BRN2 suppresses apoptosis, reprograms DNA damage repair, and is associated with a high somatic mutation burden in melanoma.

Whether cell types exposed to a high level of environmental insults possess cell type-specific prosurvival mechanisms or enhanced DNA damage repair capacity is not well understood. BRN2 is a tissue-restricted POU domain transcription factor implicated in neural development and several cancers. In melanoma, BRN2 plays a key role in promoting invasion and regulating proliferation. Here we found, surprisingly, that rather than interacting with transcription cofactors, BRN2 is instead associated with DNA damage response proteins and directly binds PARP1 and Ku70/Ku80. Rapid PARP1-dependent BRN2 association with sites of DNA damage facilitates recruitment of Ku80 and reprograms DNA damage repair by promoting Ku-dependent nonhomologous end-joining (NHEJ) at the expense of homologous recombination. BRN2 also suppresses an apoptosis-associated gene expression program to protect against UVB-, chemotherapy- and vemurafenib-induced apoptosis. Remarkably, BRN2 expression also correlates with a high single-nucleotide variation prevalence in human melanomas. By promoting error-prone DNA damage repair via NHEJ and suppressing apoptosis of damaged cells, our results suggest that BRN2 contributes to the generation of melanomas with a high mutation burden. Our findings highlight a novel role for a key transcription factor in reprogramming DNA damage repair and suggest that BRN2 may impact the response to DNA-damaging agents in BRN2-expressing cancers.

Pharmacokinetics and safety of LEAD-452, an EGFR-specific 4-1BB-agonistic trimerbody in non-human primates.

LEAD-452 is a humanized bispecific EGFR-targeted 4-1BB-agonistic trimerbody with a unique trimeric configuration compared to other 4-1BB-specific antibodies that are currently in development. Indeed, enhanced tumor-specific costimulation and very remarkable safety and efficacy profiles have been observed in mouse models. Here, we conducted for the first time a preclinical pharmacokinetic and toxicity study in non-human primates (NHP) (Macaca fascicularis). LEAD-452 exhibits comparable binding affinity for human and macaque targets, indicating its pharmacological significance for safety testing across species. The NHP were administered LEAD-452 in a series of ascending doses, ranging from 0.1 mg/kg to 10 mg/kg, and repeated doses up to 20 mg/kg. The administration of LEAD-452 was found to be clinically well tolerated, with no major related adverse effects observed. Furthermore, there have been no reported cases of liver toxicity, thrombocytopenia, and neutropenia, which are commonly associated with treatments using conventional anti-4-1BB IgG-based antibodies. In addition, neither IgM nor IgG-based anti-drug antibodies were detected in serum samples from NHP during the study, regardless of the dose of LEAD-452 administered. These results support the clinical development of LEAD-452 for the treatment of solid tumors.

RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function.

Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells. Silencing RAD51 or BRCA2 stimulated both SSA and A-EJ, but not C-NHEJ, validating the two-step model. Three different RAD51 dominant-negative forms (DN-RAD51s) repressed GC and stimulated SSA/A-EJ. However, a fourth DN-RAD51 repressed SSA/A-EJ, although it efficiently represses GC. In living cells, the three DN-RAD51s that stimulate SSA/A-EJ failed to load efficiently onto damaged chromatin and inhibited the binding of endogenous RAD51, while the fourth DN-RAD51, which inhibits SSA/A-EJ, efficiently loads on damaged chromatin. Therefore, the binding of RAD51 to DNA, rather than its ability to promote GC, is required for SSA/A-EJ inhibition by RAD51. We showed that RAD51 did not limit resection of endonuclease-induced DSBs, but prevented spontaneous and RAD52-induced annealing of complementary ssDNA in vitro. Therefore, RAD51 controls the selection of the DSB repair pathway, protecting genome integrity from nonconservative DSB repair through ssDNA occupancy, independently of the promotion of CG.

The Tumor Suppressor PALB2: Inside Out.

Partner and Localizer of BRCA2 (PALB2) has emerged as an important and versatile player in genome integrity maintenance. Biallelic mutations in PALB2 cause Fanconi anemia (FA) subtype FA-N, whereas monoallelic mutations predispose to breast, and pancreatic familial cancers. Herein, we review recent developments in our understanding of the mechanisms of regulation of the tumor suppressor PALB2 and its functional domains. Regulation of PALB2 functions in DNA damage response and repair occurs on multiple levels, including homodimerization, phosphorylation, and ubiquitylation. With a molecular emphasis, we present PALB2-associated cancer mutations and their detailed analysis by functional assays.

