A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth.

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

UMG1/CD3ε-bispecific T-cell engager redirects T-cell cytotoxicity against diffuse large B-cell lymphoma.

UMG1 is a unique epitope of CD43, not expressed by normal cells and tissues of haematopoietic and non-haematopoietic origin, except thymocytes and a minority (<5%) of peripheral blood T lymphocytes. By immunohistochemistry analysis of tissue microarray and pathology slides, we found high UMG1 expression in 20%-24% of diffuse large B-cell lymphomas (DLBCLs), including highly aggressive BCL2high and CD20low cases. UMG1 membrane expression was also found in DLBCL bone marrow-infiltrating cells and established cell lines. Targeting UMG1 with a novel asymmetric UMG1/CD3ε-bispecific T-cell engager (BTCE) induced redirected cytotoxicity against DLBCL cells and was synergistic with lenalidomide. We conclude that UMG1/CD3ε-BTCE is a promising therapeutic for DLBCLs.

Genetic Biomarkers of Sorafenib Response in Patients with Hepatocellular Carcinoma.

The identification of biomarkers for predicting inter-individual sorafenib response variability could allow hepatocellular carcinoma (HCC) patient stratification. SNPs in angiogenesis- and drug absorption, distribution, metabolism, and excretion (ADME)-related genes were evaluated to identify new potential predictive biomarkers of sorafenib response in HCC patients. Five known SNPs in angiogenesis-related genes, including VEGF-A, VEGF-C, HIF-1a, ANGPT2, and NOS3, were investigated in 34 HCC patients (9 sorafenib responders and 25 non-responders). A subgroup of 23 patients was genotyped for SNPs in ADME genes. A machine learning classifier method was used to discover classification rules for our dataset. We found that only the VEGF-A (rs2010963) C allele and CC genotype were significantly associated with sorafenib response. ADME-related gene analysis identified 10 polymorphic variants in ADH1A (rs6811453), ADH6 (rs10008281), SULT1A2/CCDC101 (rs11401), CYP26A1 (rs7905939), DPYD (rs2297595 and rs1801265), FMO2 (rs2020863), and SLC22A14 (rs149738, rs171248, and rs183574) significantly associated with sorafenib response. We have identified a genetic signature of predictive response that could permit non-responder/responder patient stratification. Angiogenesis- and ADME-related genes correlation was confirmed by cumulative genetic risk score and network and pathway enrichment analysis. Our findings provide a proof of concept that needs further validation in follow-up studies for HCC patient stratification for sorafenib prescription.

A Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) for ovarian cancer.

BACKGROUND: Pronectins™ are a new class of fibronectin-3-domain 14th-derived (14Fn3) antibody mimics that can be engineered as bispecific T cell engager (BTCE) to redirect immune effector cells against cancer. We describe here the in vitro and in vivo activity of a Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) against Epithelial Ovarian Cancer (EOC). METHODS: pAXLxCD3ε T-cell mediated cytotoxicity was evaluated by flow cytometry and bioluminescence. pAXLxCD3ε mediated T-cell infiltration, activation and proliferation were assessed by immunofluorescence microscopy and by flow cytometry. Activity of pAXLxCD3ε was also investigated in combination with poly-ADP ribose polymerase inhibitors (PARPi). In vivo antitumor activity of pAXLxCD3ε was evaluated in immunocompromised (NSG) mice bearing intraperitoneal or subcutaneous EOC xenografts and immunologically reconstituted with human peripheral blood mononuclear cells (PBMC). RESULTS: pAXLxCD3ε induced dose-dependent cytotoxicity by activation of T lymphocytes against EOC cells, regardless of their histologic origin. The addition of PARPi to cell cultures enhanced pAXLxCD3ε cytotoxicity. Importantly, in vivo, pAXLxCD3ε was highly effective against EOC xenografts in two different NSG mouse models, by inhibiting the growth of tumor cells in ascites and subcutaneous xenografts. This effect translated into a significantly prolonged survival of treated animals. CONCLUSION: pAXLxCD3ε is an active therapeutics against EOC cells providing a rational for its development as a novel agent in this still incurable disease. The preclinical validation of a first-in-class agent opens the way to the development of a new 14Fn3-based scaffold platform for the generation of innovative immune therapeutics against cancer.

Hit identification of novel small molecules interfering with MALAT1 triplex by a structure-based virtual screening.

