Targeting EZH2 Research Articles

Inhibiting EZH2 targets atypical teratoid rhabdoid tumor by triggering viral mimicry via both RNA and DNA sensing pathways

Inactivating mutations in SMARCB1 confer an oncogenic dependency on EZH2 in atypical teratoid rhabdoid tumors (ATRTs), but the underlying mechanism has not been fully elucidated. We found that the sensitivity of ATRTs to EZH2 inhibition (EZH2i) is associated with the viral mimicry response. Unlike other epigenetic therapies targeting transcriptional repressors, EZH2i-induced viral mimicry is not triggered by cryptic transcription of endogenous retroelements, but rather mediated by increased expression of genes enriched for intronic inverted-repeat Alu (IR-Alu) elements. Interestingly, interferon-stimulated genes (ISGs) are highly enriched for dsRNA-forming intronic IR-Alu elements, suggesting a feedforward loop whereby these activated ISGs may reinforce dsRNA formation and viral mimicry. EZH2i also upregulates the expression of full-length LINE-1s, leading to genomic instability and cGAS/STING signaling in a process dependent on reverse transcriptase activity. Co-depletion of dsRNA sensing and cytoplasmic DNA sensing completely rescues the viral mimicry response to EZH2i in SMARCB1-deficient tumors.

Ailanthone suppresses cell proliferation of renal cell carcinoma partially via inhibition of EZH2

BackgroundAilanthone (Ail) extracted from medicinal plants has played an anticancer role in multiple cancers, while there is no research about Ail in renal cell carcinoma (RCC).MethodsIn the present study, we performed CCK-8 and flow cytometry to assess the effect of Ail on cell viability, apoptosis and cycle. We also performed tandem mass tags (TMT)-labeled quantitative proteomic technology and bioinformatic analysis to identify the functional pathway and proteins of Ail in RCC.ResultsThe results showed Ail could inhibit cell viability and induce cell apoptosis. Proteomic profiling identified 1732 differentially expressed proteins in cells treated with Ail, compared to the negative control group. Gene ontology function annotation and Gene Set Enrichment Analysis (GSEA) were performed to identified the involved biological processes, molecular function and pathway. Results of GSEA proved the enrichment of Deps in EZH2 targets. The comparison between Deps and EZH2 co-expressed genes revealed 44 overlapped genes and we identified 4 hub genes (CDC20, CEP55, TOP2A, and UBE2C) associated with RCC progression. The molecular docking study revealed a moderate to tight binding potential of Ail and EZH2, and western blotting showed EZH2 was suppressed after cells treated with Ail.ConclusionAltogether, we identified the anticancer role of Ail in RCC, including inhibition of cell proliferation and induction of apoptosis. The results also screened the key proteins mediate the function of Ail, which have laid a theoretical foundation for elucidating the applications of Ail in clinical research.

Unveiling the therapeutic potential of IHMT-337 in glioma treatment: targeting the EZH2-SLC12A5 axis

Glioma is the most common malignant tumor of the central nervous system, with EZH2 playing a crucial regulatory role. This study further explores the abnormal expression of EZH2 and its mechanisms in regulating glioma progression. Additionally, it was found that IHMT-337 can potentially be a therapeutic agent for glioma. The prognosis, expression, and localization of EZH2 were determined using bioinformatics, IHC staining, Western blot (WB) analysis, and immunofluorescence (IF) localization. The effects of EZH2 on cell function were assessed using CCK-8 assays, Transwell assays, and wound healing assays. Public databases and RT-qPCR were utilized to identify downstream targets. The mechanisms regulating these downstream targets were elucidated using MS-PCR and WB analysis. The efficacy of IHMT-337 was demonstrated through IC50 measurements, WB analysis, and RT-qPCR. The effects of IHMT-337 on glioma cells in vitro were evaluated using Transwell assays, EdU incorporation assays, and flow cytometry. The potential of IHMT-337 as a treatment for glioma was assessed using a blood–brain barrier (BBB) model and an orthotopic glioma model. Our research confirms significantly elevated EZH2 expression in gliomas, correlating with patient prognosis. EZH2 facilitates glioma proliferation, migration, and invasion alongside promoting SLC12A5 DNA methylation. By regulating SLC12A5 expression, EZH2 activates the WNK1-OSR1-NKCC1 pathway, enhancing its interaction with ERM to promote glioma migration. IHMT-337 targets EZH2 in vitro to inhibit WNK1 activation, thereby suppressing glioma cell migration. Additionally, it inhibits cell proliferation and arrests the cell cycle. IHMT-337 has the potential to cross the BBB and has successfully inhibited glioma progression in vivo. This study expands our understanding of the EZH2-SLC12A5 axis in gliomas, laying a new foundation for the clinical translation of IHMT-337 and offering new insights for precision glioma therapy.

