ZEB1 promotes non-homologous end joining double-strand break repair.

Repair of DSB induced by IR is primarily carried out by Non-Homologous End Joining (NHEJ), a pathway in which 53BP1 plays a key role. We have discovered that the EMT-inducing transcriptional repressor ZEB1 (i) interacts with 53BP1 and that this interaction occurs rapidly and is significantly amplified following exposure of cells to IR; (ii) is required for the localization of 53BP1 to a subset of double-stranded breaks, and for physiological DSB repair; (iii) co-localizes with 53BP1 at IR-induced foci (IRIF); (iv) promotes NHEJ and inhibits Homologous Recombination (HR); (v) depletion increases resection at DSBs and (vi) confers PARP inhibitor (PARPi) sensitivity on BRCA1-deficient cells. Lastly, ZEB1's effects on repair pathway choice, resection, and PARPi sensitivity all rely on its homeodomain. In contrast to the well-characterized therapeutic resistance of high ZEB1-expressing cancer cells, the novel ZEB1-53BP1-shieldin resection axis described here exposes a therapeutic vulnerability: ZEB1 levels in BRCA1-deficient tumors may serve as a predictive biomarker of response to PARPis.

Protein kinase CK2 phosphorylation of SAPS3 subunit increases PP6 phosphatase activity with Aurora A kinase.

Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.

Local control of 1-5 fraction radiotherapy regimens for spinal metastases: an analysis of the impacts of biologically effective dose and primary histology.

BACKGROUND: This analysis evaluates the impacts of biologically effective dose (BED) and histology on local control (LC) of spinal metastases treated with highly conformal radiotherapy to moderately-escalated doses. MATERIALS AND METHODS: Patients were treated at two institutions from 2010-2020. Treatments with less than 5 Gy per fraction or 8 Gy in 1 fraction were excluded. The dataset was divided into three RPA classes predictive of survival (1). The primary endpoint was LC. RESULTS: 223 patients with 248 treatments met inclusion criteria. Patients had a median Karnofsky Performance Status (KPS ) of 80, and common histologies included breast (29.4%), non-small cell lung cancer (15.7%), and prostate (13.3%). A median 24 Gy was delivered in 3 fractions (BED: 38.4 Gy) to a median planning target volume (PTV) of 37.3 cc. 2-year LC was 75.7%, and 2-year OS was 42.1%. Increased BED was predictive of improved LC for primary prostate cancer (HR = 0.85, 95% CI: 0.74-0.99). Patients with favorable survival (RPA class 1) had improved LC with BED ≥ 40 Gy (p = 0.05), unlike the intermediate and poor survival groups. No grade 3-5 toxicities were reported. CONCLUSIONS: Moderately-escalated treatments were efficacious and well-tolerated. BED ≥ 40 Gy may improve LC, particularly for prostate cancer and patients with favorable survival.

Stereotactic radiosurgery for the treatment of bulky spine metastases.

INTRODUCTION: Stereotactic radiosurgery (SRS) has shown durable local control for the treatment of metastatic diseasespinal metastases. Multilevel disease or epidural or paraspinal involvement present challenges to achieving local control, and this study aims to analyze treatment outcomes for such lesions. METHODS: Patients treated at a single institution with SRS to the spine from 2010-2018 were retrospectively reviewed. Inclusion criteria required clinical follow-up with either a pain assessment or imaging study. Bulky spine metastasis was defined as consisting of multilevel disease or epidural or paraspinal tumor involvement. RESULTS: 54 patients treated for 62 lesions met inclusion criteria. 42 treatments included at least two vertebrae, and 21 and 31 had paraspinal and epidural involvement, respectively. Treatment regimens had a median 24 Gy in 3 fractions to a volume of 37.75 cm3. Median follow-up was 14.36 months, with 5 instances (8%) of local failure. Median overall survival was 13.32 months. Pain improvement was achieved in 47 treatments (76%), and pain improved with treatment (p < 0.0001). Severe pain (HR = 3.08, p = 0.05), additional bone metastases (HR = 4.82, p = 0.05), and paraspinal involvement (HR = 3.93, p < 0.005) were predictive for worse overall survival. Kaplan-Meier analysis demonstrated that prior chemotherapy (p = 0.03) and additional bone metastases (p = 0.02) were predictive of worse overall survival. Grade < 3 toxicity was observed in 19 cases; no grade ≥ 3 side effects were observed. CONCLUSIONS: SRS can effectively treat bulky metastases to the spine, resulting in improvement of pain with minimal toxicity. Severe pain independently predicts for worse overall survival, indicating that treatment prior to worsening of pain is strongly recommended.

