Mutations In Domain Research Articles

Loss of CD20 expression as a mechanism of resistance to mosunetuzumab in relapsed/refractory B-cell lymphomas

AbstractCD20 is an established therapeutic target in B-cell malignancies. The CD20 × CD3 bispecific antibody mosunetuzumab has significant efficacy in B-cell non-Hodgkin lymphomas (NHLs). Because target antigen loss is a recognized mechanism of resistance, we evaluated CD20 expression relative to clinical response in patients with relapsed and/or refractory NHL in the phase 1/2 GO29781 trial investigating mosunetuzumab monotherapy. CD20 was studied using immunohistochemistry (IHC), RNA sequencing, and whole-exome sequencing performed centrally in biopsy specimens collected before treatment at predose, during treatment, or upon progression. Before treatment, most patients exhibited a high proportion of tumor cells expressing CD20; however, in 16 of 293 patients (5.5%) the proportion was <10%. Analyses of paired biopsy specimens from patients on treatment revealed that CD20 levels were maintained in 29 of 30 patients (97%) vs at progression, where CD20 loss was observed in 11 of 32 patients (34%). Reduced transcription or acquisition of truncating mutations explained most but not all cases of CD20 loss. In vitro modeling confirmed the effects of CD20 variants identified in clinical samples on reduction of CD20 expression and missense mutations in the extracellular domain that could block mosunetuzumab binding. This study expands the knowledge about the occurrence of target antigen loss after anti-CD20 therapeutics to include CD20-targeting bispecific antibodies and elucidates mechanisms of reduced CD20 expression at disease progression that may be generalizable to other anti-CD20 targeting agents. These results also confirm the utility of readily available IHC staining for CD20 as a tool to inform clinical decisions. This trial was registered at www.ClinicalTrials.gov as #NCT02500407.

Thrombomodulin Regulates PTEN/AKT Signaling Axis in Endothelial Cells.

We recently demonstrated that deletion of thrombomodulin gene from endothelial cells results in upregulation of proinflammatory phenotype. In this study, we investigated the molecular basis for the altered phenotype in thrombomodulin-deficient (TM-/-) cells. Different constructs containing deletions or mutations in the cytoplasmic domain of thrombomodulin were prepared and introduced to TM-/- cells. The phenotype of cells expressing different derivatives of thrombomodulin and tissue samples of thrombomodulin-knockout mice were analyzed for expression of distinct regulatory genes in established signaling assays. The phosphatase and tensin homolog were phosphorylated and its recruitment to the plasma membrane was impaired in TM-/- cells, leading to hyperactivation of AKT (protein kinase B) and phosphorylation-dependent nuclear exclusion of the transcription factor, forkhead box O1. The proliferative/migratory properties of TM-/- cells were enhanced, and cells exhibited hypersensitivity to stimulation by angiopoietin 1 and vascular endothelial growth factor. Reexpression of wild-type thrombomodulin in TM-/- cells normalized the cellular phenotype; however, thrombomodulin lacking its cytoplasmic domain failed to restore the normal phenotype in TM-/- cells. Increased basal permeability and loss of VE-cadherin were restored to normal levels by reexpression of wild-type thrombomodulin but not by a thrombomodulin construct lacking its cytoplasmic domain. A thrombomodulin cytoplasmic domain deletion mutant containing 3-membrane-proximal Arg-Lys-Lys residues restored the barrier-permeability function of TM-/- cells. Enhanced phosphatase and tensin homolog phosphorylation and activation of AKT and mTORC1 (mammalian target of rapamycin complex 1) were also observed in the liver of thrombomodulin-KO mice. These results suggest that the cytoplasmic domain of thrombomodulin interacts with the actin cytoskeleton and plays a crucial role in regulation of phosphatase and tensin homolog/AKT signaling in endothelial cells.

