Developing an educational "hub": impact of a distance-learning curriculum in a multinational cohort.

PURPOSE: To address a gap in radiation oncology education in low- and middle-income countries (LMICs), we sought to evaluate the effectiveness and generalizability of a refined curriculum on intensity modulated radiotherapy (IMRT) offered to existing radiation therapy (RT) clinics across Africa and Latin America (LATAM) at no cost. METHODS: A curriculum was created based on prior needs assessments and adapted for participating medical physicists, radiation oncologists, radiation therapists, and trainees in LMICs. English-speaking and Spanish-speaking teams of volunteer educators delivered 27 hour-long sessions 1-2 times weekly for 4 months using video conferencing to African and LATAM cohorts, respectively. Pre- and post-course multiple-choice examinations were administered to LATAM participants, and pre- and post-course self-confidence (1-5 Likert-scale) and open-ended feedback were collected from all participants. RESULTS: Twenty-five centers across Africa (13) and LATAM (12) participated, yielding a total of 332 enrolled participants (128 African, 204 LATAM). Sessions were delivered with a mean of 44 (22.5) and 85 (25.4) participants in the African and LATAM programs, respectively. Paired pre and post-course data demonstrated significant (p < 0.001) improvement in knowledge from 47.9 to 89.6% and self-confidence across four domains including foundations (+ 1.1), commissioning (+ 1.3), contouring (+ 1.7), and treatment planning (+ 1.0). Attendance was a significant predictor of change in self-confidence in "high attendance" participants only, suggesting a threshold effect. Qualitative data demonstrates that participants look forward to applying their knowledge in the clinical setting. CONCLUSION: A specialized radiation oncology curriculum adapted for LMIC audiences was effective for both African and LATAM participants. Participant feedback suggests that the refined IMRT course empowered clinics with knowledge and confidence to help train others. This feasible "Hub and Spokes" approach in which a distance-learning course establishes a hub to be leveraged by spokes (learners) may be generalizable to others aiming to reduce global health care disparities through training efforts.

Frameworks for Radiation Oncology Global Health Initiatives in US Residency Programs.

PURPOSE: To understand trends, pathways, and experiences and to establish a framework for radiation oncology (RO) programs interested in developing global health (GH) initiatives. METHODS: An in-depth interview was conducted of all US RO programs with established GH initiatives. Programs were identified by reviewing results of the 2018 Association of Residents in Radiation Oncology Global Health Resident Survey and individualized outreach to screen for additional programs meeting the following criteria: (1) active resident involvement in RO-specific GH opportunities, (2) active faculty involvement in these initiatives, and (3) department chair or program director awareness and support for ongoing opportunities. Among 88 residency programs, 11 were identified. Standardized questions explored the type of initiative, planning, staff and resident involvement, challenges, components to success, and history of programs through December 2018. RESULTS: Between 2010 and 2018, 11 programs started initiatives. Total resident participants ranged from one to 13 (median = 3) in each program's history. Initiatives spanned education (n = 9 [82%]), clinical mentorship (73%), innovative technology (55%), bilateral hosting programs (45%), clinical development and equipment (45%), promotion of local research (36%), clinical care (36%), industry partnerships (27%), and remote tumor board (18%). Faculty involvement included radiation oncologists (91%), medical physicists (55%), and non-RO department faculty (27%). Six programs (55%) had faculty with prior GH experience. Four (36%) programs reported medical student involvement in projects. Barriers included international communication (36%), time for faculty (18%), funding (9%), and legal (9%) concerns. Commonest components of success included fostering relationships with international sites and identifying needs before solutions. CONCLUSION: RO GH initiatives were reported as positive, educational, and feasible across 11 US residency programs. Growth is expected, representing opportunities for innovation and service among US programs.

Leveraging Telehealth and Medical Student Volunteers to Bridge Gaps in Education Access for Providers in Limited-Resource Settings.

