National Cancer Institute (NCI) state of the science: Targeted radiosensitizers in colorectal cancer.

Colorectal cancer (CRC) represents a major public health problem as the second leading cause of cancer-related mortality in the United States. Of an estimated 140,000 newly diagnosed CRC cases in 2018, roughly one-third of these patients will have a primary tumor located in the distal large bowel or rectum. The current standard-of-care approach includes curative-intent surgery, often after preoperative (neoadjuvant) radiotherapy (RT), to increase rates of tumor down-staging, clinical and pathologic response, as well as improving surgical resection quality. However, despite advancements in surgical techniques, as well as sharpened precision of dosimetry offered by contemporary RT delivery platforms, the oncology community continues to face challenges related to disease relapse. Ongoing investigations are aimed at testing novel radiosensitizing agents and treatments that might exploit the systemic antitumor effects of RT using immunotherapies. If successful, these treatments may usher in a new curative paradigm for rectal cancers, such that surgical interventions may be avoided. Importantly, this disease offers an opportunity to correlate matched paired biopsies, radiographic response, and molecular mechanisms of treatment sensitivity and resistance with clinical outcomes. Herein, the authors highlight the available evidence from preclinical models and early-phase studies, with an emphasis on promising developmental therapeutics undergoing prospective validation in larger scale clinical trials. This review by the National Cancer Institute's Radiation Research Program Colorectal Cancer Working Group provides an updated, comprehensive examination of the continuously evolving state of the science regarding radiosensitizer drug development in the curative treatment of CRC.

A case report of typhlitis during novel use of ropidoxuridine-capecitabine-radiotherapy for treatment-naïve rectal cancer.

BACKGROUND: Rectal carcinomas are tumors that arise from the last 12 cm of the large intestine closest to the anus. They generally have a modest prognosis exacerbated by a high local recurrence rate if radiosensitizing chemotherapy is not given during radiotherapy. This case report discusses the clinical trial treatment of a patient with rectal adenocarcinoma by a new ropidoxuridine-capecitabine-radiotherapy combination. This case report is novel due to the patient's participation in an accelerated titration phase I clinical trial and the resultant rare adverse event of treatment-related sigmoid typhlitis. CASE PRESENTATION: The patient was an 82-year-old female who noticed hematochezia and change in stool caliber over a period of 3 months. A rectal mass was identified by biopsy as a microsatellite stable adenocarcinoma. A planned total neoadjuvant treatment involved eight cycles of leucovorin calcium (folinic acid)-fluorouracil-oxaliplatin (mFOLFOX6) chemotherapy, followed by a clinical trial combination of ropidoxuridine-capecitabine-radiotherapy, prior to definitive surgery. The patient began daily intensity modulated pelvic radiotherapy with concurrent twice-daily oral ropidoxuridine and twice-daily oral capecitabine to be given over 6 weeks. After 14 days of ropidoxuridine-capecitabine-radiotherapy, the patient developed sigmoid typhlitis requiring a 10-day hospitalization and 14-day disruption of treatment. The patient died 27 days after the start of ropidoxuridine-capecitabine-radiotherapy. This adverse event was listed as a definite attribution to the ropidoxuridine-capecitabine treatment; pharmacokinetic and pharmacodynamic data showed low ropidoxuridine metabolite DNA incorporation and high capecitabine metabolite concentration. The accelerated titration phase I clinical trial has been subsequently closed to accrual (NCT04406857). CONCLUSIONS: We believe this case report demonstrates the decision-making process for terminating a phase I accelerated titration designed clinical trial. The report also presents the rare complication of sigmoid typhlitis as a treatment-attributed adverse event. In this case, a ropidoxuridine-capecitabine combination was used as an investigational radiosensitizing treatment now with a narrower future clinical development pathway.

Coordination of DNA mismatch repair and base excision repair processing of chemotherapy and radiation damage for targeting resistant cancers.

