Gain-of-function Mutants Research Articles

Discovery of Potent and Selective Blockers Targeting the Epilepsy-Associated KNa1.1 Channel.

Gain-of-function (GOF) mutations of the sodium-activated potassium channel KNa1.1 (Slack, Slo2.2, or KCa4.1) induce severe, drug-resistant forms of epilepsy in infants and children. Although quinidine has shown promise in treating KCNT1-related epilepsies compared to other drugs, its limited efficacy and substantial side effects necessitate the development of new KNa1.1 channel inhibitors. In this study, we developed a novel class of KNa1.1 inhibitors using combined silico approaches and structural optimization. Among these inhibitors, compound Z05 was identified as a selective potential KNa1.1 inhibitor, especially against the hERG channel. Moreover, its binding site and potential counteraction to a GOF mutant Y796H were identified by the mutation studies. Our data also showed that Z05 had significant pharmacological profiles, including high brain penetration and moderate oral bioavailability, offering a valuable in vitro tool compound for further drug development in treating KCNT1-related epilepsies.

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

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

Abstract 6986: Oncogenic CARD11 enhances marginal zone B cell survival by potentiating the non-canonical NF-κB pathway

Abstract Dysregulation of antigen receptor-mediated signaling contributes to the oncogenesis of lymphoid malignancies. The activated B cell-like (ABC) subtype of Diffuse Large B cell Lymphoma (DLBCL) is characterized by receptor-independent constitutive activation of the canonical nuclear factor-kappaB (NF-κB) pathway, which is essential for the proliferation and survival of tumor cells. Caspase recruitment domain-containing protein 11 (CARD11) is a scaffolding protein that regulates B and T cell receptor signaling to canonical NF-κB activation downstream for adaptive immune response. Oncogenic gain-of-function (GOF) CARD11 mutations bypass normal pathway regulation and result in a hyperactive CARD11 that signals constitutively. Although present in ~10% of human ABC-DLBCL biopsies, we do not have a complete mechanistic understanding of how CARD11 GOF mutations drive abnormal B cell proliferation to cause lymphoma. We generated a mouse expressing the DLBCL-associated GOF mutation C49Y from the endogenous CARD11 locus. GOF CARD11 was sufficient to specifically drive B cell expansion in the mice with no significant effect on CD4+ or CD8+ T cells or NK cells. Quantification of splenic lymphocytes revealed that CARD11C49Y/C49Y mice had ~5-fold more splenic B cells when compared to CARD11+/+ mice and ~13-fold more marginal zone (MZ) B cells. CARD11C49Y/C49Y MZ B cells proliferated and survived much better than CARD11+/+ following stimulation with αIgM and importantly exhibited significant survivability prior to stimulation. Bulk mRNA sequencing revealed genes uniquely dysregulated in CARD11C49Y/C49Y MZ B cells including those associated with non-canonical NF-κB pathway signaling. CARD11C49Y/C49Y MZ B cells exhibited elevated levels of the p100 precursor NF-kB subunit leading to enhanced production of nuclear p52 following BAFF treatment. CARD11C49Y/C49Y MZ B cells also exhibited elevated levels of TRAF2, TRAF3 and cIAP1/2, components of a complex that degrades the NIK kinase that is responsible in normal cells for inducing p100 processing. Our results reveal an unexpected ability of CARD11 GOF mutants to potentiate the non-canonical NF-κB pathway through p100 accumulation, which likely contributes to the dysregulated B cell expansion observed in DLBCL cases that harbor CARD11 GOF variants. Citation Format: Anushka Ghosh, Joel Pomerantz. Oncogenic CARD11 enhances marginal zone B cell survival by potentiating the non-canonical NF-κB pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6986.

