Mucormycosis is an emerging, life-threatening human infection caused by Mucorales fungi1-3. Metabolic disorders uniquely predispose an ever-expanding group of patients to mucormycosis through poorly understood mechanisms1,2,4,5, suggesting that uncharacterized host metabolic effectors may confer protective immunity against this infection. Here we uncover a master regulatory role of albumin in host defence against Mucorales through the modulation of fungal pathogenicity. Our initial studies identified severe hypoalb uminaemia as a prominent metabolic abnormality and an independent biomarker of poor mucormycosis outcome across three distinct cohorts of patients with mucormycosis. Notably, purified albumin selectively inhibits Mucorales growth among a range of pathogens, and albumin-deficient mice display susceptibility specifically to mucormycosis. The antifungal activity of albumin is mediated by the release of bound free fatty acids (FFAs). Albumin prevents FFA oxidation, which otherwise abolishes their antifungal properties, and sera from patients with mucormycosis display high levels of oxidized FFAs. Physiologically, albumin-bound FFAs suppress the expression of key virulence factors by inhibiting protein synthesis, the reby rendering Mucorales avirulent in vivo. Overall, we identify a host defence mechanism that directs the pathogen to suppress its pathogenicity program in response to unfavourable metabolic cues regulated by albumin. These findings have major implications for the pathogenesis and management of mucormycosis.

Aging and DNA damage increase the risk of chronic inflammation and autoimmunity, yet the molecular underpinnings remain unclear. In this study, we uncover a DNA damage-driven mechanism in macrophages that triggers immune autoreactivity. Here, using Er1Lyz2/− mice with a macrophage-specific DNA repair defect in ERCC1−XPF, we demonstrate that monocyte-derived macrophages accumulate DNA damage, activate the immune system, drive polyclonal T cell responses and generate antinuclear autoantibodies. Proteomic and immunopeptidomic analyses reveal a distinct major histocompatibility complex class II (MHC-II) antigen repertoire enriched in nuclear and ribosomal peptides, relying on autophagy for nuclear cargo delivery to MHC-II. Aged macrophages exhibit a similar lysosomal cargo profile, linking autophagy-driven nuclear antigen presentation to immune activation. Notably, inhibiting autophagy in Er1Lyz2/− mice suppresses autoimmune features, pinpointing autophagy-facilitated nuclear antigen processing as a central driver of age-related autoimmunity. These findings establish DNA damage-induced autophagy in macrophages as a pivotal mechanism linking aging to autoimmunity, unveiling potential therapeutic targets to mitigate age-related immune dysregulation. Using a mouse model with a macrophage-specific DNA repair defect, Niotis, Arvanitaki et al. identify DNA damage-induced autophagy in macrophages as a link from aging to autoimmunity.

Human paraoxonase 1 (PON1), an enzyme bound to high-density lipoprotein (HDL), hydrolyzes oxidized lipids and contributes to HDL atheroprotective functions. Decreased serum paraoxonase and arylesterase activities of PON1 have been reported in patients at increased atherosclerosis risk, such as rheumatoid arthritis patients, and associated with arthritis severity and cardiovascular risk. Agents that can modulate PON1 activity and HDL-mediated effects have not been discovered. Aiming to discover chemical tools that enhance PON1 activity, we screened a library of marketed drugs (956 compounds) to identify small molecules that can increase HDL-associated PON1 activity. Screening was performed by a kinetic absorbance assay using human HDL as a source of PON1, and paraoxon and phenyl acetate as substrates to measure paraoxonase and arylesterase activities, respectively. Screening identified the drug dantrolene as a potential PON1 activator, which was confirmed by enzymatic kinetic assays using recombinant wild-type PON1, as well as the PON1[L55M] variant displaying decreased enzyme activity in humans. Furthermore, we used the collagen-induced arthritis (CIA) mouse model to examine the effect of dantrolene on HDL properties and arthritis in vivo. Administration of dantrolene in CIA mice increased paraoxonase and arylesterase activities of PON1, as well as the antioxidant capacity of HDL, and reduced arthritis severity by inhibition of naïve CD4+ T cell differentiation to effector memory cells and generation of Th1 cells. Collectively, our in vitro and in vivo findings indicate using small molecules to enhance HDL-associated PON1 activity is a tractable approach that could lead to novel therapeutics targeting immune responses and atherosclerosis.

