Collagen VI sustains cell stemness and chemotherapy resistance in glioblastoma.

Microenvironmental factors are known fundamental regulators of the phenotype and aggressiveness of glioblastoma (GBM), the most lethal brain tumor, characterized by fast progression and marked resistance to treatments. In this context, the extracellular matrix (ECM) is known to heavily influence the behavior of cancer cells from several origins, contributing to stem cell niches, influencing tumor invasiveness and response to chemotherapy, mediating survival signaling cascades, and modulating inflammatory cell recruitment. Here, we show that collagen VI (COL6), an ECM protein widely expressed in both normal and pathological tissues, has a distinctive distribution within the GBM mass, strongly correlated with the most aggressive and phenotypically immature cells. Our data demonstrate that COL6 sustains the stem-like properties of GBM cells and supports the maintenance of an aggressive transcriptional program promoting cancer cell proliferation and survival. In particular, we identified a specific subset of COL6-transcriptionally co-regulated genes, required for the response of cells to replicative stress and DNA damage, supporting the concept that COL6 is an essential stimulus for the activation of GBM cell response and resistance to chemotherapy, through the ATM/ATR axis. Altogether, these findings indicate that COL6 plays a pivotal role in GBM tumor biology, exerting a pleiotropic action across different GBM hallmarks, including phenotypic identity and gene transcription, as well as response to treatments, thus providing valuable information for the understanding of the complex microenvironmental cues underlying GBM malignancy.

Involvement of Mitochondria in the Selective Response to Microsecond Pulsed Electric Fields on Healthy and Cancer Stem Cells in the Brain.

In the last few years, pulsed electric fields have emerged as promising clinical tools for tumor treatments. This study highlights the distinct impact of a specific pulsed electric field protocol, PEF-5 (0.3 MV/m, 40 µs, 5 pulses), on astrocytes (NHA) and medulloblastoma (D283) and glioblastoma (U87 NS) cancer stem-like cells (CSCs). We pursued this goal by performing ultrastructural analyses corroborated by molecular/omics approaches to understand the vulnerability or resistance mechanisms triggered by PEF-5 exposure in the different cell types. Electron microscopic analyses showed that, independently of exposed cells, the main targets of PEF-5 were the cell membrane and the cytoskeleton, causing membrane filopodium-like protrusion disappearance on the cell surface, here observed for the first time, accompanied by rapid cell swelling. PEF-5 induced different modifications in cell mitochondria. A complete mitochondrial dysfunction was demonstrated in D283, while a mild or negligible perturbation was observed in mitochondria of U87 NS cells and NHAs, respectively, not sufficient to impair their cell functions. Altogether, these results suggest the possibility of using PEF-based technology as a novel strategy to target selectively mitochondria of brain CSCs, preserving healthy cells.

Targeting the plasticity of mesenchymal stromal cells to reroute the course of acute myeloid leukemia.

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but >30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

Effects of Ultra-Short Pulsed Electric Field Exposure on Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common brain cancer in adults. GBM starts from a small fraction of poorly differentiated and aggressive cancer stem cells (CSCs) responsible for aberrant proliferation and invasion. Due to extreme tumor heterogeneity, actual therapies provide poor positive outcomes, and cancers usually recur. Therefore, alternative approaches, possibly targeting CSCs, are necessary against GBM. Among emerging therapies, high intensity ultra-short pulsed electric fields (PEFs) are considered extremely promising and our previous results demonstrated the ability of a specific electric pulse protocol to selectively affect medulloblastoma CSCs preserving normal cells. Here, we tested the same exposure protocol to investigate the response of U87 GBM cells and U87-derived neurospheres. By analyzing different in vitro biological endpoints and taking advantage of transcriptomic and bioinformatics analyses, we found that, independent of CSC content, PEF exposure affected cell proliferation and differentially regulated hypoxia, inflammation and P53/cell cycle checkpoints. PEF exposure also significantly reduced the ability to form new neurospheres and inhibited the invasion potential. Importantly, exclusively in U87 neurospheres, PEF exposure changed the expression of stem-ness/differentiation genes. Our results confirm this physical stimulus as a promising treatment to destabilize GBM, opening up the possibility of developing effective PEF-mediated therapies.

