3086 Background: Epidermal growth factor receptor (EGFR) is a clinically validated target and is highly expressed in various solid tumors, including triple-negative breast cancer (TNBC). Clinical efficacy of EGFR-directed therapies in TNBC, however, is often limited by intratumoral heterogeneity, acquired resistance, and mutations conferring insensitivity to tyrosine kinase inhibitors. TSY-310 is a novel bispecific EGFR×ROR1 nanobody-Fc fusion antibody–drug conjugate (BsADC) designed to address these challenges by simultaneously targeting EGFR and receptor tyrosine kinase-like orphan receptor 1 (ROR1). TSY-310 carries an average of 3.6 molecules of monomethyl auristatin E (MMAE) via a protease-cleavable valine–citrulline linker, enabling potent and tumor-selective cytotoxicity, and induces strong tumor regression across multiple NSCLC PDX models representing a range of EGFR and ROR1 expression levels. Methods: Cytotoxicity was evaluated in cell lines with distinct EGFR/ROR1 expression profiles. Bystander killing was assessed in co-culture systems of antigen-positive and antigen-negative cells. Cellular internalization and trafficking were analyzed by immunofluorescence microscopy. In vivo efficacy and bystander activity are investigated in xenograft models with heterogeneous antigen expression. Results: TSY-310 exhibited sub-nanomolar cytotoxicity (IC 50 = 0.45 nM) against MDA-MB-468 (ROR1⁺/EGFR⁺) TNBC cells in correlation with dual receptor expression levels, and demonstrated robust bystander killing, eliminating neighboring antigen-negative A427-Luc and MDA-MB-453-Luc cells through MMAE diffusion in co-culture systems. Rapid internalization of TSY-310 was observed in dual-positive MDA-MB-468 cells within 6 hours, consistent with enhanced cytotoxic activity. In a EGFR⁺/ROR1⁺ HBCx-28 TNBC PDX model, it achieved complete and sustained tumor regressions in all treated mice (5/5) and demonstrated superior efficacy compared with the monospecific ROR1-directed ADC on a payload-equivalent basis. In vivo studies to assess bystander activity and efficacy in heterogeneous tumor models are ongoing. Conclusions: These findings highlight TSY-310 as a first-in-class bispecific ADC capable of overcoming tumor heterogeneity through dual binding and bystander killing, supporting its potential as an effective therapeutic agent for heterogeneous solid tumors such as TNBC, where existing EGFR-targeted therapies have shown limited benefit.

Abstract Mutations in KRAS represent the most common oncogenic event in human cancer and occur in approximately 30% of lung adenocarcinomas. The mechanisms by which lung tumours evade apoptosis induced by oncogenic KRAS-driven stress remain incompletely understood. Here, we identify the anti-apoptotic regulator FLIP ( CFLAR ) as a critical dependency in KRAS-mutant lung cancers. We demonstrate that KRAS-mutant human lung cancer cell lines exhibit elevated FLIP expression and enhanced dependence on FLIP for survival compared to KRAS wild-type counterparts. Subsequently, using genetically engineered mouse models (GEMMs), we show that FLIP is essential for Kras -driven lung tumour development in vivo . In vitro, FLIP-deficient lung cancer cells display spontaneous, caspase-8- dependent apoptosis and hyper-sensitivity to the immune/inflammatory cytokines TNFα and TRAIL. Strikingly, FLIP-null lung cancer cells fail to engraft even in highly immunodeficient orthotopic models that lack TRAIL-expressing immune cells but retain TNFα-expressing monocytes. Moreover, silencing of TNFR1 or TNFα but not TRAIL-R2 rescued constitutive caspase-8-dependent apoptosis in FLIP null lung cancer cells, implicating TNFα/TNFR1 in mediating this apoptotic response. Mechanistically, we find that mutant KRAS sustains FLIP expression via ERK1/2 signalling, thereby protecting cells from caspase-8 activation. Notably, KRAS inhibition downregulates FLIP, sensitising cells to TNFα- and TRAIL-induced apoptosis. These findings uncover a novel KRAS–ERK–FLIP axis that protects tumour cells from caspase-8-mediated apoptosis and reveal FLIP as a key survival factor co-opted by KRAS -mutant lung cancers. Beyond identifying FLIP as a promising therapeutic target in KRAS mutant lung cancer, our work also provides mechanistic insight into the pro-apoptotic effects of KRAS inhibitors and suggests that FLIP expression may serve as a predictive biomarker to enhance patient stratification and the therapeutic efficacy of these agents in lung cancer.

