KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor-deficient mice, Kdm2b-deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.
Polycomb repressive complex 2 (PRC2) components, EZH2 and its homolog EZH1, and PI3K/Akt signaling pathway are focal points as therapeutic targets for multiple myeloma. However, the exact crosstalk between their downstream targets remains unclear. We herein elucidated some epigenetic interactions following Akt inhibition and demonstrated the efficacy of the combined inhibition of Akt and PRC2. We found that TAS-117, a potent and selective Akt inhibitor, downregulated EZH2 expression at the mRNA and protein levels via interference with the Rb-E2F pathway, while EZH1 was compensatively upregulated to maintain H3K27me3 modifications. Consistent with these results, the dual EZH2/EZH1 inhibitor, UNC1999, but not the selective EZH2 inhibitor, GSK126, synergistically enhanced TAS-117-induced cytotoxicity and provoked myeloma cell apoptosis. RNA-seq analysis revealed the activation of the FOXO signaling pathway after TAS-117 treatment. FOXO3/4 mRNA and their downstream targets were upregulated with the enhanced nuclear localization of FOXO3 protein after TAS-117 treatment. ChIP assays confirmed the direct binding of FOXO3to EZH1 promoter, which was enhanced by TAS-117 treatment. Moreover, FOXO3
Introduction: A novel tubulin binding agent PTC596, which is currently in clinical trials for solid tumors, was originally identified by its ability to kill cancer stem cells and to reduce BMI1 activity. PTC596 treatment results in hyperphosphorylation of the BMI1 protein and loss of BMI1 function as demonstrated by a reduction in H2A ubiquitination levels in a range of solid tumor lines. Subsequent studies have shown that the down-regulation of BMI1 protein is due to a G2/M arrest. In this study, we aimed to investigate the in-vitro and in-vivo anti-tumor activities of PTC596 and the combination with bortezomib in multiple myeloma (MM). Methods: For in-vitro evaluation, MTS and BrdU ELISA assays were performed using human MM cell lines. Approved by the Institutional Review Committee at Chiba University, primary myeloma cells and bone marrow stromal cells (BMSCs) were collected from the bone marrow of MM patients with informed consent. For in-vivo evaluation, the MM.1S subcutaneous xenograft model in NOG mice was used. To understand the mechanisms of action and target genes of the treatments, flow cytometry (FCM), western blotting, RNA-seq, and ChIP-seq were performed. Results: PTC596 induced significant cytotoxicity in all MM cell lines tested, including bortezomib-resistant OPM-2/BTZ and KMS-11/BTZ cells (CC50: 24-98 nM). PTC596 also suppressed cell proliferation when these cell lines were co-cultured with BMSCs. As expected, PTC596 reduced the levels of BMI1 protein and uH2A in a dose-dependent manner. Of note, PTC596 induced cell cycle arrest as detected by a BrdU FCM assay in MM cells and apoptosis as detected by annexin-V FCM in MM cell lines and primary myeloma cells. Moreover, oral administration of PTC596 twice a week for three weeks significantly inhibited the growth of MM.1S tumors implanted in immunodeficient mice and improved the survival of mice as compared with mice treated with vehicle only (p=0.0021). Of interest, bortezomib appeared to transcriptionally repress the expression of BMI1 and reduce the levels of uH2A. We then tested the efficacy of the combination of PTC596 with bortezomib in MM cells and found additive or synergistic effects when MM cell lines were co-cultured with BMSCs. Reductions in the levels of BMI1 protein and uH2A by PTC596 or bortezomib alone were significantly enhanced in the combination treatment. Furthermore, apoptosis induced by bortezomib was significantly enhanced by the combination with PTC596 as evidenced by increased annexin-positive cells detected in flow cytometric analysis and increased cleavage of caspases with reduction in MCL1 protein in western blotting. RNA-seq of MM.1S cells treated with PTC596 alone or in combination with bortezomib demonstrated repression of gene sets related to the cell cycle in either setting and enrichment of gene sets related to apoptosis in the combination. Ongoing analysis of our ChIP-seq data will reveal the direct targets of BMI1 in MM cells. Remarkably, oral administration of PTC596 combined with subcutaneous injection of bortezomib twice a week for five weeks significantly reduced MM.1S tumor growth in comparison to the control or either single treatment (p<0.0001 vs. control, p=0.0003 vs. PTC596, p=0.0104 vs. bortezomib). Severe body weight loss was not observed in the combination-treated mice except one mouse which needed washout period until recovery. White blood cell count and hemoglobin level in peripheral blood of mice were not significantly changed during the treatment with PTC596 or even the combination. Survival of the host mice treated with the combination was significantly prolonged as compared with the control or either single treatment (p<0.0001 vs. control, p=0.0002 vs. PTC596, p=0.0072 vs. bortezomib). Conclusions: Our results demonstrate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options in MM. The results of this study support the clinical evaluation of this promising therapeutic approach to improve the outcome of MM patients. Disclosures Sheedy: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment.
