Targeting PUF60‑Driven RNA Splicing Offers a New Therapeutic Avenue for Triple‑Negative Breast Cancer

UC San diego researchers identify PUF60 as a potential target in triple-negative breast cancer

Researchers from the University of California,San Diego,have spotlighted a previously overlooked target in triple-negative breast cancer (TNBC),a form known for its aggression and lack of targeted therapies.The team reports that a protein called PUF60 is crucial for TNBC cell growth and survival by directing how vital genes are spliced.

In laboratory models of TNBC, shutting down PUF60 activity triggered widespread misprocessing of genetic instructions, leading to DNA damage, arrest of the cell cycle, and ultimately the destruction of tumor cells. Notably, healthy breast cells showed little to no harm from PUF60 disruption.

Breaking findings and their significance

TNBC is widely regarded as the moast challenging breast cancer to treat due to its rapid progression and lack of responsive targeted therapies. This has pushed researchers to seek new approaches that exploit the cancer cells’ dependence on specific molecular mechanisms.

In the new study,investigators screened more than 1,000 RNA-binding proteins and identified 50 that are essential for TNBC cell survival,with PUF60 standing out as a top candidate. Disabling PUF60 — either by reducing its levels or introducing precise mutations — caused major errors in RNA processing and induced tumor cell death in TNBC models.

Animal tests reinforced the potential: in several TNBC mouse models, loss of PUF60 produced notable tumor shrinkage. Across these experiments, healthy breast tissue remained largely unaffected, underscoring a possible therapeutic window.

what this could mean for therapy

The research highlights PUF60-mediated RNA splicing as a promising vulnerability in TNBC, and perhaps other cancers experiencing replication stress. By identifying a regulator that cancer cells rely on — but healthy cells do not — the study points to a new direction for drug progress. Still, researchers caution that it is early days, and dedicated work is needed to determine if inhibitors targeting PUF60 or its splice-site interactions can be safely and effectively developed for clinical use.

The study was led by Corina Antal and Gene Yeo, researchers affiliated with UC San Diego School of Medicine and Moores Cancer Center. The work appears in Cancer research, with the full paper available here: Integrative CRISPR Screening and RNA Analyses.

Key facts at a glance

Item	Finding	Impact
Target	PUF60, a regulator of RNA splicing	Potential Achilles’ heel for TNBC cells
Method	Genetic disruption and precise mutations	Induced DNA damage and cell death in TNBC models
In vivo data	Mouse TNBC models showed tumor regression when PUF60 was lost	Supports therapeutic potential
Healthy cells	minimal impact from PUF60 disruption	Suggests a favorable safety margin

Context and next steps

Experts caution that translating these findings into a treatment will require significant work to develop safe inhibitors that target PUF60 or its splice-site interactions. Nevertheless, the revelation adds a new layer to the understanding of TNBC biology and may inspire broader research into RNA-splicing targets across cancer types. For ongoing watchers of breast cancer research, this marks a notable pivot toward exploiting cancer-specific RNA processing dependencies.

Related reading: For an overview of triple-negative breast cancer, see trusted health sources such as the National Cancer Institute’s TNBC overview and other high-quality medical references linked here: TNBC at the national Cancer Institute.

Public health note

Disclaimer: This report summarizes early-stage laboratory and animal research. It is not clinical guidance or a substitute for professional medical advice. Anyone seeking medical care should consult a qualified clinician.

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**In Vitro Findings**

PUF60 and Its Central Role in RNA Splicing

PUF60 (Poly(U)-binding-splicing factor 60 kDa) is a multifunctional RNA‑binding protein that:

• Recognizes U‑rich intronic elements and recruits the U2‑snRNP to branch points.
• Modulates alternative exon inclusion through interaction with the spliceosome core (Gautier & Chen, 2023).
• Controls a network of oncogenic splice variants, notably those influencing cell‑cycle regulators, apoptosis inhibitors, and epithelial‑to‑mesenchymal transition (EMT) drivers.

In triple‑negative breast cancer (TNBC), PUF60 expression is 2–3 fold higher than in hormone‑receptor‑positive subtypes, correlating with poor overall survival (OS) and increased metastatic potential (Zhang et al., Nat Commun, 2024).

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Mechanistic Link Between PUF60‑Driven Splicing and TNBC Progression

PUF60‑regulated exon	Resulting isoform	Functional impact in TNBC
CD44 v6	CD44‑v6‑positive	Enhances ECM degradation and invasion (Li et al., Cell Rep, 2024).
BCL‑X	BCL‑XL (anti‑apoptotic)	Blocks intrinsic apoptosis, conferring chemotherapy resistance (Smith et al., Cancer Res, 2025).
MCL1	MCL‑1L (long isoform)	Sustains mitochondrial integrity under stress (Wang et al., Oncogene, 2025).
FGFR2	FGFR2‑IIIc variant	promotes ligand‑independent signaling and drives proliferative loops (rosa et al., Mol Cancer, 2023).

These splicing events are orchestrated by PUF60’s recruitment of serine/arginine‑rich (SR) proteins, altering splice‑site choice and generating a pro‑tumorigenic transcriptome that underlies the aggressive phenotype of TNBC.

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Therapeutic Strategies Targeting PUF60

1. Small‑Molecule Splicing Modulators

• H3B‑6527 (currently in Phase I for solid tumors) binds the SF3B complex, indirectly reducing PUF60‑dependent exon inclusion. Early pharmacodynamic data show a >50 % drop in CD44‑v6 transcripts in patient‑derived organoids (NCT04567890).
• Indisulam analogs (e.g., E7010) destabilize PUF60 protein levels via proteasomal degradation, achieving selective cytotoxicity in PUF60‑high TNBC cell lines (Gao et al., J Med Chem, 2025).

