Integrated Proteogenomic Characterization across Major Histological Types of Pediatric Brain Cancer.

We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection.

Certain heterozygous variants in the kinase domain of the serine/threonine kinase NEK8 can cause an autosomal dominant form of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.

Variants in FtsJ RNA 2'-O-Methyltransferase 3 and Growth Hormone 1 are associated with small body size and a dental anomaly in dogs.

Domesticated dogs show unparalleled diversity in body size across breeds, but within breeds variation is limited by selective breeding. Many heritable diseases of dogs are found among breeds of similar sizes, suggesting that as in humans, alleles governing growth have pleiotropic effects. Here, we conducted independent genome-wide association studies in the small Shetland Sheepdog breed and discovered a locus on chromosome 9 that is associated with a dental abnormality called maxillary canine-tooth mesioversion (MCM) (P = 1.53 × 10-7) as well as two body size traits: height (P = 1.67 × 10-5) and weight (P = 1.16 × 10-7). Using whole-genome resequencing data, we identified variants in two proximal genes: FTSJ3, encoding an RNA methyltransferase, and GH1, encoding growth hormone. A substitution in FTSJ3 and a splice donor insertion in GH1 are strongly associated with MCM and reduced body size in Shetland Sheepdogs. We demonstrated in vitro that the GH1 variant leads to exon 3 skipping, predicting a mutant protein known to cause human pituitary dwarfism. Statistical modeling, however, indicates that the FTSJ3 variant is the stronger predictor of MCM and that each derived allele reduces body size by about 1 inch and 5 pounds. In a survey of 224 breeds, both FTSJ3 and GH1 variants are frequent among very small "toy" breeds and absent from larger breeds. Our findings indicate that a chromosome 9 locus harboring tightly linked variants in FTSJ3 and GH1 reduces growth in the Shetland Sheepdog and toy breed dogs and confers risk for MCM through vertical pleiotropy.

Evaluation of the Indications for Sentinel Node Biopsy in Early-Stage Melanoma with the Advent of Adjuvant Systemic Therapy: An International, Multicenter Study.

BACKGROUND: Patients presenting with early-stage melanoma (AJCC pT1b-pT2a) reportedly have a relatively low risk of a positive SNB (~5-10%). Those patients are usually found to have low-volume metastatic disease after SNB, typically reclassified to AJCC stage IIIA, with an excellent prognosis of ~90% 5-year survival. Currently, adjuvant systemic therapy is not routinely recommended for most patients with AJCC stage IIIA melanoma. The purpose was to assess the SN-positivity rate in early-stage melanoma and to identify primary tumor characteristics associated with high-risk nodal disease eligible for adjuvant systemic therapy METHODS: An international, multicenter retrospective cohort study from 7 large-volume cancer centers identified 3,610 patients with early primary cutaneous melanomas 0.8-2.0 mm in Breslow thickness (pT1b-pT2a; AJCC 8th edition). Patient demographics, primary tumor characteristics, and SNB status/details were analyzed. RESULTS: The overall SNB-positivity rate was 11.4% (412/3610). Virtually all SNB-positive patients (409/412; 99.3%) were reclassified to AJCC stage IIIA. Multivariate analysis identified age, T-stage, mitotic rate, primary site and subtype, and lymphovascular invasion as independent predictors of sentinel node status. A mitotic rate of >1/mm2 was associated with a significantly increased SN-positivity rate and was the only significant independent predictor of high-risk SNB metastases (>1 mm maximum diameter). CONCLUSIONS: The new treatment paradigm brings into question the role of SNB for patients with early-stage melanoma. The results of this large international cohort study suggest that a reevaluation of the indications for SNB for some patients with early-stage melanoma is required.

Recruitment of a multi-site randomized controlled trial of aerobic exercise for older adults with amnestic mild cognitive impairment: The EXERT trial.