Generation of THRB-GS(E125G_G126S) and THRB-KO human iPSC lines to study noncanonical thyroid hormone signalling

THRB is a nuclear receptor, regulating gene expression dependent on thyroid hormone (TH) binding. The same receptor mediates signaling pathway activation in the cytosol. The challenge is to distinguish which of the two mechanisms is responsible for physiological effects of TH. We established an iPSC cell line with two mutations (E125G_G126S) in the THRB DNA-binding domain, which abrogates nuclear action and, thus, allows to study signaling pathway activation exclusively. We also generated a THRB knockout cell line to abolish all THRB effects. Comparison of WT and these two cell lines allows attribution of thyroid hormone effects to the underlying mechanism.

Interplay of mutations, alternate mechanisms, and treatment breaks in leukaemia: Understanding and implications studied with stochastic models

Bcr-Abl1 kinase domain mutations are the most prevalent cause of treatment resistance in chronic myeloid leukaemia (CML). Alternate resistance pathways nevertheless exist, and cell line experiments show certain patterns in the gain, and loss, of some of these alternate adaptations. These adaptations have clinical consequences when the tumour develops mechanisms that are beneficial to its growth under treatment, but slow down its growth when not treated. The results of temporarily halting treatment in CML have not been widely discussed in the clinic and there is no robust theoretical model that could suggest when such a pause in therapy can be tolerated. We constructed a dynamic model of how mechanisms such as Bcr-Abl1 overexpression and drug transporter upregulation evolve to produce resistance in cell lines, and investigate its behaviour subject to different treatment schedules, in particular when the treatment is paused (‘drug holiday’). Our study results suggest that the presence of additional resistance mechanisms creates an environment which favours mutations that are either preexisting or occur late during treatment. Importantly, the results suggest the existence of tumour drug addiction, where cancer cells become dependent on the drug for (optimal) survival, which could be exploited through a treatment holiday. All simulation code is available at https://github.com/Sandalmoth/dual-adaptation.

Binding Thermodynamics of Fourth-Generation EGFR Inhibitors Revealed by Absolute Binding Free Energy Calculations.

The overexpression or mutation of the kinase domain of the epidermal growth factor receptor (EGFR) is strongly associated with non-small-cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) have proven to be effective in treating NSCLC patients. However, EGFR mutations can result in drug resistance. To elucidate the mechanisms underlying this resistance and inform future drug development, we examined the binding affinities of BLU-945, a recently reported fourth-generation TKI, to wild-type EGFR (EGFRWT) and its double-mutant (L858R/T790M; EGFRDM) and triple-mutant (L858R/T790M/C797S; EGFRTM) forms. We compared the binding affinities of BLU-945, BLU-945 analogues, CH7233163 (another fourth-generation TKI), and erlotinib (a first-generation TKI) using absolute binding free energy calculations. Our findings reveal that BLU-945 and CH7233163 exhibit binding affinities to both EGFRDM and EGFRTM stronger than those of erlotinib, corroborating experimental data. We identified K745 and T854 as the key residues in the binding of fourth-generation EGFR TKIs. Electrostatic forces were the predominant driving force for the binding of fourth-generation TKIs to EGFR mutants. Furthermore, we discovered that the incorporation of piperidinol and sulfone groups in BLU-945 substantially enhanced its binding capacity to EGFR mutants. Our study offers valuable theoretical insights for optimizing fourth-generation EGFR TKIs.

Uncovering the Structural and Binding Insights of Dual Inhibitors Simultaneously Targeting Two Distinct Sites on EGFR Kinase.

Epidermal growth factor receptor (EGFR) is the first growth factor receptor identified in normal cells that is related to the receptor tyrosine kinase, which causes regular cell division. A point mutation in EGFR intracellular kinase domain forces the abnormal cell divisions throughout time, leading to non-small cell lung cancer (NSCLC) transformation. Thus, competitive inhibitors that bind to the ATP binding pocket have been developed as a targeted therapy for NSCLC. The third-generation kinase inhibitor Osimertinib is currently playing a very vital role in the treatment of NSCLC. However, it is not effective toward the C797S kinase domain mutation. For this reason, fourth-generation kinase noncompetitive inhibitors are introduced which work through binding to an allosteric pocket near the ATP binding region and act as a better binding agent for this mutated kinase domain. However, the problem is that these single fourth-generation kinase inhibitors may not be as effective as a single agent. The aim of this work was to apply combinations of these two inhibitors together in different binding regions of EGFR without overlapping the resistance mechanism to obtain the key direct and indirect interactions occurring between them. Moreover, the free energy of dissociation of an inhibitor from its binding sites in the presence of a second inhibitor immobilized in another binding site was also the focus of the study. To realize this aim, we performed conventional molecular dynamics simulations and principal component analysis and dynamic cross-correlation matrices along with umbrella sampling. Our results demonstrated that binding of dual inhibitors triggered conformational changes of the protein more toward the inactive state. Furthermore, allosteric inhibitors bound more strongly to protein kinase EGFR than the orthosteric inhibitors in the presence of dual inhibitors. Finally, the binding mechanism and important hydrogen-bonding residues during unbinding of the inhibitors were fully elucidated. This study provides insight into the binding of the receptor-orthosteric inhibitor-allosteric inhibitor, which can be helpful for further design of novel inhibitors that have a better inhibitory action.