Intracranial disease control for EGFR-mutant and ALK-rearranged lung cancer with large volume or symptomatic brain metastases.

PURPOSE/OBJECTIVE(S): Tyrosine kinase inhibitors (TKIs) are commonly employed for patients with brain metastases from lung cancer and specific driver mutations. We sought to identify the correlation between intracranial tumor burden and outcomes in patients with brain metastases treated with TKIs. MATERIALS/METHODS: We identified and retrospectively reviewed cases of EGFR-mutant or ALK-rearranged lung cancer with brain metastases at any time during their cancer course. Clinical characteristics and treatment information were abstracted from the medical records. Brain metastases were contoured to calculate total volume of disease at diagnosis and after initial therapy. High intracranial burden was defined as either > 10 brain metastases, volume of brain metastases > 15 cc, or largest lesion > 3 cm. Intracranial response was determined according to Response Assessment in Neuro-Oncology (RANO) criteria on the patient level. We determined the correlation between clinical and imaging characteristics and intracranial progression free survival (IC-PFS) and overall survival (OS). RESULTS: Fifty-seven patients with EGFR (n = 49) and ALK (n = 8) alterations were identified. Median follow-up from initial brain metastasis diagnosis was 17 months. Neurological symptoms were present in 54% at brain metastasis diagnosis. For those receiving TKIs alone or TKIs with radiation, at least a partial intracranial response (≥ 65% volume reduction) at 3 months from starting therapy was achieved in 94% and 58%. Progressive intracranial disease at 3 months occurred in 6.3% and 8.3%. Patients with high intracranial burden (n = 21) had a median 17 brain metastases, 6.5 cc volume, and 1.9 cm maximal tumor diameter. Median IC-PFS and OS for patients with high intracranial burden was 13.9 and 35.4 months. Patients with high intracranial burden and neurological symptoms at diagnosis had similar IC-PFS and OS compared to those with low burden and absence of neurological symptoms (p > 0.05 for each). CONCLUSION: Most patients receiving TKIs as part of their initial therapy achieve an early and durable volumetric intracranial response, irrespective of presenting disease burden or neurologic symptoms.

A single-institutional experience with low dose stereotactic body radiation therapy for liver metastases.

AIM: This study reports a single-institutional experience treating liver metastases with stereotactic body radiation therapy (SBRT). MATERIALS AND METHODS: 107 patients with 169 lesions were assessed to determine factors predictive for local control, radiographic response, and overall survival (OS). Machine learning techniques, univariate analysis, and the Kaplan-Meier method were utilized. RESULTS: Patients were treated with a relatively low median dose of 30 Gy in 3 fractions. Fractions were generally delivered once weekly. Median biologically effective dose (BED) was 60 Gy, and the median gross tumor volume (GTV) was 12.16 cc. Median follow-up was 7.36 months. 1-year local control was 75% via the Kaplan-Meier method. On follow-up imaging, 43%, 40%, and 17% of lesions were decreased, stable, and increased in size, respectively. 1-year OS was 46% and varied by primary tumor, with median OS of 34.3, 25.1, 12.5, and 4.6 months for ovarian, breast, colorectal, and lung primary tumors, respectively. Breast and ovarian primary patients had better OS (p < 0.0001), and lung primary patients had worse OS (p = 0.032). Higher BED values, the number of hepatic lesions, and larger GTV were not predictive of local control, radiographic response, or OS. 21% of patients suffered from treatment toxicity, but no grade ≥3 toxicity was reported. CONCLUSION: Relatively low-dose SBRT for liver metastases demonstrated efficacy and minimal toxicity, even for patients with large tumors or multiple lesions. This approach may be useful for patients in whom higher-dose therapy is contraindicated or associated with high risk for toxicity. OS depends largely on the primary tumor.

Functions of protein phosphatase-6 in NF-κB signaling and in lymphocytes.