Metastastic disease affects up to 50% of colorectal cancer (CRC) patients and is associated with particularly poor outcomes in the presence of the BRAF V600E mutation. Herein, we report a patient with initial diagnosis of stage IIIc CRC, who presented during follow-up (adjuvant phase) with dysphagia, left-sided lagophthalmos and multiple skin nodules. The ensuing work-up revealed disseminated metastatic disease from the primary CRC, which was BRAF V600E-mutated (retrospective tissue analysis), affecting, besides the lungs, multiple uncommon sites, such as the skin and parotid gland. The patient’s rapid disease progression did not allow for any therapeutic interventions. This is only the second report of concomitant metastatic infiltration of the skin and parotid gland by CRC, and the first with a documented molecular background of BRAF V600E mutation. BRAF V600E-mutated CRC can follow an aggressive and often unpredictable clinical course in the metastatic setting that physicians should be aware of, and the molecular profile of the tumor at diagnosis could be useful for comprehensive and timely management.

Mucormycosis is an emerging, life-threatening infection caused by Mucorales fungi with incompletely understood immunopathogenesis. We found that unlike other fungal pathogens, Mucorales trigger coordinated neutrophil clustering (swarming) upon swelling of fungal spores. Neutrophil swarms efficiently eliminate germinating Mucorales via ROS production, upon sensing of β-glucan by complement receptor 3 (CR3). Impaired neutrophil clustering in mucormycosis-predisposing conditions, allows for mucoricin toxin production by germinating Mucorales. Released mucoricin selectively binds to β-glucan on the surface of Mucorales, creating a molecular trap that induces neutrophil apoptosis and swarming disruption, leading to invasive fungal disease. Suppression of mucoricin, either genetically or physiologically by albumin-bound free fatty acids, stabilizes neutrophil clusters leading to abrogation of fungal pathogenicity. Additionally, prophylactic GM-CSF administration prevents mucoricin-induced neutrophil death, and protects mice from mucormycosis. Our work identifies neutrophil swarming as the primary target of mucoricin and provides compelling evidence for therapeutic harnessing of this pathway to improve mucormycosis outcome.

Mucormycosis is an emerging, life-threatening human infection caused by fungi of the order Mucorales. Metabolic disorders uniquely predispose an ever-expanding group of patients to mucormycosis via poorly understood mechanisms. Therefore, it is highly likely that uncharacterized host metabolic effectors confer protective immunity against mucormycosis. Here, we uncover a master regulatory role of albumin in host defense against Mucorales through the modulation of the fungal pathogenicity program. Our initial studies identified severe hypoalbuminemia as a prominent metabolic abnormality and a biomarker of poor outcome in independent cohorts of mucormycosis patients. Strikingly, we found that purified albumin selectively inhibits Mucorales growth among a range of human pathogens, and albumin-deficient mice display susceptibility specifically to mucormycosis. The antifungal activity of albumin is mediated by the release of bound free fatty acids (FFAs). Importantly, albumin prevents FFA oxidation, which results in loss of their antifungal properties. A high degree of FFA oxidation is found in the sera of patients with mucormycosis. Physiologically, albumin-bound FFAs blocks the expression of the mycotoxin mucoricin and renders Mucorales avirulent in vivo. Overall, we discovered a novel host defense mechanism that directs the pathogen to suppress its growth and the expression of virulence factors in response to unfavorable metabolic cues regulated by albumin. These findings have major implications for the pathogenesis and management of mucormycosis.

T-cell prolymphocytic leukemia (T-PLL) presents unique treatment challenges because of its rarity and aggressiveness. Allogeneic hematopoietic stem cell transplantation offers a potentially curative option, but its safety in patients with concurrent invasive fungal infections and solid malignancies remains uncertain. We present a case of a 68-year-old male with T-PLL who developed disseminated cryptococcal disease with prostate involvement and concurrent prostate cancer (PCa). Despite the challenges, successful control of the infection and radical prostatectomy enabled the patient to proceed safely to allogeneic transplantation. The case highlights the importance of vigilance for unusual infections, such as Cryptococcus, in immunocompromised patients presenting with lower urinary tract symptoms. Clinicians should consider the possibility of PCa in this population, particularly in the context of chronic leukemia. Concurrently, the potential association between fungal prostate infections and PCa warrants further investigation.

