Incorporating immunotherapy in the management of gastric cancer: molecular and clinical implications

Gastric cancer has a median survival of 11 months, and this poor prognosis has not improved over the last 30 years. Recent pre-clinical data suggest that there is high tumour-related neoantigen expression in gastric cancer cells, suggesting that a clinical strategy that enhances the host’s immune system against cancer cells may be a successful approach to improve clinical outcomes. Additionally, there has been an increasing amount of translational evidence highlighting the relevance of PD-L1 expression in gastric cancer cells, indicating that PD-1/PD-L1 inhibitors may be useful. Several molecular subgroups of gastric cancer have been identified to respond with excellent outcomes to immunotherapy, including microsatellite instable tumours, tumours bearing a high tumour mutational burden, and tumours related to a chronic EBV infection. In gastric cancer, immunotherapy has produced durable responses in chemo-refractory patients; however, most recently there has been a lot of enthusiasm as several large-scale clinical trials highlight the improved survival noted from the incorporation of immunotherapy in the first line setting for advanced gastric cancer. Our review aims to discuss current pre-clinical and clinical data supporting the innovative role of immunotherapy in gastric cancer

Phosphorus and sulfur SAD phasing of the nucleic acid-bound DNA-binding domain of interferon regulatory factor 4

Pivotal to the regulation of key cellular processes such as the transcription, replication and repair of DNA, DNA-binding proteins play vital roles in all aspects of genetic activity. The determination of high-quality structures of DNA-binding proteins, particularly those in complexes with DNA, provides crucial insights into the understanding of these processes. The presence in such complexes of phosphate-rich oligonucleotides offers the choice of a rapid method for the routine solution of DNA-binding proteins through the use of long-wavelength beamlines such as I23 at Diamond Light Source. This article reports the use of native intrinsic phosphorus and sulfur single-wavelength anomalous dispersion methods to solve the complex of the DNA-binding domain (DBD) of interferon regulatory factor 4 (IRF4) bound to its interferon-stimulated response element (ISRE). The structure unexpectedly shows three molecules of the IRF4 DBD bound to one ISRE. The sole reliance on native intrinsic anomalous scattering elements that belong to DNA-protein complexes renders the method of general applicability to a large number of such protein complexes that cannot be solved by molecular replacement or by other phasing methods

Cell-specific pattern of berberine pleiotropic effects on different human cell lines

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-Tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types

Protopine/Gemcitabine Combination Induces Cytotoxic or Cytoprotective Effects in Cell Type-Specific and Dose-Dependent Manner on Human Cancer and Normal Cells

The natural alkaloid protopine (PRO) exhibits pharmacological properties including anticancer activity. We investigated the effects of PRO, alone and in combination with the chemotherapeutic gemcitabine (GEM), on human tumor cell lines and non-tumor human dermal fibroblasts (HDFs). We found that treatments with different PRO/GEM combinations were cytotoxic or cytoprotective, depending on concentration and cell type. PRO/GEM decreased viability in pancreatic cancer MIA PaCa-2 and PANC-1 cells, while it rescued the GEM-induced viability decline in HDFs and in tumor MCF-7 cells. Moreover, PRO/GEM decreased G1, S and G2/M phases, concomitantly with an increase of subG1 phase in MIA PaCa-2 and PANC-1 cells. Differently, PRO/GEM restored the normal progression of the cell cycle, altered by GEM, and decreased cell death in HDFs. PRO alone increased mitochondrial reactive oxygen species (ROS) in MIA PaCa-2, PANC-1 cells and HDFs, while PRO/GEM increased both intracellular and mitochondrial ROS in the three cell lines. These results indicate that specific combinations of PRO/GEM may be used to induce cytotoxic effects in pancreatic tumor MIA PaCa-2 and PANC-1 cells, but have cytoprotective or no effects in HDFs

Dissecting the impact of bromodomain inhibitors on the IRF4-MYC oncogenic axis in multiple myeloma

