Different Mechanisms Of Action Research Articles

AIOLOS haploinsufficiency is associated with immunodeficiency, autoimmunity, and allergy

AIOLOS is a transcription factor of the IKAROS family, that is highly expressed in the lymphoid lineage and plays an important role in lymphocyte differentiation and development. Recently, two heterozygous AIOLOS germline mutations (G159R and N160S) were reported in primary immunodeficiency patients. Both variants associated distinctive and unique T and B cell differentiation defects, hematologic malignancies, and infectious disease susceptibility patterns through different dominant negative (DN) mechanisms. Here we report seven individuals from three unrelated families carrying AIOLOS variants Q402* (nonsense, 1 family) and E82K (missense, 2 families), leading to AIOLOS haploinsufficiency through different mechanisms of action. Clinically, patients with AIOLOS haploinsufficiency showed hypogammaglobulinemia, recurrent respiratory tract infections, and immune dysregulation manifested by autoimmunity and allergy, with incomplete clinical penetrance. Mechanistically, while the nonsense mutant Q402* is devoid of its C-terminal zinc-finger (ZF) dimerization domain (ZF5-6), it is still able to homodimerize with wildtype (WT) AIOLOS although unable to heterodimerize with WT IKAROS or HELIOS. We introduce new insights into AIOLOS biology as we found that AIOLOS ZF1, a part of AIOLOS DNA binding domain (ZF1-4), was associated with noncanonical dimerization and negative regulation of DNA binding. Further studies revealed that the mutant Q402* displayed abnormalities of DNA binding, pericentromeric targeting, post-transcriptional modification, and transcriptome regulation by haploinsufficiency. In contrast, mutant E82K showed overall normal AIOLOS functions; however, by affecting a newly defined AIOLOS protein stability domain, it also led to AIOLOS haploinsufficiency. Unlike the previously reported AIOLOS mutations, heterozygous variants Q402* and E82K do not have a DN effect over other IKAROS family proteins. Our data extend the spectrum of pathogenic variants of AIOLOS-associated diseases and provide a novel mechanism of homodimerization and protein stability that, when disrupted, could affect human health.

New syndromic combined immunodeficiency with severe neurodevelopmental defects caused by biallelic null variants in the PPM1D gene

PPM1D (protein phosphatase Mn2+/Mg2+-dependent 1D) is a phosphatase that targets proteins related to the DNA damage repair system. C-terminal variants in PPM1D have been associated with: i) malignancy as somatic variants; and ii) Jansen-de-Vries (JdV) syndrome as germline heterozygous variants. JdV syndrome curses with moderate neurodevelopmental alterations and behavioral problems, such as autistic spectrum disorders (ASD), without reports of immunodeficiency.We describe two siblings (9 and 11 years old) born to consanguineous parents with progressive B and NK cell lymphopenia, agammaglobulinemia, non-ulcerative colitis and severe neurodevelopmental defects including microcephaly, severe encephalopathy, profound cognitive deficiency, and refractory epilepsy. Exome analysis revealed a homozygous N-terminal truncating variant in PPM1D (p.Thr174fs*6) in the two siblings inherited from their healthy heterozygous parents. The siblings’ clinical phenotype was markedly more severe than in the reported JdV patients, suggesting a different mechanism of action. To test this, we included a pediatric patient with classical JdV syndrome carrying an heterozygous PPM1D variant (p. Ser403fs*30). Using primary cells and exogenous overexpression we showed that mutation p.T174fs*6 abrogated protein expression by increased proteasome degradation, leading to no protein expression in a homozygous patient and a reduction in a haplosufficient parent. In contrast, the JdV variant (p.Ser403fs*30) led to accumulation of the truncated protein with conserved phosphatase activity, resulting in a previously undescribed gain-of-function (GOF) mechanism.We also demonstrated that chemical inhibition of WT-PPM1D in-vitro dramatically decreased B cell viability. This data, similar to the reported B cell deficiency in PPM1D-KO mice, helps to address the humoral deficiency in the patients, while not observed in the heterozygous parents nor JdV patient. To further explore NK cell deficiency, we will evaluate PPM1D-KO mice. We have generated EBV-B cell-derived iPSCs from PPM1D-null, PPM1D-haplosufficient an GOF-JdV individuals, to generate artificial cerebral organoids that will allow us to explore PPM1D gene-dosage effect in neurodevelopment and its potential manipulation.Here we report a novel biallelic PPM1D-null deficiency impairing B cell development. Besides, PPM1D dysregulation either by no protein expression or GOF appears to cause neurodevelopment abnormalities. Future studies will help to understand pathophysiology of PPM1D and better targeted clinical management.PPM1D research interest group:JF Quesada1, Mercè Bolasell2, Julie E. Niemela3, Jennifer L. Stoddard3, Agustin Gil Silva3, Brigette Boast3, José R Regueiro4, Pierre Martine5, David Lopez Perez5, Ettore Appella5, Laia Alsina6,7,8, Harry L Malech9, Hye Sun Kuehn3, Sergio D. Rosenzweig31. Department of Genetics, Hospital Universitario 12 de Octubre, Madrid, Spain.2. Department of Genetic and Molecular Medicine IPER, Institut de Recerca, Hospital Sant Joan de Déu Barcelona, Barcelona, Spain.3. Immunology Service, Department of Laboratory Medicine, Clinical Center, NIH, Bethesda, Maryland, USA4. Department of Immunology, Complutense University School of Medicine and Hospital 12 de Octubre Health Research Insitute, Madrid, Spain5. Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.6. Clinical Immunology and Primary Immunodeficiencies Unit. Allergy and Clinical Immunology Department. Hospital Sant Joan de Déu. Barcelona, Spain.7. Clinical Immunology Unit. Hospital Sant Joan de Déu-Hospital Clinic. Barcelona, Spain.8. Study Group for Immune Dysfunction Diseases in Children (GEMDIP). Institut de Recerca Sant Joan de Déu. Barcelona, Spain.9. Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD.

