Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

90Y Contamination in the Interventional Radiology Suite: VARSKIN Estimation of Skin and Eye Injury and Review of Mitigation Strategies.

Our objective was to demonstrate, through computer simulations, radiation exposure levels from a 90Y contamination event during radioembolization procedures to calculate the radiation doses from various contamination scenarios. We also provide reasonable safety protocols to prevent contamination and minimize radiation exposure during decontamination. Methods: Simulations were performed using the computer code VARSKIN+, version 1.0, to determine the amount of radiation exposure resulting from different contamination scenarios. Results: The annual radiation dose limit to the skin and the lens of the eye was exceeded within 23 s of exposure to a 44-MBq droplet. Double layers of surgical gloves and level 3 gowns provided some attenuation of radiation from 90Y contamination by reducing the dose rate by 39% and 44%, respectively. Two layers of surgical gloves offered the best ratio of radiation protection without compromising dexterity. Conclusion: This study demonstrated that radiation exposures during 90Y spills or contamination events can be considerable. Interventional radiology and nuclear medicine personnel must be mindful of the risks, follow strategies to prevent spills, and be familiar with recommended decontamination procedures for spills in the interventional radiology suite.

Slow breathing for reducing stress: The effect of extending exhale.

INTRODUCTION: Slow breathing techniques are commonly used to reduce stress. While it is believed by mind-body practitioners that extending the exhale time relative to inhale increases relaxation, this has not been demonstrated. METHODS: We conducted a 12-week randomized, single-blinded trial among 100 participants to compare if yoga-based slow breathing with an exhale greater inhale versus an exhale equals inhale produces measurable differences in physiological and psychological stress among healthy adults. RESULTS: Participants mean individual instruction attendance was 10.7 ± 1.5 sessions out of 12 offered sessions. The mean weekly home practice was 4.8 ± 1.2 practices per week. There was no statistical difference between treatment groups for frequency of class attendance, home practice, or achieved slow breathing respiratory rate. Participants demonstrated fidelity to assigned breath ratios with home practice as measured by remote biometric assessments through smart garments (HEXOSKIN). Regular slow breathing practice for 12 weeks significantly reduced psychological stress as measured by PROMIS Anxiety (-4.85 S.D. ± 5.53, confidence interval [-5.60, -3.00], but not physiological stress as measured by heart rate variability. Group comparisons showed small effect size differences (d = 0.2) with further reductions in psychological stress and physiological stress from baseline to 12 weeks for exhale greater than inhale versus exhale equals inhale, however these differences were not statistically significant. CONCLUSION: While slow breathing significantly reduces psychological stress, breath ratios do not have a significant differential effect on stress reduction among healthy adults.

Clinical consensus statement: Establishing the roles of locoregional and systemic therapies for the treatment of intermediate-stage hepatocellular carcinoma in Canada.

BACKGROUND: Hepatocellular carcinoma (HCC) a leading cause of cancer mortality worldwide and approximately one-third of patients present with intermediate-stage disease. The treatment landscape of intermediate-stage HCC is rapidly evolving due to developments in local, locoregional and systemic therapies. Treatment recommendations focused on this heterogenous disease stage and that take into account the Canadian reality are lacking. To address this gap, a pan-Canadian group of experts in hepatology, transplant, surgery, radiation therapy, nuclear medicine, interventional radiology, and medical oncology came together to develop consensus recommendations on management of intermediate-stage HCC relevant to the Canadian context. METHODS: A modified Delphi framework was used to develop consensus statements with strengths of recommendation and supporting levels of evidence graded using the AHA/ACC classification system. Tentative consensus statements were drafted based on a systematic search and expert input in a series of iterative feedback cycles and were then circulated via online survey to assess the level of agreement. RESULTS & CONCLUSION: The pre-defined ratification threshold of 80 % agreement was reached for all statements in the areas of multidisciplinary treatment (n = 4), intra-arterial therapy (n = 14), biologics (n = 5), radiation therapy (n = 3), surgical resection and transplantation (n = 7), and percutaneous ablative therapy (n = 4). These generally reflected an expansion in treatment options due to developments in previously established or emergent techniques, introduction of new and more active therapies and increased therapeutic flexibility. These developments have allowed for greater treatment tailoring and personalization as well as a paradigm shift toward strategies with curative intent in a wider range of disease settings.