Lactococcus lactis mutants resistant to lactococcin A and garvicin Q reveal missense mutations in the sugar transport domain of the mannose phosphotransferase system.

Many bacteriocins target the sugar transporter mannose phosphotransferase system (man-PTS) to exert their antibacterial activity. The elucidation in recent years of the structure of man-PTS has facilitated our understanding of how bacteriocins might interact with the receptor and which domains of the transporter are involved in bacteriocin resistance. Here, we show that missense mutations in the sugar-binding domain of the man-PTS not only impede the uptake of sugars but also prevent the antibacterial activity of the bacteriocins lactococcin A and garvicin Q.

A study of the synthetic route and resistance of gilteritinib

Gilteritinib is used to treat acute myeloid leukemia (AML) carrying FMS-like tyrosine kinase 3 (FLT3) mutations. As it is a new drug, there are still many aspects that need improvement. This article will be written according to the following three points: Analysis of its mechanism of action with the receptor; Analysis of the three main synthetic routes of gilteritinib and reasonable suggestions are given; Causes and solutions to the emergence of drug resistance. The following 4 points have been found that:(1) FLT3-internal tandem duplication (ITD) mutation and FLT3-tyrosine kinase domain (TKD) mutation, leukemic cells' ability to proliferate, differentiate, and survive aberrant changes due to both kinds of mutations. (2) Gilteritinib combines with FLT3's ATP pocket, and the compound shaped from FLT3 to gilteritinib was synthesized by the juxtamembrane (JM) domain. (3) Synthesis route 3 in this paper is most suitable for industrial mass production. (4) Resistance to gilteritinib is divided into primary resistance and secondary resistance, research in the future should focus on finding new therapeutic targets.

Myo10 tail is crucial for promoting long filopodia

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.

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.

Abstract B085: Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer

Abstract Purpose: All patients with metastatic breast cancer (BC) expressing estrogen receptor α (ESR1) will eventually develop resistance to endocrine therapies. In up to 40% of cases, this resistance is caused by activating mutations in the ligand-binding domain (LBD) of ESR1. Clinical evidence suggests adverse outcomes for these patients. Our hypothesis was that ESR1 mutations may confer resistance to subsequent treatment lines, specifically chemotherapy. Experimental Design: MCF-7 cells harboring ESR1 mutations (Y537S and D538G) were used to characterize proliferation, apoptosis, and tumor response to chemotherapy in vivo. JNK/c-Jun/MDR1 pathway was studied using qRT-PCR, western-blot, gene-reporter, ChIP assays and pharmacological inhibition. Results: Our results revealed that the mutated-ER (mut-ER) cells displayed relative chemoresistance, demonstrated by higher viability and reduced apoptosis. In vivo mouse model confirmed these findings. To elucidate the underlying mechanism, we examined MDR1 expression and JNK pathway activity. We observed elevated MDR1 expression in mut-ER cells, which was also reflected in clinical BC samples. Additionally, increased occupancy of the MDR1 promoter by c-Jun was observed in mut-ER cells. Importantly, inhibition of the JNK pathway resulted in decreased MDR1 expression, particularly in D538G cells. Moreover, JNK inhibition increased apoptosis and reduced viability in all cells, with a more pronounced effect in D538G cells. Conclusions: Taken together, our data indicate that ESR1 mutations confer chemoresistance in BC through the activation of the JNK/MDR1 axis. These findings suggest that targeting this pathway could be a potential strategy for novel treatments aimed at restoring chemotherapy sensitivity and improving outcomes for patients with metastatic BC. Citation Format: Marwa Taya, Keren Merenbakh-Lamin, Asia Zubkov, Zohar Honig, Ori Mayer, Noam Shomron, Ido Wolf, Tami Rubinek. Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer [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 B085.

