Unravelling the mechanisms of resistance to imatinib mesylate in chronic myeloid leukemia: a proteomic approach

Imatinib mesylate is a potent inhibitor of the Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. The aim of this study was to obtain further insights into the Bcr-Abl activity-independent mechanisms underlying imatinib resistance, in chronic myeloid leukemia. The imatinib-resistant KCL22R and sensitive KCL22S cells were used as experimental model. None of the already described resistance mechanisms has been detected so far in KCL22R cells; therefore additional mechanisms independent of Bcr-Abl kinase activity could be envisaged. Moreover, KCL22S cells exhibited typical features of the quiescent hematopoietic Ph+ stem cells, thereby representing a good experimental model to investigate imatinib resistance. To this aim differentially expressed proteins between KCL22S and KCL22R cells were characterized using a proteomic approach: two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Tandem Mass Spectrometry. 51 proteins were identified: 27 over-expressed and 24 under-expressed in KCL22R cells versus KCL22S cells. Bioinformatic analysis with GeneSpring and Ingenuity Pathway Analysis (IPA) softwares showed that several of these proteins were involved in the modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-kB and Ras-MAPK signaling. Since the Erk pathway has been shown to influence chemotherapeutic drug resistance of hematopoietic cells, the level of activation of Erk in KCL22R and KCL22S cells was investigated. This analysis demonstrated that continuous activation of Erk occurred in KCL22R cells as compared to sensitive cells. Interestingly, examination of the most statistically significant protein network showed that several differentially expressed proteins, between KCL22R and KCL22S cells, were directly or indirectly connected with Erk. In particular, among them, this study focused on two SH2-containing, non-receptor protein tyrosine phosphatases: Shp1 (PTPN6) and Shp2 (PTPN11). It has been shown that Shp2 positively regulates the Ras-Erk pathway and is activated by phosphorylation. This study demonstrated that the level of phosphorylation and hence of activation of Shp2 in KCL22R cells was higher than in KCL22S cells. In addition the knock-down of Shp2 expression, in combination with imatinib treatment, significantly reduced the activation of Erk 1/2 in KCL22R cells and produced a reversion of the KCL22R phenotype, suggesting that Shp2 plays a role in the Bcr-Abl activity-independent mechanisms of imatinib resistance. Interestingly this study also demonstrated that Shp1, that was found down-regulated in KCL22R cells, interacted with Shp2 and that Shp1 played a negative role in the Shp2 activation in KCL22S cells. Moreover Annexin A1 and Hsp70, belonging to the same protein network, were found down-regulated in KCL22R cells. They could also play a role in imatinib resistance by the direct or indirect interaction with Shp2. Taken together these results suggest that a reduced Shp1 expression in KCL22R cells could contribute to a continuous Shp2 activation, sustaining a Bcr-Abl activity-independent pathway of proliferation and survival to imatinib treatment. These two proteins could be used as putative biomarkers to evaluate the efficacy of imatinib treatment and to develop new combinatorial therapeutic approaches

Detection of colonic dysplasia in patients with ulcerative colitis using a targeted fluorescent peptide and confocal laser endomicroscopy: A pilot study

Targeted molecular probes have been used to detect sporadic colonic dysplasia during confocal laser endomicroscopy (CLE) with promising results. This is a feasibility pilot study aiming to assess the potential role of CLE combined with a fluorescent-labeled peptide to stain and detect dysplasia associated with Ulcerative Colitis

An ancestral host defence peptide within human beta-defensin 3 recapitulates the antibacterial and antiviral activity of the full-length molecule

Host defence peptides (HDPs) are critical components of innate immunity. Despite their diversity, they share common features including a structural signature, designated “γ-core motif”. We reasoned that for each HDPs evolved from an ancestral γ-core, the latter should be the evolutionary starting point of the molecule, i.e. it should represent a structural scaffold for the modular construction of the full-length molecule, and possess biological properties. We explored the γ-core of human β-defensin 3 (HBD3) and found that it: (a) is the folding nucleus of HBD3; (b) folds rapidly and is stable in human serum; (c) displays antibacterial activity; (d) binds to CD98, which mediates HBD3 internalization in eukaryotic cells; (e) exerts antiviral activity against human immunodeficiency virus and herpes simplex virus; and (f) is not toxic to human cells. These results demonstrate that the γ-core within HBD3 is the ancestral core of the full-length molecule and is a viable HDP per se,since it is endowed with the most important biological features of HBD3. Notably, the small, stable scaffold of the HBD3 γ-core can be exploited to design disease-specific antimicrobial agents

