Non-canonical Binding Research Articles

The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

RNA binding proteins play critical roles in tumor progression by participating in the post-transcriptional regulation of RNA. However, the levels and function of RBPs in nasopharyngeal carcinoma (NPC) remain elusive. Here we identified a non-canonical RNA binding protein RAN that has the most significant role in NPC progression by a small siRNA pool screening. Functionally, RAN facilitates NPC proliferation and metastasis in vitro and in vivo. High levels of RAN are associated with poor prognosis of NPC patients and can be performed as a prognostic biomarker. Mechanistically, RAN increases the nucleus import of TDP43 and enhances TDP43 nuclear distribution. On the other hand, RAN is directly bound to the coding sequence of G3BP1 mRNA and serves as an adapter to facilitate TDP43 interacting with G3BP1 mRNA 3′ untranslated region. These contribute to increasing G3BP1 mRNA stability in the nucleus and lead to up-regulation of G3BP1, which further enhances AKT and ERK signaling and ultimately promotes NPC proliferation and metastasis. These findings reveal that RAN stabilizes intranuclear G3BP1 mRNA by dual mechanisms: recruiting TDP43 into the nucleus and enhancing its interaction with G3BP1 mRNA, suggesting a critical role of RAN in NPC progression and providing a new regulation framework of RBP-RNA.

AP-1 contributes to endosomal targeting of ubiquitin ligase RNF13 via a secondary and novel non-canonical binding motif.

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. Ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis, and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within RNF13's C-terminal region that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular, and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by variant of RNF13's dileucine sorting motif.

New targets and designed inhibitors of ASAP Arf-GAPs derived from structural characterization of the ASAP1/440-kD ankyrin-B interaction

ASAP1 and its paralog ASAP2 belong to a PI4,5P2-dependent Arf GTPase-activating protein (Arf-GAP) family capable of modulating membrane and cytoskeletal dynamics. ASAPs regulate cell adhesive structures such as invadosomes and focal adhesions during cell attachment and migration. Malfunctioning of ASAP1 has been implicated in the malignant phenotypes of various cancers. Here, we discovered that the SH3 domain of ASAP1 or ASAP2 specifically binds to a 12-residue, positively charged peptide fragment from the 440 kDa giant ankyrin-B, a neuronal axon specific scaffold protein. The high-resolution structure of the ASAP1-SH3 domain in complex with the gAnkB peptide revealed a non-canonical SH3-ligand binding mode with high affinity and specificity. Structural analysis of the complex readily uncovered a consensus ASAP1-SH3 binding motif, which allowed the discovery of a number of previously unknown binding partners of ASAP1-SH3 including Clasp1/Clasp2, ALS2, β-Pix, DAPK3, PHIP, and Limk1. Fittingly, these newly identified ASAP1 binding partners are primarily key modulators of the cytoskeletons. Finally, we designed a cell-penetrating, highly potent ASAP1 SH3 domain binding peptide with a Kd ∼7 nM as a tool for studying the roles of ASAPs in different cellular processes.

The RNA binding protein Arid5a drives IL-17-dependent autoantibody-induced glomerulonephritis.

Autoantibody-mediated glomerulonephritis (AGN) arises from dysregulated renal inflammation, with urgent need for improved treatments. IL-17 is implicated in AGN and drives pathology in a kidney-intrinsic manner via renal tubular epithelial cells (RTECs). Nonetheless, downstream signaling mechanisms provoking kidney pathology are poorly understood. A noncanonical RNA binding protein (RBP), Arid5a, was upregulated in human and mouse AGN. Arid5a-/- mice were refractory to AGN, with attenuated myeloid infiltration and impaired expression of IL-17-dependent cytokines and transcription factors (C/EBPβ, C/EBPδ). Transcriptome-wide RIP-Seq revealed that Arid5a inducibly interacts with conventional IL-17 target mRNAs, including CEBPB and CEBPD. Unexpectedly, many Arid5a RNA targets corresponded to translational regulation and RNA processing pathways, including rRNAs. Indeed, global protein synthesis was repressed in Arid5a-deficient cells, and C/EBPs were controlled at the level of protein rather than RNA accumulation. IL-17 prompted Arid5a nuclear export and association with 18S rRNA, a 40S ribosome constituent. Accordingly, IL-17-dependent renal autoimmunity is driven by Arid5a at the level of ribosome interactions and translation.

