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Figure S1: HuR inhibition was successfully achieved by CRISPR/cas9 system. Figure S2: HuR inhibition sensitizes cells to drugs. Figure S3: PDA xenografts with HuR inhibition failed to form tumors. Table S1: Lists the three guide RNAs used to generate CRISPR knockout of HuR in MIA PaCa-2 and Hs 766T cell lines. Table S2: Lists the primers used to detect the mutation created by all three guide RNAs in CRISPR knockout of HuR MIA PaCa-2 and Hs 766T cell lines. Table S3: Lists the clones along with their genotypes obtained for all three guide RNAs.

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PDF file - 28K, S1. Cell growth in a high USP11 expression cell line (PL5) following siRNA treatment targeting USP11.

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.

The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPR-Cas9–mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 3′ untranslated region. HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP-1 protein binds and posttranslationally modifies HuR in PARPi-treated PDAC cells. In a mouse xenograft model of human PDAC, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared with PARPi therapy alone. Our results highlight the HuR–PARG axis as an opportunity to enhance PARPi-based therapies. Cancer Res; 77(18); 5011–25. ©2017 AACR.

HuR regulates all isoforms of PARG by binding a discrete minimal binding region (MBR) within its 3'UTR in both DDR- proficient MIA PaCa-2 and DDR- deficient Hs 766t cells. Such PARG mRNA and protein expression is also increased with other non-PARPi stress such as gemcitabine and oxaliplatin treatment, which simultaneously induce HuR cytoplasmic translocation.

Table S5: Results of RNA sequencing in PDA cell lines (HS-766T and MiaPaCa2) with each modulated by CRISPR/Cas9 to delete HuR expression.

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.

PARG rescue in HuR silenced cells restores PARPi resistance. Small-molecule inhibition of HuR prevents its cytoplasmic accumulation, abrogates PARG mRNA regulation and increases PARPi sensitivity, independent of DDR status.

Doxycycline induction of HuR silencing in MIA PaCa-2 cells (sh290) downregulates PARG expression in vitro and tumor growth in vivo.

HuR silencing results in downregulation of PARG expression, which prevents efficient removal of PAR polymers with or without PARPi stress. Expression and function of other PAR removing enzymes is not affected with HuR silencing.

HuR directly binds and regulates poly-ADP ribose glycohydrolase mRNA expression particularly under PARPi stress, but doesn't affect expression of other PAR polymerases or hydrolyzing enzymes.

HuR expression regulates short-term cell survival and long-term anchorage-independent growth in PDA cells, irrespective of DDR proficiency. PARPi stress induces HuR translocation in a time-dependent manner.

Supplementary.Figures 1-8

Supplemental Methods and Supplemental Figure Legends

PARPi treatment mediated apoptosis and chromatin- associated trapping of PARP1 is significantly enhanced upon HuR and PARG inhibition.

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AVI file - 8881KB, siRNA HuR cells progressing through mitosis after MMC treatment.

PDF file - 658KB, HuR manipulation sensitizes PDA cells to chemotherapeutics agents.

AVI file - 8883KB, siRNA Control cells progressing through mitosis after MMC treatment.

PDF file - 2070KB, WEE1 is post-transcriptionally regulated by HuR upon DNA damage.

PDF file - 471KB, HuR silencing induced DNA damage breaks in PL5 cells.

PDF file - 2204KB, Different DNA damaging agents activate HuR.

AVI file - 8882KB, Untreated siRNA HuR cells progressing through mitosis.

PDF file - 318K, Figure S1. DNA binding affinity of full-length PARP-1 and various domain combinations. Figure S2. DNA binding affinities of deltaZn2 PARP-1 variants. Figure S3. Cell viability dose response curves of MIA PaCa-2 cells treated with non-platinum based agents. Figure S4. DNA binding affinities for mutants of 1-366deltaZn2. Figure S5. Confirmation of genomic incorporation of the plasmids. Figure S6. Western blot analysis for PARP-1 in the AR reporter assay. Figure S7. Validation of protein concentration by SDS-PAGE gel analysis.

PDF file - 350KB, WEE1 is a novel HuR target in DDR pathway.

