Contingent intramuscular boosting of P2XR7 axis improves motor function in transgenic ALS mice.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder that leads to progressive degeneration of motor neurons and severe muscle atrophy without effective treatment. Most research on the disease has been focused on studying motor neurons and supporting cells of the central nervous system. Strikingly, the recent observations have suggested that morpho-functional alterations in skeletal muscle precede motor neuron degeneration, bolstering the interest in studying muscle tissue as a potential target for the delivery of therapies. We previously showed that the systemic administration of the P2XR7 agonist, 2'(3')-O-(4-benzoylbenzoyl) adenosine 5-triphosphate (BzATP), enhanced the metabolism and promoted the myogenesis of new fibres in the skeletal muscles of SOD1G93A mice. Here we further corroborated this evidence showing that intramuscular administration of BzATP improved the motor performance of ALS mice by enhancing satellite cells and the muscle pro-regenerative activity of infiltrating macrophages. The preservation of the skeletal muscle retrogradely propagated along with the motor unit, suggesting that backward signalling from the muscle could impinge on motor neuron death. In addition to providing the basis for a suitable adjunct multisystem therapeutic approach in ALS, these data point out that the muscle should be at the centre of ALS research as a target tissue to address novel therapies in combination with those oriented to the CNS.

Evaluation of peripherin in biofluids of patients with motor neuron diseases.

Peripherin (PRPH), a type III intermediate filament, assembles with neurofilaments in neurons of the peripheral nervous system, including lower motor neurons (LMN). To evaluate the role of PRPH in LMN degeneration, we assessed PRPH and neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and serum of 91 patients with motor neuron diseases (MND) and 69 controls. Overall, we found PRPH to be more concentrated in serum than in CSF. Serum PRPH resulted significantly increased in MND patients but it was unrelated to CSF-NfL or survival in the amyotrophic lateral sclerosis (ALS) subset. PRPH might represent a marker of LMN involvement.

Prognostic and Predictive Effect of IGHV Mutational Status and Load in Chronic Lymphocytic Leukemia: Focus on FCR and BR Treatments.

BACKGROUND: Most important markers in chronic lymphocytic leukemia (CLL) are TP53 abnormalities, including mutations and deletions, and the mutational status of immunoglobulin heavy chain (IGHV) genes. However, some recent publications suggest that the IGHV mutational load could have a prognostic effect on CLL patients. PATIENTS AND METHODS: We performed a single-center retrospective study on 459 patients with productive rearrangement of the B-cell receptor to evaluate the prognostic and predictive role of IGHV mutational status and burden within the germline sequence. In particular we focused on FCR (fludarabine with cyclophosphamide, and rituximab)- (64 naive and 30 relapsed) and BR (bendamustine with rituximab)-treated patients (17 naive and 61 relapsed). A cutoff value of 2% of difference within the IGHV germline was used to define the IGHV mutational status. RESULTS: We reported that unmutated IGHV (U-IGHV) patients were characterized by a significant shorter progression-free survival (PFS) and overall survival (P < .0001) compared with mutated IGHV (M-IGHV) patients. Moreover, treatment-naive M-IGHV patients experienced a long-term disease control after FCR or BR, with PFS reaching a plateau regardless of mutational load. In our series the extent of IGHV gene mutation did not provide further relevant prognostic data over the mutational status. Relapsed patients showed dismal outcome with chemoimmunotherapy regardless of IGHV status or load. CONCLUSION: Our data, together with from those from the literature, confirmed the cutoff value of 2% to define the mutational status of IGHV gene and suggest that FCR/BR are good first-line treatment strategies for M-IGHV patients, whereas U-IGHV patients should be managed with B-cell receptor and/or B-cell lymphoma 2 (BCL2) inhibitors.

HSP70/HSF1 axis, regulated via a PI3K/AKT pathway, is a druggable target in chronic lymphocytic leukemia.

