Squamous cell carcinoma of the lung is a difficult-to-treat cancer with high prevalence in the US, and particularly in Kentucky. Here, we sought to test if the EZH1/2 inhibitor valemetostat improves anti-PD1 responses in squamous cell lung cancer models, and to develop ex vivo models to test immunotherapy drug combinations. We found that valemetostat augmented anti-tumor responses to anti-PD1 therapy, led to up-regulation of tumor cell specific Major Histocompatibility Complex Class II (MHC Class II), and drove systemic neutrophil maturation. Likewise, Ezh2 knock-out mice produced neutrophils that were more apoptotic, less migratory, and less able to produce extracellular nets, but had similar ability to kill bacteria as Ezh2-WT neutrophils. To test tumor responses to differing neutrophil populations, we engineered three-dimensional air-liquid interface cultures with tumoroids, lung mesenchymal cells, and T cells, with and without bone marrow containing neutrophils and myeloid progenitors from distinct donors. Bone marrow from tumor-naïve or mice with actively growing untreated tumors boosted tumoroid growth, while bone marrow from spontaneously tumor-rejected or mice with tumors treated with valemetostat was anti-tumor. MHC Class II blockade lowered the ability of bone marrow to boost tumor growth, and reduced the ability of valemetostat with anti-PD1 to reduce tumoroid growth. Patient samples revealed a strong negative correlation between EZH2 and MHC Class II, suggesting that targeting EZH2 activity could lead to marked increase in MHC Class II and improve treatment responses in lung squamous cell carcinomas.

Myelodysplastic syndromes and acute myeloid leukemia (MDS/AML) with both inversion/translocation of chromosome 3 (inv(3)/t(3;3)) and monosomy 7 (-7) is an extremely poor prognostic entity. To explore potential therapeutic target of MDS/AML harboring both inv(3)/t(3;3) and -7, we performed drug screen using YCU-AML1, a high-risk MDS/AML cell line harboring t(3;3) and -7 (Kunimoto et al. Hemasphere 2020), as well as OCI-AML20, another AML cell line with inv(3) and -7, and found that both YCU-AML1 and OCI-AML20 showed high response to EZH2 inhibitors valemetostat and tazemetostat. Previous study has shown that EVI1, an oncogenic transcription factor highly expressed in MDS/AML with inv(3)/t(3;3), directly binds to EZH2 and thereby recruits PRC2 complex to PTEN locus, leading to epigenetic silencing of PTEN expression and activation of PI3K/AKT/mTOR pathway in leukemia with 3q rearrangement (Yoshimi et al. Blood 2011). Together with the fact that EZH2 locus is on chromosome 7q, we hypothesized that the survival of MDS/AML cells with inv(3)/t(3;3) and -7 may be highly dependent on residual allelic EZH2-mediated silencing of specific targets which drive cell death. We further validated in colony-forming unit and cell growth assays that YCU-AML1 and OCI-AML20 are both highly sensitive to valemetostat and tazemetostat, whereas FKH-1, Kasumi-3 and SKM-1, MDS/AML cell line with -7, 3q rearrangement, and complex karyotype without chromosome 3 and 7 abnormalities respectively, are resistant to these drugs. Apoptosis analysis revealed that valemetostat and tazemetostat efficiently induced apoptosis in YCU-AML1 and OCI-AML20 but not in FKH-1, Kasumi-3 and SKM-1. To seek molecular basis of EZH2 inhibitor-mediated apoptosis induction, we performed CUT&Tag sequence for H3K27me3 using vehicle or valemetostat-treated cells. Strikingly, promoter region of GADD45 γ was the most robustly and significantly decreased annotated peak locus of H3K27me3 in valemetostat-treated OCI-AML20. H3K27me3 peak in GADD45 γ locus was also significantly decreased in valemetostat-treated YCU-AML1 but not in FKH-1 and Kasumi-3. As expected, valemetostat treatment induced increased expression of GADD45 γ in OCI-AML20. Moreover, transcriptomic analysis also demonstrated GADD45 γ as the most robustly upregulated gene in valemetostat-treated YCU-AML1 compared to vehicle-treated cells. GADD45γ is known to be an upstream regulator of stress-activated protein kinases such as p38 and JNK in which signaling pathways activation induce apoptosis upon various cellular stresses. GADD45 γ promoter region possessed putative EVI1 binding site predicted by rVista2.0 software, indicating that EVI1 may directly bind and recruit PRC2 complex to GADD45 γ locus. We further confirmed phosphorylation of Thr180/Tyr182 (T180/Y182) residues of p38α in valemetostat-treated YCU-AML1 and OCI-AML20 but not in SKM-1 and Kasumi-3. p38 is known to phosphorylate Ser33 (S33) and Ser46 residues of TP53, leading to apoptosis in lung cancer cells (Yogosawa et al. Cancer Sci. 2018). In line with this finding, valemetostat treatment induced phosphorylation of Ser33 residue of TP53 in YCU-AML1 and OCI-AML20. Phosphorylations of p38α (T180/Y182) and TP53 (S33) were also noted in tazemetostat-treated YCU-AML1 and OCI-AML20, suggesting that EZH2 inhibition directly activates GADD45γ-p38α-TP53 axis leading to apoptosis preferentially in MDS/AML cells with inv(3)/t(3;3) and -7. As a proof of concept, p38MAPK inhibitor SB203580 restored valemetostat-induced colony growth inhibition as well as apoptosis induction in YCU-AML1 and OCI-AML20. Importantly, valemetostat treatment significantly reduced leukemic burden and improved overall survival in xenotransplant mouse model of YCU-AML1. Moreover, primary MDS/AML patient bone marrow (BM) sample harboring inv(3) and -7 exhibited preferential sensitivity to valemetostat compared to BM samples derived from healthy control or MDS/AML patients with or without -7 in vitro. Taken together, our study unraveled PRC2-mediated inactivation of GADD45γ-p38α-TP53 axis as a molecular basis for evasion of apoptosis in MDS/AML with inv(3)/t(3;3) and -7, which can be preferentially abrogated by EZH2 inhibition leading to efficient induction of apoptosis in this high-risk MDS/AML.

