Multiple Mutants Research Articles

Mutations across Diverse Domains of CjXDR1 Lead to Multidrug Resistance in Clarireedia jacksonii.

Recently, Clarireedia jacksonii has emerged as a significant pathogen threatening turfgrass, and its escalating resistance to multiple drugs often undermines field interventions. This study highlighted the critical role of the fungus-specific transcription factor CjXDR1 (formerly ShXDR1) in regulating multidrug resistance (MDR) in C. jacksonii. This was demonstrated through experiments involving CjXDR1-knockout and CjXDR1-complemented strains. Our sequence analysis revealed five mutations in CjXDR1: G445D, K453E, S607F, D676H, and V690A. All five gain-of-function (GOF) mutations were confirmed to directly contribute to MDR against three different classes of fungicides (propiconazole: demethylation inhibitor, boscalid: succinate dehydrogenase inhibitor, and iprodione: dicarboximide) using the genetic transformation system and in vitro fungicide-sensitivity assay. Comparative transcriptome analysis revealed that CjXDR1 and its GOF mutations led to the overexpression of downstream genes encoding a Phase I metabolizing enzyme (CYP68) and two Phase III transporters (CjPDR1 and CjAtrD) previously reported. Knockout mutants of CYP68, CjPDR1, CjAtrD, and double-knockout mutants of CjPDR1 and CjAtrD exhibited increased sensitivity to all three fungicides tested. Among these, the CYP68-knockout mutants displayed the highest sensitivity to propiconazole, while the CjPDR1 knockout mutant exhibited significantly increased sensitivity to all three fungicides. Double-knockout mutants of CjPDR1 and CjAtrD displayed greater sensitivity than the single knockouts. In conclusion, multiple GOF mutants in CjXDR1 contribute to MDR by upregulating the expression of CjPDR1, CjAtrD, and CYP68. This study enhances our understanding of the molecular mechanisms underlying MDR in plant pathogenic fungi, providing valuable insights into GOF mutation structures and advancing the development of antifungal drugs.

Kinetic Studies on the Interaction of HIV-1 Gag Protein with the HIV-1 RNA Packaging Signal.

During HIV-1 virus assembly, the genomic RNA (vRNA) is selected for packaging by the viral protein Gag because it contains a specific packaging signal, Psi. While there have been numerous studies of Gag-Psi interactions, there is almost no information on the kinetic aspects of this interaction. We investigated the kinetics of Gag binding to different RNAs using switchSENSE DRX2 technology. We measured the association rate of Gag binding to monomeric Psi, to a "Multiple Binding Site Mutant" Psi that is inactive for genome packaging in vivo, and to a scrambled Psi. We discovered that Gag binds more rapidly to Psi RNA than to the mutant or scrambled RNAs. Furthermore, rapid Gag association kinetics are retained within sub-regions of Psi: Gag associates more rapidly with RNA containing only the 3' two of the three Psi stem-loops than with monomeric RNA containing the 5' two stem-loops or a scrambled RNA. No differences were detectable with individual Psi stem-loops. Interestingly, the rate of binding of Gag molecules to Psi increases with increasing Gag concentration, suggesting cooperativity in binding. The results are consistent with the hypothesis that selectivity in packaging derives from kinetic differences in initiation of particle assembly.

Spatiotemporal EP4–fibulin-1 expression is associated with vascular intimal hyperplasia

