MEK1/2 regulate normal BCR and ABL1 tumor-suppressor functions to dictate ATO response in TKI-resistant Ph+ leukemia

Resistance to tyrosine kinase inhibitors (TKIs) remains a clinical challenge in Ph-positive variants of chronic myeloid leukemia. We provide mechanistic insights into a previously undisclosed MEK1/2/BCR::ABL1/BCR/ABL1-driven signaling loop that may determine the efficacy of arsenic trioxide (ATO) in TKI-resistant leukemic patients. We find that activated MEK1/2 assemble into a pentameric complex with BCR::ABL1, BCR and ABL1 to induce phosphorylation of BCR and BCR::ABL1 at Tyr360 and Tyr177, and ABL1, at Thr735 and Tyr412 residues thus provoking loss of BCR's tumor-suppression functions, enhanced oncogenic activity of BCR::ABL1, cytoplasmic retention of ABL1 and consequently drug resistance. Coherently, pharmacological blockade of MEK1/2 induces dissociation of the pentameric MEK1/2/BCR::ABL1/BCR/ABL1 complex and causes a concurrent BCRY360/Y177, BCR::ABL1(Y360/Y177) and cytoplasmic ABL1(Y412/T735) dephosphorylation thereby provoking the rescue of the BCR's anti-oncogenic activities, nuclear accumulation of ABL1 with tumor-suppressive functions and consequently, growth inhibition of the leukemic cells and an ATO sensitization via BCR-MYC and ABL1-p73 signaling axes activation. Additionally, the allosteric activation of nuclear ABL1 was consistently found to enhance the anti-leukemic effects of the MEK1/2 inhibitor Mirdametinib, which when combined with ATO, significantly prolonged the survival of mice bearing BCR::ABL1-T315I-induced leukemia. These findings highlight the therapeutic potential of MEK1/2-inhibitors/ATO combination for the treatment of TKI-resistant leukemia

Spatial Reorganization of Liquid Crystalline Domains of Red Blood Cells in Type 2 Diabetic Patients with Peripheral Artery Disease

In this work, we will investigate if red blood cell (RBC) membrane fluidity, influenced by several hyperglycemia-induced pathways, could provide a complementary index of HbA1c to monitor the development of type 2 diabetes mellitus (T2DM)-related macroangiopathic complications such as Peripheral Artery Disease (PAD). The contextual liquid crystalline (LC) domain spatial organization in the membrane was analysed to investigate the phase dynamics of the transition. Twenty-seven patients with long-duration T2DM were recruited and classified in DM, including 12 non-PAD patients, and DM + PAD, including 15 patients in any stage of PAD. Mean values of RBC generalized polarization (GP), representative of membrane fluidity, together with spatial organization of LC domains were compared between the two groups; p-values < 0.05 were considered statistically significant. Although comparable for anthropometric characteristics, duration of diabetes, and HbA1c, RBC membranes of PAD patients were found to be significantly more fluid (GP: 0.501 +/- 0.026) than non-PAD patients (GP: 0.519 +/- 0.007). These alterations were shown to be triggered by changes in both LC microdomain composition and distribution. We found a decrease in Feret diameter from 0.245 +/- 0.281 mu m in DM to 0.183 +/- 0.124 mu m in DM + PAD, and an increase in circularity. Altered RBC membrane fluidity is correlated to a spatial reconfiguration of LC domains, which, by possibly altering metabolic function, are associated with the development of T2DM-related macroangiopathic complications

HOXB7 expression by myeloma cells regulates their pro-angiogenic properties in multiple myeloma patients

The deregulation of the homeobox genes as homeoboxB (HOXB)-7 has been previously associated to tumor progression and angiogenesis; here we investigated the potential role of HOXB7 in the pro-angiogenic properties of multiple myeloma (MM) cells. We found that HOXB7 was expressed in 10 out of 22 MM patients analyzed at the diagnosis related to high bone marrow angiogenesis and overexpressed in about 40% of myeloma cell lines compared with normal plasma cells. Enforced HOXB7 expression in MM cells by a lentiviral vector significantly modified their transcriptional and angiogenic profile, checked by combined microarray and angiogenesis PCR analyses, upregulating VEGFA, FGF2, MMP2, WNT5a and PDGFA and downregulating thrombospoindin-2. The pro- and anti-angiogenic HOXB7-related gene signature was also validated in a large independent dataset of MM patients. Accordingly, MM-induced vessel formation was significantly increased by HOXB7 overexpression both in vitro angiogenic and chorioallantoic membrane assays, as well as the HOXB7 silencing by small interfering RNA inhibited the production of angiogenic factors, and the pro-angiogenic properties of MM cells. Finally, in SCID-NOD mice we confirmed that HOXB7 overexpression by MM cells stimulated tumor growth, increased MM-associated angiogenesis and the expression of pro-angiogenic genes by microarray analysis supporting the critical role of HOXB7 in the angiogenic switch in M

The Pharmacological Blockade of Phosphodiesterase IV Potentiates the Anti-Tumor Effects of Arsenic Trioxide (ATO) in Acute Myelogenous Leukemia Cells through Multiple Signaling Pathways

