Khanh Do

Identification of Potential mRNA Biomarkers in Peripheral Blood Lymphocytes for Human Exposure to Ionizing Radiation

Abstract Amundson, S. A., Do, K. T., Shahab, S., Bittner, M., Meltzer, P., Trent, J. and Fornace, A. J., Jr. Identification of Potential mRNA Biomarkers in Peripheral Blood Lymphocytes for Human Exposure to Ionizing Radiation. Since early in the Atomic Age, biological indicators of radiation exposure have been sought, but currently available methods are not entirely satisfactory. Using cDNA microarray hybridization to discover new potential biomarkers, we have identified genes expressed at increased levels in human peripheral blood lymphocytes after ex vivo irradiation. We recently used this technique to identify a large set of ionizing radiation-responsive genes in a human cell line (Oncogene 18, 3666–3672, 1999). The present set of radiation markers in peripheral blood lymphocytes was identified 24 h after treatment, and while the magnitude of mRNA induction generally decreased over time, many markers were still significantly elevated up to 72 h after irradiation. In all donors, the most highly responsive gene identified was DDB2, which codes for the p48 subunit of XPE, a protein known to play a crucial role in repair of ultraviolet (UV) radiation damage in DNA. Induction of DDB2, CDKN1A (also known as CIP1/WAF1) and XPC showed a linear dose–response relationship between 0.2 and 2 Gy at 24 and 48 h after irradiation, with less linearity at earlier or later times. These results suggest that relative levels of gene expression in peripheral blood cells may provide estimates of environmental radiation exposures.

Induction of Stress Genes by Low Doses of Gamma Rays

Using cells of a human myeloid tumor cell line (ML-1), we have detected induction of several stress-responsive genes by doses of gamma rays below 50 cGy. We found a linear dose-response relationship for induction of CDKN1A (formerly known as CIP1/WAF1) and GADD45 mRNA levels over the range of 2-50 cGy, with no evidence of a threshold for induction. Although exposures to 2 and 5 cGy did not result in any detectable reduction in cloning efficiency or increased apoptosis in ML-1 cells, these exposures did produce a transient delay of cells in the phases of the cell cycle in addition to the observed up-regulation of CDKN1A and GADD45. The relative induction of genes such as CDKN1A by radiation doses that produce little toxicity indicates that surviving cells do contribute significantly to the observed stress responses. These studies should provide insight into the molecular responses to physiologically relevant doses that cannot necessarily be extrapolated from high-dose studies.

Integrating global gene expression and radiation survival parameters across the 60 cell lines of the National Cancer Institute Anticancer Drug Screen

The 60 cell lines of the National Cancer Institute Anticancer Drug Screen (NCI-60) constitute the most extensively characterized in vitro cancer cell model. They have been tested for sensitivity to more than 100,000 potential chemotherapy agents and have been profiled extensively at the DNA, RNA, protein, functional, and pharmacologic levels. We have used the NCI-60 cell lines and three additional lines to develop a database of responses of cancer cells to ionizing radiation. We compared clonogenic survival, apoptosis, and gene expression response by microarray. Although several studies have profiled relative basal gene expression in the NCI-60, this is the first comparison of large-scale gene expression changes in response to genotoxic stress. Twenty-two genes were differentially regulated in cells with low survival after 2-Gy gamma-rays; 14 genes identified lines more sensitive to 8 Gy. Unlike reported basal gene expression patterns, changes in expression in response to radiation showed little tissue-of-origin effect, except for differentiating the lymphoblastoid cell lines from other cell types. Basal expression patterns, however, discriminated well between radiosensitive and more resistant lines, possibly being more informative than radiation response signatures. The most striking patterns in the radiation data were a set of genes up-regulated preferentially in the p53 wild-type lines and a set of cell cycle regulatory genes down-regulated across the entire NCI-60 panel. The response of those genes to gamma-rays seems to be unaffected by the myriad of genetic differences across this diverse cell set; it represents the most penetrant gene expression response to ionizing radiation yet observed.

Stress-specific signatures: expression profiling of p53 wild-type and -null human cells

Gene expression responses of human cell lines exposed to a diverse set of stress agents were compared by cDNA microarray hybridization. The B-lymphoblastoid cell line TK6 (p53 wild-type) and its p53-null derivative, NH32, were treated in parallel to facilitate investigation of p53-dependent responses. RNA was extracted 4 h after the beginning of treatment when no notable decrease in cell viability was evident in the cultures. Gene expression signatures were defined that discriminated between four broad general mechanisms of stress agents: Non-DNA-damaging stresses (heat shock, osmotic shock, and 12-O-tetradecanoylphorbol 13-acetate), agents causing mainly oxidative stress (arsenite and hydrogen peroxide), ionizing radiations (neutron and γ-ray exposures), and other DNA-damaging agents (ultraviolet radiation, methyl methanesulfonate, adriamycin, camptothecin, and cis-Platinum(II)diammine dichloride (cisplatin)). Within this data set, non-DNA-damaging stresses could be discriminated from all DNA-damaging stresses, and profiles for individual agents were also defined. While DNA-damaging stresses showed a strong p53-dependent element in their responses, no discernible p53-dependent responses were triggered by the non-DNA-damaging stresses. A set of 16 genes did exhibit a robust p53-dependent pattern of induction in response to all nine DNA-damaging agents, however.

