The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

Identifying Protein Interactions with Viral DNA Genomes during Virus Infection.

Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing primarily on herpesviruses and adenoviruses, which replicate in the nucleus of infected cells, and vaccinia virus, which replicates in the cytoplasm. We discuss experimental approaches used to discover and validate interactions of host proteins with viral genomes and how these interactions impact processes that occur during infection, including the host DNA damage response and viral genome replication, repair, and transcription. We highlight the current state of knowledge regarding virus-host protein interactions and also outline emerging areas and future directions for research.

Acupuncture in cancer care: recommendations for safe practice (peer-reviewed expert opinion).

BACKGROUND: Up-to-date recommendations for the safe practice of acupuncture in integrative oncology are overdue with new cancer treatments and an increase in survivors with late effects of disease; 17 years have elapsed since Filshie and Hester's 2006 guidelines. During 2022/2023 an expert panel assembled to produce updated recommendations aiming to facilitate safe and appropriate care by acupuncturists working with people with cancer. METHODS: A core development team comprising three integrative oncology professionals comprehensively updated pre-existing unpublished recommendations. Twelve invited international experts (senior acupuncturists with and without experience of working in oncology settings, oncologists, physicians and nurses trained in integrative oncology, researchers, academics, and professional body representatives) reviewed the recommendations. In multiple iterations, the core team harmonised comments for final ratification. To aid dissemination and uptake the panel represents national and international integrative oncology associations and major cancer treatment centres in Europe, USA, Australia, and the Middle East. RESULTS: These recommendations facilitate safe care by articulating contra-indications, cautions, and risks for patients both on and off treatment (surgery, SACT, radiotherapy). Situations where acupuncture may be contra-indicated or practices need adapting are identified. "Red and Amber Flags" highlight where urgent referral is essential. CONCLUSION: These are the first international, multidisciplinary peer-reviewed recommendations for safe acupuncture practice in integrative oncology. Concerns about safety remain a significant barrier to appropriate referral from oncology teams, to use by acupuncturists and to uptake by patients. Disseminating trustworthy, widely accessible guidance should facilitate informed, confident practice of acupuncture in and outside of oncology healthcare settings.

Characteristics and Challenges of Providing Acupuncture and Chinese Herbal Medicine in Oncology Treatment: Report of Survey Data and Experience of Five Unique Clinical Settings.

Acupuncture is an integrative therapy with strong evidence to support its use in the oncology setting, yet barriers exist for implementation into conventional medical clinics. Though acupuncture is recommended in clinical practice guidelines for oncology, there is little data in the literature showing how acupuncture and other related therapies, including herbal medicine are successfully implemented in some oncology clinics, while others experience barriers to care. To characterize the current use of acupuncture (ACU) and herbal medicine (HM) in oncology clinics, we collected general demographic and usage data from 5 example clinics. In addition, to better understand the barriers faced by ACU and HM clinics in implementing acupuncture as a treatment modality, a survey was deployed to 2320 members of the Society for Integrative Oncology. This article examines the characteristics of oncology settings around the world, and shares data from the survey on the use of these therapies in the field of oncology. The primary barrier to acupuncture care, as reported by providers, was cost. With just under 70% of the oncologists reporting it as the most important obstacle. Additional barriers to implementation included concerns about competency and training, accessibility and safety of herbal medicine during treatment. Though acupuncture is being incorporated into more conventional oncology settings, organized strategies for implementation involving payers and policymakers is needed.

Feasibility and Usability of EnergyPoints: A Mobile Health App to Guide Acupressure Use for Cancer Symptom Management.

OBJECTIVE: To examine the feasibility and usability of EnergyPoints™, an innovative mobile health app that teaches and guides people with cancer to implement daily acupressure to self-manage their fatigue and sleep disturbances. METHODS AND INTERVENTION: The study used an integrated agile, human-centered approach. Adults (age 18 years and over) with cancer experiencing at least moderate fatigue, and living in the Greater New York City community, were recruited from social media, patient advocacy groups, and referrals. Twenty participants (in 3 sprints of 3, 5, and 12) were video-recorded thinking aloud while using the app for the first time. They then used the app at home to self-administer acupressure (twice daily for 1 week) while continuously wearing a fitness tracker. Each participant completed an exit interview and modified Computer System Usability Questionnaire post-participation. RESULTS: Participants were ages 40 to 76 years and 65% female; 65% were non-Hispanic white. Mean pass rates per ritual exceeded 80%. Users completed (totally or partially) greater than 90% of stimulating acupressure and 70% of relaxing acupressure rituals. Sprint 3 SPs totally completed at least 1 ritual 87% of the time. The majority agreed or strongly agreed the app was easy to use (90%), easy to learn (85%), easy to understand (75%), and effective in helping perform self-acupressure (85%). In an analysis of ease of completing 5 key tasks, all successfully completed the tasks; 3 users required some assistance. Of 654 usability statements, those coded as personal experience/context (197), content related to acupressure learning (105), and content related to the onboarding/profile (71) were most frequent. The design team integrated recommendations into the app before the next sprint. CONCLUSIONS: Findings supported feasibility and usability, as well as acceptability, and led to significant alterations and improvements. EnergyPoints™ offers an opportunity to mainstream acupressure and help cancer survivors self-manage their symptoms.

Phosphorylation regulates viral biomolecular condensates to promote infectious progeny production.

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication.

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

Battlefield acupuncture for chronic musculoskeletal pain in cancer survivors: a novel care delivery model for oncology acupuncture.

Introduction: Battlefield Acupuncture (BFA), a standardized auricular acupuncture protocol, is widely used for pain in the military but is not well-studied in oncology. This study examined cancer survivors who received BFA for pain. Methods: This is a secondary analysis of a randomized trial that compared the effectiveness of BFA and electroacupuncture vs. usual care for chronic musculoskeletal pain in cancer survivors. This study focused on participants randomized to BFA. Participants received 10 weekly treatments. Needles were placed until one of these stop conditions were satisfied: ten needles were administered; pain severity decreased to ≤1 out of 10; patient declined further needling, or vasovagal reaction was observed. Pain severity was assessed using Brief Pain Inventory. Responders were those with ≥30% pain severity reduction. We examined pain location, BFA stop reason, and pain reduction of participants during the first session. We also examined which factors predicted responder status after the first session (week 1) or the full treatment (week 12). Results: Among 143 randomized to BFA, most common pain locations were lower back (30.8%) and knee/leg (18.2%). Of 138 who initiated treatment, 41 (30.0%) received ten needles; 81 (59.1%) achieved pain ≤1; 14 (10.2%) declined further needling; and 1 (0.7%) had vasovagal reaction. BFA reduced pain severity by 2.9 points (95% CI 2.6 to 3.2) after the first session (P < 0.001). After adjusting for baseline pain severity, responders at week 1 were 2.5 times more likely to be responders at week 12, compared to those who were non-responders at week 1 (AOR 2.5, 95% CI 1.02 to 6.11, P = 0.04). Among those who achieved pain ≤1, 74% were responders at week 12, a higher proportion compared to the proportion of responders among those who received ten needles (39.5%), those who declined further needling (50%), and those with vasovagal reaction (0.0%) (P = 0.001). Those with pain in proximal joints had a higher proportion of responders at week 12, compared to those with pain in distal joints (64.2% vs. 20%, P = 0.008). Conclusion: Specific factors may predict the likelihood of achieving meaningful pain reduction from BFA. Understanding these predictors could inform precision pain management and acupuncture delivery models.

Shared sequence characteristics identified in non-canonical rearrangements of HSV-1 genomes.

IMPORTANCE: Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells.

Intestinal transit-amplifying cells require METTL3 for growth factor signaling and cell survival.

Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells' critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.

Double-Strand Break Repair Pathways Differentially Affect Processing and Transduction by Dual AAV Vectors.

Recombinant adeno-associated viral vectors (rAAV) are a powerful tool for gene delivery but have a limited DNA carrying capacity. Efforts to expand this genetic payload have focused on engineering the vector components, such as dual trans-splicing vectors which double the delivery size by exploiting the natural concatenation of rAAV genomes in host nuclei. We hypothesized that inefficient dual vector transduction could be improved by modulating host factors which affect concatenation. Since factors mediating concatenation are not well defined, we performed a genome-wide screen to identify host cell regulators. We discovered that Homologous Recombination (HR) is inhibitory to dual vector transduction. We demonstrate that depletion or inhibition of HR factors BRCA1 and Rad51 significantly increase reconstitution of a large split transgene by increasing both concatenation and expression from rAAVs. Our results define new roles for DNA damage repair in rAAV transduction and highlight the potential for pharmacological intervention to increase genetic payload of rAAV vectors.

SWAMNA: a comprehensive platform for analysis of nucleic acid modifications.

The interest in MS-based analysis of modified nucleic acids is increasing due to the application of nucleic acids in therapeutics. However, there are few available integrated platforms for characterizing nucleic acid modifications. Herein, we report a general mass spectrometry-based SWATH platform to identify and quantify both RNA and DNA modifications, which we call SWATH analysis of modified nucleic acids (SWAMNA). SWAMNA incorporates the search engine, NuMo finder, enabling the analysis of modifications in native and permethylated form. SWAMNA will aid discoveries that provide new insights into nucleic acid modifications.

Electro-acupuncture versus battle field auricular acupuncture in breast cancer survivors with chronic musculoskeletal pain: subgroup analysis of a randomized clinical trial.

PURPOSE: Chronic musculoskeletal pain is common and debilitating among breast cancer survivors. The PEACE trial demonstrated that electro-acupuncture (EA) and battle field auricular acupuncture (BFAA) both reduced pain more than usual care (UC) in cancer survivors. However, the comparative effectiveness between EA and BFAA among breast cancer survivors is unknown. METHODS: EA and BFAA received ten weekly treatments. UC was offered ten EA treatments after week 12. The primary endpoint was change in mean Brief Pain Inventory (BPI) pain severity from baseline to week 12. We analyzed the subset of 165 (46%) trial participants with a breast cancer primary diagnosis. We conducted constrained linear mixed model analyses, which constrained all arms to a common pre-randomization baseline mean. Model-based mean estimates at weeks 12 and 24 were compared between arms using model contrasts. RESULTS: Among 165 breast cancer survivors, common pre-randomization mean pain severity was 5.35 [95% Confidence Interval (CI) 5.04, 5.66]. At week 12, BPI pain severity score was 2.69 (2.26. 3.13) in EA, 3.60 (3.17, 4.02) in BFAA, and 5.06 (4.47, 5.65) in UC. EA reduced pain severity significantly more than BFAA at weeks 12 [- 0.90 (- 1.45, - 0.36), p = 0.001] and 24 [- 0.82, (- 1.38, - 0.27), p = 0.004]. EA and BFAA significantly improved both Patient-Reported Outcomes Measurement Information System (PROMIS) - Global Health physical health and mental health component scores at week 12 compared to UC. Mild toxicities were reported. CONCLUSION: EA was more effective than BFAA at reducing pain severity, but both similarly improved physical and mental health scores. Breast cancer survivors with chronic musculoskeletal pain may consider EA before BFAA. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02979574. https://clinicaltrials.gov/ct2/show/NCT02979574.

Intestinal transit amplifying cells require METTL3 for growth factor signaling, KRAS expression, and cell survival.

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration.

A viral biomolecular condensate coordinates assembly of progeny particles.

Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes1,2. Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation3-8. Although linked to several viral processes3-5,9,10, evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection.

Addressing the benefits of inhibiting APOBEC3-dependent mutagenesis in cancer.

Mutational signatures associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC)3 cytosine deaminase activity have been found in over half of cancer types, including some therapy-resistant and metastatic tumors. Driver mutations can occur in APOBEC3-favored sequence contexts, suggesting that mutagenesis by APOBEC3 enzymes may drive cancer evolution. The APOBEC3-mediated signatures are often detected in subclonal branches of tumor phylogenies and are acquired in cancer cell lines over long periods of time, indicating that APOBEC3 mutagenesis can be ongoing in cancer. Collectively, these and other observations have led to the proposal that APOBEC3 mutagenesis represents a disease-modifying process that could be inhibited to limit tumor heterogeneity, metastasis and drug resistance. However, critical aspects of APOBEC3 biology in cancer and in healthy tissues have not been clearly defined, limiting well-grounded predictions regarding the benefits of inhibiting APOBEC3 mutagenesis in different settings in cancer. We discuss the relevant mechanistic gaps and strategies to address them to investigate whether inhibiting APOBEC3 mutagenesis may confer clinical benefits in cancer.

Novel viral splicing events and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts.

Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and RNA cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts that meet stringent criteria for expression. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORFs), six novel ORF-containing transcripts, and 15 transcripts encoding for messages that could alter protein functions through truncation or fusion of canonical ORFs. In addition, we detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking distinct gene transcription units. Among these chimeric proteins we detected an evolutionarily conserved protein containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies combined with mass spectrometry can reveal further complexity within viral transcriptomes and resulting proteomes.

Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy.

Herpes Simplex Virus-2 Variation Contributes to Neurovirulence During Neonatal Infection.

Herpes simplex virus (HSV) infection of the neonatal brain causes severe encephalitis and permanent neurologic deficits. However, infants infected with HSV at the time of birth follow varied clinical courses, with approximately half of infants experiencing only external infection of the skin rather than invasive neurologic disease. Understanding the cause of these divergent outcomes is essential to developing neuroprotective strategies. To directly assess the contribution of viral variation to neurovirulence, independent of human host factors, we evaluated clinical HSV isolates from neonates with different neurologic outcomes in neurologically relevant in vitro and in vivo models. We found that isolates taken from neonates with encephalitis are more neurovirulent in human neuronal culture and mouse models of HSV encephalitis, as compared to isolates collected from neonates with skin-limited disease. These findings suggest that inherent characteristics of the infecting HSV strain contribute to disease outcome following neonatal infection.