Mutations In Domain Research Articles

Identification of a crosstalk between ClC-1 C-terminal CBS domains and the transmembrane region.

CLC channels and transporters have large C-terminal regions which contain two cystathionine β-synthetase (CBS) domains. It has been hypothesized that conformational changes in these domains upon nucleotide binding modulate the gating of the CLC dimer. It is not clear how rearrangements that occur in the CBS domains are transmitted to the ion pathway, as CBS domains interact with the rest of the channel at multiple locations and some of these sites are not visible in recent solved cryogenic electron microscopy structures or are difficult to model using the AlphaFold server. Using ClC-1 as a model, we started working with a described ClC-1 mutation (H835R) located in the first alpha helix of the CBS2 domain which changes the voltage dependence of gating. We then identified several residues located in the disorganized loop after helix R (R-linker) that revert the phenotype of this mutation. We additionally proved that R-linker's function is connected to the CBS2 domain as current intensity, plasma membrane levels and gating defects of several R-linker variants were corrected by adding the mutation H835R. Furthermore, cross-linking studies using newly developed split-cysless ClC-1 channels containing specific cysteine mutants in the R-linker and the CBS2 domain indicate that these two regions are in close contact. Considering these new results, we propose that conformational changes occurring in the CBS domains could be transmitted to the CLC intracellular chloride binding site by means of its interaction with the R-linker. KEY POINTS: CBS domains, which are present as pairs in CLC proteins, are involved in the regulation by nucleotides of CLC gating. It is not clear how CBS domains interact with different regions of the transmembrane (TM) region to regulate gating. Using ClC-1 as a model, we investigated how a mutation in the second CBS domain dramatically changes the voltage dependence of gating taking advantage of recently solved structures and AlphaFold models of CLC proteins. Thus, we identified in the linker after helix R (R-linker) several revertant mutations of the gating defects caused by a mutation in the second CBS2 domain and vice versa, indicating that these two regions functionally interact. These interactions were biochemically proven by employing cysteine cross-linkings using a newly developed split-cysless ClC-1 channel. Based on these findings we experimentally prove that the R-linker is a key element for the transmission of the CBS conformational rearrangements to the TM region.

Exploring the role of ESR1 mutations in metastatic hormone receptor-positive breast cancer T cell immune surveillance disruption

BackgroundBreast Cancer (BC) is the most common type of cancer in women around the world and 70% of cases are hormone-receptor positive (HR+). In 40% of cases, a key mechanism of endocrine resistance to the standard first line is a mutation of the ligand-binding domain (LBD) of Estrogen Receptor 1 (ESR1) encoding estrogen receptor α (ER). Most common ESR1 mutations that occur at positions 537 and 538 have been associated with poor clinical outcomes. ESR1 mutations have the potential to provide neoantigens. This study aims to identify if ESR1 mutations generate specific T cell responses against ESR1 neoantigens in patients with HR+ HER2− BC, and to investigate if ESR1 mutations might correlate with a gene expression profile related to immune surveillance disruption.MethodsWe identified candidate ESR1-derived peptides by predictive software (SYFPEITHI and NetMHCpan 3.0). Then the immunogenicity of ESR1-derived peptides was assessed in Peripheral-Blood-Mononuclear-Cells from 31 healthy donors (HD) and 25 patients with metastatic HR-positive BC by IFN-γ ELISpot assay. A vaccination assay on a humanized mouse model (HLA-A2/DR1) was used to validate the immunogenicity and the presentation of these peptides. Finally, we used Bulk RNA-Seq sequencing along with MCPcounter, a cellular deconvolution method, to investigate the immune contexture of ESR1-mutated BC.ResultsPreliminary results showed recognition of ESR1-derived peptides by women HD lymphocytes but not in men. Frequencies and intensities of such immune responses were increased in patients with BC. Our results showed that 40% of patients had specific immune responses. In addition, we demonstrated the HLA-A2 ESR1 peptide immunogenicity in humanized HLA-A2/DR1 mice. In a data set generated from BC patients refractory to conventional therapy we showed that ESR1 mutations are correlated in advanced diseases with downregulation of molecules involved in antigen presentation and with loss HLA Class I gene expression. ESR1-mutated BC had a decrease in immune cell infiltration.ConclusionThese results support that common ESR1 mutations generate neoantigens in hormone-receptor positive metastatic breast cancers. If ESR1 peptides-restricted lymphocytes were detectable in BC patients, ESR1 mutations promote immune escape at advanced stages.Trial registrationClinicalTrials.gov, NCT02838381. Registered on June 2012.

