An improved method for generating human spinal cord neural stem cells.

Neural stem cells have exhibited efficacy in pre-clinical models of spinal cord injury (SCI) and are on a translational path to human testing. We recently reported that neural stem cells must be driven to a spinal cord fate to optimize host axonal regeneration into sites of implantation in the injured spinal cord, where they subsequently form neural relays across the lesion that support significant functional improvement. We also reported methods of deriving and culturing human spinal cord neural stem cells derived from embryonic stem cells that can be sustained over serial high passage numbers in vitro, providing a potentially optimized cell source for human clinical trials. We now report further optimization of methods for deriving and sustaining cultures of human spinal cord neural stem cell lines that result in improved karyotypic stability while retaining anatomical efficacy in vivo. This development improves prospects for safe human translation.

Stereotactic Radiation Therapy in Early Non-Small Cell Lung Cancer and Interstitial Lung Disease: A Nonrandomized Clinical Trial.

Importance: Patients with interstitial lung disease (ILD) and early-stage non-small cell lung cancer (NSCLC) have been reported to be at high risk of toxic effects after stereotactic ablative radiotherapy (SABR), but for many patients, there are limited alternative treatment options. Objective: To prospectively assess the benefits and toxic effects of SABR in this patient population. Design, Setting, and Participants: This prospective cohort study was conducted at 6 academic radiation oncology institutions, 5 in Canada and 1 in Scotland, with accrual between March 7, 2019, and January 12, 2022. Patients aged 18 years or older with fibrotic ILD and a diagnosis of T1-2N0 NSCLC who were not candidates for surgical resection were enrolled. Intervention: Patients were treated with SABR to a dose of 50 Gy in 5 fractions every other day. Main Outcomes and Measures: The study prespecified that SABR would be considered worthwhile if median overall survival-the primary end point-was longer than 1 year, with a grade 3 to 4 risk of toxic effects less than 35% and a grade 5 risk of toxic effects less than 15%. Secondary end points included toxic effects, progression-free survival (PFS), local control (LC), quality-of-life outcomes, and changes in pulmonary function. Intention-to-treat analysis was conducted. Results: Thirty-nine patients enrolled and received SABR. Median age was 78 (IQR, 67-83) years and 59% (n = 23) were male. At baseline, 70% (26 of 37) of patients reported dyspnea, median forced expiratory volume in first second of expiration was 80% (IQR, 66%-90%) predicted, median forced vital capacity was 84% (IQR, 69%-94%) predicted, and median diffusion capacity of the lung for carbon monoxide was 49% (IQR, 38%-61%) predicted. Median follow-up was 19 (IQR, 14-25) months. Overall survival at 1 year was 79% (95%, CI 62%-89%; P < .001 vs the unacceptable rate), and median overall survival was 25 months (95% CI, 14 months to not reached). Median PFS was 19 months (95% CI, 13-28 months), and 2-year LC was 92% (95% CI, 69%-98%). Adverse event rates (highest grade per patient) were grade 1 to 2: n = 12 (31%), grade 3: n = 4 (10%), grade 4: n = 0, and grade 5: n = 3 (7.7%, all due to respiratory deterioration). Conclusions and Relevance: In this trial, use of SABR in patients with fibrotic ILD met the prespecified acceptability thresholds for both toxicity and efficacy, supporting the use of SABR for curative-intent treatment after a careful discussion of risks and benefits. Trial Registration: ClinicalTrials.gov Identifier: NCT03485378.

CCR2 monocytes as therapeutic targets for acute disc herniation and radiculopathy in mouse models.