DNA damage processing by mismatch repair (MMR) and/or base excision repair (BER) can determine the therapeutic index following treatment of human cancers using radiation therapy and several classes of chemotherapy drugs. Over the last decade, basic and translational cancer research in DNA repair has led to an increased understanding of how these two DNA repair pathways can modify cytotoxicity to chemotherapy and/or ionizing radiation treatments in both normal and malignant tissues. This Molecular Pathways article provides an overview of the current understanding of mechanisms involved in MMR and BER damage processing, including insights into possible coordination of these two DNA repair pathways after chemotherapy and/or ionizing radiation damage. It also introduces principles of systems biology that have been applied to better understand the complexities and coordination of MMR and BER in processing these DNA damages. Finally, it highlights novel therapeutic approaches to target resistant (or DNA damage tolerant) human cancers using chemical and molecular modifiers of chemotherapy and/or ionizing radiation including poly (ADP-ribose) polymerase inhibitors, methoxyamine and iododeoxyuridine (and the prodrug, 5-iodo-2-pyrimidinone-2'-deoxyribose).

Understanding DNA damage response and DNA repair pathways: applications to more targeted cancer therapeutics.

Radiation therapy and many of the commonly used cancer chemotherapeutic drugs target DNA for cytotoxicity. Indeed, the subsequent DNA damage response (DDR) to these cancer treatments in both malignant and normal cells/tissues determines the therapeutic index (TI) of the treatment. The DDR is a complex set of cell processes involving multiple DNA repair, cell cycle regulation, and cell death/survival pathways (or networks) with both damage specificity and coordination of the DDR to different types of DNA damage. Over the last decade, significant progress has been made in elucidating these complex cellular and molecular networks involved in the DDR in human tumor and normal tissues. Based on what has been learned about these processes using experimental in vitro and in vivo models, DDR and DNA pathways are now potential targets for cancer therapy. This article presents an overview of our current understanding of the DDR, including the key DNA repair pathways involved in determining the cytotoxicity to several classes of chemotherapy drugs (CT) as well as ionizing radiation (IR). Since many different types of human cancers can arise from genetic or epigenetic changes in the DDR and DNA repair pathways, this article also covers recent developments in cancer therapeutics that attempt to target these specific tumor-related DDR/DNA repair defects as monotherapy or, more commonly, when combined with conventional cancer treatments.

BRCA1 activates a G2-M cell cycle checkpoint following 6-thioguanine-induced DNA mismatch damage.

Human DNA mismatch repair (MMR) is involved in the response to certain chemotherapy drugs, including 6-thioguanine (6-TG). Consistently, MMR-deficient human tumor cells show resistance to 6-TG damage as manifested by a reduced G(2)-M arrest and decreased apoptosis. In this study, we investigate the role of the BRCA1 protein in modulating a 6-TG-induced MMR damage response, using an isogenic human breast cancer cell line model, including a BRCA1 mutated cell line (HCC1937) and its transfectant with a wild-type BRCA1 cDNA. The MMR proteins MSH2, MSH6, MLH1, and PMS2 are similarly detected in both cell lines. BRCA1-mutant cells are more resistant to 6-TG than BRCA1-positive cells in a clonogenic survival assay and show reduced apoptosis. Additionally, the mutated BRCA1 results in an almost complete loss of a G(2)-M cell cycle checkpoint response induced by 6-TG. Transfection of single specific small interfering RNAs (siRNA) against MSH2, MLH1, ATR, and Chk1 in BRCA1-positive cells markedly reduces the BRCA1-dependent G(2)-M checkpoint response. Interestingly, ATR and Chk1 siRNA transfection in BRCA1-positive cells shows similar levels of 6-TG cytotoxicity as the control transfectant, whereas MSH2 and MLH1 siRNA transfectants show 6-TG resistance as expected. DNA MMR processing, as measured by the number of 6-TG-induced DNA strand breaks using an alkaline comet assay (+/-z-VAD-fmk cotreatment) and by levels of iododeoxyuridine-DNA incorporation, is independent of BRCA1, suggesting the involvement of BRCA1 in the G(2)-M checkpoint response to 6-TG but not in the subsequent excision processing of 6-TG mispairs by MMR.

Toxicology and pharmacokinetic study of orally administered 5-iodo-2-pyrimidinone-2'deoxyribose (IPdR) x 28 days in Fischer-344 rats: impact on the initial clinical phase I trial design of IPdR-mediated radiosensitization.

