Littérature scientifique sur le sujet « FISH, cathepsin K, target therapy »

The new category of MiT family translocation renal cell carcinoma has been included into the World Health Organization (WHO) classification in 2016. The MiT family translocation renal cell carcinoma comprises Xp11 translocation renal cell carcinoma harboring TFE3 gene fusions and t(6;11) renal cell carcinoma harboring TFEB gene fusion. At the beginning, they were recognized in childhood; nevertheless, it has been demonstrated that these neoplasms can occur in adults as well. In the nineties, among Xp11 renal cell carcinoma, ASPL, PRCC, and SFPQ (PSF) were the first genes recognized as partners in TFE3 rearrangement. Recently, many other genes have been identified, and a wide spectrum of morphologies has been described. For this reason, the diagnosis may be challenging based on the histology, and the differential diagnosis includes the most common renal cell neoplasms and pure epithelioid PEComa/epithelioid angiomyolipoma of the kidney. During the last decades, many efforts have been made to identify immunohistochemical markers to reach the right diagnosis. To date, staining for PAX8, cathepsin K, and melanogenesis markers are the most useful identifiers. However, the diagnosis requires the demonstration of the chromosomal rearrangement, and fluorescent in situ hybridization (FISH) is considered the gold standard. The outcome of Xp11 translocation renal cell carcinoma is highly variable, with some patients surviving decades with indolent disease and others dying rapidly of progressive disease. Despite most instances of t(6;11) renal cell carcinoma having an indolent clinical course, a few published cases demonstrate aggressive behavior. Recently, renal cell carcinomas with TFEB amplification have been described in connection with t(6;11) renal cell carcinoma. Those tumors appear to be associated with a more aggressive clinical course. For the aggressive cases of MiT family translocation carcinoma, the optimal therapy remains to be determined; however, new target therapies seem to be promising, and the search for predictive markers is mandatory.

Cathepsin K is a papain-like cysteine protease with high matrix-degrading activity. Among several cathepsins, cathepsin K is the most potent mammalian collagenase, mainly expressed by osteoclasts. This review summarizes most of the recent findings of cathepsin K expression, highlighting its role in renal tumors for diagnostic purposes and as a potential molecular target. Indeed, cathepsin K is a recognized diagnostic tool for the identification of TFE3/TFEB-rearranged renal cell carcinoma, TFEB-amplified renal cell carcinoma, and pure epithelioid PEComa/epithelioid angiomyolipoma. More recently, its expression has been observed in a subgroup of eosinophilic renal neoplasms molecularly characterized by TSC/mTOR gene mutations. Interestingly, both TSC mutations or TFE3 rearrangement have been reported in pure epithelioid PEComa/epithelioid angiomyolipoma. Therefore, cathepsin K seems to be a downstream marker of TFE3/TFEB rearrangement, TFEB amplification, and mTOR pathway activation. Given the established role of mTOR inhibitors as a pharmacological option in renal cancers, cathepsin K could be of use as a predictive marker of therapy response and as a potential target. In the future, uropathologists may implement the use of cathepsin K to establish a diagnosis among renal tumors with clear cells, papillary architecture, and oncocytic features.

Dental caries, one of the most prevalent infectious diseases worldwide, affects approximately 80% of children and the majority of adults. Dental caries may result in endodontic disease, leading to dental pulp necrosis, periapical inflammation and bone resorption, severe pain, and tooth loss. Periapical inflammation may also increase inflammation in other parts of the body. Although many studies have attempted to develop therapies for this disease, there is still an urgent need for effective treatments. In this study, we applied a novel gene therapeutic approach using recombinant adeno-associated virus (AAV)-mediated RNAi knockdown of Cathepsin K (Ctsk) gene expression, to target osteoclasts and periapical bone resorption in a mouse model. We found that AAV-sh-Cathepsin K (AAV-sh-Ctsk) impaired osteoclast function in vivo and furthermore reduced bacterial infection-stimulated bone resorption by 88%. Reduced periapical lesion size was accompanied by decreases in mononuclear leukocyte infiltration and inflammatory cytokine expression. Our study shows that AAV-RNAi silencing of Cathepsin K in periapical tissues can significantly reduce endodontic disease development, bone destruction, and inflammation in the periapical lesion. This is the first demonstration that AAV-mediated RNAi knockdown gene therapy may significantly reduce the severity of endodontic disease.

