A reversible carnitine palmitoyltransferase 1 (CPT1) inhibitor offsets chronic lymphocytic leukaemia cell proliferation

Crucial for chronic lymphocytic leukaemia (CLL) development and progression are the iterative cycles of cell re-activation and proliferation that take place in lymphoid tissues. These iterative cycles are fundamental for the development and the progression of the disease. Since cellular fatty acid (FA) import and oxidation (FAO) were recently reported to be upregulated in CLL, compared to normal B lymphocytes, we explored the in vitro effects of ST1326, a reversible inhibitor of carnitine-palmitoyl transferase 1A (CPT1A), on leukaemic cells subject to activating microenvironment-mimicking stimuli. ST1326 induced dose-dependent mitochondrial dysfunction and cell death, which were remarkably higher in activated/proliferating than quiescent CLL cells. Drug sensitivity was observed irrespective of the presence of TP53 alterations or chromosomal abnormalities, which are known to cause chemoresistance in CLL patients. The treatment of normal B lymphocytes with ST1326, at doses lethal to CLL cells, causes only a slight inhibition of cell activation/proliferation, indicating a modest cytostatic, not cytotoxic, effect. ST1326 cytotoxicity in CLL was associated with decreased levels of intracellular Acetyl-CoA and down-regulation of signalling pathways that are crucial for leukaemic cell survival, activation and proliferation. In particular, environment-induced activation of STAT3 and STAT6 transcription factors, known to upregulate anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL, was impaired by ST1326. As a consequence, drug combination experiments with the BH3-mimetic ABT-199/Venetoclax, whose effects are counteracted by Mcl-1/Bcl-xL and cell proliferation, showed strong ST1326-mediated potentiation of ABT-199 cytotoxicity in activated/proliferating CLL cells. We also observed that ST1326 showed a synergic effect with Fludarabine in activated/proliferating CLL cells. The data indicate that CLL cells turning to an activated/proliferating state become more dependent on FAO and more sensitive to FAO-antagonists, and pave the way for ST1326 as an adjuvant tool in anti-CLL drug-combination regimens with drugs that lose efficacy on proliferating leukaemic cells

Relevance of fatty acid metabolism in proliferating CLL cells

Chronic lymphocytic leukemia (CLL) cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. We previously demonstrated that the antidiabetic drug metformin, known to also inhibit oxidative phosphorylation (OXPHOS), inhibits cell cycle entry of leukemic cells derived ex-vivo from the peripheral blood of CLL patients and stimulated in vitro by cell culture systems that recreate a microenvironment to drive their proliferation (Bruno et al, Oncotarget, 2015). However, overtly proliferating CLL cells were resistant to the cytostatic effects of metformin. Since metformin switched the energetic metabolism of activated, not yet proliferating, CLL cells from OXPHOS to accelerated glycolysis, in the present study we asked whether combining metformin with glycolysis impairment could inhibit also proliferating CLL cells. Still, CLL cells recovered from a transitory block and rescued in vitro proliferation. What kind of energetic reprogramming was involved in the resistance of proliferating CLL cells to glucose utilization? Recent studies highlight on the role of fatty acid utilization of CLL cells. We asked 1) whether inhibitors of lipid metabolism could impair proliferation of in vitro stimulated CLL cells; 2) whether impairing glucose energetic pathways could act synergistically with beta oxidation inhibitors.We found that inhibitors of critical steps of fatty acid metabolisms, such as carnitine-palmitoyl transferase 1A (CPT1A) -rate-limiting enzyme for fatty acid import into mitochondria- or Peroxisome Proliferator-Activated Receptor (PPAR)-alpha -regulator of beta-oxidation- administered at clinically achievable doses, were ineffective on quiescent CLL cells and on CLL cells stimulated by the microenvironment during the first stages of activation. Conversely, remarkable susceptibility to undergo apoptosis was observed at later stages of cell activation and during overt proliferation. Synergism with impairment of other energetic pathways occurred depending on the stage of activation of the in vitro stimulated CLL cells.The results suggest that energetic metabolic pathways could be relevant targets for CLL treatment, provided that the complex metabolic reprogramming network during the transition of leukemic cells from quiescence to proliferation, and back, are clearly elucidated. This work was supported by grants from AIRC IG15426

