WITHDRAWN: Oncogenic KRAS G12D extrinsically induces an immunosuppressive microenvironment in lung adenocarcinoma.

This manuscript has been withdrawn by the authors due to a dispute over co-first authorship that is currently being arbitrated by the medical school at our institution. Therefore, the authors do not wish this work to be cited as reference for the project. Upon completion of the arbitration process, we will take steps to revert the current withdrawn status. If you have any questions, please contact the corresponding author.

Targeting DNA Repair and Survival Signaling in Diffuse Intrinsic Pontine Gliomas to Prevent Tumor Recurrence.

Therapeutic resistance remains a major obstacle to successful clinical management of diffuse intrinsic pontine glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. On the basis of these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrated that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Because radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of Ataxia Telangiectasia Mutated to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease.

Modeling Molecular Pathogenesis of Idiopathic Pulmonary Fibrosis-Associated Lung Cancer in Mice.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive, often fatal loss of lung function due to overactive collagen production and tissue scarring. Patients with IPF have a sevenfold-increased risk of developing lung cancer. The COVID-19 pandemic has increased the number of patients with lung diseases, and infection can worsen prognoses for those with chronic lung diseases and disease-associated cancer. Understanding the molecular pathogenesis of IPF-associated lung cancer is imperative for identifying diagnostic biomarkers and targeted therapies that will facilitate prevention of IPF and progression to lung cancer. To understand how IPF-associated fibroblast activation, matrix remodeling, epithelial-to-mesenchymal transition (EMT), and immune modulation influences lung cancer predisposition, we developed a mouse model to recapitulate the molecular pathogenesis of pulmonary fibrosis-associated lung cancer using the bleomycin and Lewis lung carcinoma models. We demonstrate that development of pulmonary fibrosis-associated lung cancer is likely linked to increased abundance of tumor-associated macrophages and a unique gene signature that supports an immune-suppressive microenvironment through secreted factors. Not surprisingly, preexisting fibrosis provides a pre-metastatic niche and results in augmented tumor growth, and tumors associated with bleomycin-induced fibrosis are characterized by a dramatic loss of cytokeratin expression, indicative of EMT. IMPLICATIONS: This characterization of tumors associated with lung diseases provides new therapeutic targets that may aid in the development of treatment paradigms for lung cancer patients with preexisting pulmonary diseases.

Top-Down Proteomics of Human Saliva, Analyzed with Logistic Regression and Machine Learning Methods, Reveal Molecular Signatures of Ovarian Cancer.

Ovarian cancer (OC) is the most lethal of all gynecological cancers. Due to vague symptoms, OC is mostly detected at advanced stages, with a 5-year survival rate (SR) of only 30%; diagnosis at stage I increases the 5-year SR to 90%, suggesting that early diagnosis is essential to cure OC. Currently, the clinical need for an early, reliable diagnostic test for OC screening remains unmet; indeed, screening is not even recommended for healthy women with no familial history of OC for fear of post-screening adverse events. Salivary diagnostics is considered a major resource for diagnostics of the future. In this work, we searched for OC biomarkers (BMs) by comparing saliva samples of patients with various stages of OC, breast cancer (BC) patients, and healthy subjects using an unbiased, high-throughput proteomics approach. We analyzed the results using both logistic regression (LR) and machine learning (ML) for pattern analysis and variable selection to highlight molecular signatures for OC and BC diagnosis and possibly re-classification. Here, we show that saliva is an informative test fluid for an unbiased proteomic search of candidate BMs for identifying OC patients. Although we were not able to fully exploit the potential of ML methods due to the small sample size of our study, LR and ML provided patterns of candidate BMs that are now available for further validation analysis in the relevant population and for biochemical identification.

Histone H3 K27M-mediated regulation of cancer cell stemness and differentiation in diffuse midline glioma.

