The 12th Tuscany Retreat on Cancer Research and Apoptosis was held on August 19-26, 2023. The biennial retreat aims to bring together scientists who advance research in cancer, cell death, and neurodegenerative diseases. Topics covered ranged from drug resistance in cancer to insights into novel molecular cell signaling mechanisms and targets, all related to the pathways and molecules that regulate programmed cell death and the diseases that result from the dysregulation of programmed cell death. In this meeting review, we summarize the content of the most recent retreat.

AbstractMYC is a central regulator of gene transcription and is frequently dysregulated in human cancers. As targeting MYC directly is challenging, an alternative strategy is to identify specific proteins or processes required for MYC to function as a potent cancer driver that can be targeted to result in synthetic lethality. To identify potential targets in MYC-driven cancers, we performed a genome-wide CRISPR knockout screen using an isogenic pair of breast cancer cell lines in which MYC dysregulation is the switch from benign to transformed tumor growth. Proteins that regulate R-loops were identified as a potential class of synthetic lethal targets. Dysregulated MYC elevated global transcription and coincident R-loop accumulation. Topoisomerase 1 (TOP1), a regulator of R-loops by DNA topology, was validated to be a vulnerability in cells with high MYC activity. Genetic knockdown of TOP1 in MYC-transformed cells resulted in reduced colony formation compared with control cells, demonstrating synthetic lethality. Overexpression of RNaseH1, a riboendonuclease that specifically degrades R-loops, rescued the reduction in clonogenicity induced by TOP1 deficiency, demonstrating that this vulnerability is driven by aberrant R-loop accumulation. Genetic and pharmacologic TOP1 inhibition selectively reduced the fitness of MYC-transformed tumors in vivo. Finally, drug response to TOP1 inhibitors (i.e., topotecan) significantly correlated with MYC levels and activity across panels of breast cancer cell lines and patient-derived organoids. Together, these results highlight TOP1 as a promising target for MYC-driven cancers.Significance:CRISPR screening reveals topoisomerase 1 as an immediately actionable vulnerability in cancers harboring MYC as a driver oncoprotein that can be targeted with clinically approved inhibitors.

Includes Supplemental Figures S1-S6 and Supplemental Tables S1-S6.

Cardiotoxicity is a major complication of chemotherapy, which leads in time to progressive electro-mechanical dysfunction, irreversible myocardial tissue remodeling and eventual heart failure. In this pilot study, we seek to find key biomarkers of early cardiotoxic effects. For this, a weekly dose of doxorubicin, DXO was injected in swine during a four-week treatment. Longitudinal MR imaging was performed pre- and post-DXO (at: 1, 5 and 9 weeks after ending the DXO-therapy) using a scanning protocol at 3T that included: functional CINE imaging; T2 and T1 mapping, and 3D late gadolinium enhancement (LGE) for tissue characterization. Our results showed that, compared to the mean baseline values pre-DXO, ejection fraction gradually decreased from a mean 47% to ~34% post-DXO, indicating that the cardiac biomechanical function started to deteriorate within weeks post-DXO. The initially increased T2-derived edema appeared to resolve by week 5. Furthermore, a gradual deposition of diffuse fibrosis post-DXO was identified by T1 values and LGE, and confirmed by collagen-sensitive histological stains. Our preliminary results will help establish MR imaging protocols and models that predict early DXO-induced cardiotoxicity, which could be used by clinicians to develop cardioprotective strategies for preventing heart failure progression post-chemotherapy.