Background: Several studies have shown that the anti-inflammatory/anti-oxidant properties of HDL are impaired in the inflammatory environment of rheumatic diseases, including rheumatoid arthritis (RA). HMG-CoA reductase inhibitors beside their role in decreasing cholesterol levels also display anti-inflammatory and immune-modulating properties both in vivo and in vitro. Nevertheless, the effects of statin use in RA patients are rather contradictory. Objectives: In the present study, we aimed to assess the effect of established and novel HDL-targeting agents on HDL properties in the context of systemic inflammatory responses. Methods: Screening of an FDA approved drug library for their potency in enhancing or restoring the structure and function of HDL in vitro highlighted that two substances, lovastatin and dantrolene could have a positive effect in regulating inflammation in RA. In this context, we used the collagen-induced arthritis (CIA) model in the DBA/1J mouse strain and we administrated lovastatin, dantrolene or their vehicle therapeutically to study their effect in disease progression. We also utilized the atherosclerosis-prone transgenic mouse model of spontaneous arthritis K/BxAg7 to investigate the effect of a common lipid-lowering drug (simvastatin) in arthritis progression and atherosclerosis development. Results: We found that treatment with lovastatin worsened arthritis phenotype by reducing T regulatory cells (Tregs) in the draining lymph nodes and by increasing infiltration of myeloid cells in the spleen of CIA mice. On the contrary, dantrolene administration improved arthritis severity in the CIA model by reducing macrophage and dendritic cell infiltration of the spleen, and by blocking differentiation of naive CD4+ T cells into effector memory cells. Additionally, we found an increased paraoxonase and arylesterase activity of PON1 and an improved anti-oxidant capacity of HDL in the dantrolene-treated group, which was in line with our in vitro findings. Regarding the effect of simvastatin in the K/BxAg7 model, we found that inhibition of cholesterol biosynthesis blocked CD8+ T cell differentiation into effector memory cells and increased Tregs in the spleen. Additionally, simvastatin restored the anti-oxidant capacity of HDL in mice receiving atherogenic diet, although it had no beneficial impact on arthritis progression. Conclusion: Altogether, even though blockade of cholesterol biosynthesis did not seem to have a clear role in arthritis severity in both mouse models of experimental arthritis herein, the administration of dantrolene moderated arthritis by altering key pathogenic cell subsets and improved PON1 activity in the CIA model. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.

Adjuvant Temozolomide is considered the front-line Glioblastoma chemotherapeutic treatment; yet not all patients respond. Latest trends in clinical trials usually refer to Doxorubicin; yet it can lead to severe side-effects if administered in high doses. While Glioblastoma prognosis remains poor, little is known about the combination of the two chemotherapeutics. Patient-derived spheroids were generated and treated with a range of Temozolomide/Doxorubicin concentrations either as monotherapy or in combination. Optical microscopy was used to monitor the growth pattern and cell death. Based on the monotherapy experiments, we developed a probabilistic mathematical framework in order to describe the drug-induced effect at the single-cell level and simulate drug doses in combination assuming probabilistic independence. Doxorubicin was found to be effective in doses even four orders of magnitude less than Temozolomide in monotherapy. The combination therapy doses tested in vitro were able to lead to irreversible growth inhibition at doses where monotherapy resulted in relapse. In our simulations, we assumed both drugs are anti-mitotic; Temozolomide has a growth-arrest effect, while Doxorubicin is able to cumulatively cause necrosis. Interestingly, under no mechanistic synergy assumption, the in silico predictions underestimate the in vitro results. In silico models allow the exploration of a variety of potential underlying hypotheses. The simulated-biological discrepancy at certain doses indicates a supra-additive response when both drugs are combined. Our results suggest a Temozolomide–Doxorubicin dual chemotherapeutic scheme to both disable proliferation and increase cytotoxicity against Glioblastoma.