Abstract Aims Cyclooxygenase-2–derived prostaglandin E2 (PGE2) is thought to promote vascular intimal hyperplasia (IH). It has been reported that the PGE2 receptor EP4 is upregulated in injured vessels and that EP4 signalling in vascular smooth muscle cells (VSMCs) promotes IH. In contrast, EP4 in endothelial cells has been demonstrated to restrain IH. We aimed to investigate spatiotemporal expression of EP4 and whether modulating EP4 signalling could be a viable therapeutic strategy. Methods and results We generated EP4 reporter mice (Ptger4-IRES-nlsLacZ) and found temporary but prominent EP4 expression in VSMCs of the proliferative neointima 2 weeks after femoral artery wire injury. Injury-induced IH was diminished in VSMC-targeted EP4 heterozygous deficient mice (Ptger4fl/+;SM22-Cre) 2 and 4 weeks after vascular injury compared to that in SM22-Cre, whereas injury-induced IH was exacerbated in VSMC-targeted EP4-overexpressing mice (Ptger4-Tg) compared to controls (non-Tg). We then investigated the downstream signalling of EP4 in VSMCs. Stimulation of EP4 increased mRNA and protein levels of the glycoprotein fibulin-1 in Ptger4-Tg VSMCs. Fibulin-1C recombinant proteins increased VSMC proliferation and migration through transforming growth factor (TGF)- β/Smad3, and EP4-mediated proliferation and migration were attenuated in Fbln1fl/fl;SM22-Cre VSMCs and in CRISPR/Cas9-mediated Fbln1 knockdown in Ptger4-Tg VSMCs. We generated multiple deletion mutants of fibulin-1C and found that EGF-like modules 6–8 appear to be involved in fibulin-1-mediated proliferation. Among binding partners of fibulin-1, extracellular matrix protein 1 (ECM1) was also upregulated by EP4 stimulation, and fibulin-1C and ECM1 proteins additively enhanced VSMC proliferation and migration. Injury-induced IH was attenuated in VSMC-targeted fibulin-1 deletion mice (Fbln1fl/fl;SM22-Cre) compared to Fbln1fl/fl. Furthermore, systemic EP4 antagonist administration reduced injury-induced IH in wild-type mice. Conclusion EP4 was upregulated in VSMCs of proliferative IH, and EP4 signalling promoted IH, at least in part through fibulin-1. An EP4 antagonist might be considered as a therapeutic strategy for IH.

Role of male gonad-enriched microRNAs in sperm production in Caenorhabditis elegans.

Germ cell development and gamete production in animals require small RNA pathways. While studies indicate that microRNAs (miRNAs) are necessary for normal sperm production and function, the specific roles for individual miRNAs are largely unknown. Here, we use small RNA sequencing (RNA-seq) of dissected gonads and functional analysis of new loss-of-function alleles to identify functions for miRNAs in the control of fecundity and sperm production in Caenorhabditis elegans males and hermaphrodites. We describe a set of 29 male gonad-enriched miRNAs and identify a set of individual miRNAs (mir-58.1 and mir-235) and a miRNA cluster (mir-4807-4810.1) that are required for optimal sperm production at 20°C and a set of miRNAs (mir-49, mir-57, mir-83, mir-261, and mir-357/358) that are required for sperm production at 25°C. We observed defects in meiotic progression in mutants missing mir-58.1, mir-83, mir-235, and mir-4807-4810.1, which may contribute to the observed defects in sperm production. Further, analysis of multiple mutants of these miRNAs suggested genetic interactions between these miRNAs. This study provides insights on the regulatory roles of miRNAs that promote optimal sperm production and fecundity in males and hermaphrodites.

Functional Investigations of p53 Acetylation Enabled by Heterobifunctional Molecules.

Post-translational modifications (PTMs) dynamically regulate the critical stress response and tumor suppressive functions of p53. Among these, acetylation events mediated by multiple acetyltransferases lead to differential target gene activation and subsequent cell fate. However, our understanding of these events is incomplete due to, in part, the inability to selectively and dynamically control p53 acetylation. We recently developed a heterobifunctional small molecule system, AceTAG, to direct the acetyltransferase p300/CBP for targeted protein acetylation in cells. Here, we expand AceTAG to leverage the acetyltransferase PCAF/GCN5 and apply these tools to investigate the functional consequences of targeted p53 acetylation in human cancer cells. We demonstrate that the recruitment of p300/CBP or PCAF/GCN5 to p53 results in distinct acetylation events and differentiated transcriptional activities. Further, we show that chemically induced acetylation of multiple hotspot p53 mutants results in increased stabilization and enhancement of transcriptional activity. Collectively, these studies demonstrate the utility of AceTAG for functional investigations of protein acetylation.

Rational Design of High Affinity Interaction Between CC Chemokine Binding Protein vCCI and CCL17/TARC.

The poxvirus-derived protein vCCI (viral CC chemokine inhibitor) binds almost all members of the CC chemokine family with nanomolar affinity, inhibiting their pro-inflammatory actions. Understanding the affinity and specificity of vCCI could lead to new anti-inflammatory therapeutics. CCL17, also known as TARC, is unusual among CC chemokines by having only micromolar binding to vCCI. We have used sequence analysis and molecular simulations to determine the cause of this weak binding, which identified several locations in CCL17 where mutations seemed likely to improve binding to vCCI. Based on the aforementioned analysis, we expressed and tested multiple mutants of CCL17. We found two single point mutants V44K and Q45R that increased binding affinity to vCCI by 2-3-fold and, in combination, further improved affinity by 7-fold. The CCL17 triple mutant G17R/V44K/Q45R yielded a Kd of 0.25 ± 0.13 μM, a 68-fold improvement in affinity compared to the complex with wild-type CCL17. A quadruple mutant G17R/V44K/Q45R/R57W showed high affinity (0.59 ± 0.09 μM) compared to the wild type but lower affinity than the triple mutant. This work demonstrates that sequence comparisons and molecular simulations can predict chemokine mutations that increase the level of binding to vCCI, an important first step in developing engineered chemokine inhibitors useful for anti-inflammatory therapy.