Epigenetic dysregulation contributes to the development and progression of various cancers. Enhancer of zeste homolog (EZH)2 and EZH1 are enzymes that promote trimethylation of histone H3 at lysine 27 (H3K27me3), a repressive transcriptional mark associated with gene silencing. Valemetostat tosylate (valemetostat; DS-3201) is a first-in-class, oral, dual inhibitor of EZH2 and EZH1 that has demonstrated a greater potential to reduce H3K27me3 than EZH2-selective inhibitors and has strong antitumor activity in in vitro and in vivo studies. Dual inhibition of EZH2 and EZH1 could potentially alter the expression of genes involved in drug sensitivity and resistance in cancer cells, making valemetostat a candidate for combination therapies with established agents. This study assessed the preclinical activity of valemetostat combined with trastuzumab deruxtecan (T-DXd; DS-8201), a human epidermal growth factor receptor 2 (HER2)-directed antibody-drug conjugate (ADC), and with datopotamab deruxtecan (Dato-DXd; DS-1062), a trophoblast cell surface antigen 2 (TROP2)-directed ADC. Both T-DXd and Dato-DXd include a plasma-stable, selectively cleavable tetrapeptide-based linker and a topoisomerase I inhibitor payload (DXd, an exatecan derivative). The combined effects of valemetostat with DXd was tested in vitro in breast cancer (BC) and gastric cancer (GC) cell lines. Changes in the expression of various proteins and gene signatures were assessed by Western blot and RNA-sequencing analysis. The combined effects of valemetostat with each DXd-ADC were examined in in vivo mouse models bearing BC, GC, and non-small-cell lung cancer (NSCLC) tumors. In vitro, valemetostat enhanced cell-killing activities of the DXd payload in BC and GC cells in a concentration-dependent manner. Valemetostat upregulated the expression of Schlafen 11 (SLFN11) and HER2, in multiple HER2-low BC cell lines, and increased phosphorylation of H2AX, a marker of DNA damage, in a GC cell line. Gene signature analysis revealed that valemetostat reduced DNA damage response (DDR) signatures and increased major histocompatibility complex (MHC) signatures in BC, GC, and NSCLC in vitro models. Furthermore, valemetostat enhanced in vivo antitumor activities of T-DXd and Dato-DXd in human tumor xenografted models. Results from in vitro and in vivo analyses suggest that valemetostat potentiates the antitumor effect of DXd-ADCs via multiple cellular mechanisms, including increased expression of SLFN11, neoantigen presenting molecules (eg, MHC), ADC targets (eg, HER2), and reduced expression of genes involved in the DDR. A phase 1b, open-label, Master Protocol trial is currently investigating the clinical safety, tolerability, and preliminary efficacy of valemetostat in combination with T-DXd and Dato-DXd in patients with various solid tumors (NCT06244485). Daisuke Honma, Akiko Toyota, Yasuhiro Hama, Yasuki Kamai, Daisuke Okajima, Yoshiyuki Hizukuri, Maiko Narahara, Yuka Yamamoto, Emi Nosaka, Katsuki Usami, Kazumi Sato, Hiroki Goto, Ayumi Tezuka, Ayako Kato, Chisa Wada, Naoya Wada, Megumi Minami, Hideki Kobayashi, Mayumi Kitagawa. Preclinical assessment of valemetostat, a dual inhibitor of EZH2 and EZH1, combined with trastuzumab deruxtecan and datopotamab deruxtecan for multiple solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3790.

