Biology subjects

Gavathiotis, E.

Publications and source records attributed to Gavathiotis, E..

10 recordsLinked to original sources

bioRxiv2026

Binding kinetics enable tumor-selective MAPK and growth suppression by a Type 1 RAF Inhibitor

Therapeutic inhibition of the RAS/MAPK cascade, an oncogenic driver in over one-third of human cancers, has been limited by a core trade-off: effective pathway blockade in tumors versus dose-limiting toxicity in normal tissues. Although Type 1.5 RAF inhibitors successfully mitigated this trade-off in BRAF-monomer-driven tumors, an equivalent strategy has been lacking for RAF-dimer-driven cancers, including those with RAS mutations. Here, we characterize ELV-3111, a next-generation, potent, and selective Type 1 RAF inhibitor that overcomes this limitation via a distinct mechanism of action. ELV-3111 induces paradoxical MAPK hyperactivation in normal tissues by binding RAF and promoting the active conformation (priming) followed by rapid dissociation (fast off-rate), a biochemical feature that spares RAS-mutant tumors, where RAF is already primed. In these and other RAF dimer-driven tumor models, ELV-3111 achieves potent and selective MAPK and growth suppression. When combined with a MEK inhibitor, ELV-3111 enables complementary pharmacology: additive inhibition in tumors and compensatory effects in normal tissues. The combination produced durable regressions across RAS-mutant and BRAF-mutant models, including a RAS-mutant model refractory to current therapies, while maintaining favorable tolerability. Thus, we uncover a generalizable mechanism that enables Type 1 RAF inhibitor plus MEK inhibitor combinations to achieve tumor-selective MAPK suppression, with the potential to inform targeted therapy design.

cancer biology↗

bioRxiv2026

Spatial modulation of RAF by RAF/MEK glue enables full-dose combination with pan-RAF inhibitor and potent RAS-mutant tumor-selective MAPK and growth inhibition

The clinical benefit of MAPK-targeted therapies depends on greater pathway inhibition in tumors than normal tissues. Although pan-RAF inhibitors are active in RAS-mutant cancers, combining them with MEK inhibitors requires dose reductions due to toxicity, limiting efficacy. We show the toxicity results from MEK inhibitor-mediated feedback relief, which promotes RAF activation and pan-RAF inhibitor engagement in normal cells, narrowing the therapeutic index. We further demonstrate that MEK is exclusively cytosolic, and RAF/MEK glues overcome this limitation through spatial trapping. By stabilizing cytosolic RAF-MEK complexes, RAF/MEK glues prevent feedback-driven RAF activation in normal cells while maintaining inhibition of oncogenic RAF signaling in RAS-mutant tumors, where RAF is constitutively activated at the plasma membrane. Consequently, this enables full-dose combination with pan-RAF inhibitors, resulting in deeper MAPK suppression and robust tumor regressions in RAS-mutant models. Thus, by spatially controlling wild-type effectors, drug-induced proximity can be harnessed to increase tumor selectivity of pathway-targeted therapies. SignificanceMAPK-targeted therapies rarely achieve durable responses in RAS-mutant cancers due to dose-limiting toxicities. We show that RAF/MEK glues, by spatially trapping RAF, can be combined with pan-RAF inhibitors at full dose, yielding tumor-selective MAPK inhibition and tumor regressions in RAS-mutant models. Thus, drug-induced proximity can be exploited for tumor-selective therapy.

