The IRF2/CENP-N/AKT signaling axis promotes proliferation, cell cycling and apoptosis resistance in nasopharyngeal carcinoma cells by increasing aerobic glycolysis

Background Centromere protein N (CENP-N) has been reported to be highly expressed in malignancies, but its role and mechanism in nasopharyngeal carcinoma (NPC) are unknown. Methods Abnormal CENP-N expression from NPC microarrays of GEO database was analyzed. CENP-N expression level was confirmed in NPC tissues and cell lines. Stable CENP-N knockdown and overexpression NPC cell lines were established, and transcriptome sequencing after CENP-N knockdown was performed. In vitro and in vivo experiments were performed to test the impact of CENP-N knockdown in NPC cells. ChIP and dual luciferase reporter assays were used to verify the combination of IRF2 and CENP-N. Western blot analysis, cellular immunofluorescence, immunoprecipitation and GST pulldown assays were used to verify the combination of CENP-N and AKT. Results CENP-N was confirmed to be aberrantly highly expressed in NPC tissues and cell lines and to be associated with high 18F-FDG uptake in cancer nests and poor patient prognosis. Transcriptome sequencing after CENP-N knockdown revealed that genes with altered expression were enriched in pathways related to glucose metabolism, cell cycle regulation. CENP-N knockdown inhibited glucose metabolism, cell proliferation, cell cycling and promoted apoptosis. IRF2 is a transcription factor for CENP-N and directly promotes CENP-N expression in NPC cells. CENP-N affects the glucose metabolism, proliferation, cell cycling and apoptosis of NPC cells in vitro and in vivo through the AKT pathway. CENP-N formed a complex with AKT in NPC cells. Both an AKT inhibitor (MK-2206) and a LDHA inhibitor (GSK2837808A) blocked the effect of CENP-N overexpression on NPC cells by promoting aerobic glycolysis, proliferation, cell cycling and apoptosis resistance. Conclusions The IRF2/CENP-N/AKT axis promotes malignant biological behaviors in NPC cells by increasing aerobic glycolysis, and the IRF2/CENP-N/AKT signaling axis is expected to be a new target for NPC therapy.

The Tetraspanin CD53 Protects Hematopoietic Stem Cells Following Cellular Stress through Induction of DREAM Complex-Mediated Quiescence

Maintenance of quiescence is necessary for optimal hematopoietic stem cell (HSC) function. The absence of fine-tuned cycling regulation of HSCs can result in impaired hematopoiesis, bone marrow (BM) failure, or malignant transformation. While various factors, including inflammatory cytokines and chemotherapy, have been identified to induce HSC cycling, the mechanisms regulating HSC return to quiescence are unclear. Herein, we profiled HSCs in response to a variety of inflammatory stressors (cytokines, toll-like receptor agonists, mobilizing agents, and chemotherapeutics) and found markedly upregulated expression of CD53 in both mouse and human HSCs, in some cases by as much as 70-fold higher expression over vehicle-treated controls. CD53 is a tetraspanin, a type of transmembrane protein involved in plasma membrane organization and regulation of processes such as cellular migration, adhesion, and signaling. CD53 has been shown to be asymmetrically segregated in HSCs, with CD53-enriched HSCs believed to be more stem-like; however, there is no current proposed mechanism to explain the association between CD53 and stem cell quality. To understand the role of CD53 in HSC quality, we generated a CD53 knockout mouse, and profiled HSC phenotype and function. Under homeostatic conditions, Cd53 -/- HSC number and frequency are normal as compared to wild type (WT) mice. However, we found Cd53 -/-HSCs to have significantly impaired function, particularly in response to inflammatory stimuli. Cd53 -/- BM failed to engraft as well as WT BM (45% chimerism vs 63%, p<0.05) in competitive transplants, and this deficit was exacerbated when G-CSF-mobilized HSCs from the spleen were transplanted (47% vs 77%, p<0.0001). Analysis of cycling status during and after G-CSF stimulation found that Cd53 -/- HSCs, particularly those that mobilize to the spleen, undergo cell division twice as frequently as WT HSCs (22% vs 10%, p<0.05). Treatment of WT and Cd53 -/- mice with serial doses of the chemotherapeutic 5-fluorouracil resulted in significantly accelerated death in Cd53 -/- mice (median survival 14 days vs 28 days, p<0.0001), supporting that loss of CD53 is associated with dysregulated HSC quiescence. Transcriptomic sequencing revealed significant upregulation of genes associated with cell cycling and division in G-CSF-treated Cd53 -/- HSCs compared to WT controls. Notably, these differentially expressed genes are targets of the dimerization partner, RB-like, E2F and multi-vulval class B (DREAM) complex, a newly described transcriptional regulator that represses cell cycling-associated genes, suggesting that CD53 promotes DREAM-mediated quiescence in stressed HSCs. We performed CUT&Tag profiling of Rbl2/p130, a DREAM complex subunit, in HSCs and found that loss of CD53 was associated with decreased complex binding (consistent with enhanced transcription of cell cycle genes). Proximity labeling studies demonstrated that CD53 interacts with cell cycle machinery proteins, including Rbl2/p130, and Western blots of sorted HSCs from Cd53 -/- and WT mice treated with G-CSF showed decreased expression of DREAM complex components (Rbl1/p107 and Rbl2/p130) in the absence of CD53. Finally, enforced activation of DREAM and HSC quiescence using the CDK4/6 inhibitor palbociclib rescued the Cd53 -/-HSC repopulating defect. Together, these data derive a novel mechanism whereby CD53 regulates HSC quiescence through regulating DREAM complex binding during stress-induced cycling. Disclosures No relevant conflicts of interest to declare.

