Point Mutations In Domain Research Articles

The NEDD4-binding protein N4BP1 degrades mRNA substrates through the coding sequence independent of nonsense-mediated decay

3'-Untranslated regions (3'UTRs) are recognized for their role in regulating mRNA turnover while the turnover of a specific group of mRNAs mediated by coding sequences (CDS) remains poorly understood. N4BP1 is a critical inflammatory regulator in vivo with a molecular mechanism that is not yet clearly defined. Our study reveals that N4BP1 efficiently degrades its mRNA targets via CDS rather than the 3'-UTR. This CDS-dependent mRNA turnover mechanism appears to be a general feature of N4BP1, as evidenced by testing multiple mRNA substrates, such as Fos-C, Fos-B, Jun-B and CXCL1. Detailed mapping of the motif identified a crucial 33nt (289-322) sequence near the 5'-end of Fos-C-CDS, where the presence of polyC is necessary for N4BP1-mediated degradation. Functional studies involving domain deletion and point mutations showed that both the KH and NYN domains are essential for N4BP1 to restrict mRNA substrates. The function of N4BP1 in mRNA turnover is not dependent on nonsense-mediated decay as it efficiently restricts mRNA substrates even in cells deficient in UPF1, UPF3A, and UPF3B. Additionally, the function of N4BP1 is not reliant on LUC7L3 despite its known association with this protein. Our findings suggest that N4BP1 acts as an endoribonuclease to degrade mRNA substrates primarily through coding sequences containing a C-rich motif.

The Molecular and Biological Patterns Underlying Sustained SARS-CoV-2 Circulation in the Human Population

For four years, SARS-CoV-2, the etiological agent of COVID-19, has been circulating among humans. By the end of the second year, an absence of immunologically naive individuals was observed, attributable to extensive immunization efforts and natural viral exposure. This study focuses on delineating the molecular and biological patterns that facilitate the persistence of SARS-CoV-2, thereby informing predictions on the epidemiological trajectory of COVID-19 toward refining pandemic countermeasures. The aim of this study was to describe the molecular biological patterns identified that contribute to the persistence of the virus in the human population. For over three years since the beginning of the COVID-19 pandemic, molecular genetic monitoring of SARS-CoV-2 has been conducted, which included the collection of nasopharyngeal swabs from infected individuals, assessment of viral load, and subsequent whole-genome sequencing. We discerned dominant genetic lineages correlated with rising disease incidence. We scrutinized amino acid substitutions across SARS-CoV-2 proteins and quantified viral loads in swab samples from patients with emerging COVID-19 variants. Our findings suggest a model of viral persistence characterized by 1) periodic serotype shifts causing substantial diminutions in serum virus-neutralizing activity (> 10-fold), 2) serotype-specific accrual of point mutations in the receptor-binding domain (RBD) to modestly circumvent neutralizing antibodies and enhance receptor affinity, and 3) a gradually increasing amount of virus being shed in mucosal surfaces within a single serotype. This model aptly accounts for the dynamics of COVID-19 incidence in Moscow. For a comprehensive understanding of these dynamics, acquiring population-level data on immune tension and antibody neutralization relative to genetic lineage compositions is essential.

Geospatial share of fungicide resistant Botrytis cinerea mutations in the Tokaj and Eger wine regions according to local pest management strategies

AbstractBotrytis cinerea is one of the fungal pathogens with the widest host plant spectrum, causing serious yield losses and significant economic damage in vineyards from year to year. As an ubiquitous, polyphagous fungal pathogen, with both saprophytic and parasitic lifestyle. The sequential use of active substances belonging to the same chemical family to protect vineyards can lead to an increase in fungal chemical resistance, which is reflected in the enrichment of point mutations in the genomic regions coding proteins involved in the mechanism of action of different pesticides. The aim of our studies was to compare the sensitivity to different fungicides of B. cinerea populations in two wine regions with different pest management practices: the Tokaj region, where the presence of B. cinerea is necessary to produce noble rot wines, and the adjacent Eger Region, where a total protection against B. cinerea is desired. Our study is the first Hungarian report of some previously studied resistance mutations in ERG27 and SDHB protein-coding genes. We identified point mutations in ERG27 transmembrane domain that have not been previously described but may affect the emergence of resistance to certain fungicides. Our study shows that the B. cinerea population of the Northern Hungary region is consistently characterized by an increase in fenhexamid resistance.

