Abstract IA26: Aberrant histone methylation in multiple myeloma.

Abstract

Abstract MMSET (Multiple Myeloma SET domain) is a histone methyltransferase (HMT) overexpressed as a result of the translocation t(4;14). MMSET contains a SET domain, which encodes histone methyltransferase activity, and protein and DNA interaction domains, including PHD and PWWP domains. Overexpression of MMSET induces a global increase in H3K36 methylation with concomitant loss of global H3K27 methylation. The HMT activity of MMSET is essential for growth stimulation by MMSET as re-expression of MMSET in a t(4;14) myeloma cell line which the rearranged MMSET allele was disrupted (KMS11-TKO), rescued growth only when the HMT activity of the protein was intact. The complete H3K36/H3K27 switch mediated by MMSET requires all PHD finger domains of the protein, the second PWWP domain and the functional SET domain. Despite the global change in histone methylation in response to MMSET, microarray and RNA-Seq analysis showed that only ~1000 genes are appreciably changed in response to MMSET. Many genes activated by MMSET display a peak of H3K27me3 near the transcription start site in MMSET low cells, which is absent in MMSET overexpressing cells, displaced by a broad pattern of H3K36me2 modification. We also found a subset of genes repressed in response to MMSET overexpression. While on a genome-wide basis, H3K27 methylation is decreased in MMSET overexpressing cells, at repressed genes, H3K27me3 levels were increased in association with increased occupancy by EZH2. These regions did not show an increase in H3K36 methylation and are enriched for GC-rich elements, representing putative polycomb complex recruitment sites. The role of MMSET in malignancies extends beyond MM as a recurrent heterozygous point of the gene is found in the Broad Cell Line Encyclopedia, as well as in cases of CLL, ALL and MM. Cell lines harboring such mutations had elevated H3K36 and depressed H3K27 methylation indicating that the mutation leads to a gain of function. MMSET overexpression/increased function is also linked to advanced, metastatic solid tumors and is associated with epithelial-mesenchymal transition and up-regulation of TWIST1. Given that MMSET gain of function is associated with malignancy- it is an attractive therapeutic target. In mouse xenografts the efficacy of chemotherapy against a t(4;14) cell line was enhanced by knockdown of MMSET. Using the previously identified structure of the SET domain of NSD1, an MMSET homologue, we performed an in silico screen against a library of compounds to identify putative MMSET inhibitors. We tested the in silico hits in a high throughput screen (HTS) that combines Self-Assembled Monolayer targets and matrix-assisted laser Desorption Ionization time-of-flight (SAMDI). From this screen several potential hits have been identified. These compounds have IC50 values in the micromolar range and Differential Scanning Fluorimetry (DSF) confirmed the binding of the small molecule to MMSET. Other mutations in MM lead to altered histone methylation. H3K27 methylation a repressive mark that is “erased” by the UTX/KDM6A H3K27 demethylase. The UTX/KDM6A gene is mutated/deleted in > 30% of multiple myeloma. shRNA mediated depletion of UTX in cells wild-type for the protein slowed growth and changed cell adhesion while re-expression of UTX slowed myeloma cell growth. Examination of the Broad Cell line encyclopedia dataset showed evidence of disruption of the MLL3 and MLL2 proteins in MM. Examination of gene expression array datasets showed that patients with very low expression of UTX and presumably mutation/deletion of the protein had a distinct pattern of gene expression. Remarkably when patients were analyzed based upon very low expression of MLL2 or MLL3 they showed a gene expression pattern that highly coincided with that of UTX low patients. This suggests that UTX, MLL2 and MLL3 work in concert and deletion of UTX, MLL2 or MLL3 may drive aberrant expression of a common set of genes. Pathways affected included p53, Wnt and JAK/STAT signaling. Collectively, these data indicate that aberrant histone methylation, generally affecting H3K27 is a common theme in multiple myeloma and other lymphoid malignancies. Citation Format: Jonathan D. Licht. Aberrant histone methylation in multiple myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA26.