Hook loop dynamics engineering transcended the barrier of activity-stability trade-off and boosted the thermostability of enzymes

The improvement of enzyme thermostability often accompanies the decreased activity due to the loss of the key regions' flexibility. As a representative structure, unlocking the potential of loop dynamics will not only provide new ideas for stabilization strategies, but also help to deepen the understanding of the relationship between enzyme structural dynamics and function. In this study, a creative “hook loop dynamics engineering” (HLoD) strategy was successfully proposed for simultaneously improving the thermostability and maintaining activity of the model enzyme, Candida Antarctica lipase B. A small and smart mutant library involving five key residues located at the “hook loop” was meticulously identified and systematically investigated and thus yielded a five-point multiple mutant M1 (L147S/T244P/S250P/T256D/N292D), demonstrating a remarkable 7.0-fold increase in thermostability at 60 °C compared to the wild-type (WT). Furthermore, the activity of M1 remained comparable to that of WT, effectively transcending the barrier of activity-stability trade-off. Molecular dynamics simulations revealed that the precise regulation of hook loop dynamics via intermolecular interactions, such as salt bridges and hydrogen bonding, curbed the excessive flexibility of the pivotal regions α5 and α10 at high temperatures, thus driving the substantial enhancement of the thermostability of M1. Refining the dynamics of the flexible region via HLoD, which transcended the barrier of activity-stability trade-off, exhibited to be a robust and potentially universal strategy for designing enzymes with outstanding thermostability and activity.

Exploring the Mutated Kinases for Chemoenzymatic Synthesis of N4-Modified Cytidine Monophosphates.

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK.

Multiple genes deletion based on Cre-loxP marker-less gene deletion system for the strains from the genus of Pectobacterium.

To introduce the Cre-loxP system for constructing marker-less multiple-gene deletion mutants in Pectobacterium, overcoming limitations of antibiotic markers and enhancing the understanding of pathogenic mechanisms. Firstly, a plasmid named pEX18-Cre, containing a sacB sucrose suicide gene, was constructed to express Cre recombinase in Pectobacterium. Secondly, a mutant in which the loxP-Km fragment replaced the target gene was obtained through homologous recombination double-crossover with the chromosome. Finally, pEX18-Cre was introduced into the mutant to excise the DNA between the loxP sites, thereby removing the markers and achieving multiple gene deletions. By utilizing the Cre-loxP system, we successfully constructed multiple marker-less gene deletion mutants in Pectobacterium strains. The Cre-loxP system efficiently creates marker-less multiple-gene deletion mutants, enhancing the study of Pectobacterium pathogenic mechanisms by overcoming antibiotic marker limitations.

Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells.

Since Robert Feulgen first stained DNA in the cell, visualizing genome chromatin has been a central issue in cell biology to uncover how chromatin is organized and behaves in the cell. To approach this issue, we have developed single-molecule imaging of nucleosomes, a basic unit of chromatin, to unveil local nucleosome behavior in living cells. In this study, we investigated behaviors of nucleosomes with various histone H4 mutants in living HeLa cells to address the role of H4 tail acetylation, including H4K16Ac and others, which are generally associated with more transcriptionally active chromatin regions. We ectopically expressed wild-type (wt) or mutated H4s (H4K16 point; H4K5,8,12,16 quadruple; and H4 tail deletion) fused with HaloTag in HeLa cells. Cells that expressed wtH4-Halo, H4K16-Halo mutants, and multiple H4-Halo mutants had euchromatin-concentrated distribution. Consistently, the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains (or topologically associating domains, TADs)with active chromatin marks (A-compartment). Utilizing single-nucleosome imaging, we found that none of the H4 deacetylation or acetylation mimicked H4 mutants altered the overall local nucleosome motion. This finding suggests that H4 mutant nucleosomes embedded in the condensed euchromatic domains with excess endogenous H4 nucleosomes cannot cause an observable change in the local motion. Interestingly, H4 with four lysine-to-arginine mutations displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4 with a truncated N-terminal tail was incorporated in heterochromatic regions as well as euchromatin. Our study indicates the power of single-nucleosome imaging to understand individual histone/nucleosome behavior reflecting chromatin environments in living cells.

