Dual inhibition of EZH1/2 induces cell cycle arrest of B cell acute lymphoblastic leukemia cells through upregulation of CDKN1C and TP53INP1.

Disease-risk stratification and development of intensified chemotherapy protocols have substantially improved the outcome of acute lymphoblastic leukemia (ALL). However, outcomes of relapsed or refractory cases remain poor. Previous studies have discussed the oncogenic role of enhancer of zeste homolog 1 and 2 (EZH1/2), and the efficacy of dual inhibition of EZH1/2 as a treatment for hematological malignancy. Here, we investigated whether an EZH1/2 dual inhibitor, DS-3201 (valemetostat), has antitumor effects on B cell ALL (B-ALL). DS-3201 inhibited growth of B-ALL cell lines more significantly and strongly than the EZH2-specific inhibitor EPZ-6438, and induced cell cycle arrest and apoptosis in vitro. RNA-seq analysis to determine the effect of DS-3201 on cell cycle arrest-related genes expressed by B-ALL cell lines showed that DS-3201 upregulated CDKN1C and TP53INP1. CRIPSR/Cas9 knockout confirmed that CDKN1C and TP53INP1 are direct targets of EZH1/2 and are responsible for the antitumor effects of DS-3201 against B-ALL. Furthermore, a patient-derived xenograft (PDX) mouse model showed that DS-3201 inhibited the growth of B-ALL harboring MLL-AF4 significantly. Thus, DS-3201 provides another option for treatment of B-ALL.

Identification of a Musashi2 translocation as a novel oncogene in myeloid leukemia.

Myeloid leukemias, diseases marked by aggressiveness and poor outcomes, are frequently triggered by oncogenic translocations. In the case of chronic myelogenous leukemia (CML) the BCR-ABL fusion initiates chronic phase disease with second hits allowing progression to blast crisis. Although Gleevec has been transformative for CML, blast crisis CML remains relatively drug resistant. Here we show that MSI2-HOXA9, a translocation with an unknown role in cancer, can serve as a second hit in driving bcCML. Compared to BCR-ABL, BCR-ABL/MSI2-HOXA9 led to a more aggressive disease in vivo with decreased latency, increased lethality and a differentiation blockade that is a hallmark of blast crisis. Domain mapping revealed that the MSI2 RNA binding domain RRM1 had a preferential impact on growth and lethality of bcCML relative to RRM2 or the HOXA9 domain. Mechanistically, MSI2-HOXA9 triggered global downstream changes with a preferential upregulation of mitochondrial components. Consistent with this, BCR-ABL/MSI2-HOXA9 cells exhibited a significant increase in mitochondrial respiration. These data suggest that MSI2-HOXA9 acts, at least in part, by increasing expression of the mitochondrial polymerase Polrmt and augmenting mitochondrial function and basal respiration in blast crisis. Collectively, our findings demonstrate for the first time that translocations involving the stem and developmental signal MSI2 can be oncogenic, and suggest that MSI, which we found to be a frequent partner for an array of translocations, could also be a driver mutation across solid cancers.

CDKN1C-mediated growth inhibition by an EZH1/2 dual inhibitor overcomes resistance of mantle cell lymphoma to ibrutinib.

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.

An in vivo genome-wide CRISPR screen identifies the RNA-binding protein Staufen2 as a key regulator of myeloid leukemia.

Aggressive myeloid leukemias such as blast crisis chronic myeloid leukemia and acute myeloid leukemia remain highly lethal. Here we report a genome-wide in vivo CRISPR screen to identify new dependencies in this disease. Among these, RNA-binding proteins (RBPs) in general, and the double-stranded RBP Staufen2 (Stau2) in particular, emerged as critical regulators of myeloid leukemia. In a newly developed knockout mouse, loss of Stau2 led to a profound decrease in leukemia growth and improved survival in mouse models of the disease. Further, Stau2 was required for growth of primary human blast crisis chronic myeloid leukemia and acute myeloid leukemia. Finally, integrated analysis of CRISPR, eCLIP and RNA-sequencing identified Stau2 as a regulator of chromatin-binding factors, driving global alterations in histone methylation. Collectively, these data show that in vivo CRISPR screening is an effective tool for defining new regulators of myeloid leukemia progression and identify the double-stranded RBP Stau2 as a critical dependency of myeloid malignancies.