Non-Canonical Targets of MicroRNAs: Role in Transcriptional Regulation, Disease Pathogenesis and Potential for Therapeutic Targets.

MicroRNAs are a class of regulatory, non-coding small ribonucleic acid (RNA) molecules found in eukaryotes. Dysregulated expression of microRNAs can lead to downregulation or upregulation of their target gene. In general, microRNAs bind with the Argonaute protein and its interacting partners to form a silencing complex. This silencing complex binds with fully or partial complementary sequences in the 3'-UTR of their cognate target mRNAs and leads to degradation of the transcripts or translational inhibition, respectively. However, recent developments point towards the ability of these microRNAs to bind to the promoters, enhancers or coding sequences, leading to upregulation of their target genes. This review briefly summarizes the various non-canonical binding sites of microRNAs and their regulatory roles in various diseased conditions.

A phase 1 dose-escalation and expansion study of an intratumorally administered dual STING agonist (ONM-501) alone and in combination with cemiplimab in patients with advanced solid tumors and lymphomas.

TPS2693 Background: The Stimulator of Interferon Genes (STING) pathway has garnered significant interest as a target for anticancer interventions due to its role in driving the production of pro-inflammatory cytokines and activating tumor-specific cell killing. Cyclic dinucleotides (CDNs) such as cGAMP, the endogenous STING agonist, are not ideal clinical candidates for exogenous routes of delivery. Early trials showed limited efficacy with CDNs due to rapid clearance, degradation and/or off-target toxicity. ONM-501 is comprised of a STING-activating pH-sensitive PC7A polymer conjugated with cGAMP. At physiologic pH, ONM-501 forms a micelle, preventing degradation of cGAMP. In the acidic tumor microenvironment, ONM-501 effects STING agonism through a multi-faceted mode of action: 1) endocytosis of nanoparticles and pH-activated micelle dissociation and payload release in endolysosomes enhances intracellular delivery of cGAMP 2) the combination of cGAMP canonical binding, and PC7A polymer noncanonical binding on STING yields synergistic STING activation and 3) stabilization of STING by PC7A polymers delays its degradation and prolongs STING activation. In pre-clinical models, these factors combine to help generate a stronger and more prolonged innate immune response that more effectively translates to a robust adaptive immune response. ONM-501 is being investigated in this first-in-human Phase 1 trial as monotherapy and combination therapy with the anti-PD1 antibody, cemiplimab, for patients with solid tumors and lymphomas. Methods: ONM5001 (NCT06022029) is a first-in-human, open-label, multi-center clinical trial. Adult patients with solid tumors or lymphomas for which no standard therapy exists or for which patients decline palliative standard therapy who have at least one injectable lesion are eligible for this study. This Phase 1 study consists of three parts: monotherapy dose escalation; combination therapy dose finding; and combination therapy dose expansion in specific tumor indication(s) that will be selected based on data from mono- and combination therapy dose finding. Each dosing cycle of ONM-501 is 21 days: patients receive an intratumoral injection of ONM-501 weekly for three weeks, followed by three weeks without injection. Cemiplimab is administered intravenously during combination therapy arms according to standard administration protocol, once every three weeks. The primary endpoint is to evaluate safety of ONM-501 monotherapy as evidenced by the number of dose limiting toxicities and treatment-emergent adverse events, and to determine the recommended dose for expansion. Secondary endpoints include characterization of pharmacokinetics and pharmacodynamics, as well as initial efficacy based on RECIST evaluation criteria. Enrollment began in November 2023. Clinical trial information: NCT06022029 .

