Biology subjects

Stuani, L.

Publications and source records attributed to Stuani, L..

7 recordsLinked to original sources

bioRxiv2025

Multi-omics characterization of IDH-mutant astrocytoma-derived cell lines reveals NOTCH-regulated plastic quiescent astrocyte-like state

Diffuse IDH-mutant astrocytomas are brain tumors typically diagnosed as low-grade but capable of progressing to higher grades. They exhibit three cellular states resembling astrocytes, oligodendrocytes, and neural progenitor (NPC) cells. Understanding their biology has been challenging due to the lack of relevant in vitro models. Here we established and extensively characterized four astrocytoma cell lines (LGG275, LGG336, LGG85, LGG349) derived from IDH-mutant astrocytoma at different grades, cultured in defined media and analyzed by multi-omics. These lines display growth rates in vitro and in vivo consistent with tumor grade and recapitulate key molecular alterations observed in patient tumors, including IDH1, ATRX, and TP53 mutations, activation of the alternative lengthening of telomeres (ALT) pathway and, in the most aggressive line, amplification of MET and PDGFRA. Single-cell RNA sequencing showed that the 4 astrocytoma lines maintain the three major cellular states observed in patient tumors. A hallmark of higher-grade-derived lines (LGG85, LGG349) is the persistence of NPC-like populations without growth factors, reflecting tumor progression. The LGG275 line most accurately mirrors slow-growing astrocytomas. Using CD44 and GLAST, we isolated astrocyte-like (CD44+/GLAST+) cells from LGG275 that preferentially adopt a quiescent state yet retain remarkable plasticity, generating oligodendrocyte-like cells (CD44-/GLAST-). Transcriptomic and proteomic analyses revealed that astrocyte-like and oligodendrocyte-like cells populations resemble quiescent and activated neural stem (NSC) cells from the adult subventricular zone (SVZ). Finally, we found that NOTCH signaling regulates the balance between astrocytic and oligodendrocytic states, while DLL3, expressed by oligodendrocyte-like cells, modulates both proliferation and phenotype. These cell lines represent valuable resources for dissecting lineage dynamics, heterogeneity, and progression mechanisms in IDH-mutant astrocytomas. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/696808v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@953108org.highwire.dtl.DTLVardef@c6234aorg.highwire.dtl.DTLVardef@3703fcorg.highwire.dtl.DTLVardef@1e6c3a9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightO_LIWe constituted a richly annotated biobank derived from 4 astrocytomas, showing similar characteristics to those found in patients, providing valuable tools to investigate cellular heterogeneity and plasticity, and link with tumor progression. C_LIO_LIscRNA-sequencing revealed three cell states (oligodendrocyte-like, astrocyte-like, and stem cell-like cells) akin to those found in tumors. C_LIO_LIAstrocyte-like cells are quiescent cells, plastic and similar to quiescent neural stem cell (qNSC) from the sub-ventricular zone (SVZ), while oligodendrocyte-like cells are similar to active NSC (aNSC). C_LIO_LIThe Notch pathway plays a role in cell plasticity, enabling a shift towards an astrocyte-like state. C_LI

cancer biology↗

bioRxiv2025

Uridine Metabolism as a Targetable Metabolic Achilles' Heel for chemo-resistant B-ALL

Relapse continues to limit survival for patients with B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have independently implicated activation of B-cell developmental signaling pathways and increased glucose consumption with chemo-resistance and relapse risk. Here, we connect these observations, demonstrating that B-ALL cells with active signaling, defined by high expression of phosphorylated ribosomal protein S6 ("pS6+ cells"), are metabolically unique and glucose dependent. Isotope tracing and metabolic flux analysis confirm that pS6+ cells are highly glycolytic and notably sensitive to glucose deprivation, relying on glucose for de novo nucleotide synthesis. Uridine, but not purine or pyrimidine, rescues pS6+ cells from glucose deprivation, highlighting uridine is essential for their survival. Active signaling in pS6+ cells drives uridine production through activating phosphorylation of carbamoyl phosphate synthetase (CAD), the enzyme catalyzing the initial steps of uridine synthesis. Inhibition of signaling abolishes glucose dependency and CAD phosphorylation in pS6+ cells. Primary pS6+ cells demonstrate high expression of uridine synthesis proteins, including dihydroorotate dehydrogenase (DHODH), the rate-limiting catalyst of de novo uridine synthesis. Gene expression demonstrates that increased expression of DHODH is associated with relapse and inferior event-free survival after chemotherapy. Further, the majority of B-ALL genomic subtypes demonstrate activity of DHODH. Inhibiting DHODH using BAY2402232 effectively kills pS6+ cells in vitro, with its IC50 correlated with the strength of pS6 signaling across 14 B-ALL cell lines and patient-derived xenografts (PDX). In vivo DHODH inhibition prolongs survival and decreases leukemia burden in pS6+ B-ALL cell line and PDX models. These findings link active signaling to uridine dependency in B-ALL cells and an associated risk of relapse. Targeting uridine synthesis through DHODH inhibition offers a promising therapeutic strategy for chemo-resistant B-ALL as a novel therapeutic approach for resistant disease.

cancer biology↗

bioRxiv2023

Inosine Induces Stemness Features in CAR T cells and Enhances Potency

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing. Statement of SignificanceAdenosine is well known to inhibit T cell function and substantial effort has focused on inhibiting adenosine generation and signaling. Here, we show that exhausted T cells are suppressed by adenosine, which is only modestly impacted by inhibiting adenosine generation or signaling. In contrast, metabolism of adenosine to inosine augmented T cell function and culture of T cells with inosine induced multi-level reprogramming leading to stemness and improved anti-tumor potency. We demonstrate the feasibility of introducing inosine during GMP cell manufacturing as a novel strategy to generate enhanced CAR-T cells.