PURPOSE: A toxicology and pharmacokinetic study of orally administered (po) IPdR (5-3iodo-2-pyrimidinone-2'deoxyribose, NSC-726188) was performed in Fischer-344 rats using a once daily (qd) x 28 days dosing schedule as proposed for an initial phase I clinical trial of IPdR as a radiosensitizer. METHODS: For the toxicology assessment, 80 male and female rats (10/sex/dosage group) were randomly assigned to groups receiving either 0, 0.2, 1.0 or 2.0 g kg(-1)day(-1) of po IPdR x 28 days and one-half were observed to day 57 (recovery group). Animals were monitored for clinical signs during and following treatment with full necropsy of one-half of each dosage group at day 29 and 57. For the plasma pharmacokinetic assessment, 40 rats (10/sex/dosage group) were randomly assigned to groups receiving either 0.2 or 1.0 g kg(-1)day(-1) of po IPdR x 28 days with multiple blood samplings on days 1 and 28 and single blood sampling on days 8 and 15. RESULTS: No drug-related deaths occurred. Higher IPdR doses resulted in transient weight loss and transient decreased hemoglobins but had no effect on white cells or platelets. Complete serum chemistry evaluation showed transient mild decreases in total protein, alkaline phosphatase, and serum globulin. Necropsy evaluation at day 29 showed minimal to mild histopathologic changes in bone marrow, lymph nodes and liver; all reversed by day 59. There were no sex-dependent differences in plasma pharmacokinetics of IPdR noted and the absorption and elimination kinetics of IPdR were found to be linear over the dose range studied. CONCLUSIONS: A once-daily dosing schedule of po IPdR for 28 days with doses up to 2.0 g kg(-1)day(-1) appeared to be well tolerated in Fischer-344 rats. Drug-related weight loss and microscopic changes in bone marrow, lymph nodes and liver were observed. These changes were all reversed by day 57. IPdR disposition was linear over the dose range used. However, based on day 28 kinetics it appears that IPdR elimination is enhanced following repeated administration. These toxicology and pharmacokinetic data were used when considering the design of our initial phase I trial of po IPdR as a clinical radiosensitizer.

Comparison of helical tomotherapy versus conventional radiation to deliver craniospinal radiation.

The purpose of this study was to investigate whether helical tomotherapy would better dose-limit growing vertebral ring apophyses during craniospinal radiation as compared to conventional techniques. Four pediatric patients with M0 medulloblastoma received tomotherapy craniospinal radiation (23.4 Gy, 1.8 Gy/fx) by continuous helical delivery of 6 MV photons. Weekly blood counts were monitored. For comparison, conventional craniospinal radiation plans were generated. To assist in tomotherapy planning, a cross-sectional growth study of 52 children and young adults was completed to evaluate spine growth and maturation. Vertebral ring apophyses first fused along the posterolateral body-pedicle synostosis, proceeding circumferentially toward the anterior vertebral body such that the cervical and lumbar vertebrae fused early and mid-thoracic vertebrae fused late. For the four pediatric patients, tomotherapy resulted between 2% and 14% vertebral volume exceeding 23 Gy. Conventional craniospinal radiation predicted between 33% and 44% exceeding 23 Gy. Cumulative body radiation doses exceeding 4 Gy were between 50% and 57% for tomotherapy and between 25% and 37% for conventional craniospinal radiation. Tomotherapy radiation reduced neutrophil, platelet, and erythrocyte hemoglobin levels during treatment. Tomotherapy provides improved dose avoidance to growing vertebrae as compared to conventional craniospinal radiation. However, the long-term effects of tomotherapy dose avoidance on spine growth and large volume low dose radiation in children are not yet known.

DNA mismatch repair initiates 6-thioguanine--induced autophagy through p53 activation in human tumor cells.