PDF file - 64KB, Class of drugs used in the drug sensitivity assays performed in MiaPaCa2 cells against siRNA Control and HuR, respective IC50 of each drug identified.

DNA-damaging poisons and replicative stress commonly result in increased double-strand breaks (DSBs) in tumor cells. Non-homologous end joining (NHEJ), driven by the enzymatic function of DNA-PK, is one of the critical pathways employed to repair such DSBs. Inhibition of DNA-PK kinase activity impairs NHEJ, potentiates the activity of DNA-damaging agents, and inhibits survival of tumor cells incapable of repairing DSBs via other means, including homologous recombination (HR). We have developed an orally bioavailable, highly potent, and selective pharmacological inhibitor of DNA-PK activity, ZL-2201, for the treatment of cancer. ZL-2201 demonstrates enzymatic inhibition of DNA-PK in the single-digit nM range, with nearly 500-fold or greater selectivity against other phosphatidylinositol 3-kinase-related kinases (PIKKs). In addition, ZL-2201 showed high selectivity for DNA-PK kinase activity in a kinase panel screening study and a clean off-target profile with no appreciable inhibition against a panel of diverse, safety-relevant targets. We demonstrate that ZL-2201 effectively inhibits DNA-PK autophosphorylation and its downstream target phospho-RPA in multiple cell lines in a concentration- and time-dependent manner. DNA-PK has been shown previously to have synthetically lethal interactions with ATM. Therefore, we profiled the impact of ATM expression on the antiproliferative effects of ZL-2201. Cancer cell lines possessing defects in ATM displayed greater sensitivity to ZL-2201 compared to ATM-WT cancer cell lines. These observations were recapitulated in two ATM knockout isogenic cell lines. Moreover, ZL-2201 displayed minimal effects in a DNA-PK deficient model or in normal human and canine colon and kidney cell lines. Independent of ATM status, ZL-2201 showed strong synergy in vitro with topoisomerase II inhibitors. Following oral administration of ZL-2201 in vivo, ZL-2201 demonstrated dose-dependent and exposure-dependent antitumor activity against multiple models, with the greatest activity against ATM deficient human cancer xenografts. Antitumor activity correlated with duration of ZL-2201 exposure and level of target engagement. In addition, ZL-2201 significantly enhanced the activity of DNA-damaging agents in vivo, even in human cancer xenograft models with limited monotherapy antitumor activity for ZL-2201. The projected human PK properties, based on the ADME properties across species and PK properties in preclinical species of ZL-2201, suggest that ZL-2201 is suitable for oral administration. In conclusion, ZL-2201 demonstrates potent and selective inhibition of DNA-PK enzymatic activity, strong synergy with DNA damaging agents in vitro and in vivo, antitumor activity against multiple human xenograft models, and a favorable projected human PK profile. ZL-2201 is expected to enter clinical development in 2022. Citation Format: Shruti Lal, Bee-Chun Sun, Yuping Chen, Tom Huang, Neil Bhola, Vivian Morton, Kevin Chen, Shanghua Xia, Haoyu Zhang, Qiuping Ye, Petter Veiby, David I. Bellovin, Yuhua Ji. Discovery and characterization of ZL-2201, a potent, highly-selective, and orally bioavailable small-molecule DNA-PK inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2594.

Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated metabolic enzyme in human malignancy. A heterozygous genetic alteration, arginine 132, promotes the conversion of α-ketoglutarate to D-2-hydroxyglutarate (2-HG). Although pharmacologic inhibitors of mutant IDH1 are promising, resistance mechanisms to targeted therapy are not understood. Additionally, the role of wild-type IDH1 (WT.IDH1) in cancer requires further study. Recently, it was observed that the regulatory RNA-binding protein, HuR (ELAVL1), protects nutrient-deprived cancer cells without IDH1 mutations, by stabilizing WT.IDH1 transcripts. In the present study, a similar regulatory effect on both mutant (Mut.IDH1) and WT.IDH1 transcripts in heterozygous IDH1-mutant tumors is observed. In ribonucleoprotein immunoprecipitation assays of IDH1-mutant cell lines, wild-type and mutant IDH1 mRNAs each bound to HuR. Both isoforms were profoundly downregulated at the mRNA and protein levels after genetic suppression of HuR (siRNAs or CRISPR deletion) in HT1080 (R132C IDH1 mutation) and BT054 cells (R132H). Proliferation and invasion were adversely affected after HuR suppression and metabolomic studies revealed a reduction in Pentose Phosphate Pathway metabolites, nucleotide precursors, and 2-HG levels. HuR-deficient cells were especially sensitive to stress, including low glucose conditions or a mutant IDH1 inhibitor (AGI-5198). IDH1-mutant cancer cells were rescued by WT.IDH1 overexpression to a greater extent than Mut.IDH1 overexpression under these conditions. This study reveals the importance of HuR's regulation of both mutant and wild-type IDH1 in tumors harboring a heterozygous IDH1 mutation with implications for therapy. Implications: This study highlights the HuR–IDH1 (mutant and wild-type IDH1) regulatory axis as a critical, actionable therapeutic target in IDH1-mutated cancer, and incomplete blockade of the entire HuR–IDH1 survival axis would likely diminish the efficacy of drugs that selectively target only the mutant isoenzyme.

The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPR-Cas9–mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 3′ untranslated region. HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP-1 protein binds and posttranslationally modifies HuR in PARPi-treated PDAC cells. In a mouse xenograft model of human PDAC, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared with PARPi therapy alone. Our results highlight the HuR–PARG axis as an opportunity to enhance PARPi-based therapies. Cancer Res; 77(18); 5011–25. ©2017 AACR.

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.

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.

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.

Deoxycytidine kinase (dCK) and human antigen R (HuR) have been associated with response to gemcitabine in small studies. The present study investigates the prognostic and predictive value of dCK and HuR expression levels for sensitivity to gemcitabine and 5-fluorouracil (5-FU) in a large phase III adjuvant trial with chemoradiation backbone in pancreatic ductal adenocarcinoma (PDA). The dCK and HuR expression levels were determined by immunohistochemistry on a tissue microarray of 165 resected PDAs from the Radiation Therapy Oncology Group (RTOG) 9704 trial. Association with overall survival (OS) and disease-free survival (DFS) status were analyzed using the log-rank test and the Cox proportional hazards model. Experiments with cultured PDA cells were performed to explore mechanisms linking dCK and HuR expression to drug sensitivity. dCK expression levels were associated with improved OS for all patients analyzed from RTOG 9704 (HR: 0.66, 95% CI [0.47-0.93], P = 0.015). In a subset analysis based on treatment arm, the effect was restricted to patients receiving 5-FU (HR: 0.53, 95% CI [0.33-0.85], P = 0.0078). Studies in cultured cells confirmed that dCK expression rendered cells more sensitive to 5-FU. HuR cytoplasmic expression was neither prognostic nor predictive of treatment response. Previous studies along with drug sensitivity and biochemical studies demonstrate that radiation interferes with HuR's regulatory effects on dCK, and could account for the negative findings herein based on the clinical study design (i.e., inclusion of radiation). Finally, we demonstrate that 5-FU can increase HuR function by enhancing HuR translocation from the nucleus to the cytoplasm, similar to the effect of gemcitabine in PDA cells. For the first time, in the pre-treatment tumor samples, dCK and HuR cytoplasmic expression were strongly correlated (chi-square P = 0.015). This dual-institutional follow up study, in a multi-institutional PDA randomized clinical trial, observed that dCK expression levels were prognostic and had predictive value for sensitivity to 5-FU.

The majority of pancreatic ductal adenocarcinomas (PDA) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here we show that CRISPR-Cas9-mediated silencing of the HuR locus increases the relative sensitivity of PDA cells to PARP inhibitors (PARPi). PDA cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, polyADP-ribose glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 3′ untranslated region (UTR). HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP1 protein binds and post-translationally modifies HuR in PARPi-treated PDA cells. In a mouse xenograft model of human PDA, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared to PARPi therapy alone. Our results highlight the HuR-PARG axis as an opportunity to enhance PARPi-based therapies.

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.

