Cell-based Functional Assays Research Articles

Site-specific sulfations regulate the physicochemical properties of papillomavirus-heparan sulfate interactions for entry.

Certain human papillomaviruses (HPVs) are etiological agents for several anogenital and oropharyngeal cancers. During initial infection, HPV16, the most prevalent cancer-causing type, specifically interacts with heparan sulfates (HSs), not only enabling initial cell attachment but also triggering a crucial conformational change in viral capsids termed structural activation. It is unknown, whether these HPV16-HS interactions depend on HS sulfation patterns. Thus, we probed potential roles of HS sulfations using cell-based functional and physicochemical assays, including single-molecule force spectroscopy. Our results demonstrate that N-sulfation of HS is crucial for virus binding and structural activation by providing high-affinity sites, and that additional 6O-sulfation is required to mechanically stabilize the interaction, whereas 2O-sulfation and 3O-sulfation are mostly dispensable. Together, our findings identify the contribution of HS sulfation patterns to HPV16 binding and structural activation and reveal how distinct sulfation groups of HS synergize to facilitate HPV16 entry, which, in turn, likely influences the tropism of HPVs.

Discovery of GNE-6893, a Potent, Selective, Orally Bioavailable Small Molecule Inhibitor of HPK1.

Hematopoietic progenitor kinase 1 (HPK1) serves a key immunosuppressive role as a negative regulator of T-cell receptor (TCR) signaling. HPK1 loss-of-function is associated with augmentation of immune function and has demonstrated synergy with immune checkpoint inhibitors in syngeneic mouse cancer models. These data offer compelling evidence for the use of selective small molecule inhibitors of HPK1 in cancer immunotherapy. We identified a novel series of isoquinoline HPK1 inhibitors through fragment-based screening that displayed promising levels of biochemical potency and activity in functional cell-based assays. We used structure-based drug design to introduce key selectivity elements while simultaneously addressing pharmacokinetic liabilities. These efforts culminated in a molecule demonstrating subnanomolar biochemical inhibition of HPK1 and strong in vitro augmentation of TCR signaling in primary human T-cells. Further profiling of this molecule revealed excellent kinase selectivity (347/356 kinases <50% inhibition @ 0.1 μM), a favorable in vitro safety profile, and good projected human pharmacokinetics.

Novel potent molecular glue degraders against broad range of hematological cancer cell lines via multiple neosubstrates degradation

BackgroundTargeted protein degradation of neosubstrates plays a crucial role in hematological cancer treatment involving immunomodulatory imide drugs (IMiDs) therapy. Nevertheless, the persistence of inevitable drug resistance and hematological toxicities represents a significant obstacle to their clinical effectiveness.MethodsPhenotypic profiling of a small molecule compounds library in multiple hematological cancer cell lines was conducted to screen for hit degraders. Molecular dynamic-based rational design and cell-based functional assays were conducted to develop more potent degraders. Multiple myeloma (MM) tumor xenograft models were employed to investigate the antitumor efficacy of the degraders as single or combined agents with standard of care agents. Unbiased proteomics was employed to identify multiple therapeutically relevant neosubstrates targeted by the degraders. MM patient-derived cell lines (PDCs) and a panel of solid cancer cell lines were utilized to investigate the effects of candidate degrader on different stage of MM cells and solid malignancies. Unbiased proteomics of IMiDs-resistant MM cells, cell-based functional assays and RT-PCR analysis of clinical MM specimens were utilized to explore the role of BRD9 associated with IMiDs resistance and MM progression.ResultsWe identified a novel cereblon (CRBN)-dependent lead degrader with phthalazinone scaffold, MGD-4, which induced the degradation of Ikaros proteins. We further developed a novel potent candidate, MGD-28, significantly inhibited the growth of hematological cancer cells and induced the degradation of IKZF1/2/3 and CK1α with nanomolar potency via a Cullin-CRBN dependent pathway. Oral administration of MGD-4 and MGD-28 effectively inhibited MM tumor growth and exhibited significant synergistic effects with standard of care agents. MGD-28 exhibited preferentially profound cytotoxicity towards MM PDCs at different disease stages and broad antiproliferative activity in multiple solid malignancies. BRD9 modulated IMiDs resistance, and the expression of BRD9 was significant positively correlated with IKZF1/2/3 and CK1α in MM specimens at different stages. We also observed pronounced synergetic efficacy between the BRD9 inhibitor and MGD-28 for MM treatment.ConclusionsOur findings present a strategy for the multi-targeted degradation of Ikaros proteins and CK1α against hematological cancers, which may be expanded to additional targets and indications. This strategy may enhance efficacy treatment against multiple hematological cancers and solid tumors.

