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T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and often incurable disease. To uncover therapeutic vulnerabilities, we first developed T-ALL patient–derived tumor xenografts (PDXs) and exposed PDX cells to a library of 433 clinical-stage compounds in vitro. We identified 39 broadly active drugs with antileukemia activity. Because endothelial cells (ECs) can alter drug responses in T-ALL, we developed an EC/T-ALL coculture system. We found that ECs provide protumorigenic signals and mitigate drug responses in T-ALL PDXs. Whereas ECs broadly rescued several compounds in most models, for some drugs the rescue was restricted to individual PDXs, suggesting unique crosstalk interactions and/or intrinsic tumor features. Mechanistically, cocultured T-ALL cells and ECs underwent bidirectional transcriptomic changes at the single-cell level, highlighting distinct “education signatures.” These changes were linked to bidirectional regulation of multiple pathways in T-ALL cells as well as in ECs. Remarkably, in vitro EC-educated T-ALL cells transcriptionally mirrored ex vivo splenic T-ALL at single-cell resolution. Last, 5 effective drugs from the 2 drug screenings were tested in vivo and shown to effectively delay tumor growth and dissemination thus prolonging overall survival. In sum, we developed a T-ALL/EC platform that elucidated leukemia-microenvironment interactions and identified effective compounds and therapeutic vulnerabilities.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors. ispartof: MOLECULAR CANCER THERAPEUTICS vol:20 issue:12 pages:2317-2328 ispartof: location:United States status: published

In the hemato-oncology field, remarkable scientific progress has been achieved, primarily propelled by the discovery of new technologies, improvement in genomics, and novel in vitro and in vivo models. The establishment of multiple cell line collections and the development of instrumental mouse models enhanced our ability to discover effective therapeutics. However, cancer models that faithfully mimic individual cancers are still imperfect. Patient-derived tumor xenografts (PDTXs) have emerged as a powerful tool for identifying the mechanisms which drive tumorigenesis and for testing potential therapeutic interventions. The recognition that PDTXs can maintain many of the donor samples’ properties enabled the development of new strategies for discovering and implementing therapies. Described in this article are protocols for the generation and characterization of lymphoma PDTXs that may be used as the basis of shared procedures. Universal protocols will foster the model utilization, enable the integration of public and private repositories, and aid in the development of shared platforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Tissue handling and cryopreservation of primary and PDTX samples. Basic Protocol 2: Performing tumor implant in immunocompromised mice PDTX models. Alternate Protocol 1: Intra-medullary femoral injection. Alternate Protocol 2: Intravenous injection. Alternate Protocol 3: Intraperitoneal injection. Support Protocol 1: Phenotypical characterization of PDTXs by flow cytometry. Support Protocol 2: Biological and molecular characterization of PDTX tumors by PCR detection of IGK, IGH, and TCR rearrangements. Basic Protocol 3: Harvesting PDTX-derived tumor cells for ex vivo experiments. Basic Protocol 4: In vivo testing of multiple compounds in a PDTX mouse model.

Breast implant-associated lymphoma (BIA-ALCL) has recently been recognized as an independent peripheral T-cell lymphoma (PTCL) entity. In this study, we generated the first BIA-ALCL patient-derived tumor xenograft (PDTX) model (IL89) and a matching continuous cell line (IL89_CL#3488) to discover potential vulnerabilities and druggable targets. We characterized IL89 and IL89_CL#3488, both phenotypically and genotypically, and demonstrated that they closely resemble the matching human primary lymphoma. The tumor content underwent significant enrichment along passages, as confirmed by the increased variant allele frequency (VAF) of mutations. Known aberrations (JAK1 and KMT2C) were identified, together with novel hits, including PDGFB, PDGFRA, and SETBP1. A deep sequencing approach allowed the detection of mutations below the Whole Exome Sequencing (WES) sensitivity threshold, including JAK1G1097D, in the primary sample. RNA sequencing confirmed the expression of a signature of differentially expressed genes in BIA-ALCL. Next, we tested IL89&rsquo, s sensitivity to the JAK inhibitor ruxolitinib and observed a potent anti-tumor effect, both in vitro and in vivo. We also implemented a high-throughput drug screening approach to identify compounds associated with increased responses in the presence of ruxolitinib. In conclusion, these new IL89 BIA-ALCL models closely recapitulate the primary correspondent lymphoma and represent an informative platform for dissecting the molecular features of BIA-ALCL and performing pre-clinical drug discovery studies, fostering the development of new precision medicine approaches.

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models., T- and NK-cell lymphomas (TCL) are a group of lymphoid malignancies characterized by poor prognosis, but the absence of appropriate pre-clinical models has hampered the development of effective therapies. Here the authors establish several pre-clinical models and identify vulnerabilities that could be further exploited to treat patients afflicted by these diseases.