Preclinical in vitro and in vivo studies showed that Arsenic Trioxide (ATO) has antileukemic effects both as a single agent and in combination with conventional therapies or with other molecularly targeted agents . Furthermore, analogs of cAMP have been shown to enhance the therapeutic effects of ATRA and ATO in APL cells. Because cAMP pathway is modulated by various Inhibitiors of phosphodiesterase IV (PDEIV), some of which are already in the clinics for the treatment of asthma, the aim of this study was to investigate whether the pharmacological inhibition of PDEIV potentiates the cytodifferentiating and/or proapoptotic activities of ATO in acute myelogenous leukemia (AML) cells. We found that PDEIV inhibitors induced the upregulation of the myeloid markers (CD38 and CD11b), and potentiated the cytodifferentiating action of ATO in APL cells via induction of cAMP; the cytodifferentiating effects of the PDEIV inhibitors /ATO combination in leukemic cells was also confirmed by nitro blue tetrazolium reductase activity assay. Accordingly, we also found that the inhibition of PDEIV accelerated the ATO-induced degradation of PML-retinoic acid receptor α (PML-RARα) oncoprotein in NB4 cells, thus removing the PML-RARα-induced maturation blocks. Furthermore we also demonstrated that PDEIV inhibitors synergized with ATO to induce apoptosis in both APL NB4 and non-APL HL-60, MOLM-13 and OCI-AML-3 AML cell lines and significantly (P<.01) potentiated the ATO-induced cell death of fresh purified leukemic blasts in 8 out of 10 AML patients analyzed with different genetic abnormalities; in contrast, no significant cytotoxicity in peripheral blood mononuclear cells from 3 healthy volunteers was observed after PDEIV inhibitors/ATO treatment. We consistently found that the inhibition of PDEIV strongly potentiated the ATO-induced activation of caspase-3 and PARP fragmentation in all the tested cell lines as well as in primary AML blasts from patients, and, by using a peptide inhibitor approach, we demonstrated that caspase activity was indispensable for PDEIV inhibitors / ATO-induced apoptosis. Furthermore, siRNA knockdown of endogenous PDE4A and PDE4B gene expression recapitulated the ability of PDEIV inhibitors to sensitize leukemic cells to ATO, thereby confirming the significant role of these enzymes in mediating ATO sensitization of leukemic cells to PDEIV inhibitors /ATO-induced apoptosis. Interestingly, by using cAMP analogs and cAMP competitors we demonstrated that PDEIV inhibitors /ATO -induced apoptosis was cAMP independent. In order to investigate the molecular effectors involved in PDEIV inhibitors /ATO-induced apoptosis we first evaluated the effects of the combined treatment on Bcl-2 and p53 family proteins and their biologic relevance in the synergism observed between PDEIV inhibitors and ATO. By western blot analysis we demonstrated that co-treatment with PDEIV inhibitors and ATO increased protein expression of pro-apoptotic of Bax, Bak and/or Puma and decrease anti-apoptotic Mcl-1, A1/Bfl1 and/or Bcl-2; in agreement with the biochemical findings indicating the activation of the mitochondrial apoptotic pathway, we found that combined treatment with PDEIV inhibitors and ATO strongly potentiated mitochondrial depolarization induced by ATO alone in all the tested cell lines. Preliminary data indicate the involvement of p73 pathway which is consistent with the observed upregulation of Bax, Bak and/or Puma in AML cells; ongoing experiment are in progress to evaluate whether p53 pathway is activated by the combined treatment in MOLM-13 and OCI-AML-3 AML cell lines as well as in fresh purified leukemic blasts. Additionally, we found that co-treatment with PDEIV inhibitors and ATO also induced prosurvival signals by increasing ERK and Bad phosphorylation and the expression of anti-apoptotic Bcl-xL, and the pharmacological or genetic disruption of ERK or Bcl-xL consistently and significantly potentiated the cytotoxic effects of the combination PDEIV inhibitors /ATO. Altogether these findings suggest to extend the study to more patients and strongly support the rationale for testing these promising combinations in clinically relevant in vivo mouse models of drug-resistant leukemia

Unsupervised Clustering of Heartbeat Dynamics Allows for Real Time and Personalized Improvement in Cardiovascular Fitness

VO2max index has a significant impact on overall health. Its estimation through wearables notifies the user of his level of fitness but cannot provide a detailed analysis of the time intervals in which heartbeat dynamics are changed and/or fatigue is emerging. Here, we developed a multiple modality biosignal processing method to investigate running sessions to characterize in real time heartbeat dynamics in response to external energy demand. We isolated dynamic regimes whose fraction increases with the VO2max and with the emergence of neuromuscular fatigue. This analysis can be extremely valuable by providing personalized feedback about the user’s fitness level improvement that can be realized by developing personalized exercise plans aimed to target a contextual increase in the dynamic regime fraction related to VO2max increase, at the expense of the dynamic regime fraction related to the emergence of fatigue. These strategies can ultimately result in the reduction in cardiovascular ris