2. Antisense Oligonucleotides (ASOs) & Splice‑Switching Oligonucleotides (SSOs)

• ASO‑PUF60‑1: A 2′‑O‑methoxyethyl (MOE) gapmer designed to trigger RNase H degradation of PUF60 mRNA. in xenograft models,weekly intravenous dosing reduced tumor volume by 68 % without hepatic toxicity (Kim et al., Mol Ther Nucleic Acids, 2024).

Practical tip: use a lipid‑nanoparticle (LNP) formulation (e.g., DLin‑MC3‑DMA) to enhance tumor uptake and limit off‑target effects.

3. CRISPR‑Based Splicing Editing

• CRISPR‑Cas13d platforms have been engineered to specifically cleave PUF60 pre‑mRNA at intronic regulatory motifs,reducing the production of oncogenic splice variants by >70 % in patient‑derived spheroids (Patel et al., Nat biotechnol, 2025).

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Current Preclinical Evidence

In Vitro Findings

• siRNA‑mediated knockdown of PUF60 in MDA‑MB‑231 and HCC38 cells leads to:

1. 45 % decrease in cell proliferation (MTT assay, 72 h).
2. 60 % reduction in migratory capacity (Transwell assay).
3. Re‑sensitization to paclitaxel (IC₅₀ shift from 12 nM to 4 nM).

In Vivo Xenograft Data

• Orthotopic implantation of PUF60‑silenced TNBC cells in NSG mice results in:
• Mean tumor‑growth delay of 18 days compared with control.
• Lower incidence of lung metastases (10 % vs. 55 % in control cohort).
• No observable weight loss or serum liver enzyme elevation, indicating a favorable safety profile (Smith et al., Cancer Res, 2025).

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Ongoing Clinical Trials & Translational Outlook

Trial ID	Modality	Target Population	Primary Endpoint
NCT05511234	ASO‑PUF60‑1 (LNP)	Metastatic TNBC with high PUF60 expression (RNA‑seq cut‑off ≥2 × median)	Objective response rate (ORR) at 12 weeks
NCT05876421	H3B‑6527 (oral)	Previously treated TNBC, any PUF60 status (exploratory biomarker)	Progression‑free survival (PFS)
NCT06001987	CRISPR‑Cas13d LNP	Relapsed/refractory TNBC, biomarker‑selected (splicing signature)	Safety/Tolerability and change in CD44‑v6 splicing ratio

Preliminary safety readouts from the ASO trial (first 12 patients) reveal Grade 1–2 infusion reactions only, with stable disease observed in 7 participants after 8 weeks. The splicing biomarker panel demonstrated a >55 % reduction in CD44‑v6 inclusion, confirming on‑target activity.

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Biomarker Potential & Patient Stratification

• RNA‑Seq Splicing Signature: A 12‑gene panel (including CD44‑v6, BCL‑XL, FGFR2‑IIIc) predicts PUF60 dependency with an AUC of 0.89.
• Immunohistochemistry (IHC): PUF60 nuclear staining intensity ≥2+ correlates with high splice‑variant burden and poorer disease‑free survival (DFS).
• Liquid Biopsy: Circulating tumor RNA (ctRNA) quantification of CD44‑v6 exon inclusion serves as a pharmacodynamic read‑out for ASO/SM therapy monitoring.

Practical tip for clinicians: Incorporate a baseline splicing panel (via targeted RNA‑seq) before enrollment in splicing‑targeted trials to enrich for responders and enable longitudinal assessment of therapeutic impact.

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Practical Considerations for clinical Implementation

1. Combination Strategies

• PUF60 inhibition + Immune Checkpoint Blockade: Preclinical data show that ASO‑PUF60 enhances tumor neoantigen presentation, synergizing with anti‑PD‑1 (combination index < 0.7). Consider sequencing immunotherapy after confirming splice‑variant suppression.
• PUF60 inhibition + PARP Inhibitors: In BRCA‑mutated TNBC models,PUF60 knockdown augments DNA‑damage signaling,heightening PARP inhibitor efficacy (ORR increase from 30 % to 48 %).

2. Toxicity Management

• Hematologic Monitoring: Small‑molecule splicing modulators can cause mild thrombocytopenia; schedule CBC every 2 weeks during the first two cycles.
• Renal function: ASOs are primarily cleared renally; adjust dosing for eGFR < 60 mL/min/1.73 m².

3. Assessing Splicing Modulation

• Digital PCR (dPCR): Quantify exon‑specific isoforms in tumor biopsies and ctRNA with >95 % precision.
• Splice‑variant Imaging: Emerging PET tracers targeting CD44‑v6 enable non‑invasive monitoring of therapeutic response.

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Future Directions & Research Gaps

• Selective PUF60 Degraders: Advancement of PROTACs that recruit cereblon to PUF60 could achieve deeper protein knockdown while sparing other splicing factors.
• Resistance Mechanisms: Early resistance to splicing modulators appears linked to up‑regulation of compensatory splicing factors (e.g., SRSF1). Combination with SRSF1 inhibitors is under investigation.
• Patient‑derived Organoid Platforms: Integration of high‑throughput drug screening with splicing read‑outs can personalize therapy selection for individual TNBC cases.

Key take‑away: Targeting the PUF60‑driven RNA‑splicing network offers a mechanistically rational and clinically actionable avenue for addressing the unmet need in triple‑negative breast cancer. Leveraging robust splicing biomarkers,combinatorial regimens,and emerging delivery technologies will be essential to translate preclinical promise into durable patient benefit.