Arid5a-dependent RNA translation underlies pathology in IL-17-driven autoantibody-induced glomerulonephritis

Abstract Autoantibody-mediated glomerulonephritis (AGN) results from kidney-damaging autoimmune responses, and there is urgent need for better treatment. Emerging studies implicate Th17 cells in AGN. IL-17 signals in part through the noncanonical RNA binding protein Arid5a, which we show is induced in renal epithelial cells (RTECs) in an IL-17-dependent manner during experimental AGN. Arid5a is undetectable in healthy kidney but elevated in human glomerulonephritis biopsies. Arid5a-/- mice were refractory to AGN. Mice lacking C/EBPd but not IkBz were protected from AGN. Although Arid5a bound to Cebpd mRNA, Arid5a-deficiency did not alter cellular Cebpd mRNA concentrations, whereas C/EBPd protein levels were substantially suppressed. In line with this, global protein synthesis was suppressed in RTECs lacking Arid5a. Transcriptome-wide RIP-Seq analysis in RTECs and stromal cells showed that IL-17 prompts Arid5a to bind to inflammatory mRNAs. Additionally and unexpectedly, Arid5a interacted with ribosomal RNAs (rRNAs) and other transcripts involved in protein translation. After IL-17 treatment, Arid5a was enriched in the cytoplasm and associated with the ribosome through interactions with 40S and 60S rRNAs. Accordingly, Arid5a drives IL-17-induced renal autoimmunity through post-transcriptional control of RNA at the level of translation and ribosome interactions, providing mechanistic insight into how cytokines mediate inflammation.

Detergents with Scalable Properties Identify Noncanonical Lipopolysaccharide Binding to Bacterial Inner Membrane Proteins.

Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear. Here, we leverage the control of the hydrophilic-lipophilic balance and packing parameter concepts to chemically tune detergents that can be used to qualitatively differentiate the degree to which proteins copurify with phospholipids (PLs) and/or LPS. Given the scalable properties of these detergents, we demonstrate a detergent fine-tuning that enables the facile investigation of intact proteins and their complexes with lipids by native mass spectrometry (nMS). We conclude that LPS, a lipid that is believed to be important for outer membranes, can also affect the activity of membrane proteins that are currently not assigned to be involved in outer membrane biogenesis. Our results deliver a scalable detergent chemistry for a streamlined biophysical characterization of protein-lipid interactions, provide a rationale for the high affinity of LPS-protein binding, and identify noncanonical associations between LPS and inner membrane proteins with relevance for membrane biology and antibiotic research.

Abstract 3582: PPAR gamma acetylation governs mammary adenocarcinoma tumor growth via acetylated residues that determine DNA sequence-specific binding

Abstract Background: HER2/neu overexpression in breast cancer confers a lipogenic phenotype. Although the introduction of anti-Her2 therapies has led to dramatic improvements in survival, nearly all patients with metastatic Her2-positive breast cancer will progress on treatment suggesting the importance of developing coextinction approaches targeting multiple pathways. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. PPARγ binds additional DNA cis elements associated with other transcription factors, including C/EBPs, NFκB and AP-1 proteins, to promote non-canonical signaling. Defining the mechanisms governing the selection of canonical versus non-canonical PPARγ binding sequences may provide the opportunity to design regulators with distinct functions and side effects. PPARγ1 acetylation at K268/293 participates in the regulation of adipose tissue differentiation (1), and the conserved lysine residues (K154/155) govern lipogenesis in breast cancer cells (2). Although Cre based Pparγ1 gene deletion in mammary tumor oncomice showed Pparγ1 participates in the onset and progression of ErbB2-induced mammary tumorigenesis (3), the molecular mechanisms and the post-translational modifications of PPARγ governing Pparγ1 tumorigenic function remained to be determined. Methods: Herein, we defined the role of PPARγ1 acetylation in breast cancer growth in immune-deficient mice using distinct breast cancer cell lines (MCF10-Ras, MCF10A-NeuT). Furthermore, using ChIP and ChIP-Seq we show that the acetylated residues of Pparγ1 altered preference of cis-element binding in chromatin to augment Pparγ non-canonical binding. Results: Herein, the PPARγ1 acetylated residues K154/155 were shown to be essential for the induction of transcriptional modules governing growth factor signaling, cellular apoptosis, and autophagy in mice. The K154/155 residues determined the selection of genome-wide DNA binding sites, altering the selection from canonical to non-canonical (C/EBP) DNA sequence-specific binding. The gene signature reflecting the acetylation-dependent genomic occupancy provided predictive value in patient survival outcomes. Conclusions: Endogenous Pparγ1 promotes mammary tumor onset and progression. PPARγ1 breast tumor induction involves K154/155, which governs transcriptional programs (growth, apoptosis, and autophagy) via transcriptional complex binding to canonical vs. non-canonical regulatory elements. Pparγ1 acetylation participates in ErbB2-induced breast cancer tumor growth and inflammation and may represent a relevant target for therapeutic coextinction.