Targeting Control of Cell Cycle Enhances the Activity of Conventional Chemotherapy in Chemotherapy-Resistant Acute Myeloid Leukemia

Chemotherapy-resistant acute myeloid leukemia (AML) manifesting as primary refractory or relapsed disease carries a dismal prognosis and is often driven by clonal evolution. We have performed a genome-wide CRISPR knockout screen investigating resistance to conventional AML therapy, cytarabine and doxorubicin (AraC/Dox), in 2 independent human AML cell lines. Chemoresistant populations were enriched with gRNAs disrupting cell cycle arrest genes, including cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53. Here, we validate the direct contribution of these genes to chemoresistance and demonstrate that rationally designed therapeutic regimens targeting cell cycle enhances chemotherapy response in AML. Using CRISPR-mediated gene editing, we individually inactivated CDKN2A and CHEK2 in Cas9-expressing OCI-AML3 and MV4-11 cells. CDKN2A- and CHEK2-deficient cells demonstrated proliferative advantage in the presence of AraC/Dox in co-culture competition assays, confirming direct contribution of these gene knockouts to chemoresistance. Nil to modest reductions in apoptosis were seen in CDKN2A- and CHEK2-deficient cells treated with AraC/Dox for 72 hours compared to unedited controls. However, failure of accumulation of cells in the non-cycling G 0/G 1 proportion was seen in these edited cells after chemotherapy, corresponding with maintained DNA synthesis, as measured by BrdU incorporation, and a failure to downregulate phospho-Rb protein expression, indicating ongoing active cell cycling in spite of chemotherapy. These results confirm failure of cell cycle arrest as the major mechanism of resistance with inactivation of CDKN2A or CHEK2. To assess relevance of these genes in chemotherapy response in human AML, we analysed the effect of CDKN2A expression on prognosis. Reduced expression of CDKN2A conferred inferior overall survival in 3 independent clinical cohorts. Additionally, downregulation of CDKN2A and an increase in downstream cell cycling effector, cyclin-dependent kinase 6 (CDK6) was seen at relapse in paired diagnosis-relapse AML samples (Li et al. 2016, Nature Medicine). Further, CHEK2 mutations in clonal hematopoiesis are enriched in patients with solid organ cancers following chemo- or radiotherapy, functionally demonstrating chemoresistance over non-mutated cells. These data suggest CDKN2A and CHEK2 loss-of-function is relevant in promoting chemoresistance in human hematopoiesis. We therefore investigated whether therapeutically targeting cell cycle control pathways that converge on the G 1/S restriction point could synergise with cytotoxic chemotherapy, potentially circumventing chemoresistance. The addition of MDM2 inhibitor, nutlin-3a with AraC/Dox achieved striking synergism in TP53-competent cell lines in reducing viability and promoting apoptosis. Inhibition of CDK4/6 with palbociclib alone induced cell cycle arrest without apoptosis, however combination therapy with AraC/Dox significantly potentiated apoptosis. To functionally validate the role of CDKN2A itself, we examined the inhibitor of histone acetyltransferase KAT6A, WM-1119 (Baell et al. 2018, Nature), which transcriptionally upregulates CDKN2A. WM-1119 had anti-leukemic activity across multiple cell lines, corresponding with down regulation of cell cycling and upregulation of senescence signatures. The anti-leukemic activity was prevented by CDKN2A inactivation, indicating that CDKN2A upregulation is essential for WM-1119's anti-leukemic effect. WM-1119 enhanced activity of AraC/Dox in multiple cell contexts, mediated by cooperative induction of CDKN2A. Its efficacy and synergy with AraC/Dox was further demonstrated in MLL-AF9 primary murine AML ex vivo, demonstrating broader applicability of the approach. Our findings reveal defects in cell cycle arrest pathways as clinically relevant contributors to chemoresistance in AML. Combining agents to target cell cycle components improved in vitro anti-leukemic activity of chemotherapy and warrants further exploration of their translational potential. Disclosures Bullinger: Amgen: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Menarini: Consultancy; Hexal: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Consultancy, Honoraria; Astellas: Honoraria; Gilead: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Seattle Genetics: Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Honoraria; Abbvie: Consultancy, Honoraria. Lane: BMS: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Novartis: Consultancy; Geron: Consultancy.

