Direct Mapping of Transcription Factor Binding Reveals a Resistance Mechanism

To identify regulatory changes associated with TEAD inhibitor resistance, researchers first compared chromatin accessibility between drug-sensitive and drug-resistant mesothelioma cells using ATAC-seq. Regions becoming more accessible in resistant cells displayed an enrichment for TEAD and AP-1 transcription factor motifs, suggesting altered transcription factor activity.

The AP-1 (Activator Protein-1) transcription factor complex constitutes a family of regulatory proteins that includes FOS and JUN. AP-1 factors are frequently activated downstream of signaling pathways such as MAPK and play major roles in regulating proliferation, stress responses, and tumor progression. Single-cell ATAC-seq and RNA-seq analyses further supported this observation, revealing the increased activity of FOSL1, another member of the AP-1 transcription factor family.

However, these approaches provided only indirect evidence of transcription factor engagement. Motif enrichment suggests potential binding, while RNA-seq captures transcriptional consequences that occur downstream of regulatory events. To directly map transcription factor occupancy on chromatin in drug-resistant cancer cells, the investigators turned to CUT&RUN.

Using CUT&RUN, the researchers profiled genome-wide binding of TEAD1, YAP, and FOSL1 in drug-sensitive and drug-resistant cancer cells, revealing increased recruitment of all three transcription factors to regulatory regions associated with therapeutic resistance. Using CRISPR-mediated FOSL1 knockout cells, CUT&RUN demonstrated that FOS1L is required for YAP/TEAD recruitment to chromatin. Together, these results revealed a direct mechanistic link between signaling pathway activation and transcription factor binding in drug-resistant cancer cells.

Orthogonal Validation of Chromatin Changes Using ChIP-seq

The authors also generated ChIP-seq profiles for YAP and FOSL1, which closely matched the CUT&RUN results. Such concordance demonstrates that CUT&RUN robustly profiles transcription factor binding, addressing a common concern among researchers transitioning from traditional ChIP-based approaches. Importantly, CUT&RUN achieved this feat while profiling protein-DNA interactions directly in native chromatin using a simplified in situ workflow, all while producing higher signal-to-noise with far lower input requirements than ChIP-seq.

For drug development teams, these advantages of CUT&RUN translate into several enabling capabilities:

• The ability to generate mechanistic insights from limited samples (including primary cells, xenografts, or precious patient samples).
• More robust identification of transcriptional mechanisms driving drug response or therapeutic resistance.
• Assay automation for standardized, high throughput studies.

These capabilities are particularly valuable for directly verifying adaptive transcriptional programs that emerge during targeted cancer therapy, and support the emergence of CUT&RUN as the new gold standard approach for chromatin mapping.

Chromatin Profiling Guides Rational Combination Therapy

Integrated pathway analysis of the multiomic data implicated MAPK signaling, a known upstream activator of AP-1 transcription factors. These findings suggested that MAPK-driven activation of FOSL1 enables resistant cells to recruit YAP/TEAD back to chromatin, restoring oncogenic transcriptional programs despite TEAD inhibition and creating a MAPK-dependent escape mechanism.

These insights had immediate therapeutic implications, suggesting that MAPK inhibition could potentially restore sensitivity to TEAD inhibitors. Indeed, MAPK inhibition resensitized resistant cancer cells to TEAD-targeted therapy and improved tumor control in a mouse mesothelioma xenograft model. These findings highlighted the value of chromatin profiling in cancer drug development. By directly measuring transcription factor binding, CUT&RUN can reveal how regulatory networks change during drug treatment and identify signaling pathways that drive therapeutic escape.