Functional expression and characterization of CAPA receptor in the digestive tract and life stages of Drosophila suzukii, and differential activities with insect PRXamide peptides.

Spotted-wing drosophila, Drosophila suzukii (Matsumura), is an invasive vinegar fly that is a major threat to the small fruits industries globally. Insect capa genes encode multiple neuropeptides, including CAPA-periviscerokinin (CAPA-PVK) peptides, that are specifically known to cause diuresis or anti-diuresis in various organisms. Here we identified and characterized a corresponding G protein-coupled receptor (GPCR) of the D. suzukii CAPA-PVK peptides: CAPA receptor (CAPA-R). To better characterize the behavior of D. suzukii CAPA-R, we used insect cell-based functional expression assays to evaluate responses of CAPA-RagainstD. suzukii CAPA-PVKs, CAPA-PVKs from five species in Insecta, one species from Mollusca, modified CAPA-PVK peptides, and some PRXamide family peptides: pyrokinin (PK), diapause hormone (DH), and ecdysis-triggering hormone (ETH). Functional studies revealed that the D. suzukii CAPA-R is strongly activated by both of its own natural D. suzukii CAPA-PVKs, and interestingly, it was strongly activated by other CAPA-PVK peptides from Frankliniella occidentallis (Thysanoptera), Solenopsis invicta (Hymenoptera), Helicoverpa zea (Lepidoptera) and Plutella xylostella (Lepidoptera). However, D. suzukii CAPA-R was not activated by Mollusca CAPA-PVK or the other PRXamide peptides. Gene expression analyses showed that the CAPA-R was highly expressed in the Malpighian tubules and moderately in hindgut compared to other digestive organs or the rest of body, supporting diuretic/antidiuretic functionality. When compared across life stages of D. suzukii, expression of CAPA-R was approximately 1.5x greater in the third instar than the other stages and minimally detected in the eggs, 4-day old pupae and 3-day old adults. Our results functionally characterized the D. suzukii CAPA-R and a few short peptides were identified as potential biological targets to exploit the CAPA-R for D. suzukii management.

Development of a Cellular Assay as a Personalized Model for Testing Chronic Wound Therapeutics

Exudates of non-healing wounds contain drivers of pathogenicity. We utilized >800 exudates from non-healing and healing wounds of diverse etiologies, collected by three different methods, to develop a wound-specific, cell-based functional biomarker assay. Human dermal fibroblast proliferation served as readout to a) to differentiate between healing and non-healing wounds, b) follow the healing process of individual patients, and c) assess the effects of therapeutics for chronic wounds ex vivo. We observed a strong correlation between wound chronicity and inhibitory effects of individual exudates on fibroblast proliferation, with good diagnostic sensitivity (76-90%, depending on the sample collection method). Transition of a clinically non-healing to a healing phenotype restored fibroblast proliferation and extracellular matrix formation while reducing inflammatory cytokine production. Transcriptional analysis of fibroblasts exposed to ex vivo non-healing wound exudates revealed an induction of inflammatory cytokine- and chemokine pathways and the unfolded protein response, indicating that these changes may contribute to the pathology of non-healing wounds. Testing the wound therapeutics platelet derived growth factor and silver sulfadiazine yielded responses in line with clinical experience and indicate the usefulness of the assay to search for and profile new therapeutics.

