Periodic Reporting for period 4 - InflaPML (Promyelocytic leukemia protein (PML) outside the tumor: a new player in the control of inflammation)
Periodo di rendicontazione: 2024-12-01 al 2025-05-31
The innate immune response plays a crucial yet paradoxical role: while essential for defending the body against external pathogens, its inappropriate activation—such as after post-ischemic reperfusion or in neurodegenerative diseases (NDDs)—can exacerbate disease progression and limit therapeutic efficacy. NDDs affect millions of EU citizens and, with the aging population, their prevalence is increasing. Yet, effective therapeutic strategies remain limited. These disorders are consistently characterized by elevated levels of inflammation, for which current scientific and pharmacological approaches provide only partial solutions.
This project investigated the role of the Promyelocytic Leukemia Protein (PML) in modulating innate immune responses in NDDs. We hypothesized that loss or delocalization of PML exacerbates disease by enhancing the release of the pro-inflammatory cytokine IL-1β, rather than mitigating inflammation. IL-1β production and extracellular release are regulated by two key molecular players: NLRP3 and P2X7. Together with PML, they form a functional axis located at the endoplasmic reticulum–mitochondria contact sites (ER/MAMs), which emerged as a critical hub for sterile inflammation across several nervous system disorders—including ischemic stroke (IS), multiple sclerosis (MS), and status epilepticus (SE).
During the project, we demonstrated that PML physically and functionally interacts with NLRP3 and P2X7 at MAMs, acting as a regulatory brake on inflammasome activation. We observed that in pathological conditions, PML is delocalized or reduced in expression, leading to a loss of control over NLRP3 activation via P2X7 and contributing to a hyperinflammatory microenvironment that sustains disease progression.
We further confirmed that this PML–NLRP3–P2X7 axis represents a shared inflammatory mechanism across seemingly distinct NDDs. Through in vitro and in vivo studies, we elucidated how PML modulates the activity of key inflammatory mediators and influences IL-1β release. In addition, we explored the potential of using IL-1β levels, PML expression, and its subcellular localization as biomarkers for disease stratification and monitoring.
Finally, the project laid the groundwork for innovative preclinical strategies to modulate this axis, including the development of novel NLRP3-targeting compounds. The findings generated a strong foundation for future therapeutic and diagnostic research, with potential implications for personalized treatment approaches in neuroinflammation and broader neurodegenerative contexts.
This project investigated the role of the Promyelocytic Leukemia Protein (PML) in modulating innate immune responses in NDDs. We hypothesized that loss or delocalization of PML exacerbates disease by enhancing the release of the pro-inflammatory cytokine IL-1β, rather than mitigating inflammation. IL-1β production and extracellular release are regulated by two key molecular players: NLRP3 and P2X7. Together with PML, they form a functional axis located at the endoplasmic reticulum–mitochondria contact sites (ER/MAMs), which emerged as a critical hub for sterile inflammation across several nervous system disorders—including ischemic stroke (IS), multiple sclerosis (MS), and status epilepticus (SE).
During the project, we demonstrated that PML physically and functionally interacts with NLRP3 and P2X7 at MAMs, acting as a regulatory brake on inflammasome activation. We observed that in pathological conditions, PML is delocalized or reduced in expression, leading to a loss of control over NLRP3 activation via P2X7 and contributing to a hyperinflammatory microenvironment that sustains disease progression.
We further confirmed that this PML–NLRP3–P2X7 axis represents a shared inflammatory mechanism across seemingly distinct NDDs. Through in vitro and in vivo studies, we elucidated how PML modulates the activity of key inflammatory mediators and influences IL-1β release. In addition, we explored the potential of using IL-1β levels, PML expression, and its subcellular localization as biomarkers for disease stratification and monitoring.
Finally, the project laid the groundwork for innovative preclinical strategies to modulate this axis, including the development of novel NLRP3-targeting compounds. The findings generated a strong foundation for future therapeutic and diagnostic research, with potential implications for personalized treatment approaches in neuroinflammation and broader neurodegenerative contexts.