immunology↗

bioRxiv2022

Cytidine deaminase regulates mitochondrial biogenesis in pancreatic cancer cells

Despite tremendous efforts from the scientific community, pancreatic ductal adenocarcinoma (PDAC) is still a deadly disease and will soon become the second cause of death by cancer worldwide1. When surgery is not possible, therapeutic options are few and ineffective2. Patients are most often treated with Folfirinox or gemcitabine chemotherapy, that extends survival in weeks to months. Cytidine deaminase (CDA) catalyzes the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine to fuel RNA and DNA synthesis3. CDA also deaminates and neutralizes deoxycytidine-based therapies, and as such, has been identified as a major contributor of tumor chemoresistance, especially to gemcitabine in PDAC3. We previously identified that CDA is elevated in PDAC tumors at diagnosis and that CDA exerts an unexpected role on DNA replication that can be exploited for therapeutic intervention4. Very recently, CDA was associated with cellular metabolism5. Here, we show that CDA promotes mitochondrial biogenesis and oxidative phosphorylation independently of its deaminase activity. This uncloaks novel therapeutic vulnerabilities in primary cancer cells that overexpress this protein. This study shines a new light on the tumoral potential of CDA in PDAC.

cancer biology↗

bioRxiv2020

Adrenomedullin-CALCRL Axis Controls Relapse-Initiating Drug Tolerant Acute Myeloid Leukemia Cells

Drug tolerant leukemic cell subpopulations may explain frequent relapses in acute myeloid leukemia (AML), suggesting that these Relapse-Initiating Cells (RICs) persistent after chemotherapy represent bona fide targets to prevent drug resistance and relapse. We uncovered that the G-protein coupled receptor CALCRL is expressed in leukemic stem cells (LSCs) and RICs, and that the overexpression of CALCRL and/or of its ligand adrenomedullin (ADM) and not CGRP correlates to adverse outcome in AML. CALCRL knockdown impairs leukemic growth, decreases LSC frequency and sensitizes to cytarabine in patient-derived xenograft (PDX) models. Mechanistically, the ADM-CALCRL axis drives cell cycle, DNA repair and mitochondrial OxPHOS function of AML blasts dependent on E2F1 and BCL2. Finally, CALCRL depletion reduces LSC frequency of RICs post-chemotherapy in vivo. In summary, our data highlight a critical role of ADM-CALCRL in post-chemotherapy persistence of these cells, and disclose a promising therapeutic target to prevent relapse in AML.

cancer biology↗

bioRxiv2020

Mitochondrial determinants of response and resistance to venetoclax plus cytarabine duplet therapy in acute myeloid leukemia

The development of resistance to conventional and targeted therapy represents a major clinical barrier in treatment of acute myeloid leukemia (AML). We show that the resistance to cytarabine (AraC) and its associated mitochondrial phenotype were reversed by genetic silencing or pharmacological inhibition of BCL2 in a caspase-dependent manner. BCL2-inhibitor venetoclax (VEN) enhancement of AraC efficacy was independent of differentiation phenotype, a characteristic of response to another combination of VEN with hypomethylating agents (HMA). Furthermore, transcriptional profiles of patients with low response to VEN+AraC mirrored those of low responders to VEN+HMA in clinical trials. OxPHOS was found to be a patient stratification marker predictive of effective response to VEN+AraC but not to VEN+AZA. Importantly, whereas three cell subpopulations specifically emerged in VEN+AraC residual disease and were characterized by distinct developmental and transcriptional programs largely driven by MITF, E2F4 and p53 regulons, they each encoded proteins involved in assembly of NADH dehydrogenase complex. Notably, treatment of VEN+AraC-persisting AML cells with an ETCI inhibitor significantly increased the time-to-relapse in vivo. These findings provide the scientific rationale for new clinical trials of VEN+AraC combinations, especially in patients relapsing or non-responsive to chemotherapy, or after failure of frontline VEN+HMA regimen.

cancer biology↗

bioRxiv2019

Combinatory therapy targeting mitochondrial oxidative phosphorylation improves efficacy of IDH mutant inhibitors in acute myeloid leukemia

Isocitrate dehydrogenases (IDH) are involved in redox control and central metabolism. Mutations in IDH induce epigenetic and transcriptional reprogramming, differentiation bias, BCL-2 dependence and susceptibility to mitochondrial inhibitors in cancer cells. Here we show that high sensitivity to mitochondrial oxidative phosphorylation (OxPHOS) inhibitors is due to an enhanced mitochondrial oxidative metabolism in cell lines, PDX and patients with acute myeloid leukemia (AML) harboring IDH mutation. Along with an increase in TCA cycle intermediates, this AML-specific metabolic behavior mechanistically occurs through the increase in methylation-driven CEBP- and CPT1a-induced fatty acid oxidation, electron transport chain complex I activity and mitochondrial respiration in IDH1 mutant AML. Furthermore, an IDH mutant inhibitor that significantly and systematically reduces 2-HG oncometabolite transiently reverses mitochondrial FAO and OxPHOS gene signature and activities in patients who responded to the treatment and achieved the remission. However, at relapse or in patients who did not respond, IDH mutant inhibitor failed to block these mitochondrial properties. Accordingly, OxPHOS inhibitors such as IACS-010759 improve anti-AML efficacy of IDH mutant inhibitors alone and in combination with chemotherapy in vivo. This work provides a scientific rationale for combinatory mitochondrial-targeted therapies to treat IDH mutant-positive AML patients, especially those unresponsive to or relapsing from IDH mutant-specific inhibitors.

cancer biology↗