For the eradication of human immunodeficiency virus type 1 (HIV-1) provirus from people living with HIV-1, reactivation of latently HIV-1-infected cells is essential. Although several latency reversing agents have been identified, eradication of HIV-infected cells has been a challenge. Here, we investigated whether the novel enhancer of zeste homolog 1/2 (EZH1/2) dual inhibitor valemetostat/DS-3201/(R)-OR-S2 could efficiently reactivate latently HIV-1-infected cells in vitro and ex vivo. People living with HIV-1 who were on suppressive combined antiretroviral therapy and with plasma HIV-1 virus levels consistently below 50 copies/mL were enrolled in this study. ACH2 cells were treated with valemetostat for 7-14 days and with suberoylanilide hydroxamic acid (SAHA). CD4+ T cells were treated with valemetostat or the EZH2-selective inhibitors GSK126 and E7438 for 22 days alone or in combination with SAHA. HIV-1 expression in CD4+ T cells was determined. Valemetostat more effectively induced HIV-1 mRNA expression in ACH-2 cells when administered for 14 days than when administered for 7 days. Valemetostat reversed latently HIV-l-infected CD4+ T cells isolated from patients with HIV-1 and induced HIV-1 mRNA expression more potently than GSK126 and E7438. In addition, valemetostat induced HIV-1 mRNA expression more strongly when used in combination with SAHA compared with GSK126 and E7438. Expression levels of 21 hub genes were markedly increased after treatment with valemetostat. Gene Ontology analysis revealed that proteins encoded by these 21 genes were localized to the cell membrane and involved in the immune response. Kyoto Encyclopedia of Genes and Genomes enrichment pathway analysis showed that these 21 hub genes contributed to various signaling pathways, including the JAK-STAT signaling pathway. This study provides novel insights for the development of treatments to reactivate latently HIV-1-infected cells.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) harboring both inv(3)/t(3;3) and monosomy 7 (−7) are highly aggressive myeloid cancers of which molecular pathogenesis and therapeutic vulnerability remain elusive. High throughput drug screens, CUT&Tag/RNA sequence, and functional assays using human MDS/AML cells revealed that EZH2 inhibitors efficiently induce apoptosis preferentially in MDS/AML with inv(3)/t(3;3) and −7 through the activation of GADD45γ‐p38‐p53 axis. EVI1 activated in 3q‐rearranged MDS/AML was responsible for GADD45γ silencing by direct binding to its consensus sequence within GADD45γ promoter and recruitment of PRC2 complex via interaction with EZH2, which can be therapeutically targeted by EZH2 inhibition. MDS/AML with inv(3)/t(3;3) and −7 showed preferential sensitivity to EZH2 inhibition in both mouse model and patient samples. Thus, MDS/AML cells with inv(3)/t(3;3) and −7 possess apoptosis evasion mechanism through EVI1‐PRC2‐mediated repression of GADD45γ‐p38‐p53 axis, which is a potential therapeutic vulnerability in MDS/AML patients with these high‐risk cytogenetic lesions.