PURPOSE: We investigate the roles of DNA mismatch repair (MMR) and p53 in mediating the induction of autophagy in human tumor cells after exposure to 6-thioguanine (6-TG), a chemotherapy drug recognized by MMR. We also examine how activation of autophagy affects apoptosis (type I cell death) after MMR processing of 6-TG. EXPERIMENTAL DESIGN: Using isogenic pairs of MLH1(-)/MLH1(+) human colorectal cancer cells (HCT116) and MSH2(-)/MSH2(+) human endometrial cancer cells (HEC59), we initially measure activation of autophagy for up to 3 days after 6-TG treatment using LC3, a specific marker of autophagy. We then assess the role of p53 in autophagic signaling of 6-TG MMR processing using both pifithrin-alpha cotreatment to chemically inhibit p53 transcription and small hairpin RNA inhibition of p53 expression. Finally, we use Atg5 small hairpin RNA inhibition of autophagy to assess the effect on apoptosis after MMR processing of 6-TG. RESULTS: We find that MMR is required for mediating autophagy in response to 6-TG treatment in these human tumor cells. We also show that p53 plays an essential role in signaling from MMR to the autophagic pathway. Finally, our results indicate that 6-TG-induced autophagy inhibits apoptosis after MMR processing of 6-TG. CONCLUSIONS: These data suggest a novel function of MMR in mediating autophagy after a chemical (6-TG) DNA mismatch damage through p53 activation. The resulting autophagy inhibits apoptosis after MMR processing of 6-TG.

The interaction between two radiosensitizers: 5-iododeoxyuridine and caffeine.

5-Iododeoxyuridine (IUdR) and caffeine are recognized as potential radiosensitizers with different mechanisms of interaction with ionizing radiation (IR). To assess the interaction of these two types of radiosensitizers, we compared treatment responses to these drugs alone and in combination with IR in two p53-proficient and p53-deficient pairs of human colon cancer cell lines (HCT116 versus HCT116 p53-/- and RKO versus RKO E6). Based on clonogenic survival, the three single agents (IR, IUdR, and caffeine) as well as IUdR or caffeine combined with IR are less or equally effective in p53-deficient human tumor cells compared with p53-proficient tumor cells. However, using both radiosensitizers, a significantly greater radiosensitization was found in p53-deficient human tumor cells. To better understand the interaction of these two radiosensitizers, additional studies on DNA repair and cell cycle regulation were done. We found that caffeine enhanced IUdR-DNA incorporation and IUdR-mediated radiosensitization by partially inhibiting repair (removal) of IUdR in DNA. The repair of IR-induced DNA double-strand breaks was also inhibited by caffeine. However, these effects of caffeine on IUdR-mediated radiosensitization were not found in p53-proficient cells. Cell cycle analyses also showed a greater abrogation of IR-induced S- and G2-phase arrests by caffeine in p53-deficient cells, particularly when combined with IUdR. Collectively, these data provide the mechanistic bases for combining these two radiosensitizers to enhance tumor cytotoxicity. This differential dual mode of radiosensitization by combining IUdR and caffeine-like drugs (e.g., UCN-01) in p53-deficient human tumors may lead to a greater therapeutic gain.

The DNA N-glycosylase MED1 exhibits preference for halogenated pyrimidines and is involved in the cytotoxicity of 5-iododeoxyuridine.

The base excision repair protein MED1 (also known as MBD4), an interactor with the mismatch repair protein MLH1, has a central role in the maintenance of genomic stability with dual functions in DNA damage response and repair. MED1 acts as a thymine and uracil DNA N-glycosylase on T:G and U:G mismatches that occur at cytosine-phosphate-guanine (CpG) methylation sites due to spontaneous deamination of 5-methylcytosine and cytosine, respectively. To elucidate the mechanisms that underlie sequence discrimination by MED1, we did single-turnover kinetics with the isolated, recombinant glycosylase domain of MED1. Quantification of MED1 substrate hierarchy confirmed MED1 preference for mismatches within a CpG context and showed preference for hemimethylated base mismatches. Furthermore, the k(st) values obtained with the uracil analogues 5-fluorouracil and 5-iodouracil were over 20- to 30-fold higher than those obtained with uracil, indicating substantially higher affinity for halogenated bases. A 5-iodouracil precursor is the halogenated nucleotide 5-iododeoxyuridine (5IdU), a cytotoxic and radiosensitizing agent. Cultures of mouse embryo fibroblasts (MEF) with different Med1 genotype derived from mice with targeted inactivation of the gene were evaluated for sensitivity to 5IdU. The results revealed that Med1-null MEFs are more sensitive to 5IdU than wild-type MEFs in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Furthermore, high-performance liquid chromatography analyses revealed that Med1-null cells exhibit increased levels of 5IdU in their DNA due to increased incorporation or reduced removal. These findings establish MED1 as a bona fide repair activity for the removal of halogenated bases and indicate that MED1 may play a significant role in 5IdU cytotoxicity.

