REGULATION OF THE OXIDATIVE STRESS RESPONSE BY ARID1A

SWI/SNF is mutated in about 20% of all human cancers; in particular ARID1A is the most frequently mutated SWI/SNF subunit. ARID1A is a tumor suppressor gene, inactivating mutations in ARID1A are most frequently found in ovarian and endometrial cancers, specifically uterine corpus endometrioid carcinomas (UCEC), ovarian clear cell carcinomas (OCCC) and ovarian endometrioid carcinomas (OEC). The functional roles of ARID1A are not completely understood and there are limited therapeutic strategies that specifically target ARID1A-mutant cancers. Given that ARID1A expression is lost in cancer, ARID1A mutations cannot be targeted directly and novel therapeutic strategies are required to target ARID1A-mutant cancers. In this study, drug responses between ARID1A-wildtype and ARID1A-mutant cell lines were compared using the ‘Genomics of Drug Sensitivities in Cancer’ database. From this analysis, I found that ARID1A-mutant cell lines are more sensitive to elesclomol, which is a reactive oxygen species (ROS)-inducing agent. This finding was validated using a panel of ovarian and endometrial cancer cell lines, where ARID1A-mutant cell lines exhibited lower IC50 values and higher apoptotic rates when treated with elesclomol. Knockdown and re-expression of ARID1A in ovarian cancer cells showed that ARID1A is required to protect cancer cells from oxidative stress. In the absence of ARID1A, intracellular ROS levels were increased, and this increase was required for increased cell growth upon ARID1A depletion. Next, I investigated the relationship between ARID1A and NRF2, the major regulator of the antioxidant response in the cell. I found that ARID1A negatively regulates the expression of NRF2. Knockdown of NRF2 in ovarian cancer cell lines revealed that NRF2 expression may be preferentially required for protection from oxidative stress and cell proliferation in ARID1A-mutant cells. Analysis using The Cancer Genome Atlas (TCGA) UCEC dataset revealed that ARID1A-mutant tumor samples have higher expression of NRF2 and NRF2-target genes. In summary, this study revealed novel roles of ARID1A in protecting ovarian cancer cells against oxidative stress. In the absence of ARID1A, NRF2 is up-regulated and may be required to compensate for ARID1A deficiency. These findings suggest that ROS-inducing agents and NRF2 inhibitors may be used as therapeutic strategies in targeting ARID1A-mutant ovarian cancer cells

Depletion of TRRAP induces p53-independent senescence in liver cancer by downregulating mitotic genes

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with few effective treatments and the underlying mechanisms that drive HCC pathogenesis remain poorly characterized. Identifying genes and pathways essential for HCC cell growth will aid the development of new targeted therapies for HCC. Using a kinome CRISPR screen in three human HCC cell lines, we identified transformation/transcription domain-associated protein (TRRAP) as an essential gene for HCC cell proliferation. TRRAP has been implicated in oncogenic transformation, but how it functions in cancer cell proliferation is not established. Here, we show that depletion of TRRAP or its co-factor, histone acetyltransferase KAT5, inhibits HCC cell growth via induction of p53- and p21-independent senescence. Integrated cancer genomics analyses using patient data and RNA-sequencing identified mitotic genes as key TRRAP/KAT5 targets in HCC, and subsequent cell cycle analyses revealed that TRRAP- and KAT5-depleted cells are arrested at G2/M phase. Depletion of TOP2A, a mitotic gene and TRRAP/KAT5 target, was sufficient to recapitulate the senescent phenotype of TRRAP/KAT5 knockdown. CONCLUSION: Our results uncover a role for TRRAP/KAT5 in promoting HCC cell proliferation via activation of mitotic genes. Targeting the TRRAP/KAT5 complex is a potential therapeutic strategy for HCC

YAP1 withdrawal in hepatoblastoma drives therapeutic differentiation of tumor cells to functional hepatocyte-like cells