OBJECTIVE: Back pain and radiculopathy caused by disc herniation are major health issues worldwide. While macrophages are key players in disc herniation induced inflammation, their roles and origins in disease progression remain unclear. We aim to study the roles of monocytes and derivatives in a mouse model of disc herniation. METHODS: Using a CCR2-CreER; R26R-EGFP (Ai6) transgenic mouse strain, we fate-mapped C-C chemokine receptor type 2 (CCR2) expressing monocytes and derivatives at disc herniation sites, and employed a CCR2RFP/RFP mouse strain and a CCR2-specific antagonist to study the effects of CCR2+ monocytes on local inflammatory responses, pain level, and disc degeneration by immunostaining, flow cytometry, and histology. RESULTS: CCR2+ monocytes (GFP+) increased at the sites of disc hernia over postoperative day 4, 6, and 9 in CCR2-CreER; Ai6 mice. F4/80+ cells increased, and meanwhile, CD11b+ cells trended downward. Co-localization analysis revealed that both GFP+CD11b+ and GFP+F4/80+ constituted the majority of CD11b+ and F4/80+ cells at disc hernia sites. Fluorescence activated cell sorter purified GFP+ cells exhibited higher cytokine expressions than GFP- cells. Inhibition of CCR2 signaling reduced infiltration of monocytes and macrophages, alleviated pain, maintained disc height, and reduced osteoclast activity in adjacent cortical bone for up to 1 month. CONCLUSION: Our findings suggest that circulating CCR2+ monocytes play important roles in initiating and promoting the local inflammatory responses, pain sensitization, and degenerative changes after disc herniation, and thus may serve as therapeutic targets for disc herniation induced back and leg pain.

APOBEC3 activity promotes the survival and evolution of drug-tolerant persister cells during acquired resistance to EGFR inhibitors in lung cancer.

APOBEC mutagenesis is one of the most common endogenous sources of mutations in human cancer and is a major source of genetic intratumor heterogeneity. High levels of APOBEC mutagenesis are associated with poor prognosis and aggressive disease across diverse cancers, but the mechanistic and functional impacts of APOBEC mutagenesis on tumor evolution and therapy resistance remain relatively unexplored. To address this, we investigated the contribution of APOBEC mutagenesis to acquired therapy resistance in a model of EGFR-mutant non-small cell lung cancer. We find that inhibition of EGFR in lung cancer cells leads to a rapid and pronounced induction of APOBEC3 expression and activity. Functionally, APOBEC expression promotes the survival of drug-tolerant persister cells (DTPs) following EGFR inhibition. Constitutive expression of APOBEC3B alters the evolutionary trajectory of acquired resistance to the EGFR inhibitor gefitinib, making it more likely that resistance arises through de novo acquisition of the T790M gatekeeper mutation and squamous transdifferentiation during the DTP state. APOBEC3B expression is associated with increased expression of the squamous cell transcription factor ΔNp63 and squamous cell transdifferentiation in gefitinib-resistant cells. Knockout of ΔNp63 in gefitinibresistant cells reduces the expression of the p63 target genes IL1a/b and sensitizes these cells to the thirdgeneration EGFR inhibitor osimertinib. These results suggest that APOBEC activity promotes acquired resistance by facilitating evolution and transdifferentiation in DTPs, and suggest that approaches to target ΔNp63 in gefitinib-resistant lung cancers may have therapeutic benefit.

Broader functions of TIR domains in Arabidopsis immunity.

TIR domains are NAD-degrading enzymes that function during immune signaling in prokaryotes, plants, and animals. In plants, most TIR domains are incorporated into intracellular immune receptors termed TNLs. In Arabidopsis, TIR-derived small molecules bind and activate EDS1 heterodimers, which in turn activate RNLs, a class of cation channel-forming immune receptors. RNL activation drives cytoplasmic Ca2+ influx, transcriptional reprogramming, pathogen resistance, and host cell death. We screened for mutants that suppress an RNL activation mimic allele and identified a TNL, SADR1. Despite being required for the function of an autoactivated RNL, SADR1 is not required for defense signaling triggered by other tested TNLs. SADR1 is required for defense signaling initiated by some transmembrane pattern recognition receptors and contributes to the unbridled spread of cell death in lesion simulating disease 1. Together with RNLs, SADR1 regulates defense gene expression at infection site borders, likely in a non-cell autonomous manner. RNL mutants that cannot sustain this pattern of gene expression are unable to prevent disease spread beyond localized infection sites, suggesting that this pattern corresponds to a pathogen containment mechanism. SADR1 potentiates RNL-driven immune signaling not only through the activation of EDS1 but also partially independently of EDS1. We studied EDS1-independent TIR function using nicotinamide, an NADase inhibitor. Nicotinamide decreased defense induction from transmembrane pattern recognition receptors and decreased calcium influx, pathogen growth restriction, and host cell death following intracellular immune receptor activation. We demonstrate that TIR domains can potentiate calcium influx and defense and are thus broadly required for Arabidopsis immunity.