Abstract P05: Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2

Abstract Overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance, and poor survival outcomes. Previously, we have established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes in NKTL. As EZH2 enzymatic inhibitors have proven ineffective against NKTL, we embarked on the development of novel EZH2 Proteolysis Targeting Chimera (PROTAC) compounds. EZH2 PROTACs consist of an EZH2- and E3-ligase binding domain which promotes targeted ubiquitination of the EZH2 protein thereby marking it for proteasomal degradation. Our initial efforts focused on the iterative synthesis of EZH2 PROTAC candidates optimizing factors such as permeability, solubility and stable ternary complex formation. Through extensive screening, we identified C2911 and C1311, which successfully trigger the dose-dependent degradation of EZH2 and PRC2 components concomitant with a significant loss of survival and induction of apoptosis in both NKTL and DLBCL. These effects were rescued by pre-treatment with proteasome inhibitor Bortezomib or when E3 ligase activity was disrupted through siRNA knockdown. In NKTL, we show that EZH2 PROTAC treatment decreased JAK3 and phospho-STAT5 protein expression and identified the suppression of the JAK-STAT-MYC axis as a potential mechanism of action. EZH2 PROTAC treatment effectively reactivates transcription of canonical EZH2 target genes such as MYT1 and ANPEP and downregulates expression of oncogenes c-Myc and E2F1. Pharmacokinetic studies in vivo demonstrate that these PROTACs exhibit good plasma stability, half-life (>4h) and low clearance thereby establishing potential for future clinical development. The efficacy of EZH2 PROTACs in xenograft mice are currently being evaluated. Collectively, our data supports the discovery of novel EZH2 PROTACs and the critical role it plays in successfully targeting the oncogenic role of EZH2 in NKTL or other non-canonical EZH2-driven cancers through protein depletion where enzymatic-only inhibition was ineffective. We are further investigating the mechanisms stabilizing EZH2 protein levels to enhance PROTAC-mediated cytotoxicity. Citation Format: Nurulhuda Mustafa, Zubaida Talal Alnaseri, Darrel Tan Egk Jian, David Quach Thanh Truong, Wan Hsin Lim, Wee Joo Chng. Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2 [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P05.

Repressive H3K27me3 drives hyperglycemia-induced oxidative and inflammatory transcriptional programs in human endothelium

BackgroundHistone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium.MethodsWe studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2−) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice.ResultsIncubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2− generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice.ConclusionsEZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes.Graphical

Single-cell transcriptomics reveals cell atlas and identifies cycling tumor cells responsible for recurrence in ameloblastoma

Ameloblastoma is a benign tumor characterized by locally invasive phenotypes, leading to facial bone destruction and a high recurrence rate. However, the mechanisms governing tumor initiation and recurrence are poorly understood. Here, we uncovered cellular landscapes and mechanisms that underlie tumor recurrence in ameloblastoma at single-cell resolution. Our results revealed that ameloblastoma exhibits five tumor subpopulations varying with respect to immune response (IR), bone remodeling (BR), tooth development (TD), epithelial development (ED), and cell cycle (CC) signatures. Of note, we found that CC ameloblastoma cells were endowed with stemness and contributed to tumor recurrence, which was dominated by the EZH2-mediated program. Targeting EZH2 effectively eliminated CC ameloblastoma cells and inhibited tumor growth in ameloblastoma patient-derived organoids. These data described the tumor subpopulation and clarified the identity, function, and regulatory mechanism of CC ameloblastoma cells, providing a potential therapeutic target for ameloblastoma.

FTO protein regulates the TGF-β signalling pathway through RNA N6-methyladenosine modification to induce unexplained recurrent spontaneous abortion.

