4 Supplementary Figures from Small Interfering RNA–Mediated Polo-Like Kinase 1 Depletion Preferentially Reduces the Survival of p53-Defective, Oncogenic Transformed Cells and Inhibits Tumor Growth in Animals

Objectives: Veliparib (V) is an oral inhibitor of poly(ADP-ribose) polymerases (PARP)-1 and -2. V delays the repair of DNA damage induced by chemotherapeutic agents and increases the sensitivity of tumor cells to DNA-damaging agents in vitro. BRCA-deficient tumors are more sensitive to PARP inhibitors when used as monotherapy or in combination with DNA-damaging agents. Preclinically, V has demonstrated single-agent activity in BRCA-deficient tumor models and has shown robust potentiation of carboplatin (C), gemcitabine (G), and the combination of C/G. This study established the maximum tolerated dose (MTD) for V in combination with C/G and evaluated safety and tolerability of combination therapy followed by maintenance V in a cohort of patients with advanced ovarian cancer.

This study sought to evaluate a 99mTc-labeled trimeric cyclic RGD peptide (99mTc-4P-RGD3) as the new radiotracer for tumor imaging. The objective was to compare its biological properties with those of 99mTc-3P-RGD2 in the same animal model.HYNIC-4P-RGD3 was prepared by reacting 4P-RGD3 with excess HYNIC-OSu in the presence of diisopropylethylamine. 99mTc-4P-RGD3 was prepared using a kit formulation, and evaluated for its tumor-targeting capability and biodistribution properties in the BALB/c nude mice with U87MG human glioma xenografts. Planar and SPECT imaging studies were performed in athymic nude mice with U87MG glioma xenografts. For comparison purpose, 99mTc-3P-RGD2 (a αvβ3-targeted radiotracer currently under clinical evaluation for tumor imaging in cancer patients) was also evaluated in the same animal models. Blocking experiments were used to demonstrate the αvβ3 specificity of 99mTc-4P-RGD3.99mTc-4P-RGD3 was prepared with > 95% RCP and high specific activity (~ 200 GBq/μmol). 99mTc-4P-RGD3 and 99mTc-3P-RGD2 shared almost identical tumor uptake and similar biodistribution properties. 99mTc-4P-RGD3 had higher uptake than 99mTc-3P-RGD2 in the intestines and kidneys; but it showed better metabolic stability. The U87MG tumors were clearly visualized by SPECT with excellent contrast with 99mTc-4P-RGD3 and 99mTc-3P-RGD2.Increasing peptide multiplicity from 3P-RGD2 to 4P-RGD3 offers no advantages with respect to the tumor-targeting capability. 99mTc-4P-RGD3 is as good a SPECT radiotracer as 99mTc-3P-RGD2 for imaging αvβ3-positive tumors.

The serine/threonine kinases, Akt1/PKBα, Akt2/PKBβ, Akt3/PKBγ, have been implicated in preventing cells from undergoing apoptosis. Although several small molecule inhibitors of Akt have been reported to induce apoptosis in cancer cells, these inhibitors may have additional targets. In the current study, we used an Akt3 small interfering RNA (Akt3 siRNA) to analyze apoptosis induction in Akt1 and Akt2 double knock out MEF cells (MEF-Akt1,2-DKO). Our data indicate that Akt3 siRNA inhibits Akt3 protein expression in a dose-dependent manner. As a result, phosphorylation of Akt and its down-stream targets, including FKHRL1 and GSK3α/β, are reduced accordingly. The treatment also induces apoptosis in MEF-Akt1,2-DKO cells. However, apoptosis induction is significant only when more than 80% of Akt3 protein was depleted. Reintroducing Akt3 totally rescued Akt3siRNA induced apoptosis in MEF-Akt1,2DKO cells. In addition, re-introducing Akt1 also inhibited apoptosis induced by Akt3 siRNA. Moreover, Akt3 siRNA potentiates different stress-induced apoptosis in MEF-Akt1,2-DKO cells at a lower dose when compared to what is required for apoptosis induction by itself. Our study suggests that only a small portion of Akt is active in wild-type MEF cells and a threshold of Akt inhibition is required to induce apoptosis by pure Akt inhibitors. In addition, our data indicate that cells under stress require more Akt for its survival.

Chk1 (checkpoint kinase 1) is a serine-threonine kinase that is critical for G2/M arrest in response to DNA damage. Chk1 phosphorylates Cdc25C at serine-216, a major regulatory site, in response to DNA damage. Furthermore, Chk1 also phosphorylates Cdc25A on serine 123 which accelerates its degradation through the ubiquitin-proteasome pathway and arrests cells in late G2-phase after DNA damage. In the present study, we demonstrated that Chk1 phosphorylates pro-apoptotic protein BAD (Bcl-2/Bcl-XL-Antagonist, causing cell Death) in vitro. In vitro phosphorylation analysis with various mouse BAD peptides has revealed two phosphorylation sites for Chk1 at serine-155 and serine-170. When wild-type and mutant BAD (S155A) constructs were transfected into 293T cells, an association between BAD and Chk1 was observed by co-immunoprecipitation. In addition, there was an increase in the phosphorylation of serine-155 following DNA damage by adriamycin treatment. Our results suggest that Chk1 associates with BAD and phosphorylates the BAD protein at serine-155. Taken together, our results suggest that Chk1 may inactivate BAD by associating with and phosphorylating residues critical for BAD function in response to DNA damage.