Abstract Cathepsin S (Cat S), expressed predominately on antigen presenting cells, has been proposed as a therapeutic target for asthma. We used genetic and pharmacological tools to investigate the role of Cat S in murine models of allergic asthma. Mice null for Cat S were protected from OVA-induced pulmonary inflammation, but exhibited no protection from OVA-induced airway hyper-reactivity. To determine the role of Cat S during the challenge phase, we identified a potent and selective Cat S inhibitor, Compound A (Cpd A, IC50 mCat S = 0.6 nM, ≥470 fold selective vs mCat B, K, L), which inhibited antigen presentation in a mouse cell-based assay (IC50 = 44 nM). The prodrug of Cpd A, Cpd B, gave excellent plasma levels of Cpd A when dosed in mice by gavage, or in food. In vivo competition in mice with an irreversible pan-selective cysteine cathepsin probe showed that Cpd B gave selective inhibition of lung and spleen Cat S at 1 mpk, but lost selectivity at 50 mpk. Cpd B was dosed in mice (10, 100 mpk in food) over 4 days of the challenge period in the murine ovalbumin model. Both doses had no effect on bronchoalveolar lavage infiltrating cells, despite showing high levels of Cat S inhibition. Thus, Cat S inhibition in a therapeutic mode does not attenuate airway inflammation in antigen sensitised mice suggesting that anti-Cat S therapy would not be an effective asthma treatment.

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia, mononuclear cell infiltration, bone erosion and joint destruction. Recently we have shown that changes in energy generation-related gene expressions in the peripheral blood of tofacitinib (TOFA)-naïve patients with RA are associated with clinical response to treatment [1]. Therefore, considering metabolic status of patients with RA, may be useful for identification of prognostic biomarkers for personal responsiveness to TOFA therapy in TOFA-naïve RA patients by baseline gene expression analysis in peripheral blood mononuclear cells (PBMCs) cultured with TOFA prior to therapy.ObjectivesTo examine the association of changes in expression of the genes responsible for cell growth and proliferation [mammalian target of rapamycin, mTOR], bone and articular cartilage resorption [matrix metalloproteinase (MMP)-9, cathepsin K], and inflammation [tumour necrosis factor (TNF)α] in the PBMCs obtained at baseline from patients with RA and cultured with TOFA with therapy outcome.Methods12 patients with RA (median age 55 years), median disease duration 28.5 months (without previous history of therapy with TOFA) were examined. 6 of these patients with RA gained remission after 3 months of TOFA therapy while 6 patients, maintained high and moderate disease activity. Control group consisted of 26 healthy subjects. PBMCs were isolated prior to therapy using Ficoll density gradient and cultured with 10 nM tofacitinib citrate (TOFA) during 48h. Cell viability was monitored with 0.2% Trypan blue staining. Total RNA isolated from these cells was used for mTOR, MMP-9, cathepsin K, and TNFα gene expression studies performed with quantitative real-time RT-PCR.ResultsTOFA was capable of modifying gene expression in cultured PBMCs from the examined patients with RA compared with untreated cells. Baseline expression of all the examined genes was significantly upregulated in cultured with TOFA PBMCs from patients who maintained high and moderate disease activity after TOFA therapy compared with untreated counterparts. In contrast, TNFα gene expression was significantly downregulated in cultured with TOFA PBMCs of patients who gained remission compared with untreated cells. No significant changes in the expression of other examined genes was observed in cultured with TOFA PBMCs from the examined patients with RA who gained remission after TOFA therapy compared with untreated counterparts.ConclusionDownregulation of TNFα gene expression in cultured with TOFA PBMCs compared with untreated counterparts in TOFA-naïve patients with RA prior to therapy might serve a prognostic biomarker of personal positive response to TOFA therapy while upregulation in the expression of the same gene suggests non-response to TOFA therapy.References[1]Tchetina EV, Satybaldyev AM, Markova GA, Samarkina EYu, Lila AM. Putative association between low baseline gene expression in the peripheral blood and clinical remission in rheumatoid arthritis patients treated with tofacitinib. Life (MDPI Basel) 2021, 11(12), 1385; doi:10.3390/life11121385AcknowledgementsThis study was funded by Russian Ministry of Education and Science (Project no. 2022-009-1021062512064-0).Disclosure of InterestsNone declared