Protein phosphatase-6 (PP6) is a member of the PPP family of Ser/Thr phosphatases involved in intracellular signaling. PP6 is conserved among all eukaryotes, and genetics in model organisms indicates it has non-redundant functions relative to other PPP phosphatases. PP6 functions in association with conserved SAPS subunits and, in vertebrate species, forms heterotrimers with Ankrd subunits. Multiple studies have demonstrated how PP6 exerts negative control at different steps of nuclear factor kappaB signaling. Expression of PP6 catalytic subunit and the PPP6R1 subunit is especially high in hematopoietic cells and lymphoid tissues. Recent efforts at conditionally knocking out genes for PP6c or PP6R1 (SAPS1) have revealed distinctive effects on development of and signaling in lymphocytes.

Brainstem metastases treated with stereotactic radiosurgery: safety, efficacy, and dose response.

The safety and efficacy of stereotactic radiosurgery (SRS) in the brainstem is questioned by some over concern of violating historical brainstem SRS dose tolerance. Our purpose was to report on the clinical outcomes of patients treated at our institution with radiosurgery for brainstem metastases. Patients with metastatic tumors within or directly abutting the brainstem from 1992 to 2014 were analyzed. Patient and tumor characteristics, SRS parameters, and toxicity were recorded and analyzed for associations with local control and survival. Multivariate statistical analysis was performed using Cox proportional hazards modeling. One-hundred and eighty-nine (189) brainstem metastases from 161 patients were included in our analysis. Whole brain irradiation was administered prior to SRS in 52 % of patients. The median margin dose was 18 Gy prescribed to the 50 % isodose line. Median imaging follow up was 5.4 months and median survival was 5.5 months after SRS. At last follow up, local control was achieved in 87.3 % of brainstem lesions treated. There were 3 recorded events of grade 3-5 toxicity (1.8 %). On multivariate analysis, a margin dose ≥16 Gy was associated with improved local control (p = 0.049) and greater KPS score was associated with improved overall survival following SRS (p = 0.024). Patients with brainstem metastases who have limited intracranial disease and/or who have received whole brain irradiation should be considered for SRS. Margin doses of at least 16 Gy are associated with superior local control, and serious radiation toxicity in SRS for brainstem metastasis appears rare.

PP6 regulatory subunit R1 is bidentate anchor for targeting protein phosphatase-6 to DNA-dependent protein kinase.

DNA-dependent protein kinase (DNA-PK) becomes activated in response to DNA double strand breaks, initiating repair by the non-homologous end joining pathway. DNA·PK complexes with the regulatory subunit SAPSR1 (R1) of protein phosphatase-6 (PP6). Knockdown of either R1 or PP6c prevents DNA-PK activation in response to ionizing radiation-induced DNA damage and radiosensitizes glioblastoma cells. Here, we demonstrate that R1 is necessary for and bridges the interaction between DNA-PK and PP6c. Using R1 deletion mutants, DNA-PK binding was mapped to two distinct regions of R1 spanning residues 1-326 and 522-700. Either region expressed alone was sufficient to bind DNA-PK, but only deletion of residues 1-326, not 522-700, eliminated interaction of R1 with DNA-PK. We assign 1-326 as the dominant domain and 522-700 as the supporting region. These results demonstrate that R1 acts as a bidentate anchor to DNA-PK and recruits PP6c. Targeting the dominant interface with small molecule or peptidomimetic inhibitors could specifically prevent activation of DNA-PK and thereby sensitize cells to ionizing radiation and other genotoxic agents.

Racial and Treatment Center Differences on Time to Treatment Initiation for Nonsmall Cell Lung Cancer Patients Receiving Radiation Therapy As an Initial Treatment.

Objective: Because time to treatment has been shown to be associated with increase in the risk of death for Non Small Cell Lung Cancer (NSCLC) patients, we examined the prevalence and magnitude of racial disparities in mean time to radiation therapy (TTRT) for Stage I-III non-small cell lung cancer patients across a variety of treatment facilities. Methods: Utilizing the United States National Cancer Database (NCDB), we determined differences in TTRT between different races and different treatment facilities. Results: Concordant with past research, we found that non-White patients and patients treated at academic facilities, regardless of race, have longer mean TTRT, and that racial disparities in TTRT extend across all treatment facilities (all p<0.05). Conclusions: These findings shed light on the potential presence of and impact of structural racism on patients seeking cancer treatment, and the need for further investigation behind the reasonings behind longer TTI for non-White patients. To elucidate the real-world applicability of these results, further investigation into the societal determinants that perpetuate disparity in time to radiation therapy, and potential interventions in the clinical setting to improve cultural and racial sensitivity among healthcare professionals is recommended.