Anterior Hook Plating of Patella Fractures: A Biomechanical Analysis and Clinical Series.

OBJECTIVES: To compare anterior hook plating with established fixation constructs biomechanically and report outcomes and complications in a cohort of patella fractures treated with the technique. DESIGN: Laboratory-based biomechanical study and clinical multicenter retrospective cohort study. SETTING: 2 US Level 1 trauma centers. PATIENTS/PARTICIPANTS: 51 patients (28 M and 23 F) with 30 simple transverse and 21 comminuted patella fractures. Thirty-six cadaveric patellae were used for the biomechanical study. INTERVENTION: Biomechanical-dorsal plating was compared with cerclage wiring and modified tension band cable fixation in a comminuted patella fracture model in 36 cadaveric patellae. Constructs were tested at 0° and 45 degrees of flexion. Clinical-we reviewed a consecutive series of patella fractures in 2 centers for outcome and complications. MAIN OUTCOME MEASUREMENTS: Biomechanical-construct stiffness. Clinical-reduction, union, complications, and range of motion. RESULTS: Stiffness was greatest in dorsal plating at both 0° and 45 degrees. Dorsal plating (976 N, 1643 N) > modified tension band (317 N, 297 N) > cerclage (89.8 N, 150.3 N) at 0 and 45 degrees, respectively. 51 patients with patella fractures had them fixed with dorsal 2.7-mm mini fragment plates including a distal to proximal lag screw through the plate from the nose of the patella. 9 cases were small distal fragments not easily managed with screws and cables. All patients were followed up to union. There were 2 infections (1 superficial and 1 deep with nonunion), and 5 had implant removal (9.8%). CONCLUSIONS: Dorsal plating is biomechanically and clinically superior to modified tension band and cerclage techniques in comminuted patella fractures. This method allows for fixation of small distal pole fractures. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

A cost-consequence analysis of eLearning videos designed to supplement the consent process in lower limb arthroplasty.

INTRODUCTION: Since the Montgomery ruling in 2015 surgeons have been tasked with identifying material risk when taking informed consent. Despite this, there has been limited uptake of technological aids to supplement the consent process although such aids are shown to improve patient knowledge and satisfaction. ConsentPLUS is a free-to-access website with bite-sized educational videos designed to clearly explain lower limb arthroplasty procedures to patients and aid their consent. METHODS: The authors performed a prospective cost-consequence analysis, outlining any costs associated with the intervention and any quantitative or qualitative impacts the intervention may have on patients. RESULTS: A total of 3,143 consecutive patients were identified who were undergoing total knee or hip replacement in 25 elective NHS orthopaedic units. The total cost of development and projected 10-year running fees for ConsentPLUS total £75,000. Health Foundation support means the service is free-to-access for centres throughout the UK. Mean exposure time per patient was 10min 29s, equivalent to £185,437 of additional contact time according to the National Tariff. Mean clinic time was reduced by 17min owing to the earlier identification of material risk. Patient knowledge on pre- and post-video quizzes increased from 7.01 to 9.08 following eLearning (paired t-test = 0.998). The process had an overall satisfaction rate of 97%. CONCLUSION: Educational eLearning videos are an accessible and digestible way to supplement the consent process. This enables earlier identification of material risk in clinics owing to improved patient knowledge, leading to increased patient satisfaction with arthroplasty consenting.

Breaking the limit: Biological countermeasures for space radiation exposure to enable long-duration spaceflight.

Concerns over the health effects of space radiation exposure currently limit the duration of deep-space travel. Effective biological countermeasures could allow humanity to break this limit, facilitating human exploration and sustained presence on the Moon, Mars, or elsewhere in the Solar System. In this issue, we present a collection of 20 articles, each providing perspectives or data relevant to the implementation of a countermeasure discovery and development program. Topics include agency and drug developer perspectives, the prospects for repurposing of existing drugs or other agents, and the potential for adoption of new technologies, high-throughput screening, novel animal or microphysiological models, and alternative ground-based radiation sources. Long-term goals of a countermeasures program include reduction in the risk of radiation-exposure induced cancer death to an acceptable level and reduction in risks to the brain, cardiovascular system, and other organs.