Tubulin glycylation controls ciliary motility through modulation of outer-arm dyneins.

Tubulins undergo several kinds of posttranslational modifications (PTMs) including glutamylation and glycylation. The contribution of these PTMs to the motilities of cilia and flagella is still unclear. Here, we investigated the role of tubulin glycylation by examining a novel Chlamydomonas mutant lacking TTLL3, an enzyme responsible for initiating glycylation. Immunostaining of cells and flagella revealed that glycylation is only restricted to the axonemal tubulin composing the outer-doublet but not the central-pair microtubules. Furthermore, the flagellar localization of TTLL3 was found to be dependent on intraflagellar transport. The mutant, ttll3(ex5), completely lacks glycylation and consequently exhibits slower swimming velocity compared with the wild-type strain. By combining the ttll3(ex5) mutation with multiple axonemal dynein-deficient mutants, we found that the lack of glycylation does not affect the motility of the outer-arm dynein lacking mutations. Sliding disintegration assay using isolated axonemes revealed that the lack of glycylation decreases microtubule sliding velocity in the normal axoneme but not in the axoneme lacking the outerarm dyneins. Based on our recent study that glycylation occurs exclusively on β-tubulin in Chlamydomonas, these findings suggest that tubulin glycylation controls flagellar motility through modulating outer-arm dyneins, presumably by neutralizing the negative charges of glutamate residues at the C-terminus region of β-tubulin.

ARX regulates cortical interneuron differentiation and migration.

Mutations in aristaless-related homeobox ( ARX ) are associated with neurodevelopmental disorders including developmental epilepsies, intellectual disabilities, and autism spectrum disorders, with or without brain malformations. Aspects of these disorders have been linked to abnormal cortical interneuron (cIN) development and function. To further understand ARX's role in cIN development, multiple Arx mutant mouse lines were interrogated. We found that ARX is critical for controlling cIN numbers and distribution, especially, in the developing marginal zone (MZ). Single cell transcriptomics and ChIP-seq, combined with functional studies, revealed ARX directly or indirectly regulates genes involved in proliferation and the cell cycle (e.g., Bub3 , Cspr3 ), fate specification (e.g., Nkx2.1 , Maf , Mef2c ), and migration (e.g., Nkx2.1 , Lmo1 , Cxcr4 , Nrg1 , ErbB4 ). Our data suggest that the MZ stream defects primarily result from disordered cell-cell communication. Together our findings provide new insights into the mechanisms underlying cIN development and migration and how they are disrupted in several disorders.

WPA1 encodes a vWA domain protein that regulates wheat plant architecture

Plant height, spike, leaf, stem and grain morphologies are key components of plant architecture and related to wheat yield. A wheat (Triticum aestivum L.) mutant, wpa1, displaying temperature-dependent pleiotropic developmental anomalies, was isolated. The WPA1 gene, encoding a von Willebrand factor type A (vWA) domain protein, was located on chromosome arm 7DS and isolated by map-based cloning. The functionality of WPA1 was validated by multiple independent EMS-induced mutants and gene editing. Phylogenetic analysis revealed that WPA1 is monocotyledon-specific in higher plants. The identification of WPA1 provides opportunity to study the temperature regulated wheat development and grain yield.

Safety and efficacy of ifebemtinib (IN10018) combined with D-1553 in non-small-cell lung cancer (NSCLC) with KRAS G12C mutation: Results from a phase Ib/II study.