PROBLEM: High-quality training opportunities for providers in limited-resource settings are often scarce or nonexistent. This can lead to a dearth of boots-on-the-ground workers capable of translating knowledge into effective action. The tested telehealth education model of Project ECHO (Extension for Community Healthcare Outcomes) can help address this disparity. However, the planning and logistical coordination required can be limiting. APPROACH: Medical student volunteers interested in health disparities and global health can be leveraged to reduce the costs of administration for Project ECHO programs. From mid-2018 to present (2020), student organizations have been formed at Vanderbilt University School of Medicine, University of California, San Francisco, School of Medicine, and Albert Einstein College of Medicine. These organizations have recruited and trained volunteers, who play an active role in assessing the needs of local clinics and providers, developing curricula, and coordinating the logistical aspects of programs. OUTCOMES: In the first 4 student-coordinated Project ECHO cohorts (2019-2020), 25 clinics in 14 countries participated, with a potential impact on over 20,000 cancer patients annually. Satisfaction with the telehealth education programs was high among local clinicians and expert educators. Students' perceived ability to conduct activities important to successfully orchestrating a telehealth education program was significantly greater among students who had coordinated one or more Project ECHO programs than among students who had yet to participate for 7 of 9 competencies. There also appears to be an additive effect of participating in additional Project ECHO programs on perceived confidence and career path intentions. NEXT STEPS: The student-led model of coordinating telehealth education programs described here can be readily expanded to medical schools across the nation and beyond. With continued expansion, efforts are needed to develop assessments that provide insights into participants' learning, track changes in patient outcomes, and provide continuing medical education credits to local clinicians.

RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif.

RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a "minor groove sensor." Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy.

Replication stress by Py-Im polyamides induces a non-canonical ATR-dependent checkpoint response.

Pyrrole-imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.

Animal toxicity of hairpin pyrrole-imidazole polyamides varies with the turn unit.

A hairpin pyrrole-imidazole polyamide (1) targeted to the androgen receptor consensus half-site was found to exert antitumor effects against prostate cancer xenografts. A previous animal study showed that 1, which has a chiral amine at the α-position of the γ-aminobutyric acid turn (γ-turn), did not exhibit toxicity at doses less than 10 mg/kg. In the same study, a polyamide with an acetamide at the ß-position of the γ-turn resulted in animal morbidity at 2.3 mg/kg. To identify structural motifs that cause animal toxicity, we synthesized polyamides 1-4 with variations at the α- and ß-positions in the γ-turn. Weight loss, histopathology, and serum chemistry were analyzed in mice post-treatment. While serum concentration was similar for all four polyamides after injection, dose-limiting liver toxicity was only observed for three polyamides. Polyamide 3, with an α-acetamide, caused no significant evidence of rodent toxicity and retains activity against LNCaP xenografts.

Activity of a Py-Im polyamide targeted to the estrogen response element.

Pyrrole-imidazole (Py-Im) polyamides are a class of programmable DNA minor groove binders capable of modulating the activity of DNA-binding proteins and affecting changes in gene expression. Estrogen receptor alpha (ERα) is a ligand-activated hormone receptor that binds as a homodimer to estrogen response elements (ERE) and is a driving oncogene in a majority of breast cancers. We tested a selection of structurally similar Py-Im polyamides with differing DNA sequence specificity for activity against 17ß-estadiol (E2)-induced transcription and cytotoxicity in ERα positive, E2-stimulated T47DKBluc cells, which express luciferase under ERα control. The most active polyamide targeted the sequence 5'-WGGWCW-3' (W = A or T), which is the canonical ERE half site. Whole transcriptome analysis using RNA-Seq revealed that treatment of E2-stimulated breast cancer cells with this polyamide reduced the effects of E2 on the majority of those most strongly affected by E2 but had much less effect on the majority of E2-induced transcripts. In vivo, this polyamide circulated at detectable levels following subcutaneous injection and reduced levels of ER-driven luciferase expression in xenografted tumors in mice after subcutaneous compound administration without significant host toxicity.

Antitumor activity of a pyrrole-imidazole polyamide.

Many cancer therapeutics target DNA and exert cytotoxicity through the induction of DNA damage and inhibition of transcription. We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Polyamide treatment activates p53 signaling in LNCaP prostate cancer cells without detectable DNA damage. Genome-wide mapping of RNAP2 binding shows reduction of occupancy, preferentially at transcription start sites, but occupancy at enhancer sites is unchanged. Polyamide treatment results in a time- and dose-dependent depletion of the RNAP2 large subunit RPB1 that is preventable with proteasome inhibition. This polyamide demonstrates antitumor activity in a prostate tumor xenograft model with limited host toxicity.