Local failure and vertebral body fracture risk using multifraction stereotactic body radiation therapy for spine metastases.

PURPOSE: Single-fraction radiation surgery for spine metastases is highly effective. However, a high rate (20-39%) of vertebral body fracture (VBF) has been associated with large, single-fraction doses. We report our experience using multifraction stereotactic body radiation therapy (SBRT). METHODS AND MATERIALS: All patients who were treated with multifraction SBRT for spine metastases at our institution between 2009 and 2017 were retrospectively analyzed. SBRT was delivered in 2 to 5 fractions using the Cyberknife System (Accuray, Sunnyvale, CA). Patients were followed clinically and with magnetic resonance imaging every 3 to 6 months. Local control, complications (including VBF), and overall survival were evaluated. Patient, disease, and treatment variables were analyzed for a statistical association with outcomes. RESULTS: A total of 83 patients were treated to 98 spine lesions with a median follow-up of 7.6 months. Histologies included non-small cell lung cancer (NSCLC; 24%), renal cell carcinoma (RCC; 18%), and breast cancer (12%). Surgery or vertebroplasty were performed before SBRT in 21% of cases. Patients received a median SBRT dose of 24 Gy in a median of 3 fractions. Local control was 93% at 6 months and 84% at 1 year. Higher prescribed dose, higher biologic effective dose, higher minimum dose to 90% of the planning target volume, tumor histology, and smaller tumor volume predicted improved local control. The cumulative dose was 23 Gy versus 26 Gy for patients with and without failure (P = .02), higher biologic effective dose 39 Gy versus 46 Gy, (P = .01), and higher minimum dose to 90% of the planning target volume 23 Gy versus 26 Gy (P = .03). VBF occurred in 4.2% of all cases and 5.3% of those without surgery or vertebroplasty prior to SBRT. Only preexisting VBF predicted risk of post-SBRT VBF (P < .01). CONCLUSIONS: Multifraction SBRT results in a high local control rate for metastatic spinal disease with a low VBF rate, which suggests a favorable therapeutic ratio compared with single-fraction SBRT.

5-iodo-2-pyrimidinone-2'-deoxyribose-mediated cytotoxicity and radiosensitization in U87 human glioblastoma xenografts.

PURPOSE: 5-iodo-2-pyrimidinone-2'-deoxyribose (IPdR) is a novel orally administered (p.o.) prodrug of 5-iododeoxyuridine. Because p.o. IPdR is being considered for clinical testing as a radiosensitizer in patients with high-grade gliomas, we performed this in vivo study of IPdR-mediated cytotoxicity and radiosensitization in a human glioblastoma xenograft model, U87. METHODS AND MATERIALS: Groups of 8 or 9 athymic male nude mice (6-8 weeks old) were implanted with s.c. U87 xenograft tumors (4 x 10(6) cells) and then randomized to 10 treatment groups receiving increasing doses of p.o. IPdR (0, 100, 250, 500, and 1000 mg/kg/d) administered once daily (q.d.) x 14 days with or without radiotherapy (RT) (0 or 2 Gy/d x 4 days) on days 11-14 of IPdR treatment. Systemic toxicity was determined by body weight measurements during and after IPdR treatment. Tumor response was assessed by changes in tumor volumes. RESULTS: IPdR alone at doses of > or =500 mg/kg/d resulted in moderate inhibition of tumor growth. The combination of IPdR plus RT resulted in a significant IPdR dose-dependent tumor growth delay, with the maximum radiosensitization using > or =500 mg/kg/d. IPdR doses of 500 and 1000 mg/kg/d resulted in transient 5-15% body weight loss during treatment. CONCLUSIONS: In U87 human glioblastoma s.c. xenografts, p.o. IPdR given q.d. x 14 days and RT given 2 Gy/d x 4 days (days 11-14 of IPdR treatment) results in a significant tumor growth delay in an IPdR dose-dependent pattern. The use of p.o. IPdR plus RT holds promise for Phase I/II testing in patients with high-grade gliomas.

