Either enhancer of zeste homolog 2 (EZH2) or its homolog EZH1, the enzymatic core subunit of polycomb repressive complex 2 (PRC2), acts an essential role for the maintenance of transcriptional repression by the methylation of histone H3 lysine 27 (H3K27). EZH2 gain-of-function (GOF) mutations (e.g. Y641, A677, and A687) or wild-type activation cause an increased activity leading to hyperinduce trimethylation of H3K27 (H3K27me3). Meanwhile, loss-of-function (LOF) mutation of major components (e.g. ARID1A, PBRM1, INI1) in chromatin remodeling complex, SWItch/Sucrose Non-Fermentable (SWI/SNF), results in a loss of its ability to oppose PRC2 and subsequently activation of EZH2. Accordingly, the dysfunctions of EZH2 or the alterations of regulatory SWI/SNF complex proteins have been reported to be associated with the development and progression of a variety of malignant tumors. Although PRC2 is suppressed by EZH2 inhibition, the activity of EZH1 is complementarily increased to replace the role of EZH2. Consequently, dual inhibition of EZH1 and EZH2 could be more effective than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Thus, we have developed a novel EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. Herein, we presented that HM97662, having enhanced EZH1 inhibitory activity compared to EZH2 inhibitors currently approved or in clinical development, potently and dose-dependently decreased global H3K27me3 in cancer cells. HM97662 showed broader and stronger anti-proliferative activities against various hematological cancer cell lines with EZH2 activating mutations as well as solid tumor cell lines with negatively mutated components of regulatory protein complexes. At this time, it was confirmed in the MCL cell line that HM97662, unlike the EZH2 selective inhibitor, does not cause the elevation of EZH1. HM97662 was observed to effectively modulate cell cycle arrest and induce apoptotic markers in a dose-dependent manner, as well as decrease intracellular H3K27me3, a pharmacodynamic marker, even at low concentration. As a result, HM97662 inhibited tumor growth more effectively than known EZH2 selective inhibitor without abnormal clinical signs at once daily dose in an EZH2 GOF mutant lymphoma cell xenograft mice model as well as in a bladder cancer cell xenograft mice model harboring a genetically mutated regulatory SWI/SNF complex protein (e.g. ARID1A). Taken together, the present studies demonstrated that HM97662 is a novel and potent EZH1/2 dual inhibitor and has the promising potential for the treatment of patients with several types of cancers. Clinical trials to prove the effectiveness of HM97662 identified in preclinical studies need to be carried out immediately. Citation Format: Seung Hyun Jung, DongJin Hong, Jiyoung Hwang, Somin Park, Jooyun Byun, Miyoung Lee, Kyounghwa Koo, Gunwoo Lee, Yu-Yon Kim, Yesol Bak, Young Gil Ahn, YoungHoon Kim, Kwee Hyun Suh. A novel and potent EZH1/2 dual inhibitor, HM97662, demonstrates antitumor activity in malignant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1142.

In mammals, either enhancer of zeste homologue 2 (EZH2) or its homolog EZH1 functions as the catalytic subunit of polycomb repressive complex 2 (PRC2), and plays an essential role for the maintenance of transcriptional repression by the trimethylation of histone H3 lysine 27 (H3K27me3). Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677 and A687) has been associated with lymphoma and myeloma progression. Many publications suggest that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Up to date, there are known a few EZH2 selective inhibitors with showing anti-tumor activity in some preclinical and clinical studies. However, the inhibition of EZH2 can complementarily induce EZH1 activation, which reactivates PRC2 function in turn. Therefore, dual inhibition of EZH1 and EZH2 could give more effective than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we introduce a novel EZH1/2 dual inhibitor, HM97594, which simultaneously inhibits the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. HM97594 potently repressed trimethylation of H3K27 in lymphoma and myeloma cell lines. As a result, HM97594 showed broader and stronger activities than EZH2 selective inhibitors in in vitro studies of inhibiting the proliferation of various hematological cancer cell lines. HM97594 induced the differentiation of DLBCL to plasma cells with an increment of cell lineage specific markers (e.g. PRDM1, IRF4 and CD38) and decreased the colony forming ability. Also, HM97594 caused cell cycle G0/1 arrest and apoptosis in KARPAS-422 cells harboring EZH2 Y641 GOF mutation. In addition, HM97594 showed potent antitumor activity in KARPAS-422 lymphoma cell xenograft mouse model. Once daily orally administered HM97594 greatly inhibited tumor growth in a dose-dependent manner without body weight loss compared to the known EZH2 inhibitor. Collectively, it was demonstrated in the present studies that HM97594, EZH1/2 dual inhibitor, has promising potential as an anticancer drug for patients with subtypes of hematological malignancies. Further preclinical studies will be performed and reported soon after the establishment of a preclinical candidate. Disclosures No relevant conflicts of interest to declare.