Background. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with few innovative treatment options. This is also contributed by the lack of models capable of capturing the complexity of the tumor and its microenvironment. Aims. To identify patient-specific vulnerabilities and novel therapeutic strategies in T-ALL and interrogate the mechanisms of the crosstalk between leukemic and stromal elements. Methods. We established a drug-testing platform using patient-derived-tumor-xenografts (PDTX) and a mixed-culture approach using E4ORF1-transduced endothelial cells (ECs) (Seandel M et al, PNAS 2008) to overcome host-mediated chemoresistance. We performed functional experiments using total and single-cell RNA sequencing. Results. First, we established a battery of 22 T-ALL PDTX models that matched both phenotypically (immune-histochemistry, flow cytometry) and genotypically (TCR rearrangement, transcriptome) with the primary patients' samples. We then challenged these models (n=14 samples belonging to different PDTX and serial passages within each model) with a library of compounds (n=433) targeting redundant proteins (n=634). Unsupervised clustering and Principal Component Analysis (PCA) demonstrated two clusters of T-ALL samples based on differential drug susceptibility. We could at least partially correlate these differences to specific transcriptomic signatures predictive of drug response (Figure 1A). We then defined a group of pan-active compounds across all models (n=40), which we validated using an independent screening with/without ECs (Figure 1B). We found that ECs counteracted the activity of selected compounds (i.e. TSA, THZ1 and MLN2238). By PCA, we observed distinct response profiles based on different T-ALL models. We vectorized the EC-rescue and found that the direction was the same across all 3 models tested, indicating that it relied on similar mechanisms regardless of model identity. Based on the known role of IGF1-IGFR1 as a supportive EC-rescue axis (Medyouf H et al, J Exp Med 2011), we performed the same screening with/without recombinant IGFBP-7 (500 ng/mL), a decoy IGF1 molecule. Remarkably, IGFBP-7 completely or partially abrogated the EC-mediated rescue of selected compounds [enzastaurin (PKC-β inhibitor), SC144 (GP130 inhibitor), CHIR124 (Chk1 inhibitor) and YM155 (Survivin inhibitor)] (Figure 1B). Drugs not rescued by ECs (n=30) were considered positive hits and 5 of them (ruxolitinib, tofacitinib, panobinostat, bortezomib, irinotecan) ultimately proved to be effective in vivo in randomized pre-clinical trials either alone or in combination (Figure 1C). Our stepwise endothelial-leukemia platform led to the discovery of "public" and "private" vulnerabilities and the proof-of-principle of prediction-guided in vivo pre-clinical trials. We propose a list of compounds that could be readily translated into T-ALL clinical trials (Figure 1D). We finally proved the validity of our platform using other disease models (i.e. B and T-lymphoma PDTXs). Mechanistically, at single-cell resolution, in vitro interacting T-ALL cells and ECs underwent reciprocal transcriptome changes, with T-ALL shifting towards stemness/undifferentiation and ECs towards tumor-ECs (TECs) phenotypes. Furthermore, in vitro EC-educated T-ALL cells mimicked distinct T-ALL subsets of the leukemic spleen of corresponding PDTX mice (Figure 1E). Conclusions. These data demonstrate that our EC-T-ALL culture system simulates in vivo conditions, offering a robust platform to study drug response, leukemia-host interactions and cell plasticity. This approach will improve the pre-clinical predictability of novel drugs/combinations for T-ALL, as well as for other hematologic malignancies, and propel the development of patient-tailored treatments. Figure 1 Figure 1. Disclosures Melnick: Janssen Pharmaceuticals: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Research Funding; Epizyme: Consultancy; Constellation: Consultancy; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Elemento: AstraZeneca: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Owkin: Consultancy, Other: Current equity holder; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Janssen: Research Funding. Chiaretti: amgen: Consultancy; pfizer: Consultancy; novartis: Consultancy; Incyte: Consultancy. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

Introduction: Immunotherapy is a promising therapeutic intervention for cancer treatment. Activation of the immune system via checkpoint blockade has been shown to produce antitumor responses in patients with both solid and hematological tumors. However, many patients do not respond to checkpoint inhibitors, and additional therapies are needed to treat these patients. Testing immunotherapies requires a functional human immune system; thus, it is difficult to evaluate their effectiveness using conventional experimental models. For this reason, establishing in vivo models that closely reproduce not only human tumors, but also their interactions with the human immune system, has become mandatory. Methods: We developed a humanized mouse model and combined it with a patient-derived tumor xenograft (PDTX). Humanized mice (HuMice) were generated by transplantation of cord blood or mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells into preconditioned immunodeficient mice. We compared human engraftment in 3 different mouse strains: NSG (NOD.Cg-Prkdc scidIl2rg tm1Wjl/SzJ), NSGS (NOD.Cg-Prkdc scidIl2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) and NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl/ThomJ). Immune cell profiling and distribution was performed using flow cytometry and immunohistochemistry. The B cell receptor (BCR) repertoire was evaluated using an RNA-based NGS assay. To evaluate the maturation and functionality of T cells developing in HuMice we performed proliferation, degranulation and intracellular cytokine staining. Results: Two months after CD34+ cell transplantation, we observed high levels of human hematopoietic chimerism in all the 3 strains. NSGS mice supported high-level chimerism as early as 1 month after transplantation, with more than 25% of human CD45+ cells in the blood. In all mice the majority of human circulating leukocytes were CD19+ B cells. An early appearance of CD3+ human T cells was detected in NSGS mice as compared to the other strains. Notably, the T cell expansion correlated with a decrease in relative B cell abundance while the myeloid cell contribution to the graft remained steady. We documented the differentiation of CD4+ and CD8+ human T cells at a 2:1 ratio. The characterization of the T cell subsets revealed that the majority was represented by CD45RA-CCR7- effector memory cells in both the spleen and the blood of HuMice. Nevertheless, recipient mice did not exhibit overt signs of graft-versus-host disease. We also evaluated the cytotoxic potential of T cells isolated from the spleen of HuMice: ex vivo peptide antigen (i.e. EBV) presentation let to generation of effective and specific cytotoxic T-cells. After assessing a functional human immune system reconstitution in HuMice, we challenged them in vivo with low-passage tumor fragments from a diffuse large B cell lymphoma (DLBCL) PDTX. All tumor implants were successfully engrafted in both HuMice and non-humanized controls. Remarkably, all the 3 HuMice strains showed a significant reduction in the tumor volume and/or eradication compared to matched non-humanized controls. Flow cytometry analysis of the peripheral blood of humanized PDTX revealed that the tumor engraftment elicited a significant expansion of CD3+ T cells and cytotoxic CD8+ lymphocytes. Moreover, tumors developing in HuMice exhibited intermediate to high levels of tumor infiltrating T lymphocytes commingling with the neoplastic B cells, as determined by immunohistochemistry. Large areas of necrosis were often observed in PDTX of HuMice. Infiltrating CD3+ cells were TIGIT, PD-1 and Lag-3 positive, and did not efficiently proliferate ex vivo: all features consistent with an exhaustion phenotype. PDTX of HuMice often displayed larger areas of necrosis. Conclusions: Collectively, our data demonstrate that a robust reconstitution can be achieved in different strains of immunocompromised mice and that HuMice elicit effective anti-lymphoma responses. PDTX HuMice represent a powerful platform to study host-tumor interactions, and to test novel immune-based strategies (CAR-T, bifunctional Abs) and new pharmacological approaches to counteract T-cell exhaustion. Figure 1 Figure 1. Disclosures Scandura: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation: Research Funding; MPN-RF (Foundation): Research Funding; CR&T (Foudation): Research Funding; European Leukemia net: Honoraria, Other: travel fees . Roth: Janssen: Consultancy; Merck: Consultancy.