The Need for Biological Countermeasures to Mitigate the Risk of Space Radiation-Induced Carcinogenesis, Cardiovascular Disease, and Central Nervous System Deficiencies.

NASA's currently planned long-duration, deep space exploration missions outside of low Earth orbit (LEO) will result in the exposure of astronauts to relatively high lifetime doses of ionizing radiation (IR), exceeding what humans have previously encountered in space. Of concern to this exposure are the long-term health consequences of radiation carcinogenesis, cardiovascular and degenerative disease, and central nervous system decrements. Existing engineering solutions are insufficient to decrease the lifetime accumulated IR exposure to levels currently allowable by agency standards, therefore appropriate countermeasure and mitigation strategies must be developed to enable long duration missions. Emerging discoveries in the fields of radiation oncology and the mitigation of Acute Radiation Syndrome (ARS) have demonstrated the potential for compound-based/biological radiomodifiers to drastically improve clinical outcomes and represent a promising strategy for space radiation countermeasure development. This review outlines the unique challenges posed by the space radiation environment, defines the limits of terrestrial radiation protection strategies in space, describes a brief overview of current space radiation countermeasure development strategies, highlights potential new approaches for countermeasure identification and development, and speculates on the potential benefits beyond space exploration.

Intramolecular tetrazine-acryloyl cycloaddition: chemistry and applications.

An unprecedented intramolecular [4 + 2] tetrazine-olefin cycloaddition with α,ß-unsaturated substrates was discovered. The reaction produces unique coumarin-dihydropyridazine heterocycles that exhibited strong fluorescence with large Stokes shifts and excellent photo- and pH-stability. This property can be used for reaction analysis. The rate of cycloaddition was found to be solvent dependent and was determined using experimental data with a kinetic modeling software (COPASI) as well as DFT calculations (k 1 = 0.64 ± 0.019 s-1 and 4.1 s-1, respectively). The effects of steric and electronic properties of both the tetrazine and α,ß-unsaturated carbonyl on the reaction were studied and followed the known trends characteristic of the intermolecular reaction. Based on these results, we developed a "release-then-click" strategy for the ROS triggered release of methylselenenic acid (MeSeOH) and a fluorescent tracer. This strategy was demonstrated in HeLa cells via fluorescence imaging.

Pre-exposure Prophylaxis Discontinuation During the COVID-19 Pandemic Among Men Who Have Sex With Men in a Multisite Clinical Cohort in the United States.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic continues to put strain on health systems in the United States, leading to significant shifts in the delivery of routine clinical services, including those offering HIV pre-exposure prophylaxis (PrEP). We aimed to assess whether individuals discontinued PrEP use at higher rates during the COVID-19 pandemic and the extent to which disruptions to usual clinical care were mitigated through telehealth. METHODS: Using data from an ongoing prospective cohort of men who have sex with men (MSM) newly initiating PrEP in 3 mid-sized cities (n = 195), we calculated the rate of first-time discontinuation of PrEP use in the period before the COVID-19 pandemic and during the COVID-19 pandemic and compared these rates using incidence rate ratios (IRRs). Furthermore, we compared the characteristics of patients who discontinued PrEP use during these periods with those who continued to use PrEP during both periods. RESULTS: Rates of PrEP discontinuation before the COVID pandemic and during the COVID-19 pandemic were comparable [4.29 vs. 5.20 discontinuations per 100 person-months; IRR: 1.95; 95% confidence interval (CI): 0.83 to 1.77]. Although no significant differences in the PrEP discontinuation rate were observed in the overall population, the rate of PrEP discontinuation increased by almost 3-fold among participants aged 18-24 year old (IRR: 2.78; 95% CI: 1.48 to 5.23) and by 29% among participants covered by public insurance plans at enrollment (IRR: 1.29; 95% CI: 1.03 to 5.09). Those who continued to use PrEP were more likely to have had a follow-up clinical visit by telehealth in the early months of the pandemic (45% vs. 17%). CONCLUSIONS: In this study, rates of PrEP discontinuation were largely unchanged with the onset of the COVID-19 pandemic. The use of telehealth likely helped retain patients in PrEP care and should continue to be offered in the future.