Phase I Study of Single-Agent AZD1775 (MK-1775), a Wee1 Kinase Inhibitor, in Patients With Refractory Solid Tumors

PURPOSE Wee1 tyrosine kinase phosphorylates and inactivates cyclin-dependent kinase (Cdk) 1/2 in response to DNA damage. AZD1775 is a first-in-class inhibitor of Wee1 kinase with single-agent antitumor activity in preclinical models. We conducted a phase I study of single-agent AZD1775 in adult patients with refractory solid tumors to determine its maximum-tolerated dose (MTD), pharmacokinetics, and modulation of phosphorylated Tyr15-Cdk (pY15-Cdk) and phosphorylated histone H2AX (γH2AX) levels in paired tumor biopsies. PATIENTS AND METHODS AZD1775 was administered orally twice per day over 2.5 days per week for up to 2 weeks per 21-day cycle (3 + 3 design). At the MTD, paired tumor biopsies were obtained at baseline and after the fifth dose to determine pY15-Cdk and γH2AX levels. Six patients with BRCA-mutant solid tumors were also enrolled at the MTD. RESULTS Twenty-five patients were enrolled. The MTD was established as 225 mg twice per day orally over 2.5 days per week for 2 weeks per 21-day cycle. Confirmed partial responses were observed in two patients carrying BRCA mutations: one with head and neck cancer and one with ovarian cancer. Common toxicities were myelosuppression and diarrhea. Dose-limiting toxicities were supraventricular tachyarrhythmia and myelosuppression. Accumulation of drug (t1/2 approximately 11 hours) was observed. Reduction in pY15-Cdk levels (two of five paired biopsies) and increases in γH2AX levels (three of five paired biopsies) were demonstrated. CONCLUSION This is the first report of AZD1775 single-agent activity in patients carrying BRCA mutations. Proof-of-mechanism was demonstrated by target modulation and DNA damage response in paired tumor biopsies.

Wee1 kinase as a target for cancer therapy

Wee1, a protein kinase, regulates the G2 checkpoint in response to DNA damage. Preclinical studies have elucidated the role of wee1 in DNA damage repair and the stabilization of replication forks, supporting the validity of wee1 inhibition as a viable therapeutic target in cancer. MK-1775, a selective and potent small-molecule inhibitor of wee1, is under clinical development as a potentiator of DNA damage caused by cytotoxic chemotherapies. We present a review of the role of wee1 in the cell cycle and DNA replication and summarize the clinical development to date of this novel class of anticancer agents.

A nucleotide excision repair master-switch: p53 regulated coordinate induction of global genomic repair genes.

The tumor suppressor gene p53 is mutated in many human cancers. One of its major roles is as a transcription factor, and its many effector genes control key cellular processes including cell cycle checkpoints and apoptosis. An important role in DNA repair is also emerging for both p53 itself and some of its effector genes. The products of two p53- regulated genes, GADD45a and DDB2, are now known to participate in the global genomic repair (GGR) sub-pathway of nucleotide excision repair (NER). We recently reported the induction of a third GGR gene, XPC, following exposure of normal human peripheral blood lymphocytes to g-rays. We now show that XPC is induced in a variety of human cell lines in response to both ionizing and ultra-violet (UV) radiation and alkylating agents, and that this induction requires wild-type p53.