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy (HAM), adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated uveitis, and pulmonary diseases. Although both HAM and ATL show proliferation of infected cells, their pathogeneses are quite different. In particular, the pathogenesis of HAM is characterized by hyperimmune responses to HTLV-1-infected cells. Recently, we demonstrated the overexpression of histone methyltransferase EZH2 in ATL cells and the cytotoxic effects of EZH2 inhibitors and EZH1/2 dual inhibitors on these cells. However, these phenomena have never been studied in HAM. Furthermore, what effect these agents have on the hyperimmune response seen in HAM is completely unknown.MethodsIn this study, we investigated histone methyltransferase expression levels in infected cell populations (CD4+ and CD4+CCR4+ cells) from patients with HAM using microarray and RT-qPCR analyses. Next, using an assay system that utilizes the spontaneous proliferation characteristic of peripheral blood mononuclear cells derived from patients with HAM (HAM-PBMCs), we investigated the effects of EZH2 selective inhibitors (GSK126 and tazemetostat) and EZH1/2 dual inhibitors (OR-S1 and valemetostat, also known as DS-3201), particularly on cell proliferation rate, cytokine production, and HTLV-1 proviral load. We also examined the effect of EZH1/2 inhibitors on the proliferation of HTLV-1-infected cell lines (HCT-4 and HCT-5) derived from patients with HAM.ResultsWe found elevated expression of EZH2 in CD4+ and CD4+CCR4+ cells from patients with HAM. EZH2 selective inhibitors and EZH1/2 inhibitors significantly inhibited spontaneous proliferation of HAM-PBMC in a concentration-dependent manner. The effect was greater with EZH1/2 inhibitors. EZH1/2 inhibitors also reduced the frequencies of Ki67+ CD4+ T cells and Ki67+ CD8+ T cells. Furthermore, they reduced HTLV-1 proviral loads and increased IL-10 levels in culture supernatants but did not alter IFN-γ and TNF-α levels. These agents also caused a concentration-dependent inhibition of the proliferation of HTLV-1-infected cell lines derived from patients with HAM and increased annexin-V(+)7-aminoactinomycin D(−) early apoptotic cells.ConclusionThis study showed that EZH1/2 inhibitors suppress HTLV-1-infected cell proliferation through apoptosis and the hyperimmune response in HAM. This indicates that EZH1/2 inhibitors may be effective in treating HAM.

Enhancer of zeste homologous (EZH) 2 and its close homolog EZH1 are catalytic subunits of polycomb repressive complex (PRC) 2 protein complex, and play redundant and crucial role for the maintenance of transcriptional repression by tri-methylating histone H3 lysine 27 (H3K27). Hyper trimethylation of H3K27 has been associated with malignant lymphoma and myeloma progression, thus several small molecules suppressing PRC2 complex activity has been developed for hematological malignancy therapy. We have developed valemetostat tosylate (DS-3201b, also known as valematostat), a potent dual inhibitor of EZH1/2, and demonstrated its superior anti-proliferative effect against DLBCL cells to tazemetostat (EPZ-6438, E7438) a selective EZH2 inhibitor currently in clinic. In addition, valemetostat synergized with wide variety of 1st and 2nd line drugs used in DLBCL therapy both in vitro and in vivo proposing its potential combination opportunities. However, it is still elusive how valemetostat modulates epigenetic landscape and represses malignant B-cell proliferation more potently than selective EZH2 inhibitors. Therefore, impact on epigenetic landscape between valemetostat and tazemetostat was analyzed by RNA/ChIP-sequencing. Though these two inhibitors significantly reduced cellular global H3K27me3 level, we observed ectopic EZH1/2 accumulation in several tumor suppressor gene loci after tazemetostat treatment resulting in partial reduction in H3K27me3 and de-repression of silenced gene expression. Meanwhile valemetostat treatment evidently triggered gene expression by depleting H3K27me3 and enhancing H3K27Ac mark without inducing ectopic enrichment of EZH1/2, suggesting that valemetostat has a distinct effect on genome wide distribution of EZH1/2 from tazemetostat.