Nowadays, RNA is an attractive target for the design of new small molecules with different pharmacological activities. Among several RNA molecules, long noncoding RNAs (lncRNAs) are extensively reported to be involved in cancer pathogenesis. In particular, the overexpression of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of multiple myeloma (MM). Starting from the crystallographic structure of the triple-helical stability element at the 3'-end of MALAT1, we performed a structure-based virtual screening of a large commercial database, previously filtered according to the drug-like properties. After a thermodynamic analysis, we selected five compounds for the in vitro assays. Compound M5, characterized by a diazaindene scaffold, emerged as the most promising molecule enabling the destabilization of the MALAT1 triplex structure and antiproliferative activity on in vitro models of MM. M5 is proposed as a lead compound to be further optimized for improving its affinity toward MALAT1.

The role of brain radiotherapy for EGFR- and ALK-positive non-small-cell lung cancer with brain metastases: a review.

Non-small cell lung cancer (NSCLC) is frequently complicated by central nervous system (CNS) metastases affecting patients' life expectancy and quality. At the present clinical trials including neurosurgery, radiotherapy (RT) and systemic treatments alone or in combination have provided controversial results. CNS involvement is even more frequent in NSCLC patients with EGFR activating mutations or ALK rearrangement suggesting a role of target therapy in the upfront treatment in place of loco-regionals treatments (i.e. RT and/or surgery). So far clinical research has not explored the potential role of accurate brain imaging (i.e. MRI instead of the routine total-body contrast CT and/or PET/CT staging) to identify patients that could benefit of local therapies. Moreover, for patients who require concomitant RT there are no clear guidelines on the timing of intervention with respect to innovative precision medicine approaches with Tyrosine Kinase Inhibitors, ALK-inhibitors and/or immuno-oncological therapies. On this basis the present review describes the therapeutic strategies integrating medical and radiation oncology in patients with metastatic NSCLC (mNSCLC) adenocarcinoma with CNS involvement and EGFR activating mutations or ALK rearrangement.

Exploiting DNA Ligase III addiction of multiple myeloma by flavonoid Rhamnetin.

BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.

The New Microtubule-Targeting Agent SIX2G Induces Immunogenic Cell Death in Multiple Myeloma.

Microtubule-targeting agents (MTAs) are effective drugs for cancer treatment. A novel diaryl [1,2]oxazole class of compounds binding the colchicine site was synthesized as cis-restricted-combretastatin-A-4-analogue and then chemically modified to have improved solubility and a wider therapeutic index as compared to vinca alkaloids and taxanes. On these bases, a new class of tricyclic compounds, containing the [1,2]oxazole ring and an isoindole moiety, has been synthetized, among which SIX2G emerged as improved MTA. Several findings highlighted the ability of some chemotherapeutics to induce immunogenic cell death (ICD), which is defined by the cell surface translocation of Calreticulin (CALR) via dissociation of the PP1/GADD34 complex. In this regard, we computationally predicted the ability of SIX2G to induce CALR exposure by interacting with the PP1 RVxF domain. We then assessed both the potential cytotoxic and immunogenic activity of SIX2G on in vitro models of multiple myeloma (MM), which is an incurable hematological malignancy characterized by an immunosuppressive milieu. We found that the treatment with SIX2G inhibited cell viability by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, we observed the increase of hallmarks of ICD such as CALR exposure, ATP release and phospho-eIF2α protein level. Through co-culture experiments with immune cells, we demonstrated the increase of (i) CD86 maturation marker on dendritic cells, (ii) CD69 activation marker on cytotoxic T cells, and (iii) phagocytosis of tumor cells following treatment with SIX2G, confirming the onset of an immunogenic cascade. In conclusion, our findings provide a framework for further development of SIX2G as a new potential anti-MM agent.

Exploiting MYC-induced PARPness to target genomic instability in multiple myeloma.

Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. Since we previously demonstrated that LIG3-dependent repair is involved in the genomic instability, drug resistance and survival of MM cells, we here investigated the biological relevance of PARP1, a driver component of Alternative-Non Homologous End Joining (Alt-NHEJ) pathway, in MM. We found a significant correlation between higher PARP1 mRNA expression and poor prognosis of MM patients. PARP1 knockdown or its pharmacological inhibition by Olaparib impaired MM cells viability in vitro and was effective against in vivo xenografts of human MM. Anti-proliferative effects induced by PARP1-inhibition were correlated to increase of DNA double-strand breaks, activation of DNA Damage Response (DDR) and finally apoptosis. Importantly, by comparing a gene expression signature of PARP inhibitors (PARPi) sensitivity to our plasma cell dyscrasia (PC) gene expression profiling (GEP), we identified a subset of MM patients which could benefit from PARP inhibitors. In particular, Gene Set Enrichment Analysis (GSEA) suggested that high MYC expression correlates to PARPi sensitivity in MM. Indeed, we identified MYC as promoter of PARP1-mediated repair in MM and, consistently, we demonstrate that cytotoxic effects induced by PARP inhibition are mostly detectable on MYC-proficient MM cells. Taken together, our findings indicate that MYC-driven MM cells are addicted to PARP1 Alt-NHEJ repair, which represents therefore a druggable target in this still incurable disease.

Oleil Hydroxytyrosol (HTOL) Exerts Anti-Myeloma Activity by Antagonizing Key Survival Pathways in Malignant Plasma Cells.

Polyphenols from olive oil are endowed with several biological activities. Chemical modifications have been recently applied to these compounds to improve their therapeutic activity in different pathological settings, including cancer. Herein, we describe the in vitro effects on multiple myeloma (MM) cells of oleil hydroxytyrosol (HTOL), a synthetic fatty ester of natural hydroxytyrosol with oleic acid. HTOL reduced the viability of various human MM cell lines (HMCLs), even when co-cultured with bone marrow stromal cells, triggering ER stress, UPR and apoptosis, while it was not cytotoxic against healthy peripheral blood mononuclear cells or B lymphocytes. Whole-transcriptome profiling of HTOL-treated MM cells, coupled with protein expression analyses, indicate that HTOL antagonizes key survival pathways for malignant plasma cells, including the undruggable IRF4-c-MYC oncogenic axis. Accordingly, c-MYC gain- and loss-of-function strategies demonstrate that HTOL anti-tumor activity was, at least in part, due to c-MYC targeting. Taken together, these findings underscore the anti-MM potential of HTOL, providing the molecular framework for further investigation of HTOL-based treatments as novel anti-cancer agents.

Error-prone DNA repair pathways as determinants of immunotherapy activity: an emerging scenario for cancer treatment.

Defects in DNA repair machinery play a critical role in the pathogenesis and progression of human cancer. When they occur, the tumor cells activate error-prone mechanisms which lead to genomic instability and high mutation rate. These defects represent, therefore, a cancer Achilles'heel which could be therapeutically exploited by the use of DNA damage response inhibitors. Moreover, experimental and clinical evidence indicates that DNA repair deregulation has a pivotal role also in promoting immune recognition and immune destruction of cancer cells. Indeed, immune checkpoint inhibitors have received regulatory approval in tumors characterized by high genomic instability, such as melanomas and lung cancer. Here, we discuss how deregulation of DNA repair, through activation of error-prone mechanisms, increases immune activation against cancer. Finally, we address the potential strategies to use DNA repair components as biomarkers and/or therapeutic targets to empower immune-oncology treatment of human cancer.

Therapeutic vulnerability of multiple myeloma to MIR17PTi, a first-in-class inhibitor of pri-miR-17-92.

The microRNA (miRNA) cluster miR-17-92 is oncogenic and represents a valuable therapeutic target in c-MYC (MYC)-driven malignancies. Here, we developed novel LNA gapmeR antisense oligonucleotides (ASOs) to induce ribonuclease H-mediated degradation of MIR17HG primary transcripts and consequently prevent biogenesis of miR-17-92 miRNAs (miR-17-92s). The leading LNA ASO, MIR17PTi, impaired proliferation of several cancer cell lines (n = 48) established from both solid and hematologic tumors by on-target antisense activity, more effectively as compared with miR-17-92 inhibitors. By focusing on multiple myeloma (MM), we found that MIR17PTi triggers apoptosis via impairment of homeostatic MYC/miR-17-92 feed-forward loops (FFLs) in patient-derived MM cells and induces MYC-dependent synthetic lethality. We show that alteration of a BIM-centered FFL is instrumental for MIR17PTi to induce cytotoxicity in MM cells. MIR17PTi exerts strong in vivo antitumor activity in nonobese diabetic severe combined immunodeficient mice bearing clinically relevant models of MM, with advantageous safety and pharmacokinetic profiles in nonhuman primates. Altogether, MIR17PTi is a novel pharmacological tool to be tested in early-phase clinical trials against MM and other MYC-driven malignancies.

