Epigenetic drug discovery has generated a lot of excitement in the past few years, and many stakeholders are eagerly awaiting clinical results for the first two histone methyltransferase (HMT) inhibitor drugs, which target Dot1L and EZH2. The analogies with kinase drug discovery, i.e., targeting oncogenic gain-of-function driver mutations in enzymes that catalyze post-translational modifications, helped to validate HMTs as drug targets. Likewise, it is not unreasonable to expect similar clinical challenges, such as the emergence of drug-resistant mutant enzymes. In anticipation of this, a team at Novartis Institutes for BioMedical Research developed a model for acquired resistance to EZH2 inhibitors in lymphoma cells. The study, “Development of secondary mutations in wild-type and mutant EZH2 alleles cooperates to confer resistance to EZH2,” published in Oncogene, includes the surprising result that a mutation in the wild-type EZH2 allele was critical to resistance of lymphoma cells whose tumorigenicity is driven by an oncogenic mutant form of the enzyme. This finding has important implications for development of anti-cancer agents targeting EZH2.

EZH2 is the catalytic subunit of the PRC2 complex, which methylates lysine 27 of histone H3 (H3K27) to repress transcription. Mutations in EZH2 hotspots such as Y641 drive tumorigenesis in lymphomas and solid tumors by shifting the substrate specificity from unmethylated and monomethylated H3K27 residues to the dimethylated form. This results in massive accumulation of the trimethylated histones, which drives tumorigenic pathways by suppression cell cycle checkpoint genes and other mechanisms. At least two specific EZH2 inhibitors, developed by Constellation Pharmaceuticals and Epizyme, are currently in Phase I clinical trials for lymphoma. Novartis is also pursuing EZH2 targeted drugs and researchers there used their SAM-competitive inhibitor, EI1, to generate a model for acquired drug resistance in lymphoma cells.

Treatment of lymphoma cells that carry an oncogenic mutation (Y641N) in one allele of the EZH2 gene with EI1 for several weeks resulted in development of resistance, measured by continued cell proliferation and DNA synthesis in the presence inhibitor. Ten individual clones were isolated from each of two different resistant lines, K-R2 and K-R10. Every clone isolated had a mutation in the WT allele, Y111L, and this mutation did not occur in the Y641N allele.   Conversely, 12 of the 20 clones carried a Y661D mutation specifically in the Y641N allele, and notably quite close to that mutation. There were no mutations found in any of the other subunits that comprise the active PRC2 complex; EZH2 expression did not change; and Y641N allele frequency remained at 50%. The resistance extended to two additional SAM-competitive EZH2 inhibitors as well.

What is most striking about these results is that 8 of the 20 resistant clones had a mutation only in the WT EZH2 allele, whereas the resistance-imparting mutation in the oncogenic EZH2 allele was found only in combination with the mutation in the WT allele. Thus, a compensatory mutation of the WT allele is sufficient to impart resistance even though the Y641N protein is the causative mutation for tumorigenesis.

The resistant forms of the WT and oncogenic EZH2 proteins were purified and analyzed biochemically. The Y661D mutation did not significantly alter the substrate specificity of the oncogenic mutant; i.e., the Y641N/Y661D variant still showed a strong preference for the mono- and dimethylated versus unmethylated H3K27 substrates. However it was far less affected by the three inhibitors tested, with IC₅₀ increases ranging from 30- to more than 100-fold. The resistant form of WT EZH2 protein, Y111L, was similarly unaffected in its preference for the unmethylated H3K27 substrate, and also highly refractory to inhibition by the three SAM-competitive EZH2 inhibitors tested. Isothermal calorimetry analysis confirmed that the mutations disrupt binding of the inhibitors to the variant EZH2 enzymes.

Next, the team analyzed the methylation profile of H3K27 in both parent and resistant lymphoma cells. Consistent with the model for EZH2-driven tumorigenesis, the parent lymphoma cells exhibit a high level of trimethylated H3K27 relative to unmethylated or mono-methylated histone; this profile w reversed by treatment with EZH2 inhibitors. However, in resistant cells with the Y11L and Y661D mutations the tumorigenic H3K27 methylation profile was largely unaffected by inhibitors; i.e., H3K27Me3 levels remained high. Moreover, in the cells carrying only the Y11L mutation in the WT allele, H3K27Me3 levels were also much less affected by inhibitors, and dimethylated histones showed an even greater increase relative to the parent cell line.

These results are consistent with a model that explains how mutation of the WT EZH2 allele alone can impart resistance in the context of the Y641N oncogenic mutation. Relief of inhibition of the WT EZH2 by the Y111L mutation allows high levels of dimethylated histones to accumulate, which increases production of trimethylated histone by the Y641N enzyme, even though it is significantly inhibited. In other words, flux through the WT EZH2 enzyme, not Y641N, is the rate-limiting step for production of trimethylated histone when cells harboring an oncogenic EZH2 mutation such as Y641N are treated with an EZH2 inhibitor. Therefore, targeting the WT enzyme, in addition to oncogenic mutant forms of EZH2, would be important for development of anti-cancer drugs.

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