YBX1 is required for maintaining myeloid leukemia cell survival by regulating BCL2 stability in an m6A-dependent manner.

RNA-binding proteins (RBPs) are critical regulators of transcription and translation that are often dysregulated in cancer. Although RBPs are increasingly recognized as being important for normal hematopoiesis and for hematologic malignancies as oncogenes or tumor suppressors, RBPs that are essential for the maintenance and survival of leukemia remain elusive. Here we show that YBX1 is specifically required for maintaining myeloid leukemia cell survival in an N6-methyladenosine (m6A)-dependent manner. We found that expression of YBX1 is significantly upregulated in myeloid leukemia cells, and deletion of YBX1 dramatically induces apoptosis and promotes differentiation coupled with reduced proliferation and impaired leukemic capacity of primary human and mouse acute myeloid leukemia cells in vitro and in vivo. Loss of YBX1 has no obvious effect on normal hematopoiesis. Mechanistically, YBX1 interacts with insulin-like growth factor 2 messenger RNA (mRNA)-binding proteins (IGF2BPs) and stabilizes m6A-tagged RNA. Moreover, YBX1 deficiency dysregulates the expression of apoptosis-related genes and promotes mRNA decay of MYC and BCL2 in an m6A-dependent manner, which contributes to the defective survival that results from deletion of YBX1. Thus, our findings have uncovered a selective and critical role of YBX1 in maintaining myeloid leukemia survival, which might provide a rationale for the therapeutic targeting of YBX1 in myeloid leukemia.

Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells.

BACKGROUND: Cancer/testis antigen MAGEC2 (also known as HCA587) is highly expressed in a wide variety of tumors and plays an active role in promoting growth and metastasis of tumor cells. However, little is known for the regulation of MAGEC2 expression in cancer cells. METHODS: Western blotting and quantitative RT-PCR were performed to analyze MAGEC2 expression. Co-immunoprecipitation assay was applied for detecting the endogenous interaction of MAGEC2 and TRIM28 in tumor cells. Overexpression and knockdown assays were used to examine the effects of TRIM28 on the expression of MAGEC2 protein. Immunohistochemistry (IHC) staining was performed in hepatocellular carcinoma patients to evaluate the association between the expression of MAGEC2 and TRIM28. Proteasome inhibitors MG132 or PS-341 and lysosome inhibitor Chloroquine (CQ) were used to inhibit proteasomal or lysosomal-mediated protein degradation respectively. RESULTS: We demonstrate that MAGEC2 interacts with TRIM28 in melanoma cells and MAGEC2 expression in tumor cells depends on the expression of TRIM28. The expression level of MAGEC2 protein was significantly reduced when TRIM28 was depleted in tumor cells, and no changes were observed in MAGEC2 mRNA level. Furthermore, expression levels of MAGEC2 and TRIM28 are positively correlated in MAGEC2-positive human hepatocellular carcinoma tissues (p = 0.0011). Mechanistic studies indicate that the regulatory role of TRIM28 on MAGEC2 protein expression in tumor cells depends on proteasome-mediated pathway. CONCLUSIONS: Our findings show that TRIM28 is necessary for MAGEC2 expression in cancer cells, and TRIM28 may serve as a new potential target for immunotherapy of cancer.

Fabrication of a high-accuracy phase-type computer-generated hologram by physical vapor deposition.

With the development of optical systems used in astronomical and earth observation, aspherical and free-form surfaces are increasingly used because they are lightweight and have improved image quality. As a highly accurate, aberrationless technique, computer-generated hologram (CGH) plays an important role in wavefront testing. At present, the main way to fabricate phase CGH is reactive ion etching, which suffers from low accuracy. To improve the accuracy, physical vapor deposition (PVD) is applied in the fabrication of phase CGH. The wavefront errors of PVD-fabricated phase CGH were analyzed. Testing results indicate that the wavefront error of the CGH is 0.020λ root mean square (RMS), mainly caused by the machine tool orthogonality error rather than the PVD process. The diffraction efficiency of the +1st order is 22.4%.

Establishment of MAGEC2-knockout cells and functional investigation of MAGEC2 in tumor cells.