Mycobacterium smegmatis putative Holliday junction resolvases RuvC and RuvX play complementary roles in the processing of branched DNA structures

In eubacteria, Holliday junction (HJ) resolvases (HJRs) are crucial for faithful segregation of newly replicated chromosomes, homologous recombination and repair of stalled/collapsed DNA replication forks. However, compared with the Escherichia coli HJRs, little is known about their orthologs in mycobacterial species. A genome-wide analysis of Mycobacterium smegmatis identified two genes encoding putative HJRs, namely RuvC (MsRuvC) and RuvX (MsRuvX); but whether they play redundant, overlapping, or distinct roles remains unknown. Here, we reveal that MsRuvC exists as a homodimer while MsRuvX as a monomer in solution, and both showed high-binding affinity for branched DNAs compared with unbranched DNA species. Interestingly, the DNA cleavage specificities of MsRuvC and MsRuvX were found to be mutually exclusive: the former efficiently promotes HJ resolution, in a manner analogous to the E. coli RuvC, but does not cleave other branched DNA species; whereas the latter is a versatile DNase capable of cleaving a variety of branched DNA structures, including 3' and 5' flap DNA, splayed-arm DNA and double-stranded DNA with 3' and 5' overhangs but lacks the HJ resolution activity. Point mutations in the RNase H-like domains of MsRuvC and MsRuvX pinpointed critical residues required for their DNA cleavage activities, and also demonstrated uncoupling between DNA-binding and DNA cleavage activities. Unexpectedly, we found robust evidence that MsRuvX possesses a double-strand/single-strand junction-specific endonuclease and single-stranded DNA exonucleolytic activities. Combined, our findings highlight that the RuvC and RuvX DNases play distinct complementary, and not redundant, roles in the processing of branched DNA structures in M. smegmatis.

Drosophila model to clarify the pathological significance of OPA1 in autosomal dominant optic atrophy.

Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase (OPA1) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 (hOPA1) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1. This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies.

Antimicrobial resistance pattern of Helicobacter pylori in patients evaluated for dyspeptic symptoms in North-Eastern India with focus on detection of clarithromycin resistance conferring point mutations A2143G and A2142G within bacterial 23S rRNA gene

PurposeIn India there is evidence of antimicrobial resistance in Helicobacter pylori, a definitive pathobiont whose only known niche is human gastric mucosa. This in turn can lead to failure of treatment, persistence or chronicity of infection. This hospital based, prospective, observational study investigates the presence of antimicrobial resistance in the organism with focus on detection of A2143G and A2142G major point mutations in domain V of H. pylori 23S rRNA gene as a molecular mechanism of conferring resistance. MethodsEndoscopic gastric biopsy samples from 52 patients presenting with dyspeptic symptoms from January 2016 to December 2016 were subjected to culture in a microaerophilic environment using Campylobacter agar with for 2–5 days. Isolates were identified using gram-staining, motility test and biochemical reactions. Modified Kirby-Bauer Disc diffusion method was used to determine antimicrobial susceptibility against Clarithromycin, Metronidazole, Amoxycillin, Levofloxacin, Tetracycline, Cotrimoxazole and Erythromycin. Additionally, detection of A2143G and A2142G point mutations conferring Clarithromycin resistance was carried out using real time PCR following extraction and quantification of bacterial DNA. Histopathological examination was carried out on all biopsy samples. Descriptive and inferential statistical analytical methods were used. Differences were considered significant for p < 0.05. ResultsCulture positivity for H. pylori by phenotypic method was found to be 36.54%. Histopathologic Examination detected H. pylori in 55.7% and PCR detected 48.08% for either the wild type or one of two mutant strains A2143G and A2142G. No sample was found positive for both mutations. Metronidazole showed the highest resistance among antibiotics (78.9%) followed by Clarithromycin (47.3%). ConclusionPrevalence of antimicrobial resistance in H. pylori in North-Eastern India is substantially high with A2143G mutation being clinically most important in conferring Clarithromycin resistance. This resistance might be associated with low eradication rates despite initiation of therapy. ROC analysis of PCR proved it to be a good diagnostic tool.

C. elegans touch receptor neurons direct mechanosensory complex organization via repurposing conserved basal lamina proteins

The sense of touch is conferred by the conjoint function of somatosensory neurons and skin cells. These cells meet across a gap filled by a basal lamina, an ancient structure found in metazoans. Using Caenorhabditis elegans, we investigate the composition and ultrastructure of the extracellular matrix at the epidermis and touch receptor neuron (TRN) interface. We show that membrane-matrix complexes containing laminin, nidogen, and the MEC-4 mechano-electrical transduction channel reside at this interface and are central to proper touch sensation. Interestingly, the dimensions and spacing of these complexes correspond with the discontinuous beam-like extracellular matrix structures observed in serial-section transmission electron micrographs. These complexes fail to coalesce in touch-insensitive extracellular matrix mutants and in dissociated neurons. Loss of nidogen reduces the density of mechanoreceptor complexes and the amplitude of the touch-evoked currents they carry. Thus, neuron-epithelium cell interfaces are instrumental in mechanosensory complex assembly and function. Unlike the basal lamina ensheathing the pharynx and body wall muscle, nidogen recruitment to the puncta along TRNs is not dependent upon laminin binding. MEC-4, but not laminin or nidogen, is destabilized by point mutations in the C-terminal Kunitz domain of the extracellular matrix component, MEC-1. These findings imply that somatosensory neurons secrete proteins that actively repurpose the basal lamina to generate special-purpose mechanosensory complexes responsible for vibrotactile sensing.

Covalent-103: A phase 1, open-label, dose-escalation and expansion study of BMF-500, an oral covalent FLT3 inhibitor, in adults with acute leukemia (AL).

