Regulation of PRMT5-MDM4 axis is critical in the response to CDK4/6 inhibitors in melanoma.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.

Fascia as a regulatory system in health and disease.

Neurology and connective tissue are intimately interdependent systems and are critical in regulating many of the body's systems. Unlocking their multifaceted relationship can transform clinical understanding of the mechanisms involved in multisystemic regulation and dysregulation. The fascial system is highly innervated and rich with blood vessels, lymphatics, and hormonal and neurotransmitter receptors. Given its ubiquity, fascia may serve as a "watchman," receiving and processing information on whole body health. This paper reviews what constitutes fascia, why it is clinically important, and its contiguous and interdependent relationship with the nervous system. Unquestionably, fascial integrity is paramount to human locomotion, interaction with our environment, bodily sense, and general physical and emotional wellbeing, so an understanding of the fascial dysregulation that defines a range of pathological states, including hypermobility syndromes, autonomic dysregulation, mast cell activation, and acquired connective tissue disorders is critical in ensuring recognition, research, and appropriate management of these conditions, to the satisfaction of the patient as well as the treating practitioner.

Age-related fertility decline and elective oocyte cryopreservation: Knowledge, attitudes and practices in a pilot study of general practitioners.

BACKGROUND AND OBJECTIVES: Increasing numbers of women are experiencing age-related infertility. General practitioners (GPs) are ideally placed to provide initial age-related fertility counselling and fertility specialist referral. The aim of this pilot study was to document doctors' knowledge, attitudes and practices relating to proactive fertility counselling, ovarian reserve testing and oocyte cryopreservation. METHOD: Seventy-two GPs and general practice registrars were recruited through social media and completed an online survey. RESULTS: Knowledge gaps were shown relating to age-related female fertility decline, ovarian reserve testing and elective oocyte cryopreservation. GPs were more likely to discuss reproductive planning with women aged 35-44 years, compared with younger women. The majority of participants agreed fertility counselling should be provided by GPs; however, barriers identified include limited time, knowledge and concern for causing patient distress. DISCUSSION: Providing GPs with fertility-focused education and resources may enable sensitive, accurate and timely counselling to improve reproductive outcomes.

New Canadian and Provincial Records of Coleoptera Resulting from Annual Canadian Food Inspection Agency Surveillance for Detection of Non-Native, Potentially Invasive Forest Insects.

The arrival and establishment of adventive, invasive forest insects are a threat to the health, diversity, and productivity of forests in Canada and the world at large, and their early detection is essential for successful eradication and management. For that reason, the Canadian Food Inspection Agency (CFIA) conducts annual surveys at high risk sites such as international ports and freight terminals, industrial zones, and disposal sites for solid wood packaging material using two methods: (1) semiochemical-baited traps deployed in a total of about 63-80 sites per year in British Columbia (BC), Ontario (ON), Quebec (QC), New Brunswick (NB), Nova Scotia (NS), and Newfoundland and Labrador (NL); and (2) rearing of insects from bolts collected from stressed trees and incubated in modified shipping containers in four cities (Vancouver, Toronto, Montreal, and Halifax). We report 31 new Canadian provincial records of Coleoptera from surveys conducted in 2011-2021, including 13 new records for Canada and 9 species adventive to North America (indicated by †). Nine of the new Canadian records were native North American species previously detected only south of the border. All but three species belong to the Curculionidae family and most of these were in the subfamily Scolytinae. The records include: Xenomelanophila miranda (LeConte) (Canada, BC) (Buprestidae: Buprestinae); Neoclytus mucronatus mucronatus (Fabricius) (BC) (Cerambycidae: Cerambycinae); Amphicerus cornutus (Pallas) (Canada, BC) (Bostrichidae: Bostrichinae); Mecinus janthinus (Germar)† (ON) (Curculionidae: Curculioninae); Aulacobaris lepidii (Germar)† (Canada, ON); Buchananius striatus (LeConte) (ON) (Curculionidae: Baridinae); Cylindrocopturus binotatus LeConte (Canada, ON) (Curculionidae: Conoderinae); Himatium errans LeConte (ON); Phloeophagus canadensis Van Dyke (ON); Rhyncolus spretus Casey (Canada, BC); Stenomimus pallidus (Boheman) (Canada, ON); Tomolips quercicola (Boheman) (Canada, ON) (Curculionidae: Cossoninae); Strophosoma melanogrammum (Forster)† (NB) (Curculionidae: Entiminae); Conotrachelus aratus (Germar) (ON) (Curculionidae: Molytinae); Anisandrus maiche Stark† (Canada, ON, QC); Cnesinus strigicollis LeConte (Canada, ON); Cyclorhipidion pelliculosum (Eichhoff)† (Canada, ON, QC); Hylesinus fasciatus LeConte (QC); Hylesinus pruinosus Eichhoff (QC); Hypothenemus interstitialis (Hopkins) (Canada, ON); Lymantor alaskanus Wood (BC); Pityogenes bidentatus (Herbst)† (Canada, ON); Scolytus mali (Bechstein)† (BC); Scolytus schevyrewi Semenov† (QC); Trypodendron scabricollis (LeConte) (Canada, ON); Trypophloeus populi Hopkins (QC); Xylechinus americanus Blackman (NFLB); and Xylosandrus crassiusculus (Motschulsky)† (BC, QC) (Curculionidae: Scolytinae). We also provide additional data confirming the presence of the adventive Hylastes opacus Erichson† in NS. Rearing of insects from bolts accounted for two new records (H. pruinosus, R. spretus) and trapping accounted for the remainder. These surveys not only assist our efforts to manage forest insects by documenting new species introductions and apparent range expansions but also increase our knowledge of biodiversity.