The novel small molecule PTC596 inhibits microtubule polymerization and its clinical development has been initiated for some solid cancers. We herein investigated the preclinical efficacy of PTC596 alone and in combination with proteasome inhibitors in the treatment of multiple myeloma (MM). PTC596 inhibited the proliferation of MM cell lines as well as primary MM samples in vitro, and this was confirmed with MM cell lines in vivo. PTC596 synergized with bortezomib or carfilzomib to inhibit the growth of MM cells in vitro. The combination treatment of PTC596 with bortezomib exerted synergistic effects in a xenograft model of human MM cell lines in immunodeficient mice and exhibited acceptable tolerability. Mechanistically, treatment with PTC596 induced cell cycle arrest at G2/M phase followed by apoptotic cell death, associated with the inhibition of microtubule polymerization. RNA sequence analysis also revealed that PTC596 and the combination with bortezomib affected the cell cycle and apoptosis in MM cells. Importantly, endoplasmic reticulum stress induced by bortezomib was enhanced by PTC596, providing an underlying mechanism of action of the combination therapy. Our results indicate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options to improve outcomes in patients with MM.
PI3K/Akt pathway is constitutively activated in multiple myeloma (MM). A plethora of studies extensively investigated Akt inhibitors, alone or in combination; however, the outcomes in hematological malignancies were largely unsatisfactory, emphasizing the need for critical preclinical evaluations. Polycomb repressive complex 2 (PRC2) components, EZH2 and its related homolog EZH1, induce H3K27me3 to silence the transcription of target genes. Recent studies ensured that EZH2 inhibition alone is not sufficient to completely disrupt the oncogenic functions of PRC2. With the importance of PRC2 as a therapeutic target in MM, we aimed to investigate the mechanisms by which Akt inhibition may impact PRC2 function, and test whether targeting both EZH2 and EZH1 together with Akt inhibition is a promising treatment strategy for MM. We herein evaluated the cytotoxic effect of TAS-117, a potent and selective non-competitive Akt inhibitor, against different MM cell lines and found that responsive cell lines tended to have significant levels of activated Akt, coupled with low/deleted PTEN. Then, we examined signaling-epigenetic crosstalk on EZH2 level. TAS-117 significantly down-regulated EZH2 mRNA and protein in dose- and time-dependent manners, while H3K27me3 levels were rather maintained or elevated, suggesting compensation by EZH1. As EZH2 is a direct target for E2F1, we focused on Rb-E2F pathway as a regulatory mechanism for EZH2. TAS-117 induced marked down-regulation of E2F1 and E2F2. Moreover, TAS-117 induced the up-regulation of CDKN1B, in addition to the inactivation of cyclins and cyclin dependent kinases, hence, hypo-phosphorylated Rb, thereby stabilizing Rb-E2F1 complex and diminishing free E2F1 available for binding to its target genes, including EZH2 promoter. This prompted us to examine the impact of TAS-117 combination with either dual EZH2/1 inhibitor, UNC1999, or selective EZH2 inhibitor, GSK126. In agreement, UNC1999, but not GSK126, synergistically enhanced TAS-117-induced cytotoxicity, confirmed by combination index calculation, and provoked MM cell apoptosis. As we observed an increase in H3K27me3 levels after TAS-117 treatment, we hypothesized that EZH1 function was augmented. Consistently, we found that EZH1 was markedly up-regulated