A first-in-class Wiskott-Aldrich syndrome protein activator with anti-tumor activity in hematologic cancers.

Hematological cancers are among the most common cancers in adults and children. Despite significant improvements in therapies, many patients still succumb to the disease. Therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family regulates actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations: autoinhibited or activated. Here, we describe the development of EG-011, a first-in-class small molecule activator of the WASp auto-inhibited form. EG-011 possesses in vitro and in vivo anti-tumor activity as a single agent in lymphoma, leukemia, and multiple myeloma, including models of secondary resistance to PI3K, BTK, and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization and WASp binding was demonstrated using multiple techniques. Transcriptome analysis highlighted homology with drugs-inducing actin polymerization.

A Problem Space for Designing Visualizations.

Visualization researchers and visualization professionals seek appropriate abstractions of visualization requirements that permit considering visualization solutions independently from specific problems. Abstractions can help us design, analyze, organize, and evaluate the things we create. The literature has many task structures (taxonomies, typologies, etc.), design spaces, and related "frameworks" that provide abstractions of the problems a visualization is meant to address. In this Visualization Viewpoints article, we introduce a different one, a problem space that complements existing frameworks by focusing on the needs that a visualization is meant to solve. We believe it provides a valuable conceptual tool for designing and discussing visualizations.

The Role of Interactive Visualization in Fostering Trust in AI.

The increasing use of artificial intelligence (AI) technologies across application domains has prompted our society to pay closer attention to AI's trustworthiness, fairness, interpretability, and accountability. In order to foster trust in AI, it is important to consider the potential of interactive visualization, and how such visualizations help build trust in AI systems. This manifesto discusses the relevance of interactive visualizations and makes the following four claims: i) trust is not a technical problem, ii) trust is dynamic, iii) visualization cannot address all aspects of trust, and iv) visualization is crucial for human agency in AI.

Role of the Tyrosine Phosphatase SHP-2 in Mediating Adrenomedullin Proangiogenic Activity in Solid Tumors.

VE-cadherin is an essential adhesion molecule in endothelial adherens junctions, and the integrity of these complexes is thought to be regulated by VE-cadherin tyrosine phosphorylation. We have previously shown that adrenomedullin (AM) blockade correlates with elevated levels of phosphorylated VE-cadherin (pVE-cadherinY731) in endothelial cells, associated with impaired barrier function and a persistent increase in vascular endothelial cell permeability. However, the mechanism underlying this effect is unknown. In this article, we demonstrate that the AM-mediated dephosphorylation of pVE-cadherinY731 takes place through activation of the tyrosine phosphatase SHP-2, as judged by the rise of its active fraction phosphorylated at tyrosine 542 (pSHP-2Y542) in HUVECs and glioblastoma-derived-endothelial cells. Both pre-incubation of HUVECs with SHP-2 inhibitors NSC-87877 and SHP099 and SHP-2 silencing hindered AM-induced dephosphorylation of pVE-cadherinY731 in a dose dependent-manner, showing the role of SHP-2 in the regulation of endothelial cell contacts. Furthermore, SHP-2 inhibition impaired AM-induced HUVECs differentiation into cord-like structures in vitro and impeded AM-induced neovascularization in in vivo Matrigel plugs bioassays. Subcutaneously transplanted U87-glioma tumor xenograft mice treated with AM-receptors-blocking antibodies showed a decrease in pSHP-2Y542 associated with VE-cadherin in nascent tumor vasculature when compared to control IgG-treated xenografts. Our findings show that AM acts on VE-cadherin dynamics through pSHP-2Y542 to finally modulate cell-cell junctions in the angiogenesis process, thereby promoting a stable and functional tumor vasculature.

Development of potent dual PDK1/AurA kinase inhibitors for cancer therapy: Lead-optimization, structural insights, and ADME-Tox profile.

