PUF60-Mediated Splicing Emerges as a Therapeutic Vulnerability in Triple-Negative Breast Cancer

Recent research from the University of California-San Diego (UCSD) has unveiled an unexpected molecular dependency in triple-negative breast cancer (TNBC)—the RNA-binding protein PUF60—revealing an important role for RNA splicing regulation in tumor survival and progression.^1,2

In their approach, the UCSD group used an integrative genome-wide CRISPR/Cas9 screening strategy and paired this with RNA sequencing to assess in a systematic manner the contribution of RNA-binding proteins (RBPs) to the viability of TNBC cells. Among over 1000 RBPs assessed, 1 of the major RBP candidates found to be essential for the viability of TNBC cells is PUF60, which is a poly(U)-binding splicing factor. Based on these functional assays, PUF60 is implicated in splicing, as opposed to merely associating with splicing, in processes related to cell proliferation and survival.²

Significantly, the data from the integrative analysis with PUF60 were derived by combining both enhanced crosslinking immunoprecipitation (eCLIP) data with RNA-seq data from direct RNA targets as a result of disrupted PUF60 function. This showed that PUF60 is implicated in the inclusion of certain exons within certain proliferation-related transcripts. Disrupting PUF60—either by RNA-mediated knockdown or by mutations disrupting PUF60 protein interactions with 3’ splice sites—leads to a massive exon skip with a resultant decrease in gene transcripts necessary for cell survival as well as the accumulation of DNA damage along with the initiation of the apoptotic pathway among TNBC cells.²

Mechanistic Basis for Tumor Suppression

Splicing factors like PUF60 influence how pre-mRNA is processed into mature, translatable mRNA. The UCSD study demonstrated that when PUF60’s interaction with 3’ splice sites is hindered, nearly global changes in splicing occur that disproportionately affect transcripts involved in cell cycle progression and DNA repair. Because TNBC cells often exhibit heightened replication stress and DNA repair deficiencies, they appear particularly sensitive to disruptions in splicing fidelity—a dependency not shared by normal breast epithelial cells.¹

In both in vitro cell models and in vivo TNBC xenografts, loss of PUF60 function led to significant inhibition of tumor growth and even regression of established tumors, highlighting PUF60’s potential as a therapeutic target.^1,2 The specificity of this vulnerability is underscored by the observation that normal breast cells were largely unaffected by PUF60 inhibition, suggesting a therapeutic window that could be exploited with targeted agents.

Clinical Significance and Therapeutic Opportunities

The discovery of PUF60’s role in supporting TNBC tumor survival has several important clinical implications. First, it extends the framework of targetable cancer dependencies beyond receptor signaling and DNA-damaging agents, positioning RNA splicing mechanisms as viable drug targets. Small molecules that perturb spliceosome function are already in development for certain hematologic and solid tumors, and the current findings suggest that similar strategies could be explored for TNBC with a focused approach on PUF60 or its interactors.²

Additionally, combination approaches that utilize splicing modulation alongside existing chemotherapeutic agents would also improve antitumor efficacy because TNBC often relapses following chemotherapy and becomes resistant to many therapies. In such combinations, increased DNA damage from impaired splicing could then force tumor cells beyond their repair capability, causing enhanced cell death.

Challenges and Future Research Directions

Despite the promising nature of these findings, challenges remain before PUF60-targeted therapies can enter clinical, real-world use. Development of drugs that selectively disrupt PUF60’s splicing activity without harming global splicing in healthy tissues will require sophisticated medicinal chemistry and careful preclinical toxicity studies. Additionally, further research is needed to understand the full spectrum of PUF60’s RNA targets, since comprehensive mapping may reveal splicing events crucial for normal physiology that could complicate therapeutic targeting.

Another issue is whether there is a role for splicing factors in addition to TNBC and whether these have similar dependencies on PUF60 or related splicing factors. In this case, therapies targeting splicing could have broad applicability across multiple cancers characterized by high splicing flux.

Conclusion

TNBC, which is an aggressive form of breast cancer that lacks estrogen, progesterone, and hormone estrogen receptor 2, continues to challenge clinicians and patients alike because of its poor prognosis and limited targeted treatment options.

The integrative CRISPR and RNA analysis that was conducted by the UCSD research team uncovered a compelling vulnerability in TNBC centered on PUF60-mediated RNA splicing. By disrupting PUF60 function, the study demonstrated that TNBC cells accumulate critical RNA processing errors, triggering cell cycle arrest and tumor regression, while sparing normal cells.^1,2

This discovery not only enhances the mechanistic understanding of TNBC biology, but it also opens new avenues for the development of targeted therapies specifically aimed at RNA splicing—a frontier in cancer treatment that may finally address the unmet needs of patients who have aggressive breast cancer.

REFERENCES

1. Research alert: New vulnerability identified in aggressive breast cancer. EurekAlert! January 12, 2026. Accessed January 14, 2026. https://www.eurekalert.org/news-releases/1112345

2. Tankka AT, Einstein JM, Zhou CJ, et al. Integrative CRISPR Screens and RNA-Omics Discover an Essential Role for PUF60-3' Splice Site Interactions in Cancer Progression. Preprint. bioRxiv. 2025;2025.05.01.651692. Published 2025 May 7. doi:10.1101/2025.05.01.651692