Neratinib after trastuzumab-based adjuvant therapy in patients with HER2-positive breast cancer (ExteNET): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Neratinib, an irreversible tyrosine-kinase inhibitor of HER1, HER2, and HER4, has clinical activity in patients with HER2-positive metastatic breast cancer. We aimed to investigate the efficacy and safety of 12 months of neratinib after trastuzumab-based adjuvant therapy in patients with early-stage HER2-positive breast cancer. METHODS: We did this multicentre, randomised, double-blind, placebo-controlled, phase 3 trial at 495 centres in Europe, Asia, Australia, New Zealand, and North and South America. Eligible women (aged ≥18 years, or ≥20 years in Japan) had stage 1-3 HER2-positive breast cancer and had completed neoadjuvant and adjuvant trastuzumab therapy up to 2 years before randomisation. Inclusion criteria were amended on Feb 25, 2010, to include patients with stage 2-3 HER2-positive breast cancer who had completed trastuzumab therapy up to 1 year previously. Patients were randomly assigned (1:1) to receive oral neratinib 240 mg per day or matching placebo. The randomisation sequence was generated with permuted blocks stratified by hormone receptor status (hormone receptor-positive [oestrogen or progesterone receptor-positive or both] vs hormone receptor-negative [oestrogen and progesterone receptor-negative]), nodal status (0, 1-3, or ≥4), and trastuzumab adjuvant regimen (sequentially vs concurrently with chemotherapy), then implemented centrally via an interactive voice and web-response system. Patients, investigators, and trial sponsors were masked to treatment allocation. The primary outcome was invasive disease-free survival, as defined in the original protocol, at 2 years after randomisation. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00878709. FINDINGS: Between July 9, 2009, and Oct 24, 2011, we randomly assigned 2840 women to receive neratinib (n=1420) or placebo (n=1420). Median follow-up time was 24 months (IQR 20-25) in the neratinib group and 24 months (22-25) in the placebo group. At 2 year follow-up, 70 invasive disease-free survival events had occurred in patients in the neratinib group versus 109 events in those in the placebo group (stratified hazard ratio 0·67, 95% CI 0·50-0·91; p=0·0091). The 2-year invasive disease-free survival rate was 93·9% (95% CI 92·4-95·2) in the neratinib group and 91·6% (90·0-93·0) in the placebo group. The most common grade 3-4 adverse events in patients in the neratinib group were diarrhoea (grade 3, n=561 [40%] and grade 4, n=1 [<1%] vs grade 3, n=23 [2%] in the placebo group), vomiting (grade 3, n=47 [3%] vs n=5 [<1%]), and nausea (grade 3, n=26 [2%] vs n=2 [<1%]). QT prolongation occurred in 49 (3%) patients given neratinib and 93 (7%) patients given placebo, and decreases in left ventricular ejection fraction (≥grade 2) in 19 (1%) and 15 (1%) patients, respectively. We recorded serious adverse events in 103 (7%) patients in the neratinib group and 85 (6%) patients in the placebo group. Seven (<1%) deaths (four patients in the neratinib group and three patients in the placebo group) unrelated to disease progression occurred after study drug discontinuation. The causes of death in the neratinib group were unknown (n=2), a second primary brain tumour (n=1), and acute myeloid leukaemia (n=1), and in the placebo group were a brain haemorrhage (n=1), myocardial infarction (n=1), and gastric cancer (n=1). None of the deaths were attributed to study treatment in either group. INTERPRETATION: Neratinib for 12 months significantly improved 2-year invasive disease-free survival when given after chemotherapy and trastuzumab-based adjuvant therapy to women with HER2-positive breast cancer. Longer follow-up is needed to ensure that the improvement in breast cancer outcome is maintained. FUNDING: Wyeth, Pfizer, Puma Biotechnology.

MYD88-independent growth and survival effects of Sp1 transactivation in Waldenstrom macroglobulinemia.