Therapeutic resistance remains a major obstacle to preventing progression of H3K27M-altered Diffuse Midline Glioma (DMG). Resistance is driven in part by ALDH-positive cancer stem cells (CSC), with high ALDH1A3 expression observed in H3K27M-mutant DMG biopsies. We hypothesized that ALDH-mediated stemness and resistance may in part be driven by the oncohistone itself. Upon deletion of H3K27M, ALDH1A3 expression decreased dramatically and was accompanied by a gain in astrocytic marker expression and a loss of neurosphere forming potential, indicative of differentiation. Here we show that the oncohistone regulates histone acetylation through ALDH1A3 in a Wnt-dependent manner and that loss of H3K27M expression results in sensitization of DMGs to radiotherapy. The observed elevated Wnt signaling in H3K27M-altered DMG likely stems from a dramatic suppression of mRNA and protein expression of the Wnt inhibitor EYA4 driven by the oncohistone. Thus, our findings identify EYA4 as a bona fide tumor suppressor in DMG that upon suppression, results in aberrant Wnt signaling to orchestrate stemness and differentiation. Future studies will explore whether overexpression of EYA4 in DMG can impede growth and invasion. In summary, we have gained mechanistic insight into H3K27M-mediated regulation of cancer stemness and differentiation, which provides rationale for exploring new therapeutic targets for DMG.

Increasing cell culture density during a developmental window prevents fated rod precursors derailment toward hybrid rod-glia cells.

In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminally-dividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.

Optimal low-intensity pulsed ultrasound stimulation for promoting anti-inflammatory effects in macrophages.

In this paper, we stimulated M1-like macrophages (obtained from U937 cells) with low-intensity pulsed ultrasound (LIPUS) to lower pro-inflammatory cytokine production. A systematic screening of different frequencies, intensities, duty cycles, and exposure times was performed. The optimal stimulation conditions leading to a marked decrease in the release of inflammatory cytokines were determined to be 38 kHz, 250 mW/cm2, 20%, and 90 min, respectively. Using these parameters, we verified that up to 72 h LIPUS did not affect cell viability, resulting in an increase in metabolic activity and in a reduction of reactive oxygen species (ROS) production. Moreover, we found that two mechanosensitive ion channels (PIEZO1 and TRPV1) were involved in the LIPUS-mediated cytokine release modulation. We also assessed the role of the nuclear factor κB (NF-κB) signaling pathway and observed an enhancement of actin polymerization. Finally, transcriptomic data suggested that the bioeffects of LIPUS treatment occur through the modulation of p38 MAPK signaling pathway.

Predicting potentially pathogenic effects of hRPE65 missense mutations: a computational strategy based on molecular dynamics simulations.

The human retinal pigment epithelium-specific 65-kDa protein (hRPE65) plays a crucial role within the retinoid visual cycle and several mutations affecting either its expression level or its enzymatic function are associated with inherited retinal diseases such as Retinitis Pigmentosa. The gene therapy product voretigene neparvovec (Luxturna) has been recently approved for treating hereditary retinal dystrophies; however, the treatment is currently accessible only to patients presenting confirmed biallelic mutations that severely impair hRPE65 function, and many reported hRPE65 missense mutations lack sufficient evidences for proving their pathogenicity. In this context, we developed a computational approach aimed at evaluating the potential pathogenic effect of hRPE65 missense variants located on the dimerisation domain of the protein. The protocol evaluates how mutations may affect folding and conformation stability of this protein region, potentially helping clinicians to evaluate the eligibility for gene therapy of patients diagnosed with this type of hRPE65 variant of uncertain significance.

Sclerosing Paragangliomas: Correlations of Histological Features with Patients' Genotype and Vesicular Monoamine Transporter Expression.

Paragangliomas and pheochromocytomas are rare neuroendocrine tumors, carrying a germ-line mutation in 40% patients. Sclerosis is a rare histological feature in these tumors. We investigated the possible correlations between histological findings, first sclerosis, immunoreactivity for vesicular catecholamine transporters (VMAT1/VMAT2) and patients' genotype in a consecutive series of 57 tumors (30 paragangliomas and 27 pheochromocytomas) from 55 patients. The M-GAPP grading system, sclerosis (0-3 scale) and VMAT1/VMAT2 (0-6 scale) immunoreactivity scores were assessed. Germ-line mutations of Succinate Dehydrogenase genes, RET proto-oncogene and Von Hippel Lindau tumor suppressor gene were searched. A germ-line mutation was found in 25/55 (45.5%) patients, mainly with paraganglioma (N = 14/30, 46,66%). Significant (score ≥ 2) tumor sclerosis was found in 9 (16.1%) tumors, i.e., 7 paragangliomas and 2 pheochromocytomas, most of them (8/9) from patients with a germ-line mutation. M-GAPP score was higher in the mutation status (in 76% of patients involving the SDHx genes, in 12% the RET gene and in the remaining 12% the VHL gene) and in tumors with sclerosis (p < 0.05). Spearman's rank correlation showed a strong correlation of germ-line mutations with M-GAPP (p < 0.0001) and sclerosis (p = 0.0027) scores; a significant correlation was also found between sclerosis and M-GAPP scores (p = 0.029). VMAT1 expression was higher in paragangliomas than in pheochromocytomas (p = 0.0006), the highest scores being more frequent in mutation-bearing patients' tumors (p < 0.01). VMAT2 was highly expressed in all but two negative tumors. Sclerosis and VMAT1 expression were higher in paragangliomas than in pheochromocytomas; tumor sclerosis, M-GAPP and VMAT1 scores were associated to germ-line mutations. Sclerosis might represent a histological marker of tumor susceptibility, prompting to genetic investigations in paragangliomas.