Cancer/testis antigen MAGEC2, a member of the type I melanoma-associated antigen family, is expressed in a wide variety of cancer types but not in normal somatic cells. MAGEC2 has long been recognized as a tumor-specific target, however, its functions remain largely unknown. In this study, we established MAGEC2-knockout A375 melanoma cell lines using the CRISPR/Cas9 system. Seven clonal cell lines were generated by using four single guide RNAs targeting the coding region of the MAGEC2 gene, which produced indels that abolished MAGEC2 protein expression. To identify the differentially expressed protein profiles associated with MAGEC2 loss, isobaric tag for relative quantitation-based comparative proteomics experiments were carried out on the MAGEC2-knockcout and control A375 cells. Mining of the proteomics data identified a total 224 (61.6% upregulated and 38.4% downregulated) proteins to be significantly altered in expression level in MAGEC2-knockcout cells. Ingenuity Pathway Analysis indicated that the significantly altered proteins were involved in critical neoplasia-related biological functions such as cell death, proliferation, and movement. Gene ontology analysis identified "apoptosis signaling" as the top-most upregulated pathway associated with MAGEC2 loss. We showed that knockout or knockdown of the MAGEC2 gene sensitized melanoma cells to tumor necrosis factor-α-induced apoptosis. Interestingly, actin-based motility by Rho and RhoA signaling, known to promote cell migration, were also identified as the top downregulated pathways in MAGEC2-knockout A375 cells. In short, our study provides a suitable cell model for exploring the biological functions of MAGEC2 in malignant cells, and sheds light on the molecular pathway by which MAGEC2 promotes tumor development.

DNA 6mA demethylase ALKBH1 regulates DDX18 expression to promote proliferation of human head and neck squamous cell carcinoma.

PURPOSE: Human head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Currently, surgical resection plus a combination of chemotherapy and radiotherapy is the standard treatment for HNSCC, and the 5-year survival rate of patients with HNSCC remains very low because of the higher incidence of metastasis with consequent recurrence. Here, we aimed to investigate the potential role of DNA N6-methyladenine (6mA) demethylase ALKBH1 in tumor cell proliferation in HNSCC. METHODS: The expression of ALKBH1 in 10 pairs of HNSCC/normal tissues and 3 HNSCC cell lines were measured by qRT‒PCR and western blotting. Colony formation, flow cytometry, patient-derived HNSCC organoid assays were used to assess the role of ALKBH1 in HNSCC cell proliferation in cell lines and human HNSCC patients. MeDIP-seq, RNA sequencing, Dot blotting and western blotting were used to evaluate the regulatory effect of ALKBH1 on the expression of DEAD-box RNA helicase DDX18. A dual-luciferase reporter assay was used to assess the putative effect of DNA 6mA levels on DDX18 transcription. RESULTS: ALKBH1 was highly expressed in HNSCC cells and patient tissues. Functional experiments revealed that ALKBH1 knockdown in SCC9, SCC25, and CAL27 cells inhibited their proliferation in vitro. Using patient-derived HNSCC organoid assay, we found that knockdown of ALKBH1 inhibited the proliferation and colony formation of HNSCC patients-derived organoids. Moreover, we found that ALKBH1 can enhance DDX18 expression by erasing DNA 6mA level and regulating its promoter activity. ALKBH1 deficiency blocked tumor cell proliferation by inhibiting DDX18 expression. Exogenous overexpression of DDX18 rescued the cell proliferation arrest caused by ALKBH1 knockdown. CONCLUSION: Our data reveal the important role of ALKBH1 in regulating proliferation of HNSCC.

Decoding m6A RNA methylome identifies PRMT6-regulated lipid transport promoting AML stem cell maintenance.

N6-methyladenosine (m6A) is a common chemical modification for mammalian mRNA and exhibits high dynamics in various biological processes. However, dynamics of m6A RNA methylome during leukemogenesis remains unknown. Here, we delineate a comprehensive m6A landscape during acute myeloid leukemia (AML) development and identify PRMT6 as a key for maintaining AML stem cells. We observe an obvious change in m6A methylome during leukemogenesis and find that protein arginine methyltransferase PRMT6 and m6A reader IGF2BP2 maintain the function of human and murine leukemia stem cells (LSCs). Genetic deletion or pharmacological inhibition of PRMT6 damages AML development and LSC function. Mechanistically, IGF2BP2 stabilizes PRMT6 mRNA via m6A-mediated manner, which catalyzes H3R2me2a and suppresses lipid transporter MFSD2A expression. PRMT6 loss upregulates MFSD2A expression that increases docosahexaenoic acid levels and impairs LSC maintenance. Collectively, our findings reveal a critical role of PRMT6-MFSD2A signaling axis in AML development and provide a therapeutic strategy for targeting LSCs.

