The mechanism underlying extracellular adenosine-induced caspase-independent apoptosis in HuH-7 human hepatoma cells is not fully understood. The present study investigated the role for apoptosis-inducing factor (AIF)-homologous mitochondrion-associated inducer of death (AMID) in the pathway.To see the implication of AMID in adenosine-induced HuH-7 cell apoptosis, real-time reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescent cytochemistry, time-laps GFP monitoring, cell cycle analysis, flow cytometry, Western blotting, cell viability assay, and TUNEL staining were carried out.Adenosine upregulated AMID expression in HuH-7 cells, and translocated AMID from the cytosol into the nucleus. Adenosine induced HuH-7 cell apoptosis, and the effect was further enhanced by overexpressing AMID. Adenosine-induced HuH-7 cell apoptosis, alternatively, was inhibited by knocking-down AMID.The results of the present study provide evidence for AMID as a critical factor for adenosine-induced caspase-independent HuH-7 cell apoptosis.
Abstract INTRODUCTION AND HYPOTHESIS Mesenchymal cells known as myo-/fibroblasts (MFs) are critical immunosuppressors under gut mucosal homeostasis. Expression of immune checkpoint PD-L1 by MFs plays a key role in the control of T cell inflammatory responses. In Crohn’s disease (CD), MFs switch their activity from immunosuppressive to pro-inflammatory, where they are also known as Inflammatory Fibroblasts. However, the mechanisms responsible for these pathological changes in MF activity are unknown. Map-kinase-activated protein kinase 2 (MK2) is a major regulator of inflammation in the gut. MK2 is downstream of p38 signaling, it evokes a sub-pathway that directly regulates the production of key inflammatory cytokines implicated in CD (such as TNF-α, IL-1, and IL-6). Thus, we hypothesized that activation of MK2 signaling is critical to the pathological changes in MFs during the immunopathogenesis of CD. METHODS Human normal and CD tissues and derived MFs, as well as animal models relevant to CD, were used in this study. MF signaling/activity was analyzed using RNAseq, qRT-PCR, western blot (WB), cytokine/chemokine multiplex arrays, and confocal microscopy. RESULTS In situ analysis demonstrated an increase in MK2 activity within the inflamed compared to the non-inflamed CD and healthy control intestinal tissues, which was confirmed by WB and multiplex signaling array analysis. In situ increase in MK2 activity in CD intestinal mucosa was greatly associated with mesenchymal stromal cells that bear a “myofibroblast” phenotype (positive for α-SMA expression). An increase in MK2 activity was also observed in primary MF cultures isolated from CD (CD-MFs) when compared to normal (N-) MFs. MK2 activity within CD-MFs was also associated with a significant decrease in the expression of the immunosuppressive checkpoint PD-L1 and an increase in the expression of inflammatory CCL2 and IL-6. Inhibition of MK2 activity within CD-MFs through using the MK2-specific inhibitor PF-3644022 (10 μM) reversed the inflammatory activity of MFs. Remarkably, we observed a differential role of p38 and MK2 in the regulation of PD-L1 expression in MFs: while p38 was required for basal expression of PD-L1, activation of MK2 downregulates PD-L1 expression. These data indicate a unique role of MK2 activation in pathological reprogramming of MFs in CD. Use of MK2 inhibitor in a therapeutic modality in chronic DSS and IL-10 KO murine models of CD also significantly reduce MF-linked inflammatory responses in vivo. CONCLUSION Our data suggest that an increase in MK2 activity in CD is critical to the reprogramming of the MF from immunosuppressive toward pathological Inflammatory fibroblasts. Targeting MK2 activity within MFs could be a desirable strategy for improving the efficacy of current IBD therapeutic approaches.
Ikaros encodes a transcription factor that functions as a tumor suppressor in T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms through which Ikaros regulates gene expression and cellular proliferation in T-ALL are unknown. Re-introduction of Ikaros into Ikaros-null T-ALL cells resulted in cessation of cellular proliferation and induction of T-cell differentiation. We performed dynamic, global, epigenomic, and gene expression analyses to determine the mechanisms of Ikaros tumor suppressor activity. Our results identified novel Ikaros functions in the epigenetic regulation of gene expression: Ikaros directly regulates de novo formation and depletion of enhancers, de novo formation of active enhancers and activation of poised enhancers; Ikaros directly induces the formation of super-enhancers; and Ikaros demonstrates pioneering activity by directly regulating chromatin accessibility. Dynamic analyses demonstrate the long-lasting effects of Ikaros DNA binding on enhancer activation, de novo formation of enhancers and super-enhancers, and chromatin accessibility. Our results establish that Ikaros' tumor suppressor function occurs via global regulation of the enhancer and super-enhancer landscape and through pioneering activity. Expression analysis identified a large number of novel signaling pathways that are directly regulated by Ikaros and Ikaros-induced enhancers, and that are responsible for the cessation of proliferation and induction of T-cell differentiation in T-ALL cells.
