MYC Deregulation Sensitizes Cancer Cells to N-myristoyltransferase Inhibition

Gregor A. Lueg et al.

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Image summary: The image is a graphical abstract that illustrates the effect of N-myristoyltransferase inhibitors on normal cells and MYC-deregulated cancer cells. In normal cells, NMT inhibitors have little to no effect, and the cells remain viable. However, in MYC-deregulated cancer cells, NMT inhibitors lead to the degradation of NDUFAF4 and respiratory complex I, ultimately resulting in cell death. NMT inhibition causes mitochondrial dysfunction in high-MYC contexts through impaired complex I via effects on the NMT substrate and complex I assembly factor NDUFAF4. Therefore, MYC-deregulated cancer cells are more sensitive to NMT inhibitors compared to normal cells.

In brief

Lueg et al. discovered that deregulated MYC increases cancer cell sensitivity to N-mvristovltransferase inhibitors (NMTis). Mitochondrial dysfunction upon NMTi occurs selectively in high-MYC contexts through impaired complex I via effects on the NMT substrate and complex I assembly factor NDUFAF4. An approach for targeting MYC-driven cancers is revealed.

Highlights

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Deregulated MYC sensitizes cancer cells to NMT inhibitors Complex 1 proteins are depleted upon NMT inhibition in MYC deregulated cells Loss of NDUFAF4 N-myristoylation is linked to mitochondrial dysfunction NMT inhibitors are efficacious and tolerated in mouse models of MYC-driven cancer

MYC Deregulation Sensitizes Cancer Cells to N-myristoyltransferase Inhibition

Image summary: The image is a graphical abstract. It depicts how NMT inhibition affects normal cells versus MYC-deregulated cancer cells. In normal cells, NMT inhibition does not have a significant impact, and the cells remain viable. However, in MYC-deregulated cancer cells, NMT inhibition leads to a loss of NDUFAF4 N-myristoylation, degradation of respiratory complex I, and ultimately cell death. Thus, MYC-deregulated cancer cells are more sensitive to NMT inhibitors.

Image summary: This image shows the title, authors, affiliations, and summary of a research article. The article investigates how deregulated MYC sensitizes cancer cells to N-myristoyltransferase inhibition. The research indicates that inhibiting NMTs can be a promising strategy in cancer treatment, especially in MYC-driven cancers. The study found that cancer cells with deregulated MYC are more sensitive to NMT inhibitors. Proteomic analysis revealed that cell death is linked to the loss of membrane association of mitochondrial respiratory complex I, along with the degradation of NDUFAF4, leading to mitochondrial dysfunction. In vivo experiments demonstrated that NMT inhibitors effectively suppressed MYC-driven tumors without significant toxicity, suggesting a potential therapeutic approach for MYC-driven cancers.

Summary

Human N-myristoyltransferases (NMTs) catalyze N-terminal protein N-myristoylation and are promising targets in cancer, with an emerging mechanistic rationale for targeted therapy. Here, we screened 245 cancer cell lines against IMP-1320, a potent NMT inhibitor (NMTi), and conducted pathway-level analyses to identify that deregulated MYC increases cancer cell sensitivity to NMTis. Proteomics on detergent-enriched membrane fractions in MYC or MYCN-deregulated cancer cell models revealed that cell death is associated at least in part with loss of membrane association of mitochondrial respiratory complex 1. This is concurrent with loss of myristoylation and degradation of the complex 1 assembly factor NDUFAF4, and induction of mitochondrial dysfunction, driven by MYC or MYCN-deregulation. NMTis eliminated or suppressed MYCand MYCN-driven tumors in vivo without overt toxicity, suggesting that this constitutive co-translational protein modification can be targeted in MYC-driven cancers.