Photodynamic and Photothermal Therapies: Synergy Opportunities for Nanomedicine.

Tumoricidal photodynamic (PDT) and photothermal (PTT) therapies harness light to eliminate cancer cells with spatiotemporal precision by either generating reactive oxygen species or increasing temperature. Great strides have been made in understanding biological effects of PDT and PTT at the cellular, vascular and tumor microenvironmental levels, as well as translating both modalities in the clinic. Emerging evidence suggests that PDT and PTT may synergize due to their different mechanisms of action, and their nonoverlapping toxicity profiles make such combination potentially efficacious. Moreover, PDT/PTT combinations have gained momentum in recent years due to the development of multimodal nanoplatforms that simultaneously incorporate photodynamically- and photothermally active agents. In this review, we discuss how combining PDT and PTT can address the limitations of each modality alone and enhance treatment safety and efficacy. We provide an overview of recent literature featuring dual PDT/PTT nanoparticles and analyze the strengths and limitations of various nanoparticle design strategies. We also detail how treatment sequence and dose may affect cellular states, tumor pathophysiology and drug delivery, ultimately shaping the treatment response. Lastly, we analyze common experimental design pitfalls that complicate preclinical assessment of PDT/PTT combinations and propose rational guidelines to elucidate the mechanisms underlying PDT/PTT interactions.

Review and Update of Active and Passive Immunization Against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection (LRTI) in children, causing approximately 3.6 million hospitalizations per year, and has been associated with long-term pulmonary sequelae for up to 30years after infection, yet preventative strategies and active treatment options remain elusive. The associated morbidity and healthcare related costs could be decreased substantially with the development of these much-needed medications. After an initial false start in the development of an RSV vaccine, gradual progress is now being made with the development of multiple vaccine candidates using numerous different mechanisms of action. Furthermore, nirsevimab, a new monoclonal antibody for the prevention of RSV, has recently been registered in the European Union. New novel treatments for RSV infection are also in the pipeline, which would provide the clinician with much needed ammunition in the management of the acute disease. The next few years have the potential to change the landscape of LRTI forever through the prevention and management of RSV LRTI and thereby decrease the mortality and morbidity associated with it. In this review, we discuss these new approaches, current research, and clinical trials in monoclonal antibody and vaccine development against RSV.

P135 Efficacy and safety of upadacitinib in TNFi inadequate responders with rheumatoid arthritis from three Phase 3 clinical trials