Structure of an open KATP channel reveals tandem PIP2 binding sites mediating the Kir6.2 and SUR1 regulatory interface

ATP-sensitive potassium (KATP) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. KATP channel opening is stimulated by PIP2 and inhibited by ATP. Mutations that increase channel opening by PIP2 reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP2 in KATP channel function, previously solved open-channel structures have lacked bound PIP2, and mechanisms by which PIP2 regulates KATP channels remain unresolved. Here, we report the cryoEM structure of a KATP channel harboring the neonatal diabetes mutation Kir6.2-Q52R, in the open conformation, bound to amphipathic molecules consistent with natural C18:0/C20:4 long-chain PI(4,5)P2 at two adjacent binding sites between SUR1 and Kir6.2. The canonical PIP2 binding site is conserved among PIP2-gated Kir channels. The non-canonical PIP2 binding site forms at the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1-W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP2 binding and gating, explain the antagonistic regulation of KATP channels by PIP2 and ATP, and provide a putative mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.

Alternative target recognition elements for chimeric antigen receptor (CAR) T cells: beyond standard antibody fragments

Background aimsChimeric antigen receptor T (CAR-T) cells are a remarkably efficacious, highly promising and rapidly evolving strategy in the field of immuno-oncology. The precision of these targeted cellular therapies is driven by the specificity of the antigen recognition element (the “binder”) encoded in the CAR. This binder redirects these immune effector cells precisely toward a defined antigen on the surface of cancer cells, leading to T-cell receptor–independent tumor lysis. Currently, for tumor targeting most CAR-T cells are designed using single-chain variable fragments (scFvs) derived from murine or human immunoglobulins. However, there are several emerging alternative binder modalities that are finding increasing utility for improved CAR function beyond scFvs. MethodsHere we review the most recent developments in the use of non-canonical protein binding domains in CAR design, including nanobodies, DARPins, natural ligands, and de novo–designed protein elements. ResultsOverall, we describe how new protein binder formats, with their unique structural properties and mechanisms of action, may possess key advantages over traditional scFv CAR designs. ConclusionsThese alternative binder designs may contribute to enhanced CAR-T therapeutic options and, ultimately, improved outcomes for cancer patients.

Splicing analysis of STAT3 tandem donor suggests non-canonical binding registers for U1 and U6 snRNAs.

Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought.

Cbp1 and Cren7 form chromatin-like structures that ensure efficient transcription of long CRISPR arrays

CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbour extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposons. Cbp1 recruits Cren7 forming together ‘chimeric’ chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third helix-turn-helix domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays.

The noncanonical nucleotide binding site 1 of the bile salt export pump is optimized for proper function of the transporter.