Replicative Instability Drives Cancer Progression.

In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress exhibit genomic instability and poor survival rates across tumor types. Seminal studies have demonstrated that genomic instability develops following inactivation of BRCA1, BRCA2, or BRCA-related genes. However, it is recognized that many tumors exhibit genomic instability but lack BRCA inactivation. We sought to identify a pan-cancer mechanism that underpins genomic instability and cancer progression in BRCA-wildtype tumors. Methods: Using multi-omics data from two independent consortia, we analyzed data from dozens of tumor types to identify patient cohorts characterized by poor outcomes, genomic instability, and wildtype BRCA genes. We developed several novel metrics to identify the genetic underpinnings of genomic instability in tumors with wildtype BRCA. Associated clinical data was mined to analyze patient responses to standard of care therapies and potential differences in metastatic dissemination. Results: Systematic analysis of the DNA repair landscape revealed that defective single-strand break repair, translesion synthesis, and non-homologous end-joining effectors drive genomic instability in tumors with wildtype BRCA and BRCA-related genes. Importantly, we find that loss of these effectors promotes replication stress, therapy resistance, and increased primary carcinoma to brain metastasis. Conclusions: Our results have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by the accumulation of intra-chromosomal, gene-level gain and loss events at replication stress sensitive (RSS) genome sites. We find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution. Clinically, we find that RIN drives therapy resistance and distant metastases across multiple tumor types.

A Genome-Edited ERα-HiBiT Fusion Reporter Cell Line for the Identification of ERα Modulators Via High-Throughput Screening and CETSA.

The estrogen receptor α (ERα) is a target of intense pharmacological intervention and toxicological biomonitoring. Current methods to directly quantify cellular levels of ERα involve antibody-based assays, which are labor-intensive and of limited throughput. In this study, we generated a post-translational reporter cell line, referred to as MCF7-ERα-HiBiT, by fusing a small pro-luminescent nanoluciferase (NLuc) tag (HiBiT) to the C-terminus of endogenous ERα in MCF7 cells. The tag allows the luminescent detection and quantification of endogenous ERα protein by addition of the complementary NLuc enzyme fragment. This MCF7-ERα-HiBiT cell line was optimized for quantitative high-throughput screening (qHTS) to identify compounds that reduce ERα levels. In addition, the same cell line was optimized for a qHTS cellular thermal shift assay to identify compounds that bind and thermally stabilize ERα. Here, we interrogated the MCF7-ERα-HiBiT assay against the NCATS Pharmacological Collection (NPC) of 2,678 approved drugs and identified compounds that potently reduce and thermally stabilize ERα. Our novel post-translational reporter cell line provides a unique opportunity for profiling large pharmacological and toxicological compound libraries for their effect on ERα levels as well as for assessing direct compound binding to the receptor, thus facilitating mechanistic studies by which compounds exert their biological effects on ERα.

A single institutional experience with central lung stereotactic body radiation therapy demonstrating encouraging results with increased inter-fraction time.

BACKGROUND: Stereotactic body radiation therapy (SBRT) is an effective treatment modality for non-small cell lung cancer (NSCLC); however, there are concerns regarding potential toxicity for centrally located tumors. METHODS: This retrospective study considered patients with SBRT for central lung NSCLC (defined as a tumor within 2 cm of any mediastinal critical structure). The institutional protocol was that patients with central tumors received SBRT less frequently than daily-generally once or twice weekly. RESULTS: A total of 115 patients with 148 lesions were treated with SBRT to a median 45 [5-60] Gy in 4 [1-5] fractions over a median 5.3 [0-18] days. Many patients treated with this method presented with advanced disease: 58 treatments involved nodal targets, and 42 had stage 3 disease. 52% of patients had chronic obstructive pulmonary disease (COPD), and only 49% had a biopsy, often due to concerns regarding other medical comorbidities. Rates of prior chemotherapy, thoracic surgery, and thoracic radiotherapy were 32%, 21%, and 49%, respectively. Via the Kaplan-Meier method, 2-year overall survival was 65%, and 2-year local control was 77%. Two-year local-progression free survival was 53%, and 2-year progression-survival was 48%. Treatments for stage 3 disease had an impressive 82% 2-year local control that was comparable to early stage treatments. Patients with stage 3 disease had a 2-year overall survival of 59%, which trended towards decreased overall survival compared to early stage patients. There were 13 grade 1 (9%) and 14 grade 2 (9%) toxicities. There were no reported grade ≥3 acute or late toxicities and only 3 cases of pneumonitis. CONCLUSIONS: Our series demonstrates encouraging local control with low rates of toxicity for central lung SBRT, including many stage 3 patients. This may be the result of the relatively large inter-fraction interval. This interval may allow for greater tumor effects (such as reoxygenation) and improved tolerance from normal tissues.