TPS6589 Background: FLT3 mutations occur in 25-35% of AML and result from an internal tandem duplication (ITD) of amino acids in the juxtamembrane domain of FLT3 kinase or point mutations in the tyrosine kinase domain (TKD). FLT3-ITD mutations are associated with increased relapse, shorter remission, and decreased disease-free and overall survival. BMF-500 is a novel oral, highly potent and selective covalent inhibitor of FLT3 including wild-type (WT), ITD, TKD, and resistance mutations (e.g., gatekeeper F691). BMF-500 has high affinity for FLT3, lack of cKIT inhibition, and sustained cell-killing despite drug washout (Law et al., ASH 2022 Abst 2756). BMF-500 shows sustained tumor regression and improved survival in subcutaneous and disseminated xenografts of FLT3-m AML. Methods: COVALENT-103 (NCT05918692) is a multicenter, first-in-human study evaluating the safety, tolerability, and antileukemic activity of escalating doses of daily BMF-500 in patients with relapsed or refractory (R/R) AL, including AML, ALL, or MPAL, with or without FLT3-m. The trial has 2 arms that dose escalate in parallel: Arm A (without) and Arm B (with) concomitant use of a CYP3A4 inhibitor. Using an accelerated titration design (ATD), doses of BMF-500 are escalated in single-subject cohorts until one subject experiences ≥Grade 2 related adverse event or dose-limiting toxicity (DLT). At that point, the cohort will switch to a classical “3 +3” design. Patients with WT FLT3 AL may enroll up to 33% per arm. Treatment continues in 28-day cycles until progression or intolerability. Expansion cohorts will enroll additional patients to obtain further safety and efficacy data. Patients must be refractory, relapsed or have progressed on or following discontinuation of the most recent anti-cancer therapy or be ineligible for any approved standard of care, including hematopoietic stem cell transplant (HSCT). FLT3-positive AML patients must have received a FLT3 inhibitor approved for R/R FLT3-m AML. Additional inclusion criteria include ECOG PS ≤2, adequate organ function, and documented FLT3 mutation status. Key exclusion criteria include known CNS disease, clinically significant cardiovascular disease, and WBC &gt;50,000/µL (uncontrollable with cytoreductive therapy). The primary objective is to evaluate safety and tolerability and determine the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-500 monotherapy based on available pharmacokinetic/pharmacodynamic (PK/PD), safety and efficacy data. Secondary objectives include characterization of the PK/PD of BMF-500, and assessment of its antitumor activity per modified Cheson (2003) criteria or NCCN Clinical Practice Guidelines (ALL Version 1.2022) as determined by the investigator. The study was initiated in July 2023, is currently in dose escalation, and we plan to enroll approximately 110 patients total. Clinical trial information: NCT05918692 .

GATA binding protein 2 mediated ankyrin repeat domain containing 26 high expression in myeloid-derived cell lines.

Thrombocytopenia 2, an autosomal dominant inherited disease characterized by moderate thrombocytopenia, predisposition to myeloid malignancies and normal platelet size and function, can be caused by 5'-untranslated region (UTR) point mutations in ankyrin repeat domain containing 26 (ANKRD26). Runt related transcription factor 1 (RUNX1) and friend leukemia integration 1 (FLI1) have been identified as negative regulators of ANKRD26. However, the positive regulators of ANKRD26 are still unknown. To prove the positive regulatory effect of GATA binding protein 2 (GATA2) on ANKRD26 transcription. Human induced pluripotent stem cells derived from bone marrow (hiPSC-BM) and urothelium (hiPSC-U) were used to examine the ANKRD26 expression pattern in the early stage of differentiation. Then, transcriptome sequencing of these iPSCs and three public transcription factor (TF) databases (Cistrome DB, animal TFDB and ENCODE) were used to identify potential TF candidates for ANKRD26. Furthermore, overexpression and dual-luciferase reporter experiments were used to verify the regulatory effect of the candidate TFs on ANKRD26. Moreover, using the GENT2 platform, we analyzed the relationship between ANKRD26 expression and overall survival in cancer patients. In hiPSC-BMs and hiPSC-Us, we found that the transcription levels of ANKRD26 varied in the absence of RUNX1 and FLI1. We sequenced hiPSC-BM and hiPSC-U and identified 68 candidate TFs for ANKRD26. Together with three public TF databases, we found that GATA2 was the only candidate gene that could positively regulate ANKRD26. Using dual-luciferase reporter experiments, we showed that GATA2 directly binds to the 5'-UTR of ANKRD26 and promotes its transcription. There are two identified binding sites of GATA2 that are located 2 kb upstream of the TSS of ANKRD26. In addition, we discovered that high ANKRD26 expression is always related to a more favorable prognosis in breast and lung cancer patients. We first discovered that the transcription factor GATA2 plays a positive role in ANKRD26 transcription and identified its precise binding sites at the promoter region, and we revealed the importance of ANKRD26 in many tissue-derived cancers.

Current knowledge about FLT3 gene mutations, exploring the isoforms, and protein importance in AML.