Introduction

N -Myristoylation is an irreversible lipid modification of proteins at an N-terminal glycine, mediated in humans by two closely related N-myristoyltransferases, NMT1 and NMT. The substrate and acyl-CoA-binding sites of NMT1 and NMT2 are highly conserved, whereas the two isozymes differ predominantly in their N-termini, which is dispensable for catalysis but may be involved in cellular localization.2 However, current evidence suggests that cellular N-myristoylation is predominantly catalyzed by NMT1, at least in developing T cells and embryonic stem cells. N-Myristoylation modulates membrane association, ^{} protein stability, and protein-protein interactions, and proteomic and bioinformatic studies have identified over 200 substrates of NMT in the human proteome. Most substrates are modified co-translationally at the ribosome after processing by methionine aminopeptidases (MetAPs), although post-translational N -myristoylation on neo-N-termini exposed after protease cleavage has been observed. A wide range of cellular path- ways are modulated by NMT substrates, including mTORC1 signaling, proteasomal degradation, and protein trafficking, supporting the central importance of this modification to cellular homeostasis. It is therefore unsurprising that multiple N -myristoylated proteins have been associated with pro-oncogenic activity, including well-known oncoproteins such as SRC, ABL2, and PRKACA. As a result, targeting cancer through the global ablation of N -myristoylation by NMT inhibition has been proposed, although a mechanistic rationale supporting a sufficient therapeutic index for NMT inhibition has been lacking. Many prior studies were limited by a lack of potent NMT inhibitors (NMTis; Figures 1B and S1A ), which have only recently been reported. Furthermore, past studies have predominantly focused on selected individual NMT substrates rather than addressing the broad consequences of NMT inhibition, despite the pleiotropic effect of NMTi on diverse cellular pathways. MYC proteins (MYC, MYCN, and MYCL) are a family of transcription factors that are highly implicated in tumorigenesis and tumor maintenance. The MYC transcriptional program promotes protein synthesis, metabolic remodeling, and ribosomal and mitochondrial biogenesis, ultimately contributing to many of the hallmarks of cancer. Deregulation of MYC proteins is present in the majority of cancers, although the paralogs deregulated differ between types of cancer. While MYC is altered ubiquitously, MYCN deregulation is enriched in tumors of neuronal and neuroendocrinal origin, and MYCL deregulation is most commonly associated with small-cell lung cancer. Importantly, deregulation of MYC proteins is often associated with aggressive disease and poor prognosis, but MYC proteins currently remain intractable drug targets due to their unstructured nature.

Here, we combined large-scale cancer cell line screening against a potent and specific NMTi with pathway-level analysis of cell state, revealing that deregulation of MYC renders cancer cells acutely sensitive to NMT inhibition. Analyses of detergentenriched membrane fractions in separate models of MYC- and MYCN-deregulated cells showed that NMTi-induced cell death in these contexts is associated with loss of mitochondrial respiratory complex 1 proteins. This is concurrent with depletion of the N-myristoylated complex 1 assembly factor NDUFAF4 and induction of mitochondrial dysfunction selectively in MYC-deregulated contexts. NMTi inhibited or eliminated MYC- and MYCN-driven tumors in vivo without overt toxicity, providing a mechanistic framework for NMTi as a targeted cancer therapy. This work offers examples of targeting a constitutive co-translational protein modification in MYC-deregulated cancers and potentially presents an avenue to target cancers driven by this class of intractable oncoproteins.

Figure 1 summary: This figure illustrates cancer subtype sensitivity to NMTis in a cancer cell line screen and human NMT1 and NMT2 catalyze protein N-myristoylation of specific substrates. The figure presents a schematic of the N-myristoylation process, where NMT1 and NMT2 modify proteins at the ribosome, leading to various functions. It also shows the chemical structures of different NMT inhibitors along with their IC50 values for NMT1 and NMT2. The figure includes violin plots showing the distribution of NMTi sensitivity across different cancer types. Scatter plots show the relationship between NMT1 and NMT2 expression and sensitivity to one of the NMT inhibitors. Finally, the figure shows the impact of NMT1 knockout on cell viability in the presence of NMT inhibitors. The data suggests that cancer subtype is a better predictor of sensitivity to NMT inhibitors than the expression levels of NMT1 or NMT2. Additionally, knockout of NMT1 appears to increase sensitivity to NMT inhibitors.