Comparison of Bolus Versus Dual-Syringe Administration Systems on Glass Yttrium-90 Microsphere Deposition in an In Vitro Microvascular Hepatic Tumor Model.

PURPOSE: To utilize an in vitro microvascular hepatic tumor model to compare the deposition characteristics of glass yttrium-90 microspheres using the dual-syringe (DS) and traditional bolus administration methods. MATERIALS AND METHODS: The microvascular tumor model represented a 3.5-cm tumor in a 1,400-cm3 liver with a total hepatic flow of 160 mL/min and was dynamically perfused. A microcatheter was placed in a 2-mm artery feeding the tumor model and 2 additional nontarget arteries. Glass microspheres with a diameter of 20-30 µm were administered using 2 methods: (a) DS delivery at a concentration of 50 mg/mL in either a single, continuous 2-mL infusion or two 1-mL infusions and (b) bolus delivery (BD) of 100 mg of microspheres in a single 3-mL infusion. RESULTS: Overall, the degree of on-target deposition of the microspheres was 85% ± 11%, with no significant differences between the administration methods. Although the distal penetration into the tumor arterioles was approximately 15 mm (from the second microvascular bifurcation of the tumor model) for all the cases, the distal peak particle counts were significantly higher for the DS delivery case (approximately 5 × 105 microspheres achieving distal deposition vs 2 × 105 for the BD case). This resulted in significantly higher deposition uniformity within the tumor model (90% for the DS delivery case vs 80% for the BD case, α = 0.05). CONCLUSIONS: The use of this new in vitro microvascular hepatic tumor model demonstrated that the administration method can affect the deposition of yttrium-90 microspheres within a tumor, with greater distal deposition and more uniform tumor coverage when the microspheres are delivered at consistent concentrations using a DS delivery device. The BD administration method was associated with less favorable deposition characteristics of the microspheres.

Precision dosimetry in yttrium-90 radioembolization through CT imaging of radiopaque microspheres in a rabbit liver model.

PURPOSE: To perform precision dosimetry in yttrium-90 radioembolization through CT imaging of radiopaque microspheres in a rabbit liver model and to compare extracted dose metrics to those produced from conventional PET-based dosimetry. MATERIALS AND METHODS: A CT calibration phantom was designed containing posts with nominal microsphere concentrations of 0.5 mg/mL, 5.0 mg/mL, and 25.0 mg/mL. The mean Hounsfield unit was extracted from the post volumes to generate a calibration curve to relate Hounsfield units to microsphere concentration. A nominal bolus of 40 mg of microspheres was administered to the livers of eight rabbits, followed by PET/CT imaging. A CT-based activity distribution was calculated through the application of the calibration curve to the CT liver volume. Post-treatment dosimetry was performed through the convolution of yttrium-90 dose-voxel kernels and the PET- and CT-based cumulated activity distributions. The mean dose to the liver in PET- and CT-based dose distributions was compared through linear regression, ANOVA, and Bland-Altman analysis. RESULTS: A linear least-squares fit to the average Hounsfield unit and microsphere concentration data from the calibration phantom confirmed a strong correlation (r2 > 0.999) with a slope of 14.13 HU/mg/mL. A poor correlation was found between the mean dose derived from CT and PET (r2 = 0.374), while the ANOVA analysis revealed statistically significant differences (p < 10-12) between the MIRD-derived mean dose and the PET- and CT-derived mean dose. Bland-Altman analysis predicted an offset of 15.0 Gy between the mean dose in CT and PET. The dose within the liver was shown to be more heterogeneous in CT than in PET with an average coefficient of variation equal to 1.99 and 1.02, respectively. CONCLUSION: The benefits of a CT-based approach to post-treatment dosimetry in yttrium-90 radioembolization include improved visualization of the dose distribution, reduced partial volume effects, a better representation of dose heterogeneity, and the mitigation of respiratory motion effects. Post-treatment CT imaging of radiopaque microspheres in yttrium-90 radioembolization provides the means to perform precision dosimetry and extract accurate dose metrics used to refine the understanding of the dose-response relationship, which could ultimately improve future patient outcomes.

Post-administration dosimetry in yttrium-90 radioembolization through micro-CT imaging of radiopaque microspheres in a porcine renal model.