PTIP governs NAD+ metabolism by regulating CD38 expression to drive macrophage inflammation.

NAD+ metabolism is involved in many biological processes. However, the underlying mechanism of how NAD+ metabolism is regulated remains elusive. Here, we find that PTIP governs NAD+ metabolism in macrophages by regulating CD38 expression and is required for macrophage inflammation. Through integrating histone modifications with NAD+ metabolic gene expression profiling, we identify PTIP as a key factor in regulating CD38 expression, the primary NAD+-consuming enzyme in macrophages. Interestingly, we find that PTIP deletion impairs the proinflammatory response of primary murine and human macrophages, promotes their metabolic switch from glycolysis to oxidative phosphorylation, and alters NAD+ metabolism via downregulating CD38 expression. Mechanistically, an intronic enhancer of CD38 is identified. PTIP regulates CD38 expression by cooperating with acetyltransferase p300 in establishing the CD38 active enhancer with enriched H3K27ac. Overall, our findings reveal a critical role for PTIP in fine-tuning the inflammatory responses of macrophages via regulating NAD+ metabolism.

Differential m6A RNA landscapes across hematopoiesis reveal a role for IGF2BP2 in preserving hematopoietic stem cell function.

N6-methyladenosine (m6A) on mRNA plays critical roles in various cellular processes. However, the landscape and dynamics of m6A modification in hematopoietic system remain unknown. Here, we delineate a comprehensive m6A landscape across hematopoietic hierarchy and uncover that IGF2BP2 is required for preserving the function of hematopoietic stem cells (HSCs). Our data reveal a cell-type-specific m6A landscape in hematopoiesis. m6A modifications arise mostly in the early stage of hematopoiesis and prefer to play distinct roles for determining mRNA fates in HSCs and committed progenitors. Mechanistically, increased m6A-IGF2BP2 expression controls transcriptional state and maintenance of HSCs. IGF2BP2 deficiency induces quiescence loss and impairs HSC function. Moreover, IGF2BP2 loss increases mitochondrial activity of HSCs by accelerating Bmi1 mRNA decay, leading to de-repression of mitochondria-related genes. Collectively, our results present a fascinating portrait of m6A modification of hematopoietic hierarchy and reveal a key role of IGF2BP2 in maintaining HSC function by restraining mitochondrial activity.

Leukemogenic Chromatin Alterations Promote AML Leukemia Stem Cells via a KDM4C-ALKBH5-AXL Signaling Axis.

N6-methyladenosine (m6A) is a commonly present modification of mammalian mRNAs and plays key roles in various cellular processes. m6A modifiers catalyze this reversible modification. However, the underlying mechanisms by which these m6A modifiers are regulated remain elusive. Here we show that expression of m6A demethylase ALKBH5 is regulated by chromatin state alteration during leukemogenesis of human acute myeloid leukemia (AML), and ALKBH5 is required for maintaining leukemia stem cell (LSC) function but is dispensable for normal hematopoiesis. Mechanistically, KDM4C regulates ALKBH5 expression via increasing chromatin accessibility of ALKBH5 locus, by reducing H3K9me3 levels and promoting recruitment of MYB and Pol II. Moreover, ALKBH5 affects mRNA stability of receptor tyrosine kinase AXL in an m6A-dependent way. Thus, our findings link chromatin state dynamics with expression regulation of m6A modifiers and uncover a selective and critical role of ALKBH5 in AML that might act as a therapeutic target of specific targeting LSCs.

Cancer-testis antigen MAGE-C2 binds Rbx1 and inhibits ubiquitin ligase-mediated turnover of cyclin E.

Cancer-testis antigen MAGE-C2 is normally expressed in testis but aberrantly expressed in various kinds of tumors. Its functions in tumor cells are mostly unknown. Here, we show that MAGE-C2 binds directly to the RING domain protein Rbx1, and participates in Skp1-Cullin1-F box protein (SCF) complex. Furthermore, MAGE-C2 can inhibit the E3 ubiquitin ligase activity of SCF complex. Ablation of endogenous MAGE-C2 decreases the level of cyclin E and accelerates cyclin E turnover by inhibiting ubiquitin-mediated proteasome degradation. Overexpression of MAGE-C2 increases the level of cyclin E and promotes G1-S transition and cell proliferation, and the results are further confirmed by knockdown of MAGE-C2. Overall, the study indicates that MAGE-C2 is involved in SCF complex and increases the stability of cyclin E in tumor cells.