Enriching the Arsenal of Pharmacological Tools against MICAL2.

Molecule interacting with CasL 2 (MICAL2), a cytoskeleton dynamics regulator, are strongly expressed in several human cancer types, especially at the invasive front, in metastasizing cancer cells and in the neo-angiogenic vasculature. Although a plethora of data exist and stress a growing relevance of MICAL2 to human cancer, it is worth noting that only one small-molecule inhibitor, named CCG-1423 (1), is known to date. Herein, with the aim to develop novel MICAL2 inhibitors, starting from CCG-1423 (1), a small library of new compounds was synthetized and biologically evaluated on human dermal microvascular endothelial cells (HMEC-1) and on renal cell adenocarcinoma (786-O) cells. Among the novel compounds, 10 and 7 gave interesting results in terms of reduction in cell proliferation and/or motility, whereas no effects were observed in MICAL2-knocked down cells. Aside from the interesting biological activities, this work provides the first structure-activity relationships (SARs) of CCG-1423 (1), thus providing precious information for the discovery of new MICAL2 inhibitors.

Microgravity and space radiation inhibit autophagy in human capillary endothelial cells, through either opposite or synergistic effects on specific molecular pathways.

Microgravity and space radiation (SR) are two highly influential factors affecting humans in space flight (SF). Many health problems reported by astronauts derive from endothelial dysfunction and impaired homeostasis. Here, we describe the adaptive response of human, capillary endothelial cells to SF. Reference samples on the ground and at 1g onboard permitted discrimination between the contribution of microgravity and SR within the combined responses to SF. Cell softening and reduced motility occurred in SF cells, with a loss of actin stress fibers and a broader distribution of microtubules and intermediate filaments within the cytoplasm than in control cells. Furthermore, in space the number of primary cilia per cell increased and DNA repair mechanisms were found to be activated. Transcriptomics revealed the opposing effects of microgravity from SR for specific molecular pathways: SR, unlike microgravity, stimulated pathways for endothelial activation, such as hypoxia and inflammation, DNA repair and apoptosis, inhibiting autophagic flux and promoting an aged-like phenotype. Conversely, microgravity, unlike SR, activated pathways for metabolism and a pro-proliferative phenotype. Therefore, we suggest microgravity and SR should be considered separately to tailor effective countermeasures to protect astronauts' health.

Inducible Pluripotent Stem Cells to Model and Treat Inherited Degenerative Diseases of the Outer Retina: 3D-Organoids Limitations and Bioengineering Solutions.

Inherited retinal degenerations (IRD) affecting either photoreceptors or pigment epithelial cells cause progressive visual loss and severe disability, up to complete blindness. Retinal organoids (ROs) technologies opened up the development of human inducible pluripotent stem cells (hiPSC) for disease modeling and replacement therapies. However, hiPSC-derived ROs applications to IRD presently display limited maturation and functionality, with most photoreceptors lacking well-developed outer segments (OS) and light responsiveness comparable to their adult retinal counterparts. In this review, we address for the first time the microenvironment where OS mature, i.e., the subretinal space (SRS), and discuss SRS role in photoreceptors metabolic reprogramming required for OS generation. We also address bioengineering issues to improve culture systems proficiency to promote OS maturation in hiPSC-derived ROs. This issue is crucial, as satisfying the demanding metabolic needs of photoreceptors may unleash hiPSC-derived ROs full potential for disease modeling, drug development, and replacement therapies.

HCN1 channels: A versatile tool for signal processing by primary sensory neurons.