The improved prognosis of FLT3-internal tandem duplication but not tyrosine kinase domain mutations in acute myeloid leukemia in the era of targeted therapy: a real-world study using large-scale electronic health record data.

Not available.

Epsin3 promotes non-small cell lung cancer progression via modulating EGFR stability

BackgroundThe abnormal expression and overactivation of the epidermal growth factor receptor (EGFR), a typical cancer marker for non-small cell lung cancer (NSCLC), are closely related to the tumorigenesis and progression of NSCLC. However, the endocytosis mechanism of EGFR in lung cancer is not yet known. Epsin3 (EPN3), a member of the endocytic adaptor protein family, is essential for the endocytosis of multiple receptors. In this study, we aimed to investigate the role of EPN3 in modulating EGFR function, its effects on NSCLC progression, and its potential involvement in tyrosine kinase inhibitor (TKI) resistance, which remains a significant hurdle in NSCLC treatment.ResultsOur findings revealed that the expression of EPN3 is significantly up-regulated in NSCLC patients. Elevated EPN3 expression was proportional to shorter overall survival in patients with NSCLC. Functional analyses revealed that EPN3 directly interacts with EGFR, enhancing its recycling to the plasma membrane and preventing its degradation via the lysosomal pathway. This stabilization of EGFR led to sustained downstream signalling, promoting NSCLC cell proliferation and migration. Notably, mutations in the EGFR tyrosine kinase domain, which typically confer resistance to TKIs, did not alter the regulatory effect of EPN3.ConclusionsEPN3 enhances EGFR signalling by promoting its recycling and stability, contributing to NSCLC progression and TKI resistance. Targeting EPN3 could offer a novel therapeutic strategy to overcome drug resistance in EGFR-driven NSCLC.

Distribution of different classes of CSF3R mutations and co-mutational pattern in 360 myeloid neoplasia.

The colony-stimulating factor 3 receptor (CSF3R) plays an essential role in differentiation, growth, and survival of granulocytes. Driver mutations in CSF3R gene represent a diagnostic marker of chronic neutrophilic leukemia (CNL). Less commonly, these mutations are observed in other myeloid neoplasms but their pathogenetic and prognostic role is still unclear. Here, we analyzed a large cohort of myeloid neoplasms to evaluate the incidence of CSF3R mutations and co-mutational profile. Mutational analysis was performed using targeted NGS myeloid panel in a consecutive cohort of 360 patients with myeloid neoplasms. Mutations in CSF3R were identified in 20/360 (5.6%) cases. A CSF3R gene mutation was present in 13/179 AML cases (7.3%), in 2/27 (7.4%) CMML cases, in 1/94 (1.1%) MDS cases and in 4/60 (6.7%) other myeloid neoplasms. The frequencies of patients with CSF3R mutations lowered to 2.8% in all cases and 3.4% in AML, excluding cases with variants of uncertain significance (VUS). A total of 23 mutations of CSF3R gene were detected, half localized in the extracellular domain, 5 in the transmembrane region (type I) and 6 mutations in the cytoplasmic domain (type II). In AML, CSF3R mutations were more frequent in patients harboring CBF alterations (25.0%) and CEBPA mutations (11.8%). Two cases with AML harboring pathogenic CSF3R variants were primary refractory to induction therapy. CMML cases with T618I variant showed a myeloproliferative phenotype. Overall, our findings support the notion that CSF3R variants, particularly type I and II pathogenic mutations, may modulate the phenotypic features of leukemic cells in myeloid neoplasia.

Kinome reprogramming of G2/M kinases and repression of MYCN contribute to superior efficacy of lorlatinib in ALK-driven neuroblastoma.