An ITPA Enzyme with Improved Substrate Selectivity

Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in neural development which is not understood. Polymorphism of the ITPA gene affects outcomes for both ribavirin and thiopurine based therapies and nearly one third of the human population is thought to harbor ITPA polymorphism. In a previous site-directed mutagenesis alanine screen of the ITPA substrate selectivity pocket, we identified the ITPA mutant, E22A, as a gain-of function mutant with enhanced ITP hydrolysis activity. Here we report a rational enzyme engineering experiment to investigate the biochemical properties of position 22 ITPA mutants and find that the E22D ITPA has two- and four-fold improved substrate selectivity for ITP over the canonical purine triphosphates ATP and GTP, respectively, while maintaining biological activity. The novel E22D ITPA should be considered as a platform for further development of ITPA therapies.

Abstract C163: KRAS alterations combined with TP53 mutations as novel synthetic lethal genomic lesions for PKMYT1 inhibition

Abstract Background: Membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) is a cell cycle regulatory kinase that inhibits CDK1/CyclinB activity, delaying entry into mitosis in tumor cells experiencing replication stress (RS). RS is frequently induced by genetic alterations that drive premature transition from G1 to S phase, promoting genome instability and creating a synthetic lethal (SL) relationship between these specific alterations and PKMYT1 inhibition. This relationship has been demonstrated preclinically and clinically with CCNE1 amplification and FBXW7 mutations, common alterations in ovarian and colorectal cancers, respectively. Oncogenic KRAS gain of function (GOF) mutations, combined with TP53 alterations, are bona fide drivers of RS. Here we investigate the relationship between KRAS/TP53 alterations and PKMYT1 inhibition, mediated by the first-in-class, potent and selective PKMYT1 inhibitor lunresertib (RP-6306), alone or in combination with RS-inducing agents. Methods: KRAS/TP53 double mutant isogenic cell pairs, across relevant tumor origins, were genetically engineered to express multiple KRAS GOF mutant codons and alleles (G12D, G12C, G12V, G12R, G13D and Q61H). Cell cycle distribution perturbations and RS were evaluated. Sensitivity to lunresertib alone or in combination with chemotherapeutic or targeted agents was assessed by cell growth assays. In vitro findings were further evaluated in KRAS/TP53 double mutant patient-derived xenografts (PDX). Results: Lunresertib inhibited the growth of isogenic KRAS/TP53 double mutant cells, with up to a 12-fold EC50 shift compared to control parental cells. Combination of lunresertib with RS-inducing chemotherapeutic agents (eg. gemcitabine), or the ATR inhibitor camonsertib (RP-3500) led to synergistic cell growth inhibition in KRAS/TP53 mutant cells at significantly lower concentrations than in wild type lines. Mechanistically, KRAS/TP53 isogenic cells showed elongated S-phase and increased cyclin B1 levels, phenocopying the effects of CCNE1 amplification. Lunresertib induced premature mitosis and DNA damage in KRAS/TP53 mutant cells, sparing parental cell lines. PDX efficacy studies demonstrated robust combination benefit in pancreatic, lung and colorectal settings, with durable tumor regressions and complete responses observed. Conclusion: GOF KRAS mutations, when combined with TP53 alterations, show a strong SL relationship with PKMYT1 inhibition, alone or in combination with RS-inducing antitumor agents. Importantly, the SL phenotype was observed across multiple tumor indications and KRAS mutant codons/alleles, suggesting a potential broad scope of clinical utility beyond approved KRAS inhibitors. The mechanism is similar to CCNE1 amplification, characterized by enhanced G1/S transition driving heightened dependence on PKMYT1 to inhibit CDK1/CyclinB. Taken together, our data form rationale for the therapeutic evaluation of PKMYT1 inhibitors in KRAS/TP53 altered tumors and uncover a potential new patient selection biomarker for lunresertib. Citation Format: Marc L. Hyer, Jimmy Fourtounis, David Gallo, Vivek Bhaskaran, Rino Stocco, Rosie Kryczka, Sai Save, Helen Burston, Olivier Nicolas, Stephen Morris, Anne Roulston, Jordan T. F. Young, Michal Zimmermann, C. Gary Marshall, Artur Veloso, Elia Aguado-Fraile. KRAS alterations combined with TP53 mutations as novel synthetic lethal genomic lesions for PKMYT1 inhibition [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 C163.

