A bivalent lysine-acetylated small-molecule binding site in MYC

This study identifies a novel bivalent binding site formed by the bHLH and eMBII domains of the MYC protein, which is enhanced by lysine acetylation at K148, thereby enabling the selective targeting of oncogenic MYC with small-molecule inhibitors.

The Gist

The Big Problem: The "Ghost" Target

Imagine cancer cells as a city run by a chaotic, hyper-active mayor named MYC. This mayor is the boss of cell growth, telling the city to build more houses (cells) faster and faster. If you can stop this mayor, you can stop the cancer.

However, for decades, scientists have been unable to fire this mayor. Why? Because MYC is a "shape-shifter." Unlike other cancer targets that look like a solid lock with a keyhole, MYC is like a blob of jelly. It doesn't have a fixed shape or a clear "keyhole" (a binding pocket) where a drug can latch on. Because it's so floppy and changes shape constantly, traditional drugs just slide right off it. This has made MYC one of the most famous "undruggable" targets in medicine.

The Discovery: Finding the "Velcro"

In this study, the researchers discovered something surprising. Even though MYC is mostly jelly, it has two specific spots that act like sticky Velcro patches.

1. Patch A: A region near the end of the protein (called bHLH-LZ).
2. Patch B: A previously ignored region in the middle (called eMBII).

The researchers found that a drug called MYCi975 doesn't just stick to one patch; it grabs onto both at the same time.

• The Analogy: Imagine trying to hold a slippery fish with one hand. It's hard; it will wiggle free. But if you use two hands to grab it at two different points simultaneously, it's much harder to escape. The drug acts like a "two-handed grip," locking onto both patches of the MYC protein to stop it from doing its job.

The Secret Ingredient: The "Acetylation" Badge

Here is where it gets even more clever. The researchers noticed that in cancer cells, the "Patch B" (eMBII) often wears a special badge called acetylation (specifically on a spot called K148).

• The Analogy: Think of the cancer version of MYC as wearing a glowing neon vest. This vest (acetylation) is actually what makes the mayor so dangerous and helps the cancer grow.
• The Twist: The researchers found that their drug doesn't just grab the Velcro; it actually sticks tighter when the neon vest is on.
  • If the MYC protein is "normal" (no vest), the drug holds on loosely.
  • If the MYC protein is "cancerous" (wearing the neon vest), the drug grabs on super tight.

This is a huge deal because it means the drug can selectively target the cancer cells while mostly ignoring healthy cells that don't have the neon vest. It's like a security guard who only arrests people wearing a specific red hat, leaving everyone else alone.

How They Proved It

The team didn't just guess; they used several clever tricks to prove this:

1. The "CRISPR Tiling" Game: They used a gene-editing tool (CRISPR) to make tiny scratches all over the MYC gene in cancer cells. They then fed the cells the drug. The cells that survived were the ones where the scratches had broken the "Velcro patches." By looking at where the scratches were, they confirmed that the drug needs both patches to work.
2. The "Acetylation Mimic": They created a fake version of the protein that looked like it had the neon vest on, even without the actual chemical change. The drug stuck to this fake version just as well as the real thing, proving the vest is the key to the lock.
3. The Better Drug (MYCi648): They took the original drug and tweaked its shape to make it an even better "Velcro grabber." This new version (MYCi648) worked even better in mice, shrinking tumors significantly more than the old drug, even at lower doses.

Why This Matters

This paper changes the game for treating cancer in two major ways:

1. It solves the "Undruggable" mystery: It shows that even floppy, shape-shifting proteins (like MYC) can be targeted if you find the right "two-handed" grip.
2. It offers a safety net: Because the drug loves the "neon vest" (acetylation) found mostly in cancer, it might have fewer side effects on healthy people. It's a "smart bomb" rather than a "carpet bomb."

In summary: The researchers found a way to handcuff the chaotic cancer mayor (MYC) by using a two-handed grip on his body. They discovered that the drug works best when the mayor is wearing his "dangerous" neon vest (acetylation), which is common in tumors but rare in healthy tissue. This opens the door to a new generation of drugs that can finally take down this long-standing cancer boss.

Technical Summary

1. Problem Statement

The oncoprotein MYC is a master regulator of cancer hallmarks (proliferation, metabolism, immune evasion) but remains one of the most challenging targets in oncology.

• Undruggable Nature: MYC is largely an Intrinsically Disordered Protein (IDP) lacking stable tertiary structures and traditional "druggable" pockets.
• Lack of Selectivity: Unlike RAS, MYC is rarely mutated in human cancers, making it difficult to selectively target the oncogenic form over the wild-type protein.
• Current Limitations: Previous small-molecule inhibitors (e.g., MYCi975) have shown efficacy but lack a clear structural rationale for their binding mechanism and selectivity. Their affinity for the known "MycHot" region in the basic-Helix-Loop-Helix (bHLH) domain is modest (micromolar range).