The bile salt export pump (ABCB11/BSEP) is a hepatocyte plasma membrane-resident protein translocating bile salts into bile canaliculi. The sequence alignment of the four full-length transporters of the ABCB subfamily (ABCB1, ABCB4, ABCB5 and ABCB11) indicates that the NBD-NBD contact interface of ABCB11 differs from that of other members in only four residues. Notably, these are all located in the noncanonical nucleotide binding site 1 (NBS1). Substitution of all four deviant residues with canonical ones (quadruple mutant) significantly decreased the transport activity of the protein. In this study, we mutated two deviant residues in the signature sequence to generate a double mutant (R1221G/E1223Q). Furthermore, a triple mutant (E502S/R1221G/E1223Q) was generated, in which the deviant residues of the signature sequence and Q-loop were mutated concurrently to canonical residues. The double and triple mutants showed 80% and 60%, respectively, of the activity of wild-type BSEP. As expected, an increasing number of mutations gradually impair transport as an intricate network of interactions within the ABC proteins ensures proper functioning.

N-terminal ectodomain of BTNL2 inhibits T cell activation via a non-canonical interaction with its putative receptor that results in a delayed progression of DSS-induced ulcerative colitis

Butyrophilin-like 2 (BTNL2) is a T cell inhibitory molecule that interacts with unknown binding partners to modulate the immune response in a number of inflammatory and autoimmune diseases. In this study, we found that the inhibitory effects of BTNL2 on T cell activation and effector functions can be executed by its N-terminal IgV domain (BTNL2 IgV1) alone. Structure-guided mutation of key residues on BTNL2 IgV1 based on known receptor-ligand interfaces involving immunoglobulin superfamily members revealed that BTNL2 uses a non-canonical binding interface with its putative receptor. A high avidity BTNL2 IgV1 probe revealed that in an inducible model of ulcerative colitis, severe colitis was accompanied by a selective enrichment of BTNL2-receptor expressing effector-memory CD4+ and CD8+ T cells in the Peyer’s patches. Intraperitoneal administration of BTNL2 IgV1 resulted in a significant delay in the progression of DSS-induced colitis and also showed reduced activation of the BTNL2-receptor-expressing T cells in the Peyer’s patches. Thus, this study demonstrates that the BTNL2-receptor-expressing T cells in the Peyer’s patches participate in the disease pathogenesis and can serve as a novel therapeutic target in ulcerative colitis, which can be modulated by BTNL2 IgV1.

A non-canonical Puf3p-binding sequence regulates CAT5/COQ7 mRNA under both fermentable and respiratory conditions in budding yeast.

The Saccharomyces cerevisiae uses a highly glycolytic metabolism, if glucose is available, through appropriately suppressing mitochondrial functions except for some of them such as Fe/S cluster biogenesis. Puf3p, a Pumillio family protein, plays a pivotal role in modulating mitochondrial activity, especially during fermentation, by destabilizing its target mRNAs and/or by repressing their translation. Puf3p preferentially binds to 8-nt conserved binding sequences in the 3'-UTR of nuclear-encoded mitochondrial (nc-mitochondrial) mRNAs, leading to broad effects on gene expression under fermentable conditions. To further explore how Puf3p post-transcriptionally regulates nc-mitochondrial mRNAs in response to cell growth conditions, we initially focused on nc-mitochondrial mRNAs known to be enriched in monosomes in a glucose-rich environment. We unexpectedly found that one of the monosome-enriched mRNAs, CAT5/COQ7 mRNA, directly interacts with Puf3p through its non-canonical Puf3p binding sequence, which is generally less considered as a Puf3p binding site. Western blot analysis showed that Puf3p represses translation of Cat5p, regardless of culture in fermentable or respiratory medium. In vitro binding assay confirmed Puf3p's direct interaction with CAT5 mRNA via this non-canonical Puf3p-binding site. Although cat5 mutants of the non-canonical Puf3p-binding site grow normally, Cat5p expression is altered, indicating that CAT5 mRNA is a bona fide Puf3p target with additional regulatory factors acting through this sequence. Unlike other yeast PUF proteins, Puf3p uniquely regulates Cat5p by destabilizing mRNA and repressing translation, shedding new light on an unknown part of the Puf3p regulatory network. Given that pathological variants of human COQ7 lead to CoQ10 deficiency and yeast cat5Δ can be complemented by hCOQ7, our findings may also offer some insights into clinical aspects of COQ7-related disorders.