The purpose of this study is to perform post-administration dosimetry in yttrium-90 radioembolization through micro-CT imaging of radiopaque microsphere distributions in a porcine renal model and explore the impact of spatial resolution of an imaging system on the extraction of specific dose metrics. Following the administration of radiopaque microspheres to the kidney of a hybrid farm pig, the kidney was explanted and imaged with micro-CT. To produce an activity distribution, 400 MBq of yttrium-90 activity was distributed throughout segmented voxels of the embolized vasculature based on an established linear relationship between microsphere concentration and CT voxel value. This distribution was down-sampled to coarser isotropic grids ranging in voxel size from 2.5 to 15 mm to emulate nominal resolutions comparable to those found in yttrium-90 PET and Bremsstrahlung SPECT imaging. Dose distributions were calculated through the convolution of activity distributions with dose-voxel kernels generated using the GATE Monte Carlo toolkit. Contours were computed to represent normal tissue and target volumes. Dose-volume histograms, dose metrics, and dose profiles were compared to a ground truth dose distribution computed with GATE. The mean dose to the target for all studied voxel sizes was found to be within 5.7% of the ground truth mean dose.D70was shown to be strongly correlated with image voxel size of the dose distribution (r2 = 0.90).D70is cited in the literature as an important dose metric and its dependence on voxel size suggests higher resolution dose distributions may provide new perspectives on dose-response relationships in yttrium-90 radioembolization. This study demonstrates that dose distributions with large voxels incorrectly homogenize the dose by attributing escalated doses to normal tissues and reduced doses in high-dose target regions. High-resolution micro-CT imaging of radiopaque microsphere distributions can provide increased confidence in characterizing the absorbed dose heterogeneity in yttrium-90 radioembolization.

Assessment of dynamic cardiac repolarization and contractility in patients with hypertrophic cardiomyopathy.

AIMS: Arrhythmia mechanisms in hypertrophic cardiomyopathy remain uncertain. Preclinical models suggest hypertrophic cardiomyopathy-linked mutations perturb sarcomere length-dependent activation, alter cardiac repolarization in rate-dependent fashion and potentiate triggered electrical activity. This study was designed to assess rate-dependence of clinical surrogates of contractility and repolarization in humans with hypertrophic cardiomyopathy. METHODS: All participants had a cardiac implantable device capable of atrial pacing. Cases had clinical diagnosis of hypertrophic cardiomyopathy, controls were age-matched. Continuous electrocardiogram and blood pressure were recorded during and immediately after 30 second pacing trains delivered at increasing rates. RESULTS: Nine hypertrophic cardiomyopathy patients and 10 controls were enrolled (47% female, median 55 years), with similar baseline QRS duration, QT interval and blood pressure. Median septal thickness in hypertrophic cardiomyopathy patients was 18mm; 33% of hypertrophic cardiomyopathy patients had peak sub-aortic velocity >50mmHg. Ventricular ectopy occurred during or immediately after pacing trains in 4/9 hypertrophic cardiomyopathy patients and 0/10 controls (P = 0.03). During delivery of steady rate pacing across a range of cycle lengths, the QT-RR relationship was not statistically different between HCM and control groups; no differences were seen in subgroup analysis of patients with or without intact AV node conduction. Similarly, there was no difference between groups in the QT interval of the first post-pause recovery beat after pacing trains. No statistically significant differences were seen in surrogate measures for cardiac contractility. CONCLUSION: Rapid pacing trains triggered ventricular ectopy in hypertrophic cardiomyopathy patients, but not controls. This finding aligns with pre-clinical descriptions of excessive cardiomyocyte calcium loading during rapid pacing, increased post-pause sarcoplasmic reticulum calcium release, and subsequent calcium-triggered activity. Normal contractility at all diastolic intervals argues against clinical significance of altered length-dependent myofilament activation.

Direct control of CAR T cells through small molecule-regulated antibodies.

Antibody-based therapeutics have experienced a rapid growth in recent years and are now utilized in various modalities spanning from conventional antibodies, antibody-drug conjugates, bispecific antibodies to chimeric antigen receptor (CAR) T cells. Many next generation antibody therapeutics achieve enhanced potency but often increase the risk of adverse events. Antibody scaffolds capable of exhibiting inducible affinities could reduce the risk of adverse events by enabling a transient suspension of antibody activity. To demonstrate this, we develop conditionally activated, single-module CARs, in which tumor antigen recognition is directly modulated by an FDA-approved small molecule drug. The resulting CAR T cells demonstrate specific cytotoxicity of tumor cells comparable to that of traditional CARs, but the cytotoxicity is reversibly attenuated by the addition of the small molecule. The exogenous control of conditional CAR T cell activity allows continual modulation of therapeutic activity to improve the safety profile of CAR T cells across all disease indications.