8605 Background: KRAS G12C mutation is present in about 12-14% of NSCLC population. Ifebemtinib (IN10018) is a highly potent and selective oral inhibitor of focal adhesion kinase (FAK). D-1553 (garsorasib) is a novel oral and potent KRAS G12C inhibitor. Preclinical data showed that ifebemtinib in combination with KRAS G12C inhibitors had synergistic anti-cancer effect in multiple KRAS G12C mutant cancer models. This study is to evaluate the safety and antitumor activity of ifebemtinib combined with D-1553 in solid tumors with KRAS G12C mutation. Here we report the preliminary data from phase II part of front-line NSCLCs with KRAS G12C mutation. Methods: Locally advanced or metastatic NSCLC subjects without prior systemic anti-cancer therapy were enrolled. Subjects were required to have KRAS G12C mutation in tumor tissues. All subjects were assigned to the recommended phase II dose (RP2D) of ifebemtinib (100mg qd) + D-1553 (600mg bid). Results: As of 31 January 2024, 33 front-line NSCLC subjects with KRAS G12C mutation were enrolled and received the combination therapy. Median age was 65yrs (range 58, 83), 93.9% were male, and 81.8% had stage IV metastatic disease. Median study follow-up (FU) was 2.2 months (range 1.1, 9.2). The safety profile of the combination therapy is comparable to each single agent without additive toxicities. No ifebemtinib- or D-1553-related death was reported. Four subjects (12.1%) had ifebemtinib-related SAEs (diarrhea, enteritis, oedema peripheral and proteinuria) as assessed by investigators, and all SAEs were also considered D-1553 related. Six subjects (18.1%) had ≥ Grade 3 ifebemtinib-related AEs as assessed by investigators, and all were also D-1553 related AEs. The majority of ifebemtinib-related AEs were CTCAE Grade 1 or 2. There was no event leading to study treatment discontinuation. We have observed continuing tumor shrinkage in almost all subjects who had at least 2 tumor assessments. Among 16 subjects with ≥ 3 months FU, preliminary efficacy found that 14 had best overall response of partial response (PR) including 12 confirmed PRs and 2 unconfirmed but still in treatment, 1 had stable disease (SD), and 1 had progressive disease (PD). The overall response rate (ORR) was 87.5% (95%CI: 56.6, 96.2), and disease control rate (DCR) was 93.8% (95%CI: 63.6, 98.5). The median duration of response (DOR), median progression-free survival (PFS) and overall survival (OS) have not been reached. Conclusions: The combination of ifebemtinib and D-1553, as a dual-oral regimen, showed promising antitumor activity and manageable safety profile in KRAS G12C mutant front-line NSCLC population. Long term follow-up is needed to assess the potential of such novel combo. Clinical trial information: NCT06166836 ; NCT05379946 .

Prediction of SARS-CoV-2 Infection Phosphorylation Sites and Associations of these Modifications with Lung Cancer Development.

Since the emergence of SARS-CoV-2 viruses, multiple mutant strains have been identified. Infection with SARS-CoV-2 virus leads to alterations in host cell phosphorylation signal, which systematically modulates the immune response. Identification and analysis of SARS-CoV-2 virus infection phosphorylation sites enable insight into the mechanisms of viral infection and effects on host cells, providing important fundamental data for the study and development of potent drugs for the treatment of immune inflammatory diseases. In this paper, we have analyzed the SARS-CoV-2 virus-infected phosphorylation region and developed a transformer-based deep learning-assisted identification method for the specific identification of phosphorylation sites in SARS-CoV-2 virus-infected host cells. Furthermore, through association analysis with lung cancer, we found that SARS-CoV-2 infection may affect the regulatory role of the immune system, leading to an abnormal increase or decrease in the immune inflammatory response, which may be associated with the development and progression of cancer. We anticipate that this study will provide an important reference for SARS-CoV-2 virus evolution as well as immune-related studies and provide a reliable complementary screening tool for anti-SARS-CoV-2 virus drug and vaccine design.

Contributions of lignification, tissue arrangement patterns, and cross-sectional area to whole-stem mechanical properties in Arabidopsis thaliana.

Plant cells withstand mechanical stress originating from turgor pressure by robustly maintaining the mechanical properties of the cell wall. This applies at the organ scale as well; many plant stems act as pressurized cylinders, where the epidermis is under tension and inner tissues are under compression. The clavata3 de-etiolated3 (clv3-8 det3-1) double mutant of Arabidopsis thaliana displays cracks in its stems because of a conflict between the mechanical properties of the weak epidermis and over-proliferation of inner stem tissues. In this work, we conducted three-point bending tests on various Arabidopsis thaliana mutants, including those displaying the stem cracking phenotype, to examine the differences in their mechanical properties. The clv3-8 det3-1 double mutant exhibited reduced stem stiffness, consistent with reduced differentiation due to the clv3-8 mutation. Yet, in clv3-8, stem cross-sectionalarea was increased associating with the increase in vascular bundle number, and stem cross-sections displayed various shapes. To uncouple the contribution of geometry and cell-wall differentiation to the mechanical properties of the whole stems, we tested the contribution of lignified fibers to stem stiffness. In order to suppress lignin deposition in stems genetically, we generated multiple higher-order mutants by crossing clv3-8 and/or det3-1 with nst1-1 nst3-1, in which lignin deposition is suppressed. Stem stiffness was reduced markedly in all nst1-1 nst3-1 mutant backgrounds. Overall, our results suggest that stem stiffness relies on the presence of differentiated, lignified, fiber tissue as well as on the alignment or spatial distribution of vascular bundles within the stem organ.

