Translational Control in Tumour Progression and Drug Resistance

Protein biosynthesis is a multi-step process that starts with the transcription of nuclear DNA, depository of genetic information, into messenger RNA (mRNA) that is used as template for the following polypeptide chain synthesis, also known as translation. Each step of this essential process is highly controlled in order to modulate any specific protein requirement of the cell in response to different stimuli and cellular events. This regulatory process is called translational control. Deregulation of the core signalling network in translational control, the phosphatidyl inositol trisphosphate kinase (PI3K), Protein Kinase B (PKB or Akt), mammalian target of rapamycin (mTOR) and RAS mitogen-activated protein kinase (MAPK)/MAPK-interacting Kinases (MNK) pathways, frequently occurs in human cancers and leads to aberrant modulation of mRNA translation. However, investigations on the contribution of these two pathways to translational regulation led to the interesting finding that translation factors are also substrate of signalling molecules. Post-translational modifications, including cleavage and phosphorylation, usually affect translational factors activity in protein biosynthesis; on the other hand, direct interaction of translational components with signalling mediators can either activate the pathway in which the mediator is involved or redirect translation factors to other activities, such as cytoskeletal rearrangements. These findings shed light on new functions of translation factors, different from their canonical role in protein synthesis. Taken together, these new functions are an intriguing step forward to the discovery of molecular mechanisms at the base of cellular response during “special” conditions such as cancer and drug resistance

silicon based technology for ligand receptor molecular identification

One of the most important goals in the fields of biology and medicine is the possibility to dispose of efficient tools for the characterization of the extraordinary complexity of ligand-receptor interactions. To approach this theme, we explored the use of crystalline silicon (cSi) technology for the realization of a biotechnological device in which the ligand-receptor interactions are revealed by means of optical measurements. Here, we describe a chemical procedure for the functionalization of microwell etched on silicon wafers, and the subsequent anchoring of biological molecules like an antibody anti-A20 murine lymphoma cell line. The optical analysis of the interaction on the biochips between the bound biomolecule and their corresponding ligand indicated that the functionalized cSi is suitable for this application

Polar and non-polar organic binder characterization in Pompeian wall paintings: comparison to a simulated painting mimicking an a secco technique.

The use of Fourier transform infrared spectromicroscopy and mass spectrometry (MS) allowed us to characterize the composition of polar and non-polar binders present in sporadic wall paint fragments taken from Pompeii's archaeological excavation. The analyses of the polar and non-polar binder components extracted from paint powder layer showed the presence of amino acids, sugars, and fatty acids but the absence of proteinaceous material. These results are consistent with a water tempera painting mixture composed of pigments, flours, gums, and oils and are in agreement with those obtained from a simulated wall paint sample made for mimicking an ancient "a secco" technique. Notably, for the first time, we report the capability to discriminate by tandem MS the presence of free amino acids in the paint layer

Cellular Interaction of Human Eukaryotic Elongation Factor 1A Isoforms

Besides its canonical role in protein synthesis, the eukaryotic translation elongation factor 1A (eEF1A) is also involved in many other cellular processes such as cell survival and apoptosis. We showed that eEF1A phosphorylation by C-Raf in vitro occurred only in the presence of eEF1A1 and eEF1A2, thus suggesting that both isoforms interacted in cancer cells (heterodimer formation). This hypothesis was recently investigated in COS-7 cells where fluorescent recombinant eEF1A isoforms colocalized at the level of cytoplasm with a FRET signal more intense at plasma membrane level. Here, we addressed our attention in highlighting and confirming this interaction in a different cell line, HEK 293, normally expressing eEF1A1 but lacking the eEF1A2 isoform. To this end, His-tagged eEF1A2 was expressed in HEK 293 cells and found to colocalize with endogenous eEF1A1 in the cytoplasm, also at the level of cellular membranes. Moreover, FRET analysis showed, in this case, the appearance of a stronger signal mainly at the level of the plasma membrane. These results confirmed what was previously observed in COS-7 cells and strongly reinforced the interaction among eEF1A isoforms. Moreover, the formation of eEF1A heterodimer in cancer cells could also be important for cytoskeleton rearrangements rather than for phosphorylation, most likely occurring during cell survival and apoptosis

Accelerating development of engineered T cell therapies in the EU: current regulatory framework for studying multiple product versions and T2EVOLVE recommendations

To accelerate the development of Advanced Therapy Medicinal Products (ATMPs) for patients suffering from life-threatening cancer with limited therapeutic options, regulatory approaches need to be constantly reviewed, evaluated and adjusted, as necessary. This includes utilizing science and risk-based approaches to mitigate and balance potential risks associated with early clinical research and a more flexible manufacturing paradigm. In this paper, T2EVOLVE an Innovative Medicine Initiative (IMI) consortium explores opportunities to expedite the development of CAR and TCR engineered T cell therapies in the EU by leveraging tools within the existing EU regulatory framework to facilitate an iterative and adaptive learning approach across different product versions with similar design elements or based on the same platform technology. As understanding of the linkage between product quality attributes, manufacturing processes, clinical efficacy and safety evolves through development and post licensure, opportunities are emerging to streamline regulatory submissions, optimize clinical studies and extrapolate data across product versions reducing the need to perform duplicative studies. It is worth noting that this paper is focusing on CAR- and TCR-engineered T cell therapies but the concepts may be applied more broadly to engineered cell therapy products (e.g., CAR NK cell therapy products)