Results

NMT Expression Does Not Correlate With NMTi Sensitivity in a Cell Line Screen

First, we set out to discover which cancer subtypes are highly sensitive to NMT inhibition. We screened a panel of 245 cancer cell lines, spanning a range of cancer types against a range of concentrations of IMP-1 20 (Figures 1B and S1A), a highly potent NMTi sharing a similar core structure as the previously reported and commonly used tool NMTi IMP-1088, albeit with improved pharmacokinetic properties. IMP-1320 was effective in a subset of all cancer types tested, particularly in leukemia cancer cell lines (Figures 1C and S1B; Table S1), ^{} and expression of NMT1 or NMT2 did not correlate with IC_{50} values across the cell line panel (Figure 1D), consistent with previous screens against DDD86481, an NMT inhibitor from a different chemical class. Consistent with cellular N -myristoylation catalyzed predominantly by NMT1,3 whole-genome CRISPR KO screens (Cancer Dependency Map, DepMap) processed with a common pipeline identified NMT1 as required for optimal proliferation (common essential) of most cancer cell lines, whereas NMT2 lacked essentiality in any cell line tested (Figure S1C). The gene effect score of NMT1 KO is also significantly correlated with NMT2 expression, likely due to partial rescue of N-myristoylation by NMT2. However, CRISPR-Cas9 NMT1 homozygous knockout in HeLa cells (Figures S1D and S1E) conferred 1,000 fold greater sensitivity to IMP-1088, shifting the EC subscript 50 value from 10 nanoMolar to 10 picoMolar, whereas NMT2 knockout had minimal impact (Figure 1E), consistent with a negligible impact of NMT2 expression on NMTi sensitivity. Notably, all potent human NMTis reported to date are dual NMT1 and NMT2 inhibitors, due to the very high homology of these isoforms in the catalytic domain. It is therefore likely that genetic associations with differential NMT expression do not apply in the context of pharmacological inhibition of NMTs due to concurrent inhibition of both enzymes.

Image summary: The image shows a CellPress article report. The article discusses targeting a constitutive co-translational protein modification in MYC-deregulated cancers and potentially presents an avenue to target cancers driven by this class of intractable oncoproteins. The article also discusses NMT expression and MYC deregulation.

MYC Deregulation Sensitizes Cancer Cells to NMTi

To identify biologically relevant predictors for NMTi sensitivity, 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 (Figure 2A). Cell lines were classified as sensitive (IC sub 50 less than 0.2 micro Molar) or less sensitive (IC sub 50 greater than 0.2 micro Molar) to IMP-1320. Using a linear model, we then identified gene sets with significantly altered enrichment scores (FDR less than 1 percent) between sensitive and less sensitive cell lines and found 11 gene sets that correlated with sensitivity to NMT inhibition, with the majority being less expressed in sensitive cell lines (Figure 2B). These results were broadly consistent across a range of sensitivity thresholds and also with a no-threshold method to identify significant gene sets, confirming their validity. Treating each gene set as a binary operator, the area under the curve (AUC) of the receiver operating curve (ROC) confirmed that these gene sets individually predicted sensitivity to NMT inhibition (Figure S2A). Notably, transcription of MYC target V1 was both significantly enriched in sensitive lines and predictive of sensitivity (Figure 2C), a finding consistent with previously reported screens using DDD86481. Furthermore, cancer cell lines with greater dependence on NMT1 expression were also overrepresented in high MYC expression and/or structural alterations in MYC or MYCN (Figure S2B).

We next explored the hypothesis that deregulated MYC increases sensitivity of cancer cells to NMTi using IMP-1088, a thoroughly validated, highly potent, and widely used NMTi in P493-6 immortalized B cells. These cells are a model of Burkitt's lymphoma,a cancer type that has previously been shown to be highly sensitive to NMT inhibition. Importantly, they highly express MYC, which can be suppressed by a combination of doxycycline and beta-estradiol, allowing investigation of MYC-mediated effects. MYC was regulated to high or low levels over 24 hours (Figure 2D), and cells subsequently exposed to various concentrations of IMP-1088 for 5 days with cell death determined by SYTOX Green staining as a readout for NMTi sensitivity. Cell death was highly pronounced in high-MYC P493-6 cells upon treatment with 100 nM IMP-1088, a concentration sufficient to robustly inhibit cellular NMT activity as observed by metabolic labeling with myristate analogue YnMyr (Figure S2C), whereas minimal death occurred in low-MYC P493-6 cells (Figure 2E). Similar sensitivity was observed in MYCN tet-off SHEP21N cells, a model of a highly aggressive form of clinical neuroblastoma (NB) driven primarily by MYCN-amplification (Figure 2F). High-MYCN SHEP21N cells treated with NMTi experienced high levels of cytotoxicity relative to DMSO-treated controls, whereas low-MYCN SHEP21N cells were less sensitive, again at concentrations that inhibit cellular N-myristoylation (Figures 2G and S2C). However, NMTi did not deplete MYC or MYCN levels in either model, consistent with a sensitizing mechanism downstream of MYC/MYCN expression (Figure S2D). The dependency of NMTi efficacy on MYC or MYCN was confirmed by cell quantification assays (Figure S2E) in P493-6 cells and MYCN-ER-SHEP cells, a neuroblastoma cell line in which 4-hydroxytamoxifen (tam) induces MYCN and also using a chemically distinct and potent NMTi (DDD86481), confirming the role of on-target NMT inhibition in cell death. Collectively, these data support MYC deregulation as a sensitizing factor for NMTi in cancer cells.