Mutations in the tyrosine kinase domain of the Anaplastic Lymphoma Kinase (ALK) oncogene in neuroblastoma occur most frequently at one of three hotspot amino acid residues, with the F1174* and F1245* variants conferring de novo resistance to first and second generation ALK inhibitors including crizotinib and ceritinib. Lorlatinib, a third generation ALK/ROS inhibitor, overcomes de novo resistance and induces complete and sustained tumor regressions in patient-derived xenograft (PDX) models unresponsive to crizotinib. Lorlatinib has now completed Phase 1 testing in children and adults with relapsed/refractory ALK-driven neuroblastoma and entered pivotal Phase 3 testing within the Children's Oncology Group. To define mechanisms underlying the superior activity of lorlatinib, we utilized a chemical proteomics approach to quantitatively measure functional kinome dynamics in response to lorlatinib and crizotinib in clinically relevant ALK-driven neuroblastoma PDX models. Lorlatinib was a markedly more potent inhibitor of ALK and preferentially downregulated several kinases implicated in G2/M cell cycle transition compared to crizotinib. Lorlatinib treatment also led to the repression of MYCN expression and its occupancy at promoters of the same G2/M kinases. These data providing mechanistic insight into the superior efficacy of lorlatinib over crizotinib for the treatment of ALK-driven neuroblastoma.

Sam–Sam Association Between EphA2 and SASH1: In Silico Studies of Cancer-Linked Mutations

Recently, SASH1 has emerged as a novel protein interactor of a few Eph tyrosine kinase receptors like EphA2. These interactions involve the first N-terminal Sam (sterile alpha motif) domain of SASH1 (SASH1-Sam1) and the Sam domain of Eph receptors. Currently, the functional meaning of the SASH1-Sam1/EphA2-Sam complex is unknown, but EphA2 is a well-established and crucial player in cancer onset and progression. Thus, herein, to investigate a possible correlation between the formation of the SASH1-Sam1/EphA2-Sam complex and EphA2 activity in cancer, cancer-linked mutations in SASH1-Sam1 were deeply analyzed. Our research plan relied first on searching the COSMIC database for cancer-related SASH1 variants carrying missense mutations in the Sam1 domain and then, through a variety of bioinformatic tools and molecular dynamic simulations, studying how these mutations could affect the stability of SASH1-Sam1 alone, leading eventually to a defective fold. Next, through docking studies, with the support of AlphaFold2 structure predictions, we investigated if/how mutations in SASH1-Sam1 could affect binding to EphA2-Sam. Our study, apart from presenting a solid multistep research protocol to analyze structural consequences related to cancer-associated protein variants with the support of cutting-edge artificial intelligence tools, suggests a few mutations that could more likely modulate the interaction between SASH1-Sam1 and EphA2-Sam.

Missense mutations of the ephrin receptor EPHA1 associated with Alzheimer's disease disrupt receptor signaling functions.

Missense mutations in the EPHA1 receptor tyrosine kinase have been identified in Alzheimer's patients. To gain insight into their potential role in disease pathogenesis, we investigated the effects of four of these mutations. We show that the P460L mutation in the second fibronectin type III (FN2) domain drastically reduces EPHA1 cell surface localization while increasing tyrosine phosphorylation of the cell surface-localized receptor. The R791H mutation in the kinase domain abolishes EPHA1 tyrosine phosphorylation, indicating abrogation of kinase-dependent signaling. Furthermore, both mutations decrease EPHA1 phosphorylation on S906 in the kinase-SAM linker region, suggesting impairment of a noncanonical form of signaling regulated by serine/threonine kinases. The R492Q mutation, also in the FN2 domain, has milder effects than the P460L mutation while the R926C mutation in the SAM domain increases S906 phosphorylation. We also found that EPHA1 undergoes constitutive proteolytic cleavage in the FN2 domain, generating a soluble 55kDaN-terminal fragment containing the ligand-binding domain and a transmembrane 60kDaC-terminal fragment. The 60kDa WT fragment is phosphorylated on both tyrosine residues and S906, suggesting signaling functions. The P460L mutant 60kDa fragment undergoes proteasomal degradation and the R791H mutant fragment lacks tyrosine phosphorylation and has decreased S906 phosphorylation. These findings advance our understanding of EPHA1 signaling mechanisms and support the notion that alterations in EPHA1 signaling due to missense mutations contribute to Alzheimer's disease pathogenesis.