B-cell progenitor fate determinant interferon regulatory factor 4 (IRF4) exerts key roles in the pathogenesis and progression of multiple myeloma (MM), a currently incurable plasma cell malignancy. Aberrant expression of IRF4 and the establishment of a positive auto-regulatory loop with oncogene MYC, drives a MM specific gene-expression programme leading to the abnormal expansion of malignant immature plasma cells. Targeting the IRF4-MYC oncogenic loop has the potential to provide a selective and effective therapy for MM. Here we evaluate the use of bromodomain inhibitors to target the IRF4-MYC axis through combined inhibition of their known epigenetic regulators, BRD4 and CBP/EP300. Although all inhibitors induced cell death, we found no synergistic effect of targeting both of these regulators on the viability of MM cell-lines. Importantly, for all inhibitors over a time period up to 72 hours, we detected reduced IRF4 mRNA, but a limited decrease in IRF4 protein expression or mRNA levels of downstream target genes. This indicates that inhibitor-induced loss of cell viability is not mediated through reduced IRF4 protein expression, as previously proposed. Further analysis revealed a long half-life of IRF4 protein in MM cells. In support of our experimental observations, gene network modelling of MM suggests that bromodomain inhibition is exerted primarily through MYC and not IRF4. These findings suggest that despite the autofeedback positive regulatory loop between IRF4 and MYC, bromodomain inhibitors are not effective at targeting IRF4 in MM and that novel therapeutic strategies should focus on the direct inhibition or degradation of IRF4. This article is protected by copyright

IRF4 in multiple myeloma—biology, disease and therapeutic target

Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by abnormal proliferation of plasma cells. Interferon Regulatory Factor 4 (IRF4), a member of the interferon regulatory family of transcription factors, is central to the genesis of MM. IRF4 is highly expressed in B cells and plasma cells where it plays essential roles in controlling B cell to plasma cell differentiation and immunoglobulin class switching. Overexpression of IRF4 is found in MM patients’ derived cells, often as a result of activating mutations or translocations, where it is required for their survival. In this review, we rst describe the roles fi of IRF4 in B cells and plasma cells and then analyse the subversion of the IRF4 transcriptional network in MM. Moreover, we discuss current therapies for MM as well as direct targeting of IRF4 as a potential new therapeutic strategy

Targeting the IRF4 transcriptional network to subvert multiple myeloma

EMBARGOED - expected end date 21.12.202

Effetti indotti dall'alcaloide naturale berberina in tre diverse linee cellulari umane