Myositis autoantibodies detected by line blot immunoassay: clinical associations and correlation with antibody signal intensity.

The aim of this study is to assess the relationship between myositis specific (MSA) and myositis associated (MAA) antibodies and diagnosis (including idiopathic inflammatory myopathies [IIM] and other systemic autoimmune diseases [SAID]), and to explore the impact of antibody signal intensity in diagnostic accuracy. We retrospectively reviewed all the serum samples obtained from patients tested for MSA/MAA by line immunoassay (LIA) between 01/01/2018 and 31/12/2020 in Ramón y Cajal University Hospital (Spain). Clinical true positive (CTP) MSAs and MAAs were defined as those patients with IIM or SAID with phenotypes expected of that MSA/MAA. Patients who did not have a phenotype compatible with that antibody were classified as clinical false positive (CFP). One hundred and thirty positive samples were analysed. Forty-six patients (33.38%) were classified as IIM, forty-two (32.3%) as SAID and forty-two (32.3%) as non-IIM/SAID. Among these 130 patients, 164 MSA/MAA were detected. Eighty-five (51.8%) positive MSA/MAA were classified as CTP, and seventy-nine (48.2%) as CFP. Strongly positive antibodies were more frequently CTP (35/47, 74.5%) than weak positives (54/68, 36.8%), (p ˂ 0.001). Antibodies classified as CTP had a higher signal intensity than CFP (36.77 AU vs 20.00 AU, CI95% 7.79-22.09, p ˂ 0.001). The probability of a CFP was associated to negative ANA, low ANA titer, and multiple positive MSA/MAA (p Ë‚ 0.001). In this study, we confirmed that CFP results using LIA are frequent, and are associated with low signal intensity MSA/MAA, negative ANA, lower titer ANA, and with multiple positive samples.

Reflective based learning for nursing ethical competency during clinical practices.

BACKGROUND: A combination of theoretical and practical approaches is required to learn and acquire ethical competencies in caring. Occasionally, reflection on practical action differs from theoretical learning. In the context of reflective learning, issues such as ethical values can be discussed since they evoke conflict among nursing students. AIM: To identify ethical conflicts encountered by nursing students during clinical placements and to determine their cooperation strategies. RESEARCH DESIGN: Qualitative study with a content analysis according to Elo and Kinglas framework. PARTICIPANTS AND RESEARCH CONTEXT: Students enrolled in a nursing program at a Spanish university aged between 22 and 35, mainly women. METHODOLOGY: The study includes 134 ethical reflections from nursing students in the last year of the nursing program, written during their clinical practices in a variety of learning environments. The research team analyzed the reflections using an inductive content analysis method. ETHICAL CONSIDERATIONS: Ethical permission was obtained by the management center according to Law 3/2028, and all the participants accepted to participate through the informed consent form. FINDINGS: Three main categories emerged from the analysis of the ethical reflections: (1) evaluation of professional performance and patient care; (2) the student as the protagonist of the dilemma; (3) student coping. Student dilemmas and concerns are related to ignorance, student-patient communication, mistakes made and self-confidence. Some situations conflict with the autonomy of patients and their rights, and can contribute to stressful situations for patients. Stress factors include hospital routines, which the patient cannot modify, and asymmetric relationships with staff, which encourage passivity. CONCLUSION: All ethical problems detected by the students begin with the professional-patient relationship, including issues related to bad news, errors or malpractice. Reflection on the ethical values of nursing, both in the classroom and in clinical practices, allows students to develop a greater ethical awareness of care, enhancing their decision-making skills in ethical dilemmas.

T Cell-Redirecting Strategies to 'STAb' Tumors: Beyond CARs and Bispecific Antibodies.