To investigate the role of PML in neuroinflammation, the project was structured around four main goals:
- Characterize the PML–NLRP3–P2X7 axis in neuroinflammatory processes;
- Define the novel function of cytoplasmic PML in modulating immune responses in SE, IS, and MS;
- Establish IL-1β and PML as potential biomarkers of neuroinflammatory diseases;
- Design, synthesize, and evaluate novel compounds capable of attenuating the PML-dependent immune response.
To elucidate the molecular mechanisms by which PML regulates IL-1β release and NLRP3 activity, we examined the intracellular localization of NLRP3 and P2X7 in wild-type (WT) and PML-knockout (KO) models, both at baseline and under inflammasome activation. NLRP3 predominantly localized at the endoplasmic reticulum/mitochondria-associated membranes (ER/MAMs), with increased accumulation in PML-deficient cells. Unexpectedly, P2X7 was also found at MAMs, and its presence was enhanced in the absence of PML. A stronger NLRP3–P2X7 interaction was detected in PML-KO cells, suggesting PML normally limits their association.
Biochemical analyses confirmed the co-localization and interaction of PML, NLRP3, and P2X7 at the ER/MAM interface, supporting the existence of a functional tripartite complex regulated by PML to control immune signaling at the subcellular level.
To further explore PML’s role in CNS immune regulation, we developed in vitro and in vivo models of multiple sclerosis (MS), status epilepticus (SE), and ischemic stroke (IS). In all models, PML deficiency led to markedly increased neuroinflammation, confirming its role as a negative regulator of pathological immune activation in the brain.
We synthesized a new series of aryl sulfonamide derivatives (ASDs) targeting NLRP3. In vitro assays on macrophages identified three lead compounds capable of significantly reducing IL-1β release. Molecular docking simulations, based on cryo-EM structures of NLRP3, confirmed a specific and stable binding mode.
Additionally, we developed novel proteolysis-targeting chimeras (PROTACs) that not only inhibited IL-1β release but also induced selective degradation of NLRP3. These compounds showed high potency at nanomolar concentrations, significantly outperforming traditional inflammasome inhibitors. Preliminary in vivo studies confirmed their efficacy, making them strong candidates for next-generation treatments of inflammasome-driven neuroinflammation.
As part of our biomarker investigations, IL-1β was assessed as a prognostic and predictive marker across neuroinflammatory conditions:
- In MS, IL-1β serum levels were monitored longitudinally from pre-diagnosis to one year post-treatment, showing a gradual decline and supporting its use in disease monitoring;
- In SE, in collaboration with the IRCCS Besta Institute (Milan), IL-1β levels were elevated in epileptogenic brain regions, where PML was also found to be delocalized;
- In IS, in collaboration with the Neurology Department at Ferrara Hospital, a prospective study was launched to assess IL-1β serum levels during acute and post-acute phases.
The project outcomes were disseminated through:
- Three peer-reviewed publications in PNAS, Cell Death & Differentiation, and Biomedicine & Pharmacotherapy, along with several reviews;
- Multiple presentations at international scientific conferences;
- The filing of three national patents on novel NLRP3 inhibitors, now entering the European phase;
- Submission of Proof of Concept proposals awarded the Seal of Excellence by the European Commission.
- Characterize the PML–NLRP3–P2X7 axis in neuroinflammatory processes;
- Define the novel function of cytoplasmic PML in modulating immune responses in SE, IS, and MS;
- Establish IL-1β and PML as potential biomarkers of neuroinflammatory diseases;
- Design, synthesize, and evaluate novel compounds capable of attenuating the PML-dependent immune response.
To elucidate the molecular mechanisms by which PML regulates IL-1β release and NLRP3 activity, we examined the intracellular localization of NLRP3 and P2X7 in wild-type (WT) and PML-knockout (KO) models, both at baseline and under inflammasome activation. NLRP3 predominantly localized at the endoplasmic reticulum/mitochondria-associated membranes (ER/MAMs), with increased accumulation in PML-deficient cells. Unexpectedly, P2X7 was also found at MAMs, and its presence was enhanced in the absence of PML. A stronger NLRP3–P2X7 interaction was detected in PML-KO cells, suggesting PML normally limits their association.