Unexpected effects of biphosphonates in in vitro models of activated CLL cells

Recent studies suggest that the commonly prescribed anti-osteoporosis drugs bisphosphonates (BPs) might also exhibit antitumor activity. We investigated a possible anticancer effect of BPs on B-chronic lymphocytic leukemia (CLL) cells obtained from peripheral blood of 26 CLL patients. Zoledronate, etidronate and clodronate were administered in vitro simultaneously to following activation stimuli: i) CD40L-expressing fibroblasts, ii) soluble recombinant CD40L produced in our laboratory +IL-4, iii) CpG ODN 2006+IL-15 with or without bone marrow stromal cells (BMSC). CLL cell viability, activation/proliferation were monitored by flow cytometry. We unexpectedly observed that BPs generated a protective effect from spontaneous apoptosis in 11/26 (42%) patients (viability + 18%-392%) and an augmentation in CLL cell activation/proliferation in 61% of the samples (S+G2M phase: +100%±25). Interestingly, protection from spontaneous apoptosis or increment of cell activation, required the presence of either fibroblasts, BMSC or autologous Nurse Like Cells (NLC). We thus hypothesized that supportive cells are involved in the BPs effects either through cell-cell interactions with leukemic cells or T cells, or through soluble factors release in the medium. Functional experiments with transwells suggest that stromal cells, in presence of Clodronate, release soluble factors in the medium that may probably concur to the unexpected Clodronate-mediated enhancement of CLL cell activation/proliferation. This work is in progress and several critical questions on the mechanisms are still unanswered. Nevertheless, the phenomenological data argue that caution should be taken when administering BPs against osteoporosis in elderly persons, who could have Monoclonal B Lymphocytosis or CLL

Cross border critical infrastructure. A new approach for the protection evaluation

The work proposes an operational model for the management of cross-border transport critical infrastructures. Particular focus is given to road and rail network. The main goal is to develop a Decision Support System (DSS) for the management of relevant events that may have impacts on the two countries. The SICt project - Resilience of cross-border Critical Infrastructures is part of the Interreg VA Italy-Switzerland 2014-2020. The project area includes Regione Lombardia (Italy) and Canton Ticino (Switzerland) and has been divided into two areas. The study area where the hazards (events) of both anthropic and natural origin are analyzed. Critical scenarios on critical infrastructure are also evaluated. The impact area is a larger area in which the effects, after the total or partial interruptions of a critical infrastructure in the study area, are evaluated. Specifically, the work illustrates the different operational phases of the proposed model. The first one is the analysis of anthropic and natural hazards related to the study area: road and rail accidents, rail failures, intense rainfall events, floods, landslides, debris flow, etc... The second phase concerns the detailed analysis aimed at managing the events, with particular reference to the first responders' intervention models and the emergency and traffic management plans. This phase also includes the implementation of a monitoring network and an IT platform, used to share data and information about critical infrastructures. The third phase is the test and validation of the IT platform implemented

Modeling Analysis of Automated and Connected Cars in Signalized Intersections

It is acknowledged that many of the problems related to urban congestion can be solved through the diffusion of automated vehicles capable not only of replacing drivers, but also of receiving information from the infrastructure. In this article, the effects of driverless cars (level 3–4 of automation) and of the Green Light Optimal Speed Advisory (GLOSA) system, a particular kind of Cooperative – Intelligent Transport System (C-ITS), will be evaluated at an urban signalized intersection through a set of micro-simulations. The aim of the paper is to analyze the two system as stand-alone before evaluating their jointed implementation, so to obtain their impacts and to analyze if and how they synergize for different levels of market penetration. The results of these simulations demonstrate that automated and connected cars should bring global benefits at intersections and also result in a first set of recommendations and best practices for the implementation of the systems in the short-medium term. Particular focus is given to the interaction between the equipped vehicles and traditional traffic, to frame the negative effects on the overall crossing both in Traffic Efficiency and Environment. Finally, the evaluation of a real crossing in Milan is performed and the results of the overall node are provided for different scenarios and time horizon.Peer reviewe

BRAF Mutations in an Italian Regional Population: Implications for the Therapy of Thyroid Cancer

