Biology subjects

Paggetti, J.

Publications and source records attributed to Paggetti, J..

5 recordsLinked to original sources

bioRxiv2026

The FDA-approved CDK4/6 inhibitor Abemaciclib is a potent agonist of the β-arrestin-biased atypical chemokine receptor ACKR3

Atypical chemokine receptor 3 (ACKR3/CXCR7) is an intrinsically {beta}-arrestin-biased family-A GPCR that controls chemokine availability by scavenging CXCL12 and CXCL11, and consequently plays a key role in cancer, chronic inflammation, autoimmunity, cardiovascular disease, and CNS repair. Despite its pathophysiological significance, the chemical space available to interrogate and therapeutically modulate this receptor remains limited. Here, by using a drug-repurposing strategy to screen 2,342 FDA-approved compounds in a NanoBRET-based fluorescent chemokine-binding competition assay, we identified the clinically used CDK4/6 inhibitor abemaciclib (Verzenios(R)) as a nanomolar ACKR3 ligand. Abemaciclib inhibited CXCL12 binding to ACKR3 with comparable affinity to the ACKR3-reference agonist VUF15485, whereas the related clinically used CDK4/6 inhibitors palbociclib, ribociclib, dalpiciclib and trilaciclib were inactive on this receptor. Binding and functional profiling across classical and atypical chemokine receptors revealed selectivity for ACKR3, including lack of detectable activity at the CXCL12 receptors CXCR4 and ACKR5. Abemaciclib acted as a potent ACKR3 agonist, recruiting both {beta}-arrestin-1 and 2 and promoting rapid receptor internalization with potencies and efficacies, like the reference ACKR3 agonist VUF15485. Major CYP3A4-derived abemaciclib metabolites retained ACKR3 binding and agonist activity, albeit with reduced potency. Methionine 13C-HSQC NMR confirmed direct receptor engagement and revealed a small-molecule-like ACKR3 conformational signature distinct from the CXCL12-bound state. Cryo-EM of the abemaciclib-ACKR3-CID24 complex at 3.2 [A] uncovered a unique, inverted U-shaped ligand binding pose in the orthosteric pocket and activation-associated rearrangements of TM6, TM7 and helix 8. Functionally, abemaciclib blocked ACKR3-mediated uptake of CXCL11 and CXCL12 in vitro. Moreover, oral administration of abemaciclib in mice resulted in a time-dependent increase in plasma CXCL12 levels, indicative of in vivo ACKR3 target engagement. These findings establish this FDA-approved kinase inhibitor as a potent ACKR3 modulator and reveal an unexpected pharmacological link of this CDK4/6 inhibitor to atypical chemokine receptor biology.

molecular biology↗

bioRxiv2026

Chemokine and opioid peptide scavenging through constitutive and ligand-induced release of ACKR3-bearing extracellular vesicles

Atypical chemokine receptors (ACKRs) are non-signaling GPCRs that regulate ligand availability, with ACKR3 functioning as a dual scavenger of chemokines and opioid peptides. Here, we demonstrate that following ligand stimulation, besides the canonical internalization, ACKR3 is released on extracellular vesicles (EVs). ACKR3 was also found on EVs released under basal conditions, although to a lesser extent. These observations were confirmed across multiple cellular contexts, including endogenous systems. Mechanistically, basal and ligand-induced EV release are independent of GRKs and {beta}-arrestin but each relies on distinct trafficking routes and C-terminal determinants. Ligand-induced EV release is associated with plasma membrane localization and receptor recycling pathways. In contrast, basal EV release is governed by intracellular sorting processes and influenced by receptor ubiquitination and RAMP3. Functionally, EV-associated ACKR3 retains high-affinity ligand binding, enabling sequestration of CXCL12 and opioid peptides and thereby attenuating their signaling through CXCR4 and MOR. We also show that the release on EVs, in particular under basal conditions, is observed for other receptors such as KOR, CXCR4 and several ACKRs. Collectively, these findings establish EVs as regulators in chemokine and opioid systems and as a previously underappreciated dimension of ACKR3 and more broadly GPCR biology.

Cell Biology↗

bioRxiv2025

Humanized glioblastoma patient-derived orthotopic xenografts recreate a locally immunosuppressed human immune ecosystem amenable to immunotherapeutic modulation

