Biology subjects

Brunetti, L.

Publications and source records attributed to Brunetti, L..

8 recordsLinked to original sources

bioRxiv2026

TGS1 mediates mRNA 5'-cap trimethylation to promote oxidative phosphorylation in acute myeloid leukaemia.

The Trimethyl guanosine Synthase (TGS1) is a highly conserved enzyme mediating di-methylation of the 5-cap 7-methylguanosine of RNA to generate 2,2,7-trimethylguanosine (m2,2,7G). Known TGS1 targets include snRNAs, snoRNAs, the telomeric RNA component and a limited number of mRNAs encoding selenoproteins. TGS1 is highly expressed in acute myeloid leukaemia (AML) cells, and its expression correlates with poor prognosis. Here, we report that TGS1 directly methylates the cap of more than 500 mRNAs in AML cells. Specifically, we demonstrate that TGS1 modifies nuclear mRNAs encoding mitochondrial proteins, including critical components of complexes involved in both the TCA cycle and oxidative phosphorylation, promoting their translation mediated by mitochondria associated cytosolic ribosomes. Functionally, we report that TGS1 depletion impairs mitochondrial respiration and increases oxidative stress. This, in turn, impairs the growth of AML cells causing differentiation and cell cycle arrest in vitro and in vivo. Finally, we demonstrate that TGS1 depletion sensitizes AML cells to RSL3, a small molecule promoting ferroptosis. Taken together, our findings establish TGS1 as a key regulator of oxidative phosphorylation and mitochondrial redox status of AML cells and highlight its potential as a therapeutic target in leukaemia.

cancer biology↗

bioRxiv2025

Clinical and molecular characterisation of primary refractoriness to atezolizumab plus bevacizumab in patients with unresectable hepatocellular carcinoma.

BackgroundDespite improved outcomes with atezolizumab plus bevacizumab (A+B) in hepatocellular carcinoma (HCC), primary refractoriness (PRef), characterised by early progression or short-lived disease stabilisation following treatment, remains a significant and poorly understood clinical challenge. MethodsWe analysed 1296 patients with HCC and Child-Pugh A liver cirrhosis treated with frontline A+B (AB-real) and validated findings in 645 trial participants recruited to IMbrave150 and GO30140. PRef was defined by Society for the Immunotherapy of Cancer (SITC) criteria as progressive disease in the first 6 months after treatment initiation. Patients who achieved complete response, partial response or stable disease for [≥] 6 months were classified as responders. We performed a multi-parametric analysis of pre-treatment tumour tissue including machine learning-based quantification of tumour-infiltrating lymphocytes, imaging mass cytometry and RNA sequencing (RNAseq) to evaluate differences in the tumour microenvironment (TME) of PRef versus responding patients. We employed conditional inference tree analyses to provide a hierarchical organisation of determinants of PRef. ResultsAmong 677 AB-real and 378 trial patients evaluable by SITC criteria, PRef identified inferior median OS in comparison with responding patients (AB-real: 7.3 vs. 31.5 months, HR 3.7, 95%CI 2.8-8.5, p<0.001; Trials: 10.8 vs. NR, HR 4.6, 95%CI 3.3-6.3, p<0.001). PRef patients exhibited higher baseline systemic inflammation (neutrophil-to-lymphocyte ratio, NLR [≥]3), a distinctively immunosuppressive TME enriched in CD163+ tumour-associated macrophages and a higher Treg/Teff ratio. RNAseq of tumour tissue demonstrated lower intrinsic immunogenicity in PRef samples, characterised by repressed IFN-{gamma} and Teff signatures, with elevated myeloid infiltration. Conditional inference tree analysis identified IFN-{gamma} signature downregulation combined with NLR [≥]3 as the strongest contributor of PRef. ConclusionsPRef to A+B identifies a distinct biological entity characterised by unopposed systemic inflammation, myeloid cell infiltration and T-cell depletion. Targeting myeloid-mediated immunosuppression, particularly in patients with low IFN-{gamma} signature expression and elevated NLR might enhance responsiveness to A+B. HighlightsO_LIPrimary refractoriness to atezolizumab plus bevacizumab in hepatocellular carcinoma, as defined by SITC criteria, is associated with poor clinical outcomes. C_LIO_LITumour microenvironment profiling reveals an immunosuppressive phenotype characterized by high myeloid infiltration, reduced interferon-{gamma} signalling, and T-cell depletion. C_LIO_LIThe combination of systemic inflammation and low IFN-{gamma} signature expression strongly predicts primary refractoriness and may inform therapeutic decision-making. C_LI