Peripheral T cell lymphomas (PTCLs) are a heterogeneous group of orphan neoplasms. Despite the introduction of anthracycline-based chemotherapy protocols, with or without autologous haematopoietic transplantation and a plethora of new agents, the progression-free survival of patients with PTCLs needs to be improved. The rarity of these neoplasms, the limited knowledge of their driving defects and the lack of experimental models have impaired clinical successes. This scenario is now rapidly changing with the discovery of a spectrum of genomic defects that hijack essential signalling pathways and foster T cell transformation. This knowledge has led to new genomic-based stratifications, which are being used to establish objective diagnostic criteria, more effective risk assessment and target-based interventions. The integration of genomic and functional data has provided the basis for targeted therapies and immunological approaches that underlie individual tumour vulnerabilities. Fortunately, novel therapeutic strategies can now be rapidly tested in preclinical models and effectively translated to the clinic by means of well-designed clinical trials. We believe that by combining new targeted agents with immune regulators and chimeric antigen receptor-expressing natural killer and T cells, the overall survival of patients with PTCLs will dramatically increase.

The molecular mechanisms leading to the transformation of anaplastic lymphoma kinase negative (ALK−) anaplastic large cell lymphoma (ALCL) have been only in part elucidated. To identify new culprits which promote and drive ALCL, we performed a total transcriptome sequencing and discovered 1208 previously unknown intergenic long noncoding RNAs (lncRNAs), including 18 lncRNAs preferentially expressed in ALCL. We selected an unknown lncRNA, BlackMamba, with an ALK− ALCL preferential expression, for molecular and functional studies. BlackMamba is a chromatin-associated lncRNA regulated by STAT3 via a canonical transcriptional signaling pathway. Knockdown experiments demonstrated that BlackMamba contributes to the pathogenesis of ALCL regulating cell growth and cell morphology. Mechanistically, BlackMamba interacts with the DNA helicase HELLS controlling its recruitment to the promoter regions of cell-architecture-related genes, fostering their expression. Collectively, these findings provide evidence of a previously unknown tumorigenic role of STAT3 via a lncRNA-DNA helicase axis and reveal an undiscovered role for lncRNA in the maintenance of the neoplastic phenotype of ALK−ALCL.

Background: Myeloproliferative neoplasia (MPN) and angioimmunoblastic T-cell lymphoma (AITL) are two distinct hematologic cancers. However, we have shown that patients with both malignancies probably occur with frequencies higher than expected in the background population (1,2). AITL tumors show a high frequency (30-70%) of mutations in RHOA and epigenetic modifier genes such as DNMT3A, TET2 and IDH2. The latter genomic alterations are similar to those found in myeloid diseases such as MPN. This observation has raised questions on whether the two hematologic malignancies may share genomic aberrations (e.g. at progenitor level) suggesting a possible pathogenetic relationship between these diseases. Aim: To further investigate these questions, we explored the mutational landscape in MPN and AITL tumors occurring in the same host. Methods: From a Danish cohort of 97 patients with co-existing MPN and lymphoma identified through the Danish Lymphoma and Pathology Registries and immunohistochemically validated by tertiary center hematopathologists, five patients with MPN and concurrent and/or subsequent AITL were included. Paired DNA samples obtained from lymph node biopsies (AITL component), bone marrow aspirates (MPN component) and saliva specimens (normal reference DNA) were analyzed by whole exome sequencing. A minimum variant allele frequency threshold of 10% was applied. However, mutations in epigenetic modifier genes, RHOA and JAK2, were also included, if found at slightly lower frequencies. Results: Figure 1 shows an overview of the identified mutations. At a VAF% cut-off value of 10 and a median coverage of 30, mutations shared between the MPN and AITL specimens were found within the DNMT3A, TET2, and IDH2 genes. Mutations in the JAK2 gene were identified in all MPN and in some of the AITL samples. NOTCH2 mutations were frequent and some were shared between the MPN and AITL samples. Two patients with essential thrombocytosis and AITL had the same NOTCH2 (C19W) and NCOR1 (K178N) mutation. Most of the identified genomic alterations were inactivating missense mutations. Conclusion: Co-existence of MPN and AITL occurs and may not be a random event. We studied tumor tissues from patients diagnosed with both diseases, and identified recurrent shared and non-shared mutations in samples from both types of cancers. These mutations may contribute to the development of the two malignancies in the same host, partly through common steps at precursor level (e.g. DNMT3A) and partly more downstream at a more lineage-specific level (e.g. IDH2). The functional role of the observed NOTCH2 and NCOR1 mutations is currently under investigation. Holst J et al. Blood 2017, 130 (S1): 1525 Frederiksen H et al. Blood 2011;118(25):6515-6520 Disclosures Tam: Takeda: Consultancy; Paragon Genomics: Consultancy.