Recent studies have revealed the involvement of RNA m6A modification in embryonic development; however, the relationship between aberrant RNA m6A modification and unexplained recurrent spontaneous abortion (URSA) remains unclear. In this study, we analysed the level of RNA m6A modification in trophoblasts using dot blot, RNA m6A quantification, and MeRIP assays. By integrating data from the GEO database, RNA-Seq, and MeRIP-Seq, we examined the aberrant expression of m6A methyltransferases and their downstream molecules in chorionic villus (placental) tissues. RNA pull-down, RIP, and electrophoretic mobility shift assay were used to analyse the binding relationship between the YTHDC1 protein and MEG3. Additionally, RNA stability and BrU immunoprecipitation chase assays were utilised to elucidate the regulation of MEG3 stability by YTHDC1. ChIP and DNA pull-down RNA experiments were performed to elucidate the mechanism by which MEG3 targets EZH2 to the TGF-β1 promoter. The results showed that the expression of the m6A demethylase FTO protein was significantly increased in URSA trophoblasts, leading to inhibition of the MEG3 m6A modification and weakening of the stabilising effect of the m6A binding protein YTHDC1 on MEG3. Furthermore, MEG3 was found to bind simultaneously with the EZH2 protein and the TGF-β1 gene promoter, enabling the localisation of EZH2 protein to the TGF-β1 gene promoter and subsequent inhibition of TGF-β1 gene expression. In summary, our findings elucidate the mechanism by which FTO protein regulates the MEG3-TGF-β signalling pathway, thereby suppressing trophoblast invasion and proliferation in URSA trophoblast cells. These findings provide new insights for the treatment of URSA.

Retinoids and EZH2 inhibitors cooperate to orchestrate anti-oncogenic effects on bladder cancer cells.

The highly mutated nature of bladder cancers harboring mutations in chromatin regulatory genes opposing Polycomb-mediated repression highlights the importance of targeting EZH2 in bladder cancer. Furthermore, the critical role of the retinoic acid signaling pathway in the development and homeostasis of the urothelium, and the anti-oncogenic effects of retinoids are well established. Therefore, our aim is to simultaneously target EZH2 and retinoic acid signaling in bladder cancer to potentiate the therapeutic response. Here we report that this coordinated targeting strategy stimulates an anti-oncogenic profile, as reflected by inducing a synergistic reduction in cell viability that was associated with increased apoptosis and cell cycle arrest in a cooperative and orchestrated manner. This study characterized anti-oncogenic transcriptional reprogramming centered on the transcriptional regulator CHOP by stimulating the endoplasmic reticulum stress response. We further portrayed a molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of a subset of genes involved in unfolded protein responses, reflecting the molecular mechanism underlying this co-targeting strategy. These findings highlight the importance of co-targeting the EZH2 and retinoic acid pathway in bladder cancers and encourage the design of novel treatments employing retinoids coupled with EZH2 inhibitors in bladder carcinoma.

EZH2 inhibitors promote β-like cell regeneration in young and adult type 1 diabetes donors

β-cells are a type of endocrine cell found in pancreatic islets that synthesize, store and release insulin. In type 1 diabetes (T1D), T-cells of the immune system selectively destroy the insulin-producing β-cells. Destruction of these cells leads to a lifelong dependence on exogenous insulin administration for survival. Consequently, there is an urgent need to identify novel therapies that stimulate β-cell growth and induce β-cell function. We and others have shown that pancreatic ductal progenitor cells are a promising source for regenerating β-cells for T1D owing to their inherent differentiation capacity. Default transcriptional suppression is refractory to exocrine reaction and tightly controls the regenerative potential by the EZH2 methyltransferase. In the present study, we show that transient stimulation of exocrine cells, derived from juvenile and adult T1D donors to the FDA-approved EZH2 inhibitors GSK126 and Tazemetostat (Taz) influence a phenotypic shift towards a β-like cell identity. The transition from repressed to permissive chromatin states are dependent on bivalent H3K27me3 and H3K4me3 chromatin modification. Targeting EZH2 is fundamental to β-cell regenerative potential. Reprogrammed pancreatic ductal cells exhibit insulin production and secretion in response to a physiological glucose challenge ex vivo. These pre-clinical studies underscore the potential of small molecule inhibitors as novel modulators of ductal progenitor differentiation and a promising new approach for the restoration of β-like cell function.

Metabolic switch regulates lineage plasticity and induces synthetic lethality in triple-negative breast cancer

Metabolic reprogramming is key for cancer development, yet the mechanism that sustains triple-negative breast cancer (TNBC) cell growth despite deficient pyruvate kinase M2 (PKM2) and tumor glycolysis remains to be determined. Here, we find that deficiency in tumor glycolysis activates a metabolic switch from glycolysis to fatty acid β-oxidation (FAO) to fuel TNBC growth. We show that, in TNBC cells, PKM2 directly interacts with histone methyltransferase EZH2 to coordinately mediate epigenetic silencing of a carnitine transporter, SLC16A9. Inhibition of PKM2 leads to impaired EZH2 recruitment to SLC16A9, and in turn de-represses SLC16A9 expression to increase intracellular carnitine influx, programming TNBC cells to an FAO-dependent and luminal-like cell state. Together, these findings reveal a new metabolic switch that drives TNBC from a metabolically heterogeneous-lineage plastic cell state to an FAO-dependent-lineage committed cell state, where dual targeting of EZH2 and FAO induces potent synthetic lethality in TNBC.

