MYC is a metastasis gene for non-small-cell lung cancer.

Metastasis is a process by which cancer cells learn to form satellite tumors in distant organs and represents the principle cause of death of patients with solid tumors. NSCLC is the most lethal human cancer due to its high rate of metastasis. Lack of a suitable animal model has so far hampered analysis of metastatic progression. We have examined c-MYC for its ability to induce metastasis in a C-RAF-driven mouse model for non-small-cell lung cancer. c-MYC alone induced frank tumor growth only after long latency at which time secondary mutations in K-Ras or LKB1 were detected reminiscent of human NSCLC. Combination with C-RAF led to immediate acceleration of tumor growth, conversion to papillary epithelial cells and angiogenic switch induction. Moreover, addition of c-MYC was sufficient to induce macrometastasis in liver and lymph nodes with short latency associated with lineage switch events. Thus we have generated the first conditional model for metastasis of NSCLC and identified a gene, c-MYC that is able to orchestrate all steps of this process. Potential markers for detection of metastasis were identified and validated for diagnosis of human biopsies. These markers may represent targets for future therapeutic intervention as they include genes such as Gata4 that are exclusively expressed during lung development

Choline Kinase Alpha Inhibition by EB-3D Triggers Cellular Senescence, Reduces Tumor Growth and Metastatic Dissemination in Breast Cancer

Choline kinase (ChoK) is the first enzyme of the Kennedy pathway leading to the biosynthesis of phosphatidylcholine (PtdCho), the most abundant phospholipid in eukaryotic cell membranes. EB-3D is a novel choline kinase 1 (ChoK 1) inhibitor with potent antiproliferative activity against a panel of several cancer cell lines. ChoK 1 is particularly overexpressed and hyperactivated in aggressive breast cancer. By NMR analysis, we demonstrated that EB-3D is able to reduce the synthesis of phosphocholine, and using flow cytometry, immunoblotting, and q-RT-PCR as well as proliferation and invasion assays, we proved that EB-3D strongly impairs breast cancer cell proliferation, migration, and invasion. EB-3D induces senescence in breast cancer cell lines through the activation of the metabolic sensor AMPK and the subsequent dephosphorylation of mTORC1 downstream targets, such as p70S6K, S6 ribosomal protein, and 4E-BP1. Moreover, EB-3D strongly synergizes with drugs commonly used for breast cancer treatment. The antitumorigenic potential of EB-3D was evaluated in vivo in the syngeneic orthotopic E0771 mouse model of breast cancer, where it induces a significant reduction of the tumor mass at low doses. In addition, EB-3D showed an antimetastatic effect in experimental and spontaneous metastasis models. Altogether, our results indicate that EB-3D could be a promising new anticancer agent to improve aggressive breast cancer treatment protocols.This work was supported by funds from Istituto di Ricerca Pediatrica (IRP)-Città della Speranza and Cassa di Risparmio di Padova e Rovigo—CARIPARO Foundation (project IRP13/05) and by the University of Granada, (Cei-Biotic project CEI2013-MP-1), and Associazione Italiana per la Ricerca sul Cancro (AIRC) MFAG 18459 grant (R.R.). E.M. was supported by AIRC (21101) and V.S. by FIRC (16616) fellowships

Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations

Functional relevance of circRNA aberrant expression in pediatric acute leukemia with KMT2A::AFF1 fusion

: Circular RNAs (circRNAs) are emerging molecular players in leukemogenesis and promising therapeutic targets. In KMT2A::AFF1 (MLL::AF4)-rearranged leukemia, an aggressive disease compared with other pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), data about circRNAs are limited. Here, we disclose the circRNA landscape of infant patients with KMT2A::AFF1 translocated BCP-ALL showing dysregulated, mostly ectopically expressed, circRNAs in leukemia cells. Most of these circRNAs, apart from circHIPK3 and circZNF609, previously associated with oncogenic behavior in ALL, are still uncharacterized. An in vitro loss-of-function screening identified an oncogenic role of circFKBP5, circKLHL2, circNR3C1, and circPAN3 in KMT2A::AFF1 ALL, whose silencing affected cell proliferation and apoptosis. Further study in an extended cohort disclosed a significantly correlated expression of these oncogenic circRNAs and their putative involvement in common regulatory networks. Moreover, it showed that circAFF1 upregulation occurs in a subset of cases with HOXA KMT2A::AFF1 ALL. Collectively, functional analyses and patient data reveal oncogenic circRNA upregulation as a relevant mechanism that sustains the malignant cell phenotype in KMT2A::AFF1 ALL