Irreversible inhibition of TRF2TRFH recruiting functions by a covalent cyclic peptide induces telomeric replication stress in cancer cells

The TRF2 shelterin component is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, causing telomeric DNA damage. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound, as it consistently induces a telomeric DNA damage response in cancer cell lines. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response, increased telomeric replication stress, and impaired recruitment of RTEL1 and SLX4 to telomeres. We demonstrate that APOD53 impairs cancer cell growth and find that co-treatment with APOD53 can exacerbate telomere replication stress caused by the G4 stabilizer RHPS4 and low dose aphidicolin (APH).

Inhibition potential of natural flavonoids against selected omicron (B.1.19) mutations in the spike receptor binding domain of SARS-CoV-2: a molecular modeling approach

The omicron (B.1.19) variant of contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is considered a variant of concern (VOC) due to its increased transmissibility and highly infectious nature. The spike receptor-binding domain (RBD) is a hotspot of mutations and is regarded as a prominent target for screening drug candidates owing to its crucial role in viral entry and immune evasion. To date, no effective therapy or antivirals have been reported; therefore, there is an urgent need for rapid screening of antivirals. An extensive molecular modelling study has been performed with the primary goal to assess the inhibition potential of natural flavonoids as inhibitors against RBD from a manually curated library. Out of 40 natural flavonoids, five natural flavonoids, namely tomentin A (−8.7 kcal/mol), tomentin C (−8.6 kcal/mol), hyperoside (−8.4 kcal/mol), catechin gallate (−8.3 kcal/mol), and corylifol A (−8.2 kcal/mol), have been considered as the top-ranked compounds based on their binding affinity and molecular interaction profiling. The state-of-the-art molecular dynamics (MD) simulations of these top-ranked compounds in complex with RBD exhibited stable dynamics and structural compactness patterns on 200 nanoseconds. Additionally, complexes of these molecules demonstrated favorable free binding energies and affirmed the docking and simulation results. Moreover, the post-simulation validation of these interacted flavonoids using principal component analysis (PCA) revealed stable interaction patterns with RBD. The integrated results suggest that tomentin A, tomentin C, hyperoside, catechin gallate, and corylifol A might be effective against the emerging variants of SARS-CoV-2 and should be further evaluated using in-vitro and in-vivo experiments. Communicated by Ramaswamy H. Sarma

Abstract C145: ANS014004, a novel small-molecule type II c-Met inhibitor effectively overcomes clinical-resistance MET mutations and exhibits antitumor activity in preclinical models of MET-amplified non-small cell lung cancer (NSCLC) and gastric cancer