Discovery of a non-canonical GRHL1 binding site using deep convolutional and recurrent neural networks

BackgroundTranscription factors regulate gene expression by binding to transcription factor binding sites (TFBSs). Most models for predicting TFBSs are based on position weight matrices (PWMs), which require a specific motif to be present in the DNA sequence and do not consider interdependencies of nucleotides. Novel approaches such as Transcription Factor Flexible Models or recurrent neural networks consequently provide higher accuracies. However, it is unclear whether such approaches can uncover novel non-canonical, hitherto unexpected TFBSs relevant to human transcriptional regulation.ResultsIn this study, we trained a convolutional recurrent neural network with HT-SELEX data for GRHL1 binding and applied it to a set of GRHL1 binding sites obtained from ChIP-Seq experiments from human cells. We identified 46 non-canonical GRHL1 binding sites, which were not found by a conventional PWM approach. Unexpectedly, some of the newly predicted binding sequences lacked the CNNG core motif, so far considered obligatory for GRHL1 binding. Using isothermal titration calorimetry, we experimentally confirmed binding between the GRHL1-DNA binding domain and predicted GRHL1 binding sites, including a non-canonical GRHL1 binding site. Mutagenesis of individual nucleotides revealed a correlation between predicted binding strength and experimentally validated binding affinity across representative sequences. This correlation was neither observed with a PWM-based nor another deep learning approach.ConclusionsOur results show that convolutional recurrent neural networks may uncover unanticipated binding sites and facilitate quantitative transcription factor binding predictions.

Abstract A142: Fragment based discovery of inhibitors of the eIF4E:eIF4G interaction

Abstract eIF4E is the rate limiting factor for cap-dependent protein translation, forming an essential part of the translation initiation complex, eIF4F and is a key signalling convergence point for the major oncogenic pathways MAPK and PI3K/AKT/MTOR. eIF4E has been shown to be overexpressed in cancer, which has been associated with poor prognosis and resistance to chemotherapeutics and targeted agents such as mTOR and MAPK inhibitors. eIF4E is therefore a promising target for cancer treatment but has so far been difficult to target with small molecules due to a lack of druggable pockets. Fragment based crystallographic screening is a powerful technique for probing the surface of proteins to identify potentially druggable binding sites. Here, we describe our fragment-based approach and hit optimisation campaign to generate potent compounds binding to eIF4E For the fragment screen, we generated an engineered form of eIF4E, where the N-terminal (residues 1-35) were removed and replaced a short, tethered peptide based on the canonical binding sequence of the eIF4E binding protein 4E-BP1, and screened 1400 fragments using a combination of Xray crystallography and NMR. Several low affinity (mM) fragment hits were identified, binding to both the mRNA cap- binding site and at an additional binding site of unknown functional relevance. Using a combination of biophysical and biochemical assays, subsequent rounds of iterative structure-based drug design of yielded potent lead molecules against both binding sites. For the cap-binding site, lead compounds of low nM potency were generated but due to the polar nature of the binding site, the molecules had very poor permeability properties and did not inhibit cell proliferation. For the non-canonical binding site, lead compounds with potency of 100nM alongside less potent enantiomers were generated which provided useful tools for probing the function of the binding site in vitro. We developed a MSD (Meso-scale discovery) assay to determine that the lead compound and not the inactive enantiomer could disrupt the eIF4E:eIF4G interaction in HeLa cell lysates with an IC50 of 1uM. Using a HeLa extract in vitro translation assay, we could also demonstrate that both the cap site and non-canonical site lead compounds could inhibit cap-dependent but not IRES driven protein translation. This work demonstrates the power of fragment screening to identify novel functional pockets on difficult to drug proteins such as eIF4E. Citation Format: Caroline J Richardson, Mladen Vincovic, Charlotte East, Nicola Wallis, George Ward, Andrew Woodhead. Fragment based discovery of inhibitors of the eIF4E:eIF4G interaction [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 A142.