La berberina è un alcaloide isochinolico di origine naturale estratto da specie vegetali come Berberis aristata e Berberis vulgaris. Questo alcaloide ha una varietà di attività farmacologiche senza significativi effetti collaterali. Recenti studi indicano che la berberina ha proprietà antineoplastiche agendo a diversi livelli della tumorigenesi quali sopravvivenza, proliferazione e migrazione cellulare. Il mio lavoro di tesi, svolto presso l’Unità di Biologia Cellulare dell’ Università di Pisa, analizza come differenti linee cellulari rispondono al trattamento con berberina. In particolare, ho utilizzato la linea cellulare MIA PaCa-2 di carcinoma pancreatico e la linea cellulare U343 di glioblastoma; come controllo non tumorale ho utilizzato la linea cellulare primaria HDF (Human Dermal Fibroblasts). Poichè la berberina emette luce fluorescente verde quando esposta a luce UV, ho visualizzato come differenti concentrazioni di berberina si localizzano all’interno dei tre tipi cellulari. Ho osservato che la berberina è presente nel citoplasma e/o nel nucleo in modo dose-dipendente. Allo scopo di valutare se la berberina abbia un effetto differenziale sulla vitalità delle cellule tumorali, ho effettuato una conta cellulare con il test di esclusione al colorante Trypan Blue. Ho osservato che pur essendo presente una diminuzione della vitalità in tutti e tre i tipi cellulari, il calo di vitalità è più consistente nelle cellule tumorali. Utilizzando il TMRM (tetramethylrhodamine methyl ester), marcatore del potenziale redox di membrana del mitocondrio, ho anche analizzato se la berberina causi stress mitocondriale. E’ risultato che i trattamenti con berberina provocano una riduzione del segnale del TMRM indicando un decadimento dell’attività mitocondriale in tutti e tre i tipi cellulari. E’ interessante notare come la riduzione del segnale del TMRM sia maggiore nelle due linee tumorali, suggerendo quindi una selettiva azione citotossica della berberina sul mitocondrio di queste cellule. Per comprendere quale tipo di morte cellulare venga indotto dalla berberina ho analizzato lo stato di apoptosi, di senescenza cellulare e di autofagia nelle cellule trattate con berberina. Per studiare se sia coinvolto il meccanismo di morte apoptotica ho analizzato l’attività dell’enzima CASPASI-3. Il risultato di questi esperimenti dimostra che in cellule HDF il trattamento con berberina 10 μM induce attivazione della CASPASI-3, mentre è necessaria una elevata concentrazione di berberina (150 μM) per indurre l’attivazione della CASPASI-3 nelle due linee tumorali. La senescenza cellulare, analizzata mediante il saggio della β-galattosidasi, dimostra che la berberina aumenta i livelli di senescenza in tutti e tre tipi cellulari in maniera dose-dipendente e in maniera preferenziale nelle cellule tumorali MIA PaCa-2 rispetto alle non tumorali HDF. Infine la lettura al citofluorimetro del segnale emesso dal marcatore autofagico Acridina Orange, ha evidenziato che la berberina induce anche autofagia nelle cellule tumorali, ma non nelle cellule HDF. Poichè la berberina entra nel nucleo, ho valutato se essa possa agire a livello trascrizionale interferendo con l’espressione di geni correlati con processi importanti per la carcinogenesi. Ho quindi analizzato, tramite real-time RT PCR, i livelli di espressione genica di vari marcatori molecolari. In particolare ho analizzato l’espressione dei geni DNMT1, DNMT3A, DNMT3B e MGMT coinvolti nei pathway epigenetici, dei geni P53, P21, P14, P16, e CASP-3 coinvolti nei pathway di senescenza cellulare e apoptotici e dei geni PTEN, BECN-1 e LC3II coinvolti nel pathway autofagico. I risultati dimostrano che l’azione della berberina a livello trascrizionale è diversa nei tre tipi cellulari analizzati. Ho infine valutato se la berberina interferisca con la capacità di migrazione delle cellule tumorali MIA PaCa-2 mediante il saggio del “wound healing”. E’ risultato che la berberina rallenta la migrazione delle cellule MIA PaCa-2 in maniera dose-dipendente. Gli esperimenti di real-time RT PCR mostrano un calo dei livelli di espressione di CXCR4, un gene che svolge un importante ruolo nella migrazione cellulare, nelle cellule MIA PaCa-2 trattate con berberina rispetto al controllo. In conclusione questi risultati dimostrano che la berberina induce maggiori effetti citotossici nelle cellule tumorali rispetto alle cellule non tumorali HDF. Inoltre la risposta al trattamento con la berberina è linea cellulare-specifica. Queste caratteristiche rendono sicuramente vario e non specifico l’effetto di questo alcaloide. Disporre di nuove informazioni circa i meccanismi anti-tumorali attraverso i quali la berberina agisce su specifiche popolazioni tumorali, è sicuramente importante per evitare possibili interazioni negative della berberina con i chemioterapici e per favorire invece interazioni che potenzino in modo sinergico gli effetti anti-tumorali dei farmaci stessi.

The Inhibitory Properties of a Novel, Selective LMTK3 Kinase Inhibitor

Recently, the oncogenic role of lemur tyrosine kinase 3 (LMTK3) has been well established in different tumor types, highlighting it as a viable therapeutic target. In the present study, using in vitro and cell-based assays coupled with biophysical analyses, we identify a highly selective small molecule LMTK3 inhibitor, namely C36. Biochemical/biophysical and cellular studies revealed that C36 displays a high in vitro selectivity profile and provides notable therapeutic effect when tested in the National Cancer Institute (NCI)-60 cancer cell line panel. We also report the binding affinity between LMTK3 and C36 as demonstrated via microscale thermophoresis (MST). In addition, C36 exhibits a mixed-type inhibition against LMTK3, consistent with the inhibitor overlapping with both the adenosine 5′-triphosphate (ATP)- and substrate-binding sites. Treatment of different breast cancer cell lines with C36 led to decreased proliferation and increased apoptosis, further reinforcing the prospective value of LMTK3 inhibitors for cancer therapy