Ex vivo mapping of enhancer networks that define the transcriptional program driving melanoma metastasis

: Mortality from vmelanoma is associated with metastatic disease, but the mechanisms leading to spreading of the cancer cells remain obscure. Spatial profiling revealed that melanoma is characterized by a high degree of heterogeneity, which is established by the ability of melanoma cells to switch between different phenotypical stages. This plasticity, likely a heritage from embryonic pathways, accounts for a relevant part of the metastatic potential of these lesions, and requires the rapid and efficient reorganization of the transcriptional landscape of melanoma cells. A large part of the non-coding genome cooperates to control gene expression, specifically through the activity of enhancers (ENHs). In this study, we aimed to identify ex vivo the network of active ENHs and to outline their cooperative interactions in supporting transcriptional adaptation during melanoma metastatic progression. We conducted a genome-wide analysis to map active ENHs distribution in a retrospective cohort of 39 melanoma patients, comparing the profiles obtained in primary (N = 19) and metastatic (N = 20) melanoma lesions. Unsupervised clustering showed that the profile for acetylated histone H3 at lysine 27 (H3K27ac) efficiently segregates lesions into three different clusters corresponding to progressive stages of the disease. We reconstructed the map of super-ENHs (SEs) and cooperative ENHs that associate with metastatic progression in melanoma, which showed that cooperation among regulatory elements is a mandatory requirement for transcriptional plasticity. We also showed that these elements carry out specialized and non-redundant functions, and indicated the existence of a hierarchical organization, with SEs on top as masterminds of the entire transcriptional program and classical ENHs as executors. By providing an innovative vision of how the chromatin landscape of melanoma works during metastatic spreading, our data also point out the need to integrate functional profiling in the analysis of cancer lesions to increase definition and improve interpretation of tumor heterogeneity

Ricostruzione della rete di cooperazione tra enhancers che regola la metastatizzazione del melanoma

Il melanoma cutaneo è la forma più aggressiva di tumore della pelle e la sua mortalità si associa prevalentemente alla presenza di metastasi. I determinanti molecolari necessari allo sviluppo delle metastasi nel melanoma non sono ancora del tutto noti. Studi funzionali hanno dimostrato che più dell’80% del genoma non codificante è biochimicamente attivo ed è coinvolto nel controllo dell’espressione genica. Enhancers (ENHs) e Super-ENHs (SEs) sono elementi regolatori non codificanti che agendo da soli o in cooperazione reclutano il complesso di trascrizione e controllano finemente specifici programmi trascrizionali. Come tutti i processi complessi, la tumorigenesi e la progressione tumorale necessitano di una profonda riprogrammazione a livello trascrizionale per essere eseguiti. Durante il processo di metastatizzazione, in particolare, si innescano specifici programmi trascrizionali che sostengono e supportano la plasticità delle cellule tumorali e l’acquisizione di caratteristiche di aggressività che le rendono prone alla diffusione metastatica. L’obiettivo dello studio è identificare gli elementi regolatori non codificanti che si riattivano durante la progressione metastatica e i fattori di trascrizione che li regolano, portando all’attivazione di programmi trascrizionali pro-metastatici nel melanoma. Su una corte retrospettiva di 19 melanomi primari e 20 melanomi metastatici derivati da pazienti, abbiamo applicato tecniche di sequenziamento high-throughput per studiare il profilo trascrizionale e la distribuzione a livello di tutto il genoma del marcatore istonico di cromatina attiva H3K27ac. L’unsupervised clustering dei campioni ha dimostrato che i profili di espressione genica non sono in grado di discriminare i tumori primari dalle metastasi. Al contrario, i profili di H3K27ac permettono di segregare i campioni in tre diversi gruppi che rappresentano diversi stadi di progressione del tumore. Applicando metodi bioinformatici, tra cui gli algoritmi ROSE e FIMO, abbiamo definito la complessa rete di SEs ed ENHs, nonché i loro regolatori trascrizionali, coinvolti nel controllo della progressione metastatica del melanoma, sottolineando il loro ruolo fondamentale nel mantenimento della plasticità trascrizionale. Nel complesso, i nostri risultati suggeriscono una cooperazione gerarchica tra gli elementi regolatori, che vede i SEs a capo della rete e gli ENHs come esecutori del programma trascrizionale. Il nostro approccio ha inoltre sottolineato l’importanza e la necessità di introdurre sistematicamente la profilazione funzionale del genoma nello studio dell’eterogeneità e della progressione tumorale al fine di avere una conoscenza più approfondita della complessità di questi processi.Cutaneous melanoma is the most threatening form of skin cancer and its mortality is mostly associated with metastatic disease. Nowadays, the molecular determinants required to establish overt melanoma metastasis are still poorly defined. The systematic functional analysis of the genome has revealed that more than 80% of the non-coding genome is biochemically active and controls gene expression. Enhancers (ENHs) and Super-ENHs (SEs) are non-coding regulatory elements that act alone or in concert to recruit the transcription machinery and finely tune specific transcriptional programs. Given their complexity, cancer initiation and progression require a profound reprogramming of the transcriptional regulatory landscape to guide cell malignant transformation. In particular, during metastasization cancerous cells trigger specific gene programs to sustain cell plasticity and the acquirement of aggressive features leading to metastatic spreading. In this study we aimed to identify the core of active non-coding regulatory elements and their upstream regulators mastering the activation of pro-metastatic gene expression programs in melanoma. We performed high-throughput transcriptome profiling and genome-wide analysis of the distribution of H3K27ac, histone marker of active chromatin, in a retrospective cohort of 19 primary and 20 metastatic patients-derived melanomas. Unsupervised clustering showed that gene expression profiling cannot discriminate primary tumors from metastatic samples. Conversely, H3K27ac profiles efficiently segregate lesions in three different clusters corresponding to progressive stages of the disease. We applied bioinformatics tools, like ROSE and FIMO, to outline the complex network of SEs and cooperative ENHs and their upstream transcription regulators orchestrating melanoma metastatic progression, thus highlighting their essential role in transcriptional plasticity. Taken together our findings suggested that regulatory elements act in concert in a hierarchical interplay mastered by SEs and executed by classical ENHs. Moreover, we identified a core of Transcription Factors controlling the activation of the regulatory regions essential for melanoma metastatic progression. Our approach highlighted the importance and need to systematically integrate functional profiling in the study of tumor heterogeneity and progression to have a deeper understanding of these processes complexity

