Shruti Lal

Mitoxantrone Targets Human Ubiquitin-Specific Peptidase 11 (USP11) and Is a Potent Inhibitor of Pancreatic Cancer Cell Survival

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related death in the United States, with a 95% five-year mortality rate. For over a decade, gemcitabine (GEM) has been the established first-line treatment for this disease despite suboptimal response rates. The development of PARP inhibitors that target the DNA damage repair (DDR) system in PDA cells has generated encouraging results. Ubiquitin-specific peptidase 11 (USP11), an enzyme that interacts with the DDR protein BRCA2, was recently discovered to play a key role in DNA double-strand break repair and may be a novel therapeutic target. A systematic high-throughput approach was used to biochemically screen 2,000 U.S. Food and Drug Administration (FDA)-approved compounds for inhibition of USP11 enzymatic activity. Six pharmacologically active small molecules that inhibit USP11 enzymatic activity were identified. An in vitro drug sensitivity assay demonstrated that one of these USP11 inhibitors, mitoxantrone, impacted PDA cell survival with an IC50 of less than 10 nM. Importantly, across six different PDA cell lines, two with defects in the Fanconi anemia/BRCA2 pathway (Hs766T and Capan-1), mitoxantrone is 40- to 20,000-fold more potent than GEM, with increased endogenous USP11 mRNA levels associated with increased sensitivity to mitoxantrone. Interestingly, USP11 silencing in PDA cells also enhanced sensitivity to GEM. These findings establish a preclinical model for the rapid discovery of FDA-approved compounds and identify USP11 as a target of mitoxantrone in PDA. Implications: This high-throughput approach provides a strong rationale to study mitoxantrone in an early-phase clinical setting for the treatment of PDA. Mol Cancer Res; 11(8); 901–11. ©2013 AACR.

Targeting PARP-1 allosteric regulation offers therapeutic potential against cancer

PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs, raising important questions about long-term off-target effects. Here, we propose a new strategy that targets PARP-1 allosteric regulation as a selective way of inhibiting PARP-1. We found that disruption of PARP-1 domain-domain contacts through mutagenesis held no cellular consequences on recruitment to DNA damage or a model system of transcriptional regulation, but prevented DNA-damage-dependent catalytic activation. Furthermore, PARP-1 mutant overexpression in a pancreatic cancer cell line (MIA PaCa-2) increased sensitivity to platinum-based anticancer agents. These results not only highlight the potential of a synergistic drug combination of allosteric PARP inhibitors with DNA-damaging agents in genomically unstable cancer cells (regardless of homologous recombination status), but also signify important applications of selective PARP-1 inhibition. Finally, the development of a high-throughput PARP-1 assay is described as a tool to promote discovery of novel PARP-1 selective inhibitors.

WEE1 inhibition in pancreatic cancer cells is dependent on DNA repair status in a context dependent manner

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease, in part, because of the lack of effective targeted therapeutic options. MK-1775 (also known as AZD1775), a mitotic inhibitor, has been demonstrated to enhance the anti-tumor effects of DNA damaging agents such as gemcitabine. We evaluated the efficacy of MK-1775 alone or in combination with DNA damaging agents (MMC or oxaliplatin) in PDA cell lines that are either DNA repair proficient (DDR-P) or deficient (DDR-D). PDA cell lines PL11, Hs 766T and Capan-1 harboring naturally selected mutations in DNA repair genes FANCC, FANCG and BRCA2 respectively, were less sensitive to MK-1775 as compared to two out of four representative DDR-P (MIA PaCa2 and PANC-1) cell lines. Accordingly, DDR-P cells exhibit reduced sensitivity to MK-1775 upon siRNA silencing of DNA repair genes, BRCA2 or FANCD2, compared to control cells. Only DDR-P cells showed increased apoptosis as a result of early mitotic entry and catastrophe compared to DDR-D cells. Taken together with other recently published reports, our results add another level of evidence that the efficacy of WEE1 inhibition is influenced by the DNA repair status of a cell and may also be dependent on the tumor type and model evaluated.

Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells

Cancer aggressiveness may result from the selective pressure of a harsh nutrient-deprived microenvironment. Here we illustrate how such conditions promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Glucose or glutamine withdrawal resulted in a 5- to 10-fold protective effect with chemotherapy treatment. PDAC xenografts were less sensitive to gemcitabine in hypoglycemic mice compared with hyperglycemic mice. Consistent with this observation, patients receiving adjuvant gemcitabine (n = 107) with elevated serum glucose levels (HgbA1C > 6.5%) exhibited improved survival. We identified enhanced antioxidant defense as a driver of chemoresistance in this setting. ROS levels were doubled in vitro by either nutrient withdrawal or gemcitabine treatment, but depriving PDAC cells of nutrients before gemcitabine treatment attenuated this effect. Mechanistic investigations based on RNAi or CRISPR approaches implicated the RNA binding protein HuR in preserving survival under nutrient withdrawal, with or without gemcitabine. Notably, RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation. HuR-deficient PDAC cells lacked the ability to engraft successfully in immunocompromised mice, but IDH1 overexpression in these cells was sufficient to fully restore chemoresistance under low nutrient conditions. Overall, our findings highlight the HuR-IDH1 regulatory axis as a critical, actionable therapeutic target in pancreatic cancer. Cancer Res; 77(16); 4460-71. ©2017 AACR.

MUC1 promoter-driven DTA as a targeted therapeutic strategy against pancreatic cancer

Mucin1 (MUC1) is overexpressed in pancreatic ductal adenocarcinoma (PDA) and is associated with tumor aggressiveness, suggesting that MUC1 is a promising therapeutic target for promoter-driven diphtheria toxin A (DTA). Endogenous MUC1 transcript levels were analyzed by quantitative PCR (qPCR) in multiple PDA cells (Capan1, HPAFII, Su.86.86, Capan2, Hs766T, MiaPaCa2, and Panc1). Expression levels were correlated with luciferase activity and cell death after transfection with MUC1 promoter–driven luciferase and DTA constructs. MUC1-positive (+) cells had significantly elevated MUC1 mRNA expression compared with MUC1-negative (−) cells. Luciferase activity was significantly higher in MUC1+ cells when transfected with MUC1 promoter–driven luciferase and MUC1+ cells underwent enhanced cell death after transfection with a single dose of MUC1 promoter–driven DTA. IFNγ pretreatment enhanced MUC1 expression in MUC1− cells and induced sensitivity to MUC1–DTA therapy. Matched primary and metastatic tumor lesions from clinical specimens revealed similar MUC1 IHC labeling patterns, and a tissue microarray of human PDA biopsies revealed increased immunolabeling with a combination of MUC1 and mesothelin (MSLN) antibodies, compared with either antibody alone. Combining MUC1 with MSLN-targeted DTA enhanced drug efficacy in an in vitro model of heterogeneous PDA. These data demonstrate that MUC1 promoter–driven DTA preferentially kills MUC1-expressing PDA cells and drugs that enhance MUC1 expression sensitize PDA cells with low MUC1 expression. Implications: MUC1 expression in primary and metastatic lesions provides a rationale for the development of a systemic MUC1 promoter–driven DTA therapy that may be further enhanced by combination with other promoter-driven DTA constructs. Mol Cancer Res; 13(3); 439–48. ©2014 AACR.

CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States, whereas colorectal cancer is the third most common cancer. The RNA-binding protein HuR (ELAVL1) supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and colorectal cancer tumor cohorts as compared with normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and colorectal cancer (HCT116) cell lines. HuR deficiency has a mild phenotype, in vitro, as HuR-deficient MIA PaCa-2 (MIA.HuR-KO(−/−)) cells had increased apoptosis when compared with isogenic wild-type (MIA.HuR-WT(+/+)) cells. Using this isogenic system, mRNAs were identified that specifically bound to HuR and were required for transforming a two-dimensional culture into three dimensional (i.e., organoids). Importantly, HuR-deficient MIA PaCa-2 and Hs 766T cells were unable to engraft tumors in vivo compared with control HuR-proficient cells, demonstrating a unique xenograft lethal phenotype. Although not as a dramatic phenotype, CRISPR knockout HuR HCT116 colon cancer cells (HCT.HuR-KO(−/−)) showed significantly reduced in vivo tumor growth compared with controls (HCT.HuR-WT(+/+)). Finally, HuR deletion affects KRAS activity and controls a subset of pro-oncogenic genes. Implications: The work reported here supports the notion that targeting HuR is a promising therapeutic strategy to treat GI malignancies. Mol Cancer Res; 15(6); 696–707. ©2017 AACR.

Studying RNA-Binding Protein Interactions with Target mRNAs in Eukaryotic Cells: Native Ribonucleoprotein Immunoprecipitation (RIP) Assays

Post-transcriptional regulation of mRNA can potently dictate protein expression patterns in eukaryotic cells. This mode of regulation occurs through cis-acting regulatory regions in the mRNA transcript that mediate direct interactions with trans-acting RNA-binding proteins (RBPs). This mRNA/protein interaction can be studied in numerous ways that range from in vitro to in vivo through messenger ribonucleoprotein immunoprecipitation (mRNP-IP or RIP) assays. This modified immunoprecipitation approach is an important and sensitive method to determine the regulation of gene expression by specific RBPs under different cellular stressors.