The redirection of T cell activity towards cancer cells via targeting of tumor-associated antigens (TAAs) by soluble bispecific antibodies (bsAbs) or membrane-anchored chimeric antigen receptors is one of the most promising cancer immunotherapy strategies currently in development. We review here an emerging approach that combines aspects of antibody- and cell-based therapies: STAb immunotherapy, based on the endogenous secretion of T cell-redirecting bsAbs (STAb). STAb immunotherapies use ex vivo or in vivo genetic modifications of different cell types with nucleic acids or viral vectors encoding bsAbs; these can result in effective and persistent concentrations of antibodies. After introducing core concepts, we discuss plausible ways by which STAb strategies might be further developed to improve their potential efficacy and safety in preclinical and clinical testing.

Selection of Debaryomyces hansenii isolates as starters in meat products based on phenotypic virulence factors, tolerance to abiotic stress conditions and aroma generation.

INTRODUCTION: Debaryomyces hansenii is a yeast widely used in meat fermentations as starter for the purpose of improving the aromatic quality of the final product. However, it has not been the subject of an extensive study regarding phenotypic characteristics important for starter selection, such as the capacity to grow at abiotic stress conditions occurring during fermentation, the ability to generate desirable aromas and the absence of virulence traits in yeasts. AIMS: The aim of this study was to screen 60 strains of D. hansenii isolated from assorted foods for their potential application as starters in dry-cured fermented sausages manufacture. METHODS: The abiotic stress factors tested were low aw and pH and high concentration of salt, acetic acid and lactic acid. The phenotypic virulence traits explored were growth at 37°C, pseudohyphal and biofilm generation, invasiveness and enzymatic activities present in virulent yeasts. The generation of desirable meat aromas was tested in models containing aroma precursors applying an olfactory analysis. A quantitative profiling of stress tolerance was used to test the potential performance of selected strains in meat fermentations. RESULTS: The results demonstrated that most strains displayed no virulence trait or were only positive for biofilm production. Moreover, the strains showed large heterogeneity regarding their tolerance to abiotic stress factors, although most of them could grow at intermediate to high levels of the traits. The sensory analysis was the criteria determining the selection of starter strains. CONCLUSIONS: The evaluation of the phenotypic traits demonstrates that D. hansenii is a safe yeast, it is able to tolerate the stress in meat fermentation and it is able to generate desirable aromas. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study confirm the adequacy of selected D. hansenii strains to be applied as starters in meat products.

Evaluation of flare rate and reduction strategies for bDMARDs in juvenile idiopathic arthritis: real world data from a single-centre cohort.

This study aimed to determine the flare rate (FR) in a cohort of Juvenile Idiopathic Arthritis (JIA) patients with tapered or abruptly discontinued biologic disease-modifying anti-rheumatic drugs (bDMARDs) and to identify predictors of flare. This retrospective observational study included 191 bDMARD dose-reduction events in patients with JIA followed-up at a referral hospital during the period 2000-2019. FR was analysed according to reduction strategies. To identify predictors of flare, Kaplan-Meier and Cox-regression models were plotted at 6 months (6 m), 12 months (12 m) and 24 months (24 m) following tapering (TP) or withdrawal (WD). 165 episodes of TP and 71 episodes of WD were included; 45 episodes where treatment was withdrawn after TP were included in both strategies. FR after TP was 13.4% at 6 m and increased up to 26.6% at 12 m and 51.4% at 24 m. After WD, FR was higher, 52.1% of events had a flare at 6 m and 67.6% at 12 m. Previous TP did not increase time in remission after WD of bDMARDs in the Kaplan-Meier analysis. Factors associated with flares were identified after TP at 6 m: female sex, higher number of previous bDMARDs and longer time on bDMARD treatment were positively associated with flares. Polyarticular subtype and younger age at diagnosis were associated with flares at 12 and 24 m after TP. No factors were identified in multivariable analysis after WD. TP is a successful strategy to maintain remission with lower bDMARD doses. Previous TP of bDMARDs does not seem to increase time in remission after WD.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Optimisation of 2-(N-phenyl carboxamide) triazolopyrimidine antimalarials with moderate to slow acting erythrocytic stage activity.