Abstract Background/Aims For patients with RA who are refractory to biologic disease-modifying antirheumatic drugs (bDMARDs), such as tumor necrosis factor inhibitors (TNFis), optimal disease control is less likely to be achieved with subsequent therapy. In line with recommendations from EULAR and ACR, switching to a treatment with a different mechanism of action is appropriate for these patients. Objectives To describe the efficacy and safety of upadacitinib (UPA) 15 mg once daily in patients with RA and an inadequate response or intolerance to TNFis (TNFi-IR). Methods A post hoc subgroup analysis was conducted in TNFi-IR patients who were treated with UPA 15 mg once daily in three Phase 3 clinical trials: SELECT-BEYOND, -CHOICE, and -COMPARE. For COMPARE, only patients treated with adalimumab and switched to UPA as rescue therapy were included. ≥20/50/70% improvement in ACR criteria, DAS28-CRP, Clinical Disease Activity Index, and Simple Disease Activity Index, as well as change from baseline in HAQ-DI and other patient-reported outcomes (PROs) were reported through 24 weeks. Non-responder imputation was used for all missing categorical outcomes; as observed (COMPARE) or multiple imputation (CHOICE, BEYOND) were used for missing continuous outcomes. Pooled safety results were presented as exposure-adjusted event rates (EAERs) with a cut-off of June 30, 2021. Results 568 TNFi-IR patients were included: 146 from BEYOND, 263 from CHOICE, and 159 from COMPARE. Mean duration since RA diagnosis was longer for BEYOND and CHOICE versus COMPARE; CV risk factors were common among this refractory population. ACR20/50/70 and disease activity outcomes observed in the TNFi-IR population were generally consistent with the overall BEYOND and CHOICE bDMARD-IR populations, and consistent across the three studies in the TNFi-IR subgroups. Improvements in PROs including HAQ-DI, fatigue, pain, and morning stiffness over 24 weeks were observed (data not shown). Pooled safety results reporting 1574.8 PY of exposure in the TNFi-IR subgroup showed similar results to the overall BEYOND and CHOICE bDMARD-IR study populations, with EAERs of 3.1 events/100 PY for herpes zoster and 0.8 events/100 PY for adjudicated major adverse CV events and venous thromboembolism, and malignancy excluding non-melanoma skin cancer. The EAER of any AE leading to death was 1.4 events/100 PY. Conclusion In this post hoc subgroup analysis, TNFi-IR patients treated with UPA 15 mg achieved clinically meaningful efficacy responses over 24 weeks, with safety consistent with the overall bDMARD-IR patient population in the Phase 3 program. Disclosure R. Fleischmann: Consultancies; Consultant for AbbVie, Amgen, Bristol-Myers Squibb, Eli Lilly, Galvani, Gilead, GSK, Janssen, Novartis, Pfizer Inc, and UCB. Grants/research support; Grant/research support from AbbVie, Amgen, Biosplice, Bristol-Myers Squibb, Flexion, Gilead, Horizon, Eli Lilly, Galvani, Janssen, Novartis, Pfizer Inc, Sanofi-Aventis, Selecta, Teva, UCB, Viela, and. L. Bessette: Consultancies; AbbVie, Amgen, Bristol-Meyers Squibb, Celgene, Eli Lilly, Fresenius Kabi, Gilead, Janssen, Merck, Novartis, and Pfizer, Roche, Sanofi-Aventis, Teva, and UCB. Member of speakers’ bureau; AbbVie, Amgen, Bristol-Meyers Squibb, Celgene, Eli Lilly, Fresenius Kabi, Gilead, Janssen, Merck, Novartis, and Pfizer, Roche, Sanofi-Aventis, Teva, and UCB. Grants/research support; AbbVie, Amgen, Bristol-Meyers Squibb, Celgene, Eli Lilly, Fresenius Kabi, Gilead, Janssen, Merck, Novartis, and Pfizer, Roche, Sanofi-Aventis, Teva, and UCB. S. Hall: Consultancies; AbbVie, Amgen, Bristol-Meyers Sqibb, Eli Lilly, Gilead, Janssen, Merck, Novartis, and UCB. Grants/research support; AbbVie, Amgen, Bristol-Meyers Sqibb, Eli Lilly, Gilead, Janssen, Merck, Novartis, and UCB. J. Sparks: Consultancies; Consulted for AbbVie, Amgen, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead, Inova Diagnostics, Janssen, Optum, and Pfizer. M. Jain: Consultancies; Amgen, Abbvie, Eli Lilly, Pfizer, and Novartis. Grants/research support; Amgen, Abbvie, Eli Lilly, Pfizer, and Novartis. A. Kakehasi: Consultancies; Amgen, Janssen, UCB, AbbVie, Pfizer, Eli Lilly, Novartis, Sandoz, Fresenius Kabi. Member of speakers’ bureau; Amgen, Janssen, UCB, AbbVie, Pfizer, Eli Lilly, Novartis, Sandoz, Fresenius Kabi. Grants/research support; Amgen, Janssen, UCB, AbbVie, Pfizer, Eli Lilly, Novartis, Sandoz, Fresenius Kabi. Y. Song: Shareholder/stock ownership; full-time employees of AbbVie and may own stock or options. S. Meerwein: Shareholder/stock ownership; full-time employees of AbbVie and may own stock or options. R. DeMasi: Shareholder/stock ownership; full-time employees of AbbVie and may own stock or options. J. Suboticki: Shareholder/stock ownership; full-time employees of AbbVie and may own stock or options. A. Rubbert-Roth: Consultancies; AbbVie, AbbVie Deutschland, Amgen, Bristol-Myers Squibb, Chugai Pharmaceuticals, Eli Lilly, F. Hoffman-La Roche, Gilead Sciences, Janssen Global Services, Novartis, and Sanofi Pasteur.

Consensus on the management of united airways disease with type 2 inflammation: a multidisciplinary Delphi study

BackgroundScientific evidence on patients with multimorbid type 2 asthma and chronic rhinosinusitis with nasal polyps (CRSwNP) from a united airways disease (UAD) perspective remains scarce, despite the frequent coexistence of these entities. We aimed to generate expert consensus-based recommendations for the management of UAD patients.MethodsUsing a two-round Delphi method, Spanish expert allergists, pulmonologists and otolaryngologists expressed their agreement on 32 statements (52 items) on a 9-point Likert scale, classified as appropriate (median 7–9), uncertain (4–6) or inappropriate (1–3). Consensus was considered when at least two-thirds of the panel scored within the range containing the median.ResultsA panel of 30 experts reached consensus on the appropriateness of 43 out of the 52 (82.7%) items. The usefulness of certain biomarkers (tissue and peripheral blood eosinophil count, serum total IgE, and fraction of exhaled nitric oxide [FeNO]) in the identification and follow-up of type 2 inflammation, and assessment of the response to biologics, were agreed. Some of these biomarkers were also associated with disease severity and/or recurrence after endoscopic sinus surgery (ESS). Consensus was achieved on treatment strategies related to the prescription of anti-IL-4/IL-13 or anti-IgE agents, concomitant treatment with systemic corticosteroids, and combining or switching to biologics with a different mechanism of action, considering a number of UAD clinical scenarios.ConclusionWe provide expert-based recommendations to assist in clinical decision-making for the management of patients with multimorbid type 2 asthma and CRSwNP. Specific clinical trials and real-world studies focusing on the single-entity UAD are required to address controversial items.