Abstract SY07-02: Cell competition vs cell cooperation: Unlocking oncogenic potential of mutant clones in the gut

Abstract The epithelial monolayer of the intestine is one of the fastest renewing tissues of the human body. Every week, the entire epithelium is replaced by intestinal stem cells (ISCs) that reside in crypt-like structures within the monolayer. Within these crypts, a small number of ISCs continuously divide to provide all specialized cell types necessary to maintain normal intestinal homeostasis, while simultaneously competing with each other for a position within the crypt bottom. This competition, characterized by neutral loss-and-replacement events, can be drastically disturbed when one of the ISCs acquires a mutation that confers a competitive advantage. As a result, mutant ISCs will replace all normal ISCs, colonize the intestinal crypt, and facilitate the initiation of colorectal cancer (CRC). CRC development is a stepwise process characterized by the accumulation of mutations in cancer driver genes over time. Almost 80% of all CRCs present with early mutations in the tumor suppressor gene APC, leading to constitutive activation of the Wnt pathway and the formation of pre-malignant adenomas. We have previously demonstrated that, within individual crypts, Apc-mutant ISCs have a competitive advantage over normal ISCs, although the mechanism underlying this unequal competition remained unclear. Recently, we revealed that Apc-mutant ISCs in fact act as "supercompetitors" by secreting Wnt antagonists such as NOTUM, WIF1 and DKK2, that actively disadvantage neighboring wild type ISCs by forcing them do differentiate. We demonstrated that boosting wild type fitness by downstream pharmacological activation of the Wnt signaling pathway by GSK3β-inhibitor lithium rendered wild type ISCs resistant to the Wnt antagonists, reduced the number of Apc-mutant crypts, and prevented adenoma formation. Together, this work highlights that modulating cell competition between normal and mutant cells within the crypt can provide a novel approach to prevent CRC development. Our findings are particularly relevant for familial adenomatous polyposis (FAP) patients, that carry germline mutations in APC and are predisposed to the development of CRC. Based on our fundamental discoveries, we are currently performing a clinical trial to assess the chemopreventive effect of lithium on the expansion of APC-mutant clones in FAP patients [ClinicalTrials.gov: NCT05402891]. Importantly, it is becoming increasingly evident that intestinal adenomas do not form from a single Apc-mutant crypt, but require fields of multiple mutant crypts to facilitate tumor initiation. Although field cancerization by enhanced crypt fission has been observed for several oncogenic mutants in the gut (e.g. KrasG12D, PIK3CAH1047R), Apc-mutant cells are often contained within individual crypts. This indicates that multiple independent mutant clones may act cooperatively to facilitate tumorigenesis, thereby pointing towards a polyclonal origin of intestinal adenomas. Our current work investigates how Apc-mutant clones interact by modifying their local environment through reciprocal signalling with stromal and epithelial cells to escape the anatomical constraints of the crypt, thereby unlocking their oncogenic potential in the gut. Ultimately, unravelling the molecular mechanisms underlying clonal cooperation will inform potential chemoprevention strategies for CRC. Citation Format: Sanne van Neerven. Cell competition vs cell cooperation: Unlocking oncogenic potential of mutant clones in the gut [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 SY07-02.

Abstract 4099: AMP-peptide vaccination against multiple p53 mutant epitopes promotes lymph node delivery to generate potent, functional T cell immunity