The morphology of inflorescences is regulated in part by the temporal and spatial events that regulate flower specification. In Arabidopsis, an endogenous flowering time pathway mediated by a subset of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factors, including SPL3, SPL4, and SPL5, function to specify flowers by activating floral meristem identity genes. During shoot development, SPL3, SPL4, and SPL5 are post-transcriptionally regulated by microRNA156 (miR156). The photoperiod regulated florigenic signal, FLOWERING LOCUS T (FT), promotes floral induction, in part by activating SPL3, SPL4, and SPL5. In turn, these SPLs function in parallel with FT to specify flower meristems. Two related BELL1-like homeobox genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) expressed in the shoot apical meristem are absolutely required for the specification of floral meristems. Genetic studies show that the floral specification function of FT depends upon PNY and PNF; however, the interplay between these homeodomain proteins and SPLs is not known. In this manuscript, we show that the photoperiodic floral induction of SPL3, SPL4, and SPL5 is dependent upon PNY and PNF. Further, PNY and PNF also control SPL3, SPL4, and SPL5 expression by negatively regulating miR156. Lastly, ectopic expression of SPL4 partially rescues the pny pnf non-flower-producing phenotype, while overexpression of SPL3 or SPL5 in pny pnf plants was unable to restore flower specification. These results suggest that: (1) SPL3, SPL4, and SPL5 function is dependent upon PNY and PNF, or (2) expression of multiple SPLs is required for floral specification in pny pnf plants.

Growth of the aerial part of the plant is dependent upon the maintenance of the shoot apical meristem (SAM). A balance between the self-renewing stem cells in the central zone (CZ) and organogenesis in the peripheral zone (PZ) is essential for the integrity, function, and maintenance of the SAM. Understanding how the SAM maintains a balance between stem cell perpetuation and organogenesis is a central question in plant biology. Two related BELL1-like homeodomain proteins, PENNYWISE (PNY) and POUND-FOOLISH (PNF), act to specify floral meristems during reproductive development. However, genetic studies also show that PNY and PNF regulate the maintenance of the SAM. To understand the role of PNY and PNF in meristem maintenance, the expression patterns for genes that specifically localize to the peripheral and central regions of the SAM were examined in Arabidopsis (Arabidopsis thaliana). Results from these experiments indicate that the integrity of the CZ is impaired in pny pnf plants, which alters the balance of stem cell renewal and organogenesis. As a result, pools of CZ cells may be allocated into initiating leaf primordia. Consistent with these results, the integrity of the central region of pny pnf SAMs can be partially restored by increasing the size of the CZ. Interestingly, flower specification is also reestablished by augmenting the size of the SAM in pny pnf plants. Taken together, we propose that PNY and PNF act to restrict organogenesis to the PZ by maintaining a boundary between the CZ and PZ.

Sea slugs (Gastropoda: Opisthobranchia) are characterized by extensive morphological homoplasy. In particular, reduced or absent shells are predominant throughout the group. This trend towards shell loss has resulted in a poor fossil record. DNA-based phylogenies have been helpful in improving our understanding of the evolution of this group and major clades are emerging. We report 13 new complete opisthobranch mitochondrial genomes that provide robust support for some of these emerging nodes. We name three new clades within the Opisthobranchia, the Actopleura (Acteonoidea plus Nudipleura), Placoesophaga (Cephalaspidea plus Anaspidea), and Siphoglossa (Sacoglossa plus the Siphonaria). Finally we use molecular clock dating that suggests an earlier opisthobranch divergence than previously reported. The implications of this evolutionary scenario are discussed.

In Arabidopsis floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene LEAFY. The floral specification activity of FT is dependent upon two related BELL1-like homeobox (BLH) genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) which are required for floral evocation. PNY and PNF interact with a subset of KNOTTED1-LIKE homeobox proteins including SHOOT MERISTEMLESS (STM). Genetic analyses show that these BLH proteins function with STM to specify flowers and internodes during inflorescence development. In this study, experimental evidence demonstrates that the specification of flower and coflorescence meristems requires the combined activities of FT-FD and STM. FT and FD also regulate meristem maintenance during inflorescence development. In plants with reduced STM function, ectopic FT and FD promote the formation of axillary meristems during inflorescence development. Lastly, gene expression studies indicate that STM functions with FT-FD and AGAMOUS-LIKE 24 (AGL24)-SUPPRESSOR OF OVEREXPRESSION OF CONTANS1 (SOC1) complexes to up-regulate flower meristem identity genes during inflorescence development.