Lymphodepletion – an essential but undervalued part of the chimeric antigen receptor T-cell therapy cycle

Lymphodepletion (LD) or conditioning is an essential step in the application of currently used autologous and allogeneic chimeric antigen receptor T-cell (CAR-T) therapies as it maximizes engraftment, efficacy and long-term survival of CAR-T. Its main modes of action are the depletion and modulation of endogenous lymphocytes, conditioning of the microenvironment for improved CAR-T expansion and persistence, and reduction of tumor load. However, most LD regimens provide a broad and fairly unspecific suppression of T-cells as well as other hematopoietic cells, which can also lead to severe side effects, particularly infections. We reviewed 1271 published studies (2011-2023) with regard to current LD strategies for approved anti-CD19 CAR-T products for large B cell lymphoma (LBCL). Fludarabine (Flu) and cyclophosphamide (Cy) (alone or in combination) were the most commonly used agents. A large number of different schemes and combinations have been reported. In the respective schemes, doses of Flu and Cy (range 75-120mg/m2 and 750-1.500mg/m2) and wash out times (range 2-5 days) differed substantially. Furthermore, combinations with other agents such as bendamustine (benda), busulfan or alemtuzumab (for allogeneic CAR-T) were described. This diversity creates a challenge but also an opportunity to investigate the impact of LD on cellular kinetics and clinical outcomes of CAR-T. Only 21 studies explicitly investigated in more detail the influence of LD on safety and efficacy. As Flu and Cy can potentially impact both the in vivo activity and toxicity of CAR-T, a more detailed analysis of LD outcomes will be needed before we are able to fully assess its impact on different T-cell subsets within the CAR-T product. The T2EVOLVE consortium propagates a strategic investigation of LD protocols for the development of optimized conditioning regimens

Regulatory phosphorylations on the eukaryotic elongation factor 1A mediated by RAF kinases

The elongation factor 1A has a crucial role in protein biosynthesis. EF1A belongs to the GTP-binding family of proteins and is able to deliver the aa-tRNA to the A-site of the ribosome because of its GTPase activity. Higher eukaryotes express two tissue specific isoforms of EF1A. In the last years, these isoforms were found to be involved in several different processes like senescence, apoptosis and transformation. In particular EF1A2 is overexpressed in several tumor cells and participates in drug resistence processes. Previous experiments in H1355 cells demonstrated that IFN treatment results in an antiapoptotical answer during which EF1A2 is increasingly expressed due to its RAF kinase mediated phosphorylation. As this interaction establishes a new link between the mitogenic cascade and protein biosynthesis, in vitro kinase assays were performed in presence of B- and C-RAF kinase and recombinant EF1A1/2 to investigate this important interaction. Mass spectrometry analysis identified treonine 88 (exclusively mediated by B-RAF) and serine 21 on EF1A1 and serine 21 on EF1A2 as B-/ and C-RAF mediated phosphorylation sites. Interestingly, serine 21 belongs to the consensus sequence of the GTP/GDP binding domain of EF1A. Its phosphorylation prevents the binding of the nucleotide suggesting that this potentially RAF mediated modification has a regulatory role affecting the function of the elongation factor

FT-IR spectromicroscopy of mammalian cell cultures during necrosis and apoptosis induced by drugs

FT-IR is used in the field of biology and medicine to detect bimolecular changes in disordered cells and tissues. In this report, using FT-IR microscopy, we characterize changes in apoptotic and necrotic Jurkat cells with respect to normal cells. The analysis of deconvoluted regions of the FT-IR spectra showed significant differences compared to the controls in three spectral regions. In particular, the apoptotic cells were characterized by an increase in the absorption at 2925 cm−1, due to the asymmetric CH2-stretching (νasCH2) of membrane lipids whereas the spectral areas ratio (A1654/A1629) of the amide I region indicated an increase in apoptotic cells of more α-helical structures with respect to of β-sheet content. Interestingly, apoptotic cells showed the appearance of a peak around 1743 cm−1, ν(C=O) assigned to acid ester. Because no other similar increase for lipid bands was observed, the increase of A1745 is not simply due to an increase in the number of lipid molecules or their density but could also be indicative as marker of apoptosis. These spectral changes were not observed in necrotic Jurkat cells

FT-IR spectromicroscopy of mammalian cell cultures during necrosis and apoptosis induced by drugs, Spectroscopy

Abstract. FT-IR is used in the field of biology and medicine to detect bimolecular changes in disordered cells and tissues. In this report, using FT-IR microscopy, we characterize changes in apoptotic and necrotic Jurkat cells with respect to normal cells. The analysis of deconvoluted regions of the FT-IR spectra showed significant differences compared to the controls in three spectral regions. In particular, the apoptotic cells were characterized by an increase in the absorption at 2925 cm −1 , due to the asymmetric CH 2 -stretching (νasCH 2 ) of membrane lipids whereas the spectral areas ratio (A 1654 /A 1629 ) of the amide I region indicated an increase in apoptotic cells of more α-helical structures with respect to of β-sheet content. Interestingly, apoptotic cells showed the appearance of a peak around 1743 cm −1 , ν(C=O) assigned to acid ester. Because no other similar increase for lipid bands was observed, the increase of A 1745 is not simply due to an increase in the number of lipid molecules or their density but could also be indicative as marker of apoptosis. These spectral changes were not observed in necrotic Jurkat cells