N-acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages.

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N-acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA.

Shedding Light on the Pathogenesis of Splanchnic Vein Thrombosis.

Splanchnic vein thrombosis is a rare but potentially life-threatening manifestation of venous thromboembolism, with challenging implications both at the pathological and therapeutic level. It is frequently associated with liver cirrhosis, but it could also be provoked by myeloproliferative disorders, cancer of various gastroenterological origin, abdominal infections and thrombophilia. A portion of splanchnic vein thrombosis is still classified as idiopathic. Here, we review the mechanisms of splanchnic vein thrombosis, including new insights on the role of clonal hematopoiesis in idiopathic SVT pathogenesis, with important implications from the therapeutic standpoint.

P53 vs NF-κB: the role of nuclear factor-kappa B in the regulation of p53 activity and vice versa.

The onco-suppressor p53 is a transcription factor that regulates a wide spectrum of genes involved in various cellular functions including apoptosis, cell cycle arrest, senescence, autophagy, DNA repair and angiogenesis. p53 and NF-κB generally have opposing effects in cancer cells. While p53 activity is associated with apoptosis induction, the stimulation of NF-κB has been demonstrated to promote resistance to programmed cell death. Although the transcription factor NF-κB family is considered as the master regulator of cancer development and maintenance, it has been mainly studied in relation to its ability to regulate p53. This has revealed the importance of the crosstalk between NF-κB, p53 and other crucial cell signaling pathways. This review analyzes the various mechanisms by which NF-κB regulates the activity of p53 and the role of p53 on NF-κB activity.

Inhibition of bromodomain and extra-terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia.

The development of drugs able to target BTK, PI3k-delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non-recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19+ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax-resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first-line therapy in combination with venetoclax and as second-line therapy, after the emergence of venetoclax-resistant clones.

Targeting few to help hundreds: JAK, MAPK and ROCK pathways as druggable targets in atypical chronic myeloid leukemia.

Atypical Chronic Myeloid Leukemia (aCML) is a myeloproliferative neoplasm characterized by neutrophilic leukocytosis and dysgranulopoiesis. From a genetic point of view, aCML shows a heterogeneous mutational landscape with mutations affecting signal transduction proteins but also broad genetic modifiers and chromatin remodelers, making difficult to understand the molecular mechanisms causing the onset of the disease. The JAK-STAT, MAPK and ROCK pathways are known to be responsible for myeloproliferation in physiological conditions and to be aberrantly activated in myeloproliferative diseases. Furthermore, experimental evidences suggest the efficacy of inhibitors targeting these pathways in repressing myeloproliferation, opening the way to deep clinical investigations. However, the activation status of these pathways is rarely analyzed when genetic mutations do not occur in a component of the signaling cascade. Given that mutations in functionally unrelated genes give rise to the same pathology, it is tempting to speculate that alteration in the few signaling pathways mentioned above might be a common feature of pathological myeloproliferation. If so, targeted therapy would be an option to be considered for aCML patients.

Morgana acts as an oncosuppressor in chronic myeloid leukemia.

We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana (+/-) mice spontaneously develop a lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph(+) CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph(+) CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph(+) CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, can overcome suboptimal responses in patients in which morgana is underexpressed.

The non-genomic loss of function of tumor suppressors: an essential role in the pathogenesis of chronic myeloid leukemia chronic phase.

BACKGROUND: Chronic Myeloid Leukemia was always referred as a unique cancer due to the apparent independence from tumor suppressors' deletions/mutations in the early stages of the disease. However, it is now well documented that even genetically wild-type tumor suppressors can be involved in tumorigenesis, when functionally inactivated. In particular, tumor suppressors' functions can be impaired by subtle variations of protein levels, changes in cellular compartmentalization and post-transcriptional/post-translational modifications, such as phosphorylation, acetylation, ubiquitination and sumoylation. Notably, tumor suppressors inactivation offers challenging therapeutic opportunities. The reactivation of an inactive and genetically wild-type tumor suppressor could indeed promote selective apoptosis of cancer cells without affecting normal cells. MAIN BODY: Chronic Myeloid Leukemia (CML) could be considered as the paradigm for non-genomic loss of function of tumor suppressors due to the ability of BCR-ABL to directly promote functionally inactivation of several tumor suppressors. SHORT CONCLUSION: In this review we will describe new insights on the role of FoxO, PP2A, p27, BLK, PTEN and other tumor suppressors in CML pathogenesis. Finally, we will describe strategies to promote tumor suppressors reactivation in CML.

Mechanisms of p53 Functional De-Regulation: Role of the IκB-α/p53 Complex.

TP53 is one of the most frequently-mutated and deleted tumor suppressors in cancer, with a dramatic correlation with dismal prognoses. In addition to genetic inactivation, the p53 protein can be functionally inactivated in cancer, through post-transductional modifications, changes in cellular compartmentalization, and interactions with other proteins. Here, we review the mechanisms of p53 functional inactivation, with a particular emphasis on the interaction between p53 and IκB-α, the NFKBIA gene product.