Up-regulation of Melanophilin (MLPH) gene during avian adipogenesis and decreased fat pad weights with adipocyte hypotrophy in MLPH knockout quail.

Advanced genetic and nutritional strategies aimed at modulating fat deposition can significantly reduce production costs and enhance profitability in the poultry industry. Melanophilin (MLPH) is recognized as a key gene regulating pigmentation as shown by diluted hair and feather coloration in MLPH mutant animals, including avian models. However, the effects of MLPH during fat accretion have not been studied yet. Therefore, the objectives of the current study are to measure the temporal expression of the MLPH gene during the adipocyte differentiation in vitro and in vivo and to investigate the effect of MLPH loss on fat accretion and adipocyte sizes in vivo using MLPH knockout quail model. The current in vitro studies reveal that MLPH gene expression levels were considerably elevated during adipogenesis in avian cells [101-fold in DF-1, 28.5-fold in chicken embryonic fibroblasts (CEF) and 4-fold in quail embryonic fibroblasts (QEF), compared to the undifferentiated cells of each cell type, p < 0.05]. In addition, fractionated fat cells (FC) showed increased expression levels of MLPH (5.7-fold, p < 0.05) compared to stromal-vascular cells (SVC). Using the MLPH knockout quail, disruption of the MLPH gene resulted in significantly reduced body weight (BW) and subcutaneous fat (S. Fat) pad weights compared to the wild type (WT) (p < 0.05). Further analysis through sectioning and staining of the fat tissues revealed that the mutation in Rab binding domain (RBD) of quail MLPH resulted in decreased fat cell sizes (p < 0.01). Overall, our data clearly demonstrated that MLPH can be a potential adipogenic marker gene, and MLPH may be associated with fat accretion in the gene edited quail model, highlighting the important role of MLPH in adipogenesis.

Fibrillin-1 G234D mutation in the hybrid1 domain causes tight skin associated with dysregulated elastogenesis and increased collagen cross-linking in mice

Fibrillin-1, an extracellular matrix (ECM) protein encoded by the FBN1 gene, serves as a microfibril scaffold crucial for elastic fiber formation and homeostasis in pliable tissue such as the skin. Aside from causing Marfan syndrome, some mutations in FBN1 result in scleroderma, marked by hardened and thicker skin which limits joint mobility. Here, we describe a tight skin phenotype in the Fbn1G234D/G234D mice carrying a corresponding variant of FBN1 in the hybrid1 domain that was identified in a patient with familial aortic dissection. Unlike scleroderma, skin thickness and collagen fiber abundance do not change in the Fbn1G234D/G234D mutant skin. Instead, increased collagen cross-links were observed. In addition, short elastic fibers were sparsely located underneath the panniculus muscle layer, and an abundance of thin, aberrant elastic fibers was increased within the subcutaneous fascia, which may have tightened skin attachment to the underlying skeletal muscle. Structurally, Fbn1G234D/G234D microfibrils have a disrupted shoulder region that shares similarities with hybrid1 deletion mutant microfibrils. We then demonstrate the consequence of fibrillin-1 G234D mutation on dermal fibroblast functions. Mutant primary fibroblasts produce fewer elastic fibers, exhibit slower migration and increased cell stiffness. Moreover, secretome from mutant fibroblasts are marked by enhanced secretion of ECM, ECM-modifying enzymes, proteoglycans and cytokines, which are pro-tissue repair/fibrogenic. The transcriptome of mutant fibroblasts displays an increased expression of myogenic developmental and immune-related genes. Our study proposes that imbalanced ECM homeostasis due to a fibrillin-1 G234D mutation impacts fibroblast properties with potential ramifications on skin function.

Isolation and molecular characterization of an enteric isolate of the Genotype-Ia Bovine coronavirus with notable mutations in the receptor binding domain of the spike glycoprotein.

