P. C. Mack

Impairment of the DNA repair and growth arrest pathways by p53R2 silencing enhances DNA damage-induced apoptosis in a p53-dependent manner in prostate cancer cells.

p53R2 is a p53-inducible ribonucleotide reductase that contributes to DNA repair by supplying deoxynucleotide triphosphate pools in response to DNA damage. In this study, we found that p53R2 was overexpressed in prostate tumor cell lines compared with immortalized prostatic epithelial cells and that the protein was induced upon DNA damage. We investigated the effects of p53R2 silencing on DNA damage in LNCaP cells (wild-type p53). Silencing p53R2 potentiated the apoptotic effects of ionizing radiation and doxorubicin treatment as shown by increased sub-G1 content and decreased colony formation. This sensitizing effect was specific to DNA-damaging agents. Comet assay and γ-H2AX phosphorylation status showed that the decreased p53R2 levels inhibited DNA repair. Silencing p53R2 also reduced the levels of p21WAF1/CIP1 at the posttranscriptional level, suggesting links between the p53-dependent DNA repair and cell cycle arrest pathways. Using LNCaP sublines stably expressing dominant-negative mutant p53, we found that the sensitizing effect of p53R2 silencing is mediated by p53-dependent apoptosis pathways. In the LNCaP sublines (R273H, R248W, and G245S) that have defects in inducing p53-dependent apoptosis, p53R2 silencing did not potentiate DNA damage–induced apoptosis, whereas p53R2 silencing was effective in a LNCaP subline (P151S) which retains the ability to induce p53-dependent apoptosis. This study shows that p53R2 is a potential therapeutic target that could be used to enhance the effectiveness of ionizing radiation or DNA-damaging chemotherapy in a subset of patients with prostate cancer. (Mol Cancer Res 2008;6(5):808–18)

Abstract IA20: Linking tumor genomics to patient outcomes through a large-scale patient-derived xenograft (PDX) platform.

Background: Preclinical models have generally proven suboptimal for directing clinical application of new anti-cancer therapies. Here we detail an integrated research platform engaging core resources at JAX-WEST and the clinical research and genomics facilities at UCDCCC. Pilot studies using this platform are focusing on non-small cell lung cancer (NSCLC) due to molecular targets of interest, such as epidermal growth factor receptor (EGFR), heterogeneity in NSCLC tumor biology and the complexity of related cancer signaling pathways. Methods: Clinically and demographically annotated cancer patients (pt) seen at UCDCCC and collaborating facilities undergo tumor biopsy of various types which are implanted into JAX Nod Scid Gamma (NSG) mice to develop PDXs. Pt tumors and subsequent PDXs are assessed by histomorphology, clinically applicable molecular biomarkers, gene expression arrays and genome-wide technologies (NGS). NSCLC PDXs are grouped as panels (EGFR mutant (MT), KRAS MT ALK+). PDX panels of interest undergo multi-regimen drug testing for differential efficacy, together with pre- and post-therapy NGS and timed tumor pharmacodynamics (PD) assessment, to determine mechanisms of primary and acquired resistance in individual PDX models and how to overcome them. Results: As of November 2013 over 1,200 cancer pt tumors have been xenotransplanted into NSG mice (~175 from NSCLC), including successful PDX formation from small FNA and, cell pellets and transportability of specimens by overnight shipping for implantation. NSCLC PDXs show excellent histomorphologic, gene expression and mutational fidelity to host pt tumors, including mutation status for KRAS, EGFR and gene expression levels. Pilot studies in a panel of EGFR MT PDXs with TKI-acquired resistance demonstrate differential drug activity which mimics that of the host pt to the same therapy, and tumor PD at baseline and timed intervals post-therapy provide the basis for subgrouping resistance mechanisms Conclusion: This UCDCCC-JAX collaboration has established a large resource applicable to multi-drug testing and tumor PD in a wide range of clinically and genomically characterized tumors, including PDX panels for representative oncogene-driven NSCLCs. An EGFR-directed pilot project supports the feasibility of systematically integrating data derived from these models in order to optimize drug development and treatment strategies to address drug resistance mechanisms. This approach to PDX development and testing will be prospectively integrated into a developing multi-institution clinical trial of the Southwest Oncology Group (S1403), designed to advance understanding of differences in inter- and intra-patient tumor biology and hasten the transition to personalized cancer therapy. Citation Format: David R. Gandara, T. Li, P.N. Lara, K. Kelly, D.T. Cooke, R. Gandour-Edwards, K. Yoneda, N. Goodwin, S. Kuslak-Meyer, P. Mack. Linking tumor genomics to patient outcomes through a large-scale patient-derived xenograft (PDX) platform. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr IA20.