Acute myeloid leukaemia (AML) is a complex haematological malignancy characterised by diverse genetic alterations leading to abnormal proliferation of myeloid precursor cells. One of the most significant genetic alterations in AML involves mutations in the FLT3 gene, which plays a critical role in haematopoiesis and haematopoietic homeostasis. This review explores the current understanding of FLT3 gene mutations and isoforms and the importance of the FLT3 protein in AML. FLT3 mutations, including internal tandem duplications (FLT3-ITD) and point mutations in the tyrosine kinase domain (FLT3-TKD), occur in 25-30% in AML and are associated with poor prognosis. FLT3-ITD mutations lead to constitutive activation of the FLT3 signalling pathway, promoting cell survival and proliferation. FLT3-TKD mutations affect the tyrosine kinase domain and affect AML prognosis in various ways. Furthermore, FLT3 isoforms, including shorter variants, contribute to the complexity of FLT3 biology. Additionally, nonpathological polymorphisms in FLT3 are being explored for their potential impact on AML prognosis and treatment response. This review also discusses the development of molecular treatments targeting FLT3, including first-generation and next-generation tyrosine kinase inhibitors, highlighting the challenges of resistance that often arise during therapy. The final chapter describes FLT3 protein domain rearrangements and their relevance to AML pathogenesis.

Activating Point Mutations in the MET Kinase Domain Represent a Unique Molecular Subset of Lung Cancer and Other Malignancies Targetable with MET Inhibitors.

Activating point mutations in the MET tyrosine kinase domain (TKD) are oncogenic in a subset of papillary renal cell carcinomas. Here, using comprehensive genomic profiling among >600,000 patients, we identify activating MET TKD point mutations as putative oncogenic driver across diverse cancers, with a frequency of ∼0.5%. The most common mutations in the MET TKD defined as oncogenic or likely oncogenic according to OncoKB resulted in amino acid substitutions at positions H1094, L1195, F1200, D1228, Y1230, M1250, and others. Preclinical modeling of these alterations confirmed their oncogenic potential and also demonstrated differential patterns of sensitivity to type I and type II MET inhibitors. Two patients with metastatic lung adenocarcinoma harboring MET TKD mutations (H1094Y, F1200I) and no other known oncogenic drivers achieved confirmed partial responses to a type I MET inhibitor. Activating MET TKD mutations occur in multiple malignancies and may confer clinical sensitivity to currently available MET inhibitors. Significance: The identification of targetable genomic subsets of cancer has revolutionized precision oncology and offers patients treatments with more selective and effective agents. Here, we demonstrate that activating, oncogenic MET tyrosine kinase domain mutations are found across a diversity of cancer types and are responsive to MET tyrosine kinase inhibitors.

Abstract 2751: Functional assessment of STK11 C-terminal domain variants

Abstract STK11 mutations in KRAS-driven lung adenocarcinomas (LUADs) are associated with aggressive tumor phenotypes characterized by increased risk of metastasis and lower overall and progression free survival. STK11 is a serine-threonine kinase that is comprised of an N-terminal domain containing a nuclear localization signal, a kinase domain, and a C-terminal domain (CTD). In LUAD, STK11 loss of function impacts many aspects of coordinated cell motility and promotes alterations characteristic of metastasis including cell polarity and adhesion. Both kinase activity and subcellular localization are important for STK11 tumor suppressor function. Our objective was to develop a reliable method to identify pathogenic STK11 mutations. Using archival data from the University of Vermont Medical Center, we identified 28 STK11 missense variants that had not yet been functionally characterized. We evaluated the functional impact of these variants on STK11 kinase activity through detection of autophosphorylation using an in-vitro kinase assay, as well as their ability to induce p53 activity through a luciferase reporter. Of the variants studied, 5 were CTD mutations. These variants retained kinase activity and the ability to bind STK11’s partners STRAD-alpha and MO25 in co-immunoprecipitation assays. Although the CTD of STK11 has no catalytic activity, it is thought to be integral for translocation to the cytoplasm where it can colocalize with actin and the cell membrane. STK11 dependent regulation of directional migration requires both proper localization to the actin cytoskeleton and kinase activity. Moreover, it has been reported that the C-terminal polybasic motif of STK11 (aa403-426), consisting of 3 Lysine and 5 Arginine residues is key for its localization and activation of AMPK at the plasma membrane. Therefore, when ascertaining functional status of STK11 in vivo, it is important to consider kinase independent STK11 functions. In LUAD cell lines, we show through immunofluorescent microscopy that expression of a truncated construct encoding STK11 with a CTD deletion results in nuclear sequestration. Additionally, point mutants in 3 of the polybasic motif residues (R409W, K416E and K423E) retain kinase activity and STRAD-alpha binding, but also lead to nuclear sequestration - highlighting the importance of a multipronged approach when assessing STK11 function. Future studies will assess the ability of these C-terminal domain mutants to phosphorylate cytoplasmic substrates, and their impact on cell migration and invasion. Additionally, we will utilize the TurboID proximity ligation system to assess the effect of C-terminal domain point mutations on binding partner accessibility. Citation Format: Gopika Nandagopal, Sean Lenahan, Hannah Ross, Hailey Sarausky, David Seward, Paula Deming. Functional assessment of STK11 C-terminal domain variants [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 2751.