The thiol-ene reaction between an alkene and a thiol can be exploited for selective labelling of cysteine residues in protein profiling applications. Here, we explore thiol-ene activation in systems from chemical models to complex cellular milieus, using UV, visible wavelength and redox initiators. Initial studies in chemical models required an oxygen-free environment for efficient coupling and showed very poor activation when using a redox initiator. When thiol-ene activation was performed in protein and cell lysate models, all three initiation methods were successful. Faster thiol-ene reaction was observed as the cysteine and alkene were brought into proximity by a binding event prior to activation, leading to quicker adduct formation in the protein model system than the chemical models. Furthermore, in the protein-protein coupling, none of the activators required an oxygen-free environment. Taken together, these observations demonstrate the broad potential for thiol-ene coupling to be used in protein profiling.

Ubiquitination influences a myriad of biological processes, such as the trafficking or degradation of ubiquitin-tagged target proteins. This posttranslational modification can be reversed by deubiquitinating enzymes (DUBs) that counterbalance the action of E3 ubiquitin ligases. We investigated the impact of PR-619, a membrane-permeable and broad-spectrum DUB inhibitor, on the entry of Ca2+ through native voltage-gated Ca2+ channels (VGCCs) of cultured embryonic cortical neurons. Fura-2-based Ca2+ imaging experiments showed that PR-619 reduced the cytosolic Ca2+ rises induced by depolarization by affecting mainly dihydropyridine-sensitive (L-type) VGCCs. This inhibition was sensitive to dynamin inhibitor Myr-Dip and lysosomal agents chloroquine and bafilomycin-A. PR-619 also reduced the amount of Cav1.2 proteins. A pharmacological approach was set out to better delineate the identity of the DUB responsible for this inhibitory action of PR-619. Since UCH-L1 and USP19 are two highly expressed neuronal DUBs, we investigated the effects of selective UCH-L1 (IMP1710, GK13S) and USP19 (ADC141) inhibitors. IMP1710 and GK13S depressed the Ca2+ uptake through L-type VGCCs, whereas ADC141 and the UCH-L3 inhibitor TCID had no effects. In addition, UCH-L1 inhibition impaired the neuronal Ca2+ storage capacities of neurons and reduced the Cav1.2 protein levels. Thus, UCH-L1 influences the neuronal uptake and storage of Ca2+, which is likely to have important physiological implications. Altogether, these results posit UCH-L1, the main DUB of the brain, as an important regulator of neuronal Ca2+ homeostasis and add to our understanding of its cellular functions.

The incidence of oesophageal adenocarcinoma (OAC) has risen six-fold in western countries over the last forty years but survival rates have only marginally improved. Hyperactivation of the PI3K-AKT-mTOR pathway is a common occurrence in OAC, driving cell survival, proliferation and resistance to chemotherapeutic agents. Inhibition of AKT has been explored as a treatment strategy with limited success and current inhibitors have failed to progress through clinical trials. Our study, describes a novel allosteric AKT inhibitor, ALM301, and demonstrates an enhancement of the efficacy of conventional chemotherapy when combined with ALM301 in OAC. Reduced sensitivity to ALM301 is associated with high expression of the Inhibitor of Apoptosis (IAP) family of proteins, particularly XIAP. Combined AKT and IAP inhibition synergistically enhanced OAC cell death and successfully re-sensitized ALM301 and chemotherapy resistant cell lines. A high degree of synergism was also observed in patient-derived OAC organoids indicating the potential clinical relevance of the combination. This study demonstrates the role for dual AKT/IAP inhibition in OAC and provides a strong rationale for the further investigation of this highly efficacious combination strategy.

BACKGROUND:Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts.METHODS:To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs).RESULTS:Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival.CONCLUSIONS:USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).