Abstract Mesenchymal epithelial transition (MET) proto-oncogene receptor tyrosine kinase (RTK) is a cell surface receptor selective for hepatocyte growth factor (HGF), and is involved in embryogenesis regulation, wound healing, organ regeneration, angiogenesis, and immunomodulation. Aberrant MET oncogenic alterations include: MET exon 14 skipping (MET∆ex14) mutations; activating mutations in the kinase domain; MET gene amplification; MET fusions; and MET protein overexpression. These oncogenic alterations occur in a wide range of human solid cancers. Small-molecule type I c-Met inhibitors bind to the active (Asp-Phe-Gly “DFG-in” state) of c-Met within the adenosine triphosphate (ATP) binding pocket via the Y1230 residue, resulting in down-regulation of multiple pro-oncogenic downstream factors. Presently, type I c-Met inhibitors such as Capmatinib are used as monotherapies in patients with locally advanced or metastatic NSCLC with MET∆ex14 mutations. However, development of post-treatment resistance to type I c-Met inhibitors occurs clinically, including through acquired mutations in codons D1228 and Y1230. No drugs have been approved globally for MET alteration indications other than MET∆ex14 mutations. Next generation MET inhibitors are thus needed to treat patients harboring various MET oncogenic alterations beyond MET∆ex14, including post-treatment acquired mutations. ANS014004 is a newly developed small-molecule type II inhibitor of c-Met, binding to the inactive state of c-Met (“DFG-out”) in the ATP pocket. ANS014004 showed greater kinase inhibition activity against c-Met mutant kinases Y1230H and D1228A in vitro (IC50’s = 2.596 and 2.971 nM, respectively), when compared with Capmatinib, Cabozantinib, and Merestinib (IC50 values = &amp;gt;5000, ~10, and ~5 nM respectively). Further, ANS014004 showed a stronger anti-proliferative effect in MET mutant Ba/F3 cells TPR-Met D1228A and TPR-Met-Y1230H in vitro (IC50’s = 49.34 nM and 30.32 nM, respectively), when compared with Capmatinib (IC50 values&amp;gt;10000nM) and Merestinib (IC50 values: 37~100 nM). ANS014004 also showed potent antitumor effects in vitro in the human cancer tumor cell lines harboring various pathogenetic MET alterations: Hs-746T (gastric); MKN-45 (gastric); EBC-1 (NSCLC); NCI-H1993 (NSCLC); with IC50 ranging from 6.38~62.32 nM. In several subcutaneous xenograft tumor model studies using these same cell lines implanted in BALB/c nude mice, orally administered ANS014004 produced significant dose-dependent tumor growth inhibition, and the corresponding exposures of ANS014004 in plasma, tumor and brain samples increased with increasing dose. ANS0104001 was also observed to induce tumor regression in a MET-amplified intracranial xenograft model. Overall, the non-clinical studies of ANS014004 demonstrated it is a potent, orally-bioavailable type II c-Met inhibitor with activity against various pathogenetic MET alterations and with favorable absorption, distribution, pharmacokinetic, efficacy, and tolerability profiles in vivo. The molecule will enter a Phase 1 clinical study in the US soon. Citation Format: Gong Li, Hepeng Shi, Peilong Zhang, Xiangqiu Li, Wenli Lan, Linna Li, David Chung, Kevin Schaab. ANS014004, a novel small-molecule type II c-Met inhibitor effectively overcomes clinical-resistance MET mutations and exhibits antitumor activity in preclinical models of MET-amplified non-small cell lung cancer (NSCLC) and gastric cancer [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 C145.

Abstract C165: Zurletrectinib, a next-generation TRK inhibitor shows strong intracranial activity against NTRK fusion-positive tumors with on-target resistance to first-generation drugs

