Validation of Candidate Drivers of Osteosarcomagenesis with High-Throughput In Vitro Screening

Faculty advisor: David LargaespadaThis research was supported by the Undergraduate Research Opportunities Program (UROP)

Minichromosome Maintenance 2 Bound with Retroviral Gp70 Is Localized to Cytoplasm and Enhances DNA-Damage-Induced Apoptosis

The interaction of viral proteins with host-cellular proteins elicits the activation of cellular signal transduction pathways and possibly leads to viral pathogenesis as well as cellular biological events. Apoptotic signals induced by DNA-damage are remarkably up-regulated by Friend leukemia virus (FLV) exclusively in C3H hosts; however, the mechanisms underlying the apoptosis enhancement and host-specificity are unknown. Here, we show that C3H mouse-derived hematopoietic cells originally express higher levels of the minichromosome maintenance (MCM) 2 protein than BALB/c- or C57BL/6-deriverd cells, and undergo more frequent apoptosis following doxorubicin-induced DNA-damage in the presence of the FLV envelope protein gp70. Dual transfection with gp70/Mcm2 reproduced doxorubicin-induced apoptosis even in BALB/c-derived 3T3 cells. Immunoprecipitation assays using various deletion mutants of MCM2 revealed that gp70 bound to the nuclear localization signal (NLS) 1 (amino acids 18–24) of MCM2, interfered with the function of NLS2 (amino acids 132–152), and suppressed the normal nuclear-import of MCM2. Cytoplasmic MCM2 reduced the activity of protein phosphatase 2A (PP2A) leading to the subsequent hyperphosphorylation of DNA-dependent protein kinase (DNA-PK). Phosphorylated DNA-PK exhibited elevated kinase activity to phosphorylate P53, thereby up-regulating p53-dependent apoptosis. An apoptosis-enhancing domain was identified in the C-terminal portion (amino acids 703–904) of MCM2. Furthermore, simultaneous treatment with FLV and doxorubicin extended the survival of SCID mice bearing 8047 leukemia cells expressing high levels of MCM2. Thus, depending on its subcellular localization, MCM2 plays different roles. It participates in DNA replication in the nucleus as shown previously, and enhances apoptosis in the cytoplasm

CRISPR screening identifies M1AP as a new MYC regulator with a promoter-reporter system

Background MYC is one of the proto-oncogenes contributing to tumorigenesis in many human cancers. Although the mechanism of MYC regulation is still not fully understood, learning about the comprehensive mechanism controlling the transcriptional activity of MYC will lead to therapeutic targets. The CRISPR/Cas9 library system is a simple and powerful screening technique. This study aims to identify new transcriptional upstream activators of MYC using the CRISPR activation library with new promoter-reporter systems. Methods and Results The MYC promoter-reporter system was developed with a photoconvertible fluorescent protein, Dendra2, and named “pMYC-promoter-Dendra2.” This MYC promoter-reporter system was designed to harbor a proximal MYC promoter at (3.1 kb). Both the CRISPR activation library and pMYC-promoter-Dendra2 were induced to HEK 293T cells, and Dendra2-positive cells, that are supposed that MYC should be upregulated, were collected individually by a cell sorter. Among the 169 cells collected, 12 clones were successfully established. Then, pMYC-promoter-Dendra2 was transfected again into these 12 clones, and two of 12 clones showed Dendra2 positivity. In this procedure, the cells with non-specific autofluorescence were correctly distinguished by utilizing the photoswitchable character of Dendra2. Using extracted genomic DNA of these two Dendra2 positive clones, polymerase chain reaction (PCR) was performed to amplify the guide RNA (gRNA) containing region, which was introduced by the CRISPR activation library. Eventually, PLEKHO2, MICU, MBTPS1, and M1AP were identified, and these gRNAs were transfected individually into HEK 293T cells again using the CRISPR activation system. Only M1AP gRNA transfected cells showed Dendra2-positive fluorescence. Then, the overexpression vector for M1AP with a doxycycline-inducible vector confirmed that M1AP induced high MYC expression by real-time quantitative PCR and western blot. Furthermore, the dual-luciferase assay showed a significant increase of promoter activity, and MYC mRNA was higher in M1AP- overexpressing cells. M1AP is highly expressed in several cancers, though, a positive correlation between M1AP and MYC was observed only in human acute myeloid leukemia. Conclusion The present study confirmed that the experimental method using the CRISPR library technology functions effectively for the identification of molecules that activate endogenous MYC. This method will help elucidate the regulatory mechanism of MYC expression, as well as supporting further drug research against malignant tumors

