Littérature scientifique sur le sujet « Phosphoscan »

Abstract The bone marrow microenvironment provides essential signals for the fate of normal hematopoietic and for leukemic cells. Contact with marrow stroma, which is part of the microenvironment, is generally thought to convey anti-apoptotic signals to (clonal) leukemia cells. Patients with low-grade myelodysplastic syndrome (MDS) early in the disease course show high rates of apoptosis in normal and clonal marrow cells, mediated by tumor necrosis factor alpha (TNFα) and other cytokines. As MDS advances and evolves to leukemia, clonal cells tend to become apoptosis resistant. We showed previously that the leukemia-derived cell line KG1a was resistant to TNFα-mediated apoptosis, but TNFα did induce caspase-3 activation and apoptosis in KG1a cells when co-cultured with the human marrow stroma cell line HS5 (derived from healthy marrow). Apoptosis was contact dependent and required expression of TNF receptor 1 on KG1a cells. Identical results were obtained in co-cultures with primary stroma cells. Gene expression profiling of KG1a cells showed that stroma contact resulted in significant upregulation of genes involved in apoptosis, including PYCARD and p53. To further dissect the relevant signaling pathways, we used a PhosphoScan proteomic LC-MS (Liquid chromatography-mass spectrometry) method to identify proteins that were phosphorylated in response to stroma contact. In parallel to KG1a we examined the parent cell line KG1, which is sensitive to TNFα mediated apoptosis. We determined the phosphorylation sites in proteins within the leukemic cell lines using MS2 and MS3 scans. Database searches were performed with X! Tandem and Mascot and results analyzed by PeptideProphet using data from a synthetic doubly-phosphorylated peptide as control. In KG1a cells cultured without stroma support, the peptide DJ-1/Park-7 was highly phosphorylated, and expression of p53 was inhibited as indicated by decreased levels of p53 mRNA and protein. In co-culture with stroma, KG1a cells expressed higher levels of p53 protein, and levels of phosphorylated DJ-1/ Park-7 were undetectable over a time course of 30 min to 24 hours. In apoptosis-sensitive KG1 cells constitutive DJ-1/Park-7 phosphorylation (in the absence of stroma contact) was undetectable, and p53 was expressed at higher levels than in KG1a cells, consistent with the observed activation of caspase-3 and induction of apoptosis in KG1 cells. Taken together, these data suggest that phosphorylation of DJ-1/Park-7, originally identified as an oncogene product involved in cellular transformation, oxidative stress responses, and transcriptional regulation, was associated with repression of p53 and resistance to TNFα-mediated apoptosis. The relevance of DJ-1/Park-7 (and other genes identified by the PhosphoScan proteomic method) in primary MDS cells is currently being investigated at the molecular and functional levels.

AbstractIn patients with myelodysplastic syndromes (MDS), apoptosis in hematopoietic cells is up-regulated in low-grade disease, whereas advanced disease is characterized by apoptosis resistance. We have shown that marrow stroma–derived signals convey sensitivity to tumor-necrosis-factor alpha (TNF-α)–mediated apoptosis in otherwise-resistant KG1a myeloid cells and CD34+ cells from MDS marrow. Here, we used a PhosphoScan proteomic liquid chromatography–mass spectrometry method to identify signals relevant for this effect. The transcription factor DJ-1/PARK-7 (DJ-1) was highly phosphorylated in KG1a cells cultured without stroma but dephosphorylated after stroma coculture, whereas expression of p53 increased significantly, suggesting a stroma contact-dependent effect of DJ-1 on p53. In CD34+ marrow cells from advanced MDS, expression of DJ-1 was up-regulated, whereas p53 levels were low, resulting in significantly greater DJ-1/p53 ratios than in patients with low-grade MDS (P = .01). DJ-1 levels were correlated with increasing International Prognostic Scoring System scores (P = .006). Increasing DJ-1/p53 ratios were associated with an increased risk of mortality, although the correlation did not reach statistical significance (P = .18). These data suggest that DJ-1/p53 interactions contribute to apoptosis resistance in clonal myeloid cells and may serve as a prognostic marker in patients with MDS.