Inhibition of Ca2+-permeable TRPV3 and inflammatory cytokine release by honokiol and magnolol in human epidermal keratinocytes

Transient receptor potential vanilloid-3 (TRPV3) ion channels are prominently expressed in keratinocytes, playing a vital role in skin functions. Honokiol and magnolol (H&M) the primary bioactive constituents in Magnolia officinalis extract, demonstrate anti-inflammatory and skin-protective properties. Nevertheless, the underlying mechanism regarding their effect on Ca2+-permeable ion channels remain unclear. Our purpose in this study is to investigate the effect of H&M on TRPV3 and cytokine release in normal human epidermal keratinocytes (NHEKs), including its gain-of-function (GOF) mutants (G573S and G573C) associated with Olmstead syndrome. We performed whole-cell patch-clamp, fura-2 spectrofluorimetry to investigate channels activity, CCK-8 assay to analyze cell death and enzyme-linked immunosorbent assay to assess the cytokine release from NHEKs. H&M inhibited the TRPV3 current (ITRPV3) and cytosolic calcium increase in NHEKs, HEK293T cells overexpressing hTRPV3 and its GOF mutants. Moreover, the release of pro-inflammatory cytokines (interleukin-6 and -8) from keratinocytes stimulated by TRPV3 agonist was effectively suppressed by H&M. Our findings provide insights into the mechanism underlying the anti-inflammatory effects of H&M, highlighting their potential in treating skin diseases.

A Novel and Potent EZH1/2 Dual Inhibitor, HM97662 Demonstrated a Wide Spectrum of Therapeutic Potential for Hematological Malignancies

Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homolog 2 (EZH2) and its homolog EZH1 are catalytic components of polycomb repressive complex 2 (PRC2), which tri-methylate histone H3 at lysine 27 (H3K27me3) to repress transcription of their target genes. Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677, and A687) has been associated with the progression of hematological malignancies including B-cell lymphoma, T-cell lymphoma, and multiple myeloma. Hence, it has been suggested that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Although the methyltransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. Moreover, EZH1 directly binds to chromatin and modulates its condensation. Therefore, dual inhibition of EZH1 and EZH2 can give better effect than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we presented a novel EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. SPR analysis of HM97662 on trimeric complexes (EZH1/EZH2, SUZ12, and EED) demonstrated that HM97662 binds both EZH1- and EZH2-PRC2 complexes. HM97662 potently repressed tri-methylation of H3K27 in KARPAS 422 DLBCL, HH PTCL, and MM.1S MM cell lines. Furthermore, HM97662 showed broader and stronger activities than EZH2 selective inhibitors in long-term proliferation assay against a variety of hematological cancer cell lines. HM97662 induced the differentiation of DLBCL to plasma cells with the increment of cell lineage specific markers (e.g. PRDM1 and CD38), and caused cell cycle G0/G1 arrest and apoptosis in KARPAS 422 DLBCL cells harboring EZH2 Y641N GOF mutation. Additionally, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH PTCL cell line. Two cell cycle repressors, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were significantly increased by HM97662. We further conducted a chromatin accessibility assay and identified that the chromatin structures of that genes were loosened and highly transcribed after the treatment of HM97662. As a result, HM97662 showed potent antitumor activity in various hematological cell lines xenograft mouse models including B-cell lymphoma, T-cell lymphoma, and multiple myeloma (e.g. KARPAS 422, HuT-102, and MM.1S). Once daily oral dosing of HM97662 greatly inhibited tumor growth in a dose-dependent manner without significant clinical signs compared to the known EZH2 selective or EZH1/2 dual inhibitor. In conclusion, the present preclinical studies demonstrated that HM97662, an EZH1/2 dual inhibitor, had a promising and wide spectrum of therapeutic potential for hematological malignancies. It is urgent to assess the effectiveness of HM97662 in further clinical trials.