Abstract 2854: CRISPR knockout of HuR in pancreatic cancer cells causes a xenograft lethal phenotype

Pancreatic ductal adenocarcinoma (PDA) is the most prevalent type of pancreatic cancer and will soon become the second leading cause of cancer related deaths in the U.S. Studies show that the nuclear localized mRNA-binding protein HuR (ELAVL1) is activated in PDA cells, with cytoplasmic translocation associated with increased tumor size and poor prognosis. Previous in vitro and in vivo studies have established HuR9s role as a PDA cell survival mechanism. Thus, we explored the phenotypic effect of completely eliminating HuR expression from PDA cells through the use of clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas9 technology to target and disrupt the HuR genomic sequence. Since INDELs are induced randomly, we designed 3 gRNAs to target HuR at different loci. Gene disruption was determined via sequencing and validated through protein and mRNA expression, where homozygous knockouts (HuR −/− ) had undetectable HuR expression as compared to wild-type (HuR +/+ ), heterozygotes (HuR +/− ), and CRISPR/Cas9 negative control. Sanger sequencing confirmed homozygous knockouts with a frame shift mutation on both alleles. When HuR knockout cells were exposed to chemotherapeutic stress including mitomycin C, oxaliplatin, and gemcitabine, no HuR expression (nuclear or cytoplasmic) was detected via immunofluorescence. Phenotypically, HuR −/− cells resulted in increased apoptosis and necrosis as measured via trypan blue assay, and accordingly, had increased caspase 3 activity, a marker of a cell death. HuR −/− cells, when treated with mitomycin C, oxaliplatin, gemcitabine, and glucose deprivation exhibited decreased long and short-term cell survival as compared to control cells. HuR −/− cells, pulse-labeled with bromodeoxyurdine (BrdU), had a higher proportion of cells in S phase and fewer cells in G2/M phase. HuR deletion enhanced premature mitotic entry thereby preventing efficient repair of DNA damage, leading to cell death. Importantly, CRISPR knockout of HuR showed marked impairment in tumor growth in mouse xenografts. The differences in median tumor volume with HuR −/- xenografts was significant as compared to xenografts in mice with HuR (+/+) cells (0.0 mm 3 vs 378.0 mm 3 , P Citation Format: Edwin Cheung, Shruti Lal, Mahsa Zarei, Nicole C. Mambelli-Lisboa, Saswati Chand, Carmella Romeo, Kevin O’Hayer, Eric Londin, Joseph A. Cozzitorto, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. CRISPR knockout of HuR in pancreatic cancer cells causes a xenograft lethal phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2854.

Abstract 1656: MK-1775 (WEE1 inhibition) lacks efficacy against DNA repair deficient pancreatic cancer cells

Introduction: In this study, we explored the possibility of developing a personalized approach of using MK-1775, a potent WEE1 inhibitor, as mono- or combination therapy to treat pancreatic ductal adenocarcinoma (PDA) cells with DNA repair deficiency. Experimental Methods: We treated PDA cells harboring diverse genetic backgrounds with IC50 doses of clinically-relevant DNA damaging agents: mitomycin C (MMC) and the WEE1 inhibitor MK-1775. Drug sensitivity assays were performed in isogenic PDA cells that are either proficient or deficient in Fanconi Anemia (FA)/BRCA2 pathway. In addition, proficient cell lines were treated with siRNA oligos targeted against FANCD2 and BRCA2 genes. Mechanistic studies such as Annexin V staining were performed to measure apoptotic cells upon MMC and MK-1775 treatments. The degree of DNA damage was measured by immunofluorescence (IF) assay using γH2AX antibody. Mitotic entry was analyzed by both IF and flow cytometry analyses using pH3 antibody. Results: Drug sensitivity assays demonstrated that FA/BRCA2-pathway proficient PDA cell lines (MiaPaCa2 and Panc1) are sensitive to the cytotoxic effect of MK-1775 compared to DNA repair-deficient cell lines (Capan1:BRCA2 deficient and Hs766T:FANCG deficient) which showed acute resistance to MK-1775. Immunoblotting showed that MK-1775 efficiently reduces the expression of WEE1 and accordingly phosphorylation of CDK1 in all cell lines. Annexin V staining showed a higher percentage of cell death in the FA/BRCA2-pathway proficient cell lines compared to deficient cell lines when exposed to MK-1775. Furthermore, IF experiments demonstrated that MMC treatment induces γH2AX foci in all cell lines, however, the FA/BRCA2 proficient cell lines showed a higher degree of nuclear abnormality and multi-nucleation after MK-1775 treatment compared to deficient cell lines. In addition, the FA/BRCA2 proficient cell lines showed significantly higher phospho histone 3 (pH3) staining, a marker of mitotic cells. FACS analyses validated that proficient cell lines showed a higher percentage of cells in the mitotic phase when exposed to MK-1775. In addition, we evaluated the cytotoxic effect of MK-1775 in combination with MMC and showed that FA/BRCA2 proficient cells are more sensitive to dual treatment than deficient cells. In addition, immnobloting detected cleaved caspase 3, a marker of apoptosis, after MK-1775 treatment alone or in combination with MMC, demonstrating cells undergoing mitotic catastrophe. We validated the results in the FA/BRCA2-pathway proficient cell lines pre-treated with siFANCD2 or siBRCA2 oligos as compared to the proficient control lines. Conclusions: These results support a paradigm in which identified high risk FA/BRCA2-mutated patients would not benefit from WEE1 inhibitor monotherapy; and thus, would most likely respond better to conventional DNA damaging agent-based therapies (e.g., oxaliplatin or MMC). Citation Format: Shruti Lal, Saswati N. Chand, Emanuela Dylgjeri, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. MK-1775 (WEE1 inhibition) lacks efficacy against DNA repair deficient pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2015-1656