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.

A Multiscale Map of the Stem Cell State in Pancreatic Adenocarcinoma.

Drug resistance and relapse remain key challenges in pancreatic cancer. Here, we have used RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and genome-wide CRISPR analysis to map the molecular dependencies of pancreatic cancer stem cells, highly therapy-resistant cells that preferentially drive tumorigenesis and progression. This integrated genomic approach revealed an unexpected utilization of immuno-regulatory signals by pancreatic cancer epithelial cells. In particular, the nuclear hormone receptor retinoic-acid-receptor-related orphan receptor gamma (RORγ), known to drive inflammation and T cell differentiation, was upregulated during pancreatic cancer progression, and its genetic or pharmacologic inhibition led to a striking defect in pancreatic cancer growth and a marked improvement in survival. Further, a large-scale retrospective analysis in patients revealed that RORγ expression may predict pancreatic cancer aggressiveness, as it positively correlated with advanced disease and metastasis. Collectively, these data identify an orthogonal co-option of immuno-regulatory signals by pancreatic cancer stem cells, suggesting that autoimmune drugs should be evaluated as novel treatment strategies for pancreatic cancer patients.

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.

Gene expression profiles of the cochlea and vestibular endorgans: localization and function of genes causing deafness.

OBJECTIVES: We sought to elucidate the gene expression profiles of the causative genes as well as the localization of the encoded proteins involved in hereditary hearing loss. METHODS: Relevant articles (as of September 2014) were searched in PubMed databases, and the gene symbols of the genes reported to be associated with deafness were located on the Hereditary Hearing Loss Homepage using localization, expression, and distribution as keywords. RESULTS: Our review of the literature allowed us to systematize the gene expression profiles for genetic deafness in the inner ear, clarifying the unique functions and specific expression patterns of these genes in the cochlea and vestibular endorgans. CONCLUSIONS: The coordinated actions of various encoded molecules are essential for the normal development and maintenance of auditory and vestibular function.

Essential role of PU.1 in maintenance of mixed lineage leukemia-associated leukemic stem cells.

Acute myeloid leukemia is a clonal malignant disorder derived from a small number of leukemic stem cells (LSCs). Rearrangements of the mixed lineage leukemia (MLL) gene are found in acute myeloid leukemia associated with poor prognosis. The upregulation of Hox genes is critical for LSC induction and maintenance, but is unlikely to support malignancy and the high LSC frequency observed in MLL leukemias. The present study shows that MLL fusion proteins interact with the transcription factor PU.1 to activate the transcription of CSF-1R, which is critical for LSC activity. Acute myeloid leukemia is cured by either deletion of PU.1 or ablation of cells expressing CSF-1R. Kinase inhibitors specific for CSF-1R prolong survival time. These findings indicate that PU.1-mediated upregulation of CSF-1R is a critical effector of MLL leukemogenesis.

PML-RARα and its phosphorylation regulate pml oligomerization and HIPK2 stability.

The PML gene is frequently fused to the retinoic acid receptor α (RARα) gene in acute promyelocytic leukemia (APL), generating a characteristic PML-RARα oncogenic chimera. PML-RARα disrupts the discrete nuclear speckles termed nuclear bodies, which are formed in PML, suggesting that nuclear body disruption is involved in leukemogenesis. Nuclear body formation that relies upon PML oligomerization and its stabilization of the hypoxia-inducible protein kinase (HIPK)-2 is disrupted by expression of the PML-RARα chimera. Here, we report that disruption of nuclear bodies is also mediated by PML-RARα inhibition of PML oligomerization. PKA-mediated phosphorylation of PML-RARα blocked its ability to inhibit PML oligomerization and destabilize HIPK2. Our results establish that both PML oligomerization and HIPK2 stabilization at nuclear bodies are important for APL cell differentiation, offering insights into the basis for the most common prodifferentiation therapies of APL used clinically.

