Biology subjects

Pecoraro, M.

Publications and source records attributed to Pecoraro, M..

12 recordsLinked to original sources

bioRxiv2026

Droplet single-cell CRISPR screens identify regulators of T cell-mediated target-cell killing

Cytotoxic CD8 T cells kill target cells through brief cell-cell encounters, but pooled genetic screens cannot readily link perturbations in individual T cells to the fate of the target cells they engage. We developed droplet single-cell CRISPR screening to pair individual primary human CD8 T cells with cancer cells, measure rapid target-cell death, and recover sgRNAs from phenotype-defined droplets. Applied across primary T cells from multiple donors, the platform recovered regulators of T cell receptor signaling, synapse formation, granule exocytosis and cytotoxic differentiation, and identified negative regulators of killing, including established inhibitory nodes such as PTEN, RASA2 and FOXO1, together with AFAP1L2 and components of the mTORC1 pathway. Validation across bispecific engager and TCR-engineered settings showed that selected hits modulate target-cell killing across recognition modalities and tumor models. Unexpectedly, perturbation of RPTOR or RHEB enhanced cytotoxic execution while reducing mTORC1 output, increasing AKT phosphorylation, and attenuating anabolic programs. Transient pharmacologic mTORC1 inhibition reproduced this rapid-killing state and improved antitumor activity after adoptive transfer. These results establish an interaction-resolved pooled genetic strategy for mapping cytotoxicity regulators and reveal that transient modulation of mTORC1 can shift T cells from anabolic growth toward rapid cytotoxic execution to enhance antitumor activity.

immunology↗

bioRxiv2026

Circulating extracellular vesicles drive microglial senescence and neurodegeneration in Parkinson`s disease

BackgroundExtracellular vesicles (EV), secreted membrane particles involved in cell-to-cell communication, carry important information on immunity and its dysregulation. Recent studies have demonstrated the crucial role of peripheral and central inflammation in causing Parkinsons disease (PD), as well as the involvement of EV in mediating neuron-glial interactions during neurodegeneration. However, the underlying mechanism of plasmatic EV in PD pathogenesis remains unknown. MethodsEV were isolated from pool of plasma of PD patients and age- and sex-matched healthy controls (HC) using size-exclusion chromatography and characterized by nanoparticle tracking analysis, western blot, and transmission electron microscopy. SH-SY5Y neurons and HMC3 microglia cells were treated with EV, and their impact was evaluated using flow cytometry and immunofluorescence. Conditioned medium (CM) from EV-treated HMC3 cells was applied to SH-SY5Y neurons to determine indirect neurotoxic effects. Cytokine profiling and senescence-like features of EV-treated HMC3 cells were assessed. Unbiased proteomic analysis of PD-EV and HC-EV were further performed. ResultsPD-EV induced axonal degeneration and cell death in SH-SY5Y neurons and increased levels of TNF-, IL-1{beta}, IFN-{gamma}, IL-8, and CCL11, accompanied by the expression of p16INK4a in HMC3 cells, suggesting a proinflammatory, senescence-associated secretory phenotype (SASP). Enrichment pathway analysis revealed that these changes were mainly related to inflammatory and immune responses. Moreover, CM from PD-EV-HMC3 cells increased apoptotic cell death in SH-SY5Y neurons more than direct PD-EV. Notably, proteomic analysis of PD-EV showed higher expression of proteins involved in complement cascades, immune response, phagocytosis, and post translational protein translation, further supporting the potential of EV to induce inflammatory changes in PD. ConclusionsThis study demonstrates that plasmatic PD-EV contributes to neuronal degeneration by reducing neuronal integrity and indirectly by activating microglia through the secretion of pro-inflammatory, senescence-associated mediators. Circulating EV exerts a role in bridging peripheral inflammation with microglia, modulating neuroinflammatory events.

neuroscience↗

bioRxiv2025

Targeting FOXA1 and FOXA2 Disrupts the Lineage-Specific Oncogenic Output Program in Prostate Cancer

Activation of the androgen receptor (AR) is the key lineage-specific oncogenic pathway and the primary therapeutic target in prostate cancer. While AR signaling is enabled by the pioneer transcription factor FOXA1, its homolog FOXA2 is specifically expressed in advanced lineage plasticity prostate cancers that have lost the AR signaling axis. However, their roles and utility as drug targets remain incompletely characterized. Here, we show an unexpected collaboration of FOXA1 and FOXA2 in mediating AR-independent cell proliferation in different lineage plasticity cancer subtypes. Conversely, joint loss-of-function or pharmacologic disruption of FOXA1 and FOXA2 leads to the collapse of lineage-specific oncogenic transcription factors followed by cell cycle arrest. In summary, our findings uncover a druggable dependency for AR-positive and - negative prostate cancers.