In vivo metabolic imaging identifies lipid vulnerability in a preclinical model of Her2+/Neu breast cancer residual disease and recurrence.

Recurrent cancer cells that evade therapy is a leading cause of death in breast cancer patients. This risk is high for women showing an overexpression of human epidermal growth factor receptor 2 (Her2). Cells that persist can rely on different substrates for energy production relative to their primary tumor counterpart. Here, we characterize metabolic reprogramming related to tumor dormancy and recurrence in a doxycycline-induced Her2+/Neu model of breast cancer with varying times to recurrence using longitudinal fluorescence microscopy. Glucose uptake (2-NBDG) and mitochondrial membrane potential (TMRE) imaging metabolically phenotype mammary tumors as they transition to regression, dormancy, and recurrence. "Fast-recurrence" tumors (time to recurrence ~55 days), transition from glycolysis to mitochondrial metabolism during regression and this persists upon recurrence. "Slow-recurrence" tumors (time to recurrence ~100 days) rely on both glycolysis and mitochondrial metabolism during recurrence. The increase in mitochondrial activity in fast-recurrence tumors is attributed to a switch from glucose to fatty acids as the primary energy source for mitochondrial metabolism. Consequently, when fast-recurrence tumors receive treatment with a fatty acid inhibitor, Etomoxir, tumors report an increase in glucose uptake and lipid synthesis during regression. Treatment with Etomoxir ultimately prolongs survival. We show that metabolic reprogramming reports on tumor recurrence characteristics, particularly at time points that are essential for actionable targets. The temporal characteristics of metabolic reprogramming will be critical in determining the use of an appropriate timing for potential therapies; namely, the notion that metabolic-targeted inhibition during regression reports long-term therapeutic benefit.

Benzothiazole-Derived Sulfones and Sulfoxides as Reactive Templates for Biothiols and Sulfane Sulfurs.

In this work we studied the reactions of benzothiazole sulfones and sulfoxides toward reactive sulfur species. The reaction of thiols with benzothiazole sulfones produces sulfinic acids (RSO2H), which can further react with sulfane sulfurs to form thiosulfonic acids (RSO2SH). This was used to design fluorescent sensors for hydrogen polysulfides. The reaction of thiols with benzothiazole sulfoxides produces sulfenic acids (RSOH), which can undergo fast intramolecular cyclization and be used to design thiol-triggered fluorescent sensors.

Evaluation of the Response of HNSCC Cell Lines to γ-Rays and 12C Ions: Can Radioresistant Tumors Be Identified and Selected for 12C Ion Radiotherapy?

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Thirty percent of patients will experience locoregional recurrence for which median survival is less than 1 year. Factors contributing to treatment failure include inherent resistance to X-rays and chemotherapy, hypoxia, epithelial to mesenchymal transition, and immune suppression. The unique properties of 12C radiotherapy including enhanced cell killing, a decreased oxygen enhancement ratio, generation of complex DNA damage, and the potential to overcome immune suppression make its application well suited to the treatment of HNSCC. We examined the 12C radioresponse of five HNSCC cell lines, whose surviving fraction at 3.5 Gy ranged from average to resistant when compared with a larger panel of 38 cell lines to determine if 12C irradiation can overcome X-ray radioresistance and to identify biomarkers predictive of 12C radioresponse. Cells were irradiated with 12C using a SOBP with an average LET of 80 keV/µm (CNAO: Pavia, Italy). RBE values varied depending upon endpoint used. A 37 gene signature was able to place cells in their respective radiosensitivity cohort with an accuracy of 86%. Radioresistant cells were characterized by an enrichment of genes associated with radioresistance and survival mechanisms including but not limited to G2/M Checkpoint MTORC1, HIF1α, and PI3K/AKT/MTOR signaling. These data were used in conjunction with an in silico-based modeling approach to evaluate tumor control probability after 12C irradiation that compared clinically used treatment schedules with fixed RBE values vs. the RBEs determined for each cell line. Based on the above analysis, we present the framework of a strategy to utilize biological markers to predict which HNSCC patients would benefit the most from 12C radiotherapy.