Oncogenic deubiquitination controls tyrosine kinase signaling and therapy response in acute lymphoblastic leukemia

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia

Protein kinase CK2α is overexpressed in classical hodgkin lymphoma, regulates key signaling pathways, PD-L1 and may represent a new target for therapy

IntroductionIn classical Hodgkin lymphoma (cHL), the survival of neoplastic cells is mediated by the activation of NF-κB, JAK/STAT and PI3K/Akt signaling pathways. CK2 is a highly conserved serine/threonine kinase, consisting of two catalytic (α) and two regulatory (β) subunits, which is involved in several cellular processes and both subunits were found overexpressed in solid tumors and hematologic malignancies.Methods and resultsBiochemical analyses and in vitro assays showed an impaired expression of CK2 subunits in cHL, with CK2α being overexpressed and a decreased expression of CK2β compared to normal B lymphocytes. Mechanistically, CK2β was found to be ubiquitinated in all HL cell lines and consequently degraded by the proteasome pathway. Furthermore, at basal condition STAT3, NF-kB and AKT are phosphorylated in CK2-related targets, resulting in constitutive pathways activation. The inhibition of CK2 with CX-4945/silmitasertib triggered the de-phosphorylation of NF-κB-S529, STAT3-S727, AKT-S129 and -S473, leading to cHL cell lines apoptosis. Moreover, CX-4945/silmitasertib was able to decrease the expression of the immuno-checkpoint CD274/PD-L1 but not of CD30, and to synergize with monomethyl auristatin E (MMAE), the microtubule inhibitor of brentuximab vedotin.ConclusionsOur data point out a pivotal role of CK2 in the survival and the activation of key signaling pathways in cHL. The skewed expression between CK2α and CK2β has never been reported in other lymphomas and might be specific for cHL. The effects of CK2 inhibition on PD-L1 expression and the synergistic combination of CX-4945/silmitasertib with MMAE pinpoints CK2 as a high-impact target for the development of new therapies for cH

Oncogenic deubiquitination controls tyrosine kinase signaling and therapy response in acute lymphoblastic leukemia

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL).We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia

Notch3 signalling promotes tumour growth in colorectal cancer: implication for Notch target therapy