Identification of the Factor That Leads Human Mesenchymal Stem Cell Lines into Decellularized Bone

Hematopoiesis is maintained by the interaction of hematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (MSCs) in bone marrow microenvironments, called niches. Certain genetic mutations in MSCs, not HSCs, provoke some hematopoietic neoplasms, such as myelodysplastic syndrome. An in vivo bone marrow niche model using human MSC cell lines with specific genetic mutations and bone scaffolds is necessary to elucidate these interactions and the disease onset. We focused on decellularized bone (DCB) as a useful bone scaffold and attempted to induce human MSCs (UE7T-9 cells) into the DCB. Using the CRISPR activation library, we identified SHC4 upregulation as a candidate factor, with the SHC4 overexpression in UE7T-9 cells activating their migratory ability and upregulating genes to promote hematopoietic cell migration. This is the first study to apply the CRISPR library to engraft cells into decellularized biomaterials. SHC4 overexpression is essential for engrafting UE7T-9 cells into DCB, and it might be the first step toward creating an in vivo human–mouse hybrid bone marrow niche model

Efficient Identification of the MYC Regulator with the Use of the CRISPR Library and Context-Matched Database Screenings

MYC is a major oncogene that plays an important role in cell proliferation in human cancers. Therefore, the mechanism behind MYC regulation is a viable therapeutic target for the treatment of cancer. Comprehensive and efficient screening of MYC regulators is needed, and we had previously established a promoter screening system using fluorescent proteins and the CRISPR library. For the efficient identification of candidate genes, a database was used, for which mRNA expression was correlated with MYC using datasets featuring “Similar” and “Not exactly similar” contexts. INTS14 and ERI2 were identified using datasets featuring the “Similar” context group, and INTS14 and ERI2 were capable of enhancing MYC promoter activity. In further database analysis of human cancers, a higher expression of MYC mRNA was observed in the INTS14 mRNA high-expressing prostate and liver cancers. The knockdown of INTS14 in prostate cell lines resulted in decreased MYC mRNA and protein expression and also induced G0/1 arrest. This study confirmed that CRISPR screening combined with context-matched database screening is effective in identifying genes that regulate the MYC promoter. This method can be applied to other genes and is expected to be useful in identifying the regulators of other proto-oncogenes

Schematic illustration of the structure of MCM2 and its functions in the cytoplasm and nucleus.

<p>(<b>A</b>) The various functional domains of the MCM2 protein are shown, and the domains and regions required for the activities are indicated. (<b>B</b>) Schematic of the novel role of MCM2 in apoptosis enhancement. Normally, MCM2 is recruited into the nucleus for participation in DNA replication. As a result, cellular proliferation is upregulated (proliferation signal). However, when gp70 is present in the cytoplasm, it binds to MCM2 and inhibits its nuclear entry. Furthermore, cytoplasmic gp70-MCM2-complex interacts with PP2A and inhibits its interaction with DNA-PK. Consequently, hyperphosphorylated DNA-PK enhances DNA-damage-induced apoptosis via a P53-related pathway (apoptosis signal).</p

Subcellular localization of MCM2 and the role of the NLS domains in enhancing doxorubicin-induced apoptosis.