Most primary sensory neurons (PSNs) generate a slowly-activating inward current in response to membrane hyperpolarization (Ih) and express HCN1 along with additional isoforms coding for hyperpolarization-activated channels (HCN). Changes in HCN expression may affect the excitability and firing patterns of PSNs, but retinal and inner ear PSNs do not fire action potentials, suggesting HCN channel roles may extend beyond excitability and cell firing control. In patients taking Ih blockers, photopsia triggered in response to abrupt changes in luminance correlates with impaired visual signal processing via parallel rod and cone pathways. Furthermore, in a mouse model of inherited retinal degeneration, HCN blockers or Hcn1 genetic ablation may worsen photoreceptors' demise. PSN's use of HCN channels to adjust either their firing rate or process signals generated by sensory transduction in non-spiking PSNs indicates HCN1 channels as a versatile tool with a novel role in sensory processing beyond firing control.

Monoacylglycerol lipase (MAGL) inhibitors based on a diphenylsulfide-benzoylpiperidine scaffold.

Monoacylglycerol lipase (MAGL) is an enzyme belonging to the endocannabinoid system that mainly metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG). Numerous studies have shown the involvement of this enzyme in various pathological conditions such as pain, cancer progression, Parkinson's and Alzheimer's disease, thus encouraging the development of new MAGL modulators. In this context, we developed new diphenylsulfide-benzoylpiperidine derivatives characterized by a high enzymatic MAGL inhibition activity in the low nanomolar range, a reversible mechanism of action and selectivity. The three most active compounds (15-17) induced an appreciable inhibition of cell viability in a panel of nine cancer cell lines, with IC50 values ranging between 0.32 and 10 µM, thus highlighting their potential as novel anticancer agents.

MICAL2 is essential for myogenic lineage commitment.

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

Hypergravity Activates a Pro-Angiogenic Homeostatic Response by Human Capillary Endothelial Cells.

Capillary endothelial cells are responsible for homeostatic responses to organismic and environmental stimulations. When malfunctioning, they may cause disease. Exposure to microgravity is known to have negative effects on astronauts' physiology, the endothelium being a particularly sensitive organ. Microgravity-related dysfunctions are striking similar to the consequences of sedentary life, bed rest, and ageing on Earth. Among different countermeasures implemented to minimize the effects of microgravity, a promising one is artificial gravity. We examined the effects of hypergravity on human microvascular endothelial cells of dermal capillary origin (HMEC-1) treated at 4 g for 15 min, and at 20 g for 15 min, 3 and 6 h. We evaluated cell morphology, gene expression and 2D motility and function. We found a profound rearrangement of the cytoskeleton network, dose-dependent increase of Focal Adhesion kinase (FAK) phosphorylation and Yes-associated protein 1 (YAP1) expression, suggesting cell stiffening and increased proneness to motility. Transcriptome analysis showed expression changes of genes associated with cardiovascular homeostasis, nitric oxide production, angiogenesis, and inflammation. Hypergravity-treated cells also showed significantly improved motility and function (2D migration and tube formation). These results, expanding our knowledge about the homeostatic response of capillary endothelial cells, show that adaptation to hypergravity has opposite effect compared to microgravity on the same cell type.

MICAL2 is expressed in cancer associated neo-angiogenic capillary endothelia and it is required for endothelial cell viability, motility and VEGF response.

The capacity of inducing angiogenesis is a recognized hallmark of cancer cells. The cancer microenvironment, characterized by hypoxia and inflammatory signals, promotes proliferation, migration and activation of quiescent endothelial cells (EC) from surrounding vascular network. Current anti-angiogenic drugs present side effects, temporary efficacy, and issues of primary resistance, thereby calling for the identification of new therapeutic targets. MICALs are a unique family of redox enzymes that destabilize F-actin in cytoskeletal dynamics. MICAL2 mediates Semaphorin3A-NRP2 response to VEGFR1 in rat ECs. MICAL2 also enters the p130Cas interactome in response to VEGF in HUVEC. Previously, we showed that MICAL2 is overexpressed in metastatic cancer. A small-molecule inhibitor of MICAL2 exists (CCG-1423). Here we report that 1) MICAL2 is expressed in neo-angiogenic ECs in human solid tumors (kidney and breast carcinoma, glioblastoma and cardiac myxoma, n = 67, were analyzed with immunohistochemistry) and in animal models of ischemia/inflammation neo-angiogenesis, but not in normal capillary bed; 2) MICAL2 protein pharmacological inhibition (CCG-1423) or gene KD reduce EC viability and functional performance; 3) MICAL2 KD disables ECs response to VEGF in vitro. Whole-genome gene expression profiling reveals MICAL2 involvement in angiogenesis and vascular development pathways. Based on these results, we propose that MICAL2 expression in ECs participates to inflammation-induced neo-angiogenesis and that MICAL2 inhibition should be tested in cancer- and noncancer-associated neo-angiogenesis, where chronic inflammation represents a relevant pathophysiological mechanism.