Figure 2 summary: The figure includes multiple components. It begins with a schematic outlining the process of identifying gene sets correlated with sensitivity to NMT inhibition, starting from gene expression data and NMTi sensitivity data, progressing through ssGSEA, linear model analysis, and ROC curve generation. A heatmap displays gene sets with significantly altered enrichment scores between sensitive and less sensitive cell lines. An ROC curve assesses the predictive power of MYC target V1 transcription for sensitivity. Western blots show MYC and MYCN levels under high and low regulation conditions. Finally, line plots depict cell death over time in both high and low MYC/MYCN cells upon treatment with varying concentrations of an NMT inhibitor. The figure suggests that MYC deregulation sensitizes cancer cells to NMTi. Cell lines with greater dependence on NMT1 expression are overrepresented in high MYC expression. High-MYC cells exhibit more pronounced cell death upon treatment with an NMT inhibitor compared to low-MYC cells, indicating that deregulated MYC increases the sensitivity of cancer cells to NMTi.

NMTi Drives Mitochondrial Dysfunction in High-MYC Cancer Cells

Many of the greater than 200 known human N-myristoylated proteins associate dynamically with membranes, and the myristoyl group frequently plays a critical role in mediating membrane localization. We therefore hypothesized that a key downstream consequence of NMT inhibition would be mis-localization or depletion of biologically relevant protein complexes from the membrane and that the combined effect of multiple N-myristoylated proteins on these complexes and their interactors may be enough to significantly affect their function. We therefore isolated detergent-enriched membrane fractions (detergent fractions) through Triton X-114 (TX-114) phase separation and applied LC-MS/ MS-based analysis to determine changes upon NMTi treatment (Figure 3A), which may result from changed protein abundance or localization. We first examined the effect in high-MYC P493-6 cells treated with 100 nM IMP-1088 for 24 h, just prior to the onset of significant cell death. Overall, 3,532 proteins were identified, of which 72% had UniProt membrane annotations and 87 are known to be co-translationally N-myristoylated. As expected, the predominant effect of NMT inhibition was to deplete N -myristoylated proteins in detergent fractions (Figure S3A). All significantly affected NMT substrates (p less than 0.05, 53 proteins) were depleted in this fraction by NMTi, with a majority of these highly affected (log2 fold change less than -0.585 ). Protein-protein interaction analysis of all proteins significantly depleted in the detergent fraction by NMTi ( p less than 0.05 , 273 proteins, Figure 3B) using the stringApp Cytoscape plugin revealed several distinct protein clusters, including several pathways previously reported to be affected downstream of NMT inhibition, such as mTOR signaling through the Ragulator-Rag complex. However, the cluster with the most affected proteins is related to mitochondrial respiratory complex 1, consistent with previously reported impacts on complex 1 at the whole proteome level upon NMT inhibition or NMT1 KO. An analogous experiment in high-MYCN SHEP21N cells produced similar results in terms of affected biological functions (Figures 3C and S3B), implying a conserved mechanism of action across MYC paralogues.

MYC upregulation is known to drastically increase mitochondrial biogenesis, and inhibitors of oxidative phosphorylation have demonstrated efficacy in MYC- or MYCN-expressing cancer cells including MYCN-amplified neuroblastoma and B cell lymphomas. We therefore hypothesized that disruption of complex 1, provoked by NMTi, causes a failure in mitochondrial function 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 (Figure 3D), as previously reported. and found that exposure of high-MYC or high-MYCN cells to NMTi significantly ( p less than 0.05 ) reduced respiratory parameters, whereas low-MYC or low-MYCN cells were unaffected by NMTi treatment (Figures 3E, S3C, and S3D). These effects were already observable after only 12-h NMTi treatment in high-MYC P493-6 cells (Figure S3E). Additionally, in high-MYC, but not low-MYC, P493-6 cells, IMP-1088 treatment (100 nM, 18 h) decreased mitochondrial potential and increased superoxide generation (Figure S3F). Notably, both IMP-1088 and DDD86481 induced similar impacts on mitochondrial function in patient-derived (PD) LY11212 DLBCL cancer cells (Figure S3G). PD LY11212 cells were derived from a patient with multi-chemotherapy-resistant lymphoma carrying MYC and BCL2 translocations, characteristic of so-called "double hit" lymphomas that have the least favorable clinical outcomes among DLBCL. In these cells, both IMP-1088 and DDD86481 delivered potent inhibition of N -myristoylation (Figure S3H). Taken together, these findings indicate that NMTi drives mitochondrial respiratory complex 1 defects and subsequent mitochondrial dysfunction in MYCderegulated cancer cells.

