Supplementary Figure 2 from Extracellular Signal-Related Kinase Positively Regulates Ataxia Telangiectasia Mutated, Homologous Recombination Repair, and the DNA Damage Response

Supplementary Figure Legends 1-4 from Extracellular Signal-Related Kinase Positively Regulates Ataxia Telangiectasia Mutated, Homologous Recombination Repair, and the DNA Damage Response

PDF File - 571K, Human U1242 glioma cells form highly invasive and aggressive orthotopic tumors in mice.

Supplementary Data from Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion

Supplementary Figure 3 from Extracellular Signal-Related Kinase Positively Regulates Ataxia Telangiectasia Mutated, Homologous Recombination Repair, and the DNA Damage Response

PDF File - 578K, Human U1242/luc-GFP gliomas show dose-dependent increases in p-(S824)-KAP1 foci formation after radiation which can be inhibited by KU-60019.

PDF File - 49K, Imaging of tumors that were apparently cured.

PDF file - 53K, Preparation of calibration and QC tissue matrix.

Supplementary Figure 1 from Extracellular Signal-Related Kinase Positively Regulates Ataxia Telangiectasia Mutated, Homologous Recombination Repair, and the DNA Damage Response

PDF file - 159K, U1242 tumors with 'forced' expression of mutant p53 are more sensitive to KU-60019.

PDF File - 80K, Surrogate dye show excellent distribution in the brain by osmotic pump delivery.

PDF File - 80K, KU-60019 administered by CED radiosensitizes U1242 tumors.

PDF file - 58K, Concentrations of KU-60019 in mouse brain after CED.

Supplementary Figure 4 from Extracellular Signal-Related Kinase Positively Regulates Ataxia Telangiectasia Mutated, Homologous Recombination Repair, and the DNA Damage Response

PDF file - 80K, Imaging of U87/luc-DsRed (p53-281G) bearing mice.

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65-S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM-PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

Abstract Purpose: Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of only 12 to 15 months. Current standard treatment consists of surgery followed by chemoradiation. The poor survival of patients with GBM is due to aggressive tumor invasiveness, an inability to remove all tumor tissue, and an innate tumor chemo- and radioresistance. Ataxia–telangiectasia mutated (ATM) is an excellent target for radiosensitizing GBM because of its critical role in regulating the DNA damage response and p53, among other cellular processes. As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, and growth, and potently radiosensitized human glioma cells in vitro. Experimental Design: Using orthotopic xenograft models of GBM, we now show that KU-60019 is also an effective radiosensitizer in vivo. Human glioma cells expressing reporter genes for monitoring tumor growth and dispersal were grown intracranially, and KU-60019 was administered intratumorally by convection-enhanced delivery or osmotic pump. Results: Our results show that the combined effect of KU-60019 and radiation significantly increased survival of mice 2- to 3-fold over controls. Importantly, we show that glioma with mutant p53 is much more sensitive to KU-60019 radiosensitization than genetically matched wild-type glioma. Conclusions: Taken together, our results suggest that an ATM kinase inhibitor may be an effective radiosensitizer and adjuvant therapy for patients with mutant p53 brain cancers. Clin Cancer Res; 19(12); 3189–200. ©2013 AACR.

Currently, intraperitoneal (IP) injection of D-luciferin is the preferred method of providing substrate for bioluminescent imaging (BLI); however it has a failure rate of 3-10% due to accidental intestinal injection. The present study evaluates the quality of BLI after subcutaneous (SC) injection of D-luciferin and demonstrates the effectiveness of SC injection in anatomically disparate tumor models. Mice bearing luciferase-expressing tumors underwent BLI after SC or IP injection of D-luciferin. The average time to maximal luminescence was 6 min (range 5-9 min) after SC injection and 8 min (range 5-8 min) after IP injection. Within 7 minutes of injection, SC and IP routes yielded similar luminescence in subcutaneous, intracranial, tongue, and lung xenograft tumor models. In a model of combined subcutaneous and intracranial xenografts, SC injection resulted in proportional luminescence at all sites, confirming that preferential delivery of substrate does not occur. While tumors were occasionally not visualized with IP injection, all tumors were visualized reliably with SC injection. Thus, SC injection of D-luciferin is a convenient and effective alternative to IP injection for BLI in nude mice. It may be a preferable approach, particularly for tumors with weaker signals and/or when greater precision is required.

Bioluminescence imaging (BLI) is a relatively new noninvasive technology used for quantitative assessment of tumor growth and therapeutic effect in living animal models. BLI involves the generation of light by luciferase-expressing cells following administration of the substrate luciferin in the presence of oxygen and ATP. In the present study, the effects of hypoxia, hypoperfusion, and pH on BLI signal (BLS) intensity were evaluated in vitro using cultured cells and in vivo using a xenograft model in nude mice. The intensity of the BLS was significantly reduced in the presence of acute and chronic hypoxia. Changes in cell density, viability, and pH also affected BLS. Although BLI is a convenient non-invasive tool for tumor assessment, these factors should be considered when interpreting BLS intensity, especially in solid tumors that could be hypoxic due to rapid growth, inadequate blood supply, and/or treatment.