Malaria is a devastating parasitic disease caused by parasites from the genus Plasmodium. Therapeutic resistance has been reported against all clinically available antimalarials, threatening our ability to control the disease and therefore there is an ongoing need for the development of novel antimalarials. Towards this goal, we identified the 2-(N-phenyl carboxamide) triazolopyrimidine class from a high throughput screen of the Janssen Jumpstarter library against the asexual stages of the P. falciparum parasite. Here we describe the structure activity relationship of the identified class and the optimisation of asexual stage activity while maintaining selectivity against the human HepG2 cell line. The most potent analogues from this study were shown to exhibit equipotent activity against P. falciparum multidrug resistant strains and P. knowlesi asexual parasites. Asexual stage phenotyping studies determined the triazolopyrimidine class arrests parasites at the trophozoite stage, but it is likely these parasites are still metabolically active until the second asexual cycle, and thus have a moderate to slow onset of action. Non-NADPH dependent degradation of the central carboxamide and low aqueous solubility was observed in in vitro ADME profiling. A significant challenge remains to correct these liabilities for further advancement of the 2-(N-phenyl carboxamide) triazolopyrimidine scaffold as a potential moderate to slow acting partner in a curative or prophylactic antimalarial treatment.

A novel multiple-stage antimalarial agent that inhibits protein synthesis.

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance.

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) ß5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The ß5 inhibitors synergize with a ß2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA ß5 inhibitor surprisingly harbored a point mutation in the noncatalytic ß6 subunit. The ß6 mutant was resistant to the species-selective Pf20S ß5 inhibitor but remained sensitive to the species-nonselective ß5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S ß5 inhibitor was accompanied by increased sensitivity to a Pf20S ß2 inhibitor. Finally, the ß5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S ß5 and ß2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.

Contraceptive counselling experiences in Spain in the process of creating a web-based contraceptive decision support tool: a qualitative study.

BACKGROUND: The choice of contraceptive method is a complex decision, and professionals should offer counselling based on the preferences, values and personal situation of the user(s). Some users are unsatisfied with the counselling received, which may, among other consequences, adversely affect method use adherence. In view of this situation, we propose exploring the experiences and needs of users and professionals for contraceptive counselling, in the context of creating a web-based contraceptive decision support tool. METHODS/DESIGN: Qualitative research was conducted through focus group discussions (64 users split into eight groups, and 19 professionals in two groups, in Tarragona, Spain) to explore the subjects' experiences and needs. The data were categorized and the categories were defined and classified based on the three-step protocol or framework for Quality on Contraceptive Counseling (QCC), created by experts, which reviews the quality of interactions between user and professional during the counselling process. RESULTS: In counselling, users demand more information about the different methods, in an environment of erroneous knowledge and misinformation, which lead to false beliefs and myths in the population that are not contrasted by the professional in counselling. They complain that the method is imposed on them and that their views regarding the decision are not considered. Professionals are concerned that their lack of training leads to counselling directed towards the methods they know best. They acknowledge that a paternalistic paradigm persists in the healthcare they provide, and decision support tools may help to improve the situation. CONCLUSIONS: Users feel unsatisfied and/or demand more information and a warmer, more caring approach. Professionals are reluctant to assume a process of shared decision-making. The use of a contraception DST website may solve some shortcomings in counselling detected in our environment.

High rates of severe disease and death due to SARS-CoV-2 infection in rheumatic disease patients treated with rituximab: a descriptive study.

The objective of this study is to describe the characteristics and outcomes of rheumatic and musculoskeletal disease (RMD) patients who were treated with rituximab and had suspected or confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this descriptive study, RMD patients who were treated with rituximab in the last 12 months at the Rheumatology Department of our hospital were screened for SARS-CoV-2 infection via telephone interview and a comprehensive review of clinical health records (01/02/2020-26/05/2020). Those with probable or confirmed SARS-CoV-2 infection were included. In total, 76 patients were screened. Of these, 13 (17.1%) had suspected or confirmed SARS-CoV-2 infection. With regard to these 13 patients, the median age at coronavirus disease (COVID-19) diagnosis was 68 years (range 28-76 years) and 8 (61.5%) were female. Five patients had rheumatoid arthritis, three had systemic vasculitis, two had Sjögren syndrome, and two had systemic lupus erythematosus. Additionally, seven patients (53.8%) had pulmonary involvement secondary to RMD. Eight patients (61.5%) developed severe disease leading to hospitalization, and seven developed bilateral pneumonia and respiratory insufficiency. Of the eight hospitalized patients, five (62.5%) fulfilled the acute respiratory distress syndrome criteria and three developed a critical disease and died. Our cohort had a high rate of severe disease requiring hospitalization (61.5%), with bilateral pneumonia and hyperinflammation leading to a high mortality rate (23.1%). Treatment with rituximab should be considered a possible risk factor for unfavorable outcomes in COVID-19 patients with RMD. However, further study is required to confirm this association.

