Honeybadger: A Novel Regulator of RAS-MAPK Signaling in MYC-Driven Tumors

Honeybadger: A Novel Regulator of RAS-MAPK Signaling in MYC-Driven Tumors

Uncovering a Critical Tumor-Suppressive Micropeptide

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• Introduction: PVT1 Locus & Cancer
• PVT1ts: A Novel Tumor Suppressor
• Honeybadger (HNB): Discovery & Expression
• HNB Regulation of RAS/MAPK Pathway
• HNB-KRAS Interaction Mechanism
• HNB, MYC Stabilization & Tumor Growth
• PVT1 Locus Alterations & Tumorigenesis
• Conclusion & Therapeutic Implications

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PVT1 Locus and Cancer

Genomic Rearrangements and Their Role in Tumorigenesis

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• Genomic rearrangements, beyond classical fusions, drive cancer progression.
• Long noncoding RNAs (lncRNAs) often involved, but their functional impact is opaque.
• Plasmacytoma Variant Translocation 1 (PVT1) is a frequently altered locus in MYC-driven cancers.
• PVT1 translocations produce an asymmetric architecture, preserving 5′-PVT1 region.
• This leads to circular RNA (CircPVT1) encoding Firefox (FFX), an oncoprotein activating AKT-mTORC1 signaling.

• Genomic translocations at the PVT1 locus are highly recurrent in human cancers.
• These rearrangements preferentially delete the 3′-PVT1 region while retaining the 5′-PVT1 segment.
• This asymmetry suggests a selective loss of tumor-suppressive genomic material.
• The 5'-PVT1 region encodes the oncoprotein Firefox (FFX).

• The deleted 3′-PVT1 segment encodes a tumor-suppressive micropeptide.
• This previously unrecognized transcript variant is designated PVT1ts.
• Loss of PVT1ts is a consequence of PVT1 translocations.
• PVT1ts silencing accelerates proliferation and increases MYC protein levels in MYC/PVT1-neutral cell lines.
• Depletion of residual PVT1ts in D458 cells enhanced tumor growth in vivo.

• Orthotopic transplantation assay shows accelerated tumor growth upon PVT1ts depletion.
• D458 cells with shRNA1-PVT1ts implanted into cerebella of immunocompromised mice.
• Significantly enhanced tumor growth observed at days 9, 14, and 21 post-transplantation.
• Loss of PVT1ts promotes MYC upregulation, accelerates proliferation, and enhances tumor aggressiveness.

Honeybadger: A Novel Micropeptide

Discovery and Endogenous Expression in Human Cells

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• Analysis of PVT1ts transcript revealed a short open reading frame (sORF).
• This sORF encodes a 14-amino acid micropeptide named Honeybadger (HNB).
• CRISPR/Cas9 introduced an in-frame HiBiT tag for endogenous detection.
• Lytic bioluminescence assays confirmed endogenous expression of the HNB peptide.
• Immunofluorescence further validated the presence of the tagged HNB peptide.

• Figure 2A shows the nucleotide and amino acid sequence of the 14-residue HNB micropeptide.
• CRISPR/Cas9 strategy introduced an in-frame HiBiT tag for sensitive detection (Figure 2B).
• Lytic bioluminescence assays confirmed endogenous HNB-HiBiT signal in U2OS knock-in cells (Figure 2C).
• Immunofluorescence staining with anti-HiBiT antibody further validated tagged HNB peptide presence (Figure 2D).

• PepQuery2 interrogation of human proteomics databases identified the HNB-derived peptide sequence TQLGAVK.
• High-confidence detection in multiple independent samples confirmed endogenous HNB expression.
• Figure 2E highlights the HNB peptide sequence with the TQLGAVK segment.
• Figure 2F displays a representative TMT-labeled MS/MS spectrum of the detected HNB-derived peptide.

HNB Regulation of RAS/MAPK

Defining the Tumor-Suppressive Mechanism

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• Transcriptomic profiling of D458 cells after HNB knockdown implicated enhanced KRAS pathway activity.
• Gene Set Enrichment Analysis (GSEA) showed downregulation of genes typically decreased by KRAS activation.
• HNB knockdown led to increased phosphorylation of MEK1/2 and ERK1/2.
• This indicates elevated RAS/MAPK signaling upon HNB depletion in D458 and NCI-H1792 cells.

• Western blot analysis demonstrates increased phospho-MEK1/2 and phospho-ERK1/2 levels.
• This occurs in D458 (KRAS wild type) and NCI-H1792 (KRASG12C/+) cells upon HNB depletion.
• The rise in phosphorylation signifies elevated RAS/MAPK signaling activity.
• HNB acts as a negative regulator, suppressing this critical pathway.

HNB Expression Suppresses RAS/MAPK Doxycycline-inducible HNB transduction in D458 (wild-type KRAS) and NCI-H1792 (KRASG12C/+) cell lines.

Induction of HNB significantly reduced phosphorylation of MEK1/2, ERK1/2, and p90RSK.

Direct Regulation of Pathway Activity This provides strong evidence that HNB acts as a negative regulator of RAS/MAPK signaling.

The findings confirm HNB's sufficiency in modulating this crucial pathway.

This modulation occurs in both KRAS wild-type and mutant contexts.

HNB-KRAS Interaction

Unveiling the Molecular Mechanism of Regulation

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• AlphaFold 3 modeled a high-confidence interaction interface between HNB and KRAS.
• Figure 4A shows the 14-amino acid HNB peptide structure.
• Figure 4B illustrates the per-residue predicted aligned error of the AlphaFold prediction, highlighting the interaction interface.
• IPSAE analysis indicated HNB residue F11 as a key contact point.