Somatic mutations activating Wiskott-Aldrich syndrome protein concomitant with RAS pathway mutations in juvenile myelomonocytic leukemia patients.

The WAS gene product is expressed exclusively in the cytoplasm of hematopoietic cells and constitutional genetic abrogation of WASP leads to Wiskott-Aldrich syndrome (WAS). Moreover, mutational activation of WASP has been associated with X-linked neutropenia. Although studies reported that patients with constitutional WAS mutations affecting functional WASP expression may present juvenile myelomonocytic leukemia (JMML)-like features, confounding differential diagnosis above all in the copresence of mutated RAS, an activating somatic mutation of WASP has not been previously described in JMML patients. In our ongoing studies on JMML genomics, we at first detected a somatic WAS mutation in a major clone found at two consecutive relapses in one of two twins with JMML. Both twins were treated with hematopoietic stem cell transplantation after diagnosis of JMML. The somatic WAS mutation detected here displayed an activating WASP phenotype. Screening of 46 sporadic JMML patients at disease onset for mutations in the same PBD domain of WAS revealed two additional singleton patients carrying minor mutated clones. This is the first study to associate somatically acquired WASP mutations with a hematopoietic malignancy and increases insight in the complexity of the genomic landscape of JMML that shows low recurrent mutations concomitant with general hyperactivation of RAS pathway signaling.

On the effects of 30.5 GHz sinusoidal wave exposure on glioblastoma organoids.

Introduction: Glioblastoma (grade IV) is the most aggressive primary brain tumor in adults, representing one of the biggest therapeutic challenges due to its highly aggressive nature. In this study, we investigated the impact of millimeter waves on tridimensional glioblastoma organoids derived directly from patient tumors. Our goal was to explore novel therapeutic possibilities in the fight against this challenging disease. Methods: The exposure setup was meticulously developed in-house, and we employed a comprehensive dosimetry approach, combining numerical and experimental methods. Biological endpoints included a global transcriptional profiling analysis to highlight possible deregulated pathways, analysis of cell morphological changes, and cell phenotypic characterization which are all important players in the control of glioblastoma progression. Results and discussion: Our results revealed a significant effect of continuous millimeter waves at 30.5 GHz on cell proliferation and apoptosis, although without affecting the differentiation status of glioblastoma cells composing the organoids. Excitingly, when applying a power level of 0.1 W (Root Mean Square), we discovered a remarkable (statistically significant) therapeutic effect when combined with the chemotherapeutic agent Temozolomide, leading to increased glioblastoma cell death. These findings present a promising interventional window for treating glioblastoma cells, harnessing the potential therapeutic benefits of 30.5 GHz CW exposure. Temperature increase during treatments was carefully monitored and simulated with a good agreement, demonstrating a negligible involvement of the temperature elevation for the observed effects. By exploring this innovative approach, we pave the way for improved future treatments of glioblastoma that has remained exceptionally challenging until now.

Functional relevance of circRNA aberrant expression in pediatric acute leukemia with KMT2A::AFF1 fusion.

ABSTRACT: Circular RNAs (circRNAs) are emerging molecular players in leukemogenesis and promising therapeutic targets. In KMT2A::AFF1 (MLL::AF4)-rearranged leukemia, an aggressive disease compared with other pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), data about circRNAs are limited. Here, we disclose the circRNA landscape of infant patients with KMT2A::AFF1 translocated BCP-ALL showing dysregulated, mostly ectopically expressed, circRNAs in leukemia cells. Most of these circRNAs, apart from circHIPK3 and circZNF609, previously associated with oncogenic behavior in ALL, are still uncharacterized. An in vitro loss-of-function screening identified an oncogenic role of circFKBP5, circKLHL2, circNR3C1, and circPAN3 in KMT2A::AFF1 ALL, whose silencing affected cell proliferation and apoptosis. Further study in an extended cohort disclosed a significantly correlated expression of these oncogenic circRNAs and their putative involvement in common regulatory networks. Moreover, it showed that circAFF1 upregulation occurs in a subset of cases with HOXA KMT2A::AFF1 ALL. Collectively, functional analyses and patient data reveal oncogenic circRNA upregulation as a relevant mechanism that sustains the malignant cell phenotype in KMT2A::AFF1 ALL.

