MOZ is critical for the development of MOZ/MLL fusion-induced leukemia through regulation of Hoxa9/Meis1 expression.

Monocytic leukemia zinc finger protein (MOZ, MYST3, or KAT6A) is a MYST-type acetyltransferase involved in chromosomal translocation in acute myelogenous leukemia (AML) and myelodysplastic syndrome. MOZ is established as essential for hematopoiesis; however, the role of MOZ in AML has not been addressed. We propose that MOZ is critical for AML development induced by MLL-AF9, MLL-AF10, or MOZ-TIF2 fusions. Moz-deficient hematopoietic stem/progenitor cells (HSPCs) transduced with an MLL-AF10 fusion gene neither formed colonies in methylcellulose nor induced AML in mice. Moz-deficient HSPCs bearing MLL-AF9 also generated significantly reduced colony and cell numbers. Moz-deficient HSPCs expressing MOZ-TIF2 could form colonies in vitro but could not induce AML in mice. By contrast, Moz was dispensable for colony formation by HOXA9-transduced cells and AML development caused by HOXA9 and MEIS1, suggesting a specific requirement for MOZ in AML induced by MOZ/MLL fusions. Expression of the Hoxa9 and Meis1 genes was decreased in Moz-deficient MLL fusion-expressing cells, while expression of Meis1, but not Hoxa9, was reduced in Moz-deficient MOZ-TIF2 AML cells. AML development induced by MOZ-TIF2 was rescued by introducing Meis1 into Moz-deficient cells carrying MOZ-TIF2. Meis1 deletion impaired MOZ-TIF2-mediated AML development. Active histone modifications were also severely reduced at the Meis1 locus in Moz-deficient MOZ-TIF2 and MLL-AF9 AML cells. These results suggest that endogenous MOZ is critical for MOZ/MLL fusion-induced AML development and maintains active chromatin signatures at target gene loci.

A Potent Blood-Brain Barrier-Permeable Mutant IDH1 Inhibitor Suppresses the Growth of Glioblastoma with IDH1 Mutation in a Patient-Derived Orthotopic Xenograft Model.

Gliomas are the second most common primary brain tumors in adults. They are treated with combination therapies, including surgery, radiotherapy, and chemotherapy. There are currently limited treatment options for recurrent gliomas, and new targeted therapies need to be identified, especially in glioblastomas, which have poor prognosis. Isocitrate dehydrogenase (IDH) mutations are detected in various tumors, including gliomas. Most patients with IDH mutant glioma harbor the IDH1R132H subtype. Mutant IDH catalyzes the conversion of α-ketoglutarate to the oncometabolite 2-hydroxyglutarate (2-HG), which induces aberrant epigenetic status and contributes to malignant progression, and is therefore a potential therapeutic target for IDH mutant tumors. The present study describes a novel, orally bioavailable selective mutant IDH1 inhibitor, DS-1001b. The drug has high blood-brain barrier (BBB) permeability and inhibits IDH1R132H. Continuous administration of DS-1001b impaired tumor growth and decreased 2-HG levels in subcutaneous and intracranial xenograft models derived from a patient with glioblastoma with IDH1 mutation. Moreover, the expression of glial fibrillary acidic protein was strongly induced by DS-1001b, suggesting that inhibition of mutant IDH1 promotes glial differentiation. These results reveal the efficacy of BBB-permeable DS-1001b in orthotopic patient-derived xenograft models and provide a preclinical rationale for the clinical testing of DS-1001b in recurrent gliomas.

Selective inhibition of mutant IDH1 by DS-1001b ameliorates aberrant histone modifications and impairs tumor activity in chondrosarcoma.

Chondrosarcoma is the second most common malignant bone tumor. It is characterized by low vascularity and an abundant extracellular matrix, which confer these tumors resistance to chemotherapy and radiotherapy. There are currently no effective treatment options for relapsed or dedifferentiated chondrosarcoma, and new targeted therapies need to be identified. Isocitrate dehydrogenase (IDH) mutations, which are detected in ~50% of chondrosarcoma patients, contribute to malignant transformation by catalyzing the production of 2-hydroxyglutarate (2-HG), a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Mutant IDH inhibitors are therefore potential novel anticancer drugs in IDH mutant tumors. Here, we examined the efficacy of the inhibition of mutant IDH1 as an antitumor approach in chondrosarcoma cells in vitro and in vivo, and investigated the association between the IDH mutation and chondrosarcoma cells. DS-1001b, a novel, orally bioavailable, selective mutant IDH1 inhibitor, impaired the proliferation of chondrosarcoma cells with IDH1 mutations in vitro and in vivo, and decreased 2-HG levels. RNA-seq analysis showed that inhibition of mutant IDH1 promoted chondrocyte differentiation in the conventional chondrosarcoma L835 cell line and caused cell cycle arrest in the dedifferentiated JJ012 cell line. Mutant IDH1-mediated modulation of SOX9 and CDKN1C expression regulated chondrosarcoma tumor progression, and DS-1001b upregulated the expression of these genes via a common mechanism involving the demethylation of H3K9me3. DS-1001b treatment reversed the epigenetic changes caused by aberrant histone modifications. The present data strongly suggest that inhibition of mutant IDH1 is a promising therapeutic approach in chondrosarcoma, particularly for the treatment of relapsed or dedifferentiated chondrosarcoma.