BCoV new isolate was plaque purified, isolated, and propagated in vitro using MDBK and HRT-18. The full-length genome sequencing of this new BCoV isolate (31Kbs) was drafted and deported in the GenBank. The genome organization is (5'-UTR-Gene-1-32kDa-HE-S-4.9 kDa-4.8kDa-12.7kDa-E-M-N-UTR-3'). Phylogenetic analysis based on the sequences of (the full-length genome, S, HE, and N) showed that the BCoV-13 clustered with other North American BCoV genotype I members. The sequence analysis shows several synonymous mutations among various domains of the S glycoprotein, especially the receptor binding domain. We found nine notable nucleotide deletions immediately downstream of the RNA binding domain of the nucleocapsid gene. Further gene function studies are encouraged to study the function of these mutations on the BCoV molecular pathogenesis and immune regulation. This research enhances our understanding of BCoV genomics and contributes to improved diagnostic and control measures for BCoV infections in cattle.

Blocking p85β nuclear translocation by importazole enhances Alpelisib efficacy against PIK3CA-helical-domain-mutant tumors.

PIK3CA, which encodes protein p110α, is one of the most frequently mutated oncogenes and a promising drug-target for human cancer. Previously, we demonstrate that p85β is released from PI3K complex which contain PIK3CA helical domain mutations and translocates into nucleus to regulate tri-methylation of H3K27, thereby promoting tumorigenicity. Here, we identify DIRAS2 and SOWAHB as target genes of nuclear p85β in PIK3CA-helical-domain-mutant tumors. DIRAS2 and SOWAHB are tumor suppressive genes, whose expression are repressed by nuclear p85β through histone methyltransferase EZH2. More importantly, combination of PI3K inhibitor and importin-β inhibitor effectively inhibits the growth of PIK3CA-helical-domain-mutant tumors by synchronously blocking both AKT signaling and nuclear p85β/DIRAS2 and SOWAHB axis. In this study, we evaluate the combination effect of Alpelisib and Importazole for PIK3CA helical domain mutant tumors and demonstrate its underlying mechanism.

Clinical Characteristics of Patients With Becker Muscular Dystrophy Having Pathogenic Microvariants or Duplications.

Becker muscular dystrophy (BMD) is an allelic disorder of Duchenne muscular dystrophy (DMD) in which pathogenic variants in DMD cause progressive worsening of motor dysfunction, muscle weakness and atrophy, and death due to respiratory and cardiac failure. BMD often has in-frame deletions that preserve the amino acid reading frame, but there are some cases with microvariants or duplications. In recent years, the importance of therapeutic development and care for BMD has been emphasized. Therefore, the purpose of this study was to understand the clinical characteristics of BMD patients with microvariants or duplications and to determine the genotype-phenotype relationship. The study focused on patients with pathogenic microvariants or duplications in DMD who were ambulatory after 16 years of age or had specific muscle biopsy results between June 13, 2017, and March 31, 2023. Informed consent was obtained from the patients or their surrogates. Data concerning DMD variants, muscle biopsy findings, skeletal muscle, respiratory and cardiac function, and CNS involvement were collected and analyzed statistically. Thirty-three patients with BMD had pathogenic microvariants (missense variants, nonsense variants, splice site variants, and other microvariants), and 16 patients had in-frame duplications in DMD. Many patients with microvariants had abnormal ECG findings. The effect of variant type on patient outcomes varied. Regardless of the type of microvariant, skeletal muscle and respiratory dysfunction was more severe in mutants of the cysteine-rich/C-terminal domain than in rod domain mutants. On the other hand, there was no significant difference in the complication rate of CNS disorders among the 3 domains of dystrophin. Microvariant forms, in particular, tend to vary in clinical severity according to the site of the dystrophin protein mutation rather than the type of pathogenic variant. The results of this study may be useful for genetic counseling, care, and treatment of patients with BMD.

Differential Efficacy of Alpelisib by PIK3CA Mutation Site in Head and Neck Squamous Cell Carcinoma: An Analysis from the KCSG HN 15-16 TRIUMPH Trial.