It is well known that aberrant activation of the Notch pathway plays a critical role in the pathogenesis of T cell acute lymphoblastic leukaemia (T-ALL) and of certain solid tumors including lung, breast and ovarian cancer. In particular, increased Notch1 activity has been observed in intestinal adenoma, partially accomplished by β-catenin-mediated up-regulation of the Notch ligand Jagged-1. Whether further mechanisms of Notch activation exist and other Notch receptors might be involved in colorectal cancer (CRC) has not been investigated so far. In this study we investigated the possible involvement of Notch3 signaling in CRC, and the possible therapeutic implications. Intrigued by the observation that Notch3 mRNA and protein are over-expressed in a subset (20%) of human CRC, we sought to investigate how Notch3 modulates oncogenic features of CRC cells. By exploiting xenografts of CRC cells with different tumorigenic properties in mice, we found that the aggressive phenotype was associated with altered expression of components of the Notch pathway, including augmented expression of Delta-like 4 (DLL4) and Jagged-1 ligands and increased levels of the Notch3 transcript and intracellular domain (ICD). Stimulation with immobilized recombinant DLL4 dramatically increased Notch3 expression and Notch signaling. Moreover, forced expression of an active form of Notch3 mirrored effects of DLL4 stimulation and increased tumor formation. Conversely, blocking Notch3 signaling resulted in perturbation of the cell cycle followed by reduction of cell proliferation and inhibition of tumor growth. Moreover, we observed that these CRC cells have also different metastatic potential in terms of number and dimension when injected intravenously in mice. Lung metastases formed by CRC cells expressed Notch3, but the number or size was not significantly reduced by anti-Notch2/3 treatment. Overall, these findings indicate that Notch3 receptor can modulate the tumorigenic properties of CRC cells, and that DLL4 contributes to sustain Notch activity in DLL4-expressing tumors. Further studies are necessary to clarify the role of Notch3 in metastasis and design effective target therapies.E’ ormai noto da alcuni anni che un’aberrante attivazione della via di segnalazione di Notch gioca un ruolo critico nella patogenesi della leucemia linfoblastica acuta a cellule T (T-ALL) e di alcune neoplasie solide come il cancro del polmone, della mammella e dell’ovaio. Inoltre, una marcata attivazione del recettore Notch1 è stata osservata negli adenomi intestinali ed è correlata all’aumentata espressione del ligando Jagged-1 indotta dall’attivazione del pathway Wnt-APC-β-catenina. E’ stato inoltre dimostrato che questa pathway, insieme a quella di Notch, interviene nella regolazione della proliferazione e del differenziamento delle cellule epiteliali della mucosa intestinale normale. Attualmente, non si sa ancora se altri meccanismi di attivazione della pathway di Notch, oltre a quello ligando-dipendente, siano operativi nel CRC e nemmeno se possano essere coinvolti altri recettori della stessa famiglia. In questo studio, abbiamo cercato di chiarire il possibile coinvolgimento di Notch3 nel CRC. Basandoci sull’osservazione che Notch3 risulta essere frequentemente overespresso sia a livello di mRNA che di proteina nei campioni umani di CRC, abbiamo cercato di chiarire come il recettore Notch3 potesse modulare le proprietà tumorigeniche delle cellule di CRC. A tale scopo, si sono rivelati particolarmente utili alcuni xenotrapianti di cellule umane di CRC che presentano una diversa aggressività in topi NOD/SCID. Infatti, utilizzando i tumori sperimentali, abbiamo potuto dimostrare che l’espressione dei diversi componenti del pathway di Notch risulta essere significativamente elevata nella variante aggressiva rispetto a quella dormiente. In particolare si è osservata un’ aumentata espressione dei ligandi DLL4 e Jagged-1 ed un incremento dei livelli del trascritto di Notch3 e della forma attiva del recettore nei tumori che crescono più rapidamente. Una simile up-regolazione di Notch3 con un’aumentata attivazione della via di segnalazione si è osservata in seguito a stimolazione delle cellule di CRC in vitro con il ligando DLL4 ricombinante. Analogamente, anche l’overespressione di una forma attiva del recettore Notch3 in queste stesse cellule conferisce loro una maggiore capacità proliferativa e favorisce la formazione di tumori in vivo. Al contrario, si è visto che l’inattivazione del pathway mediante silenziamento genico di Notch3 nelle cellule aggressive di CRC, determina significative alterazioni del ciclo cellulare con conseguente riduzione nella proliferazione in vitro e un ritardo nella crescita dei tumori in vivo. Complessivamente, questi risultati dimostrano che il recettore Notch3 può modulare le proprietà tumorigeniche delle cellule di CRC, in particolare contribuendo a mantenere elevata l’attivazione della via di Notch nei tumori esprimenti nel loro microambiente tumorale alti livelli di DLL4. Inoltre, l’inoculo della variante più aggressiva rispetto a quella dormiente per via endovenosa in topi NOD/SCID ha mostrato che le cellule di CRC non hanno solo una diversa capacità tumorigenica, ma anche una differente capacità metastatica a livello polmonare, sia in termine di numero che di dimensioni delle metastasi osservate. Basandoci sui risultati fin qui ottenuti, e su uno studio recentemente pubblicato in cui è stato dimostrato come il pathway di Notch sembra essere coinvolto anche nel processo di metastatizzazione, abbiamo condotto un primo esperimento pilota che prevedeva, in topi portatori di metastasi polmonari, il blocco dei recettori Notch3 e Notch2 mediante l’impiego di un anticorpo neutralizzante. Sfortunatamente i risultati ottenuti non hanno fino ad ora mostrato una riduzione significativa nel numero e nelle dimensioni delle metastasi analizzate: l’analisi dei livelli di alterazione di Notch dopo il trattamento è in corso. A conclusione dello studio le nostre osservazioni rappresentano un punto di partenza per un futuro sviluppo di terapie che abbiano Notch3 come bersaglio per il trattamento di un sottogruppo di casi di CRC