cancer biology↗

bioRxiv2025

Nuclear Phase Separation Drives NPM1-mutant Acute Myeloid Leukemia

During cancer development, mutations promote gene expression changes that cause transformation. Leukemia is frequently associated with aberrant HOXA expression driven by translocations in nucleoporin genes or KMT2A, and mutations in NPM1. How disparate mutations converge on this regulatory pathway is not understood. Here we demonstrate that mutant NPM1 (NPM1c) forms nuclear condensates in multiple human cell lines, mouse models, and primary patient samples. We show NPM1c phase separation is necessary and sufficient to coordinate the recruitment of NUP98 and KMT2A to condensates. Through extensive mutagenesis and pharmacological destabilization of phase separation, we find that NPM1c condensates are necessary for regulating gene expression, promoting in vivo expansion, and maintaining the undifferentiated leukemic state. Finally, we show that nucleoporin and KMT2A fusion proteins form condensates that are biophysically indistinguishable from NPM1c condensates. Together, these data define a new condensate underlying leukemias that we term coordinating bodies (C-bodies), and propose C-bodies as a therapeutic vulnerability.

biophysics↗

bioRxiv2025

FGF overactivation underlies reduced neurogenesis in cerebellar organoid models of neurodevelopmental ciliopathy

The primary cilium acts as a specialized signaling compartment that coordinates multiple developmental pathways, yet its role in regulating signaling activity during human brain development remains poorly understood. Biallelic mutations in ciliary genes cause neurodevelopmental ciliopathies such as Joubert syndrome, which are characterized by cerebellar hypoplasia and dysplasia. Here, we generated human cerebellar organoids carrying null or patient-derived mutations in RPGRIP1L, a ciliary transition zone gene associated with Joubert Syndrome, to investigate how ciliary dysfunction alters human cerebellar neurogenesis. While control organoids robustly express markers of cerebellar glutamatergic and GABAergic lineages including Purkinje cells, RPGRIP1L-deficient organoids display a consistent and severe reduction in Purkinje cell markers, accompanied by impaired neurogenesis and increased progenitor proliferation. These defects coincide with prolonged overactivation of the FGF/MAPK signaling pathway. We further show that the MAPK effector pMEK1/2 localizes at the base of primary cilia, where its levels are significantly increased in RPGRIP1L-deficient cerebellar progenitors. Pharmacological inhibition of FGF receptors reduces pMEK1/2 activation at cilia base and rescues both the proliferation-neurogenesis imbalance and Purkinje lineage defects, without restoring the underlying ciliary abnormalities. Together, our findings identify the primary cilium as a compartment that refines FGF/MAPK signaling in human cerebellar progenitors, and support a model in which RPGRIP1L restrains FGF pathway activity to promote neurogenesis. These results provide a mechanism linking ciliary dysfunction to altered cerebellar development and provide new insight into the developmental origin of cerebellar impairment in neurodevelopmental ciliopathies

developmental biology↗

bioRxiv2024

A DIFFERENTIAL REQUIREMENT FOR THE CILIOPATHY GENE RPGRIP1L IN HUMAN AND MOUSE SPINAL PROGENITOR FATE SPECIFICATION

Studying developmental processes in the context of the human central nervous system is essential to understand neurodevelopmental diseases. In this paper we perform a comparative functional study of the ciliopathy gene RPGRIP1L in human and mouse spinal development using in vitro 3D differentiation of pluripotent stem cells. RPGRIP1L, a causal gene of severe neurodevelopmental ciliopathies such as Joubert and Meckel syndromes, encodes a scaffolding protein of the ciliary transition zone involved in ciliary gating. Previous work has identified a major role for Rpgrip1l in mouse brain and spinal cord development, via controlling the Sonic Hedgehog (SHH)/GLI pathway. We show that spinal organoids derived from Rpgrip1l mutant mouse embryonic stem cells faithfully recapitulate the loss of motoneurons and the strong reduction of SHH signaling observed in the mutant mice. In contrast, human induced pluripotent stem cells mutant for RPGRIP1L produce motoneurons and activate the SHH pathway at levels similar to wild types, a property shared by human iPSCs mutant for another ciliopathy gene TMEM67. Moreover, we show that, in human RPGRIP1L mutant organoids, motoneurons acquire a more anterior identity, expressing HOX genes and other proteins normally present in the hindbrain and cervical spinal cord while motoneurons from wild type organoids strictly display a caudal brachial identity. By performing a temporal transcriptome analysis throughout the differentiation process, we find that the antero-posterior specification defect arises in early axial progenitors and correlates with the loss of cilia in these cells. Thus, this study uncovers distinct functions in humans and mice for ciliopathy proteins and a novel role for RPGRIP1L in human spinal antero-posterior patterning. These findings have important implications for understanding the role of cilia in human spinal cord development and the pathogenic mechanisms of neurodevelopmental ciliopathies.