Observational retrospective clinical study on clinical features of macrolide-resistant Mycoplasma pneumoniae pneumonia in Chinese pediatric cases

This study aimed to investigate differences in clinical characteristics and laboratory findings between children infected with Macrolide-Sensitive Mycoplasma pneumoniae (MSMP) and Macrolide-Resistant Mycoplasma pneumoniae (MRMP). Additionally, the research sought to identify laboratory markers for rapidly distinguishing refractory Mycoplasma pneumoniae pneumonia (RMPP) from ordinary Mycoplasma pneumoniae pneumonia (OMPP). In total, 265 Mycoplasma pneumoniae (MP) patients were included, with MRMP identified by specific point mutations in domain V of the 23S rRNA gene. A retrospective analysis compared the clinical courses and laboratory data, revealing that MRMP patients experienced prolonged febrile days (P = 0.004), elevated CRP levels (P < 0.001), and higher MP DNA loads than MSMP patients (P = 0.037). Based on clinical symptoms, MRMP was divided into RMPP (n = 56) and OMPP (n = 70), with RMPP demonstrating significantly increased IL-18, community-acquired respiratory distress syndrome (CARDS) toxins in nasopharyngeal aspirate, and serum CRP levels (P < 0.001; P = 0.006; P < 0.001). In conclusion, timely recognition of RMPP is crucial for enhancing prognosis. The identification of MRMP, coupled with proinflammatory cytokines such as IL-18, CARDS toxins, and CRP, emerges as promising markers with the potential to contribute significantly to diagnostic accuracy and prognosis assessment.

Abstract B057: Oncogenic Foxl2 directs enhancer reprogramming and changes in 3D genome structure in ovarian granulosa cell tumors

Abstract Background: Adult-type granulosa cell tumors (aGCTs) are a rare monogenic disease. Nearly all aGCTs carry a missense point mutation in the Forkhead domain containing FOXL2 (Foxl2 p.C134W) transcription factor, but the oncogenic mechanism of this mutation is unknown. Other Forkhead family transcription factors have well-described “pioneer” activity, binding to compacted, nucleosome-bound DNA and increasing accessibility for other regulatory proteins. Objectives: To investigate if oncogenic Foxl2-C134W has pioneering activity and if mutant Foxl2 operates its oncogenic activity through enhancer reprogramming. Methods: CRISPR/Cas9 editing was used to generate isogenic granulosa cell tumor cells lacking either the FOXL2 wild-type allele (single knock-out; SKO) or both the mutant and wild-type FOXL2 alleles (double knock-out; DKO). ATAC-Seq and endogenous Foxl2 ChIP-Seq were performed on these isogenic lines to determine the differential chromatin accessibility at Foxl2-bound regulatory regions across genotypes. Promoter capture HiC was performed using a bespoke genome-wide hybrid capture probe set, CHiCAGO was used to identify significant HiC interactions, and an activity-by-contact model was constructed to identify enhancer elements bound by endogenous Foxl2-C134W and determine their target promoter contact frequency (promoter capture HiC) and enhancer activity (geometric mean of H3K27ac and ATACseq signal. Results: Endogenous ChIP-seq of Foxl2-C134W from the SKO cell line identified 1147 high-confidence peaks. De novo motif discovery identified the canonical Foxl2 binding motif (TGTTTACATT, P = 1.4 × 10−7) in 44.9% of peaks, as well as a novel variant motif (TGTTTTGTCT, P = 6.7 × 10−15) in 68.5% of peaks. Median chromatin accessibility at Foxl2-C134W peak regions, as measured by ATAC-seq, was significantly decreased in the DKO cells compared to either SKO or parental cell lines (P &amp;lt; 2 × 10-16 for both comparisons). Decreased chromatin accessibility at Foxl2-C134W ChIP-seq peaks in the DKO cells was driven by peaks containing the novel variant Foxl2 binding motif. Promoter capture HiC through activity-by-contact model identified promoter-enhancer interactions lost in the DKO cell line involving several genes, including ADAMTS5, FOXF2, and PRICKLE1 (adjusted P &amp;lt; 0.05). Conclusion: Foxl2-C134W exhibits “pioneering” activity, increasing chromatin accessibility at key gene regulatory elements with associated re-programming of promoter-enhancer interactions. The oncogenic mechanism of Foxl2-C134W in granulosa cell tumors may involve changes in DNA binding specificity, re-directing this pioneering function to sites containing a novel variant Foxl2 binding motif. Citation Format: Veena K. Vuttaradhi, Eleonora Khlebus, Thomas Welte, Barrett Lawson, Robert Tyler Hillman. Oncogenic Foxl2 directs enhancer reprogramming and changes in 3D genome structure in ovarian granulosa cell tumors [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B057.