Subchondral bone lesions, as the crucial inducement for accelerating cartilage degeneration, have been considered as the initiating factor and the potential therapeutic target of knee osteoarthritis (KOA). Acupotomy, the biomechanical therapy guided by traditional Chinese meridians theory, alleviates cartilage deterioration by correcting abnormal mechanics. Whether this mechanical effect of acupotomy inhibits KOA subchondral bone lesions is indistinct. This study aimed to investigate the effects of acupotomy on inhibiting subchondral bone resorption and to define the possible mechanism in immobilization-induced KOA rabbits. After KOA modeling, 8 groups of rabbits (4w/6w acupotomy, 4w/6w electroacupuncture, 4w/6w model, and 4w/6w control groups) received the indicated intervention for 3 weeks. Histological and bone histomorphometry analyses revealed that acupotomy prevented both cartilage surface erosion and subchondral bone loss. Further, acupotomy suppressed osteoclast activity and enhanced osteoblast activity in KOA subchondral bone, showing a significantly decreased expression of tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinases-9 (MMP-9), and cathepsin K (Ctsk) and a significantly increased expression of osteocalcin (OCN); this regulation may be mediated by blocking the decrease in osteoprotegerin (OPG) and the increase in NF- κ B receptor activated protein ligand (RANKL). These findings indicated that acupotomy inhibited osteoclast activity and promoted osteoblast activity to ameliorate hyperactive subchondral bone resorption and cartilage degeneration in immobilization-induced KOA rabbits, which may be mediated by the OPG/RANKL signaling pathway. Taken together, our results indicate that acupotomy may have therapeutic potential in KOA by restoring the balance between bone formation and bone resorption to attenuate subchondral bone lesions.

ObjectivesAccelerated atherosclerotic disease typically complicates rheumatoid arthritis (RA), leading to premature cardiovascular death. Inflammatory macrophages are key effector cells in both rheumatoid synovitis and the plaques of coronary artery disease (CAD). Whether both diseases share macrophage-dependent pathogenic mechanisms is unknown.MethodsPatients with RA or CAD (at least one myocardial infarction) and healthy age-matched controls were recruited into the study. Peripheral blood CD14+ monocytes were differentiated into macrophages. Metabolic profiles were assessed by Seahorse Analyzer, intracellular ATP concentrations were quantified and mitochondrial protein localisation was determined by confocal image analysis.ResultsIn macrophages from patients with RA or CAD, mitochondria consumed more oxygen, generated more ATP and built tight interorganelle connections with the endoplasmic reticulum, forming mitochondria-associated membranes (MAM). Calcium transfer through MAM sites sustained mitochondrial hyperactivity and was dependent on inactivation of glycogen synthase kinase 3b (GSK3b), a serine/threonine kinase functioning as a metabolic switch. In patient-derived macrophages, inactivated pGSK3b-Ser9 co-precipitated with the mitochondrial fraction. Immunostaining of atherosclerotic plaques and synovial lesions confirmed that most macrophages had inactivated GSK3b. MAM formation and GSK3b inactivation sustained production of the collagenase cathepsin K, a macrophage effector function closely correlated with clinical disease activity in RA and CAD.ConclusionsRe-organisation of the macrophage metabolism in patients with RA and CAD drives unopposed oxygen consumption and ultimately, excessive production of tissue-destructive enzymes. The underlying molecular defect relates to the deactivation of GSK3b, which controls mitochondrial fuel influx and as such represents a potential therapeutic target for anti-inflammatory therapy.