Leptospira ClpP mutant variants in association with the ClpX, acyldepsipeptide, and the trigger factor displays unprecedented gain-of-function

The functionally active ClpP (LinClpP) of Leptospira interrogans is composed of two different isoforms (LinClpP1 and LinClpP2). In this study, five mutants of LinClpP (LinClpP1E170D, LinClpP1N172D, LinClpP2IG_del, LinClpP2S40AK41N, LinClpP2Y62A) targeting its critical hotspot residues were generated. The functional activity of pure LinClpP mutant variants or its heterocomplex and its effect when associated with a chaperone (LinClpX)/antibiotic acyldepsipeptide (ADEP1)/trigger factor (LinTF) was examined. The two mutants (LinClpP2S40AK41N and LinClpP2Y62A) displayed gain-of-function (GOF) in peptidase activity. The ADEP1-bound heterocomplex (LinClpP1P2S40AK41N and LinClpP1P2Y62A) measured 1.7 and 1.5-fold higher protease activity than ADEP-bound LinClpP1P2. The dynamic light scattering analysis of ADEP1-bound GOF mutants displayed increased hydrodynamic diameter. In the presence of LinTF, the heterocomplex (LinClpP1P2S40AK41N and LinClpP1P2Y62A) exhibited a 3-fold surge in peptidase activity. The deletion mutant (LinClpP2IG_del) or its heterocomplex (LinClpP1P2IG_del) displayed no activity. Similarly, the pure LinClpP1E170D and LinClpP1N172D could not cleave a model dipeptide. However, its heterocomplex (LinClpP1E170DP2 and LinClpP1N172DP2) showed 0.5-fold lower peptidase activity than the LinClpP1P2. Collectively, two mutants (LinClpP2S40AK41N and LinClpP2Y62A) have GOF and can degrade model dipeptide substrate without the aid of LinClpP1 isoform and thus provide new insights into unprecedented LinClpP activation.

Gain-of-function mutant p53 together with ERG proto-oncogene drive prostate cancer by beta-catenin activation and pyrimidine synthesis

Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and β-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. β-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals β-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.

Integrative Multi-Omics Analysis of Oncogenic EZH2 Mutants: From Epigenetic Reprogramming to Molecular Signatures.

Somatic heterozygous mutations in the active site of the enhancer of zeste homolog 2 (EZH2) are prevalent in diffuse large B-cell lymphoma (DLBCL) and acute myeloid leukemia (AML). The methyltransferase activity of EZH2 towards lysine 27 on histone H3 (H3K27) and non-histone proteins is dysregulated by the presence of gain-of-function (GOF) and loss-of-function (LOF) mutations altering chromatin compaction, protein complex recruitment, and transcriptional regulation. In this study, a comprehensive multi-omics approach was carried out to characterize the effects of differential H3K27me3 deposition driven by EZH2 mutations. Three stable isogenic mutants (EZH2Y641F, EZH2A677G, and EZH2H689A/F667I) were examined using EpiProfile, H3K27me3 CUT&Tag, ATAC-Seq, transcriptomics, label-free proteomics, and untargeted metabolomics. A discrete set of genes and downstream targets were identified for the EZH2 GOF and LOF mutants that impacted pathways involved in cellular proliferation, differentiation, and migration. Disruption of protein networks and metabolic signatures able to sustain aberrant cell behavior was observed in response to EZH2 mutations. This systems biology-based analysis sheds light on EZH2-mediated cell transformative processes, from the epigenetic to the phenotypic level. These studies provide novel insights into aberrant EZH2 function along with targets that can be explored for improved diagnostics/treatment in hematologic malignancies with mutated EZH2.

Differential Binding of NLRP3 to non-oxidized and Ox-mtDNA mediates NLRP3 Inflammasome Activation