Targeting NF-κB by the Cell-Permeable NEMO-Binding Domain Peptide Improves Albuminuria and Renal Lesions in an Experimental Model of Type 2 Diabetic Nephropathy.

Diabetic nephropathy (DN) is a multifactorial disease characterized by hyperglycemia and close interaction of hemodynamic, metabolic and inflammatory factors. Nuclear factor-κB (NF-κB) is a principal matchmaker linking hyperglycemia and inflammation. The present work investigates the cell-permeable peptide containing the inhibitor of kappa B kinase γ (IKKγ)/NF-κB essential modulator (NEMO)-binding domain (NBD) as therapeutic option to modulate inflammation in a preclinical model of type 2 diabetes (T2D) with DN. Black and tan, brachyuric obese/obese mice were randomized into 4 interventions groups: Active NBD peptide (10 and 6 µg/g body weight); Inactive mutant peptide (10 µg/g); and vehicle control. In vivo/ex vivo fluorescence imaging revealed efficient delivery of NBD peptide, systemic biodistribution and selective renal metabolization. In vivo administration of active NBD peptide improved albuminuria (>40% reduction on average) and kidney damage, decreased podocyte loss and basement membrane thickness, and modulated the expression of proinflammatory and oxidative stress markers. In vitro, NBD blocked IKK-mediated NF-κB induction and target gene expression in mesangial cells exposed to diabetic-like milieu. These results constitute the first nephroprotective effect of NBD peptide in a T2D mouse model that recapitulates the kidney lesions observed in DN patients. Targeting IKK-dependent NF-κB activation could be a therapeutic strategy to combat kidney inflammation in DN.

Nurses' emotions on care relationship: A qualitative study.

AIMS: To describe nurses' experiences regarding the care relationship built with medical-surgical patients in acute hospitalization units and the association with their clinical practice. BACKGROUND: Nurses' commitment to care and their relationships as well are the core of quality of care. Nurses consider that the emotional commitment is essential to the profession, thus accepting to be exposed to emotional distress. METHODS: In-depth interviews were conducted to explore the experience of 23 nurses from seven Spanish hospitals. Taped interviews were transcribed verbatim and analysed according to inductive content analysis. RESULTS: Six subcategories emerged from the data analysis: Fulfilling experiences, Feeling the pain of others, Emotional distress, Stepping back, Seeking professional support, and Evolution of emotions, which were combined in two main categories: Nurses' emotions and Nurses' coping strategies. Patient's suffering, work environment and interprofessional relations influence the care relationship. CONCLUSIONS: Positive and negative emotions emerge spontaneously in professional relationships of experienced nurses. Lack of time and high workloads are factors that hinder the nurse-patient relationship. This care relationship is often the reason that fulfils them. Despite their level of expertise and having coping strategies, these are not always effective and, sometimes, nurses need professional help. IMPLICATIONS FOR NURSING MANAGEMENT: Nurses will continue performing their job with commitment; therefore, nursing managers should take care of their staff and pay attention to the emotional competence related to patients' relationships. Promoting self-care and a good working environment could improve their coping mechanisms.

Cancer cell plasticity: Impact on tumor progression and therapy response.

Most tumors exhibit intra-tumor heterogeneity, which is associated with disease progression and an impaired response to therapy. Cancer cell plasticity has been proposed as being an important mechanism that, along with genetic and epigenetic alterations, promotes cancer cell diversity and contributes to intra-tumor heterogeneity. Plasticity endows cancer cells with the capacity to shift dynamically between a differentiated state, with limited tumorigenic potential, and an undifferentiated or cancer stem-like cell (CSC) state, which is responsible for long-term tumor growth. In addition, it confers the ability to transit into distinct CSC states with different competence to invade, disseminate and seed metastasis. Cancer cell plasticity has been linked to the epithelial-to-mesenchymal transition program and relies not only on cell-autonomous mechanisms, but also on signals provided by the tumor microenvironment and/or induced in response to therapy. We provide an overview of the dynamic transition for cancer cell states, the mechanisms governing cell plasticity and their impact on tumor progression, metastasis and therapy response. Understanding the mechanisms involved in cancer cell plasticity will provide insights for establishing new therapeutic interventions.

p53 controls the plasticity of mammary luminal progenitor cells downstream of Met signaling.