cancer biology↗

bioRxiv2026

The GNMT N-terminus Couples Folate Feedback to Methyl-donor Homeostasis

Maintenance of S-adenosylmethionine (SAM) homeostasis is essential for methylation of biomolecules, nucleotide and polyamine synthesis, and redox homeostasis. While all methyltransferases consume SAM, only a subset of highly tissue specific methyltransferases regulates SAM homeostasis. Among them, glycine N-methyltransferase (GNMT) is enriched in the liver and its dysregulated activity has been linked to compromised liver function. GNMT is inhibited by the methyl carrier 5-methyltetrahydrofolate (5mTHF), suggesting a negative-feedback mechanism regulating its activity. Here, we identify the GNMT N-terminal tail, and specifically phosphorylation at serine 9 (S9ph), as a regulatory modification linking folate-dependent feedback inhibition to SAM homeostasis. Structural and biochemical analyses and molecular dynamics simulations revealed that the N-terminal tail is required for catalytic turnover of SAM and for 5mTHF binding. Phosphoproteomic analysis showed that GNMT S9ph is abundant in mouse liver and further enriched in aged mice. Consistent with loss of folate-dependent negative feedback, both distal N-terminal truncation (residues 1-8) and a phosphomimetic substitution abolished 5mTHF binding while maintaining catalytic activity. In hepatocyte cell lines lacking endogenous GNMT, lentiviral overexpression of constitutively active GNMT mutants depleted SAM, increased SAH, disrupted protein methylation, impaired growth, and induced transcriptional responses consistent with methyl-donor stress. Together, these findings identify the GNMT N-terminus as a tunable phosphoregulatory domain that dynamically regulates GNMT activity and cellular methylation potential.

biochemistry↗

bioRxiv2025

Quiescent OXPHOS-high triple-negative breast cancer cells that persist after chemotherapy depend on BCL-XL for survival

The persistent residual tumor cells that survive after chemotherapy are a major cause of treatment failure, but their survival mechanisms remain largely elusive. These cancer cells are typically characterized by a quiescent state with suppressed activity of MYC and MTOR. We observed that the MYC-suppressed persistent triple-negative breast cancer (TNBC) cells are metabolically flexible and can upregulate mitochondrial oxidative phosphorylation (OXPHOS) genes and respiratory function ("OXPHOS-high" cell state) in response to DNA-damaging anthracyclines such as doxorubicin, but not to taxanes. The elevated biomass and respiratory function of mitochondria in OXPHOS-high persistent cancer cells were associated with mitochondrial elongation and remodeling suggestive of increased mitochondrial fusion. A genome-wide CRISPR editing screen in doxorubicin-persistent OXPHOS-high TNBC cells revealed BCL-XL gene as the top survival dependency in these quiescent tumor cells, but not in their untreated proliferating counterparts. Quiescent OXPHOS-high TNBC cells were highly sensitive to BCL-XL inhibitors, but not to inhibitors of BCL2 and MCL1. Interestingly, inhibition of BCL-XL in doxorubicin-persistent OXPHOS-high TNBC cells rapidly abrogated mitochondrial elongation and respiratory function, followed by caspase 3/7 activation and cell death. The platelet-sparing proteolysis targeted chimera (PROTAC) BCL-XL degrader DT2216 enhanced the efficacy of doxorubicin against TNBC xenografts in vivo without induction of thrombocytopenia that is often observed with the first-generation BCL-XL inhibitors, supporting the development of this combinatorial treatment strategy for eliminating dormant tumor cells that persist after treatment with anthracycline-based chemotherapy.

cancer biology↗

bioRxiv2024

Identification of functional rare coding variants in IGF-1 gene in humans with exceptional longevity

Diminished signaling via insulin/insulin-like growth factor-1 (IGF-1) axis is associated with longevity in different model organisms. IGF-1 gene is highly conserved across species, with only few evolutionary changes identified in it. Despite its potential role in regulating life span, no coding variants in IGF-1 have been reported in human longevity cohorts to date. This study investigated the whole exome sequencing data from 2,487 individuals in a cohort of Ashkenazi Jewish centenarians, their offspring, and controls without familial longevity to identify functional IGF-1 coding variants. We identified two likely functional coding variants IGF-1:p.Ile91Leu and IGF-1:p.Ala118Thr in our longevity cohort. Notably, a centenarian specific novel variant IGF-1:p.Ile91Leu was located at the binding interface of IGF-1 - IGF-1R, whereas IGF-1:p.Ala118Thr was significantly associated with lower circulating levels of IGF-1. We performed extended all-atom molecular dynamics simulations to evaluate the impact of Ile91Leu on stability, binding dynamics and energetics of IGF-1 bound to IGF-1R. The IGF-1:p.Ile91Leu formed less stable interactions with IGF-1Rs critical binding pocket residues and demonstrated lower binding affinity at the extracellular binding site compared to wild-type IGF-1. Our findings suggest that IGF-1:p.Ile91Leu and IGF-1:p.Ala118Thr variants attenuate IGF-1R activity by impairing IGF-1 binding and diminishing the circulatory levels of IGF-1, respectively. Consequently, diminished IGF-1 signaling resulting from these variants may contribute to exceptional longevity in humans.