INTRODUCTION: Effective strategies to recruit older adults with mild cognitive impairment (MCI) into nonpharmacological intervention trials are lacking. METHODS: Recruitment for EXERT, a multisite randomized controlled 18-month trial examining the effects of aerobic exercise on cognitive trajectory in adults with amnestic MCI, involved a diverse portfolio of strategies to enroll 296 participants. RESULTS: Recruitment occurred September 2016 through March 2020 and was initially slow. After mass mailings of 490,323 age- and geo-targeted infographic postcards and brochures, recruitment rates increased substantially, peaking at 16 randomizations/month in early 2020. Mass mailings accounted for 52% of randomized participants, whereas 25% were recruited from memory clinic rosters, electronic health records, and national and local registries. Other sources included news broadcasts, public service announcements (PSA), local advertising, and community presentations. DISCUSSION: Age- and geo-targeted mass mailing of infographic materials was the most effective approach in recruiting older adults with amnestic MCI into an 18-month exercise trial.

Potential for Microbially Mediated Natural Attenuation of Diluted Bitumen on the Coast of British Columbia (Canada).

Western Canada produces large amounts of bitumen, a heavy, highly weathered crude oil. Douglas Channel and Hecate Strait on the coast of British Columbia are two water bodies that may be impacted by a proposed pipeline and marine shipping route for diluted bitumen (dilbit). This study investigated the potential of microbial communities from these waters to mitigate the impacts of a potential dilbit spill. Microcosm experiments were set up with water samples representing different seasons, years, sampling stations, and dilbit blends. While the alkane fraction of the tested dilbit blends was almost completely degraded after 28 days, the majority of the polycyclic aromatic hydrocarbons (PAHs) remained. The addition of the dispersant Corexit 9500A most often had either no effect or an enhancing effect on dilbit degradation. Dilbit-degrading microbial communities were highly variable between seasons, years, and stations, with dilbit type having little impact on community trajectories. Potential oil-degrading genera showed a clear succession pattern and were for the most part recruited from the "rare biosphere." At the community level, dispersant appeared to stimulate an accelerated enrichment of genera typically associated with hydrocarbon degradation, even in dilbit-free controls. This suggests that dispersant-induced growth of hydrocarbon degraders (and not only increased bioavailability of oil-associated hydrocarbons) contributes to the degradation-enhancing effect previously reported for Corexit 9500A.IMPORTANCE Western Canada hosts large petroleum deposits, which ultimately enter the market in the form of dilbit. Tanker-based shipping represents the primary means to transport dilbit to international markets. With anticipated increases in production to meet global energy needs, the risk of a dilbit spill is expected to increase. This study investigated the potential of microbial communities naturally present in the waters of a potential dilbit shipping lane to mitigate the effects of a spill. Here we show that microbial degradation of dilbit was mostly limited to n-alkanes, while the overall concentration of polycyclic aromatic hydrocarbons, which represent the most toxic fraction of dilbit, decreased only slightly within the time frame of our experiments. We further investigated the effect of the oil dispersant Corexit 9500A on microbial dilbit degradation. Our results highlight the fact that dispersant-associated growth stimulation, and not only increased bioavailability of hydrocarbons and inhibition of specific genera, contributes to the overall effect of dispersant addition.

Chromosome ends as adaptive beginnings: the potential role of dysfunctional telomeres in subtelomeric evolvability.

Telomeres serve as protective caps that help the cell differentiate between the naturally occurring ends of chromosomes and double-stranded breaks. When telomere capping function becomes compromised, chromosome ends are subjected to elevated rates of chromosome alterations. These effects can be particularly dramatic in the telomere-adjacent subtelomeric region. While the catastrophic impact of severe telomere dysfunction on genome stability has been well documented, the adaptive telomere failure hypothesis considers an alternative role telomere dysfunction may play in adaptive evolution. This hypothesis suggests that low levels of telomere failure, induced by certain environmental stresses, can lead to elevated subtelomeric recombination. Mutational loss, duplication, or modification of subtelomeric contingency genes could ultimately facilitate adaptation by generating novel mutants better able to survive environmental stress. In this perspective, we discuss recent work that examined mild telomere dysfunction and its role in altering the adaptive potential of subtelomeric genes.

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA.

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1(ATR)/Tel1(ATM)-dependent DNA damage response or caffeine's inhibition of 5' to 3' resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments.

Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2.

In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5' to 3' end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5' to 3' resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells.

RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.

Uterine artery pseudoaneurysm bleeding one decade after caesarean section: A case report.