after TAS-117 treatment in dose- and time-dependent manners. Importantly, EZH1 knockdown significantly enhanced the sensitivity of myeloma cells to TAS-117-induced cytotoxicity. To clarify the molecular mechanisms underlying EZH1 up-regulation, we performed RNA-seq followed by KEGG pathway analysis for up-regulated genes in TAS-117-treated group. We focused on FOXO pathway enrichment as it is a crucial target in MM treatment using Akt inhibitors. We then focused on FOXO3 as it was the main FOXO family gene expressed in MM cells according to our RNA-seq data. We examined the nuclear localization of FOXO3 following TAS-117 treatment. We found that TAS-117 significantly enhanced the nuclear accumulation of FOXO3, as depicted by both the immunostaining images and the digital calculations of the nuclear subset of FOXO3. Murine Ezh1 promoter was shown to be bound by Foxo transcription factors (TFs) in neuronal progenitors, T-regulatory cells, CD8+ cells, and pre-B cells. More than 80% of FOXO3-binding sites share the common binding motif, GTAAACAA, which was found both in human EZH1 (+48 from the TSS) and mouse Ezh1 (+77 from the TSS) promoter regions. So, we hypothesized that FOXO3 may be a regulatory partner for human EZH1 gene in myeloma cells in response to TAS-117 treatment. To this end, we performed ChIP-qPCR analysis for TAS-117-treated and -untreated cells. TAS-117 promoted the binding of FOXO3 to EZH1 promoter, in addition to one of the canonical FOXO3 targets, BIM promoter. To further confirm our results, we expressed shRNA against FOXO3 (shFOXO3) in MM cells which, interestingly, induced the down-regulation of EZH1 mRNA. In conclusion, the present results defined novel signaling-epigenetic crosstalk between PI3K/Akt pathway and PRC2 components, EZH2 and EZH1, and demonstrated that Akt inhibition can differently modulates EZH2 and EZH1 levels via Akt downstream effectors, E2F1 and FOXO3, respectively. Therefore, targeting both EZH2 and EZH1 in addition to Akt inhibition may be a promising rationale to eradicate MM, leading to significant advances in treatment. Disclosures No relevant conflicts of interest to declare.
UTX/KDM6A, a histone H3K27 demethylase and a key component of the COMPASS complex, is frequently lost or mutated in cancer; however, its tumor suppressor function remains largely uncharacterized in multiple myeloma (MM). Here, we show that the conditional deletion of the X-linked Utx in germinal center (GC) derived cells collaborates with the activating BrafV600E mutation and promotes induction of lethal GC/post-GC B cell malignancies with MM-like plasma cell neoplasms being the most frequent. Mice that developed MM-like neoplasms showed expansion of clonal plasma cells in the bone marrow and extramedullary organs, serum M proteins, and anemia. Add-back of either wild-type UTX or a series of mutants revealed that cIDR domain, that forms phase-separated liquid condensates, is largely responsible for the catalytic activity-independent tumor suppressor function of UTX in MM cells. Utx loss in concert with BrafV600E only slightly induced MM-like profiles of transcriptome, chromatin accessibility, and H3K27 acetylation, however, it allowed plasma cells to gradually undergo full transformation through activation of transcriptional networks specific to MM that induce high levels of Myc expression. Our results reveal a tumor suppressor function of UTX in MM and implicate its insufficiency in the transcriptional reprogramming of plasma cells in the pathogenesis of MM.
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