Abstract NTRK fusions are oncogenic drivers in multiple tumor types. While treatment with TRK inhibitors is effective in ~80% of patients with NTRK fusion-positive tumors, resistance eventually occurs. The most common mechanism of resistance is the emergence of mutations in the kinase domain of the fusions that impair drug binding. Next-generation TRK inhibitors active against these mutants are in clinical development (selitrectinib) or have received breakthrough therapy designation (repotrectinib). Here, we describe the activity of zurletrectinib, a novel next-generation TRK inhibitor. Cell viability assays, in silico modeling, and western blot analyses were used to test the efficacy of zurletrectinib against NTRK fusion-positive models and the most common TRKA resistant mutations. Finally, the in vivo activity of zurletrectinib was assessed in glioma orthotopic models. Selitrectinib, zurletrectinib, and repotrectinib inhibited in vitro cell growth and TRK-mediated signaling of multiple NTRK fusion-positive cell lines with IC50s in the low nanomolar range. Next, we used in silico modeling to predict the binding of zurletrectinib to TRK resistant mutations. These include the TRKA G595R solvent front and the TRKA G667C xDFG substitutions, and the TRKA G595R/G667A or the TRKA G595R/G667C double mutations. Selitrectinib, repotrectinib and zurletrectinib were all predicted to bind the TRKA G595R, and the TRKA G595R/G667A mutants, but not the bulkier TRKA G667C or the TRKA G595R/G667C mutants. To confirm our results, we tested the activity of zurletrectinib against multiple primary human NTRK fusion-positive, TRKA-mutant cell lines. All next-generation inhibitors were active against models expressing the TRKA G595R or the TRKA G595R/G667A double mutation, but not against TRKA G595R/G667C mutant models. Similar results were obtained when the same drugs were tested against mouse NTRK fusion-positive glioma clones engineered by CRISPR to express the mouse Trka ortholog mutations. To determine the in vivo activity of zurletrectinib, mouse glioma orthotopic xenografts harboring the Trka G598R or the Trka G598R/G670A mutations were used. While no significant difference in the survival between selitrectinib, repotrectinib and zurletrectinib was observed in the Trka G598R model, zurletrectinib significantly improved survival of mice harboring the Trka G598R/G670A mutation (median survival=41.5, 66.5, and 104 days for selitrectinib, repotrectinib, and zurletrectinib respectively; P(selitrectinib/zurletrectinib=.0022; P(repotrectinib/zurletrectnib=.038). Together, our data identifies zurletrectinib as a novel, highly potent and selective next-generation TRK inhibitor with strong intracranial activity and a potentially more favorable safety profile than other similar inhibitors. Citation Format: Paola Roa, Valentina Foglizzo, Guilherme Harada, Matteo Repetto, Amanda Kulick, Elisa de Stanchina, Supipi Auwardt, Michelle de Marchena, Shaza Ahmed, Nicole Bremer, Soo-Ryum Yang, Alexander Drilon, Emiliano Cocco. Zurletrectinib, a next-generation TRK inhibitor shows strong intracranial activity against NTRK fusion-positive tumors with on-target resistance to first-generation drugs [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 C165.

Abstract C151: BLU-451 is a CNS-penetrant, wild-type-sparing EGFR inhibitor with broad coverage of uncommon EGFR mutations across structure-based subsets

Abstract Introduction: Uncommon EGFR mutations in non-small cell lung cancer (NSCLC), including exon 20 insertion and atypical mutations, are generally associated with poorer clinical outcomes than common EGFR mutations. Patients (pts) with these mutations have limited effective therapeutic options. Afatinib is the only approved agent for uncommon mutations but is associated with high rates of toxicity and limited central nervous system (CNS) penetration. Amivantamab and mobocertinib are approved for patients with relapsed EGFR exon 20 insertion NSCLC but are associated with limitations in efficacy and CNS activity. BLU-451 is an investigational, CNS-penetrant, EGFR-wild-type (WT)–sparing, selective inhibitor against exon 20 insertion and uncommon EGFR mutations that is currently being evaluated in the phase 1/2 CONCERTO study (NCT05241873). Here, we report on the prevalence of uncommon EGFR mutations from a single center, real-world data set (MD Anderson Cancer Center [MDACC]), and report preclinical activity of BLU-451 on a broad spectrum of uncommon EGFR mutations by structure classification. Methods: BLU-451 cellular activity was evaluated in engineered BaF3 cell lines expressing 62 EGFR kinase domain mutations as well as in cell lines dependent on WT EGFR, using the CellTiter-Glo® Luminescent Cell Viability Assay and a phosphorylation-specific EGFR AlphaLISA® assay. The MDACC Moon Shot™ Genomic Marker-Guided Therapy Initiative (GEMINI) database was searched for pts with NSCLC whose tumors harbored EGFR kinase domain mutations. Results: In the MDACC GEMINI database, a total of 1495 unique patients with NSCLC harboring EGFR mutations were identified, including 1025 with classical mutations (exon 19 deletion and L858R) with or without T790M, 223 with exon 20 insertion mutations, and 247 with only atypical mutations. In BaF3 cells, BLU-451 demonstrated nanomolar potency in multiple atypical and exon 20 insertion mutation cell lines with selectivity against WT EGFR. Conclusion: Atypical EGFR mutations represent a sizable NSCLC pt population, of similar magnitude to that of EGFR exon 20 insertion mutations. BLU-451 preclinical data in EGFR atypical and exon 20 insertion mutations showed broad, potent, and selective activity, supporting continued evaluation of BLU-451 in the phase 1/2 CONCERTO study in pts with advanced or metastatic NSCLC harboring uncommon EGFR mutations. Citation Format: Xiuning Le, Yves Millet, Monique Nilsson, Aditya Dhande, Jeffrey Keats, Holly Ponichtera, Joseph Kim, Junqin He, Yasir Y. Elamin, John V. Heymach, Chiara Conti, Stephanie Lee. BLU-451 is a CNS-penetrant, wild-type-sparing EGFR inhibitor with broad coverage of uncommon EGFR mutations across structure-based subsets [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 C151.