Mantle cell lymphoma (MCL) is a rare subtype of non-Hodgkin's lymphoma, which is characterized by overexpression of cyclin D1. Although novel drugs, such as ibrutinib, show promising clinical outcomes, relapsed MCL often acquires drug resistance. Therefore, alternative approaches for refractory and relapsed MCL are needed. Here, we examined whether a novel inhibitor of enhancer of zeste homologs 1 and 2 (EZH1/2), OR-S1 (a close analog of the clinical-stage compound valemetostat), had an antitumor effect on MCL cells. In an ibrutinib-resistant MCL patient-derived xenograft (PDX) mouse model, OR-S1 treatment by oral administration significantly inhibited MCL tumor growth, whereas ibrutinib did not. In vitro growth assays showed that compared with an established EZH2-specific inhibitor GSK126, OR-S1 had a marked antitumor effect on MCL cell lines. Furthermore, comprehensive gene expression analysis was performed using OR-S1-sensitive or insensitive MCL cell lines and showed that OR-S1 treatment modulated B-cell activation, differentiation, and cell cycle. In addition, we identified Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, also known as p57, KIP2), which contributes to cell cycle arrest, as a direct target of EZH1/2 and showed that its expression influenced MCL cell proliferation. These results suggest that EZH1/2 may be a potential novel target for the treatment of aggressive ibrutinib-resistant MCL via CDKN1C-mediated cell cycle arrest.

Introduction: Treatment of acute myeloid leukemia (AML) in elderly patients has been challenging largely because of their intolerance to intensive therapy and development of therapy resistance. Epigenetic alterations, including aberrant DNA methylation and alterations in histone modifications, are frequently present in leukemia cells and associated with therapy resistance. Polycomb repressive complex 2 (PRC2) subunits, enhancer of zeste homolog (EZH) 1 and 2 (EZH1/2), play a critical role in gene silencing by methylation of histone H3 on Lys27 (H3K27me3). Knowing that deregulated H3K27me3 is linked to malignant hematopoiesis and PRC2 is required for AML cell survival (Basheer et al., 2019; Neff et al., 2012), we postulated that targeting both EZH1 and 2 concomitantly will be efficacious in disrupting oncogenic signaling. A recent study using a murine AML model has demonstrated that genetic deletion of EZH1/2 depleted quiescent leukemic stem cells (LSC) and prolonged survival of leukemia bearing mice (Fujita at al., 2018).

