well to our in vivo models of lymphoma and neuroblastoma, in which IMP-1320 robustly eliminated or controlled tumors. Furthermore, although we focused here on MYC, other Hallmark gene sets were also predictive for NMTi sensitivity.

It is plausible that oncogenic deregulation of these pathways would also render cancer cells acutely sensitive to NMTi.

The advent of potent human NMTi has been essential to facilitate robust screening and system-level studies and to establish novel markers for NMTi sensitivity in cancer. NMT inhibitors have recently advanced to the clinic. Successful application of systemic NMTi in the clinic may require biomarker-based identification of the most sensitive cancers to NMTi, as the diverse effects on greater than 200 NMT substrates may lead to a relatively low therapeutic index for small molecule approaches.

Our data suggest that a significant therapeutic window exists to target MYC-driven cancers with NMTi, and we expect that future refinement of dose schedules and understanding of dose-limiting toxicity will enable clinical development of NMTi targeting high-MYC cancers.

In this study, we focused on the MYC-dependent effects of NMT inhibition through the lens of a single substrate, the complex I assembly factor NDUFAF4. While this mechanistic link provides a compelling rationale for selective sensitivity in MYC-deregulated cancers, we did not systematically assess whether other NMT substrates or the pathways they regulate are similarly affected in an MYC-dependent manner. Given the profound impact of MYC on protein synthesis and turnover, it is plausible that MYC deregulation sensitizes multiple pathways downstream of NMT by driving more rapid depletion of NMT substrates, but this hypothesis was not directly assessed in our study.

Multiple pathways beyond complex I assembly have previously been proposed to modulate NMTi sensitivity in cancer, and future studies employing unbiased proteomic and functional screening approaches may uncover the broader network of MYC-sensitized NMT substrates and their contributions to NMTi-induced cytotoxicity.

While NMTi is highly effective in vivo in animal models of cancers with deregulated MYC, we did not assess the influence of sex on the response to NMT inhibition. We also did not explore the durability of these responses, which may be relevant to MYC-deregulated cancers that often relapse following initial treatment. Extended in vivo studies could be undertaken to evaluate the potential for tumor recurrence after NMTi therapy, although in practice the value of animal studies for translation to humans remains limited.

Such insights will help define the therapeutic window and inform the rational design of biomarker-driven or targeted delivery strategies for NMTi-based therapies.

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