Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer

Azorsa, David O.; Gonzales, Irma M.; Basu, Gargi D.; Choudhary, Ashish; Arora, Shilpi; Bisanz, Kristen; Kiefer, Jeffrey; Henderson, Meredith C.; Trent, Jeffrey M.; Hoff, Daniel D. Von; Mousses, Spyro

doi:10.1186/1479-5876-7-43

Cited by 93 publications

(80 citation statements)

References 51 publications

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“…mRNA expression analysis by reverse transcription-PCR requires less material, is reproducible and fully quantitative, and thus has a higher potential for miniaturization. This effort is complementary and has similar intent as high-throughput lethality assays using gene interference to knockdown target genes in combination with conventional chemotherapy as a strategy to rationally identify combination partners (22). The ability to knockdown genes requires an in vitro platform such as a panel of cell lines.…”

Section: Discussionmentioning

confidence: 99%

A Fine-Needle Aspirate–Based Vulnerability Assay Identifies Polo-Like Kinase 1 as a Mediator of Gemcitabine Resistance in Pancreatic Cancer

Jimeno

Rubio-Viqueira

Rajeshkumar

et al. 2010

Molecular Cancer Therapeutics

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This work aimed to discover targets for combination treatment with gemcitabine in pancreatic cancer. We selected 11 tumors from our live collection of freshly generated pancreatic cancer xenografts with known degrees of varying gemcitabine sensitivity. We briefly (6 h) exposed fine-needle aspiration material to control vehicle or gemcitabine (1 μmol/L) and compared the gene expression of the treated and untreated samples using a reverse transcription-PCR-based, customized low-density array with 45 target genes of therapeutic interest. The gene expression of the untreated sample (which can be considered a baseline/static readout) was not predictive of gemcitabine efficacy in these tumors. Altogether, the only gene that differentiated sensitive versus resistant cases was polo-like kinase 1 (Plk1), showing >50% downregulation in sensitive cases and no change in the resistant cases. Inhibition of Plk1 by either small interfering RNA gene knockdown or with the Plk1 pathway modulator (ON 01910.Na) synergized with gemcitabine in gemcitabine-refractory in vitro models providing mechanistic proof of concept. In vivo experiments in gemcitabine-resistant xenografts showed synergistic activity decreasing cell proliferation and tumor regressions. A quantitative gene expression-based vulnerability assay identified Plk1 as a relevant target dictating the susceptibility of pancreatic cancer to gemcitabine. Dynamic interrogation of cancer has the potential to provide key information about mechanisms of resistance and to enhance individualization of treatment. Mol Cancer Ther; 9(2); 311-8. ©2010 AACR.

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Section: Discussionmentioning

confidence: 99%

A Fine-Needle Aspirate–Based Vulnerability Assay Identifies Polo-Like Kinase 1 as a Mediator of Gemcitabine Resistance in Pancreatic Cancer

Jimeno

Rubio-Viqueira

Rajeshkumar

et al. 2010

Molecular Cancer Therapeutics

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“…lethal partners of PNKP, representing 6.1% of genes tested, which is typical for initial screens of this type (33)(34)(35). Of these proteins, 14 are currently considered to be tumor suppressors, including PTEN and SHP-1.…”

Section: Discussionmentioning

confidence: 99%

Genetic Screening for Synthetic Lethal Partners of Polynucleotide Kinase/Phosphatase: Potential for Targeting SHP-1–Depleted Cancers

et al. 2012

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A genetic screen using a library of 6,961 siRNAs led to the identification of SHP-1 (PTPN6), a tumor suppressor frequently mutated in malignant lymphomas, leukemias, and prostate cancer, as a potential synthetic lethal partner of the DNA repair protein polynucleotide kinase/phosphatase (PNKP). After confirming the partnership with SHP-1, we observed that codepletion of PNKP and SHP-1 induced apoptosis. A T-cell lymphoma cell line that is SHP-1 deficient (Karpas 299) was shown to be sensitive to a chemical inhibitor of PNKP, but resistance was restored by expression of wild-type SHP-1 in these cells. We determined that while SHP-1 depletion does not significantly impact DNA strand-break repair, it does amplify the level of reactive oxygen species (ROS) and elevate endogenous DNA damage. The ROS scavenger WR1065 afforded protection to SHP-1-depleted cells treated with the PNKP inhibitor. We propose that codisruption of SHP-1 and PNKP leads to an increase in DNA damage that escapes repair, resulting in the accumulation of cytotoxic double-strand breaks and induction of apoptosis. This supports an alternative paradigm for synthetic lethal partnerships that could be exploited therapeutically. Cancer Res; 72(22); 5934-44. Ó2012 AACR.

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“…7,12,13 Briefly, a validated siRNA library targeting 572 kinases (Kinome siRNA Library Version 1.0; QIAGEN) with 2 different siRNA sequences per kinase was printed onto 384-well plates (Fisher Scientific). Cationic lipid-based transfection reagents were diluted in Opti-MEM (Invitrogen) and added to assay plates using a Fill liquid dispenser (Bio-Tek).…”

Section: High-throughput Sirna Screensmentioning

confidence: 99%

“…We adapted a siRNAscreening platform in our laboratory 7,8,12,13 that uses transfection conditions without electroporation to myeloid suspension cells and performed the first kinome-wide siRNA-Ara-C sensitizer screen in leukemia cells. WEE-1 kinase family genes (WEE1 and PKMYT1) and CHEK1 emerged as top targets potentiating Ara-C activity.…”

Section: Introductionmentioning

confidence: 99%

RNAi screening of the kinome with cytarabine in leukemias

Tibes

Bogenberger

Chaudhuri

et al. 2012

Blood

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To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

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Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer

Cited by 93 publications

References 51 publications

A Fine-Needle Aspirate–Based Vulnerability Assay Identifies Polo-Like Kinase 1 as a Mediator of Gemcitabine Resistance in Pancreatic Cancer

A Fine-Needle Aspirate–Based Vulnerability Assay Identifies Polo-Like Kinase 1 as a Mediator of Gemcitabine Resistance in Pancreatic Cancer

Genetic Screening for Synthetic Lethal Partners of Polynucleotide Kinase/Phosphatase: Potential for Targeting SHP-1–Depleted Cancers

RNAi screening of the kinome with cytarabine in leukemias

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