Linc00941 is a novel TGFβ target that primes papillary thyroid cancer metastatic behavior by regulating the expression of Cadherin 6

Background: Papillary thyroid cancers (PTCs) are common, usually indolent malignancies. Still, a small but significant percentage of patients have aggressive tumors and develop distant metastases leading to death. Currently, it is not possible to discriminate aggressive lesions due to lack of prognostic markers. Long noncoding RNAs (lncRNAs), which are selectively expressed in a context-dependent manner, are expected to represent a new landscape to search for molecular discriminants. Transforming growth factor β (TGFβ) is a multifunctional cytokine that fosters epithelial-to-mesenchymal transition and metastatic spreading. In PTCs, it triggers the expression of the metastatic marker Cadherin 6 (CDH6). Here, we investigated the TGFβ-dependent lncRNAs that may cooperate to potentiate PTC aggressiveness. Methods: We used a genome-wide approach to map enhancer (ENH)-associated lncRNAs under TGFβ control. Linc00941 was selected and validated using functional in vitro assays. A combined approach using bioinformatic analyses of the thyroid cancer (THCA)-the cancer genome atlas (TCGA) dataset and RNA-seq analysis was used to identify the processes in which linc00941 was involved in and the genes under its regulation. Correlation with clinical data was performed to evaluate the potential of this lncRNA and its targets as prognostic markers in THCA. Results: Linc00941 was identified as transcribed starting from one of the TGFβ-induced ENHs. Linc00941 expression was significantly higher in aggressive cancer both in the TCGA dataset and in a separate validation cohort from our institution. Loss of function assays for linc00941 showed that it promotes response to stimuli and invasiveness while restraining proliferation in PTC cells, a typical phenotype of metastatic cells. From the integration of TCGA data and linc00941 knockdown RNA-seq profiling, we identified 77 genes under the regulation of this lncRNA. Among these, we found the prometastatic gene CDH6. Linc00941 knockdown partially recapitulates the effects observed upon CDH6 silencing, promoting cell cytoskeleton and membrane adhesions rearrangements and autophagy. The combined expression of CDH6 and linc00941 is a distinctive feature of highly aggressive PTC lesions. Conclusions: Our data provide new insights into the biology driving metastasis in PTCs and highlight how lncRNAs cooperate with coding transcripts to sustain these processes

Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines

Proteasome inhibitors (PIs) are extensively used for the therapy of multiple myeloma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. In addition, adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. Here, to identify compounds that can increase the efficacy of PIs, we performed a functional screening using a library of small-molecule inhibitors covering key signaling pathways. Among the best synthetic lethal interactions, the euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor UNC0642 displayed a cooperative effect with carfilzomib (CFZ) in numerous multiple myeloma (MM) cell lines, including drug-resistant models. In MM patients, EHMT2 expression correlated to worse overall and progression-free survival. Moreover, EHMT2 levels were significantly increased in bortezomib-resistant patients. We demonstrated that CFZ/UNC0642 combination exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone-marrow-derived stromal cells. To exclude off-target effects, we proved that UNC0642 treatment reduces EHMT2-related molecular markers and that an alternative EHMT2 inhibitor recapitulated the synergistic activity with CFZ. Finally, we showed that the combinatorial treatment significantly perturbs autophagy and the DNA damage repair pathways, suggesting a multi-layered mechanism of action. Overall, the present study demonstrates that EHMT2 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients

