Can Micro Capture-C Help Define How Cancer Cells Respond to Small-molecule Drug Treatment?

The long-range regulatory elements present throughout the non-coding genome – which include enhancers, silencers, and insulators - orchestrate transcription factor-regulated gene expression through interactions with gene promoters thanks to chromatin looping (Pollex et al. and Ong & Corces. These processes remain fundamental to normal cellular function and, as such, the disruption of regulatory element-promoter connections through genetic and epigenetic mechanisms can prompt disease development (Cosenza et al. and Sur & Taipale). Furthermore, these processes may play an important role in the adaptation of cancer cells to selective pressures, such as those imposed by targeted therapies (Musheyev & Alayev and Vasan et al.). Micro Capture-C – a chromatin conformation capture method - can evaluate regulatory element-promoter interactions at base-pair resolution (Hua et al. and Hamley et al.) and help to define cancer-associated interactions and explore how cancer cells respond and develop resistance to small-molecule drugs. Therefore, Micro Capture-C may help to define weaknesses that could inform the treatment of aggressive, drug-resistant tumor types.

In a recent Nature Structural & Molecular Biology article, researchers led by Bence Daniel (Genentech Inc.) applied Micro Capture-C to mesothelioma cells that display resistance to an inhibitor of the TEAD family (TEAD1–4) transcription factors (Dey et al. and Calses et al.), which drive this aggressive tumor type through binding to promoter-distal cis-regulatory elements (Kameswaran et al.). This inhibitor - GNE-7883 - functions by displacing the YAP cofactor from chromatin-bound TEAD (Hagenbeek et al.). Excitingly, this chromatin conformation analysis-driven study now reveals that TEAD inhibitor-resistant mesothelioma cells can sustain pro-tumorigenic gene expression profiles not by recovering lost regulatory element-promoter interactions associated with inhibitor treatment but instead by a newly described phenomenon named "promoter reinforcement," which replaces lost regulatory element-promoter interactions via enhanced promoter activity.

Paired-Tag technology from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Could an in-depth analysis of the histone modification and transcriptomic profiles of the same single mesothelioma cells afforded by the integration of Paired-Tag have provided more profound insight into the epigenetic alterations that accompany changes to chromatin conformation induced by drug treatment and that underpin resistance and help to define the mechanisms that lead to promoter reinforcement?

Micro Capture-C Reveals Promoter Reinforcement as a Drug Resistance Mechanism in Mesothelioma Cells

The study began by analyzing mesothelioma cells before and after GNE-7883 treatment and mesothelioma cells displaying resistance to GNE-7883 treatment by bulk RNA-seq to define transcriptional responses to treatment and resistance; these data helped to define the restoration of a parental-like transcriptional program in resistant mesothelioma cells, with a minor contribution from the expression of genes not initially expressed in parental cells. Subsequent ChIP-seq analysis of YAP revealed that drug resistance associated with the restoration and expansion of the YAP chromatin-binding profile lost during acute treatment; of note, these data provided the first evidence of promoter bias as part of a promoter reinforcement strategy.

[! Two IGV screenshots showing ChIP-seq and micro-C pileups across treatment conditions and cell lines](img/image0.png Panels a and b present genome browser tracks of MCC, YAP, and CTCF ChIP-seq signals and ACTB-normalized mRNA levels at the FOSL1 and TACC3 loci in H226 cells following TEAD-family inhibitor treatment and CRISPR deletion. The data reveal that while enhancer loops are ablated in resistant cells, YAP remains loaded at both the enhancer and promoter regions. From Figure 3 of (Hagenbeek et al.)

At this stage, the authors applied Micro Capture-C to assess the potential impact of treatment and resistance on long-range interactions between regulatory elements and gene promoters. Interestingly, they did indeed observe dysregulated interaction networks in resistant mesothelioma cells, which manifested as reduced regulatory element-gene promoter interactions and increased gene promoter activity, providing further evidence of promoter reinforcement. Indeed, additional CRISPR-based perturbations of regulatory elements and gene promoters demonstrated that genes could recover or maintain gene expression in resistant cells despite reduced distal regulatory element function, suggesting a shift toward promoter-reinforced control of gene expression and the dispensability of enhancer elements. The authors next identified the transcription factor network regulating gene expression in resistant cells, highlighting the central roles of FOSL1, KLF4, and ETV4. Interestingly, CRISPR-mediated knockdown of KLF4 impaired the proliferation of resistant cells but not that of parental cells, suggesting a potential therapeutic weakness. The study next moved to create a view of the regulatory landscape at target promoters and their interacting regulatory element through a combined analysis of transcriptomic and epigenomic datasets. These data suggested that long-term TEAD inhibition reshapes the regulatory architecture of cells by inhibiting enhancer-associated H3K27ac (thanks to the loss of p300 activity) and long-range chromatin looping, while enabling promoters to regain transcriptional activity, as evidenced by the restoration of high levels of H3K4me3. The subsequent integration of chromatin accessibility via ATAC-seq linked the promoter-centric mode of transcriptional control in inhibitor-resistant cells with an increased level of chromatin remodeling activity.

[! Multi-panel image showing ChiP-QPCR results](img/image1.png Panels b and c display quantitative data from parental and resistant cells following KLF4 knockdown, featuring a heatmap of ACTB-normalized mRNA levels for twenty genes, box plots of KLF4 ChIP–qPCR enrichment at the indicated gene promoters, and gauge plots illustrating promoter transcript levels in resistant cells under control versus knockdown conditions. Together, these data show that in cells with acquired resistance to R-G7883, the re-expression and elevated transcript levels of these genes are strongly associated with KLF4 occupancy at their promoters. From Figure 5 of (Hagenbeek et al.)

In further support of promoter reinforcement as a general strategy beyond mesothelioma, the authors note additional studies - Martin et al. in the context of acute inhibition of SWI/SNF chromatin remodeling complexes and Zhu et al., who reported the global loss of enhancer–promoter communication in the context of carcinogenesis - that describe a similar phenomenon, Overall, this body of research provides robust evidence for promoter reinforcement as a broader mode of transcriptional adaptation in cancer.

Promoter Reinforcement – A Novel and Targetable Transcriptional Adaptation in Cancer?

This exciting study – supported by the Micro Capture-C assay - now establishes promoter reinforcement as a novel mechanism of therapeutic resistance in cancer cells, which may be therapeutically targeted to improve patient outcomes in the future. Looking to the future of this research, the authors highlight how determining the generality and therapeutic implications of this mechanism across additional drug-resistant cancer models represents the next step.

The implementation of Paired-Tag technology from Epigenome Technologies, which generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays, has the potential to provide deeper insight into such research aims. Could the simultaneous single-cell analysis of histone modification and transcriptomic profiles in treatment-resistant cancer cells complement the findings of the Micro Capture-C assay and help to provide even deeper insight into promoter reinforcement?