Figure 3 summary: The figure shows protein-protein interaction networks and mitochondrial respiration in cells treated with NMT inhibitor. The protein-protein interaction networks reveal distinct protein clusters affected by NMT inhibition. Mitochondrial respiration is increased in cells with high levels of MYC or MYCN compared to those with low levels. NMT inhibition reduces respiratory parameters in cells with high MYC or MYCN expression but has minimal impact on cells with low expression.

Figure 4 summary: The figure shows the impact of NMT inhibition on mitochondrial function and complex I assembly in high-MYC cells. The figure includes a schematic representation of the experimental workflow. It also contains protein-protein interaction networks showing the quantitative depletion of specific complex I proteins upon NMT inhibition in high-MYC cells. Additionally, line plots and box plots compare the oxygen consumption rates and respiratory parameters in high-MYC and low-MYC cells treated with NMT inhibitors. The data suggest that NMT inhibition leads to a reduction in complex I proteins and impairs mitochondrial function specifically in cells with elevated MYC levels.

NMTi Impacts NDUFAF4-Associated Complex I Assembly in High-MYC Cells

Deep proteomic analysis of the fractionated mitochondrial proteome in high- and low-MYC P493-6 cells with or without 100 nM IMP-1088 treatment revealed quantitative depletion of specific complex I proteins, with NMTi only in high but not in low-MYC cells (Figures 4A and 4B), including NDUFAF4 and NDUFB, which were previously shown to be human NMT substrates. We chose to focus on NDUFAF4 as it is significantly and specifically depleted in the mitochondrial proteome in high-MYC cells treated with NMTi (Figure 4B) and in total protein extracts in both our MYC- and MYCN-inducible cell systems (Figures 4C and 4D). NDUFAF4 is a complex I assembly factor important for complex I expression and activity and is directly transcriptionally regulated by MYC and MYCN (Figure S4A). Notably, non-N-myristoylated NDUFAF4 is subject to degradation via the glycine N-degron pathway.7 Furthermore, patients carrying a single Ala3Pro mutation in NDUFAF4 were recently reported to suffer a specific mitochondrial complex I assembly defect, leading to onset of Leigh syndrome. We hypothesized that this mutation adjacent to the Gly2 N-myristoylation site phenocopies the impact of NMTi by abolishing NDUFAF4 N-myristoylation, leading to its proteasomal degradation through the glycine N-degron pathway. 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 (Figure 4E). Furthermore, we found that NDUFAF4[Ala3Pro] N-terminal peptide is not a substrate for recombinant human NMT, in contrast to efficient N-myristoylation of wild-type NDUFAF4 peptide (Figure S4B), and NDUFAF4, but not NDUFAF4[Ala3Pro], protein could be metabolically labeled with myristate analogue YnMyr in HEK293 cells (Figure S4C). Notably, the impact of NMTi on mitochondrial localization of complex I components in high-MYC P493-6 cells (Figure 4B) is clustered around the Q module, which is dependent on NDUFAF4 for incorporation into complex 1 (Figure 4F; Figure S4D), supporting the hypothesis that impaired NDUFAF4 N-myristoylation upon NMTi leads to specific complex 1 assembly defects in high MYC cells. Taken together, these data suggest that failure to N-myristoylated NDUFAF4 is sufficient to impair physiological complex 1 assembly in humans, as seen in patients with Leigh syndrome bearing the NDUFAF4 mutation.

Image summary: This image shows a CellPress OPEN ACCESS logo and the Cell Reports Article journal information.

NMTi Suppresses MYC- and MYCN-Driven Tumors

User text: We next 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 (Figure S5) at 25 milligrams/kg/day delivered intraperitoneally (i.p.) 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 (Figure 5A). No significant effect on body weight was observed, suggesting that IMP-1320 was well tolerated under this dosing schedule (Figure 5B). IMP-1320 was also efficacious in an immunecompetent neuroblastoma mouse model. The TH-MYCN genetically engineered mouse (GEM) model spontaneously develop tumors and model 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 (Figures 5C and 5D). Notably, 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 (Figure 5E). Meta-analysis further confirmed that this was the most significantly downregulated protein complex in both mouse models (Figure 5F). Furthermore, DDD86481 (Figure S6) profoundly inhibited tumor growth in NOD scid gamma (IL2R-NSG) mice subcutaneously injected with PD LY11212 cells (Figures S7A and S7B), which are highly sensitive to both IMP-1088 (EC50 5 nM) and DDD86481 (EC50 16 nM) in vitro (Figure S7C). Taken together, these data are consistent with the hypothesis that NMTi treatment could be efficacious in a range of MYC- or MYCN-driven cancers.