Biochemical analyses confirmed the co-localization and interaction of PML, NLRP3, and P2X7 at the ER/MAM interface, supporting the existence of a functional tripartite complex regulated by PML to control immune signaling at the subcellular level.
To further explore PML’s role in CNS immune regulation, we developed in vitro and in vivo models of multiple sclerosis (MS), status epilepticus (SE), and ischemic stroke (IS). In all models, PML deficiency led to markedly increased neuroinflammation, confirming its role as a negative regulator of pathological immune activation in the brain.
We synthesized a new series of aryl sulfonamide derivatives (ASDs) targeting NLRP3. In vitro assays on macrophages identified three lead compounds capable of significantly reducing IL-1β release. Molecular docking simulations, based on cryo-EM structures of NLRP3, confirmed a specific and stable binding mode.
Additionally, we developed novel proteolysis-targeting chimeras (PROTACs) that not only inhibited IL-1β release but also induced selective degradation of NLRP3. These compounds showed high potency at nanomolar concentrations, significantly outperforming traditional inflammasome inhibitors. Preliminary in vivo studies confirmed their efficacy, making them strong candidates for next-generation treatments of inflammasome-driven neuroinflammation.
As part of our biomarker investigations, IL-1β was assessed as a prognostic and predictive marker across neuroinflammatory conditions:
- In MS, IL-1β serum levels were monitored longitudinally from pre-diagnosis to one year post-treatment, showing a gradual decline and supporting its use in disease monitoring;
- In SE, in collaboration with the IRCCS Besta Institute (Milan), IL-1β levels were elevated in epileptogenic brain regions, where PML was also found to be delocalized;
- In IS, in collaboration with the Neurology Department at Ferrara Hospital, a prospective study was launched to assess IL-1β serum levels during acute and post-acute phases.
The project outcomes were disseminated through:
- Three peer-reviewed publications in PNAS, Cell Death & Differentiation, and Biomedicine & Pharmacotherapy, along with several reviews;
- Multiple presentations at international scientific conferences;
- The filing of three national patents on novel NLRP3 inhibitors, now entering the European phase;
- Submission of Proof of Concept proposals awarded the Seal of Excellence by the European Commission.
This project has revealed a previously uncharacterized mechanism of NLRP3 inflammasome regulation at mitochondria-associated membranes (MAMs), where PML functions as a negative regulator of the NLRP3–P2X7 axis. Loss of PML promotes excessive interaction between NLRP3 and P2X7 at MAMs, resulting in exaggerated IL-1β release and a sustained inflammatory response. These findings define a novel PML–P2X7–NLRP3 signaling axis, opening new avenues for therapeutic intervention in neuroinflammatory diseases.
To develop NLRP3-targeted therapies, we pursued two complementary strategies:
The synthesis of modified aryl sulfonamide derivatives (ASDs), based on MCC950, with enhanced efficacy and reduced hepatotoxicity;
The generation of a photoaffinity probe derived from velutone F to identify its binding site on NLRP3, refining our understanding of its mechanism of action.
By the end of the project, we successfully:
- Defined the extranuclear role of PML in the context of neurodegeneration;
- Established IL-1β and/or PML variants as potential biomarkers for status epilepticus (SE) and multiple sclerosis (MS);
- Investigated their prognostic value in stroke, providing a basis for potential patient stratification strategies in clinical settings.
To develop NLRP3-targeted therapies, we pursued two complementary strategies:
The synthesis of modified aryl sulfonamide derivatives (ASDs), based on MCC950, with enhanced efficacy and reduced hepatotoxicity;
The generation of a photoaffinity probe derived from velutone F to identify its binding site on NLRP3, refining our understanding of its mechanism of action.
By the end of the project, we successfully:
- Defined the extranuclear role of PML in the context of neurodegeneration;
- Established IL-1β and/or PML variants as potential biomarkers for status epilepticus (SE) and multiple sclerosis (MS);
- Investigated their prognostic value in stroke, providing a basis for potential patient stratification strategies in clinical settings.