Abstract 4T1 is a metastatic breast cancer model with fulminant accumulation of MDSCs. Survival and function of MDSCs is often reported as STAT3-dependent, and STAT3 inhibitors such as sunitinib dramatically deplete 4T1 splenic MDSCs. Paradoxically, sunitinib fails to eradicate 4T1 intratumoral MDSCs or prevent tumor progression. We hypothesized that STAT3 activation is a consistent feature of splenic but not intratumoral MDSCs. PhosphoSTAT analyses of 4T1-bearers confirmed that splenic MDSCs of all Gr1 intensities were solely pSTAT3pos. In remarkable contrast, intratumoral Gr1high MDSCs largely lacked any pSTAT activation, and intratumoral Gr1dim MDSC precursors displayed varied expression of pSTAT5 and pSTAT1 in addition to pSTAT3. We therefore sought global means to target MDSCs. We determined that 4T1 and other tumor models could be cured by repetitive administration of cyclophosphamide alternating with TLR agonists such as CpG ODN1826. While cure required participation of host CD4+ and CD8+ T cells, global targeting of MDSCs was also observed: (1) as with sunitinib, pSTAT3pos splenic MDSCs were eradicated; (2) pSTAT expression by intratumoral MDSCs was fully abolished; (3) the remaining intratumoral MDSCs, all pSTATneg, outsurvived all other cells, and were likely induced to serve as the final mediators of tumor rejection. In conclusion, strategies which globally target MDSCs and promote the endogenous anti-tumor T cell response can cure advanced metastatic tumors.

Abstract Purpose: Flavopiridol is a cyclin-dependent kinase inhibitor with preclinical activity against multiple myeloma cells in vitro and ex vivo. It can induce apoptosis in non-cycling human cell lines, including RPMI-8226, a myeloma cell line. Suggested mechanisms of cytotoxicity include down regulation of anti-apoptotic regulators, direct binding to duplex DNA, disruption of transcription factor/DNA binding, upregulation of p53, inhibition of transcription, and inhibition of angiogenesis. A Phase II multicenter trial was conducted to determine the activity of flavopiridol in patients with relapsed or refractory multiple myeloma. Experimental Design: Eighteen patients were treated with 1 hour flavopiridol infusions (50 mg/m(2)/day) for 3 consecutive days every 21 days. Responses were assessed according to IBMTR criteria each cycle. Serial bone marrow aspirates were collected before and immediately after flavopiridol treatment to measure in vivo and ex vivo effects of flavopiridol on patient plasma cells. Immunoblotting for Mcl-1, Bcl-2, p53, cyclin D, phosphoRNA polymerase II and phosphoSTAT 3 was conducted on total cellular proteins isolated from sorted plasma/myeloma cells. Ex vivo assessment of flavopiridol sensitivity of freshly collected myeloma cells was performed for 5 patients pre-therapy. Results: Median age of patients ranged from 49 to 81 years, with a median of 65 years. Patients had received a median of 3 (range, 1–5) treatment regimens prior to enrollment. Sixty-one percent of patients were refractory to prior therapy and fifty-five percent had received prior high dose therapy with hematopoietic stem cell support. The immunoglobulin isotypes of the patients were as follows: IgG (10), IgA (4), light chain (3), and non-secretory (1). At enrollment, 13 patients had a beta-2 microglobulin greater than 3.5. Seven had a plasma cell labeling index greater than or equal to 1%. Four had an elevated LDH. The trial was stopped at time of interim analysis due to a lack of clinical efficacy. Of eighteen treated patients, 15 progressed (including 1 death on study) and 3 discontinued due to toxicity. All patients have ended the active treatment phase, and the mean number of cycles administered for all enrolled patients was 1.6 cycles (range 1–3). No objective responses were observed. The most frequent adverse events were leukopenia and diarrhea (83% each), followed by thrombocytopenia, nausea, and fatigue (61% each). Ex vivo flavopiridol treatment of pre-therapy patient plasma/myeloma cells led to cytotoxicity, but only after longer exposure times at higher flavopiridol concentrations than were anticipated to be achieved in vivo. Further, immunoblotting demonstrated no indication that known in vitro cellular effects of flavopiridol were recapitulated in vivo. Conclusions: Flavopiridol has little single-agent activity in relapsed or refractory multiple myeloma when administered at this dose and schedule, which is likely due to the inability to achieve biologically relevant concentrations in patients. .