biophysics↗

bioRxiv2024

A STAT3 Degrader Demonstrates Pre-clinical Efficacy in Venetoclax resistant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that continues to have poor prognosis despite recent therapeutic advances. Venetoclax (Ven), a BCL2-inhibitor has shown a high response rate in AML; however, relapse is invariable due to mitochondrial dysregulation that includes upregulation of the antiapoptotic protein MCL1, a central mechanism of Ven resistance (Ven-res). We have previously demonstrated that the transcription factor STAT3 is upregulated in AML hematopoietic stem and progenitor cells (HSPCs) and can be effectively targeted to induce apoptosis of these aberrant cells. We now show that overexpression of STAT3 alone is sufficient to initiate a strong AML phenotype in a transgenic murine model. Phospho-proteomic data from Ven treated AML patients show a strong correlation of high total STAT3 and phospho-STAT3 [both p-STAT3(Y705) and p-STAT3(S727)] expression with worse survival and reduced remission duration. Additionally, significant upregulation of STAT3 was observed in Ven-res cell lines, in vivo models and primary patient samples. A novel and specific degrader of STAT3 demonstrated targeted reduction of total STAT3 and resulting inhibition of its active p-STAT3(Y705) and p-STAT3(S727) forms. Treatment with the STAT3 degrader induced apoptosis in parental and Ven-res AML cell lines and decreased mitochondrial depolarisation, and thereby dependency on MCL1 in Ven-res AML cell line, as observed by BH3 profiling assay. STAT3 degrader treatment also enhanced differentiation of myeloid and erythroid colonies in Ven-res peripheral blood mononuclear cells (PBMNCs). Upregulation of p-STAT3(S727) was also associated with pronounced mitochondrial structural and functional dysfunction in Ven-res cell lines, that were restored by STAT3 degradation. Treatment with a clinical-stage STAT3 degrader, KT-333 resulted in a significant reduction in STAT3 and MCL1 protein levels within two weeks of treatment in a cell derived xenograft model of Ven-res AML. Additionally, this treatment significant improvement in the survival of a Ven-res patient-derived xenograft in-vivo study. Degradation of STAT3 resulting in downregulation of MCL1 and improvements in global mitochondrial dysfunction suggests a novel mechanism of overcoming Ven-res in AML. Statement of PurposeFive-year survival from AML is dismal at 30%. Our prior research demonstrated STAT3 over-expression in AML HSPCs to be associated with inferior survival. We now explore STAT3 over-expression in Ven-res AML, explain STAT3 mediated mitochondrial perturbations and describe a novel therapeutic strategy, STAT3 degradation to overcome Ven-res.

cancer biology↗

bioRxiv2024

Alzheimer's disease-linked risk alleles elevate microglial cGAS-associated senescence and neurodegeneration in a tauopathy model