HAUSP compartmentalization in chronic myeloid leukemia.

INTRODUCTION: PTEN plays an essential role in the pathogenesis of chronic myeloid leukemia. Recently, we have shown that BCR-ABL promotes PTEN nuclear exclusion, through the modulation of HAUSP activity. OBJECTIVES: Here, we investigate HAUSP cellular compartmentalization in primary CML samples. RESULTS: While in normal CD34 positive cells HAUSP is expressed mostly in the nucleus, in CML CD34 cells HAUSP is expressed both in the nuclear bodies and in the cytoplasm. CONCLUSIONS: This observation suggests that HAUSP behaves as a shuttling protein in CML. It can bind to BCR-ABL in the cytosol, where it is phosphorylated on tyrosine residues, and it maintains the proper compartmentalization in the nuclear bodies, where it acts as part of a PML network to regulate PTEN de-ubiquitination.

miR-15a targets the HSP90 co-chaperone Morgana in chronic myeloid leukemia.

Morgana is a ubiquitous HSP90 co-chaperone protein coded by the CHORDC1 gene. Morgana heterozygous mice develop with age a myeloid malignancy resembling human atypical myeloid leukemia (aCML), now renamed MDS/MPN with neutrophilia. Patients affected by this pathology exhibit low Morgana levels in the bone marrow (BM), suggesting that Morgana downregulation plays a causative role in the human malignancy. A decrease in Morgana expression levels is also evident in the BM of a subgroup of Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients showing resistance or an incomplete response to imatinib. Despite the relevance of these data, the mechanism through which Morgana expression is downregulated in patients' bone marrow remains unclear. In this study, we investigated the possibility that Morgana expression is regulated by miRNAs and we demonstrated that Morgana is under the control of four miRNAs (miR-15a/b and miR-26a/b) and that miR-15a may account for Morgana downregulation in CML patients.

Breast tumors interfere with endothelial TRAIL at the premetastatic niche to promote cancer cell seeding.

Endothelial cells (ECs) grant access of disseminated cancer cells to distant organs. However, the molecular players regulating the activation of quiescent ECs at the premetastatic niche (PMN) remain elusive. Here, we find that ECs at the PMN coexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate death receptor 5 (DR5). Unexpectedly, endothelial TRAIL interacts intracellularly with DR5 to prevent its signaling and preserve a quiescent vascular phenotype. In absence of endothelial TRAIL, DR5 activation induces EC death and nuclear factor κB/p38-dependent EC stickiness, compromising vascular integrity and promoting myeloid cell infiltration, breast cancer cell adhesion, and metastasis. Consistently, both down-regulation of endothelial TRAIL at the PMN by proangiogenic tumor-secreted factors and the presence of the endogenous TRAIL inhibitors decoy receptor 1 (DcR1) and DcR2 favor metastasis. This study discloses an intracrine mechanism whereby TRAIL blocks DR5 signaling in quiescent endothelia, acting as gatekeeper of the vascular barrier that is corrupted by the tumor during cancer cell dissemination.

Shedding Light on NF-κB Functions in Cellular Organelles.

NF-κB is diffusely recognized as a transcriptional factor able to modulate the expression of various genes involved in a broad spectrum of cellular functions, including proliferation, survival and migration. NF-κB is, however, also acting outside the nucleus and beyond its ability to binds to DNA. NF-κB is indeed found to localize inside different cellular organelles, such as mitochondria, endoplasmic reticulum, Golgi and nucleoli, where it acts through different partners in mediating various biological functions. Here, we discuss the relationship linking NF-κB to the cellular organelles, and how this crosstalk between cellular organelles and NF-κB signalling may be evaluated for anticancer therapies.

Understanding Aberrant Signaling to Elude Therapy Escape Mechanisms in Myeloproliferative Neoplasms.

Aberrant signaling in myeloproliferative neoplasms may arise from alterations in genes coding for signal transduction proteins or epigenetic regulators. Both mutated and normal cells cooperate, altering fragile balances in bone marrow niches and fueling persistent inflammation through paracrine or systemic signals. Despite the hopes placed in targeted therapies, myeloid proliferative neoplasms remain incurable diseases in patients not eligible for stem cell transplantation. Due to the emergence of drug resistance, patient management is often very difficult in the long term. Unexpected connections among signal transduction pathways highlighted in neoplastic cells suggest new strategies to overcome neoplastic cell adaptation.

Non-Coding RNAs: The "Dark Side Matter" of the CLL Universe.

For many years in the field of onco-hematology much attention has been given to mutations in protein-coding genes or to genetic alterations, including large chromosomal losses or rearrangements. Despite this, biological and clinical needs in this sector remain unmet. Therefore, it is not surprising that recent studies have shifted from coded to non-coded matter. The discovery of non-coding RNAs (ncRNAs) has influenced several aspects related to the treatment of cancer. In particular, in chronic lymphocytic leukemia (CLL) the knowledge of ncRNAs and their contextualization have led to the identification of new biomarkers used to follow the course of the disease, to the anticipation of mechanisms that support resistance and relapse, and to the selection of novel targeted treatment regimens. In this review, we will summarize the main ncRNAs discovered in CLL and the molecular mechanisms by which they are affected and how they influence the development and the progression of the disease.