Molecular Pathways: Targeting PARP in Cancer Treatment

Poly (ADP-ribose) polymerases (PARP) are a family of nuclear protein enzymes involved in the DNA damage response. The role of PARP-1 in base excisional repair has been extensively characterized. More recent in vitro studies additionally implicate a role for PARP-1 in facilitating homologous recombination and nonhomologous end-joining. The more faithful process of homologous recombination repair of double-stranded DNA breaks involves localization of BRCA-1 and BRCA-2 to sites of DNA damage, resection of the double-stranded break, and gap-filling DNA synthesis using the homologous sister chromatid as a template. Simultaneous dysfunction of both DNA repair pathways decreases the ability of cells to compensate, and forms the basis for the concept of synthetic lethality. Treatment strategies, thus far, have focused on two main principles: (i) exploitation of the concept of synthetic lethality in homologous recombination–deficient tumors, primarily in breast and ovarian cancer patients with BRCA mutation, and (ii) as radiosensitizers and chemosensitizers. BRCA deficiency accounts for only a fraction of dysfunction in homologous recombination repair. Epigenetic alterations of BRCA function and defects within the Fanconi anemia pathway also result in defective DNA repair. Rational therapeutic combinations exploiting alternate mechanisms of defective DNA repair, abrogation of cell-cycle checkpoints, and additional functions of PARP-1 present novel opportunities for further clinical development of PARP inhibitors. On the basis of the results of clinical studies of PARP inhibitors thus far, it is imperative that future development of PARP inhibitors take a more refined approach, identifying the unique subset of patients that would most benefit from these agents, determining the optimal time for use, and identifying the optimal combination partner in any particular setting. Clin Cancer Res; 19(5); 977–84. ©2012 AACR.

Clinical Activity of the γ-Secretase Inhibitor PF-03084014 in Adults With Desmoid Tumors (Aggressive Fibromatosis)

Purpose Desmoid tumors (aggressive fibromatosis) arise from connective tissue cells or fibroblasts. In general, they are slow growing and do not metastasize; however, locally aggressive desmoid tumors can cause severe morbidity and loss of function. Disease recurrence after surgery and/or radiation and diagnosis of multifocal desmoid tumors highlight the need to develop effective systemic treatments for this disease. In this study, we evaluate objective response rate after therapy with the γ-secretase inhibitor PF-03084014 in patients with recurrent, refractory, progressive desmoid tumors. Patients and Methods Seventeen patients with desmoid tumors received PF-03084014 150 mg orally twice a day in 3-week cycles. Response to treatment was evaluated at cycle 1 and every six cycles, that is, 18 weeks, by RECIST (Response Evaluation Criteria in Solid Tumors) version 1.1. Patient-reported outcomes were measured at baseline and at every restaging visit by using the MD Anderson Symptoms Inventory. Archival tumor and blood samples were genotyped for somatic and germline mutations in APC and CTNNB1. Results Of 17 patients accrued to the study, 15 had mutations in APC or CTNNB1 genes. Sixteen patients (94%) were evaluable for response; five (29%) experienced a confirmed partial response and have been on study for more than 2 years. Another five patients with prolonged stable disease as their best response remain on study. Patient-reported outcomes confirmed clinician reporting that the investigational agent was well tolerated and, in subgroup analyses, participants who demonstrated partial response also experienced clinically meaningful and statistically significant improvements in symptom burden. Conclusion PF-03084014 was well tolerated and demonstrated promising clinical benefit in patients with refractory, progressive desmoid tumors who receive long-term treatment.

A Phase II Study of Sorafenib Combined With Cetuximab in EGFR-Expressing, KRAS-Mutated Metastatic Colorectal Cancer

BACKGROUND Mutations in the KRAS gene predict for resistance to anti-epidermal growth factor receptor (EGFR) therapies, including cetuximab. Upregulation of vascular endothelial growth factor (VEGF)-A has been implicated in resistance to anti-EGFR treatment. Abrogation of the VEGF and RAS/RAF/MEK/ERK pathways has the potential to restore cetuximab sensitivity. PATIENTS AND METHODS Adult patients with histologically documented, measurable, EGFR-expressing, KRAS-mutated metastatic colorectal cancer (mCRC) that had progressed after 5-fluorouracil-based regimens were treated with sorafenib 400 mg orally twice daily and intravenous cetuximab weekly in 28-day cycles. The primary endpoint was the response rate (complete response, partial response, and stable disease at 4 cycles). The secondary endpoints included plasma biomarker analysis of angiogenic cytokines and correlative imaging studies with dynamic contrast-enhanced magnetic resonance imaging and zirconium 89-panitumumab. RESULTS Of the 30 patients enrolled, 26 were evaluable for response. Of the 26 patients evaluated, 4 had stable disease at 4 cycles and 1 had stable disease at 8 cycles. The median progression-free survival was 1.84 months. The common toxicities were rash, diarrhea, and liver enzyme elevations. Of the angiogenic cytokines evaluated, only the placental growth factor increased significantly with treatment (P < .0001). No pharmacodynamic parameters were associated with the treatment response. CONCLUSION We report the results of a trial that combined cetuximab and sorafenib for the treatment of KRAS-mutated mCRC, with correlative imaging studies and pharmacodynamic angiogenic cytokine profiling as downstream markers of EGFR and VEGF receptor (VEGFR) signaling. No objective responses were observed. Additional development of biomarkers for patient selection is needed to evaluate combined EGFR and VEGFR blockade as a therapeutic option in KRAS-mutated CRC.