Identification of negative regulators of p53 pathway by a forward genetic screen in ovarian cancer

Abstract Ovarian cancer is the deadliest of all female gynecological cancers and the fifth leading cause of cancer-related death in women. Lack of early detection and treatment failure are the major contributors to ovarian cancer death in women. Understanding ovarian cancer at the molecular level will help provide better treatment options, such as targeted therapies. Nearly sixty percent of women are diagnosed at late stage ovarian cancer. TP53 is the most commonly mutated gene in ovarian cancer as well as in all other cancers. However, there are subsets of ovarian cancer patients with wild-type TP53, and we would like to understand the molecular and cellular mechanisms that disable the transcriptional functions of p53 in those patients. Wild-type p53 is the transcription factor that activates sets of genes to respond to various types of cellular and molecular stress and to maintain genomic stability in normal cells. The major function of wild-type p53 is the transcriptional activation of genes that are involved in cell cycle arrest to repair DNA damage and cell death if the damage is beyond repair in cells. However, the p53 transcriptional functions of cell cycle arrest and cell death are not observed in those ovarian cancer patients with wild-type TP53 and the genes inhibiting transcriptional activities of p53 are not well studied. Additionally, our understanding of negative regulators of p53 in cell activity is not complete, and identifying those regulators may provide new insights into how the p53 pathway can be deregulated in tumors without mutations in TP53. For that purpose, we performed forward genetic screening using a patient-derived pool cDNA library constructed with pRetro-LIB vector in wild-type TP53 ovarian cancer cells to identify the potential negative regulators of p53. First, we tested our method of screening in wild-type TP53 ovarian cancer cells to prove the concept of our experimental approach. We found that using modified 293T cells (Phoenix AMPHO) with spin-fection method overcame the low transfection efficiency of retroviral particles in wild-type TP53 ovarian cancer cells (A2780). In normal cells, p53 is present at relatively low levels because of the negative feedback loop between Mdm2 and p53. To induce the p53 level in ovarian cancer cells with wild-type TP53, we used the small molecule inhibitor, Nutlin-3a. Nutlin-3a blocks the interaction between Mdm2 and p53, thereby allowing the stabilization of p53 in ovarian cancer cells. Stabilized p53 transactivates genes that initiate cell cycle arrest or cell death. Accordingly, Nutlin-3a suppresses the growth of cancer cells with wild-type TP53. We used this system to screen for exogenous genes from the patient-derived cDNA library that block Nutlin-3a-mediated growth suppression in A2780 cancer cells. The screen identified three candidate genes (NIFK, GXYLT 1, and SACS) that prevent the Nutlin-3a-induced growth suppression in A2780 cancer cells, and NIFK (also known as Nucleolar Protein Interacting with the FHA Domain of MKI67 or MKI67IP) was identified in four independent screening experiments. NIFK encodes a protein that interacts with the forkhead-associated domain of the Ki-67 antigen and has been implicated in ribosome biogenesis, e.g., pre-ribosomal RNA processing and ribosome assembly. NIFK also associates with pre-mRNAs. However, the potential association between NIFK and p53 in ovarian cancer is unknown. We evaluated the level of NIFK, p53, and Mdm2 in NIFK-overexpressing ovarian cancer cells. We found that the dose-dependent effect of NIFK expression in preventing the Nutlin-3a-induced growth suppression in A2780 cells. Our results suggest that NIFK overcomes the growth arrest by functional p53 induced by Nutlin-3a, and NIFK negatively regulates p53 pathway in ovarian cancer cells. In summary, our studies showed that NIFK overexpression cells play a role in cell survival, proliferation and the negative regulation of p53 in ovarian cancer cells, and this should be further explored to understand the molecular function of NIFK

53BP1 is required for class switch recombination

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for “classic” nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked

Genome-scale CRISPR knockout screen identifies TIGAR as a modifier of PARP inhibitor sensitivity.

Treatment of cancer with poly (ADP-ribose) polymerase (PARP) inhibitors is currently limited to cells defective in the homologous recombination (HR) pathway. Identification of genetic targets that induce or mimic HR deficiencies will extend the clinical utility of PARP inhibitors. Here we perform a CRISPR/Cas9-based genome-scale loss-of-function screen, using the sensitivity of PARP inhibitor olaparib as a surrogate. We identify C12orf5, encoding TP53 induced glycolysis and apoptosis regulator (TIGAR), as a modifier of PARP inhibitor response. We show that TIGAR is amplified in several cancer types, and higher expression of TIGAR associates with poor overall survival in ovarian cancer. TIGAR knockdown enhances sensitivity to olaparib in cancer cells via downregulation of BRCA1 and the Fanconi anemia pathway and increases senescence of these cells by affecting metabolic pathways and increasing the cytotoxic effects of olaparib. Our results indicate TIGAR should be explored as a therapeutic target for treating cancer and extending the use of PARP inhibitors

p53 Binding Protein 53BP1 Is Required for DNA Damage Responses and Tumor Suppression in Mice

53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53. It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM(−/−) mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1(−/−) cells show a slight S-phase checkpoint defect and prolonged G(2)/M arrest after treatment with ionizing radiation. Moreover, 53BP1(−/−) cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression

Heterozygous mutations in valosin-containing protein (VCP) and resistance to VCP inhibitors.