Supplementary Figure 2 from Sonic Hedgehog Signaling Pathway Is Activated in ALK-Positive Anaplastic Large Cell Lymphoma

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1(+) group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1(+) NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

Supplementary Figure 3 from Sonic Hedgehog Signaling Pathway Is Activated in ALK-Positive Anaplastic Large Cell Lymphoma

Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with the suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed the induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth, and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.

Supplementary Figure Legends 1-3 from Sonic Hedgehog Signaling Pathway Is Activated in ALK-Positive Anaplastic Large Cell Lymphoma

BACKGROUND Brain metastasis of papillary thyroid cancer (PTC) is rare. Treatment of these patients is challenging due to the lack of specific guidelines. Early diagnosis is accompanied by immediate treatment and less morbidity. Total resection of brain lesions may be unattainable when they include infiltration of eloquent areas. This report is of an 81-year-old man who had undergone total thyroidectomy for goiter in the past and presented with metastatic papillary thyroid carcinoma (PTC) to the neck after a gap of 16 years. After two years, the patient developed a solitary cystic brain PTC metastasis associated with raised thyroglobulin (Tg) inside the cystic lesion aspirated during brain surgery. CASE REPORT An 81-year-old male patient was admitted for a space-occupying brain lesion in the right frontal lobe. The patient's history included metastatic disease of PTC to the neck with cervical lymph node metastasis and local recurrence after surgery and radioactive iodine-131 treatment. The patient underwent craniotomy and removal of the lesion. The aspirated fluid was sent for cytological examination and measurement of Tg levels, which were interestingly high. Pathology of the brain lesion revealed infiltration of brain parenchyma from a metastatic lesion characterized by eosinophilic cells with irregular contours forming grooves, resulting in cytoplasmic pseudo-inclusions, an oncotic variant of PTC. CONCLUSIONS This report has shown that residual tissue may be present following total thyroidectomy and may be the origin of PTC with metastasis to the brain. The patient in this study suffered from a brain lesion that could be excised. However, aspiration of cystic compartments could provide a rapid diagnosis in patients with non-removable brain lesions.

Supplementary Figure 1 from Sonic Hedgehog Signaling Pathway Is Activated in ALK-Positive Anaplastic Large Cell Lymphoma

In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and proteomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin-3a, on three lymphoma cell models following p53 activation. Our analysis revealed a system-wide nutlin-3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activation effects, involving transcription, translation, or degradation of protein components of pathways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and glycolysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mitochondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin-3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis.

Background: The JAB1 (cJun activation domain binding protein1), initially discovered as a cJun coactivator, represents the fifth component of an evolutionary highly conserved 8 subunit protein complex named COP9 signalosome (CSN5). Accumulating evidence suggests that the CSN5/JAB1 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via downregulation of CDK inhibitors. Targeting CSN5/JAB1 activity in cancer is now possible since a novel inhibitor has been developed for clinical use (CSN5i-3, Novartis). The potential role of CSN5/JAB1 in modulation of anti-tumor responses in cHL is unknown to date. Methods: The study group included 118 previously untreated cHL patients with available tissue and clinical data. Expression of CSN5/JAB1 was assessed by immunohistochemistry and a previously validated monoclonal antibody. The in vitro system included 6 cHL cell lines (MDA-V, L1236, L428, L540, KMH2, HDLM2). Expression of proteins were analysed by Western blot and gene expression (mRNA) of type 1 interferons (IFNs), including IFN-β, CXCL10 and IFN-γ, with RT-qPCR. The cHL cell lines were treated with the CSN5i-3 at different concentrations. Silencing of CSN5/JAB1 and NFkB (p65) gene was performed using transient transfection with siRNA constructs. Results: Using a 10% cutoff, CSN5/JAB1 was positive in the neoplastic HRS cells of cHL in 106 of 118 (90%) patients with a predominantly nuclear and weaker cytoplasmic pattern. Treatment of cHL cell lines with increasing concentrations of CSN5i-3 resulted in significant decrease in the cell growth and viability associated with upregulation of the CDK inhibitors p21, p27 and p57. Similarly, CSN5/JAB1 gene silencing resulted in decreased cell growth associated with increased expression of similar cell cycle inhibiting proteins. Inhibition of CSN5/JAB1 activity resulted in significantly increased gene expression of IFN-β, CXCL10 and IFN-γ at a variable degree among the cHL cell lines. Similarly, knocking down CSN5/JAB1 gene led to upregulation of type 1 IFNs. Silencing NFkB gene resulted in decreased levels of CSN5/JAB1 protein suggesting positive regulation of CSN5/JAB1 by NFkB. Conclusion: The CSN5/JAB1 is overexpressed in HRS cells of most cHL patients, promotes cell growth through cell cycle control and downregulates gene expression of type 1 IFNs in vitro. These functions are sufficiently inhibited by the CSN5i-3, designed for clinical use, in preclinical models of cHL.