CK2 inhibits apoptosis and changes its cellular localization following ionizing radiation.

In this study, we show that CK2 (casein kinase II, CKII) participates in apoptotic responses following ionizing radiation (IR). Using HeLa human cervical carcinoma cells, we find that transfection of small interfering RNA against the CK2 alpha and/or alpha' catalytic subunits results in enhanced apoptosis following IR damage as measured by flow cytometry techniques, compared with a control small interfering RNA. Within 2 to 6 hours of IR, CK2 alpha partially localizes to perinuclear structures, whereas a marked nuclear localization of alpha' occurs. Treatment with a pan-caspase inhibitor or transfection of ARC (apoptosis repressor with caspase recruitment domain) suppresses the apoptotic response to IR in the CK2-reduced cells, indicating involvement of caspases. Additionally, we find that CK2 alpha and/or alpha' reduction affects cell cycle progression independent of IR damage in this human cell line. However, the G2-M checkpoint following IR is not affected in CK2 alpha- and/or alpha'-reduced cells. Thus, our data suggest that CK2 participates in inhibition of apoptosis and negatively regulates caspase activity following IR damage.

Methoxyamine potentiates iododeoxyuridine-induced radiosensitization by altering cell cycle kinetics and enhancing senescence.

We previously reported that methoxyamine (an inhibitor of base excision repair) potentiates iododeoxyuridine (IUdR)-induced radiosensitization in human tumor cells. In this study, we investigated the potential mechanisms of this enhanced cell death. Human colorectal carcinoma RKO cells were exposed to IUdR (3 micromol/L) and/or methoxyamine (3 mmol/L) for 48 hours before ionizing radiation (5 Gy). We found that IUdR/methoxyamine altered cell cycle kinetics and led to an increased G1 population but a decreased S population before ionizing radiation. Immediately following ionizing radiation (up to 6 hours), IUdR/methoxyamine-pretreated cells showed a stringent G1-S checkpoint but an insufficient G2-M checkpoint, whereas a prolonged G1 arrest, containing 2CG1 and 4CG1 cells, was found at later times up to 72 hours. Levels of cell cycle-specific markers [p21, p27, cyclin A, cyclin B1, and pcdc2(Y15)] and DNA damage signaling proteins [gammaH2AX, pChk1(S317), and pChk2(T68)] supported these altered cell cycle kinetics. Interestingly, we found that IUdR/methoxyamine pretreatment reduced ionizing radiation-induced apoptosis. Additionally, the extent of cell death through necrosis or autophagy seemed similar in all (IUdR +/- methoxyamine + ionizing radiation) treatment groups. However, a larger population of senescence-activated beta-galactosidase-positive cells was seen in IUdR/methoxyamine/ionizing radiation-treated cells, which was correlated with the increased activation of the senescence factors p53 and pRb. These data indicate that IUdR/methoxyamine pretreatment enhanced the effects of ionizing radiation by causing a prolonged G1 cell cycle arrest and by promoting stress-induced premature senescence. Thus, senescence, a novel ionizing radiation-induced tumor suppression pathway, may be effectively targeted by IUdR/methoxyamine pretreatment, resulting in an improved therapeutic gain for ionizing radiation.

Patients with Long-Term Control of Systemic Disease Are a Favorable Prognostic Group for Treatment of Brain Metastases with Stereotactic Radiosurgery Alone.