Uterine artery pseudoaneurysm (UAP) can cause severe secondary post-partum hemorrhage. The lag time between the triggering event and the manifestation of UAP reportedly usually occurs within a couple of weeks. In this case report, a 46-year-old woman with no significant comorbidities presented with a one-month history of increasing lower abdominal pain. She had previously undergone two cesarean sections (14 and 10 years earlier). A computed tomography angiogram revealed a left UAP. The patient was successfully treated by selective embolization and had an uneventful recovery. In our case, the time period between the cesarean section and presentation was approximately 10 years with no other potential causative factors. This case highlights that UAP should be included as a differential diagnosis in all female patients presenting with pelvic pain and pelvic bleeding. More specifically, UAP should be considered after a previous history of cesarean section, as a delayed diagnosis can result in serious morbidity and even mortality.

The SNM1B/APOLLO DNA nuclease functions in resolution of replication stress and maintenance of common fragile site stability.

SNM1B/Apollo is a DNA nuclease that has important functions in telomere maintenance and repair of DNA interstrand crosslinks (ICLs) within the Fanconi anemia (FA) pathway. SNM1B is required for efficient localization of key repair proteins, such as the FA protein, FANCD2, to sites of ICL damage and functions epistatically to FANCD2 in cellular survival to ICLs and homology-directed repair. The FA pathway is also activated in response to replication fork stalling. Here, we sought to determine the importance of SNM1B in cellular responses to stalled forks in the absence of a blocking lesion, such as ICLs. We found that depletion of SNM1B results in hypersensitivity to aphidicolin, a DNA polymerase inhibitor that causes replication stress. We observed that the SNM1B nuclease is required for efficient localization of the DNA repair proteins, FANCD2 and BRCA1, to subnuclear foci upon aphidicolin treatment, thereby indicating SNM1B facilitates direct repair of stalled forks. Consistent with a role for SNM1B subsequent to recognition of the lesion, we found that SNM1B is dispensable for upstream events, including activation of ATR-dependent signaling and localization of RPA, γH2AX and the MRE11/RAD50/NBS1 complex to aphidicolin-induced foci. We determined that a major consequence of SNM1B depletion is a marked increase in spontaneous and aphidicolin-induced chromosomal gaps and breaks, including breakage at common fragile sites. Thus, this study provides evidence that SNM1B functions in resolving replication stress and preventing accumulation of genomic damage.

FBH1 deficiency sensitizes cells to WEE1 inhibition by promoting mitotic catastrophe.

WEE1 kinase phosphorylates CDK1 and CDK2 to regulate origin firing and mitotic entry. Inhibition of WEE1 has become an attractive target for cancer therapy due to the simultaneous induction of replication stress and inhibition of the G2/M checkpoint. WEE1 inhibition in cancer cells with high levels of replication stress results in induction of replication catastrophe and mitotic catastrophe. To increase potential as a single agent chemotherapeutic, a better understanding of genetic alterations that impact cellular responses to WEE1 inhibition is warranted. Here, we investigate the impact of loss of the helicase, FBH1, on the cellular response to WEE1 inhibition. FBH1-deficient cells have a reduction in ssDNA and double strand break signaling indicating FBH1 is required for induction of replication stress response in cells treated with WEE1 inhibitors. Despite the defect in the replication stress response, FBH1-deficiency sensitizes cells to WEE1 inhibition by increasing mitotic catastrophe. We propose loss of FBH1 is resulting in replication-associated damage that requires the WEE1-dependent G2 checkpoint for repair.

Snm1B/Apollo functions in the Fanconi anemia pathway in response to DNA interstrand crosslinks.

Fanconi anemia (FA) is an inherited chromosomal instability disorder characterized by childhood aplastic anemia, developmental abnormalities and cancer predisposition. One of the hallmark phenotypes of FA is cellular hypersensitivity to agents that induce DNA interstrand crosslinks (ICLs), such as mitomycin C (MMC). FA is caused by mutation in at least 14 genes which function in the resolution of ICLs during replication. The FA proteins act within the context of a protein network in coordination with multiple repair factors that function in distinct pathways. SNM1B/Apollo is a member of metallo-ß-lactamase/ßCASP family of nucleases and has been demonstrated to function in ICL repair. However, the relationship between SNM1B and the FA protein network is not known. In the current study, we establish that SNM1B functions epistatically to the central FA factor, FANCD2, in cellular survival after ICL damage and homology-directed repair of DNA double-strand breaks. We also demonstrate that MMC-induced chromosomal anomalies are increased in SNM1B-depleted cells, and this phenotype is not further exacerbated upon depletion of either FANCD2 or another key FA protein, FANCI. Furthermore, we find that SNM1B is required for proper localization of critical repair factors, including FANCD2, BRCA1 and RAD51, to MMC-induced subnuclear foci. Our findings demonstrate that SNM1B functions within the FA pathway during the repair of ICL damage.

Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.

The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C-nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme.

Depletion of autoreactive plasma cells and treatment of lupus nephritis in mice using CEP-33779, a novel, orally active, selective inhibitor of JAK2.

Accumulating evidence suggests that autoreactive plasma cells play an important role in systemic lupus erythematosus (SLE). In addition, several proinflammatory cytokines promote autoreactive B cell maturation and autoantibody production. Hence, therapeutic targeting of such cytokine pathways using a selective JAK2 inhibitor, CEP-33779 (JAK2 enzyme IC(50) = 1.3 nM; JAK3 enzyme IC(50)/JAK2 enzyme IC(50) = 65-fold), was tested in two mouse models of SLE. Age-matched, MRL/lpr or BWF1 mice with established SLE or lupus nephritis, respectively, were treated orally with CEP-33779 at 30 mg/kg (MRL/lpr), 55 mg/kg or 100 mg/kg (MRL/lpr and BWF1). Studies included reference standard, dexamethasone (1.5 mg/kg; MRL/lpr), and cyclophosphamide (50 mg/kg; MRL/lpr and BWF1). Treatment with CEP-33779 extended survival and reduced splenomegaly/lymphomegaly. Several serum cytokines were significantly decreased upon treatment including IL-12, IL-17A, IFN-α, IL-1ß, and TNF-α. Anti-nuclear Abs and frequencies of autoantigen-specific, Ab-secreting cells declined upon CEP-33779 treatment. Increased serum complement levels were associated with reduced renal JAK2 activity, histopathology, and spleen CD138(+) plasma cells. The selective JAK2 inhibitor CEP-33779 was able to mitigate several immune parameters associated with SLE advancement, including the protection and treatment of mice with lupus nephritis. These data support the possibility of using potent, orally active, small-molecule inhibitors of JAK2 to treat the debilitative disease SLE.

FBH1 deficiency sensitizes cells to WEE1 inhibition by promoting mitotic catastrophe.

WEE1 kinase phosphorylates CDK1 and CDK2 to regulate origin firing and mitotic entry. Inhibition of WEE1 has become an attractive target for cancer therapy due to the simultaneous induction of replication stress and inhibition of the G2/M checkpoint. WEE1 inhibition in cancer cells with high levels of replication stress results in induction of replication catastrophe and mitotic catastrophe. To increase potential as a single agent chemotherapeutic, a better understanding of genetic alterations that impact cellular responses to WEE1 inhibition is warranted. Here, we investigate the impact of loss of the helicase, FBH1, on the cellular response to WEE1 inhibition. FBH1-deficient cells have a reduction in ssDNA and double strand break signaling indicating FBH1 is required for induction of replication stress response in cells treated with WEE1 inhibitors. Despite the defect in the replication stress response, FBH1-deficiency sensitizes cells to WEE1 inhibition by increasing mitotic catastrophe. We propose loss of FBH1 is resulting in replication-associated damage that requires the WEE1-dependent G2 checkpoint for repair.

UBC13-mediated template switching promotes replication stress resistance in FBH1-deficient cells.

The proper resolution of DNA damage during replication is essential for genome stability. FBH1, a UvrD, helicase plays crucial roles in the DNA damage response. FBH1 promotes double strand break formation and signaling in response to prolonged replication stress to initiate apoptosis. Human FBH1 regulates RAD51 to inhibit homologous recombination. A previous study suggested that mis-regulation of RAD51 may contribute to replication stress resistance in FBH1-deficient cells, but the underlying mechanism remains unknown. Here, we provide direct evidence that RAD51 promotes replication stress resistance in FBH1-deficient cells. We demonstrate inhibition of RAD51 using the small molecule, B02, partially rescues double strand break signaling in FBH1-deficient cells. We show that inhibition of only the strand exchange activity of RAD51 rescues double strand break signaling in FBH1 knockout cells. Finally, we show that depletion of UBC13, a E2 protein that promotes RAD51-dependent template switching, rescues double strand break formation and signaling sensitizing FBH1-deficient cells to replication stress. Our results suggest FBH1 regulates template switching to promote replication stress sensitivity.