cancer biology↗

bioRxiv2025

TRIM24 Degradation Counteracts Adaptation to Androgen Receptor Inhibition in Prostate Cancer

The androgen receptor (AR) is the primary therapeutic target in prostate cancer. While androgen deprivation therapy (ADT) and androgen receptor signaling inhibitors (ARSi) are effective, the disease eventually progresses to fatal castration-resistant prostate cancer (CRPC). That said, little is known about the mechanisms in residual disease that initiates tumor relapse upon ADT/ARSi. Here, we discover a crucial role for TRIM24 in supporting the survival of residual cell clusters primed for tumor relapse in vivo. Consequently, reducing TRIM24 with bifunctional degraders (dTRIM24) significantly delays or even prevents the emergence of CRPC in the context of AR reactivation and lineage plasticity. dTRIM24 not only inhibits AR signaling but also counteracts adaptive pathways engaged by AR inhibition itself, such as STAT3 activation and EMT. Our findings underscore the potential of TRIM24 as an effective and druggable target for preventing prostate cancer progression under AR inhibition. SignificanceDespite advances in targeting AR signaling in prostate cancer, tumor relapse remains a major concern. Here, we provide evidence that more durable responses can be achieved by pharmacologically degrading TRIM24. At the molecular level, TRIM24 degradation inhibits both AR signaling and adaptive pathways that enable tumor relapse.

cancer biology↗

bioRxiv2025

Structure of ATTRv-F64S fibrils isolated from skin tissue of a living patient

Amyloid transthyretin-derived (ATTR) amyloidosis is a degenerative, systemic disease characterized by transthyretin fibril deposition in organs like the heart, kidneys, liver, and skin. We report the first cryo-EM structure of transthyretin fibrils isolated from skin tissue of a living patient carrying a rare genetic mutation (ATTRv F64S). The structure adopts a highly conserved fold previously observed in other ATTR fibrils from different tissues or genetic variants. Mass spectrometry was used to evaluate fibril content and identify post-translational modifications. The structural consistency between ATTR filaments validates non-invasive skin biopsy as a diagnostic tool.

biochemistry↗

bioRxiv2024

High-throughput proteome profiling with low variation in a multi-center study using dia-PASEF

High throughput proteomics is gaining increasing traction as it facilitates screening of large sample cohorts required in clinical research and systems biology studies. Recent developments in mass spectrometry-based proteomics resulted in improved hardware and software providing deep proteome coverage, robustness, and scale accessible to a wide range of laboratories. Here, we benchmark dia-PASEF, a data-independent acquisition scheme that integrates trapped ion mobility with high scan speed, with a high-resolution time-of-flight mass analyzer (timsTOF HT) for the deep proteome analysis of a human cell line applying short 5-minute gradients. To show intra-and interlaboratory reproducibility, we performed a multi-laboratory study including 11 sites. We demonstrate that on average 7,072 protein groups and 99,835 peptides were identified in human chronic myelogenous leukemia cells on the timsTOF HT with low variation. Our results underline that dia-PASEF data acquisition combined with reproducible chromatography enables high robustness and data consistency across instruments and laboratories, which is a prerequisite for translational biomedical insights.

systems biology↗

bioRxiv2024

A patient-derived amyotrophic lateral sclerosis blood-brain barrier cell model reveals focused ultrasound-mediated anti-TDP-43 antibody delivery.

BackgroundAmyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+MB-mediated drug delivery across ALS patients BBB has not yet been reported. Similarly, the effects of FUS+MB on human ALS BBB cells remain unexplored. MethodsHere we established the first FUS+MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+MB bioeffects in vitro. ResultsGenerated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including changes to BBB integrity, permeability and TDP-43 proteinopathy. Our results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with a lack of adverse cellular effects of FUS+MB. This was accompanied by the molecular bioeffects of FUS+MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model. ConclusionsTogether, our study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+MB and provides a fully-human platform for FUS+MB-mediated drug delivery screening on an ALS BBB in vitro model.

neuroscience↗

bioRxiv2024

Crosstalk between the RNA-binding proteins Regnase-1 and -3 shapes mast cell survival and cytokine expression

Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, while both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon mast cell activation, with Tnf being a primary target of both proteins. In mast cells, Regnase-3 directly interacts with Regnase-1 and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses.