Enhancer of zeste homolog (EZH) 1 and its close homolog EZH2 are component of polycomb repressive complex 2 (PRC2), and play a partially redundant and crucial role for the maintenance of transcriptional repression by tri-methylating histone H3 lysine 27 (H3K27). Hyper tri-methylation of H3K27 have been associated with lymphoma and myeloma progression, suggesting PRC2 is a therapeutic target for hematological malignancies. We have developed a novel EZH1 and EZH2 dual inhibitor valemetostat (DS-3201b), which simultaneously inhibited the enzymatic activity of EZH1 and EZH2 in nano-molar concentration. Valemetostat demonstrated anti-proliferative activities against the Activated B-cell-like (ABC) and Germinal Center B-cell-like (GCB) subtypes of Diffuse Large B-cell Lymphoma (DLBCL) cells. Furthermore, valemetostat induced apoptosis in DLBCL cell lines, regardless of subtype. We revealed that the pleiotropic effects of valemetostat on the expression levels of B-cell receptor signaling molecules by western blotting analysis. In particular, valemetostat suppressed the expression level of BCL6 protein, a key oncogene in B cell lymphoma. Transcriptome analysis of 16 DLBCL cell lines using RNA sequencing suggested that tumor suppressor genes, DNA damage response related genes and cell cycle related genes were affected by valemetostat treatment. In particular, valemetostat down regulated c-myc signaling in valemetostat-sensitive cells. Valemetostat also demonstrated synergistic anti-tumor activity with standard of care therapy against a DLBCL cell line KARPAS-422 xenografted model. In conclusion, our results suggested that valemetostat has therapeutic activity in DLBCL cells by inhibiting B-cell receptor signaling and c-myc signaling pathway. A phase 1 clinical study of valemetostat mono-therapy is now ongoing in patients with non-Hodgkin lymphoma including DLBCL (Clinical trial information: NCT02732275). Disclosures Hama: Daiichi Sankyo Co., Ltd.: Employment. Banjo:Daiichi Sankyo Co., Ltd.: Employment. Honma:Daiichi Sankyo Co., Ltd.: Employment. Takata:Daiichi Sankyo Co., Ltd.: Employment. Nosaka:Daiichi Sankyo Co., Ltd.: Employment. Shiroishi:Daiichi Sankyo Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Employment. Yamamoto:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Hirata:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Nakano:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Inaki:Daiichi Sankyo Co., Ltd.: Employment. Goto:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Totoki:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Kataoka:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Lim:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Wada:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Kumazawa:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Tsutsumi:Daiichi Sankyo Co., Ltd.: Employment.

Although global H3K27me3 reprogramming is a hallmark of cancer, no effective therapeutic strategy for H3K27me3-high malignancies harboring EZH2WT/WT has yet been established. We explore epigenome and transcriptome in EZH2WT/WT and EZH2WT/Mu aggressive lymphomas and show that mutual interference and compensatory function of co-expressed EZH1 and EZH2 rearrange their own genome-wide distribution, thereby establishing restricted chromatin and gene expression signatures. Direct comparison of leading compounds introduces potency and a mechanism of action of the EZH1/2 dual inhibitor (valemetostat). The synthetic lethality is observed in all lymphoma models and primary adult T cell leukemia-lymphoma (ATL) cells. Opposing actions of EZH1/2-polycomb and SWI/SNF complexes are required for facultative heterochromatin formation. Inactivation of chromatin-associated genes (ARID1A, SMARCA4/BRG1, SMARCB1/SNF5, KDM6A/UTX, BAP1, KMT2D/MLL2) and oncovirus infection (HTLV-1, EBV) trigger EZH1/2 perturbation and H3K27me3 deposition. Our study provides the mechanism-based rationale for chemical dual targeting of EZH1/2 in cancer epigenome.

Multiple myeloma (MM) is largely incurable because relapse eventually occurs, despite the recent development of novel therapies. This is mainly caused by a remaining population of drug-resistant myeloma stem cells. There is a side population (SP) enriched with myeloma stem cells. Therefore, targeting SP cells may be a promising strategy to prevent and treat MM relapse.

Enhancer of zests homologous (EZH)1 and its close homolog EZH2 are component of polycomb repressive complex (PRC) 2 protein complex, and play redundant and crucial role for the maintenance of transcriptional repression by tri-methylating histone H3 lysine 27 (H3K27). Hyper tri-methylation of H3K27 has been associated with lymphoma and myeloma progression, suggesting that PRC2 is a therapeutic target for hematological malignancies. Selective EZH2 inhibitors induce compensatory activation of EZH1 which in turn re-activates PRC2 function. We hypothesized that dual inhibition of EZH1 and EZH2 is more effective than selective EZH2 inhibition as anti-tumor therapy.