Dual inhibition of enhancer of zeste homolog 1/2 overactivates WNT signaling to deplete cancer stem cells in multiple myeloma.

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.

[IDH mutations activate Hoxa9/Meis1 and hypoxia pathways in acute myeloid leukemia model mice].

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs mediate exchanges between isocitrate and α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which causes dysregulation of a set of α-KG-dependent dioxygenases such as TET, histone demethylase and others. Because mutant IDH has no necessary functions in normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as anti-cancer agents. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH2-dependent AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted mutant IDH2 from AML mice, which resulted in the loss of leukemia stem cells and significantly delayed the progression of AML. These results indicate that mutant IDHs are promising targets for anticancer therapy.

IDH2 and NPM1 Mutations Cooperate to Activate Hoxa9/Meis1 and Hypoxia Pathways in Acute Myeloid Leukemia.

IDH1 and IDH2 mutations occur frequently in acute myeloid leukemia (AML) and other cancers. The mutant isocitrate dehydrogenase (IDH) enzymes convert α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of AML in which mice were transplanted with nucleophosmin1 (NPM)(+/-) hematopoietic stem/progenitor cells cotransduced with four mutant genes (NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD), which often occur simultaneously in human AML patients. Conditional deletion of IDH2/R140Q blocked 2-HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. IDH2/R140Q was necessary for the engraftment or survival of NPMc(+) cells in vivo. Gene expression analysis indicated that NPMc increased expression of Hoxa9. IDH2/R140Q also increased the level of Meis1 and activated the hypoxia pathway in AML cells. IDH2/R140Q decreased the 5hmC modification and expression of some differentiation-inducing genes (Ebf1 and Spib). Taken together, our results indicated that IDH2 mutation is critical for the development and maintenance of AML stem-like cells, and they provided a preclinical justification for targeting mutant IDH enzymes as a strategy for anticancer therapy.

Interactomic approach for evaluating nucleophosmin-binding proteins as biomarkers for Ewing's sarcoma.

Nucleophosmin (NPM) is a novel prognostic biomarker for Ewing's sarcoma. To evaluate the prognostic utility of NPM, we conducted an interactomic approach to characterize the NPM protein complex in Ewing's sarcoma cells. A gene suppression assay revealed that NPM promoted cell proliferation and the invasive properties of Ewing's sarcoma cells. FLAG-tag-based affinity purification coupled with liquid chromatography-tandem mass spectrometry identified 106 proteins in the NPM protein complex. The functional classification suggested that the NPM complex participates in critical biological events, including ribosome biogenesis, regulation of transcription and translation, and protein folding, that are mediated by these proteins. In addition to JAK1, a candidate prognostic biomarker for Ewing's sarcoma, the NPM complex, includes 11 proteins known as prognostic biomarkers for other malignancies. Meta-analysis of gene expression profiles of 32 patients with Ewing's sarcoma revealed that 6 of 106 were significantly and independently associated with survival period. These observations suggest a functional role as well as prognostic value of these NPM complex proteins in Ewing's sarcoma. Further, our study suggests the potential applications of interactomics in conjunction with meta-analysis for biomarker discovery.

Akt enhances Mdm2-mediated ubiquitination and degradation of p53.

p53 plays a key role in DNA damage-induced apoptosis. Recent studies have reported that the phosphatidylinositol 3-OH-kinase-Akt pathway inhibits p53-mediated transcription and apoptosis, although the underlying mechanisms have yet to be determined. Mdm2, a ubiquitin ligase for p53, plays a central role in regulation of the stability of p53 and serves as a good substrate for Akt. In this study, we find that expression of Akt reduces the protein levels of p53, at least in part by enhancing the degradation of p53. Both Akt expression and serum treatment induced phosphorylation of Mdm2 at Ser186. Akt-mediated phosphorylation of Mdm2 at Ser186 had little effect on the subcellular localization of Mdm2. However, both Akt expression and serum treatment increased Mdm2 ubiquitination of p53. The serum-induced increase in p53 ubiquitination was blocked by LY294002, a phosphatidylinositol 3-OH-kinase inhibitor. Moreover, when Ser186 was replaced by Ala, Mdm2 became resistant to Akt enhancement of p53 ubiquitination and degradation. Collectively, these results suggest that Akt enhances the ubiquitination-promoting function of Mdm2 by phosphorylation of Ser186, which results in reduction of p53 protein. This study may shed light on the mechanisms by which Akt promotes survival, proliferation, and tumorigenesis.