Preclinical mitigation of 5-HT2B agonism-related cardiac valvulopathy revisited

Cardiac valvulopathy (Cardiac Valve Disease; CVD) associated with off-target activation of the 5-hydroxytryptamine (5-HT) 2B receptor has been well recognized, but is still poorly predicted during drug development. The regulatory guidance proposes the use of 5-HT2B binding data (i.e., Ki values) and free maximum therapeutic exposure (Cmax) to calculate safety margins as a threshold of detection (>10) for eliminating the risk of drug-induced cardiac valvulopathy. In this paper, we provide additional recommendations for preclinical prediction of CVD risk based on clinical pharmacodynamic and pharmacokinetic data obtained from drugs with or without 5-HT2B receptor activation. Our investigations showed that 5-HT2B agonist affinity of molecules tested in an in vitro 5-HT2B cell-based functional assay, placed in perspective to their sustained plasma exposure (AUCs) and not to their peak plasma exposure, Cmax (i.e., maximum therapeutic exposure) provide a solid basis for interpreting 5-HT2B data, for calculating safety margins and then, accurately differentiate drugs associated with a clinical risk of CVD from those which are not (despite having some agonist 5-HT2B activity). In addition, we discuss the risk of multi-organ fibrosis linked to 5-HT2B receptor activation, often underestimated, however well reported in FAERS for 5-HT2B agonists. We believe that our recommendations have the potential to mitigate the risk for the clinical development of CVD and fibrosis.

Structural and functional characterization of IgG- and non-IgG-based T-cell-engaging bispecific antibodies.

Bispecific T-cell-engaging antibodies are a growing class of therapeutics with numerous molecules being tested in clinical trials and, currently, seven of them have received market approval. They are structurally complex and function as adaptors to redirect the cytotoxicity of T cells to kill tumor cells. T-cell-engaging bispecific antibodies can be generally divided into two categories: IgG/IgG-like and non-IgG-like formats. Different formats may have different intrinsic potencies and physiochemical properties, and comprehensive studies are needed to gain a better understanding of how the differences in formats impact on structural and functional characteristics. In this study, we designed and generated bispecific T-cell-engaging antibodies with IgG-like (DVD-Ig) and non-IgG (BiTE) formats. Both target the same pair of antigens (EGFR and CD3) to minimize the possible influence of targets on functional characterization. We performed a side-by-side comparison to assess differences in the physiochemical and biological properties of these two bispecific T-cell-engaging antibodies using a variety of breast and ovarian cancer cell-based functional assays to delineate the structural-functional relationships and anti-tumor activities/potency. We found that the Fc portion of T-cell-engaging bispecific antibodies can significantly impact antigen binding activity, potency, and stability in addition to eliciting different mechanisms of action that contribute the killing of cancer cells.

Intracellular Allosteric Antagonist of the Olfactory Receptor OR51E2.

Olfactory receptors are members of class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs). Their expression and function have been increasingly studied in nonolfactory tissues, and many have been identified as potential therapeutic targets. In this manuscript, we focus on the discovery of novel ligands for the olfactory receptor family 51 subfamily E2 (OR51E2). We performed an artificial intelligence-based virtual drug screen of a ∼2.2 million small molecule library. Cell-based functional assay identified compound 80 (C80) as an antagonist and inverse agonist, and detailed pharmacological analysis revealed C80 acts as a negative allosteric modulator by significantly decreasing the agonist efficacy, while having a minimal effect on receptor affinity for agonist. C80 binds to an allosteric binding site formed by a network of nine residues localized in the intracellular parts of transmembrane domains 3, 5, 6, 7, and H8, which also partially overlaps with a G protein binding site. Mutational experiments of residues involved in C80 binding uncovered the significance of the C2406.37 position in blocking the activation-related conformational change and keeping the receptor in the inactive form. Our study provides a mechanistic understanding of the negative allosteric action of C80 on agonist-ctivated OR51E2. We believe the identification of the antagonist of OR51E2 will enable a multitude of studies aiming to determine the functional role of this receptor in specific biologic processes. SIGNIFICANCE STATEMENT: OR51E2 has been implicated in various biological processes, and its antagonists that can effectively modulate its activity have therapeutic potential. Here we report the discovery of a negative allosteric modulator of OR51E2 and provide a mechanistic understanding of its action. We demonstrate that this modulator has an inhibitory effect on the efficacy of the agonist for the receptor and reveal a network of nine residues that constitute its binding pocket, which also partially overlaps with the G protein binding site.

Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer.