A critical chemotherapeutic complication is cardiotoxicity, often leading, in time, to heart failure. In this work, we developed a novel animal protocol using magnetic resonance (MR) imaging and electrophysiology (EP) tests, designed to detect subtle structural and functional changes associated with myocardial damage in sub-chronic phases post-chemotherapy. A weekly dose of doxorubicin (DOX) was injected in four juvenile swine throughout a four-week plan, using an intravenous approach that mimics the treatment in cancer patients. We performed cardiac MR imaging as follows: in all four pigs pre-DOX; at 1 and 5 weeks post-DOX in a group of two pigs; and, at 1 and 9 weeks post-DOX in the other two pigs, using Cine imaging to assess ejection fraction (EF) and late gadolinium enhancement to quantify collagen density in the left ventricle. Additionally, X-ray-guided voltage mapping and arrhythmia tests were conducted in the group at 9 weeks post-DOX and in a healthy pig. Tissue samples were collected for histology. The results showed that EF decreased from ~46% pre-DOX to ~34% within the first 9 weeks post-DOX. This decline in LV function was explained by a gradual increase in collagen density, especially noticeable at week 9 post-DOX as derived from MRI analysis. Furthermore, ventricular fibrillation was induced via rapid pacing at 9 weeks post-DOX, most likely caused by fibrotic patches identified in voltage maps, as confirmed by MRI and collagen-sensitive histological stains. Overall, our novel preclinical protocol was able to reveal key signs of potentially-irreversible tissue changes, along with electrical remodeling and arrhythmia risk in the early months following DOX therapy. Future work will include more datasets to statistically power the study, and will use the protocol to test cardioprotective strategies.

The transcription factor and oncoprotein MYC is a potent driver of many human cancers and can regulate numerous biological activities that contribute to tumorigenesis. How a single transcription factor can regulate such a diverse set of biological programmes is central to the understanding of MYC function in cancer. In this Perspective, we highlight how multiple proteins that interact with MYC enable MYC to regulate several central control points of gene transcription. These include promoter binding, epigenetic modifications, initiation, elongation and post-transcriptional processes. Evidence shows that a combination of multiple protein interactions enables MYC to function as a potent oncoprotein, working together in a 'coalition model', as presented here. Moreover, as MYC depends on its protein interactome for function, we discuss recent research that emphasizes an unprecedented opportunity to target protein interactors to directly impede MYC oncogenesis.

Cardiac magnetic resonance (MR) imaging can detect infarct scar, a major cause of lethal arrhythmia and heart failure. Here, we describe a robust image processing pipeline developed to quantitatively analyze collagen density and features in a pig model of chronic fibrosis. Specifically, we use ex vivo diffusion tensor imaging (DTI) ( 0.6 × 0.6 × 1.2 mm resolution) to calculate fractional anisotropy maps in: healthy tissue, infarct core (IC) and gray zone (GZ) (i.e., a mixture of viable myocytes and collagen fibrils bordering IC and healthy zones). The 3 zones were validated using collagen-sensitive histological slides co-registered with MR images. Our results showed a significant ( p< 0.05 ) reduction in the mean FA values of GZ (by 17

Cardiotoxicity is a major complication of anthracycline-based cancer therapy, which leads in time to progressive electro-mechanical dysfunction, irreversible myocardial tissue remodeling and heart failure. We are developing novel preclinical large animal models to study early structural and functional cardiotoxic effects induced by doxorubicin (DOX) chemotherapy. In this pilot study, a weekly 1mg/kg dose of DOX was injected intravenously in n=4 pigs, during a four-week treatment. MR imaging was performed pre-DOX and post-DOX therapy (i.e., at: 1, 5 and 9 weeks after ending DOX injections) using a scanning protocol at 3T that included: left ventricle function quantification (i.e., ejection fraction and strain), gadolinium-based contrast-enhanced T1-mapping MR methods at 1.4mm isotropic resolution for diffuse fibrosisdetection, and T2-mapping of edema/inflammation. Our results showed that compared to mean baseline values pre-DOX, ejection fraction gradually decreased from 47% to 34% post-DOX while global longitudinal strain decreased by ∼5%, indicating that the cardiac biomechanical function started to deteriorate within 9 weeks post-DOX. The initially increased T2-derived edema at week 5 appeared to resolve by week 9, as confirmed by H&E histological stains. Furthermore, subtle patches of diffuse fibrosis were detected by contrast-enhanced MRI only at week 9 but not at week 5, and confirmed by collagen-sensitive Masson Trichrome histological stains. Lastly, in two pigs we recorded endocardial bipolar voltage maps 9 weeks post-DOX and revealed the presence of small scattered tissue patches with low voltages (<1.5mV). In these 2 pigs we also induced ventricular fibrillation via rapid pacing, suggesting that gradual deposition of DOX-induced diffuse fibrosis increases vulnerability to arrhythmia. These novel results will help us establish image-based biophysical models to predict early DOX-mediated cardiotoxicity, and will help clinicians conduct studies on cardioprotective strategies to prevent malignant arrhythmia and heart failure progression.