The TRIUMPH trial was a biomarker-driven umbrella trial for patients with recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC). This analysis focuses on the PIK3CAɑ inhibitor alpelisib (arm 1) in patients with phosphoinositide 3-kinase (PI3K) pathway alterations. Patients with PI3K pathway altered tumors were enrolled in the alpelisib arm of the TRIUMPH study. We conducted a detailed analysis of the correlation between PI3K pathway mutations and treatment outcomes including disease control rate (DCR), overall survival (OS), and progression-free survival (PFS). From Oct 2017 and Aug 2020, 203 were enrolled, with 42 treated with alpelisib. Response evaluation was possible for 33 patients. Genomic profiles revealed PIK3CA amplifications in 26.2%, and point mutations in E542K (26.2%), E545K (23.8%), and H1047R (9.5%). Neither PIK3CA amplification nor co-occurring TP53 mutations had a notable influence on alpelisib response or survival outcomes. Although the overall response rates were similar between helical domain mutations (E542, E545) and kinase domain mutations (H1047), patients with H1047 mutations exhibited significantly poorer PFS compared to those with non-H1047 PIK3CA alterations (1.6 vs. 7.3 months, p=0.017). OS in patients with H1047 kinase domain mutations showed a trend toward being shorter compared to others, though this difference did not reach statistical significance. Alpelisib showed differential efficacy based on PI3K pathway alterations in patients with R/M HNSCC and was well-tolerated. These findings suggest the usefulness of NGS testing-based decision-making when using the targeted agents in R/M HNSCC. We need to confirm results in larger cohorts.

ATP-CITRATE LYASEB1 supplies materials for sporopollenin biosynthesis and microspore development in Arabidopsis.

Acetyl-CoA is the main substrate of lipid metabolism and functions as an energy source for plant development. In the cytoplasm, acetyl-CoA is mainly produced by ATP-citrate lyase (ACL), which is composed of ACLA and ACLB subunits. In this study, we isolated the restorer-4 (res4) of the thermosensitive genic male sterile mutant reversible male sterile-2 (rvms-2) in Arabidopsis (Arabidopsis thaliana). RES4 encodes ACLB1, and res4 harbors a point mutation (Gly584 to Arg) in the citryl-CoA lyase domain. Both the ACLA and ACLB subunits are expressed in the tapetal layer of anthers. RES4 is regulated by MS188, and the res4 point mutation leads to pollen with a defective exine structure. In res4, lipid accumulation was significantly reduced within the tapetum and locules. These results indicate that acetyl-CoA synthesized by ACL is used for sporopollenin biosynthesis in the tapetum. Microspore diameter was significantly smaller in res4 than in wild type, indicating that acetyl-CoA from the tapetum supplies microspore development. Previous studies have shown that delayed degradation of the tetrad wall in res2 and res3 provides additional protection for rvms-2 microspores. The reduced volume of res4 microspores may lessen the requirement for cell wall protection to restore rvms-2 fertility. This study reveals the function of ACL in anther development and the mechanisms of fertility restoration in photoperiod- and thermo-sensitive genic male sterile lines.

PD-L1-CD80 interactions are required for intracellular signaling necessary for dendritic cell migration.

Programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) interactions are targets for immunotherapies aimed to reinvigorate T cell function. Recently, it was documented that PD-L1 regulates dendritic cell (DC) migration through intracellular signaling events. In this study, we find that both preclinical murine and clinically available human PD-L1 antibodies limit DC migration. We show that cis interactions between PD-L1 and CD80 are critical for promoting migration and define specific regions within these proteins necessary for migration. Furthermore, we demonstrate that αPD-L1 significantly impedes DC migration in a B16 melanoma tumor model. Last, we outline how blocking cis PD-L1:CD80 interactions or mutation of the intracellular domain of PD-L1, in an imiquimod-induced murine model of psoriasis, limits DC migration to the lymph node, decreases interleukin-17 production by CD4+ T cells in the lymph node, and reduces epidermal thickening. Therefore, PD-L1 and CD80 interactions are important regulators of DC migration to the draining lymph node.

An mTOR inhibitor discovery system using drug-sensitized yeast.