New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and DI) domains. In proof-of-concept studies, we demonstrated that a monoclonal antibody (mAb) that simultaneously inhibits both domains represents a promising therapeutic approach. Here, we screened a hybridoma library using a multistep selection strategy, including flow cytometry for Ab binding to native conformation protein and in vitro cell-based functional assays to isolate a novel panel of highly specific human ADAM8 dual MP and DI inhibitory mAbs, called ADPs. The screening of four top candidates for in vivo anti-cancer activity in an orthotopic MDA-MB-231 TNBC model of ADAM8-driven primary growth identified two lead mAbs, ADP2 and ADP13. Flow cytometry, hydrogen/deuterium exchange-mass spectrometry (HDX-MS) and alanine (ALA) scanning mutagenesis revealed that dual MP and DI inhibition was mediated via binding to the DI. Further testing in mice showed ADP2 and ADP13 reduce aggressive TNBC characteristics, including locoregional regrowth and metastasis, and improve survival, demonstrating strong therapeutic potential. The continued development of these mAbs into an ADAM8-targeted therapy could revolutionize TNBC treatment.

Abstract LB128: A novel tri-specific T cell engager targeting BCMA and GPRC5D for treatment of multiple myeloma

Abstract Introduction Multiple myeloma (MM) accounts for 10% of all hematologic cancers. Recent advances in MM therapy have greatly increased the overall response and survival rate. However, almost all patients eventually relapse. The prognosis still remains poor. BCMA and GPRC5D are overexpressed in myeloma cells. Although CAR-T and T cell engager (TCE) targeting BCMA or GPRC5D have been efficacious in MM patients, resistance does occur. Since the expression of BCMA and GPRC5D in MM are heterogeneous, to further improve the overall response and survival, we have recently generated a novel tri-specific T-cell engager, GBD218, targeting both BCMA and GPRC5D. GBD218 has demonstrated potent in vitro and in vivo activity against myeloma cells. Methods Anti-BCMA and GPRC5D nanobodies were screened from alpaca immune libraries, and anti-CD3 antibody was engineered from mouse hybridoma clone. The tri-specific antibodies were constructed in a “1+1+1” format through “knob into hole” technology fused with silenced IgG1 Fc. The format of the tri-specific antibodies was optimized by multiple rounds of in vitro activity and druggability evaluation. The in vivo tumor growth inhibition effects were evaluated in PBMC-humanized xenograft mouse models. Results GBD218 has been designed to potently bind hBCMA (KD=0.4nM) and hGPRC5D (cell binding EC50 ~ 2nM). To reduce CRS and other potential AEs associated with TCEs, a low affinity of anti-CD3 Fab was used. In cell-based functional assays, GBD218 showed efficient cytotoxicity against single and double positive MM cell lines with various expression levels of BCMA and GPRC5D. T cell activation and cytokine release induced by GBD218, in the presence or absence of MM cancer cells, is nicely balanced for great killing efficacy and the low risk of CRS. Importantly, the results showed that GBD218 exhibited superior in vitro killing activity compared to benchmarks, including teclistamab, talquetamab, the combination of teclistamab and talquetamab, suggesting a synergistic effect of GBD218 by targeting BCMA and GPRC5D. In xenograft models, GBD218 showed excellent anti-tumor activity, indicating great potential for GBD218 as a promising therapeutics for MM. Conclusion GBD218 is a novel tri-specific antibody that showed potent in vitro and in vivo anti-tumor activity. GBD218 efficiently kills both BCMA and/or GPRC5D expressing MM cells, which may hold promise to increase response rate and improve survival in MM patients in clinic. Citation Format: Yongcong Tan, Xuezhen Li, Fan Yu, Juehua Xu, Zhen Qian, Yiqi Cao, Xueyan Yang, Qinglin Du, Fei Peng, Shuhua Han, Qian Ding. A novel tri-specific T cell engager targeting BCMA and GPRC5D for treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB128.