Silymarin in combination with ATRA enhances apoptosis induction in human acute promyelocytic NB4 cells

Although all-trans retinoic acid (ATRA) is an efficient pattern in acute promyelocytic leukemia (APL) therapy, further studies are required due to the extant clinical limitations of ATRA. It has been reported that Silymarin, an anti-cancer herbal substance extracted from milk thistle (Silybum marianum), is able to regulate apoptosis in various types of cancer cells through different mechanisms of action. This study investigated the apoptosis-inducing effect of Silymarin (SM) alone and in combination with ATRA on human acute promyelocytic NB4 cells. Examination using MTT assay indicated that SM treatment leads to growth inhibition in NB4 cells in a dose-dependent manner. The IC50 values of SM and ATRA were calculated 90 μM and 2 μM, respectively. Cell cycle analysis by flow cytometry revealed that a more increase in the sub-G1 phase (a sign of apoptosis) when cells were exposed to SM in combination with ATRA. The incidence of apoptosis was confirmed through Hoechst 33258 staining and Annexin V-FITC analysis. The results showed that Silymarin enhances ATRA-induced apoptosis. The flow cytometric analysis also indicated an enhancement in levels of ROS in the treated cells with both compounds. The real-time PCR illustrated that SM targets apoptosis by down-regulation in Survivin and Bcl-2 while up-regulation in Bax. The findings showed that the combination of the two compounds is more effective in the induction of apoptosis in NB4 cells. Molecular docking studies indicated that Sylibin, as a primary compound of the SM, binds to the BH3 domain of Bcl-2 and the BIR domain of Survivin with various affinities. Based on the findings, it seems that SM used alone and in combination with ATRA may be beneficial for inducing apoptosis in APL cells.

Therapeutic Development of Levosimendan in Acute and Advanced Heart Failure: A Systematic Review.

Levosimendan (LS) has been progressively used for the treatment of patients developing acute as well as chronic or advanced cardiac dysfunction. It has proven to be a better inotropic agent than its counterparts in terms of its ability to increase the cardiac output in an acutely or chronically decompensated heart without an increase in the myocardial oxygen demand. The purpose of this systematic review, which was carried out in accordance with Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) 2020, was to determine the efficacy and advantages of utilizing LS in patients with both acute and chronic heart failure. We collected and reviewed articles, including clinical trials, literature reviews, randomized and non-randomized control trials, case-control and cohort studies, and systematic reviews and meta-analyses published between January 1, 2012, and November 27, 2022. The databases that were used to collect these articles included Pubmed, Pubmed Central, Cochrane Library, and Google Scholar. After applying appropriate filters, a total of 143 reports were identified from these four databases. They were further screened and subjected to quality assessment tools which finally yielded 21 studies that were included in this systematic review. This review provides strong evidence that the pharmacological properties and different mechanisms of action of LS give it an upper hand over other inotropic agents for its successful administration in patients with either acute or advanced cardiac failure, which consists of left as well as right ventricular failure, either individually or in combination.

Multifunctional Nutraceutical Composition Based on Fermented Spirulina, Apple Cider Vinegar, Jerusalem Artichoke, and Bovine Colostrum.

The main purpose of this experiment was to develop a multifunctional nutraceutical composition based on ingredients of different origins (Spirulina powder (SP), bovine colostrum (BC), Jerusalem artichoke powder (JAP), and apple cider vinegar (ACV)) which possess different health benefits through their different mechanisms of action. In order to improve the functional properties of Spirulina and bovine colostrum, fermentation with the Pediococcus acidilactici No. 29 and Lacticaseibacillus paracasei LUHS244 strains, respectively, was carried out. These LAB strains were chosen due to their good antimicrobial properties. The following parameters were analysed: for Spirulina (non-treated and fermented)-pH, colour coordinates, fatty acid profile, and contents of L-glutamic and GABA acids; for bovine colostrum (non-treated and fermented)-pH, colour coordinates, dry matter, and microbiological parameters (total LAB, total bacteria, total enterobacteria, Escherichia coli, and mould/yeast counts); for the produced nutraceuticals-hardness, colour coordinates, and overall acceptability. It was established that fermentation reduced the pH of the SP and BC and affected their colour coordinates. Fermented SP contained a greater concentration of gamma-aminobutyric and L-glutamic acids (by 5.2 times and 31.4% more, respectively), compared to the non-treated SP and BC. In addition, the presence of gamma-linolenic and omega-3 fatty acids was observed in fermented SP. Fermentation of BC reduces Escherichia coli, total bacteria, total enterobacteria, and total mould/yeast counts in samples. The obtained three-layer nutraceutical (I layer-fermented SP; II-fermented BC and JAP; III-ACV) demonstrated a high overall acceptability. Finally, our finding suggest that the selected nutraceutical combination has immense potential in the production of a multifunctional product with improved functionality and a high acceptability.

Treatment of Persistent Left Atrial Appendage Thrombus in Patients with Atrial Fibrillation on Adequate Oral Anticoagulation: Pathways of Care for All-comers and Heart Failure Patients.

In patients with AF, the presence of left atrial/left atrial appendage (LA/LAA) thrombus is related to an increased risk of thromboembolic events. Anticoagulation therapy, either with vitamin K antagonists or novel oral anticoagulants (NOACs) is therefore mandatory in AF with LA/LAA thrombus in order to lower the risk of stroke or other systemic embolic events. Despite the efficacy of these treatments, some patients will have persistent LAA thrombus remaining or may have contraindications to oral anticoagulation. Currently, little is known about the occurrence, risk factors and resolution rate of LA/LAA thrombus in patients who are already under optimal chronic oral anticoagulation, including vitamin K antagonists or NOACs. The common action in clinical practice in this scenario is switching from one to another anticoagulant drug exhibiting a different mechanism of action. Repeated cardiac imaging is then advised within several weeks to visually verify thrombus dissolution. Finally, there is a substantial scarcity of data on the role and optimal use of NOACs after LAA occlusion. The aim of this review is to critically evaluate data and provide up-to-date information on the best antithrombotic strategies in this challenging clinical scenario.