BACKGROUND: Stereotactic radiosurgery (SRS) alone is an attractive option for treatment of brain metastases. SRS avoids whole-brain radiotherapy (WBRT)-associated morbidity, but is limited by regional central nervous system (CNS) failures and short survival in some patients. We evaluated a subgroup of patients with controlled systemic disease that could represent a favorable patient population for SRS alone. METHODS: All patients with brain metastases treated with SRS without WBRT at our institution between 2004 and 2014 were grouped into two cohorts: those with controlled systemic disease (CSD) for 1 year or longer before prior to presentation with brain metastases and those without (i.e., uncontrolled systemic disease [USD]). Rates of local and regional CNS failure, and overall survival were assessed with χ2 and Student t tests. Cox regression analysis was performed to evaluate independent predictors of regional control and overall survival. RESULTS: Two hundred ninety-four patients underwent SRS to 697 lesions, of which 65 patients had CSD. Median follow-up was 9.7 months. There was no difference in local control between the two cohorts (P = 0.795). Regional CNS control was significantly better for patients with CSD (68% vs. 48%; P = 0.001). Overall survival at 1 and 5 years for CSD were 65% and 13% with USD yielding 41% and 7%, respectively (P < 0.001). Multivariate analysis demonstrated that USD (relative CSD) independently predicts regional failure (hazard ratio [HR], 1.75; P = 0.008) and shorter overall survival (HR, 1.55; P = 0.007). CONCLUSIONS: Patients with brain metastases after 1 year or longer of primary and systemic disease control represent a particularly favorable cohort, with lower regional CNS failure and prolonged survival, for an approach of SRS alone.

Casein kinase 2 regulates both apoptosis and the cell cycle following DNA damage induced by 6-thioguanine.

PURPOSE: The purine antimetabolite, 6-thioguanine (6-TG), is an effective drug in the management of acute leukemias. In this study, we analyze the mechanisms of apoptosis associated with 6-TG treatment and casein kinase 2 (CK2 or CKII) in human tumor cells. EXPERIMENTAL DESIGN: Small interfering RNA and chemical CK2 inhibitors were used to reduce CK2 activity. Control and CK2 activity-reduced cells were cultured with 6-TG and assessed by flow cytometry to measure apoptosis and cell cycle profiles. Additionally, confocal microscopy was used to assess localization of CK2 catalytic units following 6-TG treatment. RESULTS: Transfection of small interfering RNA against the CK2 alpha and/or alpha' catalytic subunits results in marked apoptosis of HeLa cells following treatment with 6-TG. Chemical inhibitors of CK2 also induce apoptosis following 6-TG treatment. Apoptosis induced by 6-TG is similarly observed in both mismatch repair-proficient and -deficient HCT116 and HeLa cells. Concomitant treatment with a pan-caspase inhibitor or transfection of apoptosis repressor with caspase recruitment domain markedly suppresses the apoptotic response to DNA damage by 6-TG in the CK2-reduced cells, indicating caspase regulation by CK2. CK2 alpha relocalizes to the endoplasmic reticulum after 6-TG treatment. Additionally, transfection of Cdc2 with a mutation at Ser(39) to Ala, which is the CK2 phosphorylation site, partially inhibits cell cycle progression in G(1) to G(2) phase following 6-TG treatment. CONCLUSION: CK2 is essential for apoptosis inhibition following DNA damage induced by 6-TG, controlling caspase activity.

Schedule-dependent drug effects of oral 5-iodo-2-pyrimidinone-2'-deoxyribose as an in vivo radiosensitizer in U251 human glioblastoma xenografts.