Abstract A143: Coupling fragment-based screening with targeted protein degradation and genetic rescue to identify and explore the function of a non-canonical pocket on eIF4E

Abstract The translation initiation factor eIF4E binds the mRNA m7G-cap to initiate recruitment and binding of eIF4F components such as eIF4G. eIF4E exhibits oncogenic activity in in vitro and in vivo assays and high eIF4E expression in cancers is associated with poor prognosis and resistance to chemotherapeutics and targeted agents. Targeting eIF4E has long been considered a promising anticancer strategy but this target has remained undruggable using conventional screening approaches. In an accompanying submission we describe a fragment-based screening approach that discovered a non-canonical binding site on eIF4E with unknown functional relevance. Lead compounds with potency of 100nM were generated and provided tools for probing the function of the non-canonical site. Here we have used the tool compounds in combination with a targeted protein degradation (dTAG) to explore the function of this non-canonical site. H1299 NSCLC clones expressing an eIF4E-FKBP12F36V fusion protein but lacking endogenous eIF4E were established. Treatment of clones with a heterobifunctional dTAGV-1 molecule, that selectively binds to FKBP12F36V and recruits the VHL E3 ligase, induced rapid degradation of the eIF4E-FKBP12F36V protein leading to inhibition of cell growth and reduced expression of the MCL1 protein biomarker. We then expressed wild-type or eIF4E mutants and determined their ability to rescue the phenotype associated with eIF4E-loss following treatment with dTAGV-1. Expression of wild-type eIF4E rescued the phenotype associated with eIF4E-loss. The W73F mutation, that is reported to block canonical eIF4G binding, disrupted binding to eIF4G in cell lysate assays, but surprisingly the W73F mutant retained the ability to rescue the eIF4E degradation phenotype. Similar to our lead compound, mutations that impacted on the non-canonical pocket (L85R and L134R) disrupted the eIF4E:eIF4G interaction. However, L85R remained able to rescue the cellular and molecular responses, in contrast to the L134R mutant that could not rescue eIF4E loss. Interestingly, a W73F/L85R double mutant had a synergistic effect with a loss of function. Despite disrupting eIF4E:eIF4G binding and protein synthesis in lysates, treatment of H1299 cells with our lead compound did not inhibit proliferation. In contrast, compound treatment of the W73F mutant recapitulated the effects of combining W73F with the L85R non-canonical pocket mutation. In summary, targeting the non-canonical pocket was sufficient to disrupt the eIF4E:eIF4G binding in lysate assays. However, the differential impact of compounds or mutation on eIF4G binding in cells suggest that the eIF4E:eIF4G interaction in intact cells may be more complex than that predicted by the in vitro models. This observation may also explain the challenges associated with developing cellularly potent inhibitors of the eIF4E:eIF4G interaction. Our approach demonstrates the power of coupling fragment-based screening with target-degradation and genetic rescue approaches to find and explore novel functional pockets on difficult to drug proteins. Citation Format: Swee Y Sharp, Marianna Martella, Christopher I Milton, George Ward, Mladen Vinkovic, Caroline Richardson, Andrew Woodhead, Paul A Clarke. Coupling fragment-based screening with targeted protein degradation and genetic rescue to identify and explore the function of a non-canonical pocket on eIF4E [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 A143.