We report the synthesis of novel first-in-class 2-oxindole-based derivatives as dual PDK1-AurA kinase inhibitors as a novel strategy to treat Ewing sarcoma. The most potent compound 12 is suitable for progression to in vivo studies. The specific attributes of 12 included nanomolar inhibitory potency against both phosphoinositide-dependent kinase-1 (PDK1) and Aurora A (AurA) kinase, with acceptable in vitro ADME-Tox properties (cytotoxicity in 2 healthy and 14 hematological and solid cancer cell-lines; inhibition of PDE4C1, SIRT7, HDAC4, HDAC6, HDAC8, HDAC9, AurB, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and hERG). X-ray crystallography and docking studies led to the identification of the key AurA and PDK1/12 interactions. Finally, in vitro drug-intake kinetics and in vivo PK appear to indicate that these compounds are attractive lead-structures for the design and synthesis of PDK1/AurA dual-target molecules to further investigate the in vivo efficacy against Ewing Sarcoma.

Sensor Fusion and Convolutional Neural Networks for Indoor Occupancy Prediction Using Multiple Low-Cost Low-Resolution Heat Sensor Data.

Indoor occupancy prediction is a prerequisite for the management of energy consumption, security, health, and other systems in smart buildings. Previous studies have shown that buildings that automatize their heating, lighting, air conditioning, and ventilation systems through considering the occupancy and activity information might reduce energy consumption by more than 50%. However, it is difficult to use high-resolution sensors and cameras for occupancy prediction due to privacy concerns. In this paper, we propose a novel solution for predicting occupancy using multiple low-cost and low-resolution heat sensors. We suggest two different methods for fusing and processing the data captured from multiple heat sensors and we use a Convolutional Neural Network for predicting occupancy. We conduct experiments to assess both the performance of the proposed solutions and analyze the impact of sensor field view overlaps on the prediction results. In summary, our experimental results show that the implemented solutions show high occupancy prediction accuracy and real-time processing capabilities.

Multi-assignment clustering: Machine learning from a biological perspective.

A common approach for analyzing large-scale molecular data is to cluster objects sharing similar characteristics. This assumes that genes with highly similar expression profiles are likely participating in a common molecular process. Biological systems are extremely complex and challenging to understand, with proteins having multiple functions that sometimes need to be activated or expressed in a time-dependent manner. Thus, the strategies applied for clustering of these molecules into groups are of key importance for translation of data to biologically interpretable findings. Here we implemented a multi-assignment clustering (MAsC) approach that allows molecules to be assigned to multiple clusters, rather than single ones as in commonly used clustering techniques. When applied to high-throughput transcriptomics data, MAsC increased power of the downstream pathway analysis and allowed identification of pathways with high biological relevance to the experimental setting and the biological systems studied. Multi-assignment clustering also reduced noise in the clustering partition by excluding genes with a low correlation to all of the resulting clusters. Together, these findings suggest that our methodology facilitates translation of large-scale molecular data into biological knowledge. The method is made available as an R package on GitLab (https://gitlab.com/wolftower/masc).

Occupancy Prediction Using Low-Cost and Low-Resolution Heat Sensors for Smart Offices.

Solving the challenge of occupancy prediction is crucial in order to design efficient and sustainable office spaces and automate lighting, heating, and air circulation in these facilities. In office spaces where large areas need to be observed, multiple sensors must be used for full coverage. In these cases, it is normally important to keep the costs low, but also to make sure that the privacy of the people who use such environments are preserved. Low-cost and low-resolution heat (thermal) sensors can be very useful to build solutions that address these concerns. However, they are extremely sensitive to noise artifacts which might be caused by heat prints of the people who left the space or by other objects, which are either using electricity or exposed to sunlight. There are some earlier solutions for occupancy prediction that employ low-resolution heat sensors; however, they have not addressed nor compensated for such heat artifacts. Therefore, in this paper, we presented a low-cost and low-energy consuming smart space implementation to predict the number of people in the environment based on whether their activity is static or dynamic in time. We used a low-resolution (8×8) and non-intrusive heat sensor to collect data from an actual meeting room. We proposed two novel workflows to predict the occupancy; one that is based on computer vision and one based on machine learning. Besides comparing the advantages and disadvantages of these different workflows, we used several state-of-the-art explainability methods in order to provide a detailed analysis of the algorithm parameters and how the image properties influence the resulting performance. Furthermore, we analyzed noise resources that affect the heat sensor data. The experiments show that the feature classification based method gives high accuracy when the data are clean from noise artifacts. However, when there are noise artifacts, the computer vision based method can compensate for those artifacts providing robust results. Because the computer vision based method requires an empty room recording, the feature classification based method should be chosen either when there is no expectancy of seeing noise artifacts in the data or when there is no empty recording available. We hope that our analysis brings light into understanding how to handle very low-resolution heat images in these environments. The presented workflows could be used in various domains and applications other than smart offices, where occupancy prediction is essential, e.g., for elderly care.