Molecular and functional profiling of chemotolerant cells unveils nucleoside metabolism-dependent vulnerabilities in medulloblastoma.

Chemotherapy resistance is considered one of the main causes of tumor relapse, still challenging researchers for the identification of the molecular mechanisms sustaining its emergence. Here, we setup and characterized chemotherapy-resistant models of Medulloblastoma (MB), one of the most lethal pediatric brain tumors, to uncover targetable vulnerabilities associated to their resistant phenotype. Integration of proteomic, transcriptomic and kinomic data revealed a significant deregulation of several pathways in resistant MB cells, converging to cell metabolism, RNA/protein homeostasis, and immune response, eventually impacting on patient outcome. Moreover, resistant MB cell response to a large library of compounds through a high-throughput screening (HTS), highlighted nucleoside metabolism as a relevant vulnerability of chemotolerant cells, with peculiar antimetabolites demonstrating increased efficacy against them and even synergism with conventional chemotherapeutics. Our results suggest that drug-resistant cells significantly rewire multiple cellular processes, allowing their adaptation to a chemotoxic environment, nevertheless exposing alternative actionable susceptibilities for their specific targeting.

Identification of Homoharringtonine as a potent inhibitor of glioblastoma cell proliferation and migration.

We previously demonstrated that Annexin A2 (ANXA2) is a pivotal mediator of the pro-oncogenic features displayed by glioblastoma (GBM) tumors, the deadliest adult brain malignancies, being involved in cell stemness, proliferation and invasion, thus negatively impacting patient prognosis. Based on these results, we hypothesized that compounds able to revert ANXA2-dependent transcriptional features could be exploited as reliable treatments to inhibit GBM cell aggressiveness by hampering their proliferative and migratory potential. Transcriptional signatures obtained by the modulation of ANXA2 activity/levels were functionally mapped through the QUADrATiC bioinformatic tool for compound identification. Selected compounds were screened by cell proliferation and migration assays in primary GBM cells, and we identified Homoharringtonine (HHT) as a potent inhibitor of GBM cell motility and proliferation, without affecting their viability. A further molecular characterization of the effects displayed by HHT, confirmed its ability to inhibit a transcriptional program involved in cell migration and invasion. Moreover, we demonstrated that the multiple antitumoral effects displayed by HHT are correlated to the inhibition of a platelet derived growth factor receptor α (PDGFRα)-dependent intracellular signaling through the impairment of Signal transducer and activator of transcription 3 (STAT3) and Ras homolog family member A (RhoA) axes. Our results demonstrate that HHT may act as a potent inhibitor of cancer cell proliferation and invasion in GBM, by hampering multiple PDGFRα-dependent oncogenic signals transduced through the STAT3 and RhoA intracellular components, finally suggesting its potential transferability for achieving an effective impairment of peculiar GBM hallmarks.

Long-term proliferation of immature hypoxia-dependent JMML cells supported by a 3D in vitro system.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal stem cell disorder that occurs in early childhood and is characterized by the hyperactivation of the RAS pathway in 95% of the patients. JMML is characterized by a hyperproliferation of granulocytes and monocytes, and little is known about the heterogeneous nature of leukemia-initiating cells, as well as of the cellular hierarchy of the JMML bone marrow. In this study, we report the generation and characterization of a novel patient-derived three-dimensional (3D) in vitro JMML model, called patient-derived JMML Atypical Organoid (pd-JAO), sustaining the long-term proliferation of JMML cells with stem cell features and patient-specific hallmarks. JMML cells brewed in a 3D model under different microenvironmental conditions acquired proliferative and survival advantages when placed under low oxygen tension. Transcriptomic and microscopic analyses revealed the activation of specific metabolic energy pathways and the inactivation of processes leading to cell death. Furthermore, we demonstrated the pd-JAO-derived cells' migratory, propagation, and self-renewal capacities. Our study contributes to the development of a robust JMML 3D in vitro model for studying and defining the impact of microenvironmental stimuli on JMML disease and the molecular mechanisms that regulate JMML initiating and propagating cells. Pd-JAO may become a promising model for compound tests focusing on new therapeutic interventions aimed at eradicating JMML progenitors and controlling JMML disease.