Characterization of a Novel Mutation in the COPI Vesicle on Binding to Dilysine Motifs

Background/Objective: The heptameric COPI coatomer complex is involved in the formation of vesicles and the intracellular trafficking of proteins between the Golgi and Endoplasmic Reticulum as well as throughout the cytoplasm. Members of the COPI complex bind dilysine motifs found in the C-terminal domain of the cargo protein, particularly KKxx or KxKxx. We generated a point mutation in the WD40 domain of the COPI alpha subunit (⍺-COP). We hypothesized that the E269V mutant ⍺-COP would not co-immunoprecipitate (co-IP) COPI cargo proteins terminating with a dilysine domain of KxKxx (Nucleolin and Stasimon/Tmem41b), but would bind cargo proteins terminating in KKxx (FLAGSyntaxin17). We predicted that a mutation in the ⍺-COP C-terminus, which impairs interaction with ε–COP, would not affect its ability to co-IP dilysine-containing cargo.&#x0D; Experimental Design or Project Methods: HEK-293TT cells were transfected with Myctagged wild-type, E269V, and triple mutant (3X) ⍺-COP. The E269V ⍺-COP mutant has an amino acid change at position 269 from glutamic acid to valine. The triple ⍺-COP mutant has three amino acid changes that eliminate binding with the ε–COP COPI subunit. Transfected ⍺-COP was immunoprecipitated using magnetic anti-Myc beads. Endogenous Nucleolin was immunoprecipitated using magnetic Protein A beads conjugated to rabbit polyclonal anti- Nucleolin antibody. Western blots of inputs and immunoprecipitates of each experiment were conducted to determine the ability of ⍺-COP to co-IP C-terminal dilysine-containing proteins.&#x0D; Results: Endogenous Nucleolin and Stasimon co-immunoprecipitated with WT and 3X ⍺-COP, but not E269V ⍺-COP.&#x0D; Conclusions and Potential Impact: The inability of mutation E269V to co-IP dilysine proteins implies that the WD40 domain of the COPI ⍺-COP protein is required for binding to KxKxxterminating proteins, as typified by Nucleolin and Stasimon.The C-terminal 3X mutation shows that ε-COP is not necessary for dilysine recognition and implies that ⍺-COP directly binds to this KxKxx motif.

Abstract B129: A bispecific antibody inhibits FGFR3 dimerization and is highly effective in FGFR3-driven tumor models

Abstract Background: The receptor tyrosine kinase (RTK) FGFR3 is frequently mutated in bladder cancer (~15-20% in muscle invasive bladder cancer, MIBC) and is a validated therapeutic target. Oncogenic alterations in FGFR3 include point mutations in the extracellular domain (S249C, R248C, G372C, Y375C) and gene fusions that place an oligomerization domain from a fusion partner at the FGFR3 C-terminus (e.g., FGFR3-TACC3). Although pan-FGFR tyrosine kinase inhibitors (TKI) have demonstrated clinical efficacy, toxicity and acquired resistance limit the benefit of these agents. While antibody-based therapy can offer superior selectivity compared to TKIs, conventional ligand-blocking antibodies are usually ineffective inhibitors of constitutively active RTKs. Further, the existence of multiple oncogenic variants of FGFR3 presents an additional challenge for antibody-mediated blockade. Results: We generated a tetravalent bispecific FGFR3xFGFR3 antibody that potently inhibits both FGFR3 point mutants and fusions with better efficacy than the conventional antibodies that we produced and a clinical-stage comparator antibody. Mechanistically, our bispecific antibody binds FGFR3 through two different epitopes located in immunoglobulin-like domains D1 and D2/3 respectively. It potently blocks the dimerization of FGFR3 S249C, the most common oncogenic variant, inhibiting receptor phosphorylation and downstream signaling. Additionally, our bispecific antibody inhibits the function of FGFR3 gatekeeper mutations (V557M/L) that are resistant to TKIs. In FGFR3-driven tumor models, our antibody is highly effective, providing efficacy similar to that of the FDA-approved TKI erdafitinib. Conclusion: We have characterized the properties of a tetravalent bispecific FGFR3xFGFR3 antibody that shows strong anti-tumor activity in models driven by FGFR3 S249C as well as by FGFR3-TACC3 fusion, outperforming a conventional antibody that was previously in clinical development. The activity of the bispecific antibody is superior to the mixture of the two parental antibodies and results from strong inhibition of FGFR3 dimerization. Our bispecific antibody provides a novel approach to achieve selective and potent inhibition of multiple oncogenic variants of FGFR3 in bladder cancer, including FGFR3 variants that are resistant to FGFR TKIs. Citation Format: Yan Yang, Naga Suhasini Avvaru, Zaoli Jiang, Nina Liu, Michael Rosconi, Bojie Zhang, Yinyin Li, Erica Ullman, John Chia-Yang Lin, Yang Shen, Christopher Daly. A bispecific antibody inhibits FGFR3 dimerization and is highly effective in FGFR3-driven tumor models [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 B129.