Immune checkpoint blockades (ICBs) are known as a promising treatment option against in advanced non-small-cell lung cancer (NSCLC). And KRAS is the most frequently mutated oncogene in NSCLC. However, KRAS mutation is not a significantly associated with survival benefit of ICBs. Mutations in LKB1 (aka STK11) and frequently co-occurring KRAS mutations are accompanied with poor survival in metastatic NSCLC immuno-oncology trials. Even tumor mutational burden is a proposed-potential biomarker for response to ICBs, there are therapeutic unmet needs in NSCLC patient with KRAS/LKB1 (KL) mutation. Epigenetic regulation lead to enhanced anti-tumor immunity of innate immune responses via inducing type I interferons. Also it drives to enhance adaptive immune responses by targeting T cell recognition factors as well as recruitment immune cells to tumor microenvironments (TMEs). Thus, we demonstrated the pharmacological inhibition of Enhancer of zeste homolog (EZH)1/2 enhance the efficacy of clinically available immunotherapies in KRAS/LKB1 mutated NSCLC. Previously, we reported that a novel EZH1/2 dual inhibitor, HM97662, has showed potently decreased global H3K27me3 and strong anti-proliferative activities against various hematological cancer cell lines with EZH2 activating mutations as well as solid tumor cell lines with negatively mutated components of regulatory protein complexes. In this study, we evaluated that HM97662 could induce of STING expression and STING-dependent cytokine production in KL mutant NSCLC cell lines. Furthermore, we explored anticancer effect by inducing immune modulation by EZH2 inhibitors in combination with an anti-PD-(L)1 agent was evaluated. As a result, HM97662 potently restored STING expression in KL mutated cell lines such as A549 and H460, but did not KRAS/p53 (KP) mutated H358 cells. HM97662 also dose-dependently increased the mRNA expression of CXCL10, IFN-α and IFN-β and secretion of CXCL10, IFN-α, CCL2, CCL3 and CCL5 at nanomolar concentrations in H460 cells. In accordance with this, HM97662 showed potent growth inhibition against H460 cells under co-culture condition with activated T cells. Moreover, HM97662 had superior anti-proliferation efficacy among the tested compounds in H460 cells with activated T cells, when it combined with anti-PD-L1 antibody avelumab. In our exploratory study, HM97662 increments the sensitivity of immune checkpoint inhibitor by restoration of STING expression and STING pathway related chemo/cytokine production in KRAS/LKB1 mutated NSCLC cell lines. These results demonstrate that HM97662 drives anti-cancer effect by immune modulation in combination with anti-PD-(L)1 agent, suggesting therapeutic potency to combination with ICBs by turning non-inflamed to inflamed TME. Clinical trials to prove the effectiveness of HM97662 identified in preclinical studies need to be carried out immediately. Citation Format: Jooyun Byun, DongJin Hong, Miyoung Lee, Soonho Kweon, Seung Hyun Jung, Yu-Yon Kim, Hyunjin Park, Junghwa Park, Young Gil Ahn, Young Hoon Kim, Kwee Hyun Suh. Overcoming immune checkpoint blockade resistance via EZH1/2 dual inhibition by HM97662 in KRAS/LKB1 mutated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3276.