Inhibition of the target of rapamycin (TOR/mTOR) protein kinase by the drug rapamycin extends lifespan and health span across diverse species. However, rapamycin has potential off-target and side effects that warrant the discovery of additional TOR inhibitors. TOR was initially discovered in Saccharomyces cerevisiae (yeast) which contains two TOR paralogs, TOR1 and TOR2. Yeast lacking functional Tor1 are viable but are hypersensitive to growth inhibition by TORC1 inhibitors, which is a property of yeast that can be exploited to identify TOR inhibitors. Additionally, yeast lacking FK506-sensitive proline rotamase (FPR1) or containing a tor1-1 allele (a mutation in the Fpr1-rapamycin binding domain of Tor1) are robustly and selectively resistant to rapamycin and analogs that allosterically inhibit TOR activity via an FPR1-dependent mechanism. To facilitate the identification of TOR inhibitors, we generated a panel of yeast strains with mutations in TOR pathway genes combined with the removal of 12 additional genes involved in drug efflux. This creates a drug-sensitive strain background that can sensitively and effectively identify TOR inhibitors. In a wild-type yeast strain background, 25µM of Torin1 and 100µM of GSK2126458 (omipalisib) are necessary to observe TOR1-dependent growth inhibition by these known TOR inhibitors. In contrast, 100nM Torin1 and 500nM GSK2126458 (omipalisib) are sufficient to identify TOR1-dependent growth inhibition in the drug-sensitized background. This represents a 200-fold and 250-fold increase in detection sensitivity for Torin1 and GSK2126458, respectively. Additionally, for the TOR inhibitor AZD8055, the drug-sensitive system resolves that the compound results in TOR1-dependent growth sensitivity at 100µM, whereas no growth inhibition is observed in a wild-type yeast strain background. Our platform also identifies the caffeine analog aminophylline as a TOR1-dependent growth inhibitor via selective tor1 growth sensitivity. We also tested nebivolol, isoliquiritigenin, canagliflozin, withaferin A, ganoderic acid A, and taurine and found no evidence for TOR inhibition using our yeast growth-based model. Our results demonstrate that this system is highly effective at identifying compounds that inhibit the TOR pathway. It offers a rapid, cost-efficient, and sensitive tool for drug discovery, with the potential to expedite the identification of new TOR inhibitors that could serve as geroprotective and/or anti-cancer agents.

Concurrent inhibition of p300/CBP and FLT3 enhances cytotoxicity and overcomes resistance in acute myeloid leukemia.

FMS-like tyrosine kinase-3 (FLT3), a class 3 receptor tyrosine kinase, can be activated by mutations of internal tandem duplication (FLT3-ITD) or point mutations in the tyrosine kinase domain (FLT3-TKD), leading to constitutive activation of downstream signaling cascades, including the JAK/STAT5, PI3K/AKT/mTOR and RAS/MAPK pathways, which promote the progression of leukemic cells. Despite the initial promise of FLT3 inhibitors, the discouraging outcomes in the treatment of FLT3-ITD-positive acute myeloid leukemia (AML) promote the pursuit of more potent and enduring therapeutic approaches. The histone acetyltransferase complex comprising the E1A binding protein P300 and its paralog CREB-binding protein (p300/CBP) is a promising therapeutic target, but the development of effective p300/CBP inhibitors faces challenges due to inherent resistance and low efficacy, often exacerbated by the absence of reliable clinical biomarkers for patient stratification. In this study we investigated the role of p300/CBP in FLT3-ITD AML and evaluated the therapeutic potential of targeting p300/CBP alone or in combination with FLT3 inhibitors. We showed that high expression of p300 was significantly associated with poor prognosis in AML patients and positively correlated with FLT3 expression. We unveiled that the p300/CBP inhibitors A485 or CCS1477 dose-dependently downregulated FLT3 transcription via abrogation of histone acetylation in FLT3-ITD AML cells; in contrast, the FLT3 inhibitor quizartinib reduced the level of H3K27Ac. Concurrent inhibition of p300/CBP and FLT3 enhanced the suppression of FLT3 signaling and H3K27 acetylation, concomitantly reducing the phosphorylation of STAT5, AKT, ERK and the expression of c-Myc, thereby leading to synergistic antileukemic effects both in vitro and in vivo. Moreover, we found that p300/CBP-associated transcripts were highly expressed in quizartinib-resistant AML cells with FLT3-TKD mutation. Targeting p300/CBP with A485 or CCS1477 retained the efficacy of quizartinib, suggesting marked synergy when combined with p300/CBP inhibitors in quizartinib-resistant AML models, as well as primary FLT3-ITD+ AML samples. These results demonstrate a potential therapeutic strategy of combining p300/CBP and FLT3 inhibitors to treat FLT3-ITD and FLT3-TKD AML.