Abstract 2668: CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway

Abstract Background CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Adenosine contributes to immune evasion by solid tumors by concentrating immunosuppressive adenosine in the tumor microenvironment. CBO421, a Drug Fc-Conjugate (DFC), comprises a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) for potential best-in-class efficacy. Methods CBO421’s binding affinity to human CD73 and Fc receptors was assessed by surface plasmon resonance. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using a commercial kit. Immunophenotyping and binding to cancer cells was measured by flow cytometry. Inhibition of soluble and membrane-bound CD73 on human PBMCs and mouse cancer cells was determined by measuring free phosphate levels. Functional activity was measured using PBMC rescue assays. In vivo efficacy of CBO421 monotherapy was evaluated in a syngeneic mouse model. Results CBO421 exhibited a binding affinity to human CD73 (KD = 0.8 nM) that was comparable to or greater to the affinity observed with small molecule inhibitors (AB680, OP-5244) and CD73-targeting mAbs (oleclumab, mupadolimab biosimilars). As expected, human FcRn binding of CBO421 was comparable to the unconjugated Fc and the full-length wild-type IgG1 control mAb. CBO421 binding to human Fc gamma receptors and ADCC activity was significantly reduced or abolished compared to the control mAb, such that it poses a minimal risk for undesired autoimmune activity. CBO421 potently inhibited soluble CD73 and CD73 expressed on human MDA-MB-231 and mouse EMT6 cancer cells. Immunophenotyping revealed that CD73 expression was primarily observed on CD8+ T and B cells from human PBMCs. In functional PBMC rescue assays, CBO421 demonstrated potent reactivation of AMP- or ATP-suppressed CD4+ or CD8+ T cells at levels comparable to small molecule inhibitors and greater than anti-CD73 mAb comparators. CBO421 monotherapy demonstrated a dose-dependent reduction in tumor volume in a syngeneic mouse model using MC38, a colorectal cancer cell line. Conclusions CBO421 exhibited strong binding and potent inhibition of both soluble and membrane-bound CD73, differentiating it from mAb CD73 inhibitors. The proprietary immune-silent Fc of CBO421 did not affect FcRn binding but substantially reduced Fc gamma receptor binding, leading to the elimination of ADCC activity to prevent undesired immune cell depletion in host tissues. CBO421 demonstrated high potency in functional cell-based assays and robust antitumor efficacy in a syngeneic mouse model. Currently, CBO421 is being advanced as a clinical development candidate for the treatment of solid cancers. Citation Format: Amanda Almaguer, Doug Zuill, Simon Döhrmann, James Levin, Nicholas Dedeic, Elizabeth Abelovski, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2668.

Abstract 1994: Drug-induced cancer cell secretome promotes resistance in colon cancer

Abstract Colorectal cancer (CRC) is the third most common cancer in the world, with a projection of increased lethality in the upcoming years. Adjuvant chemotherapy with biological agents is the most practiced standard of care today. However, patients with metastatic CRC (mCRC) do not benefit from the wide range of therapies available for CRC, which either owe to unresectability or co-morbidity or more importantly resistance to therapy. 5-fluorouracil (5-FU) combined with leucovorin, oxaliplatin, or irinotecan (FOLFOX or FOLFIRI) has been routinely employed as the first-line therapy in colon cancer, but patients often develop resistance to 5-FU. Additionally, tumor cell dormancy plays a major role in tumor relapse which leads to poor prognosis in the patients. Despite many advances, the understanding of therapy-induced resistance is still in its infancy. Here, we show that 5-FU-induced tumor cell secretome helps in the outgrowth and metastasis of 5-FU-resistant clones in colon cancer. We used parental and 5-FU-resistant colon cancer cell lines, and transgenic zebrafish embryos to test the hypothesis and perform the colon cancer cell-based functional assays. We observed that 5-FU resistant (5-FU-R) colon cancer cells showed an increase in proliferation and survival when cultured with the secretome of 5-FU exposed colon cancer cells compared to the DMSO-induced secretome. Interestingly, we also noted elevated migration and invasion in the 5-FU-R colon cancer cells exposed to 5-FU-induced secretome compared to the DMSO counterpart. Additionally, in zebrafish xenograft, we found that 5-FU-R colon cancer cells cultured with 5-FU-induced secretome showed higher tail-vein metastatic burden compared to the 5-FU-R cells cultured with DMSO-induced secretome. This provides a background to further study in detail the therapy-induced resistance as well as dormancy in colorectal cancer which will help to develop approaches to prevent or reverse chemoresistance in patients who receive systemic therapy for mCRC. Citation Format: Shakti Ranjan Satapathy, Souvik Ghatak, Anita Sjölander. Drug-induced cancer cell secretome promotes resistance in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1994.