Sustained mTORC1 activity during palbociclib-induced growth arrest triggers senescence in ER+ breast cancer cells.

Palbociclib, a selective CDK4/6 kinase inhibitor, is approved in combination with endocrine therapies for the treatment of advanced estrogen receptor positive (ER+) breast cancer. In pre-clinical cancer models, CDK4/6 inhibitors act primarily as cytostatic agents. In two commonly studied ER+ breast cancer cell lines (MCF7 and T47D), CDK4/6 inhibition drives G1-phase arrest and the acquisition of a senescent-like phenotype, both of which are reversible upon palbociclib withdrawal (incomplete senescence). Here we identify an ER+ breast cancer cell line, CAMA1, in which palbociclib treatment induces irreversible cell cycle arrest and senescence (complete senescence). In stark contrast to T47D and MCF7 cells, mTORC1 activity is not stably suppressed in CAMA1 cells during palbociclib treatment. Importantly, inhibition of mTORC1 signaling either by the mTORC1 inhibitor rapamycin or by knockdown of Raptor, a unique component of mTORC1, during palbociclib treatment of CAMA1 cells blocks the induction of complete senescence. These results indicate that sustained mTORC1 activity promotes complete senescence in ER+ breast cancer cells during CDK4/6 inhibitor-induced cell cycle arrest. Consistent with this mechanism, genetic depletion of TSC2, a negative regulator of mTORC1, in MCF7 cells resulted in sustained mTORC1 activity during palbociclib treatment and evoked a complete senescence response. These findings demonstrate that persistent mTORC1 signaling during palbociclib-induced G1 arrest is a potential liability for ER+ breast cancer cells, and suggest a strategy for novel drug combinations with palbociclib.

Anti-tumor immunity influences cancer cell reliance upon ATG7.

Macroautophagy (autophagy) is an essential cellular catabolic process required for survival under conditions of starvation. The role of autophagy in cancer is complex, context-dependent and at times contradictory, as it has been shown to inhibit, promote or be dispensable for tumor progression. In this study, we evaluated the contribution of the immune system to the reliance of tumors on autophagy by depleting autophagy-related 7 (ATG7) in murine tumor cells and grafting into immunocompetent versus immunodeficient hosts. Although loss of ATG7 did not affect tumor growth in vitro or in immunodeficient mice, our studies revealed that cancer cell reliance on autophagy was influenced by anti-tumor immune responses, including those mediated by CD8+ T cells. Furthermore, we provide insights into possible mechanisms by which autophagy disruption can enhance anti-tumor immune responses and suggest that autophagy disruption may further benefit patients with immunoreactive tumors.

Premature battery depletion due to compromised low-voltage capacitor in a family of defibrillators.

BACKGROUND: Boston Scientific (Marlborough, MA, USA) implantable cardioverter-defibrillators (ICDs) and cardiac resynchronization therapy defibrillators (CRT-Ds) manufactured between 2008 and 2014 are potentially subject to premature battery depletion through a low-voltage capacitor malfunction occurring as a result of hydrogen buildup within the device. Although some of these devices are currently under advisory, other devices manufactured during this timeframe carry a lower risk of the same malfunction. These same devices are known to have superior longevity in general, and the overall mean lifespan of the devices remains long. METHODS: All patients implanted or followed at our two centers who experienced premature battery depletion and had a Boston Scientific ICD or CRT-D potentially at risk for low-voltage capacitor malfunction were studied retrospectively. RESULTS: Nineteen out of 838 patients (2.3%) with devices potentially at risk have had premature battery depletion: 5.7% of those under advisory and 1.1% of those not under advisory. None of our patients had compromised therapy, and all had >27 days of projected battery longevity remaining. CONCLUSIONS: Undetected premature battery depletion in this population of ICDs has the potential to expose a patient to an interval of time where the device is unable to provide therapy. However, with enrollment in remote monitoring, regular follow-up, and attention to audible alerts, the risk of therapy loss due to low-voltage state can be effectively mitigated. For these reasons, prophylactic generator replacement is not recommended.