Clinical Outcomes and Predictors of Lung Toxicity After Multiple Courses of Lung Stereotactic Body Radiotherapy for Early-Stage Non-Small Cell Lung Cancer.

BACKGROUND: The clinical outcomes of multicourse lung stereotactic body radiotherapy (SBRT) have yet to be validated in a prospective study, and there are a lack of data on allowable composite dosimetry. PATIENTS AND METHODS: Forty-four patients underwent multicourse lung SBRT for recurrent or metachronous NSCLC. The median biologically effective dose (BED10) for the first course and subsequent courses were 132 and 100 Gy, respectively. Patient and treatment characteristics were evaluated to determine the correlation with the development of radiation pneumonitis (RP). RESULTS: The local control rate was 91%. A total of 13.6% developed a grade 2+ RP, and 4.5% developed a grade 3+ RP, including one grade 5. On univariable analysis, multiple composite dosimetric factors (V5 [proportion of lung structure receiving at least 5 Gy], V10, V20, V40, and mean lung dose) were correlated with the development of RP. When comprised of the first and second course of SBRT, a composite lung V5 of < 30% and > 50% was associated with a 0 and 75% incidence of grade 2+ RP, respectively. We identified no significant correlation on multivariable analysis but observed a strong trend between composite lung V5 and the development of grade 2+ RP (hazard ratio, 1.157; P = .058). Evaluation of multiple clinical factors also identified a significant correlation between the timing of repeat lung SBRT and the development of grade 2+ RP after the second course (P = .0028). CONCLUSION: Subsequent courses of lung SBRT, prescribed to a median BED10 of 100 Gy, can provide a high rate of local control with a 4.5% incidence of grade 3+ toxicity. Composite lung V5 and the timing of the second course of lung SBRT may be correlated to the development of RP.

Recursive Partitioning Analysis for Local Control Achieved With Stereotactic Body Radiation Therapy for the Liver, Spine, or Lymph Nodes.

PURPOSE: This study aims to develop a local control risk stratification using recursive partitioning analysis (RPA) for patients receiving stereotactic body radiation therapy (SBRT) for metastatic cancer. METHODS AND MATERIALS: A single institutional database of 397 SBRT treatments to the liver, spine, and lymph nodes was constructed. All treatments required imaging follow-up to assess for local control. Cox proportional hazards analysis was implemented before the decision tree analysis. The data were split into training (70%), validation (10%), and testing (20%) sets for RPA to optimize the training set. RESULTS: In the study, 361 treatments were included in the local control analysis. Two-year local control was 71%. A decision tree analysis was used and the resulting model demonstrated 93.10% fidelity for the validation set and 87.67% for the test set. RPA class 3 was composed of patients with non-small cell lung cancer (NSCLC) primary tumors and treatment targets other than the cervical, thoracic, and lumbar spines. RPA class 2 included patients with primary cancers other than NSCLC or breast and treatments targets of the sacral spine or liver. RPA class 1 consisted of all other patients (including lymph node targets and patients with primary breast cancer). Classes 3, 2, and 1 demonstrated 3-year local controls rates of 29%, 50%, and 83%, respectively. On subgroup analysis using the Kaplan-Meier method, treatments for lymph nodes and primary ovarian disease demonstrated improved local control relative to other treatment targets (P < .005) and primary disease sites (P < .005), respectively. CONCLUSIONS: A local control risk stratification model for SBRT to sites of metastatic disease was developed. Treatment target and primary tumor were identified as critical factors determining local control. NSCLC primary lesions have increased local failure for targets other than the cervical, thoracic, or lumbar spines, and improved local control was identified for lymph node sites and breast or ovarian primary tumors.