Abstract Background p53 is the most commonly mutated gene in human cancer, prevalent in nearly all tumor types. Despite its significant medical relevance and the knowledge that human T cells recognize these mutations[1,2], immunotherapies designed to promote responses against mutated p53 (mp53) have not had the hoped-for clinical impact[3]. The Amphiphile (AMP) platform improves the potency of vaccine immunotherapy by programming the delivery of vaccine components to the lymph nodes where efficient uptake by immune cells initiates tumor-targeted immune responses. AMP-modification of vaccine components (peptide antigens, molecular adjuvants) results in covalent conjugation to albumin-binding lipids. Upon injection, AMP-vaccines associate with tissue-resident albumin which efficiently distributes to draining lymph nodes. This approach was shown to promote activation of polyfunctional, cytotoxic T cells with promising safety and improved clinical outcomes in a Phase 1 trial of ELI-002, an mKRAS AMP therapeutic vaccine (AMPLIFY-201 NCT05726864). Application of this strategy to p53 mutations with ELI-008 offers the potential for improved immunotherapeutic activity in a setting of significant unmet need. Methods Following immunization of C57BL/6J mice with AMP-modified or soluble comparator vaccines consisting of mp53 peptides and CpG adjuvant, analyses were performed 7 days after the third bi-weekly dose. To assess antigen-specific T cell responses, ELISpot (IFNγ, GzmB), multiplexed proteomic, and flowcytometric analysis of effector cytokines (IFNγ, TNFα, IL-2) were performed following antigenic stimulation. Responses were determined in secondary lymphoid tissues and lung. Cytolytic capabilities of antigen-specific T cells were evaluated by monitoring specific killing of IV-transferred antigen-pulsed target cells. Results AMP-immunization generated robust immune responses yielding strong T cell activation against common p53 hot spot mutations (R248W, R248Q, R175H, G245S, R273H, Y220C, C135Y, R158H, H214R). Responses to AMP-vaccination were characterized by the generation of increased frequency of polyfunctional T cells (IFNγ, TNFα, IL2) specific to mp53 epitopes. Induced T cells demonstrated significant levels of cytolytic activity including GzmB production and elimination of target cells in vivo. Non-lymph targeted vaccines using soluble comparators were inactive. Conclusions AMP-conjugation permits efficient delivery directly to the lymph nodes and thus improves the immunogenicity of peptide vaccination. These substantially improved immune responses induced by ELI-008 represent a promising therapeutic opportunity for targeting cancer in a large fraction of human tumors. The AMP-platform technology is simple, rapid and scalable, promising broad clinical, off-the-shelf application for treating p53 mutated tumors. Citation Format: Martin P. Steinbuck, Xavier Cabana-Puig, Erica Palmer, Mimi M. Jung, Thomas Williams, Kristen Osaer, Jeff Zhang, Christopher M. Haqq, Peter C. DeMuth. AMP-peptide vaccination against multiple p53 mutant epitopes promotes lymph node delivery to generate potent, functional T cell immunity [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 4099.

Abstract 6060: Discovery of potent SHP2 protein degraders with strong anti-tumor activities in KRAS mutant cancers

Abstract Background: SHP2 plays a pivotal role in modulating the RTK/RAS/MAPK signaling pathway, which is frequently dysregulated in various cancers. Multiple allosteric SHP2 inhibitors have been under active development for the treatment of RTK/RAS/MAPK-dependent cancers, while certain limitations have come to light, such as their inability to target the active form of SHP2 and narrow therapeutic index necessitating intermittent dosing to mitigate toxicity. To overcome these challenges, we have discovered SHP2 protein degraders as next generation SHP2 targeting agents. Here, we present compelling profiles of our SHP2 degraders. Methods: We employed a variety of cancer cell lines harboring mutations in RTK, KRAS, or SHP2 for assessing in vitro activity of SHP2 degraders. For in vivo assessment of pharmacological profile, we utilized an NCI-H358 KRAS G12C mutant NSCLC xenograft. SHP2 protein levels in cell lysates and tumors were analyzed to assess SHP2 degradation efficiency. Simultaneously, ERK phosphorylation and DUSP6 mRNA levels were analyzed to evaluate downstream effects. Immunoprecipitation assays were used to assess the impact on SHP2's scaffolding roles. Results: A series of SHP2 degraders have been identified, demonstrating highly efficient SHP2 degradation activity with DC50 values in subnanomolar ranges. Robust inhibition of ERK phosphorylation, followed by remarkable anti-cancer effects in multiple KRAS mutant cancer cell lines were observed. Interestingly, in vitro cell growth inhibition by SHP2 degraders are 10 times more potent than SHP2 inhibitors currently under clinical development. Further characterization reveals several advantages of SHP2 degraders compared to SHP2 inhibitors. First, we observed a more efficient disruption of SHP2 scaffolding function, such as the formation of the SHP2-GRB2-GAB1 complex. Second, we detected effective degradation of activating mutants of SHP2, such as E76K and T507K, which are typically not targeted by SHP2 inhibitors. Experiments using a KRAS mutant CDX model demonstrate compelling in vivo profiles. A single administration of lead compounds resulted in Dmax values exceeding 95% in tumors, accompanied by the significant reduction of downstream signaling markers, such as p-ERK and DUSP6 mRNA. Notably, the reduced levels of SHP2 and downstream markers persist for several days. This suggests that utilizing intermittent dosing could be a viable approach with SHP2 degraders. Once-weekly intravenous administration achieved strong tumor growth inhibition without causing significant body weight loss. Conclusion: We have successfully discovered novel SHP2 degraders exhibiting remarkable activities. We demonstrate that SHP2 degraders have potential to overcome the limitations associated with allosteric SHP2 inhibitors and provide a promising therapeutic option for RTK/RAS/MAPK-dependent cancer patients. Citation Format: So Hyun Kim, Jihye Lee, Jiyoung Song, Geon-Tae Park, Jieun Jung, Jungtae Na, Dong Ho Lee, Soohyun Lee, Ji Youn Park, Jiyoung Kim, Onnuri Bae, Jeonghwa Han, Ju Young Kang, Mijin Moon, Yongje Shin, Je Ho Ryu, Song Hee Lee, Sook-Kyung Park. Discovery of potent SHP2 protein degraders with strong anti-tumor activities in KRAS mutant cancers [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 6060.