BACKGROUND:Simulation-based training (SBT) provides a safe environment and effective means to enhance skills development. Simulation-based curricula have been developed for a number of procedures, including gastrointestinal endoscopy. Gamification, which is the application of game-design principles to non-game contexts, is an instructional strategy with potential to enhance learning. No studies have investigated the effects of a comprehensive gamification curriculum on the acquisition of endoscopic skills among novice endoscopists. METHODS AND ANALYSIS:Thirty-six novice endoscopists will be randomised to one of two endoscopy SBT curricula: (1) the Conventional Curriculum Group, in which participants will receive 6 hours of one-on-one simulation training augmented with expert feedback and interlaced with 4 hours of small group teaching on the theory of colonoscopy or (2) the Gamified Curriculum Group, in which participants will receive the same curriculum with integration of the following game-design elements: a leaderboard summarising participants' performance, game narrative, achievement badges and rewards for top performance. In line with a progressive learning approach, simulation training for participants will progress from low to high complexity simulators, starting with a bench-top model and then moving to the EndoVR virtual reality simulator. Performance will be assessed at three points: pretraining, immediately post-training and 4-6 weeks after training. Assessments will take place on the simulator at all three time points and transfer of skills will be assessed during two clinical colonoscopies 4-6 weeks post-training. Mixed factorial ANOVAs will be used to determine if there is a performance difference between the two groups during simulated and clinical assessments. ETHICS AND DISSEMINATION:Ethical approval was obtained at St. Michael's Hospital. Results of this trial will be submitted for presentation at academic meetings and for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER:NCT03176251.

Computational models are powerful tools in electrophysiology (EP), helping us understand and predict arrhythmia associated with heart attack (i.e., myocardial infarction), a major cause of sudden cardiac death. Our broad aim is to combine novel scar imaging methods with fast computational models to enable accurate predictions of electrical wave propagation, and then to test these models in preclinical frameworks prior to clinical translation. In this work we used n = 3 swine with chronic infarct, which underwent MR followed by conventional x-ray guided electro-anatomical EP mapping. For scar imaging, we employed our T1-mapping MR method based on multi-contrast late enhancement (MCLE) at 1 × 1 mm in-plane resolution and 5 mm slice thickness. Next, we used the MCLE images as input to a fuzzy-logic algorithm and segmented the infarcted area into two zones: infarct core IC (dense fibrosis) and grey-zone, GZ (i.e., arrhythmia substrate). We further built 3D heart models from the stack of segmented 2D MCLE images, integrating tissue zones (healthy, IC and GZ) into detailed tetrahedral heart meshes (~1.5 mm element size). Finally, we investigated the accuracy of model predictions by comparing measured maps of activation times (i.e., depolarization times) with simulated maps obtained by employing a macroscopic formalism and reaction-diffusion equations. We obtained an acceptable small mean absolute error between the simulated and measured depolarization times (~12 ms, in average). Future work will focus on refining MR imaging resolution and use the models to guide ablation procedures.

