Hiroyuki Nobusue

Poor survival of patients with osteosarcoma means that novel therapeutic targets are needed. A previously developed osteosarcoma mouse model revealed that HIF‑1 target genes are upregulated under anchorage‑independent growth conditions. HIF‑1α is highly expressed at the hypoxic invasion front in vivo. Knockout of HIF‑1α attenuates cell growth under hypoxic and non‑adherent conditions in vitro, as well as growth of primary and metastatic osteosarcoma in C57BL/6 mice, suggesting key roles for HIF‑1α in osteosarcoma progression. However, tumors with a rich vasculature develop in the absence of HIF‑1α. Thus, the HIF‑independent survival pathways on which HIF‑KO clones depend needs to be identified. The present study revealed that expression of glycolysis‑related genes, which are targets of HIF, decreased in HIF‑KO clones, but the sensitivity of each clone to inhibitors varied: Some were less sensitive than HIF wild‑type cells under hypoxic conditions. Compound screening revealed that the pathways upon which KO clones depend for survival differ. Indeed, inhibiting the mitochondrial electron transport chain, PI3K or mTOR further reduced growth of KO clones under hypoxic conditions, although one clone was less sensitive to these treatments and retained high proliferation capacity under hypoxic conditions. This clone was extremely sensitive to inhibition of the mevalonate synthesis pathway, suggesting that this might be the mechanism underlying resistance to HIF‑targeted therapies. Thus, although HIF‑1 is an attractive therapeutic target for osteosarcoma, it is necessary to identify and inhibit heterogenous HIF‑independent pathways upon which individual tumor cells rely.

The in vitro growth transformation of primary B cells by Epstein-Barr virus (EBV) is the initial step in the development of posttransplant lymphoproliferative disorder (PTLD). We performed electron microscopic analysis and immunostaining of primary B cells infected with wild-type EBV. Interestingly, the nucleolar size was increased by two days after infection. A recent study found that nucleolar hypertrophy, which is caused by the induction of the IMPDH2 gene, is required for the efficient promotion of growth in cancers. In the present study, RNA-seq revealed that the IMPDH2 gene was significantly induced by EBV and that its level peaked at day 2. Even without EBV infection, the activation of primary B cells by the CD40 ligand and interleukin-4 increased IMPDH2 expression and nucleolar hypertrophy. Using EBNA2 or LMP1 knockout viruses, we found that EBNA2 and MYC, but not LMP1, induced the IMPDH2 gene during primary infections. IMPDH2 inhibition by mycophenolic acid (MPA) blocked the growth transformation of primary B cells by EBV, leading to smaller nucleoli, nuclei, and cells. Mycophenolate mofetil (MMF), which is a prodrug of MPA that is approved for use as an immunosuppressant, was tested in a mouse xenograft model. Oral MMF significantly improved the survival of mice and reduced splenomegaly. Taken together, these results indicate that EBV induces IMPDH2 expression through EBNA2-dependent and MYC-dependent mechanisms, leading to the hypertrophy of the nucleoli, nuclei, and cells as well as efficient cell proliferation. Our results provide basic evidence that IMPDH2 induction and nucleolar enlargement are crucial for B cell transformation by EBV. In addition, the use of MMF suppresses PTLD. IMPORTANCE EBV infections cause nucleolar enlargement via the induction of IMPDH2, which are essential for B cell growth transformation by EBV. Although the significance of IMPDH2 induction and nuclear hypertrophy in the tumorigenesis of glioblastoma has been reported, EBV infection brings about the change quickly by using its transcriptional cofactor, EBNA2, and MYC. Moreover, we present here, for the novel, basic evidence that an IMPDH2 inhibitor, namely, MPA or MMF, can be used for EBV-positive posttransplant lymphoproliferative disorder (PTLD).

Novel therapeutic targets are needed to better treat osteosarcoma, which is the most common bone malignancy. We previously developed mouse osteosarcoma cells, designated AX (accelerated bone formation) cells from bone marrow stromal cells. AX cells harbor both wild-type and mutant forms of p53 (R270C in the DNA-binding domain, which is equivalent to human R273C). In this study, we showed that mutant p53 did not suppress the transcriptional activation function of wild-type p53 in AX cells. Notably, AXT cells, which are cells derived from tumors originating from AX cells, lost wild-type p53 expression, were devoid of the intact transcription activation function, and were resistant to doxorubicin. ChIP-seq analyses revealed that this mutant form of p53 bound to chromatin in the vicinity of the transcription start sites of various genes but exhibited a different binding profile from wild-type p53. The knockout of mutant p53 in AX and AXT cells by CRISPR-Cas9 attenuated tumor growth but did not affect the invasion of these cells. In addition, depletion of mutant p53 did not prevent metastasis in vivo. Therefore, the therapeutic potency targeting R270C (equivalent to human R273C) mutant p53 is limited in osteosarcoma. However, considering the heterogeneous nature of osteosarcoma, it is important to further evaluate the biological and clinical significance of mutant p53 in various cases.