Abstract C171: Immune desert microenvironment is associated with poor outcome in Wilms tumors

Abstract Background: Presence of anaplasia is a potent marker for adverse outcome in patients with Wilms tumors with diffuse anaplasia (DAWT). Patients with WT with focal anaplasia (FAWT) are considered as intermediate risk. Besides TP53 mutations thought to arise in anaplastic components, the crosstalk between genomic alterations and immune landscape remains unknown. Methods: We performed whole-exome and/or RNA sequencing on genomic DNA derived from 12 matched tumor-normal WTs and extended analysis in an independent dataset of 9 cases. TP53 mutations and tumor infiltrating lymphocytes (TILs: CD3, CD4 and CD8) were also assessed. Validation was performed using TARGET-WT (n=96) and GEO-WT (n=12) cohorts. Prognostic role of TILs was investigated in another 55 pre-treated WTs. Results: TP53 (50%) and DROSHA (33.3%) were the sole recurrent mutations identified. TP53 mutations occurred in mutually exclusive manner with chromatin-modifying genes which were altered in both FAWT (100%) and DAWT (33.3%). Microenvironment analysis revealed a reproducible immune desert WTs with poor outcome that enriched in DAWT cases and TP53 mutation, which mirrored the inversion of CD4:CD8 ratio in TP53-mutated comparing to TP53-wild tumors (P=0.003). Immune desert WTs were also characterized by overexpression of DNA repair pathways, PRC2, HDCA, and polycomb targets of EZH2, leading to higher sensitivity to HDCA inhibitor of vorinostat. Additionally, the immunophenotypes were tightly associated with replication stress subtypes; WTs with high replication stress were susceptible to ATR and WEE1 inhibitors. High ratio of CD4/CD8 was considered as independent recurrence-free and overall survival factors for WTs (both P<0.05). Conclusions: Our results reveal that WT with anaplastic features are characterized by either TP53 alterations or chromatin-modifying genes. These data implicate TP53 as a tumor cell-intrinsic driver of an immune desert phenotype, in contrast with adult TP53 tumors. TP53 inactivation might therefore causes epigenetic deregulation across cancer sites, and has implications for immunotherapy. Citation Format: Xiaoping Su, Gabriel Malouf. Immune desert microenvironment is associated with poor outcome in Wilms tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C171.

Translation Inhibition through eIF4A1 Targeting in Multiple Myeloma Is a Promising Therapeutic Strategy