The RNA-binding protein Human Antigen R (HuR) is upregulated in pancreatic ductal adenocarcinoma (PDA), where it promotes tumorigenesis via its mRNA pro-survival targets. PDA cells exposed to DNA damage upregulate the mitotic inhibitor kinase, WEE1, in a HuR-dependent manner to induce cell cycle arrest and facilitate drug-resistance (1). Herein, we further evaluate a 56 base-pair (bp) region within WEE1's 3'UTR (labeled WEE1.3UTR) where HuR binds and stabilizes expression. Within this regulatory site, we observed that a 10-thymidine (T) track contains frequent polymorphisms (mean allele frequency 8.67%) of thymidine insertions (i.e, an INDEL). Using a combined approach of Sanger sequencing and a more sensitive capillary-electrophoresis (CE) assay, we screened this region in various cancer cell lines and patient samples. Results revealed three distinct alleles between individual cohorts: the wild-type (10-T, 56 bps), a 1-T insertion (11-T, 57 bps), and a 2-T insertion (12-T, 58 bps). Luciferase reporter constructs were subcloned with the HuR regulatory region embedded in the WEE1.3'UTR. In response to stress, constructs with the wild-type allele reported a higher level of expression compared to the 11-T and 12-T alleles (p Reference: 1. Lal et al., Cancer Res 2014;74(4):1128-40. Citation Format: Samantha Z. Brown, Avinoam Nevler, Henry T. Thomsett, Shruti Lal, Mahsa Zarei, Fernando Blanco, Joseph A. Cozzitorto, Alexis L. Norris-Kirby, Charles J. Yeo, Jordan M. Winter, James R. Eshleman, Jonathan R. Brody. Functional and clinical implications of an INDEL within the HuR regulatory region of the mitotic kinase inhibitor WEE1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4441.

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.

HuR (ELAV1), an RNA-binding protein abundant in cancer cells, primarily resides in the nucleus, but under specific stress (e.g., gemcitabine), HuR translocates to the cytoplasm in which it tightly modulates the expression of mRNA survival cargo. Here, we demonstrate for the first time that stressing pancreatic ductal adenocarcinoma (PDA) cells by treatment with DNA-damaging anticancer agents (mitomycin C, oxaliplatin, cisplatin, carboplatin, and a PARP inhibitor) results in HuR's translocation from the nucleus to the cytoplasm. Importantly, silencing HuR in PDA cells sensitized the cells to these agents, whereas overexpressing HuR caused resistance. HuR's role in the efficacy of DNA-damaging agents in PDA cells was, in part, attributed to the acute upregulation of WEE1 by HuR. WEE1, a mitotic inhibitor kinase, regulates the DNA damage repair pathway, and therapeutic inhibition of WEE1 in combination with chemotherapy is currently in early phase trials for the treatment of cancer. We validate WEE1 as a HuR target in vitro and in vivo by demonstrating (i) direct binding of HuR to WEE1′s mRNA (a discrete 56-bp region residing in the 3′ untranslated region) and (ii) HuR siRNA silencing and overexpression directly affects the protein levels of WEE1, especially after DNA damage. HuR's positive regulation of WEE1 increases γ-H2AX levels, induces Cdk1 phosphorylation, and promotes cell-cycle arrest at the G2–M transition. We describe a novel mechanism that PDA cells use to protect against DNA damage in which HuR posttranscriptionally regulates the expression and downstream function of WEE1 upon exposure to DNA-damaging agents. Cancer Res; 74(4); 1128–40. ©2014 AACR.

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. Cancer Res; 74(1); 31–37. ©2013 AACR.

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.