Abstract 6511: BPI-520105, a highly potent, CNS-penetrant 4th generation EGFR inhibitor overcoming major EGFR resistance mutations in NSCLC

Abstract EGFR activating mutations are prevalent in 10-50% of NSCLC patients. The most common EGFR mutations, L858R (L) and Del19 (D), initially respond to first-, second-, or third-generation (1G, 2G and 3G) EGFR-TKIs. However, on-target resistance, primarily by T790M (T) and C797X(C), inevitably emerges post-treatment and leads to disease progression. Different treatment sequence give rise to different complex mutations. In patients resistant to the 3G TKI osimertinib, double mutations L858R/C797X or Del19/C797X (LC or DC) were observed at a frequency of up to 12% after first-line osimertinib, and triple mutations L858R/T790M/C797S or Del19/T790M/C797S (LTC or DTC) were up to 20% after second-line osimertinib. There are no approved targeted therapy options for these complex resistant mutations. In this study, we developed BPI-520105, a 4th generation EGFR inhibitor capable of targeting all aforementioned major forms of EGFR activating and resistance mutations in NSCLC. It might be an optimal way to address this urgent medical need. In vitro, BPI-520105 potently inhibited the activity of a variety of EGFR kinases, as well as inhibited the proliferation and EGFR phosphorylation in cell lines bearing relative mutations, including single (D, L), double (DT, LT, and DC, LC) and triple (LTC, DTC) mutations. Meanwhile, BPI-520105 exhibited relative weakness towards wild type EGFR and IGF1R, demonstrating good selectivity in kinase and cell-based assays. Despite being a reversible EGFR inhibitor, BPI-520105 sustained the inhibition of pEGFR and pERK for up to 24 hours in the western blot assay. In vivo, oral administration of BPI-520105 displayed significant dose-dependent antitumor effect in multiple EGFR mutant xenograft models, including HCC827 (D), NCI-H1975 (LT), BaF3-EGFR-LC/DC, and BaF3-EGFR-LTC models. Remarkably, BPI-520105 significantly prolonged the lifespan of mice with brain orthotropic BaF3-EGFR-DTC-Luc2 allografts, demonstrating its activity towards brain metastasis. Additionally, BPI-520105 exhibited favorable ADME properties, with high oral exposure across various preclinical species. In summary, BPI-520105 is a highly potent, CNS-penetrant, wide type sparing, and orally bioavailable 4th generation EGFR inhibitor, holding promise for the treatment of NSCLC patients with resistance mutations post 3G progression or even at front line. Citation Format: Jing Guo, Lijia Liu, Xiangyong Liu, Bang Fu, Guangcan Bai, Changyong Qiu, Wei Ren, Xiaodong Song, Guolong Du, Xiao Sun, Yongchun Zhang, Weidong Cai, Pingping Wang, Haibo Chen, Xiaoyun Liu, Zhengyao Zou, Jiayu Zhao, Xuepeng Jv, Hong Lan, Lieming Ding, Jiabing Wang. BPI-520105, a highly potent, CNS-penetrant 4th generation EGFR inhibitor overcoming major EGFR resistance mutations in NSCLC [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 6511.