The NLRP3 inflammasome is a key mediator of the innate immune response to sterile tissue injury and is involved in many chronic and acute diseases. Physically and chemically diverse agents activate the NLRP3 inflammasome. Here, we show that NLRP3 binds non-oxidized and Ox-mtDNA differentially, with a half maximum inhibitory concentration (IC50) for non-oxidized and Ox-mtDNA of 4 nM and 247.2 nM, respectively. The NLRP3 Neonatal-Onset Multisystem Inflammatory Disease (NOMID) gain of function mutant could bind non-oxidized mtDNA but had higher affinity for Ox-mtDNA compared to WT with an IC50 of 8.1 nM. NLRP3 lacking the pyrin domain can bind both oxidized and non-oxidized mtDNA. Isolated pyrin domain prefers Ox-mtDNA. The NLRP3 pyrin domain shares a protein fold with DNA glycosylases and generate a model for DNA binding based on the structure and sequence alignment to Clostridium acetobutylicum and human OGG1, an inhibitor of Ox-mtDNA generation, 8-oxoguanine DNA glycosylases. We provide a new model for how NLRP3 interacts with Ox-mtDNA supported by DNA binding in the presence of a monoclonal antibody against the pyrin domain. These results give new insights into the mechanism of inflammasome assembly, and into the function of reactive oxygen species in establishing a robust immune response.

Tight hydrophobic core and flexible helices yield MscL with a high tension gating threshold and a membrane area mechanical strain buffer.

The mechanosensitive (MS) channel of large conductance, MscL, is the high-tension threshold osmolyte release valve that limits turgor pressure in bacterial cells in the event of drastic hypoosmotic shock. Despite MscL from Mycobacterium tuberculosis (TbMscL) being the first structurally characterized MS channel, its protective mechanism of activation at nearly-lytic tensions has not been fully understood. Here, we describe atomistic simulations of expansion and opening of wild-type (WT) TbMscL in comparison with five of its gain-of-function (GOF) mutants. We show that under far-field membrane tension applied to the edge of the periodic simulation cell, WT TbMscL expands into a funnel-like structure with trans-membrane helices bent by nearly 70°, but does not break its 'hydrophobic seal' within extended 20μs simulations. GOF mutants carrying hydrophilic substitutions in the hydrophobic gate of increasing severity (A20N, V21A, V21N, V21T and V21D) also quickly transition into funnel-shaped conformations but subsequently fully open within 1-8μs. This shows that solvation of the de-wetted (vapor-locked) constriction is the rate-limiting step in the gating of TbMscL preceded by area-buffering silent expansion. Pre-solvated gates in these GOF mutants reduce this transition barrier according to hydrophilicity and the most severe V21D eliminates it. We predict that the asymmetric shape-change of the periplasmic side of the channel during the silent expansion provides strain-buffering to the outer leaflet thus re-distributing the tension to the inner leaflet, where the gate resides.

Abstract 2585: Mutant p53 gains oncogenic functions through a cytosolic DNA response

Abstract TP53 mutations are the most common cancer driver mutations among all cancers. Although some TP53 mutations lead to a loss of function of wild-type p53, many other TP53 mutations confer gain-of-function (GOF) activities, which promote cancer cell metastasis and pro-tumorigenic inflammation. Despite that many functional models of GOF mutant p53 (mutp53) have been proposed previously, the mechanisms involved in mutp53 GOF still remain largely elusive. Here we show that by directly targeting minichromosome maintenance complex component 5 (MCM5), a component of the hexametric DNA helicase MCM2-7 complex, GOF mutp53 predisposes cancer cells to replication stress and chromosomal instability, which leads to a tumor cell-autonomous and stimulator of interferon genes (STING)-dependent cytosolic DNA response that activates downstream non-canonical nuclear factor kappa light chain enhancer of activated B cell (NC-NF-κB) signaling. Furthermore, our results demonstrate that GOF mutp53-activated tumor cell-intrinsic STING-NC-NF-κB signaling not only stimulates tumor cell metastasis, but also promotes tumor immune resistance through fostering an immunosuppressive tumor microenvironment. Therefore, our findings that mutp53 exerts its GOF role through pro-tumorigenic MCM5-CIN-STING-NC-NF-κB signaling highlight the importance of TP53 and its inactivation in cancer genome evolution of genomic instability that drives tumor development and progression. Citation Format: Mei Zhao, Tianxiao Wang, Frederico O. Gleber-Netto, Zhen Chen, Daniel J. McGrail, Javier A. Gomez, Wutong Ju, Mayur A. Gadhikar, Wencai Ma, Li Shen, Ximing Tang, Sen Pathak, Maria G. Raso, Jared Burks, Shiaw-Yih Lin, Jing Wang, Asha S. Multani, Curtis R. Pickering, Junjie Chen, Jeffrey N. Myers, Ge Zhou. Mutant p53 gains oncogenic functions through a cytosolic DNA response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2585.