The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.

Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines; this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting in human activity.

IκBα targeting promotes oxidative stress-dependent cell death.

BACKGROUND: Oxidative stress is a hallmark of many cancers. The increment in reactive oxygen species (ROS), resulting from an increased mitochondrial respiration, is the major cause of oxidative stress. Cell fate is known to be intricately linked to the amount of ROS produced. The direct generation of ROS is also one of the mechanisms exploited by common anticancer therapies, such as chemotherapy. METHODS: We assessed the role of NFKBIA with various approaches, including in silico analyses, RNA-silencing and xenotransplantation. Western blot analyses, immunohistochemistry and RT-qPCR were used to detect the expression of specific proteins and genes. Immunoprecipitation and pull-down experiments were used to evaluate protein-protein interactions. RESULTS: Here, by using an in silico approach, following the identification of NFKBIA (the gene encoding IκBα) amplification in various cancers, we described an inverse correlation between IκBα, oxidative metabolism, and ROS production in lung cancer. Furthermore, we showed that novel IκBα targeting compounds combined with cisplatin treatment promote an increase in ROS beyond the tolerated threshold, thus causing death by oxytosis. CONCLUSIONS: NFKBIA amplification and IκBα overexpression identify a unique cancer subtype associated with specific expression profile and metabolic signatures. Through p65-NFKB regulation, IκBα overexpression favors metabolic rewiring of cancer cells and distinct susceptibility to cisplatin. Lastly, we have developed a novel approach to disrupt IκBα/p65 interaction, restoring p65-mediated apoptotic responses to cisplatin due to mitochondria deregulation and ROS-production.

Targeting the Extracellular HSP90 Co-Chaperone Morgana Inhibits Cancer Cell Migration and Promotes Anticancer Immunity.

HSP90 is secreted by cancer cells into the extracellular milieu, where it exerts protumoral activities by activating extracellular substrate proteins and triggering autocrine signals through cancer cell surface receptors. Emerging evidence indicates that HSP90 co-chaperones are also secreted and may direct HSP90 extracellular activities. In this study, we found that the HSP90 co-chaperone Morgana is released by cancer cells and, in association with HSP90, induces cancer cell migration through TLR2, TLR4, and LRP1. In syngeneic cancer mouse models, a mAb targeting Morgana extracellular activity reduced primary tumor growth via macrophage-dependent recruitment of CD8+ T lymphocytes, blocked cancer cell migration, and inhibited metastatic spreading. Overall, these data define Morgana as a new player in the HSP90 extracellular interactome and suggest that Morgana may regulate HSP90 activity to promote cancer cell migration and suppress antitumor immunity. SIGNIFICANCE: This work suggests the potential therapeutic value of targeting the extracellular HSP90 co-chaperone Morgana to inhibit metastasis formation and enhance the CD8+ T-cell-mediated antitumor immune response.

Tumor Suppressors in Chronic Lymphocytic Leukemia: From Lost Partners to Active Targets.

Tumor suppressors play an important role in cancer pathogenesis and in the modulation of resistance to treatments. Loss of function of the proteins encoded by tumor suppressors, through genomic inactivation of the gene, disable all the controls that balance growth, survival, and apoptosis, promoting cancer transformation. Parallel to genetic impairments, tumor suppressor products may also be functionally inactivated in the absence of mutations/deletions upon post-transcriptional and post-translational modifications. Because restoring tumor suppressor functions remains the most effective and selective approach to induce apoptosis in cancer, the dissection of mechanisms of tumor suppressor inactivation is advisable in order to further augment targeted strategies. This review will summarize the role of tumor suppressors in chronic lymphocytic leukemia and attempt to describe how tumor suppressors can represent new hopes in our arsenal against chronic lymphocytic leukemia (CLL).

A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity.

Rapid and sensitive screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to limit the spread of the global pandemic we are facing. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is currently used for the clinical diagnosis of SARS-CoV-2 infection using nasopharyngeal swabs, tracheal aspirates, or bronchoalveolar lavage (BAL) samples. Despite the high sensitivity of the qRT-PCR method, false negative outcomes might occur, especially in patients with a low viral load. Here, we developed a multiplex qRT-PCR methodology for the simultaneous detection of SARS-CoV-2 genome (N gene) and of the human RNAse P gene as internal control. We found that multiplex qRT-PCR was effective in detecting SARS-Cov-2 infection in human specimens with 100% sensitivity. Notably, patients with few copies of SARS-CoV-2 RNA (<5 copies/reaction) were successfully detected by the novel multiplex qRT-PCR method. Finally, we assessed the efficacy of multiplex qRT-PCR on human nasopharyngeal swabs without RNA extraction. Collectively, our results provide evidence of a novel and reliable tool for SARS-CoV-2 RNA detection in human specimens, which allows the testing capacity to be expanded and the RNA extraction step to be bypassed.