developmental biology↗

bioRxiv2021

Prolonged XPO1 inhibition is essential for optimal anti-leukemic activity in NPM1-mutated AML

NPM1 encodes for a nucleolar multifunctional protein and is the most frequently mutated gene in adult acute myeloid leukemia (AML). NPM1 mutations cause the aberrant accumulation of mutant NPM1 (NPM1c) in the cytoplasm of leukemic cells, that is mediated by the nuclear exporter Exportin-1 (XPO1). Recent work has demonstrated that the interaction between NPM1c and XPO1 promotes high homeobox (HOX) genes expression, which is critical for maintaining the leukemic state of NPM1-mutated cells. However, the XPO1 inhibitor Selinexor administered once or twice/week in early-phase clinical trials did not translate into clinical benefit for NPM1-mutated AML patients. Here, we demonstrate that this dosing strategy results in only temporary disruption of the XPO1-NPM1c interaction and transient HOX genes downregulation, limiting the efficacy of Selinexor in the context of NPM1-mutated AML. Since second-generation XPO1 inhibitors can be administered more frequently, we compared intermittent (twice/week) versus prolonged (5 days/week) XPO1 inhibition in NPM1-mutated AML models. Integrating in vitro and in vivo data, we show that only prolonged XPO1 inhibition results in stable HOX downregulation, cell differentiation and remarkable anti-leukemic activity. This study lays the groundwork for the accurate design of clinical trials with second-generation XPO1 inhibitors in NPM1-mutated AML.

cancer biology↗

bioRxiv2020

Modeling IKZF1 lesions in B-ALL reveals distinct chemosensitivity patterns and potential therapeutic vulnerabilities

IKAROS family zinc finger 1 (IKZF1) alterations represent a diverse group of genetic lesions that are associated with an increased risk of relapse in B-lymphoblastic leukemia (B-ALL). Due to the heterogeneity of concomitant lesions it remains unclear how IKZF1 abnormalities directly affect cell function and therapy resistance and whether their consideration as a prognostic indicator is valuable in improving outcome. We used CRISPR/Cas9 to engineer multiple panels of isogeneic lymphoid leukemia cell lines with a spectrum of IKZF1 lesions in order to measure changes in chemosensitivity, gene expression, cell cycle, and in vivo engraftment dynamics that can be directly linked to loss of IKAROS protein. IKZF1 knockout and heterozygous null cells displayed relative resistance to a number of commonly employed therapies for B-ALL including dexamethasone, vincristine, asparaginase, and daunorubicin. Transcription profiling revealed a stem/myeloid cell-like phenotype and JAK/STAT upregulation after IKAROS loss. We also used a CRISPR homology-directed repair (HDR) strategy to knock-in the dominant-negative IK6 isoform tagged with GFP into the endogenous locus and observed a similar drug resistance profile with the exception of retained sensitivity to dexamethasone. Interestingly, IKZF1 knockout and IK6 knock-in cells both have significantly increased sensitivity to cytarabine, suggesting intensification of nucleoside analog therapy may be specifically effective for IKZF1-deleted B-ALL. Both types of IKZF1 lesions decreased survival time of xenograft mice, with higher numbers of circulating blasts and increased organ infiltration. Given these findings, exact specification of IKZF1 status in patients may be a beneficial addition to risk stratification and could inform therapy. Key pointsO_LIEngineered IKZF1 perturbations result in a stem-cell like expression signature, enhanced engraftment in vivo, and multi-drug resistance C_LIO_LILoss of IKAROS may result in new vulnerabilities due to increased sensitivity to cytarabine and upregulation of JAK/STAT and mAb targets C_LI

cancer biology↗

bioRxiv2020

CIS checkpoint deletion enhances the fitness of cord blood derived natural killer cells transduced with a chimeric antigen receptor

Immune checkpoint therapy has produced remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible SH2-containing (CIS) protein, a key negative regulator of interleukin (IL)-15 signaling, with chimeric antigen receptor (CAR) engineering of natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell anti-tumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that combining CIS checkpoint deletion with CAR engineering promotes the metabolic fitness of NK cells in an otherwise suppressive tumor microenvironment. This approach, together with the prolonged survival afforded by CAR modification, represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

immunology↗