CDK4/6 Inhibition Reprograms Mitochondrial Metabolism in BRAFV600 Melanoma via a p53 Dependent Pathway.

Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are being tested in numerous clinical trials and are currently employed successfully in the clinic for the treatment of breast cancers. Understanding their mechanism of action and interaction with other therapies is vital in their clinical development. CDK4/6 regulate the cell cycle via phosphorylation and inhibition of the tumour suppressor RB, and in addition can phosphorylate many cellular proteins and modulate numerous cellular functions including cell metabolism. Metabolic reprogramming is observed in melanoma following standard-of-care BRAF/MEK inhibition and is involved in both therapeutic response and resistance. In preclinical models, CDK4/6 inhibitors overcome BRAF/MEK inhibitor resistance, leading to sustained tumour regression; however, the metabolic response to this combination has not been explored. Here, we investigate how CDK4/6 inhibition reprograms metabolism and if this alters metabolic reprogramming observed upon BRAF/MEK inhibition. Although CDK4/6 inhibition has no substantial effect on the metabolic phenotype following BRAF/MEK targeted therapy in melanoma, CDK4/6 inhibition alone significantly enhances mitochondrial metabolism. The increase in mitochondrial metabolism in melanoma cells following CDK4/6 inhibition is fuelled in part by both glutamine metabolism and fatty acid oxidation pathways and is partially dependent on p53. Collectively, our findings identify new p53-dependent metabolic vulnerabilities that may be targeted to improve response to CDK4/6 inhibitors.

Enhancing Adoptive Cell Transfer with Combination BRAF-MEK and CDK4/6 Inhibitors in Melanoma.

Despite the success of immune checkpoint inhibitors that target cytotoxic lymphocyte antigen-4 (CTLA-4) and programmed-cell-death-1 (PD-1) in the treatment of metastatic melanoma, there is still great need to develop robust options for patients who are refractory to first line immunotherapy. As such there has been a resurgence in interest of adoptive cell transfer (ACT) particularly derived from tumor infiltrating lymphocytes. Moreover, the addition of cyclin dependent kinase 4/6 inhibitors (CDK4/6i) have been shown to greatly extend duration of response in combination with BRAF-MEK inhibitors (BRAF-MEKi) in pre-clinical models of melanoma. We therefore investigated whether combinations of BRAF-MEK-CDK4/6i and ACT were efficacious in murine models of melanoma. Triplet targeted therapy of BRAF-MEK-CDK4/6i with OT-1 ACT led to sustained and robust anti-tumor responses in BRAFi sensitive YOVAL1.1. We also show that BRAF-MEKi but not CDK4/6i enhanced MHC Class I expression in melanoma cell lines in vitro. Paradoxically CDK4/6i in low concentrations of IFN-γ reduced expression of MHC Class I and PD-L1 in YOVAL1.1. Overall, this work provides additional pre-clinical evidence to pursue combination of BRAF-MEK-CDK4/6i and to combine this combination with ACT in the clinic.

Combined BRAF, MEK, and CDK4/6 Inhibition Depletes Intratumoral Immune-Potentiating Myeloid Populations in Melanoma.

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a-/-Pten-/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

A novel immunogenic mouse model of melanoma for the preclinical assessment of combination targeted and immune-based therapy.

Both targeted therapy and immunotherapy have been used successfully to treat melanoma, but the development of resistance and poor response rates to the individual therapies has limited their success. Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles, but requires assessment in preclinical models with the capacity to respond to both therapeutic classes. Herein, we describe the development and characterization of a novel, immunogenic variant of the BrafV600ECdkn2a-/-Pten-/- YUMM1.1 tumor model that expresses the immunogen, ovalbumin (YOVAL1.1). We demonstrate that, unlike parental tumors, YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy. Importantly, YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK, responding in a manner consistent with human BRAFV600E melanoma. The YOVAL1.1 melanoma model is transplantable, immunogenic and sensitive to clinical therapies, making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma.

Effect of polymers on the retention and aging of enzyme on bioactive papers.

The effect of polymer on the retention and the thermal stability of bioactive enzymatic papers was measured using a colorimetric technique quantifying the intensity of the enzyme-substrate product complex. Alkaline phosphatase (ALP) was used as model enzyme. Three water soluble polymers: a cationic polyacrylamide (CPAM), an anionic polyacrylic acid (PAA) and a neutral polyethylene oxide (PEO) were selected as retention aids. The model polymers increased the enzyme adsorption on paper by around 50% and prevented enzyme desorption upon rewetting of the papers. The thermal deactivation of ALP retained on paper with polymers follows two sequential first order reactions. This was also observed for ALP simply physisorbed on paper. The retention aid polymers instigated a rapid initial deactivation which significantly decreased the longevity of the enzymatic papers. This suggests some enzyme-polymer interaction probably affecting the enzyme tertiary structure. A deactivation mathematical model predicting the enzymatic paper half-life was developed.