PURPOSE: 5-Iodo-2-pyrimidinone-2'-deoxyribose (IPdR) is an oral prodrug of 5-iodo-2'-deoxyuridine (IUdR), an in vitro/in vivo radiosensitizer. IPdR can be rapidly converted to IUdR by a hepatic aldehyde oxidase. Previously, we found that the enzymatic conversion of IPdR to IUdR could be transiently reduced using a once daily (q.d.) treatment schedule and this may affect IPdR-mediated tumor radiosensitization. The purpose of this study is to measure the effect of different drug dosing schedules on tumor radiosensitization and therapeutic index in human glioblastoma xenografts. EXPERIMENTAL DESIGN: Three different IPdR treatment schedules (thrice a day, t.i.d.; every other day, q.o.d.; every 3rd day, q.3.d.), compared with a q.d. schedule, were analyzed using athymic nude mice with human glioblastoma (U251) s.c. xenografts. Plasma pharmacokinetics, IUdR-DNA incorporation in tumor and normal proliferating tissues, tumor growth delay following irradiation, and body weight loss were used as end points. RESULTS: The t.i.d. schedule with the same total daily doses as the q.d. schedule (250, 500, or 1,000 mg/kg/d) improved the efficiency of IPdR conversion to IUdR. As a result, the percentage of IUdR-DNA incorporation was higher using the t.i.d. schedule in the tumor xenografts as well as in normal small intestine and bone marrow. Using a fixed dose (500 mg/kg) per administration, the q.o.d. and q.3.d. schedules also showed greater IPdR conversion than the q.d. schedule, related to a greater recovery of hepatic aldehyde oxidase activity prior to the next drug dosing. In the tumor regrowth assay, all IPdR treatment schedules showed significant increases of regrowth delays compared with the control without IPdR (q.o.d., 29.4 days; q.d., 29.7 days; t.i.d., 34.7 days; radiotherapy alone, 15.7 days). The t.i.d. schedule also showed a significantly enhanced tumor growth delay compared with the q.d. schedule. Additionally, the q.o.d. schedule resulted in a significant reduction in systemic toxicity. CONCLUSIONS: The t.i.d. and q.o.d. dosing schedules improved the efficiency of enzymatic activation of IPdR to IUdR during treatment and changed the extent of tumor radiosensitization and/or systemic toxicity compared with a q.d. dosing schedule. These dosing schedules will be considered for future clinical trials of IPdR-mediated human tumor radiosensitization.

Both DNA topoisomerase II-binding protein 1 and BRCA1 regulate the G2-M cell cycle checkpoint.

Cell cycle checkpoints play a central role in genomic stability. The human DNA topoisomerase II-binding protein 1 (TopBP1) protein contains eight BRCA1 COOH terminus motifs and shares similarities with Cut5, a yeast checkpoint Rad protein. TopBP1 also shares many features with BRCA1. We report that, when expression of TopBP1 protein is inhibited in BRCA1 mutant cells, mimicking a TopBP1, BRCA1 double-negative condition, the G(2)-M checkpoint is strongly abrogated and apoptosis is increased after ionizing radiation. However, a BRCA1-negative or a TopBP1-negative background resulted in only partial abrogation of the G(2)-M checkpoint. The BRCA1 mutant and TopBP1-reduced condition specifically destroys regulation of the Chk1 kinase but not the Chk2 kinase, suggesting involvement in the ataxia telangiectasia-related pathway. These results indicate that both TopBP1 and BRCA1 specifically regulate the G(2)-M checkpoint, partially compensating each function.

Role of MutSalpha in the recognition of iododeoxyuridine in DNA.

We have previously demonstrated that both the MLH1 and MSH2 status impact the DNA levels of the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd), and thereby radiosensitization induced by these analogues, indirectly implicating both mismatch repair (MMR) proteins in the removal of these bases from DNA. More recent data from our group demonstrate that base excision repair (BER) also impacts IdUrd-DNA levels, supporting a role for the BER pathway in IdUrd removal as well. In this study, we have examined more direct interactions between the MSH2 protein and the processing of IdUrd incorporated in DNA. Our data demonstrate that the MutSalpha (MSH2/MSH6) complex binds specifically to DNA containing an IdUrd-G mismatch, using both purified human MutSalpha as well as nuclear extracts from Msh2-proficient and-deficient mouse cell lines. MutSalpha binding to a IdUrd-G is better recognized than a G-T mismatch in the same sequence context. In addition, MSH2 protein can be found colocalized with IdUrd-DNA using confocal microscopy in G(1) synchronized cells after treatment with IdUrd. Consistent with our recent publication, coadministration of IdUrd and a chemical inhibitor of BER, methoxyamine (MX), also increases the extent of MSH2 nuclear colocalization with IdUrd. Furthermore, we show that the extent of MSH2 colocalization with IdUrd in G(1)-synchronized human tumor cells varies with MLH1 status, suggesting a role for the MLH1 protein in stabilizing the interaction between the MSH2 protein and DNA containing IdUrd. These data, both in vitro and in vivo, suggest direct involvement of MSH2 in processing IdUrd in DNA.