Epigenetic targeting of SETD8 in multiple myeloma Free

Multiple myeloma (MM) is a plasma cell malignancy and the second most common hematologic cancer. Despite effective frontline therapies, including immunotherapies, relapse remains common due to drug-resistant clones and mutation-driven resistance, highlighting the need for new therapeutic targets. SETD8, the sole lysine methyltransferase responsible for monomethylation of histone H4 at lysine 20 (H4K20me), also modifies non-histone proteins like p53 and PCNA, suppressing p53 activity and promoting cancer proliferation. SETD8 is overexpressed in various cancers. We recently reported that SETD8 is overexpressed in relapsed primary MM, and high SETD8 expression is associated with poor prognosis (Herviou et al. 2021). Notably, SETD8 knockdown (KD) inhibits proliferation in SETD8-high MM cells but not in SETD8-low or normal cells. Malignant plasma cells are particularly dependent on SETD8's methyltransferase activity. We therefore hypothesized that pharmacological inhibition of SETD8 could be a novel and effective therapeutic strategy for the treatment of MM.

The prognostic impact of SETD8 was evaluated across three public datasets (MMRF CoMMpass, UAMS Total Therapy 2, and UAMS Total Therapy 3). We further evaluated SETD8 expression by IHC in primary tissues (CD138+cells) and cell lines. To assess its role, we performed SETD8 KD and analyzed downstream effects via western blot. We have developed a novel, highly potent, and selective covalent SETD8 inhibitor (MS2928) and evaluated its effects in vitro and in vivo assays. Cell viability, apoptosis, and mRNA expression were assessed by CellTiter-Glo, Annexin-V, and qPCR. In vivo efficacy and safety were tested using MM-cell lines engrafted in NSG mice.

Analysis of gene expression datasets from three MM patient cohorts revealed that high SETD8 mRNA levels correlate with shorter progression-free and overall survival. Elevated SETD8 expression was also linked to 1q21 amplification. We assessed SETD8 protein expression across 10 human MM cell lines and observed its presence in all, albeit at varying levels, as determined by western blotting. These protein levels correlated with SETD8 gene expression (R² = 0.6). IHC on bone marrow biopsies from relapsed MM patients showed heterogeneous SETD8 expression. Compared to healthy donors (n = 10), relapsed MM samples (n = 54) had significantly higher SETD8 levels in ~80% of cases (p = 0.0006). Genetic depletion of SETD8 in MM cells (OPM-2 and JJN-3) reduces H4K20me levels and significantly inhibits cell growth (p ≤ 0.05), suggesting its functional role in MM. We also developed a potent and selective covalent inhibitor of SETD8 (MS2928). A high-resolution crystal structure (2.05 Å; PDB ID: 9CR7) revealed that MS2928 covalently binds to SETD8 at the C311 residue. MS2928 effectively inhibited SETD8 methyltransferase activity, with an IC₅₀ of 0.16 ± 0.03 µM. It also suppressed the growth of multiple myeloma (MM) cells in a dose-dependent manner, with IC₅₀ values ranging from 0.5 to 2.0 µM in OPM-2, JJN-3, and MOLP-8 cells, while being non-toxic to normal peripheral blood mononuclear cells from healthy donors. Notably, MS2928 had limited efficacy in low SETD8-expressing cell lines such as KMS-20 and EJM, consistent with a dose-dependent effect. Treatment with MS2928 (1.25–5.0 µM) resulted in a significant reduction in H4K20me levels, confirming the rapid and effective inhibition of SETD8. It also decreased MM cell viability, as indicated by elevated levels of cleaved PARP and cleaved caspase-3. In MOLP-8 and OPM-2 cells, MS2928 treatment reduced p53K382me levels and increased both mRNA and protein expression of p53 and p21. In vivo, MS2928 significantly inhibited tumor growth in an OPM-2 xenograft MM model, without causing body weight loss or organ toxicity, underscoring its therapeutic promise for MM.

In conclusion, our findings demonstrate that SETD8 is upregulated in relapsed MM patients and contributes to the dysregulation of key signaling pathways. Pharmacological inhibition of SETD8 effectively suppresses MM cell proliferation in vitro and in vivo, highlighting its potential as a novel epigenetic therapeutic target, especially for high-risk patients.