Targeting Adrenomedullin in Oncology: A Feasible Strategy With Potential as Much More Than an Alternative Anti-Angiogenic Therapy.

The development, maintenance and metastasis of solid tumors are highly dependent on the formation of blood and lymphatic vessels from pre-existing ones through a series of processes that are respectively known as angiogenesis and lymphangiogenesis. Both are mediated by specific growth-stimulating molecules, such as the vascular endothelial growth factor (VEGF) and adrenomedullin (AM), secreted by diverse cell types which involve not only the cancerogenic ones, but also those constituting the tumor stroma (i.e., macrophages, pericytes, fibroblasts, and endothelial cells). In this sense, anti-angiogenic therapy represents a clinically-validated strategy in oncology. Current therapeutic approaches are mainly based on VEGF-targeting agents, which, unfortunately, are usually limited by toxicity and/or tumor-acquired resistance. AM is a ubiquitous peptide hormone mainly secreted in the endothelium with an important involvement in blood vessel development and cardiovascular homeostasis. In this review, we will introduce the state-of-the-art in terms of AM physiology, while putting a special focus on its pro-tumorigenic role, and discuss its potential as a therapeutic target in oncology. A large amount of research has evidenced AM overexpression in a vast majority of solid tumors and a correlation between AM levels and disease stage, progression and/or vascular density has been observed. The analysis presented here indicates that the involvement of AM in the pathogenesis of cancer arises from: 1) direct promotion of cell proliferation and survival; 2) increased vascularization and the subsequent supply of nutrients and oxygen to the tumor; 3) and/or alteration of the cell phenotype into a more aggressive one. Furthermore, we have performed a deep scrutiny of the pathophysiological prominence of each of the AM receptors (AM1 and AM2) in different cancers, highlighting their differential locations and functions, as well as regulatory mechanisms. From the therapeutic point of view, we summarize here an exhaustive series of preclinical studies showing a reduction of tumor angiogenesis, metastasis and growth following treatment with AM-neutralizing antibodies, AM receptor antagonists, or AM receptor interference. Anti-AM therapy is a promising strategy to be explored in oncology, not only as an anti-angiogenic alternative in the context of acquired resistance to VEGF treatment, but also as a potential anti-metastatic approach.

Epigenetic Control of Mitochondrial Fission Enables Self-Renewal of Stem-like Tumor Cells in Human Prostate Cancer.

Cancer stem cells (CSCs) contribute to disease progression and treatment failure in human cancers. The balance among self-renewal, differentiation, and senescence determines the expansion or progressive exhaustion of CSCs. Targeting these processes might lead to novel anticancer therapies. Here, we uncover a novel link between BRD4, mitochondrial dynamics, and self-renewal of prostate CSCs. Targeting BRD4 by genetic knockdown or chemical inhibitors blocked mitochondrial fission and caused CSC exhaustion and loss of tumorigenic capability. Depletion of CSCs occurred in multiple prostate cancer models, indicating a common vulnerability and dependency on mitochondrial dynamics. These effects depended on rewiring of the BRD4-driven transcription and repression of mitochondrial fission factor (Mff). Knockdown of Mff reproduced the effects of BRD4 inhibition, whereas ectopic Mff expression rescued prostate CSCs from exhaustion. This novel concept of targeting mitochondrial plasticity in CSCs through BRD4 inhibition provides a new paradigm for developing more effective treatment strategies for prostate cancer.

Fighter pilots' teamwork: a descriptive study.