Treatment of nonhuman primates and mice with a humanized antigen-binding fragment (Fab) antibody (UCBFab) inhibiting transforming growth factor β via daily inhalation for up to 13 weeks resulted in low systemic exposure but high local exposure in the lung. Target engagement was demonstrated by reduced levels of signal transducers, phosphoSMAD and plasminogen activator inhibitor-1 in the bronchoalveolar lavage fluid (BALF). Treatment was associated with a high frequency and titer of antidrug antibodies, indicating high local immunogenicity, and local pathology within the lung and draining lymph nodes. Microscopic changes were characterized by perivascular (PV) and peribronchiolar (PB) mononuclear inflammatory cell (MIC) infiltrates that were principally lymphocytic in nature and mixed inflammatory cell infiltrates and/or inflammation within the alveoli. Immunohistochemical investigation revealed a predominantly CD68-positive macrophage and CD3- and CD8>CD4-positive T-cell response in the alveoli, whereas within the airways, there was a variable mixture of CD3-positive T cells, CD20-positive B cells, and CD68-positive macrophages. Increased cellularity of the draining lymph nodes was also noted, indicating the presence of an immune response to the inhaled test article. Morphologic changes did not progress over time, and all changes partially recovered. Increased leukocytes (principally macrophages) in BALF cytology correlated with the changes seen by histopathology.

La comprensione dei meccanismi molecolari delle malattie sta alla base della medicina moderna. Questo campo di ricerca è divenuto sempre più interdisciplinare negli anni, sfruttando tecniche sviluppate inizialmente in chimica, biologia sperimentale, immunologia, matematica, informatica etc.. e proprio recentemente, con l'avvento di tecnologie high-throughput, questo tipo di approccio è diventato più una necessità che un lusso. In questo progetto abbiamo utilizzato un approccio integrato per lo studio del linfoma a cellule del mantello (MCL). Nel tentativo di ottenere un quadro più completo, e possibilmente più realistico, della patogenesi di questo linfoma abbiamo cercato di integrare le informazioni ottenute da analisi di tipo proteomico, trascrittomico e genomico per mezzo di diverse tecnologie high-throughput. Nella prima parte del progetto abbiamo sfruttato una tecnica di tipo proteomico, chiamata Phosphoscan, per determinare il proteoma tirosina attivato di un pannello di linee cellulari di MCL. Dato che la fosforilazione su residui tirosinici regola la funzione di molte proteine, questo tipo di approccio ci ha permesso di identificare le vie del segnale più attive nei campioni analizzati. L’analisi Phosphoscan ha rivelato che le cellule del MCL hanno un’attivazione molto forte del recettore delle cellule B (BCR) e inibendo Syk con piceatannolo (una molecola chiave della via di trasduzione del segnale del BCR) si provoca la morte di queste ultime. L'attivazione di questo percorso è stata quindi verificata anche nei tessuti di tumore MCL, suggerendo che potrebbe essere un obiettivo terapeutico importante. Altre chinasi, oltre a Syk, sono state trovate attivate (fosforilate) e il loro ruolo è pertanto oggetto di indagine tramite l’utilizzo di inibitori chinasici. Ad ogni modo il meccanismo che guida l’attivazione del BCR è rimasto irrisolto. La presenza di mutazioni attivanti è un meccanismo di base della patogenesi del cancro, pertanto abbiamo ipotizzato che la presenza di un’alterazione genetica in una o più delle proteine trovate attive potesse esser responsabile dell’attivazione della via del BCR. Per verificare tale ipotesi abbiamo verificato la presenza di mutazioni nelle sequenze codificanti corrispondenti alle proteine fosforilate più abbondanti, tramite sequenziamento classico di Sanger. Abbiamo così identificato quattro mutazioni missenso nei geni PRPF4B, BTK, PRKCD e CD79B ma per determinare il loro ruolo sono necessari ulteriori studi funzionali e un estesa validazione in vitro. É stato recentemente dimostrato che il concetto di stereotipia del BCR è strettamente correlato alla sua attivazione e alla patogenesi di diversi tipi di linfoma e leucemia, come la leucemia linfatica cronica (B-CLL). Per questo motivo abbiamo analizzato i recettori per l’antigene B di diverse linee cellulari di MCL, alla ricerca di un meccanismo addizionale che potesse stimolare la cascata del segnale. Abbiamo sequenziato i geni sia delle catene pesanti che di quelle leggere del BCR e abbiamo verificato la presenza di motivi stereotipati in queste ultime. Questi risultati mettono in luce il possibile ruolo delle catene leggere nella segnalazione tonica del BCR e forniscono le basi per l’utilizzo di queste linee cellulari come modello per indagare il ruolo di specifici BCR nella patogenesi del MCL. Nella seconda parte del lavoro abbiamo applicato una tecnologia NGS, e precisamente il sequenziamento del RNA, a sette casi di MCL e tre linfonodi reattivi. Questo approccio trascrittomico su larga scala ha il vantaggio di fornire, oltre a un profilo mutazionale completo di tutta la porzione trascritta del campione in analisi, numerose informazioni (profilo di espressione genica, utilizzo di varianti di splicing, trascritti chimerici etc..) che potrebbero esser utili per meglio capire la patogenesi molecolare del MCL. Un’analisi preliminare e guidata dei dati di RNA-seq ci ha permesso di individuare undici mutazioni potenzialmente rilevanti, fra quelle già annotate nel database COSMIC. Solo quattro di queste, presenti nei geni NOTCH2, ATM, GOT2 e CHPF2, sono state validate. Abbiamo inoltre dimostrato la presenza di trascritti di fusione nel MCL, sebbene sterili e rilevabili in un numero molto limitato di casi (si tratta quindi di trascritti prevalentemente “privati”). Le informazioni ottenute da quest'analisi trascrittomica sono estremamente complesse e quindi necessitano di ulteriori analisi in termini bioinformatici e biologici, che al momento sono ancora in corso. Complessivamente queste analisi hanno portato nuova conoscenza sul meccanismo alla base del processo patogenetico nel MCL, evidenziando il ruolo della via del segnale del BCR.
Understanding the mechanisms of disease is the base of modern medicine. Cancer research has become over the years more and more interdisciplinary, using techniques developed initially in other science disciplines, such as chemistry, experimental biology, immunology, mathematics, informatics and many others. In recent years, with the advent of high-throughput techniques, this multi-faceted approach has become a necessity more than a luxury. In this project we exploited an integrated approach to the study of mantle cell lymphoma (MCL). To gain a more complete, and possibly more real, picture of MCL pathogenesis we tried to integrate information coming from proteomic, transcriptomic and genomic studies acquired by means of several modern high-throughput techniques. In the first part of this project we took advantage of a shotgun proteomic technique, called Phoshoscan, to identify the tyrosine-phosphorylation profile of MCL cells. Since tyrosine phosphorylation regulates the activity of many proteins, this approach allowed us to identify the most active pathways in the analyzed samples. The Phosphoscan analysis has shown that MCL bears an aberrant activation of the B-cell receptor (BCR) signaling pathway, and that at least in vitro the inhibition of Syk (a key molecule of BCR signaling) is lethal for MCL cells. Other kinases were also found to be active (phosphorylated) and these are matter of current investigation by means of pharmacological inhibition. However, the mechanism driving BCR activation remained elusive. Since the presence of activating mutations is one basic mechanism of cancer pathogenesis, we hypothesized that a genetic lesion in one or more of the active proteins might be responsible for the abnormal activation of the BCR pathway. To test this hypothesis we looked for the presence of mutations in the coding sequences corresponding to the most abundant Tyr-phosphorilated proteins, taking advantage of the classical Sanger sequencing. We identified four missense mutations in PRPF4B, BTK, PRKCD and CD79B genes but further functional studies and an extensive validation are necessary to assess these mutations role. Since BCR stereotypy and activation have been linked to the pathogenesis of different lymphomas and leukemias, like B-CLL, we also investigated whether MCL cell lines bore stereotyped BCR, as an additional mechanism stimulating the signaling cascade. We determined the precise sequence of rearranged heavy and light chain genes in several MCL cell lines and we found no evidence of heavy chain stereotypy, but recurrent presence of stereotyped light chain. These findings highlight the possible role of light chains in tonic BCR signaling and provide the basis to use MCL cell lines as a model to investigate the role of specific BCRs in the pathogenesis of MCL. In the second part of the work we applied a next-generation sequencing (NGS) technique, RNA-sequencing, to seven cases of MCL and three non-neoplastic lymph node samples. This whole-transcriptome approach has the advantage to provide, beyond a complete mutational profile of the expressed genes, several new data (quantitative gene expression profiling, usage of splice variants, chimeric transcripts etc) which can be useful to better understand the molecular pathogenesis of MCL. A preliminary supervised analysis of RNA-seq data identified eleven putative relevant single-nucleotide variants (SNVs), already annotated in the COSMIC database. Only four of them, affecting NOTCH2, ATM, GOT2, and CHPF2 genes, were validated. Moreover we showed that although fusion transcripts are usually present in MCL, they are detectable in a very limited number of cases (i.e. they are mostly private) and do not produce any detectable protein. However the information obtained from this analysis is extremely wide and complex and deserves a deeper investigation by means of bioinformatic analysis and biological validation that is still ongoing. Overall, these analyses brought us new insights about the mechanism driving the pathogenetic process in MCL, highlighting the role of BCR signaling pathway.