BACKGROUND and AIMS: Despite surgical and chemotherapeutic advances, the five-year survival rate for Stage IV Hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. YAP1 and beta-Catenin co-activation occurs in 80% of children\u27s HB; however, a lack of conditional genetic models precludes tumor maintenance exploration. Thus, the need for a targeted therapy remains unmet. Given the predominance of YAP1 and beta-Catenin activation in HB, we sought to evaluate YAP1 as a therapeutic target in HB. APPROACH and RESULTS: We engineered the first conditional HB murine model using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1(S127A) , constitutive beta-Catenin(DelN90) , and a luciferase reporter to murine liver. Tumor regression was evaluated using bioluminescent imaging, and tumor landscape characterized using RNA and ATAC sequencing, and DNA foot-printing. Here we show that YAP1(S127A) withdrawal mediates \u3e90% tumor regression with survival for 230+ days in mice. YAP1 (S127A) withdrawal promotes apoptosis in a subset of tumor cells and in remaining cells induces a cell fate switch driving therapeutic differentiation of HB tumors into Ki-67 negative hbHep cells with hepatocyte-like morphology and mature hepatocyte gene expression. YAP1 (S127A) withdrawal drives formation of hbHeps by modulating liver differentiation transcription factor (TF) occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and rescue liver damage in mice. CONCLUSIONS: YAP1(S127A) withdrawal, without silencing oncogenic beta-Catenin, significantly regresses hepatoblastoma, providing the first in vivo data to support YAP1 as a therapeutic target for HB. YAP1(S127A) withdrawal alone sufficiently drives long-term regression in hepatoblastoma because it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes

CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in vivo cancer modeling

Abstract CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3′-UTR of mouse β-actin, allowing co-expression of reporter genes or oncogenes from the β-actin promoter. We showed that knock-in of oncogenic Ras and loss of p53 efficiently induced intrahepatic cholangiocarcinoma in mice. Further, our strategy can generate bioluminescent liver cancer to facilitate tumor imaging. This method simplifies in vivo gain-of-function genetics by facilitating targeted integration of oncogenes

Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing

Efficient genome editing with Cas9-sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9-sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications. Using lipid nanoparticle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single intravenous injection into mice induces \u3e 80% editing of Pcsk9 in the liver. Serum Pcsk9 is reduced to undetectable levels, and cholesterol levels are significantly lowered about 35% to 40% in animals. This strategy may enable non-viral, Cas9-based genome editing in the liver in clinical settings

PAX2 Expression in Ovarian Cancer

PAX2 is one of nine PAX genes that regulate tissue development and cellular differentiation in embryos. However, the functional role of PAX2 in ovarian cancer is not known. Twenty-six ovarian cancer cell lines with different histology origins were screened for PAX2 expression. Two ovarian cancer cell lines: RMUGL (mucinous) and TOV21G (clear cell), with high PAX2 expression were chosen for further study. Knockdown PAX2 expression in these cell lines was achieved by lentiviral shRNAs targeting the PAX2 gene. PAX2 stable knockdown cells were characterized for cell proliferation, migration, apoptosis, protein profiles, and gene expression profiles. The result indicated that these stable PAX2 knockdown cells had reduced cell proliferation and migration. Microarray analysis indicated that several genes involved in growth inhibition and motility, such as G0S2, GREM1, and WFDC1, were up-regulated in PAX2 knockdown cells. On the other hand, over-expressing PAX2 in PAX2-negative ovarian cell lines suppressed their cell proliferation. In summary, PAX2 could have both oncogenic and tumor suppression functions, which might depend on the genetic content of the ovarian cancer cells. Further investigation of PAX2 in tumor suppression and mortality is warranty

PAX2 Expression in Ovarian Cancer

PAX2 is one of nine PAX genes that regulate tissue development and cellular differentiation in embryos. However, the functional role of PAX2 in ovarian cancer is not known. Twenty-six ovarian cancer cell lines with different histology origins were screened for PAX2 expression. Two ovarian cancer cell lines: RMUGL (mucinous) and TOV21G (clear cell), with high PAX2 expression were chosen for further study. Knockdown PAX2 expression in these cell lines was achieved by lentiviral shRNAs targeting the PAX2 gene. PAX2 stable knockdown cells were characterized for cell proliferation, migration, apoptosis, protein profiles, and gene expression profiles. The result indicated that these stable PAX2 knockdown cells had reduced cell proliferation and migration. Microarray analysis indicated that several genes involved in growth inhibition and motility, such as G0S2, GREM1, and WFDC1, were up-regulated in PAX2 knockdown cells. On the other hand, over-expressing PAX2 in PAX2-negative ovarian cell lines suppressed their cell proliferation. In summary, PAX2 could have both oncogenic and tumor suppression functions, which might depend on the genetic content of the ovarian cancer cells. Further investigation of PAX2 in tumor suppression and mortality is warranty

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas.

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a--a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis--as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC50 values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation

Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state.

Kindlin-1, -2, and -3 directly bind integrin β cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin β cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation

Partial DNA-guided Cas9 enables genome editing with reduced off-target activity

CRISPR-Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9-sgRNA complex as a guide, the majority of the 3\u27 end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3\u27-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA-RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells