Rapid recruitment of p53 to DNA damage sites directs DNA repair choice and integrity.

SignificanceOur work focuses on the critical longstanding question of the nontranscriptional role of p53 in tumor suppression. We demonstrate here that poly(ADP-ribose) polymerase (PARP)-dependent modification of p53 enables rapid recruitment of p53 to damage sites, where it in turn directs early repair pathway selection. Specifically, p53-mediated recruitment of 53BP1 at early time points promotes nonhomologous end joining over the more error-prone microhomology end-joining. Similarly, p53 directs nucleotide excision repair by mediating DDB1 recruitment. This property of p53 also correlates with tumor suppression in vivo. Our study provides mechanistic insight into how certain transcriptionally deficient p53 mutants may retain tumor-suppressive functions through regulating the DNA damage response.

Strategies for DPYD testing prior to fluoropyrimidine chemotherapy in the US.

PURPOSE: Patients with dihydropyrimidine dehydrogenase (DPD) deficiency are at high risk for severe and fatal toxicity from fluoropyrimidine (FP) chemotherapy. Pre-treatment DPYD testing is standard of care in many countries, but not the United States (US). This survey assessed pre-treatment DPYD testing approaches in the US to identify best practices for broader adoption. METHODS: From August to October 2023, a 22-item QualtricsXM survey was sent to institutions and clinicians known to conduct pre-treatment DPYD testing and broadly distributed through relevant organizations and social networks. Responses were analyzed using descriptive analysis. RESULTS: Responses from 24 unique US sites that have implemented pre-treatment DPYD testing or have a detailed implementation plan in place were analyzed. Only 33% of sites ordered DPYD testing for all FP-treated patients; at the remaining sites, patients were tested depending on disease characteristics or clinician preference. Almost 50% of sites depend on individual clinicians to remember to order testing without the assistance of electronic alerts or workflow reminders. DPYD testing was most often conducted by commercial laboratories that tested for at least the four or five DPYD variants considered clinically actionable. Approximately 90% of sites reported receiving results within 10 days of ordering. CONCLUSION: Implementing DPYD testing into routine clinical practice is feasible and requires a coordinated effort among the healthcare team. These results will be used to develop best practices for the clinical adoption of DPYD testing to prevent severe and fatal toxicity in cancer patients receiving FP chemotherapy.

RADX interacts with single-stranded DNA to promote replication fork stability.

Single-stranded DNA (ssDNA) regions form as an intermediate in many DNA-associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide-binding (OB) fold domain. The heterotrimeric, multi-OB fold domain-containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA-binding factor in human cells. RADX binds ssDNA via an N-terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA-binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA-binding proteins.

Role of CYP2B6 pharmacogenomics in bupropion-mediated smoking cessation.

WHAT IS KNOWN AND OBJECTIVE: Pharmacogenomics holds promise in smoking cessation because of its potential to shed light on the complexity of drug metabolism and improve treatments using therapeutic agents. The cytochrome P450 2B6 gene (CYP2B6) encodes CYP2B6 enzyme that has been found to mediate the hydroxylation of bupropion, a smoking cessation aid. CYP2B6 exhibits a range of polymorphic variants that alter the pharmacokinetics and pharmacodynamics of bupropion. Genetic variations in CYP2B6 may influence the risk of adverse effects or efficacy of treatment with bupropion. The objective of this review was to investigate the influence of pharmacogenomics on smoking cessation therapy. METHODS: A thorough literature search was conducted on PubMed, SCOPUS and EMBASE using keywords related to bupropion, smoking cessation, pharmacogenomics and CYP2B6. Research and review articles, case reports and clinical and preclinical studies pertinent to the research topic were identified, evaluated and summarized. Cited articles within the above-mentioned sources also provided pertinent information. RESULTS: There is strong literature evidence to prove that CYP2B6 polymorphisms affect pharmacokinetic and pharmacodynamic properties of bupropion, thus affecting the therapeutic outcome of smoking cessation therapy. WHAT IS NEW AND CONCLUSIONS: Complete understanding of pharmacogenetic variation of bupropion pharmacokinetics and pharmacodynamics will be beneficial for designing safer and more personalized smoking cessation therapy with improved outcomes.

How the Other Half Lives: What p53 Does When It Is Not Being a Transcription Factor.

It has been four decades since the discovery of p53, the designated 'Guardian of the Genome'. P53 is primarily known as a master transcription factor and critical tumor suppressor, with countless studies detailing the mechanisms by which it regulates a host of gene targets and their consequent signaling pathways. However, transcription-independent functions of p53 also strongly define its tumor-suppressive capabilities and recent findings shed light on the molecular mechanisms hinted at by earlier efforts. This review highlights the transcription-independent mechanisms by which p53 influences the cellular response to genomic instability (in the form of replication stress, centrosome homeostasis, and transposition) and cell death. We also pinpoint areas for further investigation in order to better understand the context dependency of p53 transcription-independent functions and how these are perturbed when TP53 is mutated in human cancer.

Genetic basis for clinical response to CTLA-4 blockade in melanoma.

BACKGROUND: Immune checkpoint inhibitors are effective cancer treatments, but molecular determinants of clinical benefit are unknown. Ipilimumab and tremelimumab are antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA-4). Anti-CTLA-4 treatment prolongs overall survival in patients with melanoma. CTLA-4 blockade activates T cells and enables them to destroy tumor cells. METHODS: We obtained tumor tissue from patients with melanoma who were treated with ipilimumab or tremelimumab. Whole-exome sequencing was performed on tumors and matched blood samples. Somatic mutations and candidate neoantigens generated from these mutations were characterized. Neoantigen peptides were tested for the ability to activate lymphocytes from ipilimumab-treated patients. RESULTS: Malignant melanoma exomes from 64 patients treated with CTLA-4 blockade were characterized with the use of massively parallel sequencing. A discovery set consisted of 11 patients who derived a long-term clinical benefit and 14 patients who derived a minimal benefit or no benefit. Mutational load was associated with the degree of clinical benefit (P=0.01) but alone was not sufficient to predict benefit. Using genomewide somatic neoepitope analysis and patient-specific HLA typing, we identified candidate tumor neoantigens for each patient. We elucidated a neoantigen landscape that is specifically present in tumors with a strong response to CTLA-4 blockade. We validated this signature in a second set of 39 patients with melanoma who were treated with anti-CTLA-4 antibodies. Predicted neoantigens activated T cells from the patients treated with ipilimumab. CONCLUSIONS: These findings define a genetic basis for benefit from CTLA-4 blockade in melanoma and provide a rationale for examining exomes of patients for whom anti-CTLA-4 agents are being considered. (Funded by the Frederick Adler Fund and others.).

Association between CYP3A4, CYP3A5 and ABCB1 genotype and tacrolimus treatment outcomes among allogeneic HSCT patients.

Aim: Successful treatment with tacrolimus to prevent graft versus host disease (GVHD) and minimize tacrolimus-related toxicities among allogeneic hematopoietic cell transplantation (alloHCT) recipients is contingent upon quickly achieving and maintaining concentrations within a narrow therapeutic range. The primary objective was to investigate associations between CYP3A4, CYP3A5 or ABCB1 genotype and the proportion of patients that attained an initial tacrolimus goal concentration following initiation of intravenous (iv.) and conversion to oral administration. Materials & methods: We retrospectively evaluated 86 patients who underwent HLA-matched (8/8) related donor alloHCT and were prescribed a tacrolimus-based regimen for GVHD prophylaxis. Results & conclusion: The findings of the present study suggests that CYP3A5 genotype may impact attainment of initial therapeutic tacrolimus concentrations with oral administration in alloHCT recipients.

Preoperative immune checkpoint inhibition and cryoablation in early-stage breast cancer.

Local cryoablation can engender systemic immune activation/anticancer responses in tumors otherwise resistant to immune checkpoint blockade (ICB). We evaluated the safety/tolerability of preoperative cryoablation plus ipilimumab and nivolumab in 5 early-stage/resectable breast cancers. The primary endpoint was met when all 5 patients underwent standard-of-care primary breast surgery undelayedly. Three patients developed transient hyperthyroidism; one developed grade 4 liver toxicity (resolved with supportive management). We compared this strategy with cryoablation and/or ipilimumab. Dual ICB plus cryoablation induced higher expression of T cell activation markers and serum Th1 cytokines and reduced immunosuppressive serum CD4+PD-1hi T cells, improving effector-to-suppressor T cell ratio. After dual ICB and before cryoablation, T cell receptor sequencing of 4 patients showed increased T cell clonality. In this small subset of patients, we provide preliminary evidence that preoperative cryoablation plus ipilimumab and nivolumab is feasible, inducing systemic adaptive immune activation potentially more robust than cryoablation with/without ipilimumab.

Clinical Impact of the CYP2C19 Gene on Diazepam for the Management of Alcohol Withdrawal Syndrome.

Diazepam is a benzodiazepine widely prescribed for the management of patients with severe alcohol withdrawal syndrome to prevent agitation, withdrawal seizures, and delirium tremens. Despite standard dosing of diazepam, a subset of patients experience refractory withdrawal syndromes or adverse drug reactions, such as impaired motor coordination, dizziness, and slurred speech. The CYP2C19 and CYP3A4 enzymes play a key role in the biotransformation of diazepam. Given the highly polymorphic nature of the CYP2C19 gene, we reviewed the clinical impact of variants in the CYP2C19 gene on both the pharmacokinetics of diazepam and treatment outcomes related to the management of alcohol withdrawal syndrome.

Domain-specific p53 mutants activate EGFR by distinct mechanisms exposing tissue-independent therapeutic vulnerabilities.

Mis-sense mutations affecting TP53 promote carcinogenesis both by inactivating tumor suppression, and by conferring pro-carcinogenic activities. We report here that p53 DNA-binding domain (DBD) and transactivation domain (TAD) mis-sense mutants unexpectedly activate pro-carcinogenic epidermal growth factor receptor (EGFR) signaling via distinct, previously unrecognized molecular mechanisms. DBD- and TAD-specific TP53 mutants exhibited different cellular localization and induced distinct gene expression profiles. In multiple tissues, EGFR is stabilized by TAD and DBD mutants in the cytosolic and nuclear compartments respectively. TAD mutants promote EGFR-mediated signaling by enhancing EGFR interaction with AKT via DDX31 in the cytosol. Conversely, DBD mutants maintain EGFR activity in the nucleus, by blocking EGFR interaction with the phosphatase SHP1, triggering c-Myc and Cyclin D1 upregulation. Our findings suggest that p53 mutants carrying gain-of-function, mis-sense mutations affecting two different domains form new protein complexes that promote carcinogenesis by enhancing EGFR signaling via distinctive mechanisms, exposing clinically relevant therapeutic vulnerabilities.

Phase I Study of a Multivalent WT1 Peptide Vaccine (Galinpepimut-S) in Combination with Nivolumab in Patients with WT1-Expressing Ovarian Cancer in Second or Third Remission.

We examined the safety and immunogenicity of sequential administration of a tetravalent, non-HLA (human leukocyte antigen) restricted, heteroclitic Wilms' Tumor 1 (WT1) peptide vaccine (galinpepimut-S) with anti-PD-1 (programmed cell death protein 1) nivolumab. This open-label, non-randomized phase I study enrolled patients with WT1-expressing ovarian cancer in second or third remission from June 2016 to July 2017. Therapy included six (every two weeks) subcutaneous inoculations of galinpepimut-S vaccine adjuvanted with Montanide, low-dose subcutaneous sargramostim at the injection site, with intravenous nivolumab over 12 weeks, and up to six additional doses until disease progression or toxicity. One-year progression-free survival (PFS) was correlated to T-cell responses and WT1-specific immunoglobulin (Ig)G levels. Eleven patients were enrolled; seven experienced a grade 1 adverse event, and one experienced a grade ≥3 adverse event considered a dose-limiting toxicity. Ten (91%) of eleven patients had T-cell responses to WT1 peptides. Seven (88%) of eight evaluable patients had IgG against WT1 antigen and full-length protein. In evaluable patients who received >2 treatments of galinpepimut-S and nivolumab, the 1-year PFS rate was 70%. Coadministration of galinpepimut-S and nivolumab demonstrated a tolerable toxicity profile and induced immune responses, as indicated by immunophenotyping and WT1-specific IgG production. Exploratory analysis for efficacy yielded a promising 1-year PFS rate.

Prioritizing pharmacogenomics implementation initiates: a survey of healthcare professionals.

Aim: Characterize current perceptions, practices, preferences and barriers to integrating pharmacogenomics into patient care at an institution with an established pharmacogenomics clinic. Materials & methods: A 16-item anonymous survey was sent to healthcare professionals practicing at Tampa General Hospital and the University of South Florida Health. Results: Survey participants consisted of nine advanced practice providers, 41 pharmacists and 64 physicians. Majority of survey participants did not feel confident in their ability to interpret and apply pharmacogenomic results. In the past 12 months, 27% of physicians reported ordering a pharmacogenomic test. The greatest reported barrier to integrating pharmacogenomics was the absence of established guidelines or protocols. Conclusion: Most clinicians believed pharmacogenomics would be useful in their clinical practice but do not feel prepared to interpret pharmacogenomic results.

Lay abstract Pharmacogenomics is a field of personalized medicine where genetic information may be used to guide treatment decisions. Many advanced practice providers, pharmacists, and physicians are not as familiar with interpreting pharmacogenomic results to make medication changes. This survey was designed and administered to clinicians at Tampa General Hospital and the University of South Florida Health to characterize perceptions, practices, preferences and barriers to integrating pharmacogenomics into patient care. Survey results revealed that most clinicians believed pharmacogenomics would be useful in their clinical practice but do not feel prepared to interpret pharmacogenomic results.

A clinician's guide for counseling patients on results of a multigene pharmacogenomic panel.

PURPOSE: This article explores approaches to pharmacogenomic counseling for patients who have undergone multigene panel testing by describing the collective experience of 5 institutions. SUMMARY: Multigene panel pharmacogenomic testing has the potential to unlock a myriad of information about a patient's past, present, and future drug response. The multifaceted nature of drug response coupled with the complexity of genetic results necessitates some form of patient education through pharmacogenomic counseling. Published literature regarding disclosure of pharmacogenomic test results is limited. This article compares the counseling practices of pharmacists from 5 different institutions with pharmacogenomics clinics whose experience represents perspectives ranging from academia to community clinical environments. Overarching counseling themes discussed during result disclosure center around (1) pharmacogenomic results, (2) gene-drug interactions, (3) gene-drug-drug interactions, (4) drug changes (5) future, familial, or disease-risk implications, (6) updates in the interpretation and application of pharmacogenomic results, (7) gauging patient comprehension, and (8) sharing results and supplemental information. CONCLUSION: Dedicating time to counseling patients on the results of a multigene pharmacogenomic panel is important given the lifelong applications of a test that is generally performed only once. The content and methods of disclosing test results shared by the experiences of pharmacists at 5 different institutions serve as guide to be further refined as research addresses effective communication strategies that enhance patient comprehension of pharmacogenomic results.

Apps for Promoting Children's Oral Health: Systematic Search in App Stores and Quality Evaluation.

BACKGROUND: Increasingly, mobile apps are being used to promote oral care. Many of them are aimed at children. OBJECTIVE: This study aimed to systematically search and evaluate apps that promote oral care and hygiene for children. METHODS: A broad search strategy (13 keywords) was developed to identify apps from Apple's App Store and the Google Play Store in April 2019. After reviewing the apps' titles and summaries, potentially relevant apps were downloaded for viewing. The quality of the apps that met the inclusion criteria was assessed by the Health on the Net Foundation Code of Conduct (HONcode) criteria for medical and health websites and the Scientific Basis of Oral Self-care (SBOSC). RESULTS: More than 3000 Apps were identified and 54 relevant apps informed the review. The quality of the apps according to the HONcode criteria was generally low. The mean HONcode score was 1.8/8.0. One-quarter of the apps had a HONcode score of 0 (14/54, 26%). The SBOSC score of the apps was evaluated based on a 6-point scale. The mean SBOSC score was 1.5/6.0; 19% (10/54) of the apps had a score of 0. There was a significant and positive correlation between HONcode and SBOSC scores (r=0.37; P<.01). More recently uploaded apps had significantly higher HONcode scores (P<.05). CONCLUSIONS: There are many apps aiming to promote oral self-care among children. The quality and scientific basis of these apps are low. Newer apps are of higher quality in terms of scientific basis. There is a need to ensure high-quality and evidence-based apps are available. The effectiveness of apps in terms of oral care and clinical outcomes among children needs to be evaluated.

Pharmacogenetics: A Precision Medicine Approach to Combatting the Opioid Epidemic.

Ineffective pain control is the most commonly cited reason for misuse of prescription opioids and is influenced by genetics. In particular, the gene encoding the CYP2D6 enzyme, which metabolizes some of the most commonly prescribed opioids (e.g., tramadol, hydrocodone) to their more potent forms, is highly polymorphic and can lead to reduced concentrations of the active metabolites and decreased opioid effectiveness. Consideration of the CYP2D6 genotype may allow for predicting opioid response and identifying patients who are likely to respond well to lower potency opioids as well as those who may derive greater pain relief from non-opioid analgesics versus certain opioids. There is emerging evidence that a CYP2D6-guided approach to pain management improves pain control and reduces opioid consumption and thus may be a promising means for combating opioid misuse. Clinical practice guidelines are available for select opioids and other analgesics to support medication and dose selection based on pharmacogenetic data. This article describes the evidence supporting genotype-guided pain management as a means of improving pain control and reducing opioid misuse and clinical recommendations for genotype-guided analgesic prescribing. In addition, a "how to" guide using patient case examples is provided to demystify the process for implementing pharmacogenetics-guided pain management in order to optimize analgesia and minimize adverse effects. Optimizing pain management through genotype-guided approaches may ultimately provide safer and more effective therapy for pain control while decreasing the risk for opioid misuse.

Tissues and Tumor Microenvironment (TME) in 3D: Models to Shed Light on Immunosuppression in Cancer.

Immunosuppression in cancer has emerged as a major hurdle to immunotherapy efforts. Immunosuppression can arise from oncogene-induced signaling within the tumor as well as from tumor-associated immune cells. Understanding various mechanisms by which the tumor can undermine and evade therapy is critical in improving current cancer immunotherapies. While mouse models have allowed for the characterization of key immune cell types and their role in tumor development, extrapolating these mechanisms to patients has been challenging. There is need for better models to unravel the effects of genetic alterations inherent in tumor cells and immune cells isolated from tumors on tumor growth and to investigate the feasibility of immunotherapy. Three-dimensional (3D) organoid model systems have developed rapidly over the past few years and allow for incorporation of components of the tumor microenvironment such as immune cells and the stroma. This bears great promise for derivation of patient-specific models in a dish for understanding and determining the impact on personalized immunotherapy. In this review, we will highlight the significance of current experimental models employed in the study of tumor immunosuppression and evaluate current tumor organoid-immune cell co-culture systems and their potential impact in shedding light on cancer immunosuppression.

Synthesis of major pharmacogenomics pretest counseling themes: a multisite comparison.

The accessibility of pharmacogenomic (PGx) testing has grown substantially over the last decade and with it has arisen a demand for patients to be counseled on the use of these tests. While guidelines exist for the use of PGx results; objective determinants for who should receive PGx testing remain incomplete. PGx clinical services have been created to meet these screening and education needs and significant variability exists between these programs. This article describes the practices of four PGx clinics during pretest counseling sessions. A description of the major tenets of the benefits, limitations and risks of testing are compiled. Additional tools are provided to serve as a foundation for those wishing to begin or expand their own counseling service.

Prolonged clonazepam-induced withdrawal symptoms in an NAT2 ultraslow acetylator.

Clonazepam undergoes nitroreduction to 7-amino-clonazepam via CYP3A4/5, followed by acetylation to 7-acetamido-clonazepam via NAT2 enzyme. While no pharmacological activity is attributed to the metabolites of clonazepam, 7-amino-clonazepam has some affinity for the benzodiazepine receptor as a partial agonist for the gamma aminobutyric acid-A receptor and can compete with clonazepam. Interindividual variability in the incidence of adverse events in patients may, in part, be attributable to differences in clonazepam metabolism. Here, we report on a case of a 70-year-old Caucasian female with insomnia and difficulty weaning off long-term use of clonazepam suggesting that a slow acetylator phenotype contributing to patient's presentation. This hypothesis was confirmed by NAT2 gene sequencing. NAT2 genotyping may play a role in guiding clonazepam therapy.

Binimetinib plus Gemcitabine and Cisplatin Phase I/II Trial in Patients with Advanced Biliary Cancers.

PURPOSE: Mutations in the RAS/RAF/MEK/ERK signaling pathway are commonly found in biliary tract cancer (BTC). Binimetinib, a selective inhibitor of MEK1/2, has single-agent activity. Preclinical data support binimetinib combination with chemotherapy, when given in an interrupted dosing schedule.Patients and Methods: A phase I/II trial evaluated binimetinib in combination with gemcitabine and cisplatin in patients with untreated advanced BTC. The primary endpoints were to determine the MTD (phase I), and PFS 6 and RR (phase II). Tumor tissue for targeted gene sequencing and blood samples for peripheral blood pERK expression were evaluated. Patients received oral binimetinib twice daily with gemcitabine and cisplatin on day 8 and 15 of a 21-day cycle. Binimetinib was held for 2 days prior to and on day of each chemotherapy treatment. RESULTS: Twelve patients enrolled in the phase I showed the MTD of binimetinib at 45 mg orally twice daily with gemcitabine 800 and cisplatin 20 mg/m2. Twenty-nine patients were treated in the phase II. Six patients treated at MTD in phase I were evaluable as part of phase II. PFS 6 months was 54% and RR was 36%. Median overall survival was 13.3 months (95% CI, 9.8-16.5). MSK-IMPACT 410-gene panel showed aberrations in the RAS-RAF-MEK-ERK pathway and mutations in PIK3CA, AKT2, PIK3CG, BRAF, and MAP3K1 in responding patients. CONCLUSIONS: Binimetinib with gemcitabine and cisplatin did not show an improvement in PFS 6 and RR. Molecular profiling may help select patients who may benefit from this triplet therapy, which is not planned at this time.

A comprehensive view of sex-specific issues related to cardiovascular disease.

Cardiovascular disease (CVD) is the leading cause of mortality in women. In fact, CVD is responsible for a third of all deaths of women worldwide and half of all deaths of women over 50 years of age in developing countries. The prevalence of CVD risk factor precursors is increasing in children. Retrospective analyses suggest that there are some clinically relevant differences between women and men in terms of prevalence, presentation, management and outcomes of the disease, but little is known about why CVD affects women and men differently. For instance, women with diabetes have a significantly higher CVD mortality rate than men with diabetes. Similarly, women with atrial fibrillation are at greater risk of stroke than men with atrial fibrillation. Historically, women have been underrepresented in clinical trials. The lack of good trial evidence concerning sex-specific outcomes has led to assumptions about CVD treatment in women, which in turn may have resulted in inadequate diagnoses and suboptimal management, greatly affecting outcomes. This knowledge gap may also explain why cardiovascular health in women is not improving as fast as that of men. Over the last decades, mortality rates in men have steadily declined, while those in women remained stable. It is also becoming increasingly evident that gender differences in cultural, behavioural, psychosocial and socioeconomic status are responsible, to various degrees, for the observed differences between women and men. However, the interaction between sex-and gender-related factors and CVD outcomes in women remains largely unknown.