Combinatorial Treatment Options for Highly Resistant Compound Mutations in the Kinase Domain of the BCR::ABL1 Fusion Gene

The introduction of tyrosine kinase inhibitor (TKI)-based therapies has dramatically changed the outcome in patients with chronic myeloid leukemia (CML) and Ph-positive acute lymphoblastic leukemia (Ph+ALL). However, the occurrence of point mutations in the kinase domain (KD) of the BCR::ABL1 fusion gene can impair or inhibit the efficacy of individual TKIs. In advanced phases of CML and in Ph +ALL, patients frequently develop compound mutations (CMs), defined as the presence of more than one mutation on the same BCR::ABL1 molecule in a leukemic cell, which are often highly or even completely resistant to any individual TKI. Some CMs respond to high doses of the currently most potent available TKI, ponatinib, but the dose-related toxicity is an important impediment. The most frequently occurring CMs, which include the gatekeeper mutation T315I, are often resistant to any currently available TKI. We performed in vitro analyses using a common cell line model based on Ba/F3 cells expressing a panel of 12 different CMs (V299L/G250E, V299L/E255V, V299L/F359V, F317L/Y253H, F317L/E255V, F317L/F359V, F359V/Y253H, T315I/M244V, T315I/G250E, T315I/E255K, T315I/E255V, T315I/F359V) in BCR::ABL1 constructs introduced into the cells by a transposon-mediated approach. The presence of single insertion copies of the mutant constructs was verified by FACS analysis to preclude skewed test results potentially caused by multiple copies of the mutant construct. The cells pre-tested in this way were exposed in vitro to different concentrations of various drugs including ponatinib combined with a number of other agents such as asciminib, hydroxyurea, palbociclib, venetoclax, ibrutinib, vodobatinib, and crizotinib. The drug concentrations tested corresponded to the spectrum of the respective dosing regimens used in the clinical setting. The cell viability assays performed indicated that CMs responding only to elevated concentrations of ponatinib could be suppressed by combinatorial treatment with lower concentrations of ponatinib and different other agents tested. Moreover, CMs displaying high levels of resistance to TKIs applied as single agents mostly revealed adequate in vitro responses to individual drug combinations with ponatinib, most commonly including asciminib, hydroxyurea or crizotinib. Only the most resistant CM constellation tested, T315I/F359V, failed to respond to the compounds investigated and showed a limited response to the combination of high concentrations of ponatinib with hydroxyurea. A good response of this CM in vitro was only achieved by a combination of ponatinib with a very high concentration of crizotinib indicating the need for further testing of additional candidate drugs for this constellation. Overall, combinatorial treatment of BCR::ABL1 CMs indicated that relatively low concentrations of the drugs used are capable of effectively inhibiting in vitro survival of mutant cells in most instances, which is important with regard to the expected toxicity of combinatorial treatment approaches. Since our earlier data indicated a good correlation between in vitro test results and clinical responses, our observations may serve as a basis for novel treatment options in patients with Ph +leukemias displaying challenging BCR::ABL1 KD-mutations. [This project was supported by an educational grant from Incyte Biosciences International Sàrl]

OsDA1 positively regulates grain width in rice

The size and shape of rice grains influence their yield and commercial value. We investigated the role of OsDA1, a rice homolog of the Arabidopsis DA1 gene, in regulating grain size and shape. OsDA1 was highly expressed in young spikelets and glumes. Its overexpression led to enlarged seeds with increased width and decreased length/width ratio (LWR) and knocking out OsDA1 reduced grain width and increased grain length and LWR. A R310K point mutation in the DA1-like domain is a potential target for breeding for increased grain width and length. OsDA1 interacted with TCP gene-family proteins to regulate grain size and shape. Our findings deepen our understanding of the molecular mechanisms underlying grain size regulation and provide useful information for improving grain yield.

Covalent-103: A Phase 1, Open-Label, Dose-Escalation, and Dose-Expansion Study of Bmf-500, an Oral Covalent FLT3 Inhibitor, in Adults with Acute Leukemia (AL)