Background: The CSN5/Jab1 (cJun activation domain-binding protein 1), initially discovered as a cJun coactivator, represents the fifth component of an evolutionary highly conserved 8 subunit protein complex, named COP9 signalosome (CSN5). Accumulating evidence suggests that the CSN5/Jab1 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via CDK inhibitors. We have previously shown that CSN5/Jab1 protein is overexpressed in T-cell lymphomas with the highest frequency of positive tumors being observed in both ALK+ and ALK- anaplastic large cell lymphomas (ALCL). Targeting CSN5/Jab1 activity in cancer is now possible since a novel inhibitor, namely CSN5i-3 (Novartis), has been developed for clinical use. The potential role of CSN5/Jab1 in modulation of anti-tumor immune responses in cancer, and particularly in ALCL, is unknown to date. Aims: To investigate the effects of the selective CSN5i-3 inhibitor in cell growth and type-I interferon - associated anti-tumor immune responses in ALCL. Methods: The ALCL in vitro system included 5 cell lines (3 ALK+ and 2 ALK- ALCL cell lines). Cell growth and viability were assessed by trypan blue and MTT assays. The effects of the CSN5i-3 on G1-S cell cycle progression were assessed by BrdU incorporation assay and on apoptosis by Annexin V/AAD staining and flow cytometry. Expression of various cell cycle regulating proteins at baseline and experimental conditions were analysed by Westem blot analysis. Gene expression of type I interferons, including interferonbeta (IFN-β), CXCL10 and interferon gamma (IFN-y), as well as, STING and a control gene (GAPDH) at the RNA level, were analysed with qRT-PCR. The ALCL cell lines were treated with the specific CSN5i-3 inhibitor at different concentrations. Silencing of the CSN5/Jab1 gene was performed using transient transfection with specific siRNA construct using the Amaxa Nucleofector System (Lonza). Results: Treatment of ALCL cell lines with increasing concentrations of CSN5i-3 resulted in decreased protein levels of CSN5/Jab1, which was associated with significantly decreased cell growth but slight increase in apoptosis (10-15%). The effects on cell growth linked to downregulation of cell cycle progression were associated with upregulation of the CDK inhibitors p21, p27 and p57. Similarly, CSN5/Jab1 gene silencing resulted in decreased cell growth associated with increased expression of similar cell cycle inhibiting proteins. In addition, inhibition of CSN5/Jab1 activity by CSN5i-3 resulted in increased IFN-β and IFN-y mRNA levels in ALK+ ALCL cells, and CXCL10, IFN-y and STING mRNA levels in ALK- ALCL cells. Similarly, knocking down CSN5/Jab1 gene by transient transfection with siRNA led to upregulation of type-I interferons, particularly IFN-β and CXCL10 in ALCL, suggesting that the effects of CSN5i-3 can be attributed to specific inhibition of CSN5/Jab1 activity. Summary/Conclusion: The novel inhibitor CSN5i-3 sufficiently downregulates expression of CSN5/Jab1 oncogene, inhibits cell cycle progression and contributes to type-I interferon - associated anti-tumor immune response in ALK+ and ALK- ALCL. These preclinical findings suggest a potential role of CSN5i-3 in molecular therapeutics and immunotherapy in T-cell lymphomas.