The molecular mechanisms leading to the transformation of anaplastic lymphoma kinase negative (ALK

Anaplastic Large Cell Lymphoma (ALCL) is an aggressive subtype of T-cell lymphoma. ALCLs are stratified based on the presence of anaplastic lymphoma kinase (ALK) translocations. ALK negative (ALK-ALCLs) are heterogeneous subtypes characterized by higher aggressiveness and poorer outcome than ALK+ALCL. The molecular and genetic asset of ALK-ALCLs has recently begun to emerge (i.e. JAK/STAT3 activating mutations, DUSP22, TP63, TP53 and IRF4 rearrangements), but an exhaustive picture of the molecular drivers leading to ALK-ALCLs transformation, progression, and immune evasion is still lacking. Long non coding RNAs (LncRNAs) are transcripts longer than 200 nucleotides with different regulatory functions ranging from transcriptional regulation to structural functions, which are emerging as relevant players in many cellular processes including cancer. Using deep RNA-sequencing profiling combined with de novo transcriptome assembly, we explored and validated the potential contribution of non-coding RNAs in a large series of ALCLs. 24 lncRNAs were found specifically enriched in ALCL samples. Among these, a 70Kb chromatin associated lncRNA (BlackMamba) was identified as preferentially associated with the ALK-ALCLs subtypes and was shown to be a target of the JAK/STAT3 signaling. BlackMamba was overexpressed in ALK-ALCL patient samples as well as in patient-derived tumor xenograft (PDTX) models. Its shRNA mediated knockdown (KD) in ALK-ALCL cells reduces cell proliferation and clonogenicity. BlackMamba KD cells also showed a remarkable increase in the number of multinucleated cells (without ploidy alteration) providing evidence that this lncRNA may be required for correct cytokinesis. To better characterize the role of BlackMamba we performed RNA-sequencing profiling in BlackMamba KD ALK-ALCL cells showing that this lncRNA affects primarily the expression of genes involved in cytoskeleton organization and remodeling. Noticeably, the DNA-helicase HELLS emerged among the most relevant BlackMamba target gene in ALK-ALCL cells and patients. Loss of BlackMamba causes profound reduction of HELLS concomitant with a reorganization of chromatin markers (reduction of K4me3 and increase of K27me3) in the HELLS locus. To test whether HELLS enforces BlackMamba-mediated transcription, we silenced HELLS by shRNA approach in ALK-ALCLs. Noticeably, loss of HELLS led to a reduction in cell growth, a delay in the duplication rate and a dramatic drop of clonogenicity potential of ALK-ALCL cells. This phenotype was also associated with an increased number of multinucleated cells, phenocopying the BlackMamba KD cells. We also showed that, HELLS KD causes expression changes in a subset of cytoskeleton-related genes previously identified as BlackMamba targets confirming that HELLS is a crucial mediator of BlackMamba function. Indeed, ectopic over-expression of HELLS in BlackMamba KD cells rescued the cell growth defects induced by the loss of lncRNA mitigated the polynucleation phenotype and restored the baseline expression of BlackMamba target genes. Being established that lncRNAs affect gene expression by recruiting chromatin remodeling complexes to target promoters or enhancers we also investigated whether BlackMamba may associates with HELLS and dictates its chromatin positioning in ALK-ALCL cells. By RNA-immunoprecipitation (RIP) we showed that HELLS binds to BlackMamba at in two distinct regions the 3'-end of the lncRNA. Next, by Chromatin Immunoprecipitation (ChIP) we demonstrated that HELLS is associated to BlackMamba target gene promoters exclusively in ALK-ALCLs in which BlackMamba is expressed. Collectively, these data demonstrate the existence of a new lncRNA-dependent mechanism controlling the recruitment of HELLS on chromatin sites and its expression in lymphomas. The axis BlackMamba-HELLS sustains the neoplastic phenotype of ALK-ALCL representing a potential vulnerability of ALCL cells. Disclosures Merli: Mundipharma: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Sandoz: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Janssen: Honoraria; Takeda: Honoraria, Other: Travel Expenses; Gilead: Honoraria.