Abstract 6011: Pharmacological inhibition of PMS2 increases tumor mutational burden, induces microsatellite instability and elicits immune mediated rejection in vivo

Abstract Tumors carrying defects in DNA mismatch repair (MMR-d) display high tumor mutational burden (TMB) and increased tumor neoantigen levels. MMR-d leads to accumulation of single nucleotide variants (SNVs) and insertions or deletions (indels) amongst repetitive DNA sequences generating microsatellite instability (MSI). Cancers that harbor &amp;gt;40% microsatellite variations are described as MSI-high (MSI-H). These tumors display unique clinical features including better prognosis, increased immune infiltration and remarkable response to immune checkpoint blockade (ICB) therapy. Colorectal cancer (CRC) patients with MMR-d often respond to therapies based on immune checkpoint blockade, whilst those with mismatch repair proficient (MMR-p) tumors do not. Therefore, identifying strategies to enhance the efficacy of immunotherapeutic treatments and transform this prevalent subgroup of CRCs from immunologically cold to hot remains an urgent and an unmet clinical need. We discovered NP1867 a first-in-class, potent, selective, covalent small molecule inhibitor of the DNA Mismatch Repair protein PMS2. We show by biochemical, biophysical, and protein-ligand X-ray crystallographic methods that NP1867 binds irreversibly to the ATP binding site of the N-terminal ATPase domain of PMS2. NP1867 displays potent binding to PMS2 in a NanoBRET cellular target engagement assay and inhibits MMR activity in mechanistic and functional cell-based assays of MMR-dependent DNA repair. Continuous treatment of murine CRC cell line CT26 (MMR-p) with NP1867 increases TMB, specifically enriches gained mutations with typical MMR-d related COSMIC mutational signatures, and elicits MSI-H status in a time-dependent manner, demonstrating for the first time that inhibition of PMS2 with a small molecule phenocopies the inactivation of MMR observed in patients. Remarkably, inoculation of immune-competent mice with CT26 cells pre-treated with NP1867 elicits tumor regressions in anti-PD1-treated animals, recapitulating the unique clinical features of MMR-d/MSI-H tumors. Conversely, no response is observed in immune-competent animals inoculated with control-treated cells. The extent of in vivo response correlates with the duration of in vitro NP1867 treatment and with the MSI-H status of treated CT26 cells. In conclusion, small molecule NP1867 functionally inhibits DNA MMR by targeting PMS2, enriches gained mutations with MMR-d COSMIC signatures, elicits MSI-H status, and enhances immune surveillance in preclinical models. These findings pave the way for the development of orally bioavailable compounds suitable for long-term dosing in animal models. Citation Format: Eleonora Piumatti, Alexia Hervieu, Philippe Riou, Julian Blagg, Adam Peall, Sam Weeks, Maria T. Rodríguez-Plata, Giuseppe Rospo, Sasi Arunachalam, Bettina Meier, Paige Tongue, Tessa McLaren, Kalpesh Parmar, Pradip Patel, David Clark, Gareth Langley, Charles Nichols, Benoît Rousseau, Paul Winship, Matthew Baker, Martin Drysdale, Giovanni Germano, Alberto Bardelli. Pharmacological inhibition of PMS2 increases tumor mutational burden, induces microsatellite instability and elicits immune mediated rejection in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6011.

Is myeloid-derived growth factor a ligand of the sphingosine-1-phosphate receptor 2?

Secretory myeloid-derived growth factor (MYDGF) exerts beneficial effects on organ repair, probably via a plasma membrane receptor; however, the identity of the expected receptor has remained elusive. In a recent study, MYDGF was reported as an agonist of the sphingosine-1-phosphate receptor 2 (S1PR2), an A-class G protein-coupled receptor that mediates the functions of the signaling lipid, sphingosine-1-phosphate (S1P). In the present study, we conducted living cell-based functional assays to test whether S1PR2 is a receptor for MYDGF. In the NanoLuc Binary Technology (NanoBiT)-based β-arrestin recruitment assay and the cAMP-response element (CRE)-controlled NanoLuc reporter assay, S1P could efficiently activate human S1PR2 overexpressed in human embryonic kidney (HEK) 293T cells; however, recombinant human MYDGF, overexpressed either from Escherichia coli or HEK293 cells, had no detectable effect. Thus, the results demonstrated that human MYDGF is not a ligand of human S1PR2. Considering the high conservation of MYDGF and S1PR2 in evolution, MYDGF is also probably not a ligand of S1PR2 in other vertebrates.

Cell-based functional assays for free sialic acid storage disorder

Cell-based functional assays for free sialic acid storage disorder

Prevalence of Adeno-Associated Virus-9-Neutralizing Antibody in Chinese Patients with Duchenne Muscular Dystrophy.