The strongest risk factors for Alzheimers disease (AD) include the {chi}4 allele of apolipoprotein E (APOE), the R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), and female sex. Here, we combine APOE4 and TREM2R47H (R47H) in female P301S tauopathy mice to identify the pathways activated when AD risk is the strongest, thereby highlighting disease-causing mechanisms. We find that the R47H variant induces neurodegeneration in female APOE4 mice without impacting hippocampal tau load. The combination of APOE4 and R47H amplified tauopathy-induced cell-autonomous microglial cGAS-STING signaling and type-I interferon response, and interferon signaling converged across glial cell types in the hippocampus. APOE4-R47H microglia displayed cGAS- and BAX-dependent upregulation of senescence, showing association between neurotoxic signatures and implicating mitochondrial permeabilization in pathogenesis. By uncovering pathways enhanced by the strongest AD risk factors, our study points to cGAS-STING signaling and associated microglial senescence as potential drivers of AD risk.

neuroscience↗

bioRxiv2023

Mechanism of Dimer Selectivity and Binding Cooperativity of BRAF inhibitors

Aberrant signaling of BRAFV600E is a major cancer driver. Current FDA-approved RAF inhibitors selectively inhibit the monomeric BRAFV600E and suffer from tumor resistance. Recently, dimer-selective and equipotent RAF inhibitors have been developed; however, the mechanism of dimer selectivity is poorly understood. Here, we report extensive molecular dynamics (MD) simulations of the monomeric and dimeric BRAFV600E in the apo form or in complex with one or two dimer-selective (PHI1) or equipotent (LY3009120) inhibitor(s). The simulations uncovered the unprecedented details of the remarkable allostery in BRAFV600E dimerization and inhibitor binding. Specifically, dimerization retrains and shifts the C helix inward and increases the flexibility of the DFG motif; dimer compatibility is due to the promotion of the C-in conformation, which is stabilized by a hydrogen bond formation between the inhibitor and the C Glu501. A more stable hydrogen bond further restrains and shifts the C helix inward, which incurs a larger entropic penalty that disfavors monomer binding. This mechanism led us to propose an empirical way based on the co-crystal structure to assess the dimer selectivity of a BRAFV600E inhibitor. Simulations also revealed that the positive cooperativity of PHI1 is due to its ability to preorganize the C and DFG conformation in the opposite protomer, priming it for binding the second inhibitor. The atomically detailed view of the interplay between BRAF dimerization and inhibitor allostery as well as cooperativity has implications for understanding kinase signaling and contributes to the design of protomer selective RAF inhibitors.

biophysics↗

bioRxiv2022

Activation of targetable inflammatory immune signaling is seen in Myelodysplastic Syndromes with SF3B1 mutations

BackgroundMutations in the SF3B1 splicing factor are commonly seen in Myelodysplastic syndromes (MDS) and Acute Myeloid Leukemia (AML), yet the specific oncogenic pathways activated by missplicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood. MethodsRNA-seq data from SF3B1 mutant samples was analyzed and functional roles of IRAK4 isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in pre-clinical models of MDS/AML ResultsRNA-seq splicing analysis of innate immune mediators in SF3B1 mutant MDS samples revealed retention of full-length exon 6 of interleukin-1 receptor-associated kinase 4 (IRAK4), a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-Long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteosomal degradation. Expression of IRAK4-Long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models. ConclusionsSF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models.

cancer biology↗

bioRxiv2018

Direct Small Molecule Activation of Mitofusins

Mitochondrial fusion is a physiological process that is regulated by mitofusins on the outer mitochondrial membrane. Conformational plasticity between anti- and pro-tethering conformations of mitofusins permits mitochondrial tethering and subsequent fusion. Here we developed a pharmacophore-based model to rationally manipulate the conformational plasticity of mitofusin 2 and perfomed an in silico small-molecule screen. This enabled the discovery of a direct activator of mitofusins, MASM7, capable of potently promoting mitochondrial fusion. The specificity of the MASM7-mitofusin 2 interaction is highlighted by structure-activity relationships of MASM7 analogues, FRET, NMR and mitochondrial fusion studies using mitofusin mutants. Our study identified the first-in-class direct activator of mitofusins, demonstrating a new paradigm for chemical modulation of mitochondrial fusion and downstream processes.

biochemistry↗