The execution of teamwork varies widely depending on the domain and task in question. Despite the considerable diversity of teams and their operation, researchers tend to aim for unified theories and models regardless of field. However, we argue that there is a need for translation and adaptation of the theoretical models to each specific domain. To this end, a case study was carried out on fighter pilots and it was investigated how teamwork is performed in this specialised and challenging environment, with a specific focus on the dependence on technology for these teams. The collaboration between the fighter pilots is described and analysed using a generic theoretical model for effective teamwork from the literature. The results show that domain-specific application and modification is needed in order for the model to capture fighter pilot's teamwork. The study provides deeper understanding of the working conditions for teams of pilots and gives design implications for how tactical support systems can enhance teamwork in the domain. Practitioner summary: This article presents a qualitative interview study with fighter pilots based on a generic theoretical teamwork model applied to the fighter domain. The purpose is to understand the conditions under which teams of fighter pilots work and to provide guidance for the design of future technological aids.

BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance.

BACKGROUND: The outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials. MATERIALS AND METHODS: The activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines. RESULTS: Birabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines. CONCLUSION: Our data provide the rationale to evaluate birabresib in patients affected by MCL.

Bromodomain and extra-terminal domain inhibition modulates the expression of pathologically relevant microRNAs in diffuse large B-cell lymphoma.

Aberrant changes in microRNA expression contribute to lymphomagenesis. Bromodomain and extra-terminal domain inhibitors such as OTX015 (MK-8628, birabresib) have demonstrated preclinical and clinical activity in hematologic tumors. MicroRNA profiling of diffuse large B-cell lymphoma cells treated with OTX015 revealed changes in the expression levels of a limited number of microRNAs, including miR-92a-1-5p, miR-21-3p, miR-155-5p and miR-96-5p. Analysis of publicly available chromatin immunoprecipitation sequencing data of diffuse large B-cell lymphoma cells treated with bromodomain and extra-terminal domain (BET) inhibitors showed that the BET family member BRD4 bound to the upstream regulatory regions of multiple microRNA genes and that this binding decreased following BET inhibition. Alignment of our microRNA profiling data with the BRD4 chromatin immunoprecipitation sequencing data revealed that microRNAs downregulated by OTX015 also exhibited reduced BRD4 binding in their promoter regions following treatment with another bromodomain and extra-terminal domain inhibitor, JQ1, indicating that BRD4 contributes directly to microRNA expression in lymphoma. Treatment with bromodomain and extra-terminal domain inhibitors also decreased the expression of the arginine methyltransferase PRMT5, which plays a crucial role in B-cell transformation and negatively modulates the transcription of miR-96-5p. The data presented here indicate that in addition to previously observed effects on the expression of coding genes, bromodomain and extra-terminal domain inhibitors also modulate the expression of microRNAs involved in lymphomagenesis.

A data analysis framework for biomedical big data: Application on mesoderm differentiation of human pluripotent stem cells.

The development of high-throughput biomolecular technologies has resulted in generation of vast omics data at an unprecedented rate. This is transforming biomedical research into a big data discipline, where the main challenges relate to the analysis and interpretation of data into new biological knowledge. The aim of this study was to develop a framework for biomedical big data analytics, and apply it for analyzing transcriptomics time series data from early differentiation of human pluripotent stem cells towards the mesoderm and cardiac lineages. To this end, transcriptome profiling by microarray was performed on differentiating human pluripotent stem cells sampled at eleven consecutive days. The gene expression data was analyzed using the five-stage analysis framework proposed in this study, including data preparation, exploratory data analysis, confirmatory analysis, biological knowledge discovery, and visualization of the results. Clustering analysis revealed several distinct expression profiles during differentiation. Genes with an early transient response were strongly related to embryonic- and mesendoderm development, for example CER1 and NODAL. Pluripotency genes, such as NANOG and SOX2, exhibited substantial downregulation shortly after onset of differentiation. Rapid induction of genes related to metal ion response, cardiac tissue development, and muscle contraction were observed around day five and six. Several transcription factors were identified as potential regulators of these processes, e.g. POU1F1, TCF4 and TBP for muscle contraction genes. Pathway analysis revealed temporal activity of several signaling pathways, for example the inhibition of WNT signaling on day 2 and its reactivation on day 4. This study provides a comprehensive characterization of biological events and key regulators of the early differentiation of human pluripotent stem cells towards the mesoderm and cardiac lineages. The proposed analysis framework can be used to structure data analysis in future research, both in stem cell differentiation, and more generally, in biomedical big data analytics.