More effective targeted therapy and new combination regimens are needed for Acute myeloid leukemia (AML), owing to the unsatisfactory long-term prognosis of the disease. Here, we investigated the synergistic effect and the mechanism of a histone deacetylase inhibitor, Chidamide in combination with Cladribine, a purine nucleoside antimetabolite analog in the disease.Cell counting kit-8 assays and Chou-Talalay's combination index were used to examine the synergistic effect of Chidamide and Cladribine on AML cell lines (U937, THP-1, and MV4-11) and primary AML cells. PI and Annexin-V/PI assays were used to detect the cell cycle effect and apoptosis effect, respectively. Global transcriptome analysis, RT-qPCR, c-MYC Knockdown, western blotting, co-immunoprecipitation, and chromatin immunoprecipitation assays were employed to explore the molecule mechanisms.The combination of Chidamide with Cladribine showed a significant increase in cell proliferation arrest, the G0/G1 phase arrest, and apoptosis compared to the single drug control in AML cell lines along with upregulated p21Waf1/Cip1 expression and downregulated CDK2/Cyclin E2 complex, and elevated cleaved caspase-9, caspase-3, and PARP. The combination significantly suppresses the c-MYC expression in AML cells, and c-MYC knockdown significantly increased the sensitivity of U937 cells to the combination compared to single drug control. Moreover, we observed HDAC2 interacts with c-Myc in AML cells, and we further identified that c-Myc binds to the promoter region of RCC1 that also could be suppressed by the combination through c-Myc-dependent. Consistently, a positive correlation of RCC1 with c-MYC was observed in the AML patient cohort. Also, RCC1 and HDAC2 high expression are associated with poor survival in AML patients. Finally, we also observed the combination significantly suppresses cell growth and induces the apoptosis of primary cells in AML patients with AML1-ETO fusion, c-KIT mutation, MLL-AF6 fusion, FLT3-ITD mutation, and in a CMML-BP patient with complex karyotype.Our results demonstrated the synergistic effect of Chidamide with Cladribine on cell growth arrest, cell cycle arrest, and apoptosis in AML and primary cells with genetic defects by targeting HDAC2/c-Myc/RCC1 signaling in AML. Our data provide experimental evidence for the undergoing clinical trial (Clinical Trial ID: NCT05330364) of Chidamide plus Cladribine as a new potential regimen in AML.
Abstract Acute myeloid leukemia (AML) represents a set of heterogeneous myeloid malignancies hallmarked by mutations in epigenetic modifiers, transcription factors, and kinases that can cause epigenetic reshaping. It is unclear to what extent AML mutations drive chromatin 3D structure alteration and contribute to myeloid transformation. We first performed Hi-C and whole-genome sequencing in 25 AML patient samples and seven healthy donor samples, and identified recurrent alterations of A/B compartments, TADs, and chromatin loops that are unique to different subtypes. To investigate how altered chromatin organization contributes to transcriptional misregulation, we performed RNA-Seq, ATAC-Seq and CUT&ag for CTCF, H3K27ac, and H3K27me3 in the same AML samples. We identified extensive and recurrent AML-specific promoter-enhancer and promoter-repressor loops. We performed both CRISPR deletion and interference experiments and validated two repressor loops that downregulated cancer related genes IKZF2 and RTTN. Furthermore, by using our recently developed algorithm, we identified structural variation-induced enhancer-hijacking and repressor-hijacking events in AML samples. We further demonstrated the role of hijacked enhancers in AML cell growth by CRISPR screening, and the role of hijacked repressors by CRISPR de-repression. We performed whole-genome bisulfite sequencing in 20 AML and normal samples, and showed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Finally, by treating the AML cells with the DNA hypomethylating agent and performing triple knockdown of DNMT1/3A/3B, we demonstrated the impact of altered DNA methylation on gene expression and 3D genome organization. Overall this study provides an invaluable resource for leukemia studies and also highlighted the role of repressor-loops and hijacked cis-elements in gene regulation and human diseases. Citation Format: Jie Xu, Fan Song, Baozhen Zhang, Huijue Lyu, Mikoto Kobayashi, Ziyu Zhao, Ye Hou, Xiaotao Wang, Yu Luan, Bei Jia, Lena Stasiak, Qixuan Wang, Qi Jin, Qiushi Jin, Yihao Fu, Ross C. Hardison, Sinisa Dovat, Leonidas C. Platanias, Yue Yang, Tomoko Yamada, Aaron D. Viny, Ross L. Levine, David F. Claxton, James R. Broach, Hong Zheng, Feng Yue. Subtype-specific and structure variation induced 3D genome alteration in acute myeloid leukemia [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 2953.
