Abstract 3516: EZH2 overexpression in myeloma patients shortens survival and in-vitro data supports a potential new targeted treatment strategy
Authors: Pawlyn Charlotte , Kaiser Martin F. , Stein Caleb K. , Wardell Christopher P. , Macleod Veronica , Edmondson Rick , Barlogie Bart , Walker Brian , Morgan Gareth J. , Davies Faith E. 
Published: August 2015

Enhancer of zeste homolog 2 (EZH2) induces methylation at histone 3 lysine 27 (H3K27), thought to have a repressive effect on target gene expression. Activating mutations in EZH2 have been shown to drive lymphomas and overexpression linked to poor prognosis in solid tumors. In normal plasma cell development EZH2 expression is downregulated on exit from the germinal centre but in myeloma (MM) expression has been shown to increase with disease progression. Recent work suggests high expression of the H3K36 methyltransferase MMSET sensitizes cells to EZH2 inhibition. We are seeking to further investigate the role of EZH2 in MM.

Data were analyzed from newly diagnosed MM patients in several large clinical trials (Whole-exome sequencing, n = 463 UK Myeloma XI, Affymetrix gene expression array (GEP), n = 259 UK Myeloma IX and n = 1213 UAMS Total Therapy Protocols).

Across the datasets, high expression of EZH2 mRNA shortened patient survival (e.g. UK IX median OS, 29.9 months vs 45.1 months, Log-rank p = 0.005), remaining significant on multivariate analyses and across GEP defined molecular subgroups. Of note, the proliferative subgroup had a higher mean expression of EZH2 (9.56 vs 8.48, t-test p = 5.953e-42) as did high risk patients defined by GEP 70 score (9.61 vs 8.46, t-test p = 1.993e-40) suggesting a link to a more aggressive disease phenotype.

There were no mutations in EZH2. This contrasts with frequent mutations seen in malignancies at earlier stages of B cell development e.g. DLBCL. Of note, 3% of MM patients had a potentially inactivating mutation or deletion in the gene encoding the H3K27 demethylase, KDM6A and significantly shorter OS than those without (% alive 2 yr: Mutated 51% (CI 30-85) vs 80% (CI 77-84), Log-rank p = 0.0498). Such mutations may increase H3K27 methylation, potentially sensitizing patients to EZH2 inhibition.

To study the biological implications of our results we tested the effect of inhibition of EZH2 (EZH2i), using a small molecule EPZ005687, in a panel of MM cell lines representing the epi/genetic diversity of MM. We saw a reduction in total cell number and% viability at 6 days of EZH2i (2-4uM) in 5/8 cell lines with evidence of dose dependent induction of apoptosis. All cell lines (except LP1) had similar levels of EZH2 protein at baseline and the responding cell lines had no unifying epi/genetic anomalies, suggesting response is not specific to either MMSET high or KDM6A mut/del cell lines. 6 days of EZH2i reduced H3K27me2/me3 on Western blotting in all cell lines regardless of viability response, demonstrating the epigenetic activity of the inhibitor. Further evaluation by Chip-seq and GEP is ongoing to identify potential differences, e.g. in focal methylation marks, that may account for differing viability responses.

Our results highlight the prognostic significance of EZH2 in MM and demonstrate excellent in-vitro inhibitor activity suggesting EZH2 as a possible future target for treatment.

Citation Format: Charlotte Pawlyn, Martin F. Kaiser, Caleb K. Stein, Christopher P. Wardell, Veronica Macleod, Rick Edmondson, Bart Barlogie, Brian Walker, Gareth J. Morgan, Faith E. Davies. EZH2 overexpression in myeloma patients shortens survival and in-vitro data supports a potential new targeted treatment strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3516. doi:10.1158/1538-7445.AM2015-3516