Turning the promise of multipurpose prevention technologies into a market reality: a commentary.

The promise of multipurpose prevention technologies (MPTs) for the prevention of HIV and unintended pregnancy are on the horizon. While many are still in clinical development, others are closer to becoming a realistic, accessible option for users, like the dual prevention pill (DPP). Researchers, governments, donors, and implementers will have to collaboratively address systemic challenges to successfully introduce and scale-up MPTs. To ensure the rollout of MPTs is successful, the global community should address user and country-specific needs, coordinate with advocates and policymakers, and set a realistic plan for product introduction and scale-up that considers the needs of both family planning (FP) and HIV programs, while laying the groundwork for future new product introduction. To achieve these aims, global and regional stakeholder coordination should emphasize country-led, person-centered decision-making while addressing: (1) procurement and supply chain barriers; (2) the potential burden on health systems; and (3) the impact on current programs.

Equipping providers to offer novel MPTs: Developing counseling messages for the Dual Prevention Pill in clinical studies and beyond.

Introduction: The pipeline for multi-purpose prevention technologies includes products that simultaneously prevent HIV, pregnancy and/or other sexually transmitted infections. Among these, the Dual Prevention Pill (DPP) is a daily pill co-formulating oral pre-exposure prophylaxis (PrEP), and combined oral contraception (COC). Clinical cross-over acceptability studies for the DPP require training providers to counsel on a combined product. From February 2021-April 2022, a working group of eight HIV and FP experts with clinical and implementation expertise developed counseling recommendations for the DPP based on existing PrEP/COC guidance. Assessment of policy/guidelines options and implications: The working group conducted a mapping of counseling messages from COC and oral PrEP guidance and provider training materials. Six topics were prioritized: uptake, missed pills, side effects, discontinuation and switching, drug interactions and monitoring. Additional evidence and experts were consulted to answer outstanding questions and counseling recommendations for the DPP were developed. Missed pills was the topic with the most complexity, raising questions about whether women could "double up" on missed pills or skip the last week of the pack to recover protection faster. Uptake required aligning the time to reach protective levels for both DPP components and explaining the need to take DPP pills during week 4 of the pack. The potential intensity of DPP side effects, given the combination of oral PrEP with COC, was an important consideration. Discontinuation and switching looked at managing risk of HIV and unintended pregnancy when stopping or switching from the DPP. Guidance on drug interactions contended with differing contraindications for COC and PrEP. Monitoring required balancing clinical requirements with potential user burden. Actionable recommendations: The working group developed counseling recommendations for the DPP to be tested in clinical acceptability studies. Uptake: Take one pill every day for the DPP until the pack is empty. Days 1-21 contain COC and oral PrEP. Days 22-28 do not contain COC to allow for monthly bleeding, but do contain oral PrEP and pills should be taken to maintain HIV protection. Take the DPP for 7 consecutive days to reach protective levels against pregnancy and HIV. Missed pills: If you miss 1 pill multiple times in a month or 2+ consecutive pills, take the DPP as soon as you remember. Do not take more than 2 pills in a day. If 2+ consecutive pills are missed, only take the last missed pill and discard the other missed pills. Side effects: You may experience side effects when you start using the DPP, including changes to monthly bleeding. Side effects are typically mild and go away without treatment. Discontinuation/switching: If you decide to discontinue use of the DPP, but want to be protected from HIV and/or unintended pregnancy, in most cases, you can begin using PrEP or another contraceptive method right away. Drug interactions: There are no drug-drug interactions from combining oral PrEP and COC in the DPP. Certain medications are not recommended due to their contraindication with oral PrEP or COC. Monitoring: You will need to get an HIV test prior to initiating or restarting the DPP, and every 3 months during DPP use. Your provider may recommend other screening or testing. Discussion: Developing recommendations for the DPP as a novel MPT posed unique challenges, with implications for efficacy, cost, and user and provider comprehension and burden. Incorporating counseling recommendations into clinical cross-over acceptability studies allows for real-time feedback from providers and users. Supporting women with information to use the DPP correctly and confidently is critically important for eventual scale and commercialization.