Bone destruction is a major complication of multiple myeloma (MM). Healthy bone is constantly remodeled through bone resorption by osteoclasts and bone formation by osteoblasts. New bone formation in MM is virtually non-existent, because differentiation of osteoblasts is inhibited by DKK1, a Wnt-β-catenin signaling inhibitor secreted by MM cells, reported by our group in NEJM, 2003. MM in its early stages is totally dependent on its microenvironment and for the hyperdiploid type MM this dependence is perpetual. Based on concordant gene expression signatures, predominantly driven by recurrent translocations and hyperdiploidy, we have classified MM into 7 distinct molecular entities. One subgroup, with significantly less bone disease and superior event-free and overall survival following high-dose therapy and stem cell transplantation than the other subgroups, defined as the Low Bone (LB) disease subgroup. Consistent with the LB phenotype, we have observed a strong inverse correlation between DKK1 and CST6 expression and by analyzing gene expression profiling (GEP) and RNA-sequencing data of more than 1,000 myeloma patients, we identified CST6 as the most upregulated gene in the LB subgroup. The aim of the present study was to determine the role of Cystatin E/M (CST6) in MM biology and to apply this knowledge to prevent both bone disease and MM cell growth. CST6, a 14-17 kD secretory protein, is a lysosomal protease inhibitor and suggested tumor suppressor gene. We showed that overexpression of CST6 in human MM cell lines prevents MM cell growth in vitro and in vivo. Also, purified CST6 protein from conditioned media of CST6-overexpressing MM cells significantly inhibits MM cell growth (p<0.01) and RANKL-induced osteoclast differentiation (p<0.01), decreases MM cell-induced bone destruction (p<0.05), and extends MM mouse survival (p<0.01). Mechanistic studies indicate that CST6 abrogates the alternative NF-kB signaling pathway as evidenced by a decrease in nuclear p52 protein in CST6-treated osteoclast precursors. Cathepsin K (CTSK), an osteoclast specific cysteine protease involved in bone resorption, was inhibited by CST6. GEP studies of whole bone marrow biopsies (WBMBx) across a spectrum of samples show higher expression of CTSK in WBMBx relative to purified plasma cells, while levels in MM remission WBMBx were higher than seen in healthy adult donors, MGUS/SMM, and newly diagnosed MM. Importantly, CTSK levels where not significantly different between remission and relapsed MM WBBx. These data show that Cathepsin K levels, and therefore osteoclasts, are elevated in the bone marrow of MM in remission and that these levels are similar to that seen in relapsed MM. Based on GEP data and experimental confirmation, we conclude that CST6, secreted by MM cells could be used clinically to target MM cells and prevent bone damage in MM. Inhibiting CTSK by CST6 in MM remission may aid in the prevention of MM relapse. Disclosures van Rhee: CDCN: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotech: Consultancy; EUSA: Consultancy.

