Methyltransferase Inhibition Enables Tgfβ Driven Induction of CDKN2A and B in Cancer Cells

Area of Science:

• Oncology
• Molecular Biology
• Epigenetics

Background:

• CDKN2A/B deletion or silencing is a common event in human cancers, highlighting its role as a tumor suppressor.
• In childhood cancers like rhabdomyosarcoma (RMS) and neuroblastoma, ARF, a CDKN2A/B transcript, is consistently silenced.

Purpose of the Study:

• To investigate the mechanisms of CDKN2A/B silencing in RMS and explore strategies for its reactivation.
• To understand the interplay between TGFβ signaling, methyltransferase activity, and a remote enhancer in regulating CDKN2A/B expression.

Main Methods:

• Utilized TGFβ stimulation in RMS cell lines, with and without methyltransferase inhibitors (DZNep, EZH2 inhibitors).
• Employed CRISPR activation (dCas9/CRISPR) to target the CDKN2A/B promoter and a remote cis-regulatory element.
• Assessed H3K27Ac marks to identify active enhancer regions and measured CDKN2A/B transcript levels and cell proliferation.

Main Results:

• TGFβ alone did not induce CDKN2A/B in RMS cells; pretreatment with methyltransferase inhibitors was required.
• TGFβ-induced CDKN2A/B expression correlated with H3K27Ac peaks at a remote 20kb cis-element, which is crucial for induction.
• CRISPR activation of CDKN2A/B via promoter or enhancer targeting suppressed RMS cell propagation in vitro.

Conclusions:

• Revealed crosstalk between methyltransferase inhibition and TGFβ-dependent enhancer activation to overcome CDKN2A/B silencing.
• Suggests that this regulatory crosstalk may extend to other TGFβ-responsive genes and influence TGFβ's dual role in cancer.