Purpose of review Contrast-induced acute kidney injury (CI-AKI) accounts for approximately 10% of all causes of hospital-acquired renal failure, causes a prolonged in-hospital stay, and represents a powerful predictor of poor early and late outcome. Here, we highlight endpoints used to assess major strategies to prevent CI-AKI. Recent findings A general consensus exists on the beneficial prophylactic effect of hydration. This seems to act by increasing urine flow rate and, thereby, by limiting the time of contact between the contrast media and the epithelial tubular cells. On the contrary, both observational trials and randomized studies are often controversial in their conclusions on the efficacy of several drugs tested to prevent CI-AKI. Compounds evaluated include diuretics (furosemide), antioxidants (i.e., N-acetylcysteine and statins), and vasodilators (i.e., calcium antagonists, dopamine, and fenoldopam). Due to the negative and/or controversial clinical results, none of these drugs has been currently recommended to prevent CI-AKI. Conclusion More reliable markers of acute kidney injury and new prophylactic strategies are warranted to prevent the incidence of CI-AKI.

Cancer-associated fibroblasts (CAFs) are the major components of the tumor microenvironment. They may drive tumor progression, although the mechanisms involved are still poorly understood. Exosomes have emerged as important mediators of intercellular communication in cancer. They mediate horizontal transfer of microRNAs (miRs), mRNAs and proteins, thus affecting breast cancer progression. Differential expression profile analysis identified three miRs (miRs -21, -378e, and -143) increased in exosomes from CAFs as compared from normal fibroblasts. Immunofluorescence indicated that exosomes may be transferred from CAFs to breast cancer cells, releasing their cargo miRs. Breast cancer cells (BT549, MDA-MB-231, and T47D lines) exposed to CAF exosomes or transfected with those miRs exhibited a significant increased capacity to form mammospheres, increased stem cell and epithelial-mesenchymal transition (EMT) markers, and anchorage-independent cell growth. These effects were reverted by transfection with anti-miRs. Similarly to CAF exosomes, normal fibroblast exosomes transfected with miRs -21, -378e, and -143 promoted the stemness and EMT phenotype of breast cancer cells. Thus, we provided evidence for the first time of the role of CAF exosomes and their miRs in the induction of the stemness and EMT phenotype in different breast cancer cell lines. Indeed, CAFs strongly promote the development of an aggressive breast cancer cell phenotype.

// Assunta Adamo 1 , Danilo Fiore 1 , Fabio De Martino 1 , Giuseppina Roscigno 1, 2 , Alessandra Affinito 1 , Elvira Donnarumma 3 , Ilaria Puoti 1 , Lucia Ricci Vitiani 4 , Roberto Pallini 5 , Cristina Quintavalle 1 , Gerolama Condorelli 1, 2 1 Department of Molecular Medicine and Medical Biotechnology, “Federico II” University of Naples, Naples, Italy 2 IEOS, CNR, Naples, Italy 3 IRCCS-SDN, Naples, Italy 4 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita, Rome, Italy 5 Institute of Neurosurgery, Universita Cattolica del Sacro Cuore, Rome, Italy Correspondence to: Gerolama Condorelli, email: gecondor@unina.it Keywords: glioblastoma, stem cells, ryk, β-catenin Received: September 06, 2016 Accepted: December 27, 2016 Published: January 09, 2017 ABSTRACT Glioblastoma multiforme (GBM) is characterized by a strong self-renewal potential and a poor differentiation state. Since receptor-like tyrosine kinase (RYK) activates the WNT/β-catenin pathway essential for cancer stem cell maintenance, we evaluated its contribution in conferring stemness to GBM cells. Here, we report that Ryk (related-to-receptor tyrosine kinase), an atypical tyrosine kinase receptor, is upregulated in samples from GBM patients as well as in GSCs. Ryk overexpression confers stemness properties to GBM cells through the modulation of the canonical Wnt signaling and by promoting the activation of pluripotency-related transcription factor circuitry and neurosphere formation ability. In contrast, siRNA-mediated knockdown of Ryk expression suppresses this stem-like phenotype. Rescue experiments reveal that stemness-promoting activity of Ryk is attributable, at least in part, to β-catenin stabilization. Furthermore, Ryk overexpression improves cell motility and anchorage independent cell growth. Taken together, our findings demonstrate that Ryk promotes stem cell-like and tumorigenic features to glioma cells its essential for the maintenance of GSCs and could be a target of novel therapies.

Background— The role of statins in the prevention of contrast-induced acute kidney injury (CIAKI) is controversial. Methods and Results— First, we investigated the in vivo effects of atorvastatin on CIAKI. Patients with chronic kidney disease enrolled in the Novel Approaches for Preventing or Limiting Events (NAPLES) II trial were randomly assigned to (1) the atorvastatin group (80 mg within 24 hours before contrast media [CM] exposure; n=202) or (2) the control group (n=208). All patients received a high dose of N -acetylcysteine and sodium bicarbonate solution. Second, we investigated the in vitro effects of atorvastatin pretreatment on CM-mediated modifications of intracellular pathways leading to apoptosis or survival in renal tubular cells. CIAKI (ie, an increase >10% of serum cystatin C concentration within 24 hours after CM exposure) occurred in 9 of 202 patients in the atorvastatin group (4.5%) and in 37 of 208 patients in the control group (17.8%) ( P =0.005; odds ratio=0.22; 95% confidence interval, 0.07–0.69). CIAKI rate was lower in the atorvastatin group in both diabetics and nondiabetics and in patients with moderate chronic kidney disease (estimated glomerular filtration rate, 31–60 mL/min per 1.73 m 2 ). In the in vitro model, pretreatment with atorvastatin (1) prevented CM-induced renal cell apoptosis by reducing stress kinases activation and (2) restored the survival signals (mediated by Akt and ERK pathways). Conclusions— A single high loading dose of atorvastatin administered within 24 hours before CM exposure is effective in reducing the rate of CIAKI. This beneficial effect is observed only in patients at low to medium risk.