Genesis of Quality Measurements to Improve the Care Delivered to Patients With Brain Metastases.

PURPOSE: High-value and high-quality health care requires outcome measurements to inform treatment decisions, but, to our knowledge, no standardized measurements exist to evaluate brain metastases (BMs) care. We propose a set of measurements and report on their implementation in the care of patients with BMs. METHODS: On the basis of a stakeholders' needs assessment and review of the literature, we identified outcome and process measurements to assess the care of patients with BMs according to treatment modality. Retrospectively, we applied these indicators of care to all patients diagnosed and treated at our institution over 2 years. RESULTS: We ascertained 5 outcome and 6 process measurements of relevance in the care of BMs. When applied to 209 patients (89.7%) who received cancer treatment, 77% were alive > 90 days after diagnosis. The proportion alive at 90 days after surgery, whole-brain radiation therapy (WBRT), and stereotactic radiosurgery (SRS) was 82%, 59%, and 81%, respectively. Other performance measurements included 30-day postoperative readmission rate (6%), SRS within 30 days of surgery (79%), use of memantine with WBRT (41%), advance directives within 6 months of diagnosis (53%), and palliative care consultation for patients with poor prognosis or receiving WBRT (45%). Measurements for the 24 patients (10.3%) receiving best supportive care were advance directives documentation (67%) and referral to palliative or hospice care (83%). CONCLUSION: We propose a set of measurements to apprise quality improvement efforts, inform treatment decision-making, and to use in evaluation of the performance of interdisciplinary BMs programs. Their refinement can potentially enhance the quality and value of care delivered to patients with BMs.

Veliparib in combination with carboplatin/paclitaxel-based chemoradiotherapy in patients with stage III non-small cell lung cancer.

OBJECTIVES: Veliparib is a potent poly(ADP)-ribose polymerase (PARP) 1 and 2 inhibitor that impedes repair of DNA damage induced by cytotoxic and radiation therapies. This phase 1 study evaluated veliparib in combination with chemoradiotherapy in patients with unresectable stage III non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Patients received veliparib orally twice daily (BID) in escalating doses (60-240 mg, Day -3 to 1 day after last dose of radiation) combined with weekly carboplatin (area under the curve [AUC] 2 mg/mL/min), paclitaxel (45 mg/m2), and daily radiation therapy (60 Gy in 30 fractions), followed by two cycles of veliparib (120-240 mg BID, Days -2 through 5 of each 21-day cycle), carboplatin (AUC 6 mg/mL/min, Day 1 of each cycle), and paclitaxel (200 mg/m2, Day 1 of each cycle) consolidation. Endpoints included veliparib maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), pharmacokinetics, safety, and efficacy. RESULTS: Forty-eight patients were enrolled. The MTD/RP2D of veliparib was 240 mg BID with chemoradiotherapy followed by 120 mg BID with consolidation. The most common any-grade adverse events (AEs) in this cohort for the whole treatment period were nausea (83%), esophagitis (75%), neutropenia (75%), and thrombocytopenia (75%). Dose-proportional pharmacokinetics of veliparib were observed. Median progression-free survival (mPFS) was 19.6 months (95% CI: 9.7-32.6). Median overall survival was estimated to be 32.6 months (95% CI: 15.0-not reached). In patients treated with the RP2D, mPFS was 19.6 months (95% CI: 3.0-not reached). CONCLUSIONS: When combined with standard concurrent chemoradiotherapy and consolidation chemotherapy in patients with stage III NSCLC, veliparib demonstrated an acceptable safety profile and antitumor activity with an mPFS of 19.6 months.

Effects of breathing variation on gating window internal target volume in respiratory gated radiation therapy.