Multiple Myeloma (MM) is a plasma cell (PC) disorder without definitive treatment and associated with multiple relapses. There is therefore an urgent need to develop innovative therapeutic strategies to overcome drug resistance. mRNA translation deregulation in involved in cancer progression by promoting aberrant proliferation or cell survival, and several translation factors such as eIF4A1 are deregulated in cancer, pointing to an addiction to high protein synthesis. In this context, inhibitors of eIF4A1-mediated translation have been developed and have shown anti-tumor effects in several models of hematological malignancies. Targeting translation in MM with eIF4A1 inhibition thus represent a potential treatment strategy in MM. To evaluate the therapeutic interest of targeting eIF4A1 in MM, we analyzed its expression in Bone Marrow Plasma Cells (BMPC, n = 22), Monoclonal Gammopathy of Unknown Significance cells (MGUS, n = 44), Smoldering MM Cells (SMMC, n = 12), MM cells (MMC, n = 345), and Human Myeloma Cell Lines (HMCLs, n = 42). We found that eIF4A1 expression increased throughout the evolution of the disease with a highest expression in HMCLs, and was significantly upregulated in the molecular subgroups Proliferation and Hyperdiploid (UAMS, TT2 cohort). Importantly, eIF4A1 high expression was associated with a poor prognosis in two independent cohorts of MM, a cohort of newly diagnosed patients treated by high dose therapy and autologous stem cell transplantation (UAMS, TT2 cohort, n = 345) and a cohort of patients at relapse treated with Proteasome Inhibitors (Mulligan, n = 188). Gene Set Enrichment Analysis (GSEA) of genes expressed in eIF4A1 high patients compared to eIF4A1 low patients revealed an enrichment in pathways related to RNA metabolism, DNA repair, epigenetic regulation and plasmablastic signature. We investigated the impact of eIF4A1 inhibition using Rocaglamide (Roca) in HMCLs. Roca was highly cytotoxic for 13 HMCLs with an IC50 < 20nM, led to cell cycle deregulation and to the downregulation of MMSET, MYC, IKAROS and EZH2 expression. Importantly, using a co-culture of primary MM cells with normal cells from the bone marrow micro-environment, we found that Roca was cytotoxic for malignant PCs but not bone marrow cells (n = 5). To deeper characterize eIF4A1 targets in MM cells, we performed Polysome Profiling to measure translational efficiency depending on the level of ribosomes associated with individual mRNAs. Roca induced a strong increase in mRNAs with one ribosome (monosome) and a decrease in mRNAs with polysomes. Sequencing of mRNAs from the light fraction (monosomes and disomes) and mRNAs from the heavy fraction (> 2 ribosomes) identified 567 genes significantly enriched in the light fraction of Roca-treated cells compared to control cells (Fold change > 1.5; p value < 0.05). GSEA unveiled an enrichment for pathways such as MYC targets, cell cycle, DNA damage and of high interest, EZH2 targets and TP53 transcriptional regulation. More precisely, translation efficiency of genes frequently associated with genomic abnormalities in MM such as MYC, TP53, or CTNNB1 was significantly decreased. We also identified several genes related to MM pathophysiology, including RECQL involved in replication stress survival and resistance to chemotherapy in MM (Viziteu et al, 2017), and EED, which interacts with EZH2, whose targeting could overcome the resistance to IMIDs and increase anti-CD38 efficacy in MM (Herviou et al, 2018; Chemlal et al, in press). We investigated the link between Roca sensitivity and the response of HMCLs to MM drugs, and found that higher resistance to Bortezomib was associated with higher sensitivity to Roca (n = 10, r = -0.6, p value < 0.05). Interestingly, we found a synergy between Roca and Bortezomib, suggesting that Roca could be used to overcome Bortezomib resistance. Moreover, since Roca affects IKAROS expression, a target of Immunomodulatory Drugs (IMIDs), we investigated the therapeutic interest to combine Roca with the IMID Lenalidomide. A synergy between Roca and Lenalidomide was identified in HMCLs, suggesting that the downregulation of MM oncoproteins with Roca could be used to increase the efficacy of IMIDs. In conclusion, Rocaglamide combination with IMIDs or other targeted therapies, such as EZH2 inhibitors, could be of clinical interest and represents a promising strategy to improve the outcome of MM patients.

Targeting Epigenetic Resistance Mechanisms to PI3 Kinase Inhibition in Leukemic Stem Cells

Acute myeloid leukemia (AML) is a heterogeneous disease associated with poor survival and frequent relapse rates due to the inability to effectively target leukemic stem cells (LSCs) and acquired resistance to existing treatments. Development of treatments targeting LSCs and overcoming acquired resistance is crucial to improve the survival of AML patients. The PI3 kinase (PI3K)/AKT signaling pathway is activated in 80% of AML patients, across various AML subtypes, making PI3K an attractive therapeutic target for this disease. Several PI3K inhibitors, such as copanlisib, are clinically approved for other indications, with a reasonable toxicity profile. The effects of PI3K inhibitors on LSCs however, remains unknown. Several catalytic isoforms of PI3K are expressed in hematopoietic cells. To determine whether these PI3K isoforms play a role in LSC self-renewal, we performed colony assays on KMT2A-AF9 retrovirally transduced hematopoietic stem and progenitor cells (HSPCs) with either genetic deletion or pharmacologic inhibition of PI3K isoforms. We found that disruption of PI3K signaling reduces the self-renewal of LSCs and promotes myeloid differentiation in both mouse LSCs and human AML cell lines. KMT2A-AF9 induces leukemic transformation via aberrant upregulation of key self-renewal genes in hematopoietic progenitors, such as HoxA9 and Meis1 (PMID: 16862118). We found that pharmacologic inhibition of PI3K isoforms leads to a reduction in HoxA9 and Meis1 expression in LSC colonies. In the KMT2A-AF9 retroviral bone marrow transplantation model of AML, genetic deletion of the PI3K isoform Pik3ca leads to a reduction in LSC numbers in the bone marrow and prolonged survival (Fig 1A), although it is not curative. In addition, we observed significant prolongation of survival upon secondary transplantation of Pik3ca leukemic cells, suggesting a reduction in functional LSCs. Transcriptome analysis on LSCs isolated from pre-clinical bone marrow aspirates of KMT2A-AF9 primary transplant mice revealed downregulation of the target genes of HoxA9, an important driver of LSC self-renewal. We also observed enrichment of the targets of EZH2, a histone methyltransferase that catalyzes the H3K27 trimethylation repressive mark. AKT is known to phosphorylate EZH2 at Ser21, leading to inhibition of EZH2 function (PMID: 16224021). Therefore, we hypothesized that Pik3ca deletion can promote LSC differentiation by de-repressing EZH2 activation via AKT inactivation. To determine whether PI3K inactivation differentiates KMT2A-AF9 leukemic cells via dephosphorylation of EZH2, we performed Western blot analysis on bone marrow cells from Pik3ca KO and WT leukemic mice. As expected, Pik3ca deletion significantly reduced AKT phosphorylation. Unexpectedly, a reduction of total EZH2 protein levels was also observed in Pik3ca KO leukemic cells (Fig 1B), suggesting that EZH2 downregulation may be a non-genetic acquired resistance mechanism to PI3K inactivation. Histone proteomics analysis at two different timepoints after treatment with the PI3K inhibitor copanlisib revealed rapid and dynamic changes in histone modifications, including H3K27me3 and H3K4me3, consistent with epigenetic mechanisms of resistance to PI3K inhibition. EZH1, a homolog of EZH2, has been reported to compensate for EZH2 function (PMID: 26219303, PMID: 31189531). Thus, we hypothesize that PI3K inhibition may sensitize leukemic cells to EZH1/2 dual inhibition by reducing EZH2 levels. We found that dual inhibition with copanlisib and the EZH1/2 inhibitor valemetostat leads to a combinatorial effect to strongly decrease the proliferation of AML cell lines and AML patient samples. Additionally, we found that treatment of KMT2A-AF9 AML mice with these compounds induces myeloid differentiation and leads to a reduction in leukemia initiating cell capacity, as demonstrated by prolonged survival upon secondary transplantation. Overall, our data suggests that PI3K plays an important role in leukemic stem cells, and that EZH2 downregulation is a non-genetic mechanism of resistance to PI3K inhibition. Additionally, our findings indicate that combining PI3K inhibitors with EZH1/2 dual inhibitors could be a promising therapeutic approach to reduce LSCs, prevent resistance, and prolong survival in AML patients.