KAP1 is a new non-genetic vulnerability of malignant pleural mesothelioma (MPM)

Malignant pleural mesothelioma (MPM) is a rare and incurable cancer, which incidence is increasing in many countries. MPM escapes the classical genetic model of cancer evolution, lacking a distinctive genetic fingerprint. Omics profiling revealed extensive heterogeneity failing to identify major vulnerabilities and restraining development of MPM-oriented therapies. Here, we performed a multilayered analysis based on a functional genome-wide CRISPR/Cas9 screening integrated with patients molecular and clinical data, to identify new non-genetic vulnerabilities of MPM. We identified a core of 18 functionally-related genes as essential for MPM cells. The chromatin reader KAP1 emerged as a dependency of MPM. We showed that KAP1 supports cell growth by orchestrating the expression of a G2/M-specific program, ensuring mitosis correct execution. Targeting KAP1 transcriptional function, by using CDK9 inhibitors resulted in a dramatic loss of MPM cells viability and shutdown of the KAP1-mediated program. Validation analysis on two independent MPM-patients sets, including a consecutive, retrospective cohort of 97 MPM, confirmed KAP1 as new non-genetic dependency of MPM and proved the association of its dependent gene program with reduced patients' survival probability. Overall these data: provided new insights into the biology of MPM delineating KAP1 and its target genes as building blocks of its clinical aggressiveness

Lysin (K)-specific demethylase 1 inhibition enhances proteasome inhibitor response and overcomes drug resistance in multiple myeloma

Abstract Background Multiple myeloma (MM) is an incurable plasma cell malignancy, accounting for approximately 1% of all cancers. Despite recent advances in the treatment of MM, due to the introduction of proteasome inhibitors (PIs) such as bortezomib (BTZ) and carfilzomib (CFZ), relapses and disease progression remain common. Therefore, a major challenge is the development of novel therapeutic approaches to overcome drug resistance, improve patient outcomes, and broaden PIs applicability to other pathologies. Methods We performed genetic and drug screens to identify new synthetic lethal partners to PIs, and validated candidates in PI-sensitive and -resistant MM cells. We also tested best synthetic lethal interactions in other B-cell malignancies, such as mantle cell, Burkitt’s and diffuse large B-cell lymphomas. We evaluated the toxicity of combination treatments in normal peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs). We confirmed the combo treatment’ synergistic effects ex vivo in primary CD138+ cells from MM patients, and in different MM xenograft models. We exploited RNA-sequencing and Reverse-Phase Protein Arrays (RPPA) to investigate the molecular mechanisms of the synergy. Results We identified lysine (K)-specific demethylase 1 (LSD1) as a top candidate whose inhibition can synergize with CFZ treatment. LSD1 silencing enhanced CFZ sensitivity in both PI-resistant and -sensitive MM cells, resulting in increased tumor cell death. Several LSD1 inhibitors (SP2509, SP2577, and CC-90011) triggered synergistic cytotoxicity in combination with different PIs in MM and other B-cell neoplasms. CFZ/SP2509 treatment exhibited a favorable cytotoxicity profile toward PBMCs and BMSCs. We confirmed the clinical potential of LSD1-proteasome inhibition in primary CD138+ cells of MM patients, and in MM xenograft models, leading to the inhibition of tumor progression. DNA damage response (DDR) and proliferation machinery were the most affected pathways by CFZ/SP2509 combo treatment, responsible for the anti-tumoral effects. Conclusions The present study preclinically demonstrated that LSD1 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients and that this combination might be exploited for the treatment of other B-cell malignancies, thus extending the therapeutic impact of the project

Long non-coding RNA mitophagy and ALK- anaplastic lymphoma associated transcript: a novel regulator of mitophagy in T cell lymphoma

: Long noncoding RNAs (lncRNAs) are emerging as powerful and versatile regulators of transcriptional programs and distinctive biomarkers of T-cell Lymphoma progression disease. Their role in the aggressive ALK- Anaplastic Large Cell Lymphoma (ALCL) subtype has been only in part elucidated. Starting from our previously identified ALCL-associated lncRNA signature and performing digital gene expression profiling of a retrospective cohort of ALCLs, we defined an 11 lncRNA signature able to discriminate among ALCL subtypes. We selected a not previously characterized lncRNA, MTAAT, with an ALK- ALCL preferential expression, for molecular and functional studies. We demonstrated that lncRNA MTAAT contributes to an aberrant mitochondrial turnover restraining mitophagy and promoting cellular proliferation. Functionally, lncRNA MTAAT acts as a repressor of a set of genes related to mitochondria quality control via chromatin reorganization. Collectively, our work demonstrates the transcriptional role of lncRNA MTAAT in orchestrating a complex transcriptional program sustaining ALK- ALCL progression