Promising in vivo efficacy of the BET bromodomain inhibitor OTX015/MK-8628 in malignant pleural mesothelioma xenografts.

It has recently been reported that a large proportion of human malignant pleural mesothelioma (MPM) cell lines and patient tissue samples present high expression of the c-MYC oncogene. This gene drives several tumorigenic processes and is overexpressed in many cancers. Although c-MYC is a strategic target to restrain cancer processes, no drugs acting as c-MYC inhibitors are available. The novel thienotriazolodiazepine small-molecule bromodomain inhibitor OTX015/MK-8628 has shown potent antiproliferative activity accompanied by c-MYC downregulation in several tumor types. This study was designed to evaluate the growth inhibitory effect of OTX015 on patient-derived MPM473, MPM487 and MPM60 mesothelioma cell lines and its antitumor activity in three patient-derived xenograft models, MPM473, MPM487 and MPM484, comparing it with cisplatin, gemcitabine and pemetrexed, three agents which are currently used to treat MPM in the clinic. OTX015 caused a significant delay in cell growth both in vitro and in vivo. It was the most effective drug in MPM473 xenografts and showed a similar level of activity as the most efficient treatment in the other two MPM models (gemcitabine in MPM487 and cisplatin in MPM484). In vitro studies showed that OTX015 downregulated c-MYC protein levels in both MPM473 and MPM487 cell lines. Our findings represent the first evidence of promising therapeutic activity of OTX015 in mesothelioma.

The bromodomain inhibitor OTX015 (MK-8628) exerts anti-tumor activity in triple-negative breast cancer models as single agent and in combination with everolimus.

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subgroup of breast tumors clinically defined by the lack of estrogen, progesterone and HER2 receptors, limiting the use of the targeted therapies employed in other breast malignancies. Recent evidence indicates that c-MYC is a key driver of TNBC. The BET-bromodomain inhibitor OTX015 (MK-8628) has potent antiproliferative activity accompanied by c-MYC down-regulation in several tumor types, and has demonstrated synergism with the mTOR inhibitor everolimus in different models. The aim of this study was to evaluate the anti-tumor activity of OTX015 as single agent and in combination with everolimus in TNBC models. OTX015 was assayed in three human TNBC-derived cell lines, HCC1937, MDA-MB-231 and MDA-MB-468, all showing antiproliferative activity after 72 h (GI50 = 75-650 nM). This was accompanied by cell cycle arrest and decreased expression of cancer stem cells markers. However, c-MYC protein and mRNA levels were only down-regulated in MDA-MB-468 cells. Gene set enrichment analysis showed up-regulation of genes involved in epigenetic control of transcription, chromatin and the cell cycle, and down-regulation of stemness-related genes. In vitro, combination with everolimus was additive in HCC1937 and MDA-MB-231 cells, but antagonistic in MDA-MB-468 cells. In MDA-MB-231 murine xenografts, tumor mass was significantly (p < 0.05) reduced by OTX015 with respect to vehicle-treated animals (best T/C = 40.7%). Although everolimus alone was not active, the combination was more effective than OTX015 alone (best T/C = 20.7%). This work supports current clinical trials with OTX015 in TNBC (NCT02259114).

OTX015 (MK-8628), a novel BET inhibitor, exhibits antitumor activity in non-small cell and small cell lung cancer models harboring different oncogenic mutations.