Combined In Vivo Microdialysis and PET Studies to Validate [11C]Yohimbine Binding as a Marker of Noradrenaline Release.

The noradrenaline system attracts attention for its role in mood disorders and neurodegenerative diseases but the lack of well-validated methods impairs our understanding when assessing its function and release in vivo. This study combines simultaneous positron emission tomography (PET) and microdialysis to explore if [11C]yohimbine, a selective antagonist radioligand of the α2 adrenoceptors, may be used to assess in vivo changes in synaptic noradrenaline during acute pharmacological challenges. Anesthetised Göttingen minipigs were positioned in a head holder in a PET/CT device. Microdialysis probes were placed in the thalamus, striatum and cortex and dialysis samples were collected every 10 min. Three 90 min [11C]yohimbine scans were acquired: at baseline and at two timepoints after the administration of amphetamine (1-10 mg/kg), a non-specific releaser of dopamine and noradrenaline, or nisoxetine (1 mg/kg), a specific noradrenaline transporter inhibitor. [11C]yohimbine volumes of distribution (VT) were obtained using the Logan kinetic model. Both challenges induced a significant decrease in yohimbine VT, with time courses reflecting their different mechanisms of action. Dialysis samples revealed a significant increase in noradrenaline extracellular concentrations after challenge and an inverse correlation with changes in yohimbine VT. These data suggest that [11C]yohimbine can be used to evaluate acute variations in synaptic noradrenaline concentrations after pharmacological challenges.

How to Promote Skin Repair? In-Depth Look at Pharmaceutical and Cosmetic Strategies.

Skin repair encompasses epidermal barrier repair and wound healing which involves multiple cellular and molecular stages. Therefore, many skin repair strategies have been proposed. In order to characterize the usage frequency of skin repair ingredients in cosmetics, medicines, and medical devices, commercialized in Portuguese pharmacies and parapharmacies, a comprehensive analysis of the products' composition was performed. A total of 120 cosmetic products, collected from national pharmacies online platforms, 21 topical medicines, and 46 medical devices, collected from INFARMED database, were included in the study, revealing the top 10 most used skin repair ingredients in these categories. A critical review regarding the effectiveness of the top ingredients was performed and an in-depth analysis focused on the top three skin repair ingredients pursued. Results demonstrated that top three most used cosmetic ingredients were metal salts and oxides (78.3%), vitamin E and its derivatives (54.2%), and Centella asiatica (L.) Urb. extract and actives (35.8%). Regarding medicines, metal salts and oxides were also the most used (47.4%) followed by vitamin B5 and derivatives (23.8%), and vitamin A and derivatives (26.3%). Silicones and derivatives were the most common skin repair ingredients in medical devices (33%), followed by petrolatum and derivatives (22%) and alginate (15%). This work provides an overview of the most used skin repair ingredients, highlighting their different mechanisms of action, aiming to provide an up-to-date tool to support health professionals' decisions.

Juvenile idiopathic arthritis – classification and methods of treatment

Introduction Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease of unknown etiology that affect children. According to the definition of JIA, the disease begins before the age of 16 and lasts more than 6 weeks. The International League of Associations for Rheumatology (ILAR) has divided juvenile idiopathic arthritis into seven categories: systemic, oligoarticular, polyarticular RF (-), polyarticular RF (+), psoriatic, enthesitis-related and undifferentiated arthritis. Purpose The aim of this review is to present the classification, etiopathogenesis, diagnosis, treatment and complications of juvenile idiopathic arthritis. Methods Literature searches in PubMed, Google Scholarship, and open source books were used to gather information. Results Complex interactions between cells of the immune system are responsible for the pathophysiology of JIA and indicate the need to divide the disease into clinical subtypes, the heterogeneity of which requires different therapeutic actions. There are many groups of drugs with different mechanisms of action used in the treatment of JIA, including: T lymphocyte inhibitors, anti-TNFα, JAK inhibitors, IL-1 and IL-6 blockers. Despite the great progress and the commitment of scientists, there is still no treatment strategy to completely stop the development of the disease. Conclusions Scientific research conducted around the world has led to the recognition of numerous pathways leading to the formation of the inflammatory process and the symptoms of JIA. Knowledge of these mechanisms allows scientists to conduct research on further drugs, the aim of which is to find a treatment strategy that prevents permanent joint damage, improves treatment results, and enables sustainable remission. It is necessary to expand knowledge about the pathways responsible for the formation of the inflammatory process, the interruption of which would allow complete inhibition of the development of the disease.

Strategies to Re-Sensitize Castration-Resistant Prostate Cancer to Antiandrogen Therapy

Since prostate cancer (PCa) was described as androgen-dependent, the androgen receptor (AR) has become the mainstay of its systemic treatment: androgen deprivation therapy (ADT). Although, through recent years, more potent drugs have been incorporated, this chronic AR signaling inhibition inevitably led the tumor to an incurable phase of castration resistance. However, in the castration-resistant status, PCa cells remain highly dependent on the AR signaling axis, and proof of it is that many men with castration-resistant prostate cancer (CRPC) still respond to newer-generation AR signaling inhibitors (ARSis). Nevertheless, this response is limited in time, and soon, the tumor develops adaptive mechanisms that make it again nonresponsive to these treatments. For this reason, researchers are focused on searching for new alternatives to control these nonresponsive tumors, such as: (1) drugs with a different mechanism of action, (2) combination therapies to boost synergies, and (3) agents or strategies to resensitize tumors to previously addressed targets. Taking advantage of the wide variety of mechanisms that promote persistent or reactivated AR signaling in CRPC, many drugs explore this last interesting behavior. In this article, we will review those strategies and drugs that are able to resensitize cancer cells to previously used treatments through the use of "hinge" treatments with the objective of obtaining an oncological benefit. Some examples are: bipolar androgen therapy (BAT) and drugs such as indomethacin, niclosamide, lapatinib, panobinostat, clomipramine, metformin, and antisense oligonucleotides. All of them have shown, in addition to an inhibitory effect on PCa, the rewarding ability to overcome acquired resistance to antiandrogenic agents in CRPC, resensitizing the tumor cells to previously used ARSis.