Abstract 628: A novel chemoresistance mechanism: HuR post-trancriptionally regulates WEE1, the mitotic inhibitor, upon DNA damage in pancreatic adenocarcinoma cells.

Introduction: We explored HuR9s (an RNA binding protein) significance in the DNA damage response (DDR) in pancreatic ductal adenocarcinoma (PDA) cells. Experimental Procedures: We treated PDA cells with DNA damaging agent9s mitomycin C (MMC), oxaliplatin, cisplatin, carboplatin, gemcitabine and a PARP inhibitor (ABT-888). HuR subcellular localization was assessed by immunoblotting. Additionally, we measured γH2AX foci, a marker of DNA damage, by immunofluorescence. Drug sensitivity assays were performed in isogenic PDA cells that differed in HuR expression (siRNA oligos against HuR, cDNAs encoding HuR for overexpression, and appropriate controls). A BrdU/PI pulse chase experiment monitored the cell cycle progression after treating cells with low (150nM) and high (1μM) doses of MMC. Ribonucleoprotein-immunoprecipitation (RIP) assays were used to isolate mRNAs bound to HuR to identify HuR targets in the DDR. Results: Enhanced-HuR cytoplasmic protein expression was observed in PDA cells exposed to all DNA damaging agents. Under high doses of certain DNA damaging agents (e.g., MMC) HuR was cleaved in a caspase-dependent manner. To assess HuR9s role in the DDR, isogenic PDA cells lines manipulated with HuR levels were treated with MMC (150nM) for 2 hrs. γH2AX foci assay revealed a 3-fold increase in foci number with MMC treatment in the HuR depleted cells, as compared to the control cells, suggesting that DNA repair was delayed in the absence of HuR. Cell cycle analyses demonstrated that HuR-depleted PDA cells progressed through the G2/M checkpoint and into mitosis upon MMC treatment. A combinatorial approach with RIP-sequencing and an siRNA library screen for genes resistant to DNA damaging agents was performed, and WEE1, a mitotic inhibitor kinase, was identified as a novel HuR target. A separate RIP-qPCR experiment validated WEE1 as an HuR binder with 56-fold more expression upon MMC stress, compared to an IgG control-IP. WEE1 protein expression and Cdk1 (a target of the WEE1 kinase and G2/M phase regulator) phosphorylation was reduced in HuR depleted cells, validating HuR as an upstream post-transcriptional regulator of WEE1 and an indirect inhibitor of the cdk1-cyclin complex in the face of DNA damage. Conclusions: We demonstrate that HuR-deficiency allows PDA cells to enter the cell cycle more efficiently upon DNA damage. This event is dependent on HuR9s regulation of WEE1 leading to activation of the CDK1-cyclin complex. These data support a model where HuR is integral to the G2/M checkpoint, forcing a cell cycle recovery response which is critical for PDA cells to avoid DNA damage. Therefore, HuR promotes survival of PDA cells through its acute activation of the DDR pathway. Disruption of HuR9s molecular interaction with WEE1 represents a therapeutic opportunity to enhance clinically utilized chemotherapeutic DNA damaging agents for PDA. Citation Format: Shruti Lal, Richard A. Burkhart, Zoe Norris, Neil Beeharry, Vikram Bhattacharjee, Isidore Rigoutsos, Timothy Yen, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. A novel chemoresistance mechanism: HuR post-trancriptionally regulates WEE1, the mitotic inhibitor, upon DNA damage in pancreatic adenocarcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 628. doi:10.1158/1538-7445.AM2013-628