Background: FLT3 mutations occur in 25-35% of patients with AML and are associated with poor prognosis. FLT3 mutations are most frequently the result of an internal tandem duplication (ITD) of amino acids in the juxtamembrane region of FLT3 or point mutations in the tyrosine kinase domain (TKD). FLT3-ITD mutations are associated with increased incidence of relapse, shorter duration of remission, and decreased disease-free and overall survival. BMF-500 is a novel orally bioavailable, highly potent and selective covalent inhibitor of FLT3 including wildtype (WT), ITD, TKD, as well as a variety of additional resistance-conferring mutations such as the gatekeeper F691. BMF-500 has demonstrated high affinity for FLT3, lack of cKIT inhibition, and sustained cell-killing capacity despite drug washout (Law et al., ASH 2022 Abstract 2756). BMF-500 has shown sustained tumor regression and improved survival in both subcutaneous and disseminated xenograft models of mutant FLT3-driven AML. Study Design: COVALENT-103 (NCT05918692) is an open-label, non-randomized, multicenter, first-in-human Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of twice daily oral BMF-500 in patients with relapsed or refractory (R/R) AL, including AML, ALL, or MPAL, with or without FLT3 mutations. The trial has 2 arms that will undergo dose escalation in parallel: Arm A (without) and Arm B (with) concomitant use of a moderate or strong CYP3A4 inhibitor. Utilizing an accelerated titration design (ATD), doses of BMF-500 will be escalated in single-subject cohorts until 1 subject experiences either a Grade 2 or higher related-adverse event or dose-limiting toxicity (DLT). At that point, the cohort will switch to a classical “3 +3” design. Patients with WT FLT3 AL may be enrolled, up to a limit of 33% per arm. Treatment will continue in 28-day cycles until progression or intolerability. Expansion cohorts will enroll additional patients to obtain further safety and efficacy data. Patients must be refractory, relapsed or must have progressed on or following discontinuation of the most recent anti-cancer therapy or be ineligible for any approved standard of care therapies, including HSCT. Participants with FLT3-mutant AML must have received treatment with a FLT3 inhibitor approved for treatment of relapsed or refractory FLT3-mutant AML. Key inclusion criteria include ECOG PS ≤ 2, adequate organ function, and documented FLT3 mutation status. Key exclusion criteria include known CNS disease involvement, clinically significant cardiovascular disease, and WBC count &amp;gt;50,000/µL (uncontrollable with cytoreductive therapy). Objectives: The primary objective of the study is to evaluate safety and tolerability and to determine the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-500 oral monotherapy based on evaluation of available PK/ PD, safety and efficacy data. Secondary objectives include characterization of the pharmacodynamics and pharmacokinetics of BMF-500, and assessment of its antitumor activity per modified Cheson (2003) criteria or the NCCN Clinical Practice Guidelines (ALL Version 1.2022) as determined by the investigator. Endpoints include best overall response rate (ORR), complete remission (CRc), duration of response (DOR), relapse-free survival (RFS) and overall survival (OS). The study was initiated in July 2023 and will enroll ~110 participants at approximately 30 sites.

Myelo-Erythroid Lineage Segregation Is Regulated By the GATA-2 Interactome

Hematopoiesis arises from hematopoietic stem cell differentiating into downstream progenitors through a multistep process of sequential lineage potential restriction leading to the final production of &amp;gt;10 hematopoietic cell type.The regulation of hematopoietic lineage development involves synergistic and antagonistic interactions between transcription factors (TF) with lineage-restricted expression patterns, both through protein-protein interaction and direct transcriptional activation.Eosinophils, basophils and mast cells are key myeloid cell types involved in allergic inflammation and have been proposed to arise from a granulocyte-macrophage progenitor (GMP), that also generates neutrophils and macrophages, with GATA-2 up-regulation as a critical determinant. However, recent work identified a distinct basophil/eosinophil/mast cells progenitor (BEMP) that arises from multi-potent erythroid-primed multipotent progenitors (EMPP) where GATA-2 is already expressed ( Drissen et al., 2016). Here, we show that the GATA-2 interactome, rather than GATA-2 expression, controls myeloid lineage choice in EMPPs. To investigate the molecular mechanism instructing lineage decision in EMPP, we performed gene-expression profiling (RNA-seq) comparing its downstream progeny, early restricted erythroid/megakaryocyte progenitor (preMegE) and BEMP. This analysis identified differential expressed genes, including antagonist gene-expression pattern between the LIM-only protein 2 ( Lmo2) and protein 4 ( Lmo4), both previously shown as GATA-2 interactor. While Lmo2 function in hematopoiesis have been exhaustively studied, the role of Lmo4 remains unknown. In colony forming unit (CFU)-assay, overexpression of Lmo4 in EMPP mediated by lentiviral delivery, resulted in an increased number of mast-colonies associated with a decreased of erythroid/megakaryocyte colonies, while the total colony number was unchanged. In addition, transplantation of hematopoietic lineage-negative cells transduced with Lmo4/empty(Ctrl) lentiviral particles, showed an increased frequency of basophils and eosinophils associated with a decrease of erythroid cells in Lmo4 overexpressing cells compared to Ctrl, in both bone marrow and spleen of recipient mice, 8 weeks after transplantation. Analysis of transduced Lineage-negative Kit + (LK) compartment of recipient mice, also revealed an increased frequency of BEMP associated with a decreased frequency of preMegE in Lmo4overexpressing cells compared to Ctrl. Taken together, these data suggested that Lmo4 overexpression instruct lineage decision of EMPP toward basophil/eosinophil/mast lineages.Over-expression followed by co-immunoprecipitation in HEK-293T cell line revealed that LMO4 compete with LMO2 binding to GATA-2, suggesting a mutually exclusive transcriptional complex. More importantly, lack of interaction between LMO4 and GATA-2 mediated by a point mutation in the zinc-finger 1 domain (ZnF1) of GATA-2 (GATA2-G320D), frequently identified in AML/AEL, resulted in an absence of mast lineage potential of EMPP compared to GATA2 +/+. Moreover, over-expression of Lmo4 in EMPP from GATA2 G320D/G320D mice compared to GATA2 +/+, failed to instruct myeloid fate in vitro. Overall, our data identified a specific molecular mechanism of hematopoietic cell fate decision driven by protein-protein interaction with GATA-2. Perturbation of GATA-2 interactome through over-expression of the cofactor Lmo4 or specific point mutation in GATA2 ZnF1 domain results in lineage fate switch of EMPP toward the erythroid/megakaryocyte or basophil/eosinophil/mast lineages.