Novel Protac-Based EZH2 Degraders Effectively Target EZH2-Driven Lymphoid Malignancies By Abolishing Both Methylation-Dependent and -Independent Functions

Across various cancers, overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance and poor survival outcomes. While the tumorigenic role of EZH2 is largely attributed to the H3K27me3-mediated repression of tumor suppressor genes, reports have also established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes, particularly in aggressive cancers like Natural Killer T-cell Lymphoma (NKTL). Here, traditional EZH2 inhibitors have been ineffective. We hypothesized that targeted depletion of the EZH2 protein will more effectively disrupt oncogenic signals arising from enzyme-independent activities of EZH2. Thus, we embarked on the development of EZH2 Proteolysis Targeting Chimeras (PROTACs) to trigger targeted ubiquitination and proteasome-mediated degradation of EZH2. No clinical EZH2 PROTAC has been reported yet. Our initial efforts focused on the iterative synthesis of a series of PROTAC candidates considering factors such as permeability, solubility and stable ternary complex formation. We developed a library of 53 compounds with various permutations of EZH2- and E3 ligase-binding ligands as well as linker lengths. Through extensive screening and testing of the EZH2 degradation activity via the Nano-Glo HiBiT lytic detection system and immunoblotting, we identified three lead Protac candidates. Compared to the clinical EZH2 inhibitor Tazemetostat (Taz), EZH2 PROTACs, C2911 and C1311 demonstrated higher cytotoxic activity in diffuse large B-cell lymphoma (DLBCL), a cancer in which Taz was evaluated during clinical trial. Importantly, C2911 and C1311 also successfully triggered EZH2 degradation in a dose-dependent manner thereby qualifying for further evaluation in NKTL. Previous work has highlighted a critical role for JAK3-mediated phosphorylation of EZH2 in promoting NKTL proliferation. Treatment of NKTL with C2911 and C1311 leads to a dose and time-dependent EZH2 degradation, loss of cell viability and increased apoptosis. These effects were absent with Taz and Pomalidomide treatments. The concomitant loss of H3K27me3 levels as well as PRC2 complex subunits, SUZ12 and EED suggest effective degradation of the EZH2 protein. We also observed that induction of NKTL cell death required a longer treatment period as compared to DLBCL. This may be potentially ascribed to the longer half-life of the EZH2 protein in NKTL as determined through cycloheximide treatment. In order to validate the mechanism of action of the EZH2 PROTACs, cells were either pretreated with pomalidomide or siRNA targeting cereblon. Indeed, this mitigated PROTAC-induced degradation of the EZH2 protein and rescued cancer cell death. Similarly, inhibiting the activity of the 26S proteasome through bortezomib also prevented EZH2 degradation. Characterization of EZH2 PROTAC-induced protein expression changes through immunoblotting highlighted that there was specific loss of H3K27me3 and H3K27me2 but not H3K27me1. Of note, we detected a concomitant decrease of Jak3 protein expression and STAT5 phosphorylation levels, more pronounced than PRC2 complex subunits. This suggests that the PROTAC may act by disrupting the non-canonical binding of EZH2 to Jak3 in NKTL. Furthermore, gene expression analyses revealed that EZH2 PROTAC treatment more effectively reactivated canonical EZH2 target genes such as MYT1 and ANPEP as compared to Taz. As the JAK-STAT-MYC axis is dysregulated in NK leukemia, we also evaluated the gene expression of c-Myc and c-Myc target gene, E2F1. EZH2 PROTAC treatment reduces the transcription levels of these oncogenes corroborating with loss of c-Myc protein expression. Studying the pharmacokinetics of the lead PROTACs in a mouse model revealed good plasma stability, half-life (> 4 h), and low clearance. Collectively, our data supports a critical role for EZH2 PROTACs in abolishing both the enzyme-dependent and independent activities of EZH2 through protein degradation, resulting in enhanced anti-cancer activity against DLBCL and NKTL. This may potentially benefit other EZH2-driven cancers. Further evaluation of the EZH2 PROTACs for clinical application is in progress. EZH2 PROTACs will next be tested in vivo. An in-depth investigation of the mechanisms stabilizing EZH2 protein levels and underlying PROTAC induced-cell death will be made to identify potential mechanisms of resistance.