2. Methodology

The authors employed a multidisciplinary approach combining structural biology, chemical biology, genomics, and proteomics:

• Biophysical Binding Assays:
  • Biolayer Interferometry (BLI): Used to measure direct binding affinities ($K_d$) of MYC inhibitors (MYCi975, MYCi648) to full-length recombinant MYC, isolated domains (bHLH-LZ, eMBII), and various mutants.
  • Fluorescence Polarization: Used for initial competitive binding estimates.
  • Pull-down Assays: Utilized biotinylated-MYCi975 to isolate MYC from cell lysates, followed by Western blotting to assess binding to specific domains.
• Genetic Screening & Validation:
  • CRISPR-tiling Mutagenesis: An unbiased screen using 886 sgRNAs covering the entire c-MYC gene in PC-3M cells (high MYC copy number) treated with MYCi975 to identify resistance alleles.
  • Deep Sequencing: Targeted ultra-deep sequencing of resistant cell populations to map mutation clusters.
  • Rescue Experiments: Generation of stable cell lines expressing wild-type (WT) vs. mutant (WAL-QRA) MYC to test resistance and degradation.
• Post-Translational Modification (PTM) Analysis:
  • Acetylation Mimicry: Generation of recombinant MYC with K-to-Q (acetylation mimic) and K-to-R (non-acetylatable) mutations.
  • Site-Specific Acetylation: Genetically encoded incorporation of acetyl-lysine (Ac-K) at positions K148 and K157 in E. coli to produce authentic acetylated MYC.
  • AlphaFold 3 Modeling: Predicted structural changes in the eMBII region upon acetylation.
• Omics & Clinical Data:
  • Pan-Cancer Acetylome Analysis: Re-analysis of Clinical Proteomic Tumor Analysis Consortium (CPTAC) data to compare K148 acetylation levels in tumors vs. normal tissues.
  • Transcriptional Profiling: Re-analysis of RNA-seq data to identify gene clusters dependent on MYC acetylation and their response to MYCi treatment.
• In Vivo Studies: Pharmacokinetic (PK) analysis and tumor regression studies in immunocompetent syngeneic mouse models (MycCaP prostate and LLC1 lung tumors) comparing MYCi975 and the analog MYCi648.

3. Key Contributions

• Discovery of a Bivalent Binding Site: The study identifies that MYC inhibitors do not bind solely to the bHLH-LZ domain but engage a bivalent site formed by the cooperative interaction of the bHLH-LZ domain and a previously unrecognized extended MYC Box II (eMBII) region (amino acids 127–189).
• Acetylation-Dependent Selectivity: The authors demonstrate that acetylation of lysines K148 and K157 within the eMBII region (essential for MYC oncogenicity) induces a conformational change that increases the structural order of the region and enhances inhibitor binding affinity.
• Therapeutic Window: This mechanism provides a rationale for selectively targeting "oncogenic" acetylated MYC in tumors while sparing normal tissue, addressing the selectivity challenge.
• Novel Inhibitor Optimization: Development of MYCi648, a potent analog with improved binding to acetylated MYC and superior in vivo efficacy compared to MYCi975.

4. Key Results

• Binding Affinity & Bivalency:
  • Full-length MYC binds MYCi975 with a $K_d$ of ~262 nM, significantly tighter than the isolated bHLH-LZ domain (~2.2–2.5 µM).
  • Deletion of either the bHLH-LZ or the eMBII region drastically reduces affinity, confirming both are required for high-affinity binding.
  • A mutant linking bHLH-LZ and eMBII with a flexible linker showed even higher affinity ($K_d$ ~120 nM), suggesting the two regions cooperate to form a pocket.
• CRISPR Resistance Screen:
  • Resistance mutations clustered non-randomly in two distinct regions: the eMBII (e.g., S192W, V194A, F195L) and the bHLH-LZ (e.g., R372Q, S373R, F374A).
  • Cells expressing the double mutant (WAL-QRA) showed ~15-fold reduced binding affinity and resistance to MYCi975-induced degradation.
• Impact of Acetylation:
  • AlphaFold 3 predicted that K148/K157 acetylation stabilizes the helix in the eMBII region.
  • BLI Assays: Acetylated MYC (K148/Ac-K157) bound MYCi975 with significantly higher affinity ($K_d$ 90 nM) compared to WT (262 nM) or non-acetylatable mutants.
  • Cellular Pull-down: MYCi975 preferentially pulled down acetylated MYC from cancer cells.
• Clinical Relevance:
  • Pan-cancer analysis of CPTAC data revealed that K148 acetylation is significantly elevated in tumors (breast, lung, glioblastoma) compared to matched normal tissues.
  • Transcriptional Selectivity: MYCi975 treatment selectively repressed gene clusters dependent on acetylated MYC (cell cycle, biosynthesis) while sparing acetylation-independent clusters.
  • Inverse Correlation: Higher AcK148-MYC levels correlated with lower IC50 (higher sensitivity) to MYCi975 across 12 cancer cell lines.
• In Vivo Efficacy:
  • MYCi648, despite lower systemic exposure (lower Cmax/AUC) than MYCi975, demonstrated superior tumor growth inhibition (TGI) in mouse models (e.g., >100% regression in MycCaP vs. 52% for MYCi975).
  • MYCi648 retained cross-resistance with MYCi975 in mutant cells, confirming the shared bivalent binding mechanism.

5. Significance

• Paradigm Shift in IDP Targeting: This work challenges the notion that IDPs are undruggable by demonstrating that cooperative interactions between disordered and ordered regions can create high-affinity, druggable pockets.
• Mechanism of Selectivity: It establishes a novel mechanism for drug selectivity: targeting a post-translationally modified state (acetylated MYC) that is enriched in tumors but rare in normal tissues. This overcomes the lack of cancer-specific mutations in MYC.
• Therapeutic Implications: The findings suggest that tumors with dysregulated acetate metabolism or HDAC5 loss (leading to MYC hyperacetylation) may be particularly sensitive to MYC inhibitors.
• Future Directions: The study provides a blueprint for designing next-generation inhibitors that specifically exploit the acetylated conformation of IDPs, potentially applicable to other intrinsically disordered oncoproteins.

In summary, the paper elucidates a bivalent, acetylation-dependent binding mechanism for MYC inhibitors, offering a structural and functional basis for the selective targeting of oncogenic MYC in cancer therapy.