Abstract 2481: Human osteoclast assays for studying the effects of bone metastasis treatments with different mechanisms of action

Abstract Metastasis to bone is one of the common complications associated with different types of advanced cancer. Zoledronic acid, a third generation nitrogen containing bisphosphonate, accumulates in bones where it binds to hydroxyapatite crystals. For years, zoledronic acid has been regarded as the standard of care for patients with advanced cancer and bone metastasis to prevent skeletal complications. As an alternative therapeutic option, denosumab has also been found to be effective in delaying skeletal-related events in advanced cancer. Denosumab, a human monoclonal antibody against receptor activator of nuclear factor kappa-B ligand (RANKL), blocks the RANKL/RANK signalling pathway. In addition to the skeletal effects, both zoledronic acid and denosumab have shown direct effect on cancer cells. In this study, effects of zoledronic acid and denosumab on osteoclast differentiation and bone resorption activity were studied using human CD34+ osteoclast precursor cells that were cultured on bovine bone slices in 96-well plates for 7 days in the presence of RANKL and M-CSF, allowing them to differentiate into mature osteoclasts. In the osteoclast differentiation assay, zoledronic acid (0.0001 - 1 000 µM) and denosumab (0.01 - 10 µg/ml) were added into the culture medium at day 0. Tartrate-resistant acid phosphatase 5b (TRACP 5b) activity was measured in the culture medium at day 7 as an index of osteoclast differentiation. In the osteoclast activity assay, the test compounds were added into the culture medium at day 7, and the formed osteoclasts were allowed to resorb bone for 3 days. C-terminal crosslinked telopeptides of type I collagen (CTX-I) was measured in the culture medium at day 10 as an index of bone resorption. Denosumab and zoledronic acid showed strong concentration dependent inhibition of osteoclast differentiation and resorption activity. We conclude that this osteoclast culture system can be used for identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. The manifold effects of zoledronic acid and denosumab on cancer cells remain to be studied in more detail. Citation Format: Katja M. Fagerlund, Mervi Ristola, Mari I. Suominen, Jenni H. Mäki-Jouppila, Jukka P. Rissanen. Human osteoclast assays for studying the effects of bone metastasis treatments with different mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2481.

Abstract 6730: Preclinical testing of lestaurtinib (CEP701) as a single and combination agent for the treatment of Ewing sarcoma

Abstract Background: Multimodality treatment of Ewing sarcoma (EwS), a highly malignant bone and soft tissue tumor, provides a cure in most patients, but in the metastatic stage the prognosis remains bleak; new systemically effective therapeutic options are urgently needed. The multikinase inhibitor lestaurtinib (CEP701) is an orphan drug that has been clinically studied in various carcinomas, refractory neuroblastoma, and acute myeloid leukemia, but has not yet established itself as a standard therapy. Methods: We performed an in vitro screening of 142 compounds with different mechanisms of action at two different concentrations and measured cell viability 120 hours after the first of two days of treatment. Lestaurtinib was further tested as a lead compound in various cell biology assays in a group of EwS cell lines genetically representing clinically predominant tumor characteristics. Results: Lestaurtinib was among the 21 of 142 (14.8%) compounds tested that were sensitive, i.e., reduced cell viability to below 20% at 1 µM and/or 10 µM in at least four of five EwS cell lines tested. In an expanded group of six other EwS cell lines, 72 hours of treatment with lestaurtinib resulted in significantly reduced cell growth compared to human mesenchymal stem cells. In several colony formation assays, a significant reduction in the number or size of colonies was observed, both when treatment occurred prior to colony growth and when colonies that had already formed were treated. Lestaurtinib induced apoptosis with a significant increase in PI+/FTIC+-driven cells, significantly promoted anoikis, and prevented cell migration by FACS, low adherence flow cytometry analysis, and scratch assays, respectively. Cell cycle analyses by FACS showed significant G2-M cell cycle arrest after 24 hours of treatment. Lestaurtinib also displayed a synergistic cytotoxic effect with doxorubicin, an established agent for the treatment of EwS, and exhibited a favorable dose-reduction index in all EwS cell lines tested. Notably, treatment with lestaurtinib significantly reduced doxorubicin dose at high efficacy levels (Fa 0.97) and low combination index values. Conclusion: Our results highlight the in vitro efficacy of lestaurtinib in EwS and warrant further testing in an in vivomodel as a single agent and in combination with doxorubicin. Citation Format: Samet Dayi, Christiane Schaefer, Anne Albers, Kiruthiga Balusamy, Marc Hotfilder, Maximilian Kerkhoff, Leonhard Koch, Hatice Önder, Claudia Rössig, Uta Dirksen, Stefan K. Zöllner. Preclinical testing of lestaurtinib (CEP701) as a single and combination agent for the treatment of Ewing sarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6730.