Development of MDS in Pediatric Patients with GATA2 Deficiency: Increased Histone Trimethylation and Deregulated Apoptosis as Potential Drivers of Transformation

GATA2 deficiency is a heterogeneous, multisystem disorder associated with a high risk of developing myelodysplastic syndrome (MDS) and the progression to acute myeloid leukemia. The mechanisms underlying malignant transformation in GATA2 deficiency remain poorly understood, necessitating predictive markers to assess an individual’s risk of progression and guide therapeutic decisions. In this study, we performed a systematic analysis of bone marrow biopsies from 57 pediatric MDS patients. Focusing on hematopoiesis and the hematopoietic niche, including its microenvironment, we used multiplex immunofluorescence combined with multispectral imaging, gene expression profiling, and multiplex RNA in situ hybridization. Patients with a GATA2 deficiency exhibited a dysregulated GATA2 transcriptional network. Disease progression (GATA2-EB, n = 6) was associated with increased GATA2 mRNA levels, restored expression of the GATA2 target EZH2, and increased H3K27me3. GATA2-EB was further characterized by the high expression of the anti-apoptotic protein BCL2, a feature absent in children with a GATA2 deficiency and refractory cytopenia of childhood (GATA2-RCC, n = 24) or other pediatric MDS subgroups (RCC, n = 17; MDS-EB, n = 10). The multispectral imaging analysis of additional BCL2 family members revealed significantly elevated Mediators of Apoptosis Combinatorial (MAC) scores in GATA2-EB patients. Taken together, our findings highlight the potential drivers of disease progression in GATA2 deficiency, particularly increased histone trimethylation and dysregulated apoptosis. Furthermore, upregulated BCL2 and EZH2 and increased MAC scores provide a strong rationale for the use of venetoclax and azacitidine in therapeutic regimens for GATA2-EB.

Polycomb repressor complex 2 suppresses interferon-responsive MHC-II expression in melanoma cells and is associated with anti-PD-1 resistance

BackgroundDespite the remarkable success of immunotherapy in treating melanoma, understanding of the underlying mechanisms of resistance remains limited. Emerging evidence suggests that upregulation of tumor-specific major histocompatibility complex-II (tsMHC-II) serves...