Sp1 transcription factor controls a pleiotropic group of genes and its aberrant activation has been reported in a number of malignancies, including multiple myeloma. In this study, we investigate and report its aberrant activation in Waldenström macroglobulinemia (WM). Both loss of and gain of Sp1 function studies have highlighted a potential oncogenic role of Sp1 in WM. We have further investigated the effect of a small molecule inhibitor, terameprocol (TMP), targeting Sp1 activity in WM. Treatment with TMP inhibited the growth and survival and impaired nuclear factor-κB and signal transducer and activator of transcription activity in WM cells. We next investigated and observed that TMP treatment induced further inhibition of WM cells in MYD88 knockdown WM cells. Moreover, we observed that Bruton's tyrosine kinase, a downstream target of MYD88 signaling pathway, is transcriptionally regulated by Sp1 in WM cells. The combined use of TMP with Bruton's tyrosine kinase or interleukin-1 receptor-associated kinase 1 and 4 inhibitors resulted in a significant and synergistic dose-dependent antiproliferative effect in MYD88-L265P-expressing WM cells. In summary, these results demonstrate Sp1 as an important transcription factor that regulates proliferation and survival of WM cells independent of MYD88 pathway activation, and provide preclinical rationale for clinical development of TMP in WM alone or in combination with inhibitors of MYD88 pathway.

Systematic review and meta-analysis on targeted therapy in advanced pancreatic cancer.

AIM: A systematic review and meta-analysis from literature has been performed to assess the impact of targeted therapy in advanced pancreatic cancer. METHODS: By searching different literature databases and major cancer meetings proceedings, data from all randomized clinical trials designed to investigate molecular targeted agents in the treatment of advanced pancreatic cancer were collected. The time-frame between January 2007 and March 2015 was selected. Data on predefined end-points, including overall survival, progression-free survival in terms of Hazard Ratio and response-rate were extracted and analyzed by a random effects model. Pooled data analysis was performed according to the DerSimonian and Laird test. The occurrence of publication bias was investigated through Begg's test by visual inspection of funnel plots. RESULTS: Twenty-seven randomized clinical trials for a total of 8205 patients were selected and included in the final analysis. A significant benefit was demonstrated for anti-EGFR agents on overall survival (HR = 0.880; 95% confidence interval (CI) 0.797-0.972; p = 0.011). In the pooled analysis no benefit on overall survival (OS: pooled HR = 0.957; 95%CI 0.900-1.017; p = 0.153), or progression-free survival (PFS: pooled HR = 0.908; 95%CI 0.817-1.010; p = 0.075) for targeted-based therapies as compared to conventional treatments could be demonstrated. No advantage was reported in response-rate (OR for RR = 1.210; 95%CI 0.990-1.478; p = 0.063). Begg's funnel plot showed no evidence of publication bias. CONCLUSION: The use of molecular targeted agents does not translate into clinical benefit. Therefore, our work highlights the need to identify predictive factors for patient selection and rationally designed clinical trials.

Phase I trial of thymidylate synthase poly-epitope peptide (TSPP) vaccine in advanced cancer patients.

Thymidylate synthase (TS) poly-epitope peptide (TSPP) is a 27-mer peptide vaccine containing the amino acidic sequences of three epitopes with HLA-A2.1-binding motifs of TS, an enzyme overexpressed in cancer cells, which plays a crucial role in DNA repair and replication. Based on the results of preclinical studies, we designed a phase Ib trial (TSPP/VAC1) to investigate, in a dose escalation setting, the safety and the biological activity of TSPP vaccination alone (arm A) or in combination with GM-CSF and IL-2 (arm B) in cancer patients. Twenty-one pretreated metastatic cancer patients, with a good performance status (ECOG ≤ 1) and no severe organ failure or immunological disease, were enrolled in the study (12 in arm A, nine in arm B) between April 2011 and January 2012, with a median follow-up of 28 months. TSPP resulted safe, and its maximal tolerated dose was not achieved. No grade 4 toxicity was observed. The most common adverse events were grade 2 dermatological reactions to the vaccine injection, cough, rhinitis, fever, poly-arthralgia, gastro-enteric symptoms and, to a lesser extent, moderate hypertension and hypothyroidism. We detected a significant rise in auto-antibodies and TS-epitope-specific CTL precursors. Furthermore, TSPP showed antitumor activity in this group of pretreated patients; indeed, we recorded one partial response and seven disease stabilizations (SD) in arm A, and three SD in arm B. Taken together, our findings provide the framework for the evaluation of the TSPP anti-tumor activity in further disease-oriented clinical trials.