Background and study aims Novice endoscopists are inaccurate in self-assessment of procedures. One means of improving self-assessment accuracy is through video-based feedback. We aimed to determine the comparative effectiveness of three video-based interventions on novice endoscopists' self-assessment accuracy of endoscopic competence. Materials and methods Novice endoscopists (performed <20 previous procedures) were recruited. Participants completed a simulated esophagogastroduodenoscopy (EGD) on a virtual reality simulator. They were then randomized to one of three groups: self-video review (SVR), which involved watching a recorded video of their own performance; benchmark review (BVR), which involved watching a video of a simulated EGD completed by an expert; and self- and benchmark video (SBVR), which involved both videos. Participants then completed two additional simulated EGD cases. Self-assessments were conducted immediately after the first procedure, after the video intervention and after the additional two procedures. External assessments were conducted by two experienced endoscopists, who were blinded to participant identity and group assignment through video recordings. External and self-assessments were completed using the global rating scale component of the Gastrointestinal Endoscopy Competency Assessment Tool (GiECAT GRS). Results Fifty-one participants completed the study. The BVR group had significantly improved self-assessment accuracy in the short-term, compared to the SBVR group P=.005). The SBVR group demonstrated significantly improved self-assessment accuracy over time (P= .01 6). There were no significant effects of group or of time for the SVR group. Conclusions Video-based interventions, particularly combined use of self- and benchmark video review, can improve accuracy of self-assessment of endoscopic competence among novices.

Simulation-based training (SBT) curricula for training novices in gastrointestinal endoscopy has been shown to be effective. Gamification, which is the application of game-design elements such as competition, rankings and rewards, has been used to enhance procedural learning in healthcare, but no studies have investigated the effects of a comprehensive gamification curriculum on the acquisition of endoscopic skills amongst novice endoscopists.

Magnetic resonance imaging (MRI) can reveal subtle microstructural changes in the heart muscle following infarction, a major cause of sudden cardiac death worldwide. We have developed preclinical models of chronic infarction and used diffusion tensor methods to efficiently probe biophysical MR signals. Here our specific goal was to identify subtle characteristics of the peri-infarct zone, where the mixture of viable cells and collagen fibrils form arrhythmia substrate. We induced myocardial infarction in n=9 Yorkshire swine using an occlusion-reperfusion technique. We occluded the left circumflex artery (LCX) in 5 pigs and the left anterior descendent artery (LAD) in 4 pigs. After 5-6 weeks healing time, high resolution diffusion tensor imaging (∼0.6x0.6x1.2mm spatial resolution) was performed ex vivo in explanted hearts. We then calculated pixel-based fractional anisotropy (FA) in 3 zones: infarct core (dense fibrosis), peri-infarct (arrhythmia substrate) and remote/normal tissue. For analysis, we selected four regions of interests (ROIs) from each tissue zone. A total of 36 ROIs selected from DT images were analyzed and further registered via anatomical markers with corresponding ROIs in histopathology slides. For histology, tissue samples were stained with collagen-sensitive stain to evaluate fibrosis under bright field and polarized light microscopy, as well as CD31 (microvascular integrity) and electrical gap junctions (Cx43). Our results demonstrated a significant decrease in FA (∼25%) in peri-infarct compared to normal myocardium (p<0.05), in agreement with quantitative histopathological and immunohistochemistry findings which showed a reduced CD31 density (∼33%) and Cx43 density (∼56%). These results provide clear evidence of altered microstructure (i.e., fiber disarray due to collagen deposition) and function (i.e., reduced electrical connections and perfusion) in peri-infarct, in large animal models relevant to scar-related human pathophysiology. Future work will focus on using these novel findings to parameterize MRI-based models to predict arrhythmia inducibility.

Background: Accurate self-assessment, usually represented by a high agreement between a self-rating and an external-rating, is an important process for skill development. In colonoscopy, skills can be categorized as technical (e.g. scope navigation) or non-technical (e.g. communication). There is a paucity of research examining the accuracy of self-assessment of technical and non-technical skills in colonoscopy among novices. Aims: To investigate the accuracy of self-assessment for technical skills and non-technical skills in live colonoscopies among novice endoscopists. Methods: Novice endoscopists (performed <50 previous colonoscopies) were recruited. Each participant completed two clinical colonoscopies. Video recordings of each colonoscopy procedure were assessed by an independent expert endoscopist, who was blinded to participant identity. Technical skills were assessed using the Direct Observation of Procedural Skills (DOPS), a procedure-specific assessment tool with good validity evidence. Non-technical skills were assessed using a modification of the Objective Structured Assessment of Non-technical skills (m-OSANTS), which also has good validity evidence. Participants self-assessed their performance immediately following each colonoscopy using the same instruments. Self-assessment accuracy between participant and expert ratings was determined using the intra-class correlation coefficient (ICC1,1) and paired-sample t-tests. Results: Thirty-nine novice endoscopists participated. Agreement between participant and expert ratings as measured by the ICC was 0.36 (95% CI: 0.03-0.62) and 0.32 (95% CI: -0.01-0.59) for technical and non-technical skills, respectively. There was a significant difference between technical