Cystine-glutamate antiporter xCT deficiency suppresses tumor growth while preserving antitumor immunity.

T cell-invigorating cancer immunotherapies have near-curative potential. However, their clinical benefit is currently limited, as only a fraction of patients respond, suggesting that these regimens may benefit from combination with tumor-targeting treatments. As oncogenic progression is accompanied by alterations in metabolic pathways, tumors often become heavily reliant on antioxidant machinery and may be susceptible to increases in oxidative stress. The cystine-glutamate antiporter xCT is frequently overexpressed in cancer and fuels the production of the antioxidant glutathione; thus, tumors prone to redox stress may be selectively vulnerable to xCT disruption. However, systemic inhibition of xCT may compromise antitumor immunity, as xCT is implicated in supporting antigen-induced T cell proliferation. Therefore, we utilized immune-competent murine tumor models to investigate whether cancer cell expression of xCT was required for tumor growth in vivo and if deletion of host xCT impacted antitumor immune responses. Deletion of xCT in tumor cells led to defective cystine uptake, accumulation of reactive oxygen species, and impaired tumor growth, supporting a cancer cell-autonomous role for xCT. In contrast, we observed that, although T cell proliferation in culture was exquisitely dependent on xCT expression, xCT was dispensable for T cell proliferation in vivo and for the generation of primary and memory immune responses to tumors. These findings prompted the combination of tumor cell xCT deletion with the immunotherapeutic agent anti-CTLA-4, which dramatically increased the frequency and durability of antitumor responses. Together, these results identify a metabolic vulnerability specific to tumors and demonstrate that xCT disruption can expand the efficacy of anticancer immunotherapies.

Signal-averaged P wave area increases during respiratory events in patients with paroxysmal atrial fibrillation and obstructive sleep apnea.

PURPOSE: P wave characteristics change during simulated apneic events in individuals with atrial fibrillation (AF). This study sought to assess whether similar changes occur during nocturnal respiratory events in patients with AF and obstructive sleep apnea (OSA). METHODS: Thirty-five individuals with severe OSA who underwent formal polysomnography and subsequent AF ablation were compared to a matched group without AF. Electrocardiographic segments from each polysomnogram corresponding to the following events were identified: period of wakefulness closest to the initial onset of sleep (baseline-awake), first respiratory event, respiratory event with the lowest nadir oxygen saturation, longest respiratory event, and last respiratory event. Signal-averaged P wave duration and signal-averaged positive P wave area (amplitude*duration for positive P wave amplitudes) were extracted using custom software. P wave characteristics during respiratory events and the baseline-awake condition were compared. RESULTS: Compared to the baseline-awake condition, the signal-averaged positive P wave area was significantly greater during the longest event and the event with the lowest oxygen saturation in those with AF, but not in those without AF. There were no significant differences in signal-averaged P wave duration for any respiratory event compared to the baseline-awake condition, regardless of AF status. CONCLUSION: In patients with paroxysmal AF and obstructive sleep apnea, the signal-averaged positive P wave area is greater during certain respiratory events than during wakefulness. This finding may reflect the acute impact on right atrial volume of increased venous return associated with respiratory events and could be useful to assess AF risk in sleep apnea and to monitor response to treatment.

Improvement in Sleep-Disordered Breathing Indices Downloaded From a Positive Airway Pressure Machine Following Conversion of Atrial Fibrillation to Sinus Rhythm.

ABSTRACT: Sleep-disordered breathing (SDB) is a contributor to atrial fibrillation (AF) and treatment of obstructive sleep apnea can reduce the recurrence of AF following catheter ablation. However, the effect of AF therapies on measures of SDB severity is less robustly described. We present the case of a middle-aged man with SDB and persistent AF who exhibited improvement in SDB metrics, as characterized by data downloaded from his auto-titrating continuous positive airway pressure (AutoCPAP) machine, very shortly following procedures that restored sinus rhythm. Between procedures, when his rhythm reverted to AF, the downloaded parameters suggested more SDB events. After catheter ablation, the patient maintained sinus rhythm and the improvement in SDB metrics was sustained as well. This case provides support in favor of a bidirectional relationship between SDB and AF and suggests that data available from PAP machines may be useful in serial assessment of SDB status relative to heart rhythm.