Background: Globally, ∼5% of patients have severe asthma. Despite new treatments, many remain symptomatic (ACQ>1.5) with low levels of Type-2 inflammation (T2-low: FeNO<20ppb & Blood Eosinophil count [BEC]<0.15x109cells/L). Aims: To profile urinary eicosanoids in "T2-low" severe asthma. Methods: Urine samples were analysed, from T2-low patients, by liquid-chromatography/mass-spectrometry, during a multi-centre, 48-week RCT enabling corticosteroid optimisation. Results: Urinary concentrations of isoprostanes, thromboxane and PGD2 metabolites are elevated in "symptom-high" [SH: uncontrolled] v "symptom-low" [SL: controlled], T2-low patients (Table 1) and are associated with a reduced FEV1 (71.7% v 88.5%, P<0.0001). After adjusting for obesity, thromboxane 2,3-dinor-TXB2 (0.32 v 0.16ng/mL, P=0.0337) and isoprostane 8,12-iso-iPF2a-VI (3.73 v 2.10ng/mL, P=0.0234) remain elevated in "SH" v "SL", T2-low patients and are associated with a reduced FEV1(88.0% v 70.6%, P=0.002). Conclusion: Elevated urinary eicosanoids in "SH/T2-low" patients are in part due to obesity, however, some thromboxane and prostaglandin metabolites are independently associated with persistent symptoms. Table 1: Lung-function, eicosanoids & biomarkers in "SH" v "SL", T2-low patients *P <0.05; **P <0.0001

Cancer cachexia is a metabolic wasting syndrome characterized by weight loss, anorexia and anemia as a result of tumor burden, and affects up to 80% of advanced cancer patients #1. Cachexia is particularly prevalent in pancreatic, lung, colorectal and gastro-intestinal cancers and can lead to reduced tolerance and responsiveness to chemotherapy, increased treatment-related toxicity and morbidity, and poor overall quality of life. There are currently no approved therapies for cancer cachexia. The development and maintenance of muscle tissue is dependent on the balance between protein synthesis and protein degradation, controlled through various anabolic and catabolic signaling pathways. Dysregulation of these pathways can result in muscle atrophy, which arises in many chronic illnesses. The ubiquitin proteasome system (UPS) has a central role in regulating skeletal muscle physiology. Previous work utilizing USP19 knock out mouse models has demonstrated that USP19 plays an important role in muscle wasting and can protect against denervation-induced muscle atrophy #2. We have previously demonstrated that inhibition of USP19 enzymatic activity spares the muscle wasting observed in limb-casted and denervated mouse models of muscle wasting. Here, we report the discovery of a novel, highly potent and selective inhibitor of USP19 (ADC-846) and demonstrate its utility in a cancer-induced muscle atrophy model in vivo. Pharmacological inhibition of USP19 by ADC-846 increased lean muscle and fat mass following oral dosing in a Lewis Lung Carcinoma-induced cachexia model and reduced the cachexic index by >60% compared to controls. This data, in combination with our previous work detailing the effect of USP19 inhibition on muscle force and function, provides a much-needed novel pharmacological strategy for therapeutic intervention in muscle wasting conditions. Citation Format: Natalie Page, Vignesh Karthikaisamy, Darren O'Hara, Aaron N. Cranston, Colin R. O'Dowd, Xavier Jacq, Richard Wilkinson, Stephanie Burton, Hayley Gibson, Joana Costa, Daniel Longley, Matthew Helm, Chris McGivern, Steven Shepherd, Christina Bell, Peter Hewitt, Mary McFarland, Hugues Miel, Steven Whitehead, Lauren Proctor, Shane J. Rountree, Mark Wappett, Mauricio Berriel Diaz, Stephan Herzig, Timothy Harrison. A novel first-in-class USP19 inhibitor for the treatment of cancer-induced muscle atrophy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB022.

Response of H460 and A549 cells to IR

Combination of SAHA with IR in H460 and A549 cells using different treatment schedules