Abstract 1712: Targeting asparagine dependency as a therapy for TP53-mutated castration-resistant prostate cancer

Abstract Background: The TP53 tumor suppressor is frequently altered in lethal, castration-resistant prostate cancer (CRPC). However, to date there are no effective treatments that specifically target TP53 gain-of-function (GOF) mutants. Thus, there is an urgent need to identify and target vulnerabilities that perpetuate disease progression and treatment resistance resulting from TP53 GOF mutants. Methods: We performed RNA sequencing (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics on GEM and PDX CRPC models with or without TP53 GOF mutations. Our findings were validated using genetic and pharmacologic approaches in relevant in vitro and in vivo models. We assessed the effect of L-asparaginase (ASNase) or/and glutaminase (GLS) inhibitor CB-839 in cell lines, organoids and PDX models. Tumors were characterized by histology and immunohistochemistry for relevant markers. Results: Using transcriptomic and metabolomic analyses, we show here that TP53-mutated prostate cancer (PCa) exhibits an increased dependency on asparagine and overexpresses asparagine synthetase (ASNS, the enzyme catalyzing the synthesis of asparagine). Mechanistically, TP53 GOF mutations transcriptionally activate ASNS expression, directly as well as via ATF4, driving de novo asparagine biosynthesis to support CRPC growth. TP53-mutated CRPC cells are sensitive to asparagine restriction by knockdown of ASNS and L-asparaginase treatment to deplete the intracellular and extracellular sources of asparagine respectively. This effect was rescued by asparagine addition. Notably, pharmacological inhibition of intracellular asparagine biosynthesis using a glutaminase (GLS) inhibitor and depletion of extracellular asparagine with L-asparaginase significantly reduced asparagine production and effectively impaired CRPC growth. Conclusions: This study highlights the significance of ASNS-mediated metabolic adaptation as a synthetic vulnerability in CRPC with TP53 GOF mutations, providing a rationale for co-targeting intracellular and extracellular asparagine production to treat these lethal prostate cancers. Citation Format: Young A Yoo. Targeting asparagine dependency as a therapy for TP53-mutated castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1712.

The human MRS2 magnesium-binding domain is a regulatory feedback switch for channel activity.

Mitochondrial RNA splicing 2 (MRS2) forms a magnesium (Mg2+) entry protein channel in mitochondria. Whereas MRS2 contains two transmembrane domains constituting a pore on the inner mitochondrial membrane, most of the protein resides within the matrix. Yet, the precise structural and functional role of this obtrusive amino terminal domain (NTD) in human MRS2 is unknown. Here, we show that the MRS2 NTD self-associates into a homodimer, contrasting the pentameric assembly of CorA, an orthologous bacterial channel. Mg2+ and calcium suppress lower and higher order oligomerization of MRS2 NTD, whereas cobalt has no effect on the NTD but disassembles full-length MRS2. Mutating-pinpointed residues-mediating Mg2+ binding to the NTD not only selectively decreases Mg2+-binding affinity ∼sevenfold but also abrogates Mg2+ binding-induced secondary, tertiary, and quaternary structure changes. Disruption of NTD Mg2+ binding strikingly potentiates mitochondrial Mg2+ uptake in WT and Mrs2 knockout cells. Our work exposes a mechanism for human MRS2 autoregulation by negative feedback from the NTD and identifies a novel gain of function mutant with broad applicability to future Mg2+ signaling research.

Allosteric regulation of exocyst: Discrete activation of tethering by two spatial signals.