A phase 0 microdosing trial of an in vivo assay for predicting chemoresistance to platinum.

2578^ Background: As alkylating agents, platinum (Pt) analogs kill cancer cells mainly through induction of DNA damage. We hypothesize that low Pt-induced DNA damage is predictive of chemoresistance. We have developed a supersensitive accelerator mass spectrometry (AMS) method, with a sensitivity of 10-18~21 mole. AMS can detect carboplatin (carbo)-induced DNA damage in tissues after patients receive one subtoxic microdose (1/100th the therapeutic dose) of 14C-labeled carbo, and further allows mechanistic analysis of chemoresistance. METHODS A 2-stage accrual design was employed. In the first stage, a dose escalation/de-escalation strategy was used to determine toxicity and recommended Phase II dose (RP2D) of 14C-Carbo. Stage II was designed to determine if DNA damage levels induced by microdosing carbo correlate with chemoresistance, and to determine underlying resistance mechanisms. Patients with advanced non-small cell lung cancer or bladder cancer planning to receive Pt-based therapy are eligible. Primary endpoint is to determine and compare DNA damage levels induced by microdosing and therapeutic carbo. Secondary endpoints: pharmacokinetics (PK) of microdosing and therapeutic carbo, levels of tumoral DNA damage, and tumor response. RESULTS Stage I of the Phase 0 trial has been completed with an accrual of 5 patients. The Dose Level I of 107 dpm/kg of body weight is RP2D of 14C-carbo, which results in a desirable 14C signal-to-noise of 10~100 times background. This 14C level could be easily detected by AMS, but with minimal radiation exposure to patients (less than 1% that of an abdominal CT scan.). No acute toxicity was observed. Serum half life of carbo ranged from 1.30 to 1.58 hours-well within the reported range for the therapeutic carbo. DNA damage levels positively correlated with carbo half-life. Comparisons of the microdosing and therapeutic carbo PK, correlation of DNA damage and tumor response to subsequent chemotherapy, and the results of Stage II trial will be reported. CONCLUSIONS A Phase 0 trial as a platform to study Pt resistance can be feasibly conducted with one non-toxic microdose of 14C-labeled carbo using supersensitive AMS-based technology and has implications for personalized therapy.

Untargeted Use of Targeted Therapy

Lung cancer is the leading cause of cancer death in men and women and frequently presents as advanced disease. The majority of lung cancers are of the non-small cell type, for which chemotherapy has demonstrated modest survival benefits at all stages of disease. Clearly, more effective therapies are needed. Agents that alter critical molecular cell growth pathways, so-called targeted therapies, are a growing area of research and development. Targeted therapies including drugs directed at the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) have recently established a role in the treatment of advanced stage non-small cell lung cancer (NSCLC). These drugs and many others are undergoing investigation, either as single agents or in combination with cytotoxics or other targeted therapies, with dual goals of improved efficacy and reduced toxicity. Although progress has been made in target identification for lung cancer treatment, the ability to select groups of NSCLC patients who benefit from these therapies based on predictive markers remains a challenge. Ongoing studies correlating potential predictive biomarkers with patient outcome are designed to refine the use of developing targeted therapies, that is, to provide a rational basis for “targeted use of targeted therapies.” Despite some recent breakthroughs in identifying molecular signatures predictive of benefit, much remains to be learned. Using erlotinib and bevacizumab as prime examples, this chapter will review a dilemma currently facing both basic scientists and clinical investigators engaged in the study of NSCLC, namely how to develop and test paradigms for individualizing patient therapy.