Human Pathophysiological Adaptations to the Space Environment.

Space is an extreme environment for the human body, where during long-term missions microgravity and high radiation levels represent major threats to crew health. Intriguingly, space flight (SF) imposes on the body of highly selected, well-trained, and healthy individuals (astronauts and cosmonauts) pathophysiological adaptive changes akin to an accelerated aging process and to some diseases. Such effects, becoming manifest over a time span of weeks (i.e., cardiovascular deconditioning) to months (i.e., loss of bone density and muscle atrophy) of exposure to weightlessness, can be reduced through proper countermeasures during SF and in due time are mostly reversible after landing. Based on these considerations, it is increasingly accepted that SF might provide a mechanistic insight into certain pathophysiological processes, a concept of interest to pre-nosological medicine. In this article, we will review the main stress factors encountered in space and their impact on the human body and will also discuss the possible lessons learned with space exploration in reference to human health on Earth. In fact, this is a productive, cross-fertilized, endeavor in which studies performed on Earth yield countermeasures for protection of space crew health, and space research is translated into health measures for Earth-bound population.

Context-dependent miR-204 and miR-211 affect the biological properties of amelanotic and melanotic melanoma cells.

Despite increasing amounts of experimental evidence depicting the involvement of non-coding RNAs in cancer, the study of BRAFV600E-regulated genes has thus far focused mainly on protein-coding ones. Here, we identify and study the microRNAs that BRAFV600E regulates through the ERK pathway.By performing small RNA sequencing on A375 melanoma cells and a vemurafenib-resistant clone that was taken as negative control, we discover miR-204 and miR-211 as the miRNAs most induced by vemurafenib. We also demonstrate that, although belonging to the same family, these two miRNAs have distinctive features. miR-204 is under the control of STAT3 and its expression is induced in amelanotic melanoma cells, where it acts as an effector of vemurafenib's anti-motility activity by targeting AP1S2. Conversely, miR-211, a known transcriptional target of MITF, is induced in melanotic melanoma cells, where it targets EDEM1 and consequently impairs the degradation of TYROSINASE (TYR) through the ER-associated degradation (ERAD) pathway. In doing so, miR-211 serves as an effector of vemurafenib's pro-pigmentation activity. We also show that such an increase in pigmentation in turn represents an adaptive response that needs to be overcome using appropriate inhibitors in order to increase the efficacy of vemurafenib.In summary, we unveil the distinct and context-dependent activities exerted by miR-204 family members in melanoma cells. Our work challenges the widely accepted "same miRNA family = same function" rule and provides a rationale for a novel treatment strategy for melanotic melanomas that is based on the combination of ERK pathway inhibitors with pigmentation inhibitors.

MICAL2 is a novel human cancer gene controlling mesenchymal to epithelial transition involved in cancer growth and invasion.

The MICAL (Molecules Interacting with CasL) proteins catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. Here we show for the first time that MICAL2 mRNA is significantly over-expressed in aggressive, poorly differentiated/undifferentiated, primary human epithelial cancers (gastric and renal). Immunohistochemistry showed MICAL2-positive cells on the cancer invasive front and in metastasizing cancer cells inside emboli, but not at sites of metastasis, suggesting MICAL2 expression was 'on' in a subpopulation of primary cancer cells seemingly detaching from the tissue of origin, enter emboli and travel to distant sites, and was turned 'off' upon homing at metastatic sites. In vitro, MICAL2 knock-down resulted in mesenchymal to epithelial transition, reduction of viability, and loss of motility and invasion properties of human cancer cells. Moreover, expression of MICAL2 cDNA in MICAL2-depleted cells induced epithelial to mesenchymal transition. Altogether our data indicate that MICAL2 over-expression is associated with cancer progression and metastatic disease. MICAL2 might be an important regulator of epithelial to mesenchymal transition and therefore a promising target for anti-metastatic therapy.