e15609 Background: Pancreatic adenocarcinoma is a genetically highly complex and heterogenous tumor type with strong genetic instability which makes it resistant to therapy. Known amplifications of oncogenes such as KRAS or MYC and deletions of tumor suppresor genes such as CDKN2A and SMAD4 have demonstrated the importance of genetic alteration in this tumor type. Methods: We report the use of an Affymetrix Genome-Wide Human single nucleotide polymorphism (SNP) Array 6.0 (906,600 SNPs) to screen for gene copy number changes and allelic imbalances in 8 microdissected primary pancreatic tumors and 7 established pancreatic cancer cell lines. Gene Chip Human Genome U133 2.0 Array was used to make an RNA expression profile. Mutation analysis of KRAS and M-FISH analysis of cell lines was performed. Results: SNP arrays confirmed the presence of previously reported cytogenetic abnormalities in the cell lines and primary tumor probes, including MYC amplifikation at 8q24, gain of 17q12 (ERBB2/HER2), 7p12 (EGFR) and 12p12.1 (KRAS). KRAS mutation was seen in 71% of cell lines (5/7). We identified several alterations in signaling pathways such as Wnt/Notch Signaling and KRAS signaling. A sizeable subset ( 7 of 15 cases; 47%) showed an amplikon at 19q13.1–13.2 in which the serine/threonine kinase Mirk/Dyrk1B is localized, a downstream effector of oncogenic k-ras. There was also strong concordance between primary tumors and cell lines with respect to gains on 8q, 12p and 18q. Analysis of gene expression was used to localize potential target genes. M-FISH analysis showed complex karyotypes with chromosomal deletions in 9p and 18q, regions that are known to harbor tumor suppressor genes (CDKN2A, SMAD4 and TP53). Conclusions: Several signaling pathways mediate tumor cell survival. Analysis of gene amplification and RNA expression profile provide molecular biological characteristics and an individual gene signature of the tumor which allow us to choose more efficient drugs to an individualized treatment. Pathways activated by KRAS such as DYRK1B may offer new therapeutic targets. Further functional characterization is needed to provide evidence for the actual role of any putative target gene. No significant financial relationships to disclose.

Renal cell carcinomas with chromosome translocation are rare neoplasms which often occur in young patients. In the last World Health Organization (WHO 2016) are named as MiT family translocation renal cell carcinoma comprising two different entities: Xp11 renal cell carcinoma and t(6;11) renal cell carcinoma. Recently, renal cell carcinomas with TFEB amplification has been described in connection with t(6;11) renal cell carcinoma. We analyzed 30 MiT family translocation renal cell carcinoma and 2 renal cell carcinomas with TFEB amplification collecting data on clinical, histological, immunohistochemical and molecular features. In this study, we sought 1) immunohistochemical diagnostic markers (cathepsin K, CD68 (PG-M1), PAX8) since the differential diagnosis is challenging, especially with pure epithelioid PEComa/epithelioid angiomyolipoma; 2) fluorescence in situ hybridization diagnostic features to reach the correct diagnosis; 3) predictive markers (MET, AXL, VEGF) in tumor tissue for target therapy. Histologically, either cytological or architectural appearance was peculiar in each case. By immunohistochemistry, almost all MiT family translocation renal cell carcinomas expressed PAX8. Staining for cathepsin K was found in 65% of Xp11 renal cell carcinomas, only a few cases were positive for melanogenic markers and all cases were negative for CD68 (PG-M1 clone). All t(6;11) renal cell carcinomas labelled for cathepsin K and Melan-A and negative for CD68 (PG-M1 clone). Seven pure epithelioid PEComa /epithelioid angiomyolipomas, used as control, were positive for cathepsin K, melanocytic markers and CD68 (PG-M1) and negative for PAX8. All MiT family translocation renal cell carcinomas were negative for AXL; 61% of Xp11 renal cell carcinomas and 5 of 7 t(6;11) renal cell carcinomas expressed MET. Fluorescence in situ hybridization results showed the presence of TFEB gene translocation in all t(6;11) renal cell carcinomas and TFE3 gene translocation in all Xp11 renal cell carcinoma with a high frequency of split fluorescent signals (mean 74% and 68% respectively). Among the eight t(6;11) renal cell carcinomas, one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3-4 copies of fluorescent signals) with a concomitant increased number of CEP6. Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, three cases showed high level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, while four showed TFEB gene rearrangement without increased copy number. In conclusion, we report the high frequency of split signals by FISH in MiT family translocation renal cell carcinomas suggesting that 40% of split signals could be used as the proper cut-off to reach the correct diagnosis. We demonstrate the usefulness of CD68 (PG-M1) immunohistochemical staining in distinguishing MiT family translocation renal cell carcinoma from pure epithelioid PEComa/epithelioid angiomyolipoma. Finally, VEGF, MET but not AXL may be potential predictive marker for targeted therapy in MiT family renal cell carcinomas.