The delivery of a mini-dystrophin gene to skeletal muscles using recombinant adeno-associated virus serotype (AAV) holds great potential as a gene therapy for Duchenne muscular dystrophy (DMD). However, the presence of anti-AAV-neutralizing antibodies (NAbs) may impede the effectiveness of gene transduction. This study aimed to evaluate the prevalence of anti-AAV9 NAbs in Chinese patients with DMD, and to characterize the target population for an AAV gene therapy. A total of one hundred male patients with DMD were included in this study, and demographic and clinical data were collected. A blood specimen was obtained from each participant for the purpose of evaluating the existence of anti-AAV9 NAbs through a cell-based functional assay conducted at a central laboratory. A NAb titer exceeding 1:4 was considered positive. The positivity rates of anti-AAV9 NAb were compared among different subgroups. The median age of this DMD cohort was 8 years old, ranging from 3 to 15 years of age. Forty-two percent of patients tested positive for anti-AAV9 NAb. Notably, all samples from patients under 4 years of age tested negative, and the positivity rates of anti-AAV9 NAb differed significantly across the three age subgroups (<4 years old, ≥4 years old and <12 years old, and ≥12 years old, χ2 = 7.221, p = 0.023). Further investigation into the living environment revealed a higher positivity rate of anti-AAV9 NAb in rural patients compared with urban patients (χ2 = 3.923, p = 0.048). Moreover, the prevalence in patients from different cities/provinces varied greatly (χ2 = 16.550, p = 0.003). There was no statistically significant difference in the positivity rate of NAb among subgroups of patients with different motor functions (ambulatory or nonambulatory) and different treatment strategies (taking or not taking glucocorticoid). In Chinese DMD patients, the prevalence of anti-AAV9 NAb was found to reach 42%. Moreover, the antibody-positive rate in children <4 years of age was low and revealed notable regional discrepancies.

Abstract A095: The paradox-breaker BRAF inhibitor plixorafenib (FORE8394) efficiently inhibits non-V600 mutations and fusions

Abstract The use of several BRAF inhibitors to target BRAF V600 (Class I BRAF mutations) mutated tumors has shown clinical benefit in multiple types of cancer. Nonetheless, V600 mutations comprise about 40% of the BRAF-altered patient population, and the remainder of the BRAF mutated patients are in need of novel targeted therapies. Clinical trials aimed at evaluating the effect of these inhibitors in non-V600 patients, mostly in combination with a MEK inhibitor, have been performed with mixed results. However, the experimental sensitivity to a wide range of class 2 (RAS independent non-V600 including BRAF fusions) or class 3 (RAS dependent non-V600) alterations to different types of BRAF inhibitors has not been reported. Here we tested the efficacy of plixorafenib (a novel paradox braker BRAF inhibitor) compared with several BRAF inhibitors in BRAF V600 and non-V600 alterations. We implemented a high-throughput cell-based functional assay to profile the response of different BRAF variants to plixorafenib (paradox breaker), vemurafenib (a first-generation inhibitor) and tovorafenib (a pan-RAF inhibitor). This method quantifies MAPK signaling pathway activation using fluorescent imaging coupled with image analysis of cells expressing the mutated protein together with a fluorescently labeled ERK2 as a signaling pathway reporter. We also validated a subset of the findings using standard western blot and cell viability experiments. Comparing the response of 4 Class I, 26 Class II and 94 BRAF fusions to plixorafenib and vemurafenib Results showed that Plixorafenib was more potent in inhibiting the majority of BRAF alterations tested, i.e., a lower IC50 value was measured for plixorafenib over vemurafenib. Furthermore, of the 94 BRAF fusions proteins tested, over 75% showed at least a 50% inhibition over the concentrations tested, while less than 20% were inhibited by vemurafenib. A smaller cohort of 23 BRAF alterations was tested with the investigational pan-RAF inhibitor tovorafenib. The IC:50 values for the Class I and II BRAF alterations tested were higher with tovorafenib compared with plixorafenib, and 6 of 13 BRAF fusions tested were resistant to tovorafenib and sensitive to plixorafenib. In conclusion, we show here that plixorafenib inhibits a wide range of V600 and non-V600 BRAF alterations, more potently compared with first generation BRAFi (vemurafenib) and a pan-RAF inhibitor (tovorafenib). Importantly, this effect has the potential to overcome an unmet clinical need in cancers with non-V600 alterations. Citation Format: Limor Cohen, Natalie Filippov-Levy, Hana Billauer, Lea Birnbaum, Sara Gasasa, Roey Maor, Yakir Amrusi, Reham Abu-Liel, Eden Goldfarb, Gabi Tarcic. The paradox-breaker BRAF inhibitor plixorafenib (FORE8394) efficiently inhibits non-V600 mutations and fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A095.