The exocyst imparts spatial control during exocytic vesicle tethering through its interactions with proteins and lipids on the vesicle and the plasma membrane. One such interaction is with the vesicle tether Sro7, although the outcome of this interaction is poorly understood. Here, we describe how Sro7 binding to the Exo84 subunit results in activation of the exocyst complex which leads to an increase in avidity for the Rab GTPase Sec4 and an increase in exocyst-mediated vesicle tethering. Gain-of-function (GOF) mutations in Exo84 that mimic Sro7 activation replicate these biochemical changes and result in allosteric changes within the complex. Direct comparison of GOF mutants which mimic Sro7- and Rho/Cdc42-activation of the exocyst reveals distinct mechanisms and outcomes. We propose a model by which these two activation pathways reside within the same tethering complex but remain insulated from one another. Structural modeling suggests a related mechanism for Sro7 activation of the exocyst in yeast and Ral GTPase activation of the exocyst in animal cells.

PP01.32 Proteasome Hyperactivity as a Targetable Vulnerability in Oncogenic Missense TP53 Mutated NSCLC

Despite advances in targeted approaches and immunotherapy, lung cancer remains the leading cause of cancer mortality in the United States and worldwide. This is partly because these novel treatments are not applicable to all patients and are often associated with primary or secondary resistance. This highlights the need for continued search for new therapeutic strategies for patients with non-small cell lung cancer (NSCLC). Gain-of-function (GOF) oncogenic TP53 mutations, which are present in 20-30% of NSCLC, are harbingers of poorer prognosis and subdued responses to therapy. To date, strategies aimed at directly targeting mutant p53 have not been successful. Here, we provide evidence of proteasome hyperactivity in GOF mutant p53 NSCLC and show that this could be a targetable vulnerability specific to this subset of NSCLC.

FMRP activity and control of Csw/SHP2 translation regulate MAPK-dependent synaptic transmission.

Noonan syndrome (NS) and NS with multiple lentigines (NSML) cognitive dysfunction are linked to SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) gain-of-function (GoF) and loss-of-function (LoF), respectively. In Drosophila disease models, we find both SHP2 mutations from human patients and corkscrew (csw) homolog LoF/GoF elevate glutamatergic transmission. Cell-targeted RNAi and neurotransmitter release analyses reveal a presynaptic requirement. Consistently, all mutants exhibit reduced synaptic depression during high-frequency stimulation. Both LoF and GoF mutants also show impaired synaptic plasticity, including reduced facilitation, augmentation, and post-tetanic potentiation. NS/NSML diseases are characterized by elevated MAPK/ERK signaling, and drugs suppressing this signaling restore normal neurotransmission in mutants. Fragile X syndrome (FXS) is likewise characterized by elevated MAPK/ERK signaling. Fragile X Mental Retardation Protein (FMRP) binds csw mRNA and neuronal Csw protein is elevated in Drosophila fragile X mental retardation 1 (dfmr1) nulls. Moreover, phosphorylated ERK (pERK) is increased in dfmr1 and csw null presynaptic boutons. We find presynaptic pERK activation in response to stimulation is reduced in dfmr1 and csw nulls. Trans-heterozygous csw/+; dfmr1/+ recapitulate elevated presynaptic pERK activation and function, showing FMRP and Csw/SHP2 act within the same signaling pathway. Thus, a FMRP and SHP2 MAPK/ERK regulative mechanism controls basal and activity-dependent neurotransmission strength.

Aberrant function of pathogenic STAT3 mutant proteins is linked to altered stability of monomers and homodimers