Abstract 2694: Blockade of the cyclooxygenase-2 prevents chemotherapy-induced cognitive impairments

Abstract Chemotherapy-induced cognitive impairment (CICI, also termed “chemobrain”) is a major neurotoxic side effect exhibited by a wide range of chemotherapeutic agents. These side effects affect approximately 14 million cancer survivors. More importantly, CICI persists well after cessation of therapy, severely interfering with quality of life. Unfortunately, there is no treatment and novel therapies are urgently needed. Utilizing cisplatin, a platinum-based chemotherapy, to model chemobrain mice in vivo, we have established that cisplatin accelerates the brain aging process thus leading to long-term memory impairment in mice similar to what is clinically reported. We further show that cisplatin causes oxidative DNA damage, mitochondrial defects, impaired neurogenesis, synaptic defects, and increased gliosis in the adult hippocampus, a brain region critical for learning and memory. Mechanistically, our RNA-sequencing and qRT-PCR analysis revealed that cisplatin dramatically increases COX-2 (Ptgs2) expression and its major product, prostaglandin E2 (PGE2) in adult mouse brain and human cortical neurons derived from induced pluripotent stem cells (iPSCs). Similar as cisplatin treatment, the levels of COX-2 expression began to increase at 0.1 µM methotrexate treatment, which significantly increased at 1 µM in human cortical neurons, indicating that COX-2 induction is a common pathogenic mechanism mediating cognitive impairment associated with cisplatin and methotrexate in spite of the fact that these compounds have different mechanisms of action for cancer eradication. Most importantly, NS-398, a selective COX-2 inhibitor, effectively prevents cognitive deficits in mice and reduction in cell viability of human neurons associated with these chemotherapies, without promoting tumor growth or interfering with cisplatin’s anti-tumor activity. Taken together, our findings strongly suggest that COX-2 induction is a main cause of CICI, making COX-2 inhibition a therapeutic target for CICI. Given that COX-2 inhibitors are currently being tested in cancer prevention as well as age-related memory loss in clinical trials, inhibiting COX-2 may have far-reaching therapeutic effects on CICI and cancer treatment, which can be applied quickly, safely, and effectively to clinical trials, in the effort to improve cancer survivor quality of life. Citation Format: Mohammad Abdur Rashid, Jason J. Tang, Ki-Hyun Yoo, Ana Corujo-Ramirez, Sang Hoon Kim, Alfredo Oliveros, Peter Cole, John R. Hawse, Mi-Hyeon Jang. Blockade of the cyclooxygenase-2 prevents chemotherapy-induced cognitive impairments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2694.

Abstract 151: Optimization of screening conditions using patient-derived organoids for diagnostic purposes

Abstract Patient-derived organoids (PDOs) are increasingly adopted in preclinical research. PDOs, or HUB Organoids®, are directly established from patient tissue, faithfully recapitulate patient disease, and effectively bridge the gap between the lab and the clinic, bringing a “patient in the lab®”. PDOs can also revolutionize clinical research because they can predict patient response to standard-of-care (SoC) compounds. However, there is an urgent need to refine the assay conditions to evaluate the activity of SoC drugs with different mechanisms of action (topoisomerase inhibitors, alkylating agents, targeted agents) and various types of molecules (small molecules, antibodies) and achieve optimal results from the PDO-based assay. Specifically, for metastatic colorectal cancer (mCRC), we have optimized the media composition required for drug screening assays to test response to SoC, such as oxaliplatin and panitumumab. Our results indicate that optimal media conditions are essential to observe an in vitro drug response in PDOs similar to the patient response in the clinic. In addition, an image-based readout was developed that enables precise quantification of organoid numbers ensuring high assay quality. Optimizing screening conditions to evaluate SoC compounds in PDOs is a required step to accelerate the implementation of the HUB Organoid Technology as a diagnostic platform to accurately predict treatment response and improve clinical outcomes for cancer patients. Citation Format: Carla S. Verissimo, Lidwien Smabers, Emerens Wensink, Esmee Koedoot, Katerina-Chara Pitsa, Mayke Doorn, Inez van Weersch, Hamdy Warda, Celia Higuera Barón, Gerben ten Hag, Robert G. Vries, Jeanine Roodhart, Sylvia F. Boj. Optimization of screening conditions using patient-derived organoids for diagnostic purposes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 151.

Abstract 2526: The Bcl-2 family proteins in CLL: Effects by the cytogenetics, microenvironments and therapy