<p><i>HA-Mcm2-FL</i> and <i>HA-</i>mutant-transfected 3T3 cells (<b>A</b>), and <i>FLAG-gp70</i>/<i>HA-Mcm2-FL</i> and <i>FLAG-gp70</i>/<i>HA</i>-mutant-transfected 3T3 cells (<b>B</b>) were treated with 1 µM doxorubicin for 24 h. HA-positive cells containing the MCM2-derived proteins are shown in red (TRITC), and DAPI-stained nuclei are shown in blue. Images were acquired using a BZ-9000 microscope (KEYENCE) with a 400× objective. (<b>C</b>) Schematic diagram of the NLS deletion mutants MCM2-ΔNLS1, MCM2-ΔNLS2, and MCM2-ΔNLS1-2. (<b>D</b>) <i>Mcm2-NLS</i> deletion mutant-transfected 3T3 cells were treated with 1 µM doxorubicin for 24 h, and apoptotic cell ratios were determined with annexin V-staining. Data represent the mean and SD of 3 experiments. The asterisks (*) indicate significant differences between the control and <i>Mcm2-ΔNLS2</i>- or <i>Mcm2-ΔNLS 1-2</i>-transfected cells (*<i>p</i><0.01). Data represent the mean and 95% CI of 3 independent experiments.</p

<i>In vivo</i> anti-tumor effects of <i>gp70</i> expression and DNA-damage on the C3H-derived cells in SCID mice.

<p>Two weeks after transplantation, mice were inoculated (i.p.) with FLV. Seven days later, the mice were treated with 1.5 mg/kg of doxorubicin or PBS. (<b>A</b>) Quantitative RT-PCR analysis of <i>gp70</i> mRNA expression in the liver of SCID mice with multiple foci of leukemic infiltration. The samples from FLV-infected mice exhibit higher levels of <i>gp70</i> than those from uninfected mice (*<i>p</i><0.01). Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (<b>B</b>) Microscopic features of TUNEL-positive cells in hepatic nodules and (<b>C</b>) TUNEL-positive cell ratio in each group of mice. Note the significant increase in apoptotic 8047 cells in mice with FLV infection and doxorubicin treatment (*<i>p</i><0.01 compared with the tumor cells of “FLV (−), doxorubicin (−) mice”). Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (<b>D</b>) Subcellular localization of MCM2 in 8047 cells of the liver demonstrated by immunohistochemistry. Images were captured with a microscope at 1,000× magnification power. Note the nuclear and/or cytoplasmic localization of MCM2 in the 8047 cells from each group of mice. (<b>E</b>) The cell counts for cytoplasmic localization of MCM2. Cell counts are shown as the number of cells per 10 high-power fields (HPF). [# <i>p</i><0.01 compared with tumor cells of “FLV (−), doxorubicin (−)” mice; *<i>p</i><0.001 compared with “FLV (−) doxorubicin (−)” mice and <i>p</i><0.05 compared with “FLV (+), doxorubicin (−)” mice]. Data represent the mean and 95% CI of from 10 mice in each group and are representative of 2 independent experiments. (<b>F</b>) Kaplan-Meier survival curves for 8047-transplanted SCID mice with/without FLV-infection and doxorubicin-treatment. Note the significant elongation of survival time in mice with FLV-infection [<i>p</i><0.01 compared with “FLV (−), doxorubicin (−)” and “FLV (−), doxorubicin (+)” mice] and in mice with FLV-infection and doxorubicin-treatment [<i>p</i><0.001 compared with “FLV (−), doxorubicin (−)” and “FLV (−), doxorubicin (+)” mice, <i>p</i><0.01 compared with “FLV (+), doxorubicin (−)” mice]. The survival curves represent data from 10 mice in each group.</p

Direct interaction of MCM2 with gp70.

<p>(<b>A</b>) Schematic diagram of full-length MCM2 (MCM2-FL) and MCM2 deletion mutants, MCM2-ΔC (aa 1–703), MCM2-ΔN (aa 156–703), MCM2-N (aa 1–155) and MCM2-C (aa 704–904). The NLS domains are shown in black, and the Zn-finger domains are gray. 3T3 cells were transfected with <i>HA</i>-tagged <i>Mcm2</i> mutants along with <i>FLAG</i>-tagged <i>gp70</i>, and either left untreated (<b>B</b>) or treated with 1 µM doxorubicin for 24 h (<b>C</b>). The expression of the MCM2 mutants (<b>B</b>, <b>C</b>, left upper) and FLAG-gp70 (<b>B</b>, <b>C</b>, left middle) was confirmed in 3T3 cells. Cell lysates were subjected to a pull-down assay to detect the binding of MCM2-FL or MCM2 mutants to FLAG-gp70 (<b>B</b>, <b>C</b>, right panel).</p

<i>In vivo</i> assessment of doxorubicin-induced apoptosis and the associated changes in mRNA expression in FLV-infected mice.