Glioblastoma is the most frequent brain tumor in adults and is the most lethal form of human cancer. Despite the improvements in treatments, survival of patients remains poor. To define novel pathways that regulate susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in glioma, we have performed genome-wide expression profiling of microRNAs (miRs). We show that in TRAIL-resistant glioma cells, levels of different miRs are increased, and in particular, miR-30b/c and -21. We demonstrate that these miRs impair TRAIL-dependent apoptosis by inhibiting the expression of key functional proteins. T98G-sensitive cells treated with miR-21 or -30b/c become resistant to TRAIL. Furthermore, we demonstrate that miR-30b/c and miR-21 target respectively the 3' untranslated region of caspase-3 and TAp63 mRNAs, and that those proteins mediate some of the effects of miR-30 and -21 on TRAIL resistance, even in human glioblastoma primary cells and in lung cancer cells. In conclusion, we show that high expression levels of miR-21 and -30b/c are needed to maintain the TRAIL-resistant phenotype, thus making these miRs as promising therapeutic targets for TRAIL resistance in glioma.Oncogene advance online publication, 10 September 2012; doi:10.1038/onc.2012.410.

// Giuseppina Roscigno 1, 2 , Cristina Quintavalle 1, 2 , Elvira Donnarumma 3 , Ilaria Puoti 1 , Angel Diaz-Lagares 4 , Margherita Iaboni 1 , Danilo Fiore 1 , Valentina Russo 1 , Matilde Todaro 5 , Giulia Romano 6 , Renato Thomas 7 , Giuseppina Cortino 7 , Miriam Gaggianesi 5 , Manel Esteller 4 , Carlo M. Croce 6 , Gerolama Condorelli 1, 2 1 Department of Molecular Medicine and Medical Biotechnology, “Federico II” University of Naples, Naples, Italy 2 IEOS-CNR, Naples, Italy 3 IRCCS-SDN, Naples, Italy 4 Epigenetic and Cancer Biology Program (PEBC) IDIBELL, Hospital Duran I Reynals, Barcelona, Spain 5 Department of Surgical and Oncological Sciences, Cellular and Molecular Pathophysiology Laboratory, University of Palermo, Palermo, Italy 6 Department of Molecular Virology, Immunology and Medical Genetics, Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 7 Department of Surgical and Oncology, Clinica Mediterranea, Naples, Italy Correspondence to: Gerolama Condorelli, e-mail: gecondor@unina.it Keywords: microRNAs, breast cancer, cancer stem cells, DNMT Received: June 15, 2015 Accepted: October 09, 2015 Published: October 19, 2015 ABSTRACT Cancer stem cells (CSCs) are a small part of the heterogeneous tumor cell population possessing self-renewal and multilineage differentiation potential as well as a great ability to sustain tumorigenesis. The molecular pathways underlying CSC phenotype are not yet well characterized. MicroRNAs (miRs) are small noncoding RNAs that play a powerful role in biological processes. Early studies have linked miRs to the control of self-renewal and differentiation in normal and cancer stem cells. We aimed to study the functional role of miRs in human breast cancer stem cells (BCSCs), also named mammospheres. We found that miR-221 was upregulated in BCSCs compared to their differentiated counterpart. Similarly, mammospheres from T47D cells had an increased level of miR-221 compared to differentiated cells. Transfection of miR-221 in T47D cells increased the number of mammospheres and the expression of stem cell markers. Among miR-221’s targets, we identified DNMT3b. Furthermore, in BCSCs we found that DNMT3b repressed the expression of various stemness genes, such as Nanog and Oct 3/4 , acting on the methylation of their promoters, partially reverting the effect of miR-221 on stemness. We hypothesize that miR-221 contributes to breast cancer tumorigenicity by regulating stemness, at least in part through the control of DNMT3b expression.

Glioblastoma is the most common primary brain tumor in adults; with a survival rate of 12 months from diagnosis. However, a small subgroup of patients, termed long-term survivors (LTS), has a survival rate longer then 12–14 months. There is thus increasing interest in the identification of molecular signatures predicting glioblastoma prognosis and in how to improve the therapeutic approach. Here, we report miR-340 as prognostic tumor-suppressor microRNA for glioblastoma. We analyzed microRNA expression in > 500 glioblastoma patients and found that although miR-340 is strongly down-regulated in glioblastoma overall, it is up-regulated in LTS patients compared to short-term survivors (STS). Indeed, miR-340 expression predicted better prognosis in glioblastoma patients. Coherently, overexpression of miR-340 in glioblastoma cells was found to produce a tumor-suppressive activity. We identified NRAS mRNA as a critical, direct target of miR-340: in fact, miR-340 negatively influenced multiple aspects of glioblastoma tumorigenesis by down-regulating NRAS and downstream AKT and ERK pathways. Thus, we demonstrate that expression of miR-340 in glioblastoma is responsible for a strong tumor-suppressive effect in LTS patients by down-regulating NRAS. miR-340 may thus represent a novel marker for glioblastoma diagnosis and prognosis, and may be developed into a tool to improve treatment of glioblastoma.