Multiple myeloma (MM) is an incurable hematological malignancy caused by accumulation of abnormal clonal plasma cells. Despite the recent development of novel therapies, relapse of MM eventually occurs as a result of a remaining population of drug-resistant myeloma stem cells. Side population (SP) cells show cancer stem cell-like characteristics in MM; thus, targeting these cells is a promising strategy to completely cure this malignancy. Herein, we showed that SP cells expressed higher levels of enhancer of zeste homolog (EZH) 1 and EZH2, which encode the catalytic subunits of Polycomb repressive complex 2 (PRC2), than non-SP cells, suggesting that EZH1 as well as EZH2 contributes to the stemness maintenance of the MM cells and that targeting both EZH1/2 is potentially a significant therapeutic approach for eradicating myeloma stem cells. A novel orally bioavailable EZH1/2 dual inhibitor, OR-S1, effectively eradicated SP cells and had a greater antitumor effect than a selective EZH2 inhibitor in vitro and in vivo, including a unique patient-derived xenograft model. Moreover, long-term continuous dosing of OR-S1 completely cured mice bearing orthotopic xenografts. Additionally, PRC2 directly regulated WNT signaling in MM, and overactivation of this signaling induced by dual inhibition of EZH1/2 eradicated myeloma stem cells and negatively affected tumorigenesis, suggesting that repression of WNT signaling by PRC2 plays an important role in stemness maintenance of MM cells. Our results show the role of EZH1/2 in the maintenance of myeloma stem cells and provide a preclinical rationale for therapeutic application of OR-S1, leading to significant advances in the treatment of MM.

Abstract Mantle cell lymphoma (MCL) is a well-defined and aggressive type of B cell non-Hodgkin’s lymphoma that is genetically characterized by the t(11;14)(q13;q32) chromosomal translocation, which results in constitutive overexpression of CYCLIN D1. Although newly developed drugs such as ibrutinib show promising clinical outcomes, relapsed MCL often acquires drug resistance, which is a critical obstacle to treatment. Alternative approaches to overcoming the drug resistance of relapsed MCL are urgently needed. PRC1 and 2 are important epigenetic regulators that maintain the stemness of embryonic and hematopoietic stem cells. EZH1 and 2 are catalytic components of PRC2, which trimethylates histone H3 at lysine 27 (H3K27) to repress transcription of target genes. Mutation and overexpression of EZH1/2 are associated with cancers, including hematopoietic malignancies. Here, we used a novel dual inhibitor of EZH1/2 to show that inhibiting EZH1/2 is a promising therapeutic strategy for MCL. First, we developed a xenograft (PDX) mouse model using cells from a heavily pretreated and relapsed MCL patient, and then orally administered an inhibitor of EZH1/2, called OR-S1. OR-S1 strongly impaired proliferation of the patient-derived tumors and did not cause any serious side effects. Additionally, an in vitro assay using MCL cell lines showed that OR-S1 inhibited the growth of MCL cells, and that the effect was much more significant than that using the single EZH2 inhibitor (GSK126). The IC50 of OR-S1 was about one tenth that of GSK126. These results strongly suggest that dual inhibition of EZH1/2 could be a promising therapeutic strategy for relapsed MCL. Next, to investigate the effect induced by dual inhibition of EZH1/2, we conducted further analyses of the MCL cell lines. Cells exposed to OR-S1 showed cell cycle arrest (G1 arrest) along with a dose-dependent reduction in phospho-Rb and cell differentiation, coupled with increased cell surface expression of hCD138. We then used RNA-seq analysis of MCL cell lines to compare OR-S1-treated cells with vehicle-treated cells and found that cell cycle-related signaling was significantly affected and that a cyclin-dependent kinase inhibitor, CDKN1C (TP57), was one of the genes most markedly upregulated by OR-S1. ChIP qPCR of MCL cell lines showed that the CDKN1C locus was strongly marked by H3K27 trimethylation, and that OR-S1 induced a significant reduction in the level of this histone marker. Furthermore, administration of OR-S1 alone to PDX mice induced increased expression of CDKN1C (as in the in vitro assay). Thus, dual inhibition of EZH1/2 in MCL induces expression of CDKN1C, which in turn causes cell cycle arrest and reduced growth of MCL. Taken together, these results strongly suggested that dual inhibition of EZH1 and EZH2 is a promising therapeutic strategy for MCL, illustrating the potential of novel epigenetic approaches to overcoming drug resistance of relapsed MCL. Citation Format: Shuhei Fujita, Yuki Kagiyama, Daisuke Honma, Nobuaki Adachi, Kazushi Araki, Issay Kitabayashi. Novel epigenetic approach to relapsed mantle cell lymphoma based on dual inhibition of EZH1/EZH2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4672. doi:10.1158/1538-7445.AM2017-4672