Neutralizing antibodies to Omicron after the fourth SARS-CoV-2 mRNA vaccine dose in immunocompromised patients highlight the need of additional boosters

IntroductionImmunocompromised patients have been shown to have an impaired immune response to COVID-19 vaccines.MethodsHere we compared the B-cell, T-cell and neutralizing antibody response to WT and Omicron BA.2 SARS-CoV-2 virus after the fourth dose of mRNA COVID-19 vaccines in patients with hematological malignancies (HM, n=71), solid tumors (ST, n=39) and immune-rheumatological (IR, n=25) diseases. The humoral and T-cell responses to SARS-CoV-2 vaccination were analyzed by quantifying the anti-RBD antibodies, their neutralization activity and the IFN-γ released after spike specific stimulation.ResultsWe show that the T-cell response is similarly boosted by the fourth dose across the different subgroups, while the antibody response is improved only in patients not receiving B-cell targeted therapies, independent on the pathology. However, 9% of patients with anti-RBD antibodies did not have neutralizing antibodies to either virus variants, while an additional 5.7% did not have neutralizing antibodies to Omicron BA.2, making these patients particularly vulnerable to SARS-CoV-2 infection. The increment of neutralizing antibodies was very similar towards Omicron BA.2 and WT virus after the third or fourth dose of vaccine, suggesting that there is no preferential skewing towards either virus variant with the booster dose. The only limited step is the amount of antibodies that are elicited after vaccination, thus increasing the probability of developing neutralizing antibodies to both variants of virus.DiscussionThese data support the recommendation of additional booster doses in frail patients to enhance the development of a B-cell response directed against Omicron and/or to enhance the T-cell response in patients treated with anti-CD20

Host defense peptide-derived privileged scaffolds for anti-infective drug discovery

'Privileged scaffolds' are molecular frameworks which have been successfully exploited for small molecule drug discovery. Peptide privileged scaffolds, featuring a strictly conserved multiple-disulfide framework and high variability in the rest of the sequence, display a broad range of biological effects, including antimicrobial and antiviral activity. Unlike small molecules, however, the cost of manufacturing these peptides is high, and their synthesis challenging. We previously described a simplified privileged scaffold corresponding to the γ-core of human β-defensin-3 (HBD3). The γ-core is a common structural signature found in virtually all host defense peptides (HDPs) stabilized by multiple disulfides, and we showed that for HBD3, it represents the evolutionary starting point of the full-length molecule and, thus, is itself a primordial HDP. Accordingly, we showed that the peptide folded rapidly and was stable in human serum, and displayed many of the biological activities of HBD3. We report here that in addition to the previously reported antibacterial activity on planktonic bacteria, the γ-core peptide is active against biofilm formation and maturation. We also show that it is readily cell penetrant, like HBD3, although with a different mechanism, which is independent from CD98. Overall, the potency of the single-disulfide, 23-amino acid γ-core is comparable with the full-length peptide across the whole spectrum of examined properties, and the peptide is not toxic to human cells. The HBD3 γ-core peptide may therefore represent the first example of an economically viable lead peptide derived from a HDP privileged scaffold. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd

Membrane protein 4F2/CD98 is a cell surface receptor involved in the internalization and trafficking of human β-defensin 3 in epithelial cells

Human β-defensins play a pivotal role in the innate immune response. Although expressed by and acting at epithelial surfaces, little is known about their specific interaction with epithelial structures. Here, we identify the transmembrane protein CD98 as a cell surface receptor involved in the internalization of human β-defensin 3 (hBD3) in human epithelial A549 cells. CD98 and hBD3 extensively colocalize on the basolateral domain of A549. While verifying their direct binding by fluorescence resonance energy transfer and surface plasmon resonance, we mapped the interaction to CD98 residues 304-414, i.e. to the region known to interact with the proteins of intestinal bacteria during colonic invasion. Treatment of A549 cells with hBD3 dramatically reduces CD98 expression and conversely, knockdown of CD98 expression impairs hBD3 cell surface binding and internalization. Competition for bacterial binding to CD98 and downregulation of CD98 expression may represent novel mechanisms for the antibacterial activity of hBD3