Prognostic Factors, FLT-3 Mutations, and Treatment Outcomes with pediatric inspired protocols in adolescent and young adults (AYA) and adult patients with Acute Lymphoblastic Leukemia.

The treatment protocols of adolescent and young adults (AYA) patients with acute lymphoblastic leukemia (ALL) have evolved, with the advent of pediatric-based regimens, measurable residual disease monitoring and mutation analysis. Among the latter, previous reports have identified FLT-3 mutations in up to 5% of pediatric patients, however the full clinical significance of these mutations in the non-pediatric population is still uncertain. Our cohort includes AYA patients with ALL treated with the NY-II and BFM protocols at different time periods, allowing analysis of prognostic factors and survival outcomes. Additionally, we analyzed DNA samples for FLT-3 mutations, focusing on the potential prognostic implications and treatment responses within our cohort. No significant differences were found in overall survival (OS) or progression-free survival (PFS) between the two treatment protocols. However, a higher rate of hematopoietic stem-cell transplantation (HSCT) was noted in the NY-II patients. Older age and high WBC count at presentation were identified as adverse prognostic factors using multivariate analysis. FLT-3 mutations were identified in 4 patients (5%) of the cohort, with only one patient having FLT-3 internal tandem duplication (ITD) mutation and three patients having FLT-3-tyrosine kinase domain (TKD) mutations. The low rate and variability of FLT-3 mutations in an Israeli cohort precludes broad conclusions regarding their prognostic significance. In our cohort, age and WBC count, but not treatment protocol or FLT-3 mutations influenced survival.

Comparison of actionable alterations in cancers with kinase fusion, mutation, and copy number alteration.

Kinase-related gene fusion and point mutations play pivotal roles as drivers in cancer, necessitating optimized, targeted therapy against these alterations. The efficacy of molecularly targeted therapeutics varies depending on the specific alteration, with great success reported for such therapeutics in the treatment of cancer with kinase fusion proteins. However, the involvement of actionable alterations in solid tumors, especially regarding kinase fusions, remains unclear. Therefore, in this study, we aimed to compare the number of actionable alterations in patients with tyrosine or serine/threonine kinase domain fusions, mutations, and copy number alterations (CNAs). We analyzed 613 patients with 40 solid cancer types who visited our division between June 2020 and April 2024. Furthermore, to detect alterations involving multiple-fusion calling, we performed comprehensive genomic sequencing using FoundationOne® companion diagnostic (F1CDx) and FoundationOne® Liquid companion diagnostic (F1LCDx). Patient characteristics and genomic profiles were analyzed to assess the frequency and distribution of actionable alterations across different cancer types. Notably, 44 of the 613 patients had fusions involving kinases, transcriptional regulators, or tumor suppressors. F1CDx and F1LCDx detected 13 cases with kinase-domain fusions. We identified 117 patients with kinase-domain mutations and 58 with kinase-domain CNAs. The number of actionable alterations in patients with kinase-domain fusion, mutation, or CNA (median [interquartile range; IQR]) was 2 (1-3), 5 (3-7), and 6 (4-8), respectively. Patients with kinase fusion had significantly fewer actionable alterations than those with kinase-domain mutations and CNAs. However, those with fusion involving tumor suppressors tended to have more actionable alterations (median [IQR]; 4 [2-9]). Cancers with kinase fusions exhibited fewer actionable alterations than those with kinase mutations and CNAs. These findings underscore the importance of detecting kinase alterations and indicate the pivotal role of kinase fusions as strong drivers of cancer development, highlighting their potential as prime targets for molecular therapeutics.