IR-induced changes in cell surface expression of death receptors

Caspase-8 dependent apoptosis induced by IR

Rationale: The past 25 years have seen huge progress in understanding of the pathobiology of type-2 (T2) asthma, identification of measurable biomarkers, and the emergence of novel monoclonal antibody treatments. Although present in a minority of patients with severe asthma, very little is known about the mechanisms underlying T2-low asthma, making it a significant unmet need in asthma research. Objectives: The objective of this study was to explore the differences between study exacerbators and nonexacerbators, to describe physiological changes at exacerbation in those who are T2HIGH and T2LOW at the time of exacerbation, and to evaluate the stability of inflammatory phenotypes when stable and at exacerbation. Methods: Exacerbation assessment was a prespecified secondary analysis of data from a 48-week, multicenter, randomized controlled clinical study comparing the use of biomarkers and symptoms to adjust steroid treatment in a T2-low severe asthma-enriched cohort. Participants were phenotyped as T2LOW (fractional exhaled nitric oxide ⩽ 20 ppb and blood eosinophil count ⩽ 150 cells/µl) or T2HIGH (fractional exhaled nitric oxide > 20 or blood eosinophil count > 150) at study enrollment and at each exacerbation. Here, we report the findings of the exacerbation analyses, including comparison of exacerbators and nonexacerbators, the physiological changes at exacerbation in those who had evidence of T2 biology at exacerbation versus those that did not, and the stability of inflammatory phenotypes when stable and at exacerbation. Measurements and Main Results: Of the 301 participants, 60.8% (183) had one or more self-reported exacerbations (total of 390). Exacerbators were more likely to be female, have a higher body mass index, and have more exacerbations requiring oral corticosteroid and unscheduled primary care attendances for exacerbations. At enrollment, 23.6% (71) were T2LOW and 76.4% (230) T2HIGH. The T2LOW group had more asthma primary care attendances, were more likely to have a previous admission to HDU (high dependency unit)/ICU and to be receiving maintenance oral corticosteroids. At exacerbation, the T2LOW events were indistinguishable from T2HIGH exacerbations in terms of lung function (mean fall in T2LOW FEV1, 200 [400] ml vs. T2HIGH 200 [300] ml; P = 0.93) and symptom increase (ACQ5: T2LOW, 1.4 [0.8] vs. T2HIGH, 1.3 [0.8]; P = 0.72), with no increase in T2 biomarkers from stable to exacerbation state in the T2LOW exacerbations. The inflammatory phenotype within individual patients was dynamic; inflammatory phenotype at study entry did not have a significant association with exacerbation phenotype. Conclusions: Asthma exacerbations demonstrating a T2LOW phenotype were physiologically and symptomatically similar to T2HIGH exacerbations. T2LOW asthma was an unstable phenotype, suggesting that exacerbation phenotyping should occur at the time of exacerbation. The clinically significant exacerbations in participants without evidence of T2 biology at the time of exacerbation highlight the unmet and pressing need to further understand the mechanisms at play in non-T2 asthma. Clinical trial registered with www.clinicaltrials.gov (NCT02717689).

The serine/threonine protein kinase AKT plays a pivotal role within the PI3K pathway in regulating cellular proliferation and apoptotic cellular functions, and AKT hyper-activation via gene amplification and/or mutation has been implicated in multiple human malignancies. There are 3 AKT isoenzymes (AKT1-3) which mediate critical, non-redundant functions. We present the discovery and development of ALM301, a novel, allosteric, sub-type selective inhibitor of AKT1/2. ALM301 binds in an allosteric pocket created by the combined movement of the PH domain and the catalytic domain, resulting in a DFG out conformation. ALM301 was shown to be highly selective against a panel of over 450 kinases and potently inhibited cellular proliferation. These effects were particularly pronounced in MCF-7 cells containing a PI3KCA mutation. Subsequent cellular downstream pathway analysis in this sensitive cell line revealed potent inhibition of pAKT signalling up to 48 h post dosing. ALM301 treatment was well tolerated in an MCF-7 xenograft model and led to a dose-dependent reduction in tumour growth. Enhanced efficacy was observed in combination with tamoxifen. In summary, ALM301 is a highly specific AKT 1/2 inhibitor with an excellent pharmacological profile suitable for further clinical development.

BACKGROUND: Approximately 5% to 10% of patients with asthma have severe disease, with a consistent preponderance in females. Current asthma guidelines recommend stepwise treatment to achieve symptom control with no differential treatment considerations for either sex.OBJECTIVE: To examine whether patient sex affects outcomes when using a composite T2-biomarker score to adjust cortico-steroid (CS) treatment in patients with severe asthma compared with standard care.METHODS: This is a post hoc analysis, stratifying patient outcomes by sex, of a 48-week, multicenter, randomized controlled clinical trial comparing a biomarker-defined treat-ment algorithm with standard care. The primary outcome was the proportion of patients with a reduction in CS treatment (inhaled and oral corticosteroids). Secondary outcomes included exacerbation rates, hospital admissions, and lung function.RESULTS: Of the 301 patients randomized, 194 (64.5%) were females and 107 (35.5%) were males. The biomarker algorithm led to a greater proportion of females being on a lower CS dose versus standard care, which was not seen in males (effect estimate: females, 3.57; 95% CI, 1.14-11.18 vs males, 0.54; 95% CI, 0.16-1.80). In T2-biomarkerelow females, reducing CS dose was not associated with increased exacerbations. Females scored higher in all domains of the 7-item Asthma Control Question-naire, apart from FEV1, but with no difference when adjusted for body mass index/anxiety and/or depression. Dissociation be-tween symptoms and T2 biomarkers were noted in both sexes, with a higher proportion of females being symptom high/T2-biomarker low (22.8% vs 15.6%; P [ .0002), whereas males were symptom low/T2-biomarker high (22.3% vs 11.4%; P < .0001).CONCLUSIONS: This exploratory post hoc analysis identified that females achieved a greater benefit from biomarker-directed CS optimization versus symptom-directed treatment. (c) 2023 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). (J Allergy Clin Immunol Pract 2023;11:1233-42)