Clinical Learning, Didactic Education, and Research Experiences of Radiation Oncology Resident Physicians in Canada.

Changes in the field of radiation oncology (RO) impacts residency training. Assessing trainee experiences is essential to inform curriculum development. We aim to explore gaps and strengths in current Canadian RO training, as we move towards competency-based medical education (CBME). An online survey was distributed to residents at all Canadian RO training programs. Surveys consisted of 66 open-ended, Likert-scale, matrix-style, and multiple-choice questions, and assessed clinical exposure, didactic teaching, professional relationships, and research experiences. Statistics were calculated from anonymized, aggregate responses. Out of 128 eligible residents, 53 responded (41% response rate). Of these, 57% were male, and 77% were Canadian medical graduates. Senior residents (PGY-4 to PGY-5) perceived insufficient exposure to lymphoma and ocular malignancies, brachytherapy for breast and esophagus malignancies, and stereotactic radiotherapy of the pancreas, prostate, and adrenal gland. Half (51%) had training on image-guided radiotherapy (IGRT) challenges, and 43% had a formal staff mentor. Most residents presented at least one research project at conferences (77%) and authored ≥ 1 publications (66%) during residency. Canadian RO residents are satisfied with their clinical training and educational experience in high-volume tumor sites and high-volume brachytherapy procedures. Areas identified for potential improvement are (1) low-volume tumor sites; (2) low-volume brachytherapy procedures; (3) low-volume stereotactic radiotherapy sites; (4) IGRT challenges; and (5) mentorship opportunities. These findings will inform future CBME curriculum revisions.

Multi-ancestral origin of intestinal tumors: Impact on growth, progression, and drug efficacy.

BACKGROUND: Data are steadily accruing that demonstrate that intestinal tumors are frequently derived from multiple founding cells, resulting in tumors comprised of distinct ancestral clones that might cooperate or alternatively compete, thereby potentially impacting different phases of the disease process. AIM: We sought to determine whether tumors with a multi-ancestral architecture involving at least two distinct clones show increased tumor number, growth, progression, or resistance to drug intervention. METHODS: Mice carrying the Min allele of Apc were generated that were mosaic with only a subset of cells in the intestinal epithelium expressing an activated form of PI3K, a key regulatory kinase affecting several important cellular processes. These cells were identifiable as they fluoresced green, whereas all other cells fluoresced red. RESULTS: Cell lineage tracing revealed that many intestinal tumors from our mouse model were derived from at least two founding cells, those expressing the activated PI3K (green) and those which did not (red). Heterotypic tumors with a multi-ancestral architecture as evidenced by a mixture of green and red cells exhibited increased tumor growth and invasiveness. Clonal architecture also had an impact on tumor response to low-dose aspirin. Aspirin treatment resulted in a greater reduction of heterotypic tumors derived from multiple founding cells as compared to tumors derived from a single founding cell. CONCLUSION: These data indicate that genetically distinct tumor-founding cells can contribute to early intratumoral heterogeneity. The coevolution of the founding cells and their progeny enhances colon tumor progression and impacts the response to aspirin. These findings are important to a more complete understanding of tumorigenesis with consequences for several distinct models of tumor evolution. They also have practical implications to the clinic. Mouse models with heterogenous tumors are likely better for predicting drug efficacy as compared to models in which the tumors are highly homogeneous. Moreover, understanding how interactions among different populations in a single heterotypic tumor with a multi-ancestral architecture impact response to a single agent and combination therapies are necessary to fully develop personalized medicine.

APOBEC Mutagenesis Inhibits Breast Cancer Growth through Induction of T cell-Mediated Antitumor Immune Responses.