Endoscopists are at risk of musculoskeletal injury due to the repetitive strain of motions used during procedures. Additionally, appropriate ergonomic practice contributes to better performance in endoscopy. Despite this, gastroenterology trainees do not receive education about ergonomics. Furthermore, there is a lack of literature on effective methods to teach ergonomic principles and behaviours to trainees. To determine the effectiveness of an educational intervention for novice endoscopists designed to teach proper ergonomic principles to use in performing colonoscopy. Novice endoscopists (performed <50 previous colonoscopies) were enrolled in a simulation course in colonoscopy. The ergonomics teaching intervention consisted of the following: one hour didactic lecture; a video demonstrating ideal ergonomics during colonoscopy; and a self-reflection checklist on ergonomics in colonoscopy used after each simulated procedure. Participants were assessed at baseline (i.e. pre-test) and after the intervention (i.e. post-test) using the Rapid Upper Limb Assessment (RULA), an assessment tool of ergonomic behaviours. Higher scores on the RULA correspond to poorer ergonomic techniques. Sixteen residents completed the training intervention. The mean final scores based on the RULA for the pre-test was 6.31 (SD=0.70) and 5.31 (SD=1.58) for the post-test. All participants showed a significant decrease in their RULA scores (p<0.015). A teaching intervention for instruction of ergonomic principles in colonoscopy, involving a video, lecture, and self-reflection tool was associated with improved performance among novices. Further work should be performed to determine the most effective means of teaching proper ergonomic technique to novice endoscopists, and to determine if this training translates into improved clinical performance. None

Non-technical skills (NTS), such as communication and professionalism, contribute to safe and effective completion of procedures. Studies in the surgical literature show that NTS can be taught using simulation-based training (SBT). The impact of dedicated instruction in NTS as applied to endoscopy has yet to be evaluated.

Background and Aims Non-technical skills (NTS), involving cognitive, social and interpersonal skills that complement technical skills, are important for the completion of safe and efficient procedures. We investigated the impact of a simulation-based curriculum with dedicated NTS training on novice endoscopists' performance of clinical colonoscopies. Methods A single-blinded randomized controlled trial was conducted at a single center. Novice endoscopists were randomized to a control curriculum or a NTS curriculum. The control curriculum involved a didactic session, virtual reality (VR) simulator colonoscopy training, and integrated scenario practice using a VR simulator, a standardized patient, and endoscopy nurse. Feedback and training were provided by experienced endoscopists. The NTS curriculum group received similar training that included a small-group session on NTS, feedback targeting NTS, and access to a self-reflective NTS checklist. The primary outcome was performance during two clinical colonoscopies, assessed using the Joint Advisory Group Direct Observation of Procedural Skills (JAG DOPS) tool. Results Thirty-nine participants completed the study. The NTS group (n = 21) had superior clinical performance during their first (P < 0.001) and second clinical colonoscopies (P < .0.001), compared to the control group (n = 18). The NTS group performed significantly better on the VR simulator (P < 0.05) and in the integrated scenario (P < 0.05). Conclusion Our findings demonstrate that dedicated NTS training led to improved performance of clinical colonoscopies among novices.

Self-assessment allows for self-regulation of procedural learning, and has been identified by ASGE as important in the development and maintenance of competence in endoscopy. There is some evidence that video-based feedback can improve self-assessment accuracy of procedural skills, but the impact of video-based feedback on self-assessment of endoscopic skill is unknown.