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 HuR's 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 mm3 vs 378.0 mm3, P < 0.005). Taken together with our past work in patient samples, this pre-clinical model demonstrates that HuR is essential for PDA growth in vivo. Future work will develop strategies to target HuR either as a monotherapy or in combination with other chemotherapies. 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.

Introduction: Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancers, predominantly an arginine to histidine amino acid change at codon 132. This structural alteration drives the catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate (2HG), which enhances DNA and protein hypermethylation and cellular dedifferentiation. Pharmacologic inhibitors of the mutant IDH1 protein show promise in clinical trials, yet the regulation of IDH1 has not been fully elucidated. We recently discovered in wild type IDH1 tumors that the regulatory RNA-binding and stress response protein, HuR (ELAVL1), protects cancer cells under nutrient deprivation by regulating the expression of core metabolic enzymes, including IDH1. We mapped the regulatory HuR binding site on the IDH1 transcript to the 3’-untranslated region. Since wild type and mutant IDH1 alleles both contain this binding sequence, we hypothesize that HuR is an important regulator of both isozymes and is biologically important in mutant IDH1 tumors. Methods: HuR expression was suppressed by siRNA in a fibrosarcoma cell line (HT-1080) harboring a natural heterozygous IDH1 mutation and cell viability was determined by PicoGreen and Trypan blue exclusion assays. Sensitivity to pharmacologic inhibition of the mutant IDH1 protein was assessed. Sanger sequencing and mRNP-IP were performed to determine HuR's impact on the expression of each IDH1 allele. To further characterize the post-transcriptional regulation of IDH1 by HuR, CRISPR/CAS 9 editing of the HuR gene was performed. Results: Sanger sequencing of IDH1 after depletion of HuR in HT1080 cells, as well as after HuR mRNP-IP, revealed that HuR regulates both the mutant and wild type IDH1. Drug sensitivity assays to a mutant IDH1 inhibitor (AGI-5198) under varying glucose concentrations revealed glucose deprivation to be a novel driver of chemo-resistance. However, HuR silencing abrogated HT1080 resistance to AGI-5198 under these conditions. Targeted knockout of HuR using the CRISPR/CAS9 system resulted in potent suppression of mutant and wild type IDH1, at both the mRNA and protein levels when compared to CRISPR/Cas9 control. Conclusions: These findings reveal that harsh metabolic conditions present in the tumor microenvironment induce chemo-resistance in an IDH1 mutant cell line to a mutant IDH1 inhibitor, in an HuR dependent manner. Moreover, both mutant and wild type IDH1 alleles are potently regulated by HuR. Therapeutic strategies that target the HuR-IDH1 axis and block this resistance mechanism may enhance the efficacy of mutant IDH1 inhibitors for relevant tumors. Citation Format: Mahsa Zarei, Shruti Lal, Nicole C. Mambelli-Lisboa, Edwin Cheung, Saswati N. Chand, Charles J. Yeo, Jonathan R. Brody, Jordan M. Winter. The mRNA-binding protein HuR, regulates mutant and wild type IDH1 expression in IDH1-mutated cancer. [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 1847.

