Abstract

Epigenetic dysregulation plays an important role in myeloma pathogenesis. Histone methyltransferases have been implicated in disease initiation and progression; for example MMSET, acting at H3K36 is the oncogenic driver in the 15% of patients with the initiating event t(4;14), whilst MLL, acting at H3K4, is recurrently mutated. It is logical to presume that enzymes with the opposite action at histone methyl marks, the demethylases, also play a role in myeloma and recurrent mutations and deletions are seen in KDM6A (UTX), a H3K27 demethylase. We further investigated the role of demethylases using linked gene expression and survival data from 3 large studies of newly diagnosed patients, a small molecule 2-oxoglutarate dependent histone demethylase inhibitor (GSK-J4, inhibitor of KDM5B+KDM6A/B) and knockdown with locked nucleic acids (LNAs), to define a potential new targeted therapeutic strategy for myeloma patients.

Linked gene expression and survival data were available for the MRC Myeloma IX study (n=259), HOVON65/GMMG study (n=246) and Total Therapy 2+3 (TT2+3) studies (n=559). Patients with high expression of KDM5B, a H3K4 demethylase, had a significantly shorter overall survival (OS) than patients with lower expression. (Myeloma IX: high expression median OS 31.6 months vs 45.5 months, log rank p=0.020. HOVON65/GMMG-H4: high expression median OS 54.8 months vs median OS not reached, log rank p <0.001. TT2+3: medians not reached, same trend toward significance). No other demethylase gene had a consistent association between expression and survival.

Inhibition of viability with GSK-J4 treatment occurred in a time and dose-dependent manner in a panel of 11 myeloma cell lines; GC50s ranging from 0.5 -3uM at 72 hours. Treatment induced apoptosis was demonstrated by increased annexin/PI staining, by flow cytometry, and increased caspase activity in the Caspase-Glo® 3/7 luminescent assay. Gene expression arrays (Illumina HumanHT12-v4), using RNA extracted from JJN-3 cells treated at 2uM for 6 and 24 hours, showed induction of expression of the metallothionein family of genes including MT1X, associated with intracellular metal-ion homeostasis, and genes associated with the cellular stress response including DDIT3 and INHBE, both ATF4 downstream target genes. These genes were validated by RT-PCR in 5 myeloma cell lines (e.g. RPMI-8226 24hrs 2.5uM GSK-J4 treatment mean RQ values: MT1X 176, DDIT 45, INHBE 70).

Locked Nucleic Acid knockdown targeting KDM6A+6B reduced cell viability and induced the same transcriptional response seen with GSK-J4. Reduced expression of KDM5B was also detected following treatment with this LNA. Knockdown targeting KDM5B alone did not affect viability or induce the same transcriptional response, suggesting inhibition of all three demethylases is required for the response seen with GSK-J4. Chip-seq analysis of JJN-3 cells treated at 2uM GSK-J4 for 6 and 48 hours or LNA knockdown of KDM6A+B for 7 days, showed increased H3K4me3 marks globally, with specific peaks upstream of the metallothionein genes and ATF4 target genes, indicating that the effects seen with GSK-J4 treatment are mediated via an epigenetic mechanism.

To evaluate the relevance of our findings to human disease, viability experiments using CD138+ plasma cells from myeloma patients were performed for cells alone and in co-culture with bone marrow stroma. GSK-J4 treatment reduced the viability of CD138 selected cells both alone and in co-culture compared to control. (Mean viability at 48hrs cells alone: 4uM GSK-J4 46%, 8uM 27%, 12uM 5%, co-cultured: 4uM 76%, 8uM 59%, 12uM 26%.) The transcriptional response seen in cell lines was conserved in primary patient cells following 24hr 2uM GSK-J4 treatment (Mean RQ values MT1X 105, INHBE 13, DDIT3 4). Treatment of bone marrow stromal cells and peripheral blood mononuclear cells with GSK-J4 demonstrated a therapeutic index between normal and malignant tissue.

This work demonstrates a pro-apoptotic effect of demethylase inhibition in myeloma, a class of drug targets not previously investigated for this disease. A complex interplay between the demethylases affected by the GSK-J4 chemical probe is demonstrated and suggests that the modulation of KDM5B, KDM6A and KDM6B simultaneously delivers the apoptotic signal. The enrichment of H3K4me3 marks at genes involved in the transcriptional response along with survival data suggests KDM5B might be key to the response.

Disclosures

Lindow:Santaris: Employment. Obad:Santaris: Employment. Oerum:Santaris: Employment.

Author notes

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Asterisk with author names denotes non-ASH members.