Synthesis of cyclic Py-Im polyamide libraries.

Cyclic Py-Im polyamides containing two GABA turn units exhibit enhanced DNA binding affinity, but extensive studies of their biological properties have been hindered due to synthetic inaccessibility. A facile modular approach toward cyclic polyamides has been developed via microwave-assisted solid-phase synthesis of hairpin amino acid oligomer intermediates followed by macrocyclization. A focused library of cyclic polyamides 1-7 targeted to the androgen response element (ARE) and the estrogen response element (ERE) were synthesized in 12-17% overall yield. The Fmoc protection strategy also allows for selective modifications on the GABA turn units that have been shown to improve cellular uptake properties. The DNA binding affinities of a library of cyclic polyamides were measured by DNA thermal denaturation assays and compared to the corresponding hairpin polyamides. Fluorescein-labeled cyclic polyamides have been synthesized and imaged via confocal microscopy in A549 and T47D cell lines. The IC(50) values of compounds 1-7 and 9-11 were determined, revealing remarkably varying levels of cytotoxicity.

Single-dose pharmacokinetic and toxicity analysis of pyrrole-imidazole polyamides in mice.

PURPOSE: Pyrrole-imidazole (Py-Im) polyamides are programmable, sequence-specific DNA minor groove-binding ligands. Previous work in cell culture has shown that various polyamides can be used to modulate the transcriptional programs of oncogenic transcription factors. In this study, two hairpin polyamides with demonstrated activity against androgen receptor signaling in cell culture were administered to mice to characterize their pharmacokinetic properties. METHODS: Py-Im polyamides were administered intravenously by tail vein injection. Plasma, urine, and fecal samples were collected over a 24-h period. Liver, kidney, and lung samples were collected postmortem. Concentrations of the administered polyamide in the plasma, excretion, and tissue samples were measured using LC/MS/MS. The biodistribution data were analyzed by both non-compartmental and compartmental pharmacokinetic models. Animal toxicity experiments were also performed by monitoring weight loss after a single subcutaneous (SC) injection of either polyamide. RESULTS: The biodistribution profiles of both compounds exhibited rapid localization to the liver, kidneys, and lungs upon injection. Plasma distribution of the two compounds showed distinct differences in the rate of clearance, the volume of distribution, and the AUCs. These two compounds also have markedly different toxicities after SC injection in mice. CONCLUSIONS: The variations in pharmacokinetics and toxicity in vivo stem from a minor chemical modification that is also correlated with differing potency in cell culture. The results obtained in this study could provide a structural basis for further improvement of polyamide activity both in cell culture and in animal models.

DNA sequence selectivity of hairpin polyamide turn units.

A class of hairpin polyamides linked by 3,4-diaminobutyric acid, resulting in a beta-amine residue at the turn unit, showed improved binding affinities relative to their alpha-amino-gamma-turn analogs for particular sequences. We incorporated beta-amino-gamma-turns in six-ring polyamides and determined whether there are any sequence preferences under the turn unit by quantitative footprinting titrations. Although there was an energetic penalty for G.C and C.G base pairs, we found little preference for T.A over A.T at the beta-amino-gamma-turn position. Fluorine and hydroxyl substituted alpha-amino-gamma-turns were synthesized for comparison. Their binding affinities and specificities in the context of six-ring polyamides demonstrated overall diminished affinity and no additional specificity at the turn position. We anticipate that this study will be a baseline for further investigation of the turn subunit as a recognition element for the DNA minor groove.

Porphyrins conjugated to DNA as CD reporters of the salt-induced B to Z-DNA transition.

The porphyrin chromophore incorporated at the 5'-position of an oligonucleotide allows the simultaneous detection of the B- to Z-DNA transition via the porphyrin Soret band circular dichroism exciton couplet signal around 420 nm and the oligonucleotide CD region below 300 nm, at micromolar concentrations.