Introductory Sentence: The objective of this study is to understand the role that HuR (an RNA binding protein) plays in the DNA damage response pathway in pancreatic cancer cells. Introduction: Maintenance of genomic stability largely depends on the DNA damage response (DDR) and a failure to activate this response can initiate genomic instability, a hallmark of pancreatic ductal adenocarcinoma (PDA). Further, defects or acute changes in the DDR most likely have therapeutic implications for drug resistance mechanisms to DNA damaging agents. HuR (ELAV1), a ubiquitous member of Hu family of RNA binding proteins, influences gene expression during certain pancreatic cancer associated stressors (e.g., gemcitabine and hypoxia). Following specific stress, HuR translocates from the nucleus to the cytoplasm and either modulates the stability or translation of dozens of mRNA targets. ATM and ATR, DNA damage protein kinases, and their substrates checkpoint kinases CHK1 and CHK2 are key players in response to DNA damage. Previously, it has been shown that ATM/ ATR activates CHK1/CHK2 and together this complex influences HuR’s function. CHK1 affects HuR’s cytoplasmic export while CHK2 modulates HuR’s binding to its target mRNAs. In this study, we assessed for the first time HuR’s ability to become functional; and in turn, modulate the DDR in pancreatic cancer cells. Experimental Procedures: We determined whether HuR translocation from the nucleus to the cytoplasm (i.e., HuR activation) via western blotting and Immunoflorescence (IF) assays after PDA cells lines MiaPaCa2 and PL5 were stressed by different DNA damaging agents mitomycin C (MMC; 150 nM), oxaliplatin (1 µM), cisplatin (1.5 µM), carboplatin (8 µM), gemcitabine (1 µM) and PARP inhibitor (ABT-888; 150 µM). A cell survival assay was performed determining the IC50 concentrations of DNA damaging agents between PDA cells transfected with siRNA oligos against HuR and control sequence. We used western blotting and Immunoflorescence (IF) assays to detect phosphorylated and total histone H2AX (γH2AX), a key component of DDR, to quantify DNA double strand breaks (DSBs) between control and silenced HuR cells. Approximately 100-150 nuclei were evaluated to quantify γH2AX foci for each sample by using ImageJ software. Western blotting was performed to detect HuR cleavage in lysates from PDA cells exposed to high MMC dose treatment (0.2µM-2µM). RNA from ribonucleoprotein-immunoprecipitation (RIPs) assays using an antibody against HuR was used for quantitative-PCR (qPCR) analysis to determine DDR pathway-related HuR targets. Results: Cytoplasmic protein lysates extracted from PDA cells treated with MMC, oxaliplatin, cisplatin, carboplatin, gemcitabine and PARP-inhibitor demonstrated enhanced HuR expression in the cytoplasm (while 5-fluorouracil did not, a negative control). In addition, silencing HuR in PDA cells sensitized cell lines to MMC by 10-20% compared to the control PDA cells. Based on a determined IC50, a 150nM dose was chosen for MMC to induce DNA damage without causing significant cell death in control and HuR silenced cells. To assess whether HuR may play a role in the DNA repair, control and silenced HuR PDA cells were treated with 150nM of MMC. The IF assay revealed that MMC treatment induced γH2AX foci in both control and silenced HuR cells after 2 hrs indicating the presence of DNA damage breaks. Remarkably, after 6 hours of MMC treatment the number of foci per nuclei increased significantly in silenced HuR cells compared to the control cells, demonstrating that DNA damage persisted and DNA repair was considerably delayed in the absence of HuR. Paradoxically, treatment of silenced HuR cells with 200nM of MMC and above reduces the γH2AX foci formation indicating that higher doses might cleave HuR function leading cells to apoptosis. Accordingly, immunoblot analysis of lysates from PDA cells treated with higher doses of MMC (0.2µM-2µM) resulted in the detection of caspasedependent HuR cleavage, yielding a 26 kDa cleavage product. In addition, western blot analysis indicates that HuR affects both total H2AX and H2AX phosphorylation in PDA cells following MMC treatment. From a RIP- sequencing experiment, we identified novel HuR target genes related to the DNA damage response including WEE1 and CHK1. qPCR results validated that WEE1 expression increases up to 80 fold compared to an IgG control-IP, validating WEE1 as a novel HuR target in the DNA damage response pathway. Conclusion: We demonstrate here that HuR is vital for the PDA cells to recover from damage induced by a number of clinically relevant DNA damaging agents. HuR levels influence both gamma and total phosphoprotein H2AX levels following MMC treatment and further studies will decipher if HuR is directly regulating H2AX or other DDR proteins. In addition, HuR may control G2-M checkpoint by regulating tyrosine kinase WEE1. These data suggest that HuR is central for the acute DDR in PDA cells. Additional studies will explore the significance of HuR in regulating gross chromosomal instability as well as its importance for an unexplained chemotherapeutic resistance mechanism that happens in the majority of PDA patients. Citation Format: Shruti Lal, Vikram Bhattacharjee, Timothy Yen, Richard A. Burkhart, Danielle M. Pineda, Charles J. Yeo, Jordan M. Winter, Isidore Rigoutsos, Jonathan R. Brody. HuR, an RNA binding protein, is critical for the DNA damage response in pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A18.