A p53-dependent tumor suppressor network is induced by selective miR-125a-5p inhibition in multiple myeloma cells.

The analysis of deregulated microRNAs (miRNAs) is emerging as a novel approach to disclose the regulation of tumor suppressor or tumor promoting pathways in tumor cells. Targeting aberrantly expressed miRNAs is therefore a promising strategy for cancer treatment. By miRNA profiling of primary plasma cells from multiple myeloma (MM) patients, we previously reported increased miR-125a-5p levels associated to specific molecular subgroups. On these premises, we aimed at investigating the biological effects triggered by miR-125a-5p modulation in MM cells. Expression of p53 pathway-related genes was down-regulated in MM cells transfected with miR-125a-5p mimics. Luciferase reporter assays confirmed specific p53 targeting at 3'UTR level by miR-125a-5p mimics. Interestingly, bone marrow stromal cells (BMSCs) affected the miR-125a-5p/p53 axis, since adhesion of MM cells to BMSCs strongly up-regulated miR-125a-5p levels, while reduced p53 expression. Moreover, ectopic miR-125a-5p reduced, while miR-125-5p inhibitors promoted, the expression of tumor suppressor miR-192 and miR-194, transcriptionally regulated by p53. Lentiviral-mediated stable inhibition of miR-125a-5p expression in wild-type p53 MM cells dampened cell growth, increased apoptosis and reduced cell migration. Importantly, inhibition of in vitro MM cell proliferation and migration was also achieved by synthetic miR-125a-5p inhibitors and was potentiated by the co-expression of miR-192 or miR-194. Taken together, our data indicate that miR-125a-5p antagonism results in the activation of p53 pathway in MM cells, underlying the crucial role of this miRNA in the biopathology of MM and providing the molecular rationale for the combinatory use of miR-125a inhibitors and miR-192 or miR-194 mimics for MM treatment.

Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma.

The Hedgehog (Hh) pathway is required for cell-fate determination during the embryonic life, as well as cell growth and differentiation in the adult organism, where the inappropriate activation has been implicated in several cancers. Here we demonstrate that Hh signaling plays a significant role in growth and survival of multiple myeloma (MM) cells. We observed that CD138(+) MM cells express Hh genes and confirmed Smoothened (Smo)-dependent Hh signaling in MM using a novel synthetic Smo inhibitor, NVP-LDE225 (Novartis), which decreased MM cell viability by inducing specific down-regulation of Gli1 and Ptch1, hallmarks of Hh activity. In addition, we detected a nuclear localization of Gli1 in MM cells, which is completely abrogated by Forskolin, a Gli1-modulating compound, confirming Smo-independent mechanisms leading to Hh activation in MM. Finally, we identified that bone marrow stromal cells are a source of the Shh ligand, although they are resistant to the Hh inhibitor because of defective Smo expression and Ptch1 up-regulation. Further in vitro as well as in vivo studies showed antitumor efficacy of NVP-LDE225 in combination with bortezomib. Altogether, our data demonstrate activation of both canonical and noncanonical Hh pathway in MM, thus providing the rationale for testing Hh inhibitors in clinical trials to improve MM patient outcome.

Gut Microbiota Are a Novel Source of Biomarkers for Immunotherapy in Non-Small-Cell Lung Cancer (NSCLC).

Despite the recent availability of immune checkpoint inhibitors, not all patients affected by Non-Small-Cell Lung Cancer (NSCLC) benefit from immunotherapy. The reason for this variability relies on a variety of factors which may allow for the identification of novel biomarkers. Presently, a variety of biomarkers are under investigation, including the PD1/PDL1 axis, the tumor mutational burden, and the microbiota. The latter is made by all the bacteria and other microorganisms hosted in our body. The gut microbiota is the most represented and has been involved in different physiological and pathological events, including cancer. In this light, it appears that all conditions modifying the gut microbiota can influence cancer, its treatment, and its treatment-related toxicities. The aim of this review is to analyze all the conditions influencing the gut microbiota and, therefore, affecting the response to immunotherapy, iRAEs, and their management in NSCLC patients. The investigation of the landscape of these biological events can allow for novel insights into the optimal management of NSCLC immunotherapy.