Single-Exon Deletions of ZNRF3 Exon 2 Cause Congenital Adrenal Hypoplasia.

Primary adrenal insufficiency (PAI) is a life-threatening condition characterized by the inability of the adrenal cortex to produce sufficient steroid hormones. E3 ubiquitin protein ligase zinc and ring finger 3 (ZNRF3) is a negative regulator of Wnt/β-catenin signaling. R-spondin 1 (RSPO1) enhances Wnt/β-catenin signaling via binding and removal of ZNRF3 from the cell surface. This work aimed to explore a novel genetic form of PAI. We analyzed 9 patients with childhood-onset PAI of biochemically and genetically unknown etiology using array comparative genomic hybridization. To examine the functionality of the identified single-exon deletions of ZNRF3 exon 2, we performed three-dimensional (3D) structure modeling and in vitro functional studies. We identified various-sized single-exon deletions encompassing ZNRF3 exon 2 in 3 patients who showed neonatal-onset adrenal hypoplasia with glucocorticoid and mineralocorticoid deficiencies. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the 3 distinct single-exon deletions were commonly transcribed into a 126-nucleotide deleted mRNA and translated into 42-amino acid deleted protein (ΔEx2-ZNRF3). Based on 3D structure modeling, we predicted that interaction between ZNRF3 and RSPO1 would be disturbed in ΔEx2-ZNRF3, suggesting loss of RSPO1-dependent activation of Wnt/β-catenin signaling. Cell-based functional assays with the TCF-LEF reporter showed that RSPO1-dependent activation of Wnt/β-catenin signaling was attenuated in cells expressing ΔEx2-ZNRF3 as compared with those expressing wild-type ZNRF3. We provided genetic evidence linking deletions encompassing ZNRF3 exon 2 and congenital adrenal hypoplasia, which might be related to constitutive inactivation of Wnt/β-catenin signaling by ΔEx2-ZNRF3.

Refined control of CRISPR-Cas9 gene editing in Clostridium sporogenes: the creation of recombinant strains for therapeutic applications.

Despite considerable clinical success, the potential of cancer immunotherapy is restricted by a lack of tumour-targeting strategies. Treatment requires systemic delivery of cytokines or antibodies at high levels to achieve clinically effective doses at malignant sites. This is exacerbated by poor penetration of tumour tissue by therapeutic antibodies. High-grade immune-related adverse events (irAEs) occur in a significant number of patients (5-15%, cancer- and therapeutic-dependent) that can lead to lifelong issues and can exclude from treatment patients with pre-existing autoimmune diseases. Tumour-homing bacteria, genetically engineered to produce therapeutics, is one of the approaches that seeks to mitigate these drawbacks. The ability of Clostridium sporogenes to form spores that are unable to germinate in the presence of oxygen (typical of healthy tissue) offers a unique advantage over other vectors. However, the limited utility of existing gene editing tools hinders the development of therapeutic strains. To overcome the limitations of previous systems, expression of the Cas9 protein and the gRNA was controlled using tetracycline inducible promoters. Furthermore, the components of the system were divided across two plasmids, improving the efficiency of cloning and conjugation. Genome integrated therapeutic genes were assayed biochemically and in cell-based functional assays. The potency of these strains was further improved through rationally-conceived gene knock-outs. The new system was validated by demonstrating the efficient addition and deletion of large sequences from the genome. This included the creation of recombinant strains expressing two pro-inflammatory cytokines, interleukin-2 (IL-2) and granulocyte macrophage-colony stimulating factor (GM-CSF), and a pro-drug converting enzyme (PCE). A comparative, temporal in vitro analysis of the integrant strains and their plasmid-based equivalents revealed a substantial reduction of cytokine activity in chromosome-based constructs. To compensate for this loss, a 7.6 kb operon of proteolytic genes was deleted from the genome. The resultant knock-out strains showed an 8- to 10-fold increase in cytokine activity compared to parental strains.