Novel Protac-Based EZH2 Degraders Effectively Target EZH2-Driven Lymphoid Malignancies By Abolishing Both Methylation-Dependent and -Independent Functions

Across various cancers, overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance and poor survival outcomes. While the tumorigenic role of EZH2 is largely attributed to the H3K27me3-mediated repression of tumor suppressor genes, reports have also established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes, particularly in aggressive cancers like Natural Killer T-cell Lymphoma (NKTL). Here, traditional EZH2 inhibitors have been ineffective. We hypothesized that targeted depletion of the EZH2 protein will more effectively disrupt oncogenic signals arising from enzyme-independent activities of EZH2. Thus, we embarked on the development of EZH2 Proteolysis Targeting Chimeras (PROTACs) to trigger targeted ubiquitination and proteasome-mediated degradation of EZH2. No clinical EZH2 PROTAC has been reported yet. Our initial efforts focused on the iterative synthesis of a series of PROTAC candidates considering factors such as permeability, solubility and stable ternary complex formation. We developed a library of 53 compounds with various permutations of EZH2- and E3 ligase-binding ligands as well as linker lengths. Through extensive screening and testing of the EZH2 degradation activity via the Nano-Glo HiBiT lytic detection system and immunoblotting, we identified three lead Protac candidates. Compared to the clinical EZH2 inhibitor Tazemetostat (Taz), EZH2 PROTACs, C2911 and C1311 demonstrated higher cytotoxic activity in diffuse large B-cell lymphoma (DLBCL), a cancer in which Taz was evaluated during clinical trial. Importantly, C2911 and C1311 also successfully triggered EZH2 degradation in a dose-dependent manner thereby qualifying for further evaluation in NKTL. Previous work has highlighted a critical role for JAK3-mediated phosphorylation of EZH2 in promoting NKTL proliferation. Treatment of NKTL with C2911 and C1311 leads to a dose and time-dependent EZH2 degradation, loss of cell viability and increased apoptosis. These effects were absent with Taz and Pomalidomide treatments. The concomitant loss of H3K27me3 levels as well as PRC2 complex subunits, SUZ12 and EED suggest effective degradation of the EZH2 protein. We also observed that induction of NKTL cell death required a longer treatment period as compared to DLBCL. This may be potentially ascribed to the longer half-life of the EZH2 protein in NKTL as determined through cycloheximide treatment. In order to validate the mechanism of action of the EZH2 PROTACs, cells were either pretreated with pomalidomide or siRNA targeting cereblon. Indeed, this mitigated PROTAC-induced degradation of the EZH2 protein and rescued cancer cell death. Similarly, inhibiting the activity of the 26S proteasome through bortezomib also prevented EZH2 degradation. Characterization of EZH2 PROTAC-induced protein expression changes through immunoblotting highlighted that there was specific loss of H3K27me3 and H3K27me2 but not H3K27me1. Of note, we detected a concomitant decrease of Jak3 protein expression and STAT5 phosphorylation levels, more pronounced than PRC2 complex subunits. This suggests that the PROTAC may act by disrupting the non-canonical binding of EZH2 to Jak3 in NKTL. Furthermore, gene expression analyses revealed that EZH2 PROTAC treatment more effectively reactivated canonical EZH2 target genes such as MYT1 and ANPEP as compared to Taz. As the JAK-STAT-MYC axis is dysregulated in NK leukemia, we also evaluated the gene expression of c-Myc and c-Myc target gene, E2F1. EZH2 PROTAC treatment reduces the transcription levels of these oncogenes corroborating with loss of c-Myc protein expression. Studying the pharmacokinetics of the lead PROTACs in a mouse model revealed good plasma stability, half-life (> 4 h), and low clearance. Collectively, our data supports a critical role for EZH2 PROTACs in abolishing both the enzyme-dependent and independent activities of EZH2 through protein degradation, resulting in enhanced anti-cancer activity against DLBCL and NKTL. This may potentially benefit other EZH2-driven cancers. Further evaluation of the EZH2 PROTACs for clinical application is in progress. EZH2 PROTACs will next be tested in vivo. An in-depth investigation of the mechanisms stabilizing EZH2 protein levels and underlying PROTAC induced-cell death will be made to identify potential mechanisms of resistance.