BACKGROUND: The adult mammary epithelium is composed of basal and luminal cells. The luminal lineage comprises two major cell populations, positive and negative for estrogen and progesterone receptors (ER and PR, respectively), both containing clonogenic progenitor cells. Deregulated ER/PR- luminal progenitor cells are suspected to be at the origin of basal-type triple-negative (TNBC) breast cancers, a subtype frequently associated with loss of P53 function and MET signaling hyperactivation. Using mouse models, we recently reported that p53 restricts luminal progenitor cell amplification whereas paracrine Met activation stimulates their growth and favors a luminal-to-basal switch. Here, we analyzed how these two critical pathways interact to control luminal progenitor function. METHODS: We have (i) established and analyzed the gene expression profile of luminal progenitors isolated by ICAM-1, a robust surface marker we previously identified; (ii) purified luminal progenitors from p53-deficient and p53-proficient mouse mammary epithelium to compare their functional and molecular characteristics; and (iii) analyzed their response to HGF, the major Met ligand, in three-dimensional cultures. RESULTS: We found that luminal progenitors, compared to non-clonogenic luminal cells, overexpress Trp53 and numerous p53 target genes. In vivo, loss of Trp53 induced the expansion of luminal progenitors, affecting expression of several important p53 target genes including those encoding negative regulators of cell cycle progression. Consistently, p53-deficient luminal progenitors displayed increased proliferative and self-renewal activities in culture. However, they did not exhibit perturbed expression of luminal-specific markers and major regulators, such as Hey1, Elf5, and Gata3. Moreover, although expressing Met at higher level than p53-proficient luminal progenitors, p53-deficient luminal progenitors failed to acquire basal-specific features when stimulated by HGF, showing that p53 promotes the plastic behavior of luminal progenitors downstream of Met activation. CONCLUSIONS: Our study reveals a crosstalk between Met- and p53-mediated signaling pathways in the regulation of luminal progenitor function. In particular, it shows that neither p53 loss alone nor p53 loss combined with Met signaling activation caused an early detectable cell fate alteration in luminal progenitors. Conceivably, additional events are required to confer basal-specific characteristics to luminal-derived TNBCs.

Evaluation of 4-Amino 2-Anilinoquinazolines against Plasmodium and Other Apicomplexan Parasites In Vitro and in a P. falciparum Humanized NOD-scid IL2Rγnull Mouse Model of Malaria.

A series of 4-amino 2-anilinoquinazolines optimized for activity against the most lethal malaria parasite of humans, Plasmodium falciparum, was evaluated for activity against other human Plasmodium parasites and related apicomplexans that infect humans and animals. Four of the most promising compounds from the 4-amino 2-anilinoquinazoline series were equally as effective against the asexual blood stages of the zoonotic P. knowlesi, suggesting that they could also be effective against the closely related P. vivax, another important human pathogen. The 2-anilinoquinazoline compounds were also potent against an array of P. falciparum parasites resistant to clinically available antimalarial compounds, although slightly less so than against the drug-sensitive 3D7 parasite line. The apicomplexan parasites Toxoplasma gondii, Babesia bovis, and Cryptosporidium parvum were less sensitive to the 2-anilinoquinazoline series with a 50% effective concentration generally in the low micromolar range, suggesting that the yet to be discovered target of these compounds is absent or highly divergent in non-Plasmodium parasites. The 2-anilinoquinazoline compounds act as rapidly as chloroquine in vitro and when tested in rodents displayed a half-life that contributed to the compound's capacity to clear P. falciparum blood stages in a humanized mouse model. At a dose of 50 mg/kg of body weight, adverse effects to the humanized mice were noted, and evaluation against a panel of experimental high-risk off targets indicated some potential off-target activity. Further optimization of the 2-anilinoquinazoline antimalarial class will concentrate on improving in vivo efficacy and addressing adverse risk.