Figure 5 summary: The figure includes multiple plots and western blots related to the effects of NMT inhibition on tumor growth and protein expression. The first plot shows the effect of a specific inhibitor on MYC expression, with high MYC tumors exhibiting a different response compared to low MYC tumors. A second plot illustrates the relative depletion of various proteins upon treatment, highlighting specific proteins that are significantly affected. Western blots demonstrate the impact of the inhibitor on the expression levels of certain proteins in different cell lines. Additional western blots show the effect of the inhibitor on protein expression in cells with varying levels of MYCN. The figure indicates that NMT inhibition can lead to tumor regression and alterations in protein expression, particularly affecting proteins involved in mitochondrial respiratory complex I. The data suggests that NMT inhibition could be a viable therapeutic strategy for cancers driven by MYC or MYCN.

Image summary: The figure contains multiple plots and images related to the impact of MYC on NMTi-induced cell death. It presents data from cell models, indicating a correlation between MYC deregulation and sensitivity to NMTi. The figure includes western blots and quantification analyses, showing the effects of NMTi on mitochondrial respiration and proteomics. The data suggests that NMTi treatment mimics the effect of a pathogenic NDUFAF4 mutant, affecting complex I assembly. Overall, the figure provides evidence for the role of MYC in modulating the cellular response to NMT inhibition, particularly in the context of mitochondrial function.

Discussion

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 (Figure 1E). A small increase in sensitivity ( less than 2-fold) was recently reported in HAP1 cells (a haploid model of chronic myelogenous leukemia (CML)) upon NMT2 KO, although consistent with our data the effect of NMT1 KO was much greater. Importantly, 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 (Figures 2E and 2G).

N -myristoylation is well known to direct substrates to the membrane to regulate multiple signaling pathways, and we describe here a systems-level analysis of the impact of NMTi in detergent-enriched membrane fractions (Figure 3). 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 1 as a highly affected node, as suggested by previous global analyses. Differences in affected complexes upon NMTi were seen between each cell line investigated, such as the proteasome, a subpopulation of which is thought to rely on N -myristoylation for membrane association. 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 (Figure 4). 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. As such, it is likely that contributions from several affected pathways combine to drive enhanced anticancer activity, as recently proposed. V 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 therefore possible that MYC deregulation also sensitizes cancer cells to NMTi through its effects on proteome dynamics. Such activity would also be consistent with previous reports in Drosophila identifying Nmt as required for Myc -driven growth, with Nmt knockdown likely inducing ribosomal stress, although the synthetic lethality observed here for NMTi is both more selective and more potent than for inhibitors of previously reported protein-synthesisrelated targets in high-MYC cells. 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 in which highly MYC expressing cells were sensitized to IMP1088 translated well to our in vivo models of lymphoma and neuroblastoma, in which IMP-1320, an analogue of IMP-1088 with improved pharmacokinetic properties, robustly eliminated or controlled tumors. Furthermore, although we focused here on MYC, other Hallmark gene sets were also predictive for NMTi sensitivity (Figure S2A). These mostly overlapped with gene sets enriched in other analyses of NMTi sensitivity, supporting their biological relevance, and it is plausible that oncogenic deregulation of these pathways would also render cancer cells acutely sensitive to NMTi. Investigation of these pathways, as well as further characterizing the interplay between NMTi and MYC, would be useful in expanding our knowledge of the range of cancers in which NMTi may be most efficacious.

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, and the results of a phase 1 trial of a NMTi have been reported. NMTi was shown to be tolerated in patients at predicted efficacious doses, although the extent of clinical benefit remains to be determined, and dose-limiting toxicities were also identified. 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. An alternative approach of targeted delivery, for example, through an NMTi antibody-drug conjugate (ADC) may offer an optimal balance between high efficacy and minimal toxicity and expand the range of treatable cancers, as recently reported.

Image summary: The image contains multiple line charts and a bar chart. The line charts show the effect of IMP-1320 treatment on various parameters like tumor volume and body weight in DOHH2 and TH-MYCN cells over time compared to a vehicle control. The bar chart shows the relative abundance of various mitochondrial respiratory complex proteins in DOHH2 and TH-MYCN cells. IMP-1320 treatment significantly reduces tumor volume in both cell lines, with a more pronounced effect in DOHH2 cells. Body weight is not significantly affected by the treatment in either cell line. There are significant differences in mitochondrial respiratory complex protein abundance between the two cell lines.