Contrast-induced acute kidney injury (CI-AKI) is an impairment of renal function following contrast media administration in the absence of an alternative cause. It represents a powerful predictor of poor early and late outcomes. Here, we review the major strategies to prevent CI-AKI.Hydration represents the gold standard as a prophylactic measure to prevent CI-AKI, acting by increasing urine flow rate and, thereby, by limiting the time of contact between the contrast media and the tubular epithelial cells. An optimal hydration regimen should be defined according to predefined clinical markers, such as urine flow rate, or left ventricular end-diastolic pressure. Recently, high-dose statins pretreatment has been included in the guidelines of CI-AKI prevention. However, uncertainty still exists on the efficacy of several compounds tested in both observational trials and randomized studies to prevent CI-AKI. Compounds evaluated include diuretics (furosemide), antioxidants (i.e. N-acetylcysteine and statins) and vasodilators (i.e. calcium antagonists, dopamine and fenoldopam).Hydration still represents the most reliable strategy to prevent CI-AKI. New prophylactic strategies for acute kidney injury are still under investigation.

Cancer-associated fibroblasts (CAFs), the major components of the tumor microenvironment may drive tumor progression. Exosomes mediate horizontal transfer of miRs, mRNAs and proteins, thus affecting breast cancer progression. Differential expression profile analysis identified three miRs increased in exosomes from CAFs as compared from normal fibroblasts. Immunofluorescence indicated that exosomes may be transferred from CAFs to breast cancer cells, releasing their cargo miRs. Breast cancer cells exposed to CAF exosomes or transfected with those miRs exhibited a significant increased capacity to form mammospheres, increased stem cell and epithelial-mesenchymal transition (EMT) markers, and anchorage-independent cell growth. These effects were reverted by transfection with anti-miRs. Similarly to CAF exosomes, normal fibroblast exosomes transfected with CAFs-miRs promoted the stemness and EMT phenotype of breast cancer cells. Thus, we provided evidence of the role of CAF exosomes and their miRs in the induction of the stemness and EMT phenotype in different breast cancer cell lines. Note: This abstract was not presented at the meeting. Citation Format: Gerolama Condorelli, Elvira Donnarumma, Danilo Fiore, Martina Nappa, Giuseppina Roscigno, Assunta Adamo, Margherita Iaboni, Valentina Russo, Alessandra Affinito, Cristina Quintavalle, Anna Rienzo, Salvatore Piscuoglio, Renato Thomas. Cancer-associated fibroblasts (CAFs) release exosomal microRNAs that dictate an aggressive phenotype in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5933. doi:10.1158/1538-7445.AM2017-5933

Glioblastoma multiforme (GBM) is one of the most deadly types of cancer. To date, the best clinical approach for treatment is based on administration of temozolomide (TMZ) in combination with radiotherapy. Much evidence suggests that the intracellular level of the alkylating enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) impacts response to TMZ in GBM patients. MGMT expression is regulated by the methylation of its promoter. However, evidence indicates that this is not the only regulatory mechanism present. Here, we describe a hitherto unknown microRNA-mediated mechanism of MGMT expression regulation. We show that miR-221 and miR-222 are upregulated in GMB patients and that these paralogues target MGMT mRNA, inducing greater TMZ-mediated cell death. However, miR-221/miR-222 also increase DNA damage and, thus, chromosomal rearrangements. Indeed, miR-221 overexpression in glioma cells led to an increase in markers of DNA damage, an effect rescued by re-expression of MGMT. Thus, chronic miR-221/222-mediated MGMT downregulation may render cells unable to repair genetic damage. This, associated also to miR-221/222 oncogenic potential, may poor GBM prognosis.

Peripheral T-cell lymphomas (PTCL) are malignant and highly aggressive hematologic tumors arising from mature post thymic T-cells. The diagnosis of PTCL includes diverse lymphoma subgroups, altogether accounting for about 15% of all non-Hodgkin lymphomas. Despite much effort in developing reliable diagnostic markers, the diagnosis of PTCLs is challenging and 20-30% of cases are diagnosed as PTCL-NOS (not otherwise specified). This heterogeneous and poorly defined group of lymphomas is frequently characterized by chemotherapy resistance and a very poor prognosis. Here we report the presence of recurrent driver activating genetic alterations in the VAV1 gene in PTCL, NOS. RNA-seq analysis of a comprehensive series of 154 PTCLs and targeted sequencing identified VAV1 gene fusions with different partners including VAV1-THAP4, VAV1-MYO1F and VAV1-S100A7. In all cases the resulting oncoproteins lack the C-terminal SH3 domain of VAV1, a motif implicated in the negative regulation of VAV1 signaling, leading to increased activation of VAV1 catalytic-dependent (MAPK, JNK) and non-catalytic-dependent (NFAT) VAV1 effector pathways. In addition, and most notably, we also identified focal microdeletions at the VAV1 intron 25-exon 26 boundary, which result in the activation of an alternative intraexonic splice acceptor site and the consequent expression of mis-splicing-driven mutant transcripts harboring a recurrent VAV1 Δ778-786 in-frame deletion. Mechanistically, the VAV1 Δ778-786 mutation removes 9 amino acids proximal to the C-terminal VAV1 SH3 domain and induces in increased VAV1 activation and signaling in biochemical assays. In all, these results support a driver role for oncogenic VAV1 signaling in T-cell transformation of major importance for the design of targeted therapies for the treatment of PTCL, NOS. Disclosures No relevant conflicts of interest to declare.