<p>Uninfected or FLV-infected BALB/c (A), C57BL/6 (B), and C3H (C) mice were intraperitoneally (i.p.) administrated with 1.5 mg/kg of doxorubicin or PBS, and the apoptotic cell ratios in the bone marrow (gray bars) and spleen cells (black bars) were determined 24 h later with annexin V-staining. Note the significant increase in the proportion of annexin V-positive cells in the bone marrow and spleen of FLV-infected C3H mice after the doxorubicin treatment compared to that in the bone marrow and spleen cells of uninfected mice “FLV (−), Doxorubicin (−)” (*<i>p</i><0.01 and ^#<i>p</i><0.01). Data represent the mean and 95% confidence intervals (CI) of 3 independent experiments. (D) Quantitative RT-PCR analysis of <i>Mcm2</i> mRNA expression in the bone marrow of uninfected and FLV-infected BALB/c, C57BL/6, and C3H mice. The bone marrow cells of the C3H strain exhibit higher levels of <i>Mcm2</i> in all groups compared to the corresponding groups of BALB/c and C57BL/6 mice (*<i>p</i><0.01, for each group). (E) Quantitative RT-PCR analysis of <i>Mcm2</i> mRNA expression in the spleen of uninfected and FLV-infected BALB/c, C57BL/6, and C3H mice. Spleen <i>Mcm2</i> expression is higher in the “FLV (+), Doxorubicin (−)” and “FLV (+), Doxorubicin (+)” C3H mice than in the corresponding groups of BALB/c and C57BL/6 mice (*<i>p</i><0.01 and ^#<i>p</i><0.01, respectively). In C3H mice, FLV-infection induces higher levels of <i>Mcm2</i> expression compared to the expression in uninfected mice. Data represent the mean and 95% CI from 5 mice in each group and are representative of 2 independent experiments. The GeneChip data for <i>Mcm</i>-associated and apoptosis-associated genes were analyzed using the Percellome method. Forty-eight male C57BL/6 and C3H mice were divided into 16 groups of 3 mice each. Uninfected or FLV-infected C57BL/6 and C3H mice were administered (i.p.) with 15 mg/kg (high dose) or 1.5 mg/kg (low dose) of doxorubicin, and the spleen was sampled 0, 1, 6, and 24 h after administration. The spleen transcriptome was measured using the Affymetrix Mouse 430-2 GeneChip. (F) The Percellome data were plotted on 3-dimensional graphs for average, +1 SD, and −1 SD surfaces as demonstrated in the left schema. The scale of expression (vertical axis) is the copy number per cell. The x-axis of the 3-dimensional graph shows the experimental groups, including the C3H and C57BL/6 mice with doxorubicin treatment (high and low doses) with or without FLV-infection. The y-axis shows the time course (0, 1, 6, and 24 h) after treatment with doxorubicin and the z-axis (vertical) indicates the intensity of mRNA expression of each gene. The data of each point are connected to form a surface illustration. The expression patterns of genes are compared using the surface images. (G) The <i>Mcm2</i> expression pattern is shown in the upper right box. Of the lower columns, the first column (H) shows the data for the genes of representative <i>Mcm</i> family members, the second column (I), PI3K members, the third column (J), p53-associated genes, the fourth column (K), caspase members and fifth column (L), protein phosphatase members (PPs). <i>Mcm</i> family members, <i>Dna-pk</i>, <i>caspase-3</i> (<i>Casp3</i>), <i>Ppp2ac,</i> and <i>Ppp6</i> exhibit gene expression patterns similar to that of <i>Mcm2</i>.</p