Phospholipases D1 and D2 regulate cell cycling in primary prostate cancer cells and are differentially associated with the nuclear matrix

Phospholipases D1 and D2 (PLD1/2) have been implicated in tumorigenesis. We previously detected higher expression of PLD in the nuclei of patient-derived prostate cancer (PCa) cells and prostate cancer cell lines. Here we have examined whether PLD1 or PLD2 are associated with the nuclear matrix and influence cell cycling. PLD1/PLD2 were detected by qualitative immunofluorescence in cultured PCa cells and extracted with a standardised protocol to reveal nuclear matrix-associated proteins. The effects of isoform-specific inhibition of PLD1or PLD2 on PCa cell cycle progression were analysed by flow cytometry. PLD2 mainly co-localised with the nucleolar marker fibrillarin in PCa cells. However, even after complete extraction, some PLD2 remained associated with the nuclear matrix. Inhibiting PLD2 effectively reduced PCa cell cycling into and through S phase. In contrast, PLD1 inhibition effects were weaker, and a subpopulation of cycling patient-derived PCa cells was unaffected by PLD1 inhibition. When associated with the nuclear matrix PLD2 could generate phosphatidic acid to regulate nuclear mTOR and control downstream transcriptional events. The association of PLD2 with the nucleolus also implies a role in stress regulation. The cell cycling results highlight the importance of PLD2 inhibition as a novel potential prostate cancer therapeutic mechanism by differential regulation of cell proliferation.

A Novel Monoclonal Antibody to PL2L60 is Effective For Therapy of Various Types of Cancers in Human and Mice

Background: PL2L60 is a PIWIL2-like (PL2L) protein that is specifically and widely expressed in various types of hematopoietic and solid tumors. However, it is unknown whether PL2L60 is an effective target for cancer immunotherapy. The current study aimed to investigate the efficacy of an monoclonal antibody (mAb) to PL2L60 (clone KAO3；IgM isotype) in treatment of the cancers either from human or mouse.Methods: The expression of PL2L60 protein in the cell surface and cytoplasm were determined in a panel of human and mouse tumor cell lines by flow cytometry, immunofluorescent staining and Western Blotting. The apoptosis and the cell cycle arrest of the tumor cells treated with mAb KAO3 were evaluated by flow cytometry. The tumorigenesis of the mAb KAO3-pretreated tumor cells was determined by tumor incidence and tumor size, and efficacy of mAb KAO3 treatment on tumor growth in tumors-bearing mice were kinetically evaluated. Complement-dependent cytotoxicity tests were utilized to determine the mechanism of mAb KAO3 killing tumor cells. Results: Treatment of human or mouse tumor cells with mAb KAO3 at the time of inoculation efficiently inhibited their tumorigenesis in the immunodeficient mice. Moreover, injection of mAb KAO3 into established tumors significantly inhibited their growth, and prolonged survival of the tumor-bearing mice, including lymphoma, breast cancer, lung cancer and cervical cancer. The inhibitory effects of mAb KAO3 were likely associated with its binding to the PL2L60 expressed on tumor cell surface, which may induce cell apoptosis through either activation of complement or blocking cell cycling. The cell cycling was arrested at G2/M phase and DNA synthesis may be inhibited.Conclusion: We have identified the PL2L60 as a novel tumor-specific and broad-spectral biomarker, which is recognized by the mAb KAO3 as an efficient target for immunotherapy of both solid and hematopoietic cancers.