The APOBEC family of cytidine deaminases is one of the most common endogenous sources of mutations in human cancer. Genomic studies of tumors have found that APOBEC mutational signatures are enriched in the HER2 subtype of breast cancer and are associated with immunotherapy response in diverse cancer types. However, the direct consequences of APOBEC mutagenesis on the tumor immune microenvironment have not been thoroughly investigated. To address this, we developed syngeneic murine mammary tumor models with inducible expression of APOBEC3B. We found that APOBEC activity induced antitumor adaptive immune responses and CD4+ T cell-mediated, antigen-specific tumor growth inhibition. Although polyclonal APOBEC tumors had a moderate growth defect, clonal APOBEC tumors were almost completely rejected, suggesting that APOBEC-mediated genetic heterogeneity limits antitumor adaptive immune responses. Consistent with the observed immune infiltration in APOBEC tumors, APOBEC activity sensitized HER2-driven breast tumors to anti-CTLA-4 checkpoint inhibition and led to a complete response to combination anti-CTLA-4 and anti-HER2 therapy. In human breast cancers, the relationship between APOBEC mutagenesis and immunogenicity varied by breast cancer subtype and the frequency of subclonal mutations. This work provides a mechanistic basis for the sensitivity of APOBEC tumors to checkpoint inhibitors and suggests a rationale for using APOBEC mutational signatures and clonality as biomarkers predicting immunotherapy response in HER2-positive (HER2+) breast cancers.

Context-dependent effects of whole-genome duplication during mammary tumor recurrence.

Whole-genome duplication (WGD) generates polyploid cells possessing more than two copies of the genome and is among the most common genetic abnormalities in cancer. The frequency of WGD increases in advanced and metastatic tumors, and WGD is associated with poor prognosis in diverse tumor types, suggesting a functional role for polyploidy in tumor progression. Experimental evidence suggests that polyploidy has both tumor-promoting and suppressing effects, but how polyploidy regulates tumor progression remains unclear. Using a genetically engineered mouse model of Her2-driven breast cancer, we explored the prevalence and consequences of whole-genome duplication during tumor growth and recurrence. While primary tumors in this model are invariably diploid, nearly 40% of recurrent tumors undergo WGD. WGD in recurrent tumors was associated with increased chromosomal instability, decreased proliferation and increased survival in stress conditions. The effects of WGD on tumor growth were dependent on tumor stage. Surprisingly, in recurrent tumor cells WGD slowed tumor formation, growth rate and opposed the process of recurrence, while WGD promoted the growth of primary tumors. These findings highlight the importance of identifying conditions that promote the growth of polyploid tumors, including the cooperating genetic mutations that allow cells to overcome the barriers to WGD tumor cell growth and proliferation.

Avasopasem manganese synergizes with hypofractionated radiation to ablate tumors through the generation of hydrogen peroxide.

Avasopasem manganese (AVA or GC4419), a selective superoxide dismutase mimetic, is in a phase 3 clinical trial (NCT03689712) as a mitigator of radiation-induced mucositis in head and neck cancer based on its superoxide scavenging activity. We tested whether AVA synergized with radiation via the generation of hydrogen peroxide, the product of superoxide dismutation, to target tumor cells in preclinical xenograft models of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, and pancreatic ductal adenocarcinoma. Treatment synergy with AVA and high dose per fraction radiation occurred when mice were given AVA once before tumor irradiation and further increased when AVA was given before and for 4 days after radiation, supporting a role for oxidative metabolism. This synergy was abrogated by conditional overexpression of catalase in the tumors. In addition, in vitro NSCLC and mammary adenocarcinoma models showed that AVA increased intracellular hydrogen peroxide concentrations and buthionine sulfoximine- and auranofin-induced inhibition of glutathione- and thioredoxin-dependent hydrogen peroxide metabolism selectively enhanced AVA-induced killing of cancer cells compared to normal cells. Gene expression in irradiated tumors treated with AVA suggested that increased inflammatory, TNFα, and apoptosis signaling also contributed to treatment synergy. These results support the hypothesis that AVA, although reducing radiotherapy damage to normal tissues, acts synergistically only with high dose per fraction radiation regimens analogous to stereotactic ablative body radiotherapy against tumors by a hydrogen peroxide-dependent mechanism. This tumoricidal synergy is now being tested in a phase I-II clinical trial in humans (NCT03340974).