How a Methyltransferase Activity Assay Could Help Identify Novel Breast Cancer Treatments

As estrogen receptor (ER) positive breast cancer (BC) becomes resistant to antiestrogen therapy, novel ways to control the disease are and continue to be developed. There are different types of breast cancer, and the most effective treatment depends on knowing this difference.  For example, if the type is estrogen receptor positive (ER) meaning cells are relying on this protein receptor to grow, then a therapy blocking the receptor is imperative.¹ What happens when these types of cancer cells become resistant to the so-called antiestrogen therapies, which often occurs? Even though there is a myriad of potential causes for resistance, the bottom line is that it must be stopped or at the very least, controlled in order for patients to remain living cancer free. A recent study found a histone methyltransferase called DOT1L (disruptor of telomeric silencing-1-like) that when inhibited, silences the ERα gene making it a potential target for treating antiestrogen resistant ER-positive BCs.²

Histone methyltransferases (HMT) are enzymes that assist in the transferring of methyl groups to lysine and arginine residues of histone proteins. Histone methylation plays an essential role in a variety of biological processes such as gene expression, cell lineage development, and mitosis. Nassa et al. found that both the H3K79 methyltransferase, DOT1L, and the ERα protein associate together with chromatin.³ In addition, after further investigation of the chromatin-bound ERα-DOT1L complex, they observed other molecules involved in the progression of ER-positive BCs via cell cycle progression, cell survival, and transcription.  Moreover, they found that when DOT1L was inhibited, a decreased progression in ER-positive BCs was detected.

Being able to measure HMTs activity is necessary especially in research involving gene regulation. DOT1L, as well as other HMTs, can be calculated using a methyltransferase activity assay.

How Can a Methyltransferase Activity Assay Help?

While most methyltransferase activity assays detect the invariant product of HMT reactions, S-adenosylhomocysteine (SAH) indirectly or by the use of coupled enzymes, the AptaFluor SAH Methyltransferase Assay affords direct detection. Not only is this universal approach capable of detecting a range of HMT activity including DOT1L, it also allows for more sensitive binding to SAH making the assay remarkable for high-throughput screening (HTS).⁴   Whether researching gene regulation in breast cancer or exploring DNA repair mechanisms, the AptaFluor SAH Methyltransferase Assay can assist in evaluating the HMT of interest, finding new inhibitors faster, thus accelerating drug discovery.

A. Dot1L was titrated in the presence of 200 nM SAM and 10 ng/µL of mono/di nucleosomes to determine an optimal EC₈₀ concentration of 6 ng/µL. Reactions were incubated at 30C for two hours. B. TR-FRET values were converted to SAH production to assess reaction progress. C. Dose-response curves with two known inhibitors of Dot1L, EPZ004777 and EPZ5676 show an IC₅₀ value of 1.7 nM and 1.4 nM respectively.

References

1. Cancer B, Breast Cancer Hormone Receptor Status 2019:1-4. https://www.cancer.org/cancer/breast-cancer/understanding-a-breast-cancer-diagnosis/breast-cancer-hormone-receptor-status.html
2. Frogne T, Jepsen JS, Larsen SS, Fog CK, Brockdorff BL, Lykkesfeldt AE. Antiestrogen-resistant human breast cancer cells require activated Protein Kinase B / Akt for growth. 2005:599-614. https://europepmc.org/abstract/med/16172194
3. Nassa G, Salvati A, Tarallo R, et al. Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells. 2019;(February):1-15. https://advances.sciencemag.org/content/5/2/eaav5590
4. AptaFluor SAH: A Homogenous, Universal Methyltransferase Assay Based on a Microbial Riboswitch Meera Kumar and Robert G . Lowery. :2017. https://www.bellbrooklabs.com/wp-content/uploads/2018/01/AptaFluor-SAH-A-Homogenous-Universal-Methyltransferase-Assay-Based-on-a-Microbial-Riboswitch-DOT-2017.pdf