Image summary: The image is a line chart. The chart shows the impact of a vehicle and IMP-1320 on tumor volume. The x-axis represents the days since the treatment started, and the y-axis represents the tumor volume. The tumor volume increases more with the vehicle than with IMP-1320.

Figure ns summary: This figure is a line graph. It depicts the impact of vehicle and IMP-1320 on Th-MYCN body weight over a period. The x-axis represents the days since treatment start, while the y-axis indicates the Th-MYCN body weight as a percentage of the control. The figure suggests that there is no significant difference between the two groups.

Figure F summary: The figure includes a violin plot and bar charts. The violin plot illustrates the abundance of mitochondrial respiratory complex I proteins in two different cell lines, DoHH2 and TH-MYCN. The bar charts show the results of gene set enrichment analysis for the same two cell lines. The mitochondrial respiratory complex I proteins are less abundant in the TH-MYCN cell line compared to the DoHH2 cell line. The gene set enrichment analysis reveals different enriched pathways in the two cell lines. In the TH-MYCN cell line, respiratory chain complex I and coatomer complex are enriched, while in the DoHH2 cell line, respiratory chain complex I, spliceosome, ribosomal subunit, and other complexes are enriched.

Limitations of the study

In this study, we focused on the MYC-dependent effects of NMT inhibition through the lens of a single substrate, the complex 1 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 1 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.

Resource Availability

Materials availability

IMP-1320 will be shared upon request under MTA completion.

Stad _ METHODS

Detailed methods are provided in the online version of this paper and include the following:

Key Resources Table

Experimental Model and Study Participant Details Cell Lines

Mouse models METHOD DETAILS Enzymatic NMT assays Cell line panel Cell line screen analysis Cloning Generation of NMT1 and NMT2 CRISPR-Cas9 knockout clones in HeLa cells Verification of CRISPR-Cas9 knockouts by sequencing NMTi treatment Real-time cytotoxicity assay Western Blot TX-114 fractionated proteomics Data-dependent acquisition \circ\, Data-independent acquisition Proteomics data analysis Oxygen consumption rate measurements Mitochondrial proteomics Mitochondrial proteomics analysis NDUFAF4 biochemistry In vivo proteomics Meta-analysis SPR Public datasets YnMyr labeling for in-gel fluorescence and streptavidin pulldown Cell viability measurements Flow cytometry Cytofluorimetry for MitoTracker staining QUANTIFICATION AND STATISTICAL ANALYSIS

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Table 1 summary: This table, titled "Key Resources Table," provides a comprehensive list of reagents and resources used in the study, categorized for clarity. The "Antibodies" section details various primary and secondary antibodies, including their sources and identifiers. The "Chemicals, peptides, and recombinant proteins" section lists numerous compounds, peptides, and recombinant proteins, indicating their sources and, where applicable, catalog numbers or references to their origin. The table also includes sections for "Critical commercial assays," "Deposited data," "Experimental models: Cell lines," "Experimental models: Organisms/strains," "Oligonucleotides," "Recombinant DNA," "Software and algorithms," and "Other" equipment. Each entry specifies the reagent or resource, its source, and an identifier such as a catalog number, RRID, or reference to a publication, providing a thorough inventory of materials and tools utilized in the research.

Figure S1 summary: The figure includes several different types of plots and images. It validates NMTi and genetic factors influencing NMTi. SPR sensorgrams show the interaction of NMTi with recombinant NMT1. A plot compares the distribution of IC50 values in leukemia lines versus other lineages. Gene effect scores for NMT1 and NMT2 are shown for sgRNA libraries. Representative chromatograms display PCR products from wild type and mutant alleles in a modified HeLa cell line. A western blot shows NMT1 or NMT2 in CRISPR-Cas9 mediated knockout and wild type HeLa cells. The SPR sensorgrams show the kinetic constants for binding. The IC50 values are significantly different between leukemia lines and other lineages. The gene effect scores indicate the essentiality of NMT1 and NMT2. The chromatograms highlight targeted regions, and the western blot confirms knockout of NMT1 and NMT2.

Figure S2 summary: This figure presents multiple data types including area under curve values, representations, fluorescence assays, western blots, and viable cell number impacts. The figure explores the relationship between MYC deregulation and cancer cell sensitivity to NMTi. It shows area under curve values for gene sets, representing NMT1-dependent cell lines with high MYC expression or structural alterations. The figure also shows NMTi target engagement in cells, western blot results for MYC or MYCN with and without treatment, and the impact of NMTi on viable cell number in cell lines. The data suggests that MYC deregulation influences cancer cell sensitivity to NMTi and demonstrates the effects of NMTi on target engagement and cell viability.