immunology↗

bioRxiv2022

Myc controls IL-15-driven expansion and translational machinery of NK cells

Myc is a pleiotropic transcription factor involved in cancer, cell proliferation, and metabolism. Its regulation and function in Natural Killer (NK) cells, which are innate cytotoxic lymphocytes important to control viral infections and cancer, remain poorly defined. Here we show that mice deficient for Myc in NK cells presented a severe reduction in these lymphocytes. Myc was required for NK cell development and expansion in response to the key cytokine interleukin (IL)-15, which induced Myc through transcriptional and posttranslational mechanisms. Mechanistically, Myc ablation in vivo largely impacted NK cells ribosomagenesis, reducing their translation and expansion capacities. Similar results were obtained by inhibiting MYC in human NK cells. Impairing translation by pharmacological intervention phenocopied the consequences of deleting or blocking MYC in vitro. Notably, mice lacking Myc in NK cells exhibited defective anticancer immunity, which reflected their decreased numbers of mature NK cells exerting suboptimal cytotoxic functions. These results indicate that MYC is a central node in NK cells, connecting IL-15 to translational fitness, expansion, and anticancer immunity.

immunology↗

bioRxiv2022

Proteogenomic analysis reveals RNA as an important source for tumor-agnostic neoantigen identification correlating with T-cell infiltration

Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types by combining proteogenomics with phenotypic and functional analyses. By using an optimized computational approach, we discovered a large number of novel tumor-specific and tumor-associated antigens including shared common target candidates. To create a pipeline for the identification of neoantigens in our cohort, we combined deep DNA and RNA sequencing with MS- based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity. In fact, we could detect a broad variety of non-wild type HLA-binding peptides in the majority of patients and confirmed the immunogenicity of 24 neoantigens. Most interestingly, the majority of total and immunogenic neoantigens originated from variants identified in the RNA dataset, illustrating the importance of RNA as a still understudied source of cancer antigens. Moreover, the amount of these mainly RNA-based immunogenic neoantigens correlated positively with overall CD8+ tumor-infiltrating T cells. This study therefore underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates. Statement of significanceThe significance of this study lies not only in the potential of our optimized proteogenomic workflow for the discovery of neoantigens (in particular RNA-derived neoantigens) for clinical application, but sheds light on the entity-agnostic prevalence of HLA class I peptide presentation of RNA processing events to be used for tumor targeting.

cancer biology↗

bioRxiv2022

STAT3 restricts prostate cancer metastasis and antiandrogen resistance by controlling LKB1/CREB signaling pathway

Prostate cancer (PCa) lethality is driven by its progression to a metastatic castration-resistant state, yet the signaling mechanisms underlying metastatic spread remain unknown. Here we show that STAT3 converges with the LKB1/mTORC1 and CREB to control metastatic disease in PCa mouse models. Unexpectedly, STAT3 was found to be upregulated in diabetic PCa patients undergoing metformin therapy with a concomitant reduction in mTORC1 expression. In preclinical mouse models of PCa, genetic ablation or activation of STAT3 had opposing effects on LKB1/AMPK/mTORC1- dependent tumorigenesis. Using genetic and pharmacological approaches, we identified LKB1 as a direct STAT3 target while repressing CREB. Furthermore, PCa patients with high CREB expression had inferior clinical outcome with significantly increased risk of disease and metastatic recurrence. We observe that castration state lowers STAT3 abundance and increases AR and CREB levels, leading to castration-resistant PCa (CRPC). Our findings revealed that STAT3 controls mTORC1 and CREB in metastatic disease, suggesting CREB as a promising target for lethal CRPC.

cancer biology↗

bioRxiv2019

STAT3-dependent systems-level analysis reveals PDK4 as an independent predictor of biochemical recurrence in prostate cancer

Prostate cancer (PCa) has a broad spectrum of clinical behaviour, hence biomarkers are urgently needed for risk stratification. We previously described the protective effect of STAT3 in a prostate cancer mouse model. By utilizing a gene co-expression network in addition to laser microdissected proteomics from human and murine prostate FFPE samples, we describe STAT3-induced downregulation of the TCA cycle/OXPHOS in PCa on transcriptomic and proteomic level. We identify pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging and PSA level. Furthermore, PDK4 expression is causally linked to type 2 diabetes mellitus, which is known to have a protective effect on PCa. We conclude that this effect is related to PDK4 expression and that PDK4 loss could serve as a biomarker for PCa with dismal prognosis.

cancer biology↗