Abstract Multiple myeloma (MM) is largely incurable as the disease eventually relapses despite the recent development of novel therapies. Previous reports show that side population (SP) cells comprise myeloma stem cells. Therefore, targeting SP cells may be a promising strategy for preventing and treating MM relapse. Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important epigenetic regulators that maintain the “stemness” of ES cells and other hematopoietic stem cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) are catalytic components of PRC2, which trimethylate histone H3 at lysine 27 to repress transcription of target genes. Mutation and overexpression of EZH2 are associated with many cancers, including MM. Here, we found that SP cells expressed significantly higher levels of EZH1/2 than non-SP cells. These results suggest that overexpression of EZH1/2 is important for maintaining the stemness of MM cells and that EZH1/2 could be a potential therapeutic target. We developed a novel EZH1/2 dual inhibitor, OR-S1, and used it to investigate the effect of pharmacologic inhibition of EZH1/2 on MM. OR-S1 suppressed the proliferation of almost all MM cell lines tested, with an IC50 significantly lower than that of the specific EZH2 inhibitor, GSK126. Furthermore, flow cytometry analysis revealed that OR-S1 significantly depleted the SP cell population. RNA-seq analysis revealed that the transcriptional profiles of MM cell lines treated with OR-S1 were characterized by up-regulation of genes related to the Wnt pathway. qRT-PCR confirmed that expression of Wnt, Frizzled, and Protein kinase C family members increased markedly after exposure to OR-S1. Previous studies show that increased activation of the canonical Wnt signaling pathway down-regulates HSC self-renewal and differentiation. Therefore, we generated β-catenin-overexpressing MM cells to examine the effects of increased Wnt signaling on MM cells. Surprisingly, proliferation of these cells was significantly lower than that of control cells. These results suggested that PRC2 directly targets Wnt signaling, and that over-activation induced by EZH1/2 dual inhibition was responsible for the reduced proliferation of MM cells. Oral administration of OR-S1 to mice bearing MM xenografts led to significant impairment of subcutaneous tumors. Interestingly, long-term administration of the drug at lower doses to mice bearing orthotopic xenografts resulted in complete eradication of minimal residual disease from the bone marrow and complete cure of MM without any serious side effects. Furthermore, OR-S1 treatment of an orthotopic PDX model derived from a relapsed and heavily pretreated MM patient led to a reduction in the levels of human immunoglobulins in the serum. Taken together, these results strongly suggest that dual inhibition of EZH1/2 is a promising therapeutic approach to eradicating myeloma stem cells and could lead to important advances in the treatment of MM. Citation Format: Makoto Nakagawa, Shuhei Fujita, Daisuke Honma, Kazushi Araki, Issay Kitabayashi. Identification of a possible therapeutic candidate for multiple myeloma based on dual inhibition of EZH1/EZH2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4670. doi:10.1158/1538-7445.AM2017-4670

The neomorphic alteration of H3K27me3 patterning is a fundamental trait of cancers. We have developed a new clinical strategy; dual inhibition of EZH1 and EZH2 significantly reprogrammed the abnormal epigenome and restored the transcriptome in lymphomas (Blood, 2016; 58th ASH, 2016; NCT02732275).