Immune-based strategies have so far failed to demonstrate clinical benefit in glioblastoma (GBM), largely due to the profound immunosuppressive tumor microenvironment (TME). To achieve more predictive preclinical insights, advanced in vivo models that faithfully recapitulate the human brain immune landscape are urgently needed. Here, we established GBM patient-derived orthotopic xenografts (PDOXs) across diverse mouse strains, including humanized models. Humanization was achieved through transplantation of CD34+ hematopoietic stem cells (HU-CD34+) or peripheral blood mononuclear cells (HU-PBMC). Both models successfully reconstituted human T-cells systemically, with stronger engraftment in HU-CD34+ mice. We observed selective infiltration and spatial organization to intracranial GBM tumors, including exhausted, memory-like, and regulatory CD4+ T-cell phenotypes, TIM-3+ immunosuppressive-like myeloid cells and intratumoral B cells. Mouse microglia-derived tumor-associated macrophages (TAMs) remained the dominant immunosuppressive immune population. Anti-PD-1 therapy, but not anti-GITR, modestly modulated the infiltration dynamics, demonstrating the susceptibility of the reconstructed adaptive immunity to immunotherapeutic intervention. These findings position humanized GBM PDOXs as a relevant preclinical platform to interrogate tumor-immune interactions and evaluate immunotherapeutic strategies in a human context. Key pointsO_LIGBM PDOXs developed in HU-CD34+ and HU-PBMC mice faithfully reconstitute systemic and local human adaptive immunity. C_LIO_LIHuman immune components undergo selective infiltration, spatial organization and transition towards exhausted CD4+ T-cells and immunosuppressive CD11c+ myeloid cells. C_LIO_LIAnti-PD-1, but not anti-GITR, locally promote human immune infiltration into intracranial GBM tumors, while sparing systemic compartments. C_LIO_LIHumanized GBM PDOXs provide a powerful preclinical platform to test novel immunotherapeutic strategies. C_LI Study importanceImmune checkpoint blockade has shown limited efficacy in GBM, reflecting the highly immunosuppressive and lymphocyte-poor nature of the TME. Conventional syngeneic and GEMM models fail to recapitulate these features, contributing to the translational disconnect between preclinical success and clinical failure. Humanized mice provide a solution to interrogate human-specific immunity in vivo, but their use in GBM has remained limited. Here, we provide the first comparison of GBM PDOX modeling in two complementary modes of humanization based on CD34+ HSCs and PBMCs. We systematically profile systemic and intratumoral compartments, showing that these models faithfully reconstitute human adaptive immunity and capture the interplay with the murine brain TME. Furthermore, we demonstrate clinically-relevant responses upon treatment with checkpoint antibodies targeting PD-1 and GITR, showing modulation of human immune subsets without altering murine TAM immunosuppression, underscoring the translational value of the system. This study establishes humanized GBM PDOXs as a versatile platform for dissecting tumor-immune interactions in the brain and for preclinical evaluation and development of novel immunotherapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/689484v2_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@1b684e1org.highwire.dtl.DTLVardef@1cdfbbdorg.highwire.dtl.DTLVardef@4a570borg.highwire.dtl.DTLVardef@98b112_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

PKM2 diverts glycolytic flux in dependence on formate overflow

Throughout the metastatic cascade, cancer cells are faced with harsh metabolic environments and nutritional stresses which apply selection pressure leaving only the most metabolically resilient cells to survive and form metastases. Metabolic characterisation of such cell populations in vitro is currently challenging. Using galactose as a tool compound to mimic glycolytic limitation within the tumour microenvironment of primary and secondary neoplastic sites, we were able to uncover metabolic flexibility and plasticity of cancer cells in vitro. In contrast to the established idea that high glycolytic flux and expression of dimeric PKM2 redirects carbons towards anabolic routes such as the pentose phosphate pathway and serine synthesis pathway (SSP), we have discovered by using stable-isotope tracing that also glycolytic limitation results in metabolic rewiring. Surprisingly, despite limited carbon availability and energetic stress, cells induce a near complete block of pyruvate kinase isozyme M2 (PKM2) to divert carbons towards SSP. Simultaneously, TCA cycle flux is sustained and oxygen consumption is increased, both supported by glutamine. Glutamine not only supports TCA cycle flux but also SSP via distinct mechanisms. Due to PKM2 block, malic enzyme exclusively supports TCA cycle flux while mitochondrial phosphoenolpyruvate carboxykinase supports SSP. Moreover, by using genetic modifications of different one-carbon (1C) cycle enzymes, we are able to reverse the PKM2 block suggesting a link between mitochondrial 1C cycle and pyruvate kinase. Thus we show that PKM2 inhibition acts as a branching point to direct glycolytic and glutamine carbons into distinct routes, overall supporting the metabolic plasticity and flexibility of cancer cells.

cancer biology↗

bioRxiv2022

T cell landscape definition by multi-omics identifies Galectin-9 as novel immunotherapy target in chronic lymphocytic leukemia

Failure of cancer immunotherapy is linked to T cell exhaustion. To decipher the underlying mechanisms, we explored the T cell landscape in blood, bone marrow and lymph node samples of patients with chronic lymphocytic leukemia (CLL), and spleen samples of a CLL mouse model. By single-cell RNA-sequencing, mass cytometry (CyTOF), and multiplex image analysis of tissue microarrays, we identified a disease-specific accumulation of distinct regulatory T cell subsets and T cell exhaustion stages and their trajectories in CLL lymph nodes. Integration of T cell receptor sequencing data revealed a clonal expansion of CD8+ precursor exhausted T cells (TPEX), suggesting their CLL reactivity. Interactome analyses identified the TIM3 ligand Galectin-9 as a novel immunoregulatory molecule in CLL. Blocking of Galectin-9 in CLL-bearing mice slowed down disease development and reduced the number of TIM3-expressing T cells. Galectin-9 expression correlated with shorter survival of patients with CLL, renal cell carcinoma or glioma. Statement of significanceOur findings for the first time define the T cell landscape in CLL lymph nodes and reshape the current understanding of T cell exhaustion in this malignancy. They further introduce Galectin-9 as novel immune checkpoint with a high potential to overcome resistance to PD1 targeting drugs in CLL and beyond.

cancer biology↗