Inhibitors targeting epigenetic control points of oncogenes offer a potential mean of blocking tumor progression in small cell and non-small cell lung carcinomas (SCLC, NSCLC). OTX015 (MK-8628) is a BET inhibitor selectively blocking BRD2/3/4. OTX015 was evaluated in a panel of NSCLC or SCLC models harboring different oncogenic mutations. Cell proliferation inhibition and cell cycle arrest were seen in sensitive NSCLC cells. MYC and MYCN were downregulated at both the mRNA and protein levels. In addition, OTX015-treatment significantly downregulated various stemness cell markers, including NANOG, Musashi-1, CD113 and EpCAM in H3122-tumors in vivo. Conversely, in SCLC models, weak antitumor activity was observed with OTX015, both in vitro and in vivo. No predictive biomarkers of OTX015 activity were identified in a large panel of candidate genes known to be affected by BET inhibition. In NSCLC models, OTX015 was equally active in both EML4-ALK positive and negative cell lines, whereas in SCLC models the presence of functional RB1 protein, which controls cell progression at G1, may be related to the final biological outcome of OTX015. Gene expression profiling in NSCLC and SCLC cell lines showed that OTX015 affects important genes and pathways with a very high overlapping between both sensitive and resistant cell lines. These data support the rationale for the OTX015 Phase Ib (NCT02259114) in solid tumors, where NSCLC patients with rearranged ALK gene or KRAS-positive mutations are currently being treated.

Therapeutic efficacy of the bromodomain inhibitor OTX015/MK-8628 in ALK-positive anaplastic large cell lymphoma: an alternative modality to overcome resistant phenotypes.

Anaplastic large cell lymphomas (ALCL) represent a peripheral T-cell lymphoma subgroup, stratified based on the presence or absence of anaplastic lymphoma kinase (ALK) chimeras. Although ALK-positive ALCLs have a more favorable outcome than ALK-negative ALCL, refractory and/or relapsed forms are common and novel treatments are needed. Here we investigated the therapeutic potential of a novel bromodomain inhibitor, OTX015/MK-8628 in ALK-positive ALCLs.The effects of OTX015 on a panel of ALK+ ALCL cell lines was evaluated in terms of proliferation, cell cycle and downstream signaling, including gene expression profiling analyses. Synergy was tested with combination targeted therapies.Bromodomain inhibition with OTX015 led primarily to ALCL cell cycle arrest in a dose-dependent manner, along with downregulation of MYC and its downstream regulated genes. MYC overexpression did not compensate this OTX015-mediated phenotype. Transcriptomic analysis of OTX015-treated ALCL cells identified a gene signature common to various hematologic malignancies treated with bromodomain inhibitors, notably large cell lymphoma. OTX015-modulated genes included transcription factors (E2F2, NFKBIZ, FOS, JUNB, ID1, HOXA5 and HOXC6), members of multiple signaling pathways (ITK, PRKCH, and MKNK2), and histones (clusters 1-3). Combination of OTX015 with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib led to cell cycle arrest then cell death, and combination with suboptimal doses of the ALK inhibitor CEP28122 caused cell cycle arrest. When OTX015 was associated with GANT61, a selective GLI1/2 inhibitor, C1156Y-resistant ALK ALCL growth was impaired.These findings support OTX015 clinical trials in refractory ALCL in combination with inhibitors of interleukin-2-inducible kinase or SHH/GLI1.

Everolimus affects vasculogenic mimicry in renal carcinoma resistant to sunitinib.

Angiogenesis is hallmark of clear cell renal cell carcinogenesis. Anti-angiogenic therapies have been successful in improving disease outcome; however, most patients treated with anti-angiogenic agents will eventually progress. In this study we report that clear cell renal cell carcinoma was associated with vasculogenic mimicry in both mice and human with tumor cells expressing endothelial markers in the vicinity of tumor vessels. We show that vasculogenic mimicry was efficiently targeted by sunitinib but eventually associated with tumor resistance and a more aggressive phenotype both in vitro and in vivo. Re-challenging these resistant tumors in mice, we showed that second-line treatment with everolimus particularly affected vasculogenic mimicry and tumor cell differentiation compared to sorafenib and axitinib. Finally, our results highlighted the phenotypic and genotypic changes at the tumor cell and microenvironment levels during sunitinib response and progression and the subsequent improvement second-line therapies bring to the current renal cell carcinoma treatment paradigm.