Background. Molecular diagnostics has offered new techniques for searching for mutations in thyroid indeterminate lesions. The study's aim was to evaluate the BRAF mutations' incidence in an Italian regional population. Subjects and Methods. 70 Caucasian patients born in Liguria with indeterminate or suspicious cytological diagnoses. Results. A BRAF gene mutation was successfully analyzed in 56/70 patients. The mutation was BRAF V600E in 12/56 cases (21%) and BRAF K601E in 2/56 (4%). Of the BRAF mutated samples on cytological diagnosis (14/56 cases), 2/14 cases (14%) were benign on final histology and 12/14 (86%) were malignant. All BRAF-mutated cases on cytology that were found to be benign on histological examination carried the K601E mutation. Of the nonmutated BRAF cases (42/56, 75%) which were later found to be malignant on definitive histology, 5 cases were follicular carcinomas (36%), 3 cases were incidentally found to be papillary microcarcinomas (22%), 2 were cases papillary carcinomas (14%), 1 was case follicular variant of papillary carcinoma (7%), 1 was case medullary carcinoma (7%), 1 case was Hurtle cell tumor (7%), and 1 case was combined cell carcinoma and papillary oncocytic carcinoma (7%). Conclusions. The presence of the BRAF V600E mutation may suggest a more aggressive surgical approach. BRAF K601E mutation did not correlate with malignancy indexes

BRAF Mutations in an Italian Regional Population: Implications for the Therapy of Thyroid Cancer

Background. Molecular diagnostics has offered new techniques for searching for mutations in thyroid indeterminate lesions. The study’s aim was to evaluate the BRAF mutations’ incidence in an Italian regional population. Subjects and Methods. 70 Caucasian patients born in Liguria with indeterminate or suspicious cytological diagnoses. Results. A BRAF gene mutation was successfully analyzed in 56/70 patients. The mutation was BRAF V600E in 12/56 cases (21%) and BRAF K601E in 2/56 (4%). Of the BRAF mutated samples on cytological diagnosis (14/56 cases), 2/14 cases (14%) were benign on final histology and 12/14 (86%) were malignant. All BRAF-mutated cases on cytology that were found to be benign on histological examination carried the K601E mutation. Of the nonmutated BRAF cases (42/56, 75%) which were later found to be malignant on definitive histology, 5 cases were follicular carcinomas (36%), 3 cases were incidentally found to be papillary microcarcinomas (22%), 2 were cases papillary carcinomas (14%), 1 was case follicular variant of papillary carcinoma (7%), 1 was case medullary carcinoma (7%), 1 case was Hurtle cell tumor (7%), and 1 case was combined cell carcinoma and papillary oncocytic carcinoma (7%). Conclusions. The presence of the BRAF V600E mutation may suggest a more aggressive surgical approach. BRAF K601E mutation did not correlate with malignancy indexes

Berberine Affects Mitochondrial Activity and Cell Growth of Leukemic Cells from Chronic Lymphocytic Leukemia Patients

B-cell chronic lymphocytic leukemia (CLL) results from accumulation of leukemic cells that are subject to iterative re-activation cycles and clonal expansion in lymphoid tissues. The effects of the well-tolerated alkaloid Berberine (BRB), used for treating metabolic disorders, were studied on ex-vivo leukemic cells activated in vitro by microenvironment stimuli. BRB decreased expression of survival/proliferation-associated molecules (e.g. Mcl-1/Bcl-xL) and inhibited stimulation-induced cell cycle entry, irrespective of TP53 alterations or chromosomal abnormalities. CLL cells rely on oxidative phosphorylation for their bioenergetics, particularly during the activation process. In this context, BRB triggered mitochondrial dysfunction and aberrant cellular energetic metabolism. Decreased ATP production and NADH recycling, associated with mitochondrial uncoupling, were not compensated by increased lactic fermentation. Antioxidant defenses were affected and could not correct the altered intracellular redox homeostasis. The data thus indicated that the cytotoxic/cytostatic action of BRB at 10-30 μM might be mediated, at least in part, by BRB-induced impairment of oxidative phosphorylation and the associated increment of oxidative damage, with consequent inhibition of cell activation and eventual cell death. Bioenergetics and cell survival were instead unaffected in normal B lymphocytes at the same BRB concentrations. Interestingly, BRB lowered the apoptotic threshold of ABT-199/Venetoclax, a promising BH3-mimetic whose cytotoxic activity is counteracted by high Mcl-1/Bcl-xL expression and increased mitochondrial oxidative phosphorylation. Our results indicate that, while CLL cells are in the process of building their survival and cycling armamentarium, the presence of BRB affects this process