Introduction. T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malignancy associated with a high risk of treatment failure. Efforts to improve outcomes have focused on underlying genetic defects. However, new evidence suggests that the microenvironment can foster drug resistance/relapses. Identification of factors that contribute to microenvironment-mediated chemo-refractoriness remains an important challenge. Here, we sought to construct an in vitro platform to dissect tumor-host interactions and to optimize drug treatments using Patient-Derived Tumor Xenograft models (PDTX) of high risk adult T-ALL and engineered human endothelial cells. Methods. T-ALL PDTX were established and serially passaged in NSG mice. Engraftment was monitored by flow cytometry of peripheral blood and/or MRI. Mice were sacrificed and leukemic cells were harvested from the spleen/bone marrow. To determine the ex vivo growing conditions, we first cultured a panel of 8 "bona fide" T-ALL cell lines and 11 PDTX cells alone in complete RPMI 20% FCS supplemented with IL2, IL12, IL15 and IL7; or co-cultured with human E4-ORF1 endothelial cells (ECs) without ILs in complete RPMI 20% FCS or serum/cytokine-free media. CDK4/6, MEK, PI3K and JAK inhibitors were used at 0.1 and 1 µM alone and in combination. Cell titer glo, cell titer blue, Annexin-V and S-cell cycle analysis were used as readouts. Total RNA from cells before and after co-culture was extracted for paired-end RNA sequencing on an Illumina HiSeq2500. Results. To study the supporting role of ECs, we first co-cultured ECs with T-ALL cell lines in vitro (serum/cytokine free co-culture) and showed that ECs could reproducibly sustain the viability of 3/8 cell lines (Loucy, KOPTK1, P12 Ichikawa) serum/cytokine-free media. A partial rescue was seen with 3 additional lines (HPB-ALL, CCRF-CEM, CUTLL1), while 2 (KE37, DND41) underwent massive cell death. We next tested whether either ILs or CXCL12 could provide anti-apoptotic signals and demonstrated that KOPTK1 and Loucy were only partially rescued by IL15 or CXCL12. Conversely, IL7, although capable of inducing a robust upregulation of pSTAT5, had no effect (CCRF-CEM and CUTLL1). We then characterized 11 PDTX from 15 high-risk adult T-ALL patients. All PDTX were serially propagated and caused T-ALL in subsequent NSG mice (massive spleen and bone marrow infiltration with extensive paravertebral mass associated with paralysis and multi-organ involvement). Genomic analysis (RNA-seq) demonstrated a high concordance between primary (pre-implant) and PDTX samples. All of them were extensively studied ex vivo, demonstratingthat T-ALL PDTX cells could only survive in ILs supplemented media, even better if enriched of growth factors and supplements for the expansion of human hematopoietic cells. However, when PDTX cells were treated with targeting compounds they all underwent massive apoptosis. Conversely, individual PDTX T-ALL could be selectively rescued by ECs, allowing the construction of individual drug response profile. To extend these data, 7 PDX T-ALL samples were screened against a 430-targeted compound library in supplemented RPMI or Stem Span media. Results indicated differential cell killing and gain (NFKB, BTK) and loss (TP-53, IGF-1R) of targets. Conclusions. These data clearly demonstrate a key role of aberrantly activated vascular niche in T-ALL cell maintenance and drug resistance. We envisage that drug screening of EC+T-ALL will lead to the identification of actionable targets in each individual patient. Our report supports the potential for future personalized curative strategies aimed at targeting both tumor cells and host tissue supporting niche elements disrupting pro-tumorigenic signals within leukemia cell niches. Disclosures Foà: Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy; Pfizer: Speakers Bureau; Ariad: Speakers Bureau. Rafii:Angiocrine Bioscience: Equity Ownership, Other: Non-paid consultant.

Contrast-induced nephropathy accounts for 410% of all causes of hospital-acquired renal failure, causes a prolonged in- hospital stay and represents a powerful predictor of poor early and late outcome. Mechanisms of contrast-induced nephropathy are not completely understood. In vitro data suggests that contrast media (CM) induces a direct toxic effect on renal tubular cells through the activation of the intrinsic apoptotic pathway. It is unclear whether this effect has a role in the clinical setting. In this work, we evaluated the effects of CM both in vivo and in vitro. By analyzing urine samples obtained from patients who experienced contrast-induced acute kidney injury (CI-AKI), we verified, by western blot and immunohistochemistry, that CM induces tubular renal cells apoptosis. Furthermore, in cultured cells, CM caused a dose–response increase in reactive oxygen species (ROS) production, which triggered Jun N-terminal kinases (JNK1/2) and p38 stress kinases marked activation and thus apoptosis. Inhibition of JNK1/2 and p38 by different approaches (i.e. pharmacological antagonists and transfection of kinase- death mutants of the upstream p38 and JNK kinases) prevented CM-induced apoptosis. Interestingly, N-acetylcysteine inhibited ROS production, and thus stress kinases and apoptosis activation. Therefore, we conclude that CM-induced tubular renal cells apoptosis represents a key mechanism of CI-AKI.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas frequently associated with poor prognosis and for which genetic mechanisms of transformation remain incompletely understood. Using RNA sequencing and targeted sequencing, here we identify a recurrent in-frame deletion (VAV1 Δ778-786) generated by a focal deletion-driven alternative splicing mechanism as well as novel VAV1 gene fusions (VAV1-THAP4, VAV1-MYO1F, and VAV1-S100A7) in PTCL. Mechanistically these genetic lesions result in increased activation of VAV1 catalytic-dependent (MAPK, JNK) and non-catalytic-dependent (nuclear factor of activated T cells, NFAT) VAV1 effector pathways. These results support a driver oncogenic role for VAV1 signaling in the pathogenesis of PTCL.