PURPOSE: To investigate the effects of breathing variation on gating window internal target volume (ITVGW) in respiratory gated radiation therapy. METHOD AND MATERIALS: Two-dimensional dynamic MRI (dMRI) of lung motion was acquired in ten volunteers and eight lung cancer patients. Resorted dMRI using 4DCT acquisition method (RedCAM) was generated for selected subjects by simulating the image rebinning process. A dynamic software generated phantom (dSGP) was created by moving a solid circle (to mimic the "tumor") with dMRI-determined motion trajectories. The gating window internal target area (ITAGw, 2D counterpart of ITVGW) was determined from both RedCAM and dSGP/dMRI. Its area (A), major axis (L1), minor axis (L2), and similarity (S) were calculated and compared. RESULTS: In the phantom study of 3 cm tumor, measurements of the ITAGW from dSGP (A =10.0 +/- 1.3 cm2, L1=3.8 +/- 0.4 cm, and L2=3.3 +/- 0.1 cm) are significantly (p <0.001) greater than those from RedCAM (A=8.5 +/- 0.7 cm(2), L1 =3.5 +/- 0.2 cm, and L2=3.1 +/- 0.1 cm). Similarly, the differences are significantly greater (p <0.001) for the 1 cm tumor (A=1.9 +/- 0.5 cm(2), L1 =1.9 +/- 0.4 cm, and L2=1.3 +/- 0.1 cm in dSGP; A=l1.3 +/- 0.1 cm(2), L1=1.5 +/- 0.2 cm, and L2 = 1.1 +/- 0.1 cm in RedCAM). In patient studies, measurements of the ITAGW from dMRI (A =15.5 +/- 8.2 cm(2), Ll=5.0 +/- 1.1 cm, and L2=3.8 +/- 1.2 cm) are also significantly greater (p <0.05) than those from RedCAM (A=13.2 +/- 8.5 cm(2), L1=4.3 +/- 1.4 cm, and L2=3.7 +/- 1.2 cm). Similarities were 0.9 +/- 0.1, 0.8 +/- 0.1, and 0.8 +/- 0.1 in the 3 cm tumor phantom, 1 cm tumor phantom, and patient studies, respectively. CONCLUSION: ITVGW can be underestimated by 4DCT due to breathing variations. An additional margin may be needed to account for this potential error in generating a PTVGW. Cautions need to be taken when generating ITVGW from 4DCT in respiratory gated radiation therapy, especially for small tumors (<3 cm) with a large motion range (>1 cm).

PP2A regulates ionizing radiation-induced apoptosis through Ser46 phosphorylation of p53.

In response to ionizing radiation, p53 plays a critical role in regulating DNA repair and apoptosis. Among multiple phosphorylation sites, evidence suggests that Ser46 promotes apoptotic cell death through mitochondrial outer membrane permeabilization (MOMP) and subsequent activation of the caspase 7-PARP pathway. Therefore, we investigated which phosphatase regulates Ser46 after ionizing radiation, reasoning that the responsible phosphatase should be a target for radiosensitization. We determined that both inhibition of PP2A by the cell-permeable inhibitor calyculin A and knockdown of PP2A by RNAi (a) enhanced Ser46 phosphorylation in p53 and (b) induced coincident caspase 7 and PARP cleavage in response to ionizing radiation. Furthermore, mutation of p53 Ser46 to Ala attenuated ionizing radiation-induced apoptotic signaling. Consequently, we concluded that PP2A regulates ionizing radiation-induced apoptotic signaling through dephosphorylation of p53 Ser46.

Aurora B Kinase Promotes CHIP-Dependent Degradation of HIF1α in Prostate Cancer Cells.

Hypoxia is a major factor in tumor progression and resistance to therapies, which involves elevated levels of the transcription factor HIF1α. Here, we report that prostate tumor xenografts express high levels of HIF1α and show greatly enhanced growth in response to knockdown of the E3 ligase CHIP (C-terminus of Hsp70-interacting protein). In multiple human prostate cancer cell lines under hypoxia, taxol treatment induces the degradation of HIF1α, and this response is abrogated by knockdown of CHIP, but not by E3 ligase VHL or RACK1. HIF1α degradation is accompanied by loss of function, evidenced by reduced expression of HIF1α-dependent genes. CHIP-dependent HIF1α degradation also occurs in cells arrested in mitosis by nocodazole instead of taxol. Mitotic kinase Aurora B activity is required for taxol-induced HIF1α degradation. Purified Aurora B directly phosphorylates HIF1α at multiple sites, and these modifications enhance its polyubiquitination by CHIP in a purified reconstituted system. Our results show how activation of Aurora B promotes CHIP-dependent degradation of HIF1α in prostate cancer cells. This new knowledge may affect the use of mitotic kinase inhibitors and open new approaches for treatment of hypoxic prostate tumors.