Abstract The balance between the pro- and anti-apoptotic members of the Bcl-2 family proteins governs the fate of CLL cells and determines their response to therapeutics. Thus, we have developed an assay to quantify the Bcl-2 family proteins at the molecular level and express them as molecules/pg total protein. This set of proteins represents the 10 major Bcl-2 members in CLL: Bcl-2, Bcl-XL, Mcl-1, BIM, BID, BMF, BAX, BAK, PUMA and Noxa. This analysis enabled us to evaluation of the interplay of these proteins and how they respond to cytogenetic aberrations, drug treatment or environmental changes. In 36 CLL samples that represent 6 districted cytogenetic features: normal cytogenetics, del17p, del11q, del13q14, 11q/13q double deletion, and trisomy 12, we found that the major anti-apoptotic proteins in CLL is Bcl-2, that averaged >9,000 molecules/pg of total protein. Mcl-1 was expressed at less than one tenth of Bcl-2 and Bcl-XL was expressed less than one percent of Bcl-2 molecules. The major pro-apoptotic proteins were Bax and Puma which were expressed at similar levels to Bcl-2. Among the cytogenetic groups, there were significant increases of Bcl-2 in the 11q/13q group whereas Mcl-1 was increased in 11q and trisomy 12 groups compared to samples with normal cytogenetic characteristics. Next, we screened CLL response to different microenvironments by incubating primary CLL cells in vitro in media supplemented with either fetal bovine serum (FBS), patient autologous plasma, a layer of StromaNKtert cells representing the stroma cell support in the bone marrow, a B-cell activating media (BCA) that consisted of anti-CD40, IL-4 and anti-IgM to mimic the lymphoid microenvironment, or oligodeoxynucleotide (ODN) plus IL-15 to represent the T-cell independent stimulation in the lymph nodes. Compared to FBS, homologous patient plasma moderately promoted CLL viability. However, stroma cells, BCA media as well ODN+IL-15 greatly induced levels of Mcl-1 and Bcl-XL and were more effective in promoting CLL survival. For the pro-apoptotic proteins, BMF was induced by BCA media, and PUMA, NOXA and BID were greatly induced by ODN+IL-15. We also examined the changes in Bcl-2 family proteins in response to therapeutic agents with different mechanisms of action, such as the CDK9 inhibitor fadraciclib, the Bcl-2 antagonist venetoclax, the Mcl-1 inhibitors AZD5991 and S63845, the proteasome inhibitor carfilzomib, the BTK inhibitor ibrutinib and the splicing inhibitor herboxidiene. Fadraciclib and herboxidiene were effective in reducing Mcl-1 to induce apoptosis. While both the Mcl-1 inhibitors and carfilzomib increased the Mcl-1 level, and carfilzomib increased Noxa that counteracted its effect on Mcl-1. Puma was reduced by most of the drugs except ibrutinib. Thus, the systematic analysis of the Bcl-2 family proteins provide insights on how the interplay of members of this important family affects the destiny of the CLL cells. Citation Format: Rong Chen, Ping Xiong, Chaomei Liu, Yuling Chen, Yingjun Jiang, Varsha Gandhi, Nitin Jain, William Wierda, William Plunkett. The Bcl-2 family proteins in CLL: Effects by the cytogenetics, microenvironments and therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2526.

Abstract 1103: Theoretical basis and formula for tumor-treating fields dose-response curves

Abstract Radiotherapy dose-response curves are linear-quadratic (LQ), chemotherapy’s are generally semi-logarithmic or sigmoidal, and Tumor-Treating Fields’ (TTFields) resemble radiotherapy LQ, while the two modalities are presumed to have fundamentally different mechanisms of action (MoA) [1,2]. Radiotherapy produces single- or double-strand DNA breaks, while TTFields act on polarizable cellular structures. Thus, changing the incident electric field direction improves TTFields efficacy, killing 20% more tumor cells in vitro, with 90⁰ changes in the front-back/left-right (x,y) plane [1]. However, by the same logic, polarizable cell structures that are orthogonal to the (x,y) plane see 0 field strength and thus are unaffected by TTFields. The formula for TTFields effective dose with no direction change, given TTFields’ incident angle on the polarizable cell structure’s axis, is simply Deff = cos θ*Dnom, where Dnom is the electric field strength at the axis. The formulae for TTFields dose-response, given one, two, and three incident directions can be calculated (Mathematica v.12, WRI, Champaign, IL) as n(Dnom|cosθ1|≥k) + n(Dnom|cosθ2|≥k) + n(Dnom|cosθ3|≥k)/N, where n is the counting function, Dnom is the nominal field strength at the axis, θi the 1st, 2nd, or 3rd incident TTFields angle, k is TTFields' efficacy floor in V/cm (see below), and N is the total cell population in vivo, vitro, or silico. The formula assumes: 1) Cells are randomly-oriented in vivo [1]; 2) Polarized cell structures are straight; 3) Electric field strength is Deff not Dnom, 4) Cells are in their radiosensitive state (e.g. mitosis), 5) Reducing TTFields duty cycle/direction has no effect down to 0.25 s ([1], and unpublished data), and 6) TTFields’ minimum efficacious field strength Deff = ~0.75 V/cm for cell structures perfectly aligned with the incident TTFields angle (cf. low dose V/cm data points in MDA-MB-231, F-98, B16F1, and H1299 empirical dose-response curves [1,2]). With feasible TTFields transducer array placements on the head or torso, it is relatively easy to achieve changes of direction in one (x,y) plane, but difficult to achieve an additional change of direction in the z-plane. For a nominal TTFields dose Dnom of 2 V/cm, only a 22⁰ change of direction off the (x,y) plane is necessary to kill the cells with Deff below the efficacy threshold, with Dnom = 1.5 V/cm, a 30⁰ change is needed, and with Dnom = 1 V/cm, a 49⁰ change is needed. In sum, we have derived TTFields’ dose-response formulae from first principles, which differentiates them from those of radiotherapy and chemotherapy, and, toward achieving 100% TTFields efficacy as a monotherapy, given motivation and guidelines for adding an additional TTFields’ change of direction per duty cycle.1.Kirson, E.D., et al., Proc Natl Acad Sci U S A, 2007.2.Moser, J.C., et al.,.Cancer Res, 2022. Citation Format: Kristen Carlson. Theoretical basis and formula for tumor-treating fields dose-response curves [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1103.