E3 ligase activity of RNF168 at stake on ring domain mutation

E3 ligase activity of RNF168 at stake on ring domain mutation

Missense mutations in the central domains of cardiac myosin binding protein-C and their potential contribution to hypertrophic cardiomyopathy

Myosin binding protein-C (MyBP-C) is a multidomain protein that regulates muscle contraction. Mutations in MYBPC3, the gene encoding for the cardiac variant (henceforth called cMyBP-C), are amongst the most frequent causes of hypertrophic cardiomyopathy (HCM). Most mutations lead to a truncated version of cMyBP-C, which is most likely unstable. However, missense mutations have also been reported, which tend to cluster in the central domains of the cMyBP-C molecule. This suggests that these central domains are more than just a passive spacer between the better characterised N- and C-terminal domains. Here we investigated the potential impact of four different missense mutations, E542Q, G596R, N755K and R820Q, which are spread over the domains C3 to C6, on the function of MyBP-C on both the isolated protein level and in cardiomyocytes in vitro. Effect on domain stability, interaction with thin filaments, binding to myosin and subcellular localisation behaviour were assessed. Our studies show that these missense mutations result in slightly different phenotypes at the molecular level, which are mutation specific. The expected functional readout of each mutation provides a valid explanation for why cMyBP-C fails to work as a brake in the regulation of muscle contraction, which eventually results in a HCM phenotype. We conclude that missense mutations in cMyBP-C must be evaluated in context of their domain localisation, their effect on interaction with thin filaments and myosin, and their effect on protein stability to explain how they lead to disease.

A gain-of-function HCN4 mutant in the HCN domain is responsible for inappropriate sinus tachycardia in a Spanish family.

In a family with inappropriate sinus tachycardia (IST), we identified a mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (If) in human sinoatrial node cells. Here, we clinically study fifteen family members and functionally analyze the p.V240M variant. Macroscopic (IHCN4) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels. All p.V240M mutation carriers exhibited IST that was accompanied by cardiomyopathy in adults. IHCN4 generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels alone. The variant, which lies in the N-terminal HCN domain, increased the single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify the channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers. In computer simulations, the p.V240M gain-of-function variant increases If and beating rate and thus explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4, which stabilizes the channels in the closed state.

A single coiled-coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels.

The expression of IKCa (SK4) channel subunits overlaps with that of SK channel subunits, and it has been proposed that the two related subunits prefer to co-assemble to form heteromeric hSK1:hIKCa channels. This implicates hSK1:hIKCa heteromers in physiological roles that might have been attributed to activation of SK channels. We have used a mutation approach to confirm formation of heterometric hSK1:hIKCa channels. Introduction of residues within hSK1 that were predicted to impart sensitivity to the hIKCa current blocker TRAM-34 changed the pharmacology of functional heteromers. Heteromeric channels formed between wildtype hIKCa and mutant hSK1 subunits displayed a significantly higher sensitivity and maximum block to addition of TRAM-34 than heteromers formed between wildtype subunits. Heteromer formation was disrupted by a single point mutation within one COOH-terminal coiled-coil domain of the hIKCa channel subunit. This mutation only disrupted the formation of hSK1:hIKCa heteromeric channels, without affecting the formation of homomeric hIKCa channels. Finally, the Ca2+ gating sensitivity of heteromeric hSK1:hIKCa channels was found to be significantly lower than the Ca2+ gating sensitivity of homomeric hIKCa channels. These data confirmed the preferred formation of heteromeric channels that results from COOH-terminal interactions between subunits. The distinct sensitivity of the heteromer to activation by Ca2+ suggests that heteromeric channels fulfil a distinct function within those neurons that express both subunits.