AbstractSTAT3 mutations, predominantly in the DNA-binding domain (DBD) and Src-homology 2 domain (SH2D), cause rare cases of immunodeficiency, malignancy, and autoimmunity. The exact mechanisms by which these mutations abrogate or enhance STAT3 function are not completely understood. Here, we examined how loss-of-function (LOF) and gain-of-function (GOF) STAT3 mutations within the DBD and SH2D affect monomer and homodimer protein stability as well as their effect on key STAT3 activation events, including recruitment to phosphotyrosine (pY) sites within peptide hormone receptors, tyrosine phosphorylation at Y705, dimerization, nuclear translocation, and DNA binding. The DBD LOF mutants showed reduced DNA binding when homodimerized, whereas the DBD GOF mutants showed increased DNA binding. DBD LOF and GOF mutants showed minimal changes in other STAT3 functions or in monomer or homodimer protein stability. However, SH2D LOF mutants demonstrated reduced conformational stability as either monomers or homodimers, leading to decreased pY-peptide recruitment, tyrosine phosphorylation, dimerization, nuclear localization, and DNA binding. In contrast, cancer-causing SH2D GOF mutants showed increased STAT3 homodimer stability, which increased their DNA binding. Of note, a small-molecule inhibitor of STAT3 that targets the tyrosine phosphopeptide–binding pocket within the STAT3 SH2D potently inhibited cell proliferation driven by STAT3 SH2D GOF mutants. These findings indicate that the stability of STAT3 protein monomer and homodimer is critical for the pathogenesis of diseases caused by SH2D LOF and GOF mutations and suggest that agents that modulate STAT3 monomer and/or homodimer protein stability may have therapeutic value in diseases caused by these mutations.

Abstract 1826: Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers

Abstract Background: Disulfiram (DSF) in combination with copper pills has emerged as a safe and exciting anticancer strategy and is undergoing clinical trials in multiple human cancers. However, DSF breaks down in the gut or blood immediately and chances of cupric diethyldithiocarbamate (Cu-DDC), the active cytotoxic principle being formed at tumor sites is very low. Therefore, we have found that synthetic Cu-DDC is directly and potently cytotoxic against p53 mutant cancers. We report here for the first time the differential effects of Cu-DDC in robustly eliminating the mutant p53 proteins from tumor cells in contrast to its augmentation of the wild-type p53 protein and activation. Destabilization of Gain Of Function (GOF) p53 mutant proteins for overcoming oncogenic addiction and improving the therapeutic efficacy has been a long-sought, but unmet anticancer avenue. Methods: A panel of 7 P53 mutant cell lines (R248W, R273H,R173H), and p53 wt tumor and normal cells (HPBLs, dental pulp stem cells, IMR90 and HUVEC) were used. Immunoprecipitation, Western blotting, Annexin-V apoptotic assays, flow cytometry, immunocytochemistry were used to quantitate p53 protein. Mia-Paca2 xenografts developed in nude mice were administered Cu-DDC/BSA. Results: Cu-DDC generated peroxides but not superoxides in tumor cells. The GOF mutant p53 proteins, R248W and R273H in three different tumor cell lines were eliminated after Cu-DDC treatment in a time- and concentration-dependent manner. The loss of mutant p53 proteins was confirmed by three different procedures. The degradation occurred rapidly in 8-10 h at 100 nM Cu-DDC and was inhibited by proteasome blockers. The expression of the GRO-α oncogene, positively regulated by GOF R248W was decreased in Cu-DDC treated Mia-Paca2 cells. Cellular proteasome activity was not inhibited by Cu-DDC. Ubiquitination of mutant p53 proteins was not altered by Cu-DDC. In contrast, the wild-type p53 present in HCT116 and MCF-7 cells, and normal cell counterparts (HPBL, IMR-90 lung cells) was significantly upregulated and activated with enhanced p21cip1 expression. Cu-DDC triggered strong apoptosis irrespective of the p53 status, although the mutant p53 containing cells were 2 to 3-fold more sensitive in cytotoxicity assays (IC50 range 0.25-0.66 µM). A significant tumor growth delay accompanied by loss of mutant p53 protein was observed in MiaPaca2 xenografts. Conclusions: In moving the DSF anticancer repurposing forward, we describe the unique ability of Cu-DDC to rapidly eliminate oncogenic p53 mutant proteins and confer potent antitumor efficacy. The degradation was unrelated to the proteostasis involving the inactivation of NPL4 by Cu-DDC described previously (supported by a Cancer Prevention and Research Institute of Texas [CPRIT] grant RP170207 to KSS). Citation Format: Viswanath Arutla, Kalkunte S. Srivenugopal. Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1826.