Figure S3 summary: The figure contains several types of charts and plots. It mainly explores the impact of NMT inhibitors on mitochondrial function in different cellular contexts, especially focusing on the role of MYC. The figure compares the effects of NMT inhibitors on oxygen consumption rate, mitochondrial potential, and superoxide production in cells with varying levels of MYC expression. The data suggests that NMT inhibition induces mitochondrial dysfunction more significantly in cells with high MYC expression. Furthermore, the figure identifies potential NMT substrates and membrane-localized proteins in different cell lines, providing insights into the mechanisms underlying the observed effects on mitochondrial function.

Figure S4 summary: The figure contains a combination of plots and diagrams. It shows MYCN ChIP-Seq tracks for NDUFAF4 in cells. The figure also depicts the calculation of Km for NMT activity in synthetic wild-type or mutant NDUFAF4 peptides. The figure illustrates validation of the lack of N -myristoylation of the NDUFAF4 mutant. Additionally, the figure displays the entire assembly scheme for human mitochondrial respiratory complex I as a cartoon representation. The mutant exhibits a lack of N-myristoylation compared to the wild type. The complex I assembly scheme highlights the role and position of NDUFAF4 within the complex.

Figure S5 summary: The table presents pharmacokinetic results for IMP-001320 after intraperitoneal dosing at a consistent level. Plasma levels of IMP-001320 show an initial rapid increase, reaching peak concentrations at very early time points, and then decline steadily over time. The maximum concentration (Cmax) is relatively high, while the time to reach maximum concentration (Tmax) is very short. The area under the curve (AUC) values, both to the last measured time point and extrapolated to infinity, are substantial, indicating significant exposure to the compound. The elimination half-life is moderate, suggesting a reasonable duration of action. The clearance rate is also moderate. Overall, the data indicates a rapid absorption and distribution, followed by a gradual elimination of IMP-001320.

Figure S6 summary: The table presents the pharmacokinetic profile of DDD86481 after a single intraperitoneal dose of 25 milligrams/kg in mice. Plasma levels of DDD86481 peaked rapidly, reaching maximum concentration within a short period. The concentration declined over time, with a substantial decrease by 24 hours post-dosing. The area under the curve (AUC) values suggest good systemic exposure, and the elimination half-life indicates a relatively short duration in the body. The variability in plasma levels and pharmacokinetic parameters among individual mice was relatively low for most metrics, indicating consistent drug exposure.

Figure S7 summary: The figure includes multiple plots. The first plot is a line plot illustrating the impact of a treatment on tumor volume over time. The treatment group exhibits a significantly lower tumor volume compared to the control group. The second plot is a line plot showing the change in mouse body weight between the start and end points of the experiments. There is no significant difference in body weight change between the treated and control groups. The third plot is a line plot depicting the metabolic viability of cancer cells treated with varying concentrations of two different compounds. One compound shows slightly more effectiveness compared to the other.

Figure S8 summary: The figure shows original image data for blots and gels. The blots show expression levels of proteins such as NMT1, NMT2, HSP90, AzTB, MYC, Vinculin, alpha-tubulin, and NDUFAF4. The figure also includes coomassie staining for reference. Some blots show original and contrast adjusted versions of the same protein.

Figure S9 summary: The figure displays original image data for blots and gels. The figure contains images related to NMT1, NMT2, HSP90, AzTB, Coomassie, MYC, Vinculin, AzT, alpha-tubulin, and NDUFAF4. The figure shows the original and contrast adjusted images for MYCN. The figure provides supporting data for other figures, offering a view into the underlying experimental results.

Figure S10 summary: The figure shows multiple scatter plots that represent examples of gating strategies for flow cytometry experiments. The plots display cell populations, viable cells, and single cells. The gating strategy helps differentiate and isolate specific cell populations for further analysis.

Table S2 summary: The table provides a comprehensive list of gRNA, cloning, and sequencing primer sequences. It includes various gRNA sequences for NMT1 and NMT2, along with corresponding sequencing primers. Additionally, it lists different versions of NDUFAF4 primers, including wild-type, G2A, and A3P mutations, and a complete NDUFAF4 gene block sequence. The sequences are diverse in length and composition, reflecting their specific roles in gene manipulation and analysis.