Attenuated Salmonella-delivered PD-1 siRNA enhances the antitumor effects of EZH2 inhibitors in colorectal cancer

Immunotherapy has made significant progress in the treatment of malignant tumors. However, strategies to combine immunotherapy with anticancer drugs have attracted great attention due to the low response rate and unique toxicity profile of immunotherapies and the subsequent development of acquired resistance in some initial responders. EZH2, a histone methyl transferase subunit of a Polycomb repressor complex,is highly expressed in a variety of tumors, and targeting EZH2 has become a new strategy for tumor therapy and drug combination. Here，we studied the effect of EZH2 inhibitors on colorectal cancer cells and their combination with immunotherapy. Our results demonstrated that EZH2 inhibitors can not only significantly inhibit the survival of colorectal cancer (CRC) cells and induce apoptosis, effectively inhibit cell invasion and migration, but also cause an increase in the expression of PD-L1 receptors on the cell surface. To determine the effect of EZH2 in combination with immunotherapy, we combine EZH2 inhibitors with PD-1 siRNA delivered by attenuated Salmonella. The vivo experiments have shown that the combination of EZH2 inhibitors and Salmonella-delivered PD-1 siRNA can further inhibit the development of CRC, trigger effective anti-tumor immunity, and improve therapeutic efficacy. Its underlying mechanisms mainly involve synergistic immunomodulation and apoptosis. This study suggests an emerging strategy based on a combination of EZH2 inhibitor and immunotherapy based on PD-1 inhibition.

CUL4B-DDB1-COP1-mediated UTX downregulation promotes colorectal cancer progression

BackgroundUTX (encoded by KDM6A), a histone demethylase for H3K27me2/3, is frequently mutated in human cancers. However, its functional and regulatory mechanisms in colorectal cancer (CRC) remain unclear.MethodsImmunohistochemistry staining was used to investigate the clinical relevance of UTX in CRC. Additionally, we generated a spontaneous mouse CRC model with conditional Utx knockout to explore the role of UTX in the colorectal tumorigenesis. Post-translational regulation of UTX was determined by co-immunoprecipitation and immunoblot analyses.ResultsHerein, we identify that downregulation of UTX, mediated by the Cullin 4B-DNA Damage Binding Protein-1-Constitutive Photomorphogenesis Protein 1 (CUL4B-DDB1-COP1) complex, promotes CRC progression. Utx deletion in intestinal epithelial cells enhanced the susceptibility to tumorigenesis in AOM/DSS-induced spontaneous mouse CRC model. However, this effect is primarily alleviated by GSK126, an inhibitor of histone methyltransferase EZH2. Mechanistically, EMP1 and AUTS2 are identified as putative UTX target genes mediating UTX functions in limiting intestinal tumorigenesis. Notably, the CUL4B-DDB1-COP1 complex is identified as the functional E3 ligase responsible for targeting UTX for degradation in CRC cells. Thus, Cop1 deficiency in mouse intestinal tissue results in UTX accumulation and restricts tumorigenesis. Furthermore, patient cohort analysis reveals that UTX expression is negatively correlated with clinical stage, favorable disease outcomes, and COP1 expression.ConclusionsIn the current study, the tumor suppressor function and regulation of UTX in CRC provide a molecular basis and the rationale to target EZH2 in UTX-deficient CRC.

Ezh2 promotes mammary tumor initiation through epigenetic regulation of the Wnt and mTORC1 signaling pathways

The regulation of gene expression through histone posttranslational modifications plays a crucial role in breast cancer progression. However, the molecular mechanisms underlying the contribution of histone modification to tumor initiation remain unclear. To gain a deeper understanding of the role of the histone modifier Enhancer of Zeste homology 2 (Ezh2) in the early stages of mammary tumor progression, we employed an inducible mammary organoid system bearing conditional Ezh2 alleles that faithfully recapitulates key events of luminal B breast cancer initiation. We showed that the loss of Ezh2 severely impairs oncogene-induced organoid growth, with Ezh2-deficient organoids maintaining a polarized epithelial phenotype. Transcriptomic profiling showed that Ezh2-deficient mammary epithelial cells up-regulated the expression of negative regulators of Wnt signaling and down-regulated genes involved in mTORC1 (mechanistic target of rapamycin complex 1) signaling. We identified Sfrp1, a Wnt signaling suppressor, as an Ezh2 target gene that is derepressed and expressed in Ezh2-deficient epithelium. Furthermore, an analysis of breast cancer data revealed that Sfrp1 expression was associated with favorable clinical outcomes in luminal B breast cancer patients. Finally, we confirmed that targeting Ezh2 impairs mTORC1 activity through an indirect mechanism that up-regulates the expression of the tumor suppressor Pten. These findings indicate that Ezh2 integrates the mTORC1 and Wnt signaling pathways during early mammary tumor progression, arguing that inhibiting Ezh2 or therapeutically targeting Ezh2-dependent programs could be beneficial for the treatment of early-stage luminal B breast cancer.

EZH2 targeting induces CD38 upregulation and response to anti-CD38 immunotherapies in multiple myeloma

EZH2 targeting induces CD38 upregulation and response to anti-CD38 immunotherapies in multiple myeloma