PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target.

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose-response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.

3D Bioprinting Allows the Establishment of Long-Term 3D Culture Model for Chronic Lymphocytic Leukemia Cells.

Chronic Lymphocytic Leukemia (CLL) represents the most common leukemia in the western world and remains incurable. Leukemic cells organize and interact in the lymphoid tissues, however what actually occurs in these sites has not been fully elucidated yet. Studying primary CLL cells in vitro is very challenging due to their short survival in culture and also to the fact that traditional two-dimensional in vitro models lack cellular and spatial complexity present in vivo. Based on these considerations, we exploited for the first time three-dimensional (3D) bioprinting to advance in vitro models for CLL. This technology allowed us to print CLL cells (both primary cells and cell lines) mixed with the appropriate, deeply characterized, hydrogel to generate a scaffold containing the cells, thus avoiding the direct cell seeding onto a precast 3D scaffold and paving the way to more complex models. Using this system, we were able to efficiently 3D bioprint leukemic cells and improve their viability in vitro that could be maintained up to 28 days. We monitored over time CLL cells viability, phenotype and gene expression, thus establishing a reproducible long-term 3D culture model for leukemia. Through RNA sequencing (RNAseq) analysis, we observed a consistent difference in gene expression profile between 2D and 3D samples, indicating a different behavior of the cells in the two different culture settings. In particular, we identified pathways upregulated in 3D, at both day 7 and 14, associated with immunoglobulins production, pro-inflammatory molecules expression, activation of cytokines/chemokines and cell-cell adhesion pathways, paralleled by a decreased production of proteins involved in DNA replication and cell division, suggesting a strong adaptation of the cells in the 3D culture. Thanks to this innovative approach, we developed a new tool that may help to better mimic the physiological 3D in vivo settings of leukemic cells as well as of immune cells in broader terms. This will allow for a more reliable study of the molecular and cellular interactions occurring in normal and neoplastic conditions in vivo, and could also be exploited for clinical purposes to test individual responses to different drugs.

Three-dimensional co-culture model of chronic lymphocytic leukemia bone marrow microenvironment predicts patient-specific response to mobilizing agents.

Chronic Lymphocytic Leukemia (CLL) cells disseminate into supportive tissue microenvironments. To investigate the mechanisms involved in leukemic cell tissue retention we developed a 3D bone marrow (BM) microenvironment that recreates CLL - BM-stromal cells interactions inside a scaffold within a bioreactor. Our system allows the parallel analysis of CLL cells retained inside the scaffold and those released in the presence/absence of pharmacological agents, mimicking tissue and circulating cell compartments, respectively. CLL cells can be retained within the scaffold only in the presence of microenvironmental elements, which through direct contact down-regulate the expression of HS1 cytoskeletal protein in CLL cells. Consist with this, the expression of HS1 was lower in CLL cells obtained from patients' BM versus CLL cells circulating in the PB. Moreover, we demonstrate that CLL cells with inactive-HS1, impaired cytoskeletal activity and a more aggressive phenotype are more likely retained within the scaffold despite the presence of Ibrutinib, whose mobilizing effect is mainly exerted on those with active-HS1, ensuing dynamic cytoskeletal activity. This differential effect would not otherwise be assessable in a traditional 2D system and may underlie a distinctive resistance of single CLL clones. Notably, CLL cells mobilized in the peripheral blood of patients during Ibrutinib therapy exhibited activated HS1, underscoring that our model reliably mirrors the in vivo situation. The 3D model described herein is suitable to reproduce and identify critical CLL-BM interactions, opening the way to pathophysiological studies and the evaluation of novel targeted therapies in an individualized manner.

The Role of Autophagy in the Maintenance of Stemness and Differentiation of Mesenchymal Stem Cells.

Regulated self-consumption, also known as autophagy, is an evolutionary conserved process that degrades cellular components by directing them to the lysosomal compartment of eukaryotic cells. As a major intracellular degradation and recycling pathway, autophagy is crucial for maintaining and remodeling cellular homeostasis during normal cellular and tissue development. Recent studies have demonstrated that autophagy is necessary for the maintenance of cellular stemness and for a number of differentiation processes, including the lineage determination of mesenchymal stem cells. These are multipotent progenitor cells with self-renewal capacities that can give rise to a subset of tissues and thus hold a consistent potential in regenerative medicine. Here, we review the current literature on the complex liaison between autophagy induced by various extra- or intracellular stimuli and the molecular targets that affect mesenchymal stem cells proliferation and differentiation.

Tumor acidosis enhances cytotoxic effects and autophagy inhibition by salinomycin on cancer cell lines and cancer stem cells.

Sustained autophagy contributes to the metabolic adaptation of cancer cells to hypoxic and acidic microenvironments. Since cells in such environments are resistant to conventional cytotoxic drugs, inhibition of autophagy represents a promising therapeutic strategy in clinical oncology. We previously reported that the efficacy of hydroxychloroquine (HCQ), an autophagy inhibitor under clinical investigation is strongly impaired in acidic tumor environments, due to poor uptake of the drug, a phenomenon widely associated with drug resistance towards many weak bases. In this study we identified salinomycin (SAL) as a potent inhibitor of autophagy and cytotoxic agent effective on several cancer cell lines under conditions of transient and chronic acidosis. Since SAL has been reported to specifically target cancer-stem cells (CSC), we used an established model of breast CSC and CSC derived from breast cancer patients to examine whether this specificity may be associated with autophagy inhibition. We indeed found that CSC-like cells are more sensitive to autophagy inhibition compared to cells not expressing CSC markers. We also report that the ability of SAL to inhibit mammosphere formation from CSC-like cells was dramatically enhanced in acidic conditions. We propose that the development and use of clinically suitable SAL derivatives may result in improved autophagy inhibition in cancer cells and CSC in the acidic tumor microenvironment and lead to clinical benefits.

Carbonic anhydrase IX inhibition is an effective strategy for osteosarcoma treatment.

OBJECTIVE: Hypoxia-inducible factor 1, a regulator of CA IX activity, is often overexpressed in human osteosarcoma (OS) but not in normal tissues, and its expression levels correlate with prognosis. In this study, we investigated the therapeutic potential of newly synthesized CA IX sulfonamide inhibitors in OS. METHODS: CA IX expression was evaluated in OS cell lines and bone marrow stromal cells (BMSC). After treatment with CA IX inhibitors, cell proliferation, apoptosis, cell cycle, extracellular and cytosolic pH changes were evaluated both in vitro and in mouse OS xenografts. RESULTS: CA IX expression levels were significantly higher in OS than in BMSC. Accordingly, CA IX inhibitor 3 induced remarkable cytotoxicity on OS cells without affecting BMSC proliferation. This activity was increased under hypoxia, and was mediated by cell cycle arrest and by the modulation of cytosolic and extracellular pH. In vivo, CA IX inhibitor 3 reduced tumor growth by inducing significant necrosis. CONCLUSIONS: Our results provide a strong rationale for the clinical use of the newly synthesized CA IX inhibitor 3 in human OS.