Deregulated transcription factors in leukemia.

Specific chromosomal translocations and other mutations associated with acute myeloblastic leukemia (AML) often involve transcription factors and transcriptional coactivators. Such target genes include AML1, C/EBPα, RARα, MOZ, p300/CBP, and MLL, all of which are important in the regulation of hematopoiesis. The resultant fusion or mutant proteins deregulate the transcription of the affected genes and disrupt their essential role in hematopoiesis, causing differentiation block and abnormal proliferation and/or survival. This review focuses on such transcription factors and coactivators, and describes their roles in leukemogenesis and hematopoiesis.

PML activates transcription by protecting HIPK2 and p300 from SCFFbx3-mediated degradation.

PML, a nuclear protein, interacts with several transcription factors and their coactivators, such as HIPK2 and p300, resulting in the activation of transcription. Although PML is thought to achieve transcription activation by stabilizing the transcription factor complex, little is known about the underlying molecular mechanism. To clarify the role of PML in transcription regulation, we purified the PML complex and identified Fbxo3 (Fbx3), Skp1, and Cullin1 as novel components of this complex. Fbx3 formed SCF(Fbx3) ubiquitin ligase and promoted the degradation of HIPK2 and p300 by the ubiquitin-proteasome pathway. PML inhibited this degradation through a mechanism that unexpectedly did not involve inhibition of the ubiquitination of HIPK2. PML, Fbx3, and HIPK2 synergistically activated p53-induced transcription. Our findings suggest that PML stabilizes the transcription factor complex by protecting HIPK2 and p300 from SCF(Fbx3)-induced degradation until transcription is completed. In contrast, the leukemia-associated fusion PML-RARalpha induced the degradation of HIPK2. We discuss the roles of PML and PML-retinoic acid receptor alpha, as well as those of HIPK2 and p300 ubiquitination, in transcriptional regulation and leukemogenesis.

Inhibition of cellular adhesion in human umbilical vein endothelial cells by NF-kappaB inhibitor DHMEQ under flow.

We previously designed and synthesized DHMEQ as an inhibitor of NF-kappaB. In the present study, we looked into the effect of DHMEQ on the cell adhesion in human umbilical vein endothelial cells (HUVEC) under flow. We used freshly prepared HUVEC and human mononuclear cells throughout the experiment. DHMEQ inhibited TNF-alpha-, IL-1beta-, and LPS-induced NF-kappaB activation in HUVEC. It also inhibited TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. DHMEQ also inhibited TNF-alpha-induced mononuclear cell-HUVEC adhesion. The effect of DHMEQ was more prominent when the cells were under shear stress. DHMEQ inhibited the adhesion between HUVEC and HT-29 colon cancer cells more clearly under the flow condition than under the static condition of the culture medium. These results suggest that DHMEQ, being a unique inhibitor of NF-kappaB, may be effective in suppressing atherosclerosis and metastasis by inhibiting the expression of adhesion molecules.

Suppression of hormone-refractory prostate cancer by a novel nuclear factor kappaB inhibitor in nude mice.

We have synthesized and explored the feasibility of using a novel nuclear factor (NF) kappaB inhibitor, a dehydroxymethylepoxyquinomicin designated as DHMEQ, against prostate cancer. The activity of NFkappaB, evaluated by transient transfection of a luciferase reporter DNA containing a specific binding sequence for NFkappaB, was inhibited by DHMEQ in three human hormone-refractory prostate cancer cell lines, DU145, JCA-1, and PC-3. Statistically significant growth inhibition was achieved by 20 micro g/ml of DHMEQ, and marked levels of apoptosis were induced 48 h after DHMEQ administration in vitro. Electrophoretic mobility shift assay showed that DHMEQ completely inhibited NFkappaB DNA binding activity in JCA-1 cells. Furthermore, i.p. administrations of DHMEQ significantly inhibited pre-established JCA-1 s.c. tumor growth in nude mice without any side effects. Our result indicates the possibility of using a novel NFkappaB activation inhibitor, DHMEQ, as a new treatment strategy against hormone-refractory prostate cancer.