ADVERTISEMENT RETURN TO BOOKPREVChapterNEXTDEUBIQUITINASES AND THEIR INHIBITORS: CHALLENGES AND RECENT ADVANCESPeter R. HewittPeter R. HewittAlmac Discovery, Belfast, U.K.More by Peter R. Hewitt, Oliver BarkerOliver BarkerAlmac Discovery, Belfast, U.K.More by Oliver Barker, Matthew D. HelmMatthew D. HelmAlmac Discovery, Belfast, U.K.More by Matthew D. Helm, Xavier JacqXavier JacqAlmac Discovery, Belfast, U.K.More by Xavier Jacq, Colin R. O'DowdColin R. O'DowdAlmac Discovery, Belfast, U.K.More by Colin R. O'Dowd, Natalie PageNatalie PageAlmac Discovery, Belfast, U.K.More by Natalie Page, J. S. Shane RountreeJ. S. Shane RountreeAlmac Discovery, Belfast, U.K.More by J. S. Shane Rountree, and Timothy HarrisonTimothy HarrisonAlmac Discovery, Belfast, U.K.More by Timothy HarrisonDOI: 10.1021/mc-2022-vol57.ch15Publication Date (Web):December 21, 2022Publication History Published online21 December 2022RIGHTS & PERMISSIONS2022 Medicinal Chemistry ReviewsChapter 15pp 363-387Medicinal Chemistry ReviewsVol. 57ISBN13: 9781734427455eISBN: 9781734427455 Copyright © 2022 MEDI, Inc. Published by American Chemical Society.Article Views210Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (777 KB) Get e-Alerts

Background Oral corticosteroid (OCS) dependence among patients with severe eosinophilic asthma can cause adverse outcomes, including adrenal insufficiency. PONENTE's OCS reduction phase showed that, following benralizumab initiation, 91.5% of patients eliminated corticosteroids or achieved a final dosage ≤5 mg·day−1 (median (range) 0.0 (0.0–40.0) mg). Methods The maintenance phase assessed the durability of corticosteroid reduction and further adrenal function recovery. For ∼6 months, patients continued benralizumab 30 mg every 8 weeks without corticosteroids or with the final dosage achieved during the reduction phase. Investigators could prescribe corticosteroids for asthma exacerbations or increase daily dosages for asthma control deteriorations. Outcomes included changes in daily OCS dosage, Asthma Control Questionnaire (ACQ)-6 and St George's Respiratory Questionnaire (SGRQ), as well as adrenal status, asthma exacerbations and adverse events. Results 598 patients entered PONENTE; 563 (94.1%) completed the reduction phase and entered the maintenance phase. From the end of reduction to the end of maintenance, the median (range) OCS dosage was unchanged (0.0 (0.0–40.0) mg), 3.2% (n=18/563) of patients experienced daily dosage increases, the mean ACQ-6 score decreased from 1.26 to 1.18 and 84.5% (n=476/563) of patients were exacerbation free. The mean SGRQ improvement (–19.65 points) from baseline to the end of maintenance indicated substantial quality-of-life improvements. Of patients entering the maintenance phase with adrenal insufficiency, 32.4% (n=104/321) demonstrated an improvement in adrenal function. Adverse events were consistent with previous reports. Conclusions Most patients successfully maintained maximal OCS reduction while achieving improved asthma control with few exacerbations and maintaining or recovering adrenal function. In the ∼6-month PONENTE maintenance phase, benralizumab-treated patients sustained long-term oral corticosteroid elimination or reduction without loss of asthma control. Improved adrenal function was observed in many patients following steroid reduction. https://bit.ly/3PjOEnG