In recent years, multiple studies including ours have reported on the mechanism of resistance towards valosin-containing protein (VCP) inhibitors. While all these studies reported target alterations via mutations in VCP as the primary mechanism of resistance, discrepancies persist to date regarding the zygosity of these mutations responsible for the resistance. In addition, the extent to which resistant cells harbor additional mutations in other genes is not well described. In this study, we performed global transcript analysis of the parental and previously reported VCP inhibitor (CB-5083) resistant cells and found additional mutations in the resistant cells. However, our CRISPR-Cas9 gene editing studies indicate that specific mutations in VCP are sufficient to produce resistance to CB-5083 suggesting the importance of on-target mutations in VCP for resistance. Strikingly, our analysis indicates a preexisting heterozygous frameshift mutation at codon 616 (N616fs*) in one of the VCP alleles in HCT116 cells, and we showed that this mutant allele is subjected to the nonsense-mediated decay (NMD). Accordingly, we identified a heterozygous mutation at codon 526 (L526S) in genomic DNA sequencing but a homozygous L526S mutation in complementary DNA sequencing in our independently generated CB-5083 resistant HCT116 cells, implying that the L526S mutation occurs in the allele that does not harbor the frameshift N616fs* mutation. Our results suggest the NMD as a possible mechanism for achieving the homozygosity of VCP mutant responsible for the resistance to VCP inhibitors while resolving the discrepancies among previous studies. Our results also underscore the importance of performing simultaneous genomic and complementary DNA sequencing when attributing mutational effects on the functionality particularly for an oligomer protein like VCP

Heterozygous mutations in valosin-containing protein (VCP) and resistance to VCP inhibitors.

In recent years, multiple studies including ours have reported on the mechanism of resistance towards valosin-containing protein (VCP) inhibitors. While all these studies reported target alterations via mutations in VCP as the primary mechanism of resistance, discrepancies persist to date regarding the zygosity of these mutations responsible for the resistance. In addition, the extent to which resistant cells harbor additional mutations in other genes is not well described. In this study, we performed global transcript analysis of the parental and previously reported VCP inhibitor (CB-5083) resistant cells and found additional mutations in the resistant cells. However, our CRISPR-Cas9 gene editing studies indicate that specific mutations in VCP are sufficient to produce resistance to CB-5083 suggesting the importance of on-target mutations in VCP for resistance. Strikingly, our analysis indicates a preexisting heterozygous frameshift mutation at codon 616 (N616fs*) in one of the VCP alleles in HCT116 cells, and we showed that this mutant allele is subjected to the nonsense-mediated decay (NMD). Accordingly, we identified a heterozygous mutation at codon 526 (L526S) in genomic DNA sequencing but a homozygous L526S mutation in complementary DNA sequencing in our independently generated CB-5083 resistant HCT116 cells, implying that the L526S mutation occurs in the allele that does not harbor the frameshift N616fs* mutation. Our results suggest the NMD as a possible mechanism for achieving the homozygosity of VCP mutant responsible for the resistance to VCP inhibitors while resolving the discrepancies among previous studies. Our results also underscore the importance of performing simultaneous genomic and complementary DNA sequencing when attributing mutational effects on the functionality particularly for an oligomer protein like VCP

Targeting of mutant p53-induced FoxM1 with thiostrepton induces cytotoxicity and enhances carboplatin sensitivity in cancer cells.

FoxM1 is an oncogenic Forkhead transcription factor that is overexpressed in ovarian cancer. However, the mechanisms by which FoxM1 is deregulated in ovarian cancer and the extent to which FoxM1 can be targeted in ovarian cancer have not been reported previously. In this study, we showed that MDM2 inhibitor Nutlin-3 upregulated p53 protein and downregulated FoxM1 expression in several cancer cell lines with wild type TP53 but not in cell lines with mutant TP53. FoxM1 downregulation was partially blocked by cycloheximide or actinomycin D, and pulse-chase studies indicate Nutlin-3 enhances FoxM1 mRNA decay. Knockdown of p53 using shRNAs abrogated the FoxM1 downregulation by Nutlin-3, indicating a p53-dependent mechanism. FoxM1 inhibitor, thiostrepton, induces apoptosis in cancer cell lines and enhances sensitivity to cisplatin in these cells. Thiostrepton downregulates FoxM1 expression in several cancer cell lines and enhances sensitivity to carboplatin in vivo. Finally, FoxM1 expression is elevated in nearly all (48/49) ovarian tumors, indicating that thiostrepton target gene is highly expressed in ovarian cancer. In summary, the present study provides novel evidence that both amorphic and neomorphic mutations in TP53 contribute to FoxM1 overexpression and that FoxM1 may be targeted for therapeutic benefits in cancers

53BP1 Cooperates with p53 and Functions as a Haploinsufficient Tumor Suppressor in Mice

p53 binding protein 1 (53BP1) is a putative DNA damage sensor that accumulates at sites of double-strand breaks (DSBs) in a manner dependent on histone H2AX. Here we show that the loss of one or both copies of 53BP1 greatly accelerates lymphomagenesis in a p53-null background, suggesting that 53BP1 and p53 cooperate in tumor suppression. A subset of 53BP1(−/−) p53(−/−) lymphomas, like those in H2AX(−/−) p53(−/−) mice, were diploid and harbored clonal translocations involving antigen receptor loci, indicating misrepair of DSBs during V(D)J recombination as one cause of oncogenic transformation. Loss of a single 53BP1 allele compromised genomic stability and DSB repair, which could explain the susceptibility of 53BP1(+/−) mice to tumorigenesis. In addition to structural aberrations, there were high rates of chromosomal missegregation and accumulation of aneuploid cells in 53BP1(−/−) p53(+/+) and 53BP1(−/−) p53(−/−) tumors as well as in primary 53BP1(−/−) splenocytes. We conclude that 53BP1 functions as a dosage-dependent caretaker that promotes genomic stability by a mechanism that preserves chromosome structure and number