Abstract Cellular proliferation in T-cell acute lymphoblastic leukemia is regulated by multiple signaling pathways. The Phosphoinositide 3-kinase (PI3K)/AKT pathway is frequently dysregulated in T-ALL. Targeting the PI3K pathway has shown promise as a novel therapeutic approach for T-ALL. However, regulation of the PI3K pathway is still not well understood. Here, we report that PI3K activity in T-ALL can be controlled by transcriptional regulation of key members of this pathway, PIK3CD and PIKFYVE. DNA binding analysis of primary T-ALL using qChIP revealed that the tumor suppressor protein, Ikaros, binds the promoter regions of PIK3CD and PIKFYVE. Since Ikaros acts as a regulator of transcription, we tested whether Ikaros binding to PIK3CD and PIKFYVE affects their expression. Overexpression of Ikaros results in reduced transcription of PIK3CD and PIKFYVE in T-ALL. Targeting Ikaros with a specific shRNA, resulted in increased transcription of PIK3CD and PIKFYVE in T-ALL. Together, these results demonstrate that Ikaros functions as a transcriptional repressor of both PIK3CD and PIKFYVE, and suggest that Ikaros can regulate the PI3K pathway in T-ALL. It has been previously shown that Ikaros function in B-cell acute lymphoblastic leukemia is regulated by oncogenic Casein Kinase II (CK2). We tested whether Ikaros ability to repress transcription of PIK3CD and PIKFYVE is regulated by CK2. Inhibition of CK2 by a specific pharmacological inhibitor, CX-4945, resulted in increased Ikaros binding to the promoters of PIK3CD and PIKFYVE, as well as in transcriptional repression of both of these genes. These results suggest that Ikaros function as a repressor of PIK3CD and PIKFYVE transcription is impaired by CK2 in T-ALL. CK2 inhibition restores Ikaros-mediated transcriptional repression of PIK3CD and PIKFYVE, which results in downregulation of the PI3K pathway. In conclusion, the presented data demonstrate that the PI3K signaling pathway is regulated by transcriptional repression of PIK3CD and PIKFYVE by Ikaros in T-ALL. Results reveal interplay between two signaling pathways in T-ALL, CK2 and PI3K, where CK2 positively regulates the PI3K pathway by inhibiting Ikaros function. These data reveal novel mechanisms that regulate cellular proliferation in T-ALL. Citation Format: Tommy Hu, Mario Soliman, Malika Kapadia, Elanora Dovat, Jonathan Payne, Chunhua Song, Sinisa Dovat. Transcriptional control of signaling pathways in T-cell lymphoblastic leukemia by Ikaros tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5540. doi:10.1158/1538-7445.AM2017-5540
Abstract Ikaros (IKZF1) is a tumor suppressor whose function is impaired in high-risk pediatric B-cell acute lymphoblastic leukemia (B-ALL). IKZF1 encodes a DNA-binding, zinc finger protein that regulates expression of genes involved in important biological pathways. Using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-SEQ) we found that Ikaros binds to the upstream regulatory regions of multiple genes that regulate the phosphatidylinositol-3-Kinase (PI3K) pathway. Ikaros target genes include PIK3C2B and PI3KFYVE. We used gain-of-function and loss-of-function experiments to determine how Ikaros regulates transcription of its target genes. Overexpression of Ikaros by retroviral transduction in Nalm6 leukemia cells results in reduced transcription of PIK3C2B and PI3KFYVE as evidenced by qRT-PCR. Luciferase reporter assays with PIK3C2B and PI3KFYVE promoters showed that Ikaros can function as a transcriptional repressor of these genes. Transfection of Nalm6 cells with Ikaros shRNA resulted in increased expression of PIK3C2B and PI3KFYVE genes. These results suggest that Ikaros functions as a transcriptional repressor of PIK3C2B and PI3KFYVE genes in leukemia. Next, we studied signaling pathways that regulate the ability of Ikaros to transcriptionally repress the PIK3C2B and PI3KFYVE genes. We have previously shown that a pro-oncogenic Casein Kinase II (CK2) can directly phosphorylate Ikaros in vivo and that CK2-mediated phosphorylation impairs Ikaros function. We tested whether inhibition of CK2 activity affects Ikaros ability to regulate PIK3C2B and PI3KFYVE transcription in leukemia. Results show that molecular and pharmacological inhibition of CK2 have a very similar effect on transcription of Ikaros target genes and they result in transcriptional repression of both PIK3C2B and PI3KFYVE genes. Treatment of leukemia cell lines, as well as primary B-ALL cells, with different CK2 inhibitors resulted in enhanced Ikaros binding to its target genes, as evidenced by quantitative chromatin immunoprecipitation (qChIP). In summary, the presented data provide evidence that Ikaros and CK2 regulate the PI3K pathway via transcriptional regulation of the PIK3C2B and PI3KFYVE genes. Our results demonstrate that CK2 inhibition enhances Ikaros activity as a transcriptional repressor of genes that promote the PI3K pathway in primary B-ALL cells, and identify CK2 inhibitors as candidate drugs to therapeutically restore Ikaros function in B-ALL. Supported by the National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. Citation Format: Chandrika Gowda, Chunhua Song, Yali Ding, Sunil Muthusami, Xiaokang Pan, Dhimant Desai, Shantu G. Amin, Kimberly J. Payne, Sinisa Dovat. Ikaros and Casein kinase II (CK2) regulate PI3K pathway in pediatric leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2159. doi:10.1158/1538-7445.AM2015-2159