Considering the role played by the heat shock protein of 70 kDa (HSP70) in cancer, we characterized this protein and its major regulator, the heat shock factor 1 (HSF1), in chronic lymphocytic leukemia (CLL). We found both HSP70 and HSF1 overexpressed in CLL patients, correlated to poor prognosis and abnormally localized in the nucleus of leukemic B cells. The two proteins were strictly correlated each other and their levels decreased consensually in those patients responding to in vivo therapeutic regimens. HSP70 and HSF1 inhibition was proved to be effective in inducing a dose-dependent in vitro apoptosis of CLL B cells. Considering that HSF1 is finely regulated by kinases belonging to pathways triggered by rat sarcoma (RAS), we benefited from a previous proteomic study performed in CLL patients aiming to assess the activation/expression of key signaling proteins. We found that patients showing high levels of HSP70 also expressed high Akt-Ser473, thus activating HSF1. Inhibition of PI3K, which activates AKT, reduced the expression of HSF1 and HSP70. By contrast, HSP70-low patients displayed high activation of MEK1/2 and ERK1/2, known to negatively regulate HSF1. These data demonstrate that the HSP70 expression is regulated by the modulation of HSF1 activity through the activation of RAS-regulated pathways and suggest the HSP70/HSF1 interplay as an interesting target for antileukemic therapies. Finally, inhibition of PI3K, that activates AKT, reduced the expression of HSF1 and HSP70.

The epg5 knockout zebrafish line: a model to study Vici syndrome.

The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5-/- mutants. By aging, epg5-/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.

Abnormal regulation of BCR signalling by c-Cbl in chronic lymphocytic leukaemia.

Abnormalities of molecules involved in signal transduction pathways are connected to Chronic Lymphocytic Leukemia (CLL) pathogenesis and a critical role has been already ascribed to B-Cell Receptor (BCR)-Lyn axis. E3 ubiquitin ligase c-Cbl, working together with adapter protein CIN85, controls the degradation of protein kinases involved in BCR signaling. To investigate cell homeostasis in CLL, we studied c-Cbl since in normal B cells it is involved in the ubiquitin-dependent Lyn degradation and in the down-regulation of BCR signaling. We found that c-Cbl is overexpressed and not ubiquitinated after BCR engagement. We observed that c-Cbl did not associate to CIN85 in CLL with respect to normal B cells at steady state, nor following BCR engagement. c-Cbl association to Lyn was not detectable in CLL after BCR stimulation, as it happens in normal B cells. In some CLL patients, c-Cbl is constitutively phosphorylated at Y731 and in the same subjects, it associated to regulatory subunit p85 of PI3K. Moreover, c-Cbl is constitutive associated to Cortactin in those CLL patients presenting Cortactin overexpression and bad prognosis. These results support the hypothesis that c-Cbl, rather than E3 ligase activity, could have an adaptor function in turn influencing cell homeostasis in CLL.

Cortactin, a Lyn substrate, is a checkpoint molecule at the intersection of BCR and CXCR4 signalling pathway in chronic lymphocytic leukaemia cells.

Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP-9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP-9 release as well as a decrease of migration capability of leukaemic B cells in vitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase-inhibitor PP2 with a consequent decrease of MMP-9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both in vivo and in vitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check-point molecule between the BCR and CXCR4 signalling pathways.

Major infections, secondary cancers and autoimmune diseases occur in different clinical subsets of chronic lymphocytic leukaemia patients.

BACKGROUND: Major infections (MIs), secondary cancers (SCs) and autoimmune diseases (ADs) are the most common and relevant complications in patients with chronic lymphocytic leukaemia. METHODS: We performed a single-centre retrospective study to investigate the prevalence of the above quoted complications, the association with most important prognostic markers and their impact on survival (n = 795). RESULTS: Almost one out of three patients experienced at least one complication and only 0.9% of the cohort developed all three complications. One hundred and twenty (20%) subjects developed SC, 98 MI (12%) and 80 AD (10%); these complications seem to occur in a mutually exclusive manner. By Kaplan-Meier analysis we estimated that after 20 years from the diagnosis SC, MI and AD occurred in 48%, 42% and 29% of patients, respectively. Furthermore, we showed that some clinical and biological markers are skewed among patients with different complications and that subjects with MI and SC had a worse prognosis than those with AD and all other patients (p < 0.0001). CONCLUSIONS: This study reveals the existence of different clinical subsets of chronic lymphocytic leukaemia patients characterised by an increased and different risk for developing specifically MI, SC and AD.