Статті в журналах з теми "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.

Abstract Osteolytic bone disease in Multiple Myeloma (MM) is caused by enhanced osteoclast (OCLs) activation and inhibition of osteoblast function. Lenalidomide and bortezomib have shown promising anti-MM effects, and bortezomib has inhibitory effects on OCLs. However, the effect of lenalidomide on OCLs in MM and how bortezomib interferes with osteoclastogenesis is unknown. Here we investigated the effect of lenalidomide and bortezomib on human OCLs. Peripheral blood mononuclear cells (PBMC) from MM patients were stimulated with receptor activator of NFk-B ligand (RANKL) (50ng/ml) and M-CSF (25ng/ml) for two weeks to induce OCL formation, in the presence or absence of lenalidomide or bortezomib. OCLs were identified by flow cytometric analysis using anti-αVβ3 integrin. Lenalidomide and bortezomib inhibited OCL differentiation indicated by a decrease in αVβ3-integrin (lenalidomide at 0μM: median 69.3%; range 28.9 – 89.0%; at 2μM: median 50.4%; range 21.5 – 64.2%; at 10μM: median 39.2%; range 33.6 – 47.5%) (bortezomib at 0nM: median 69.3%; range 28.9 – 89.0%; at 2nM: median 35.0%; range 11.0 – 79.0%; at 5nM: median 11.5%; range 5.5 – 8.8%; p<0.05). Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify OCLs and confirm OCL activity. Both drugs decreased TRAP -positive cells (lenalidomide at 0μM: median 70.5%; range 50.0 – 84.0%; at 2μM: median 47.0%; range 31.0 – 77.0%; at 10μM: median 32.5%; range 14.0 – 44.0%; p<0.05) (bortezomib at 0nM: median 70.5%; range 50.0 – 84.0%; at 2nM: median 26.0%; range 11.0 – 39.0%; p<0.01; at 5nM: median 17.0%; range 13.0 – 34.0%; p<0.001). To assess bone resorption activity, OCL were cultured with dentine discs, in the presence or absence of lenalidomide and bortezomib, followed by light microscopic analysis. Both lenalidomide and bortezomib inhibited bone resorption in a dose-dependent manner. Using western blot analysis, we identified PU.1 and pERK as major targets of lenalidomide, and NFATc1 as a target of bortezomib, resulting in inhibition of osteoclastogenesis. Furthermore, downregulation of cathepsin K, essential for resorption of the bone collagen matrix, was also noted. We demonstrated a significant decrease of growth and survival factors including MIP-1α, IL-6, B-cell activation factor (BAFF), and a proliferation-inducing ligand (APRIL). Importantly, in serum from patients with refractory of relapsed MM treated with lenalidomide, the essential bone remodeling factor RANKL, as well as the RANKL/Osteoprotegerin (OPG) ratio, were significantly reduced. The median OPG at baseline was significantly lower (median 8.8pg/ml, range 7.7–12.6pg/ml) than after treatment (median 10.4pg/ml, range 8.5–15pg/ml) (p<0.05). Lenalidomide also significantly inhibited the secretion of RANKL in patient’s serum (baseline: median 17pg/ml, range 9.0–36pg/ml; two months after therapy: median 4.2pg/ml, range 2.5–36pg/ml) (p<0.05). The ratio of RANKL/OPG was significantly higher before than after therapy (baseline: median 2.2pg/ml, range 1.1–3.0pg/ml; two months after therapy: median 0.5pg/ml, range 0.3–2.6pg/ml) (p<0.05). We conclude that both agents specifically target key factors in osteoclastogenesis, thereby inhibiting OCL differentiation and function.

Abstract B-PLL is defined by the presence of prolymphocytes in peripheral blood exceeding 55% of lymphoid cells. The diagnosis, mainly based on clinical and morphological data, can be difficult because of overlap with other B-cell malignancies. Because of the rarity of the disease, only case reports and small series describe its cytogenetic features. Few prognostic markers have been identified in this aggressive leukemia usually resistant to standard chemo-immuno therapy. We report here the cytogenetic and molecular findings in a large series of B-PLL. We also studied the in vitro response to novel targeted drugs on primary B-PLL cells. The study included 34 cases with a diagnosis of B-PLL validated by morphological review performed by three independent expert cytologists. The diagnosis of mantle cell lymphoma was excluded by karyotype (K) and FISH using CCND1, CCND2 and CCND3 probes. Median age at diagnosis was 72 years [46-88]. K was complex (≥3 abnormalities) in 73%, and highly complex (HCK≥5) in 45%. Combining K and FISH data, the most frequent chromosomal aberrations were: translocation targeting the MYC gene [t(MYC)] (21/34, 62%), 17p deletion including TP53 gene (13/34, 38%), trisomy 18/18q (10/33, 30%), 13q14 deletion (10/34, 29%), trisomy 3 (8/33, 24%), trisomy 12 (8/34, 24%) and 8p deletion (7/31, 23%). Whole-Exome Sequencing analysis of paired tumor-control DNA was performed in 16 patients. The most frequently mutated genes were TP53(6/16, 38%), associated with del17p in all, MYD88 (n=4), BCOR (n=4), MYC (n=3), SF3B1 (n=3), FAT1 (n=3), SETD2 (n=2), CHD2 (n=2), CXCR4 (n=2), BCLAF1 (n=2) and NFASC (n=2). Distribution of the chromosomal aberrations is shown in Fig 1. The main group of patients (21/34, 62%) had a t(MYC) that was associated with a higher % of prolymphocytes (86 vs 76, p=0.03), CD38 expression (90% vs 15%,p<0.001), and a lower K complexity (HCK≥5 : 20% vs 85%, p=0.0004). Mutations in MYC and in genes involved in RNA metabolism and chromatin remodeling were almost exclusively observed with t(MYC). Principal component analysis of gene expression data in 12 cases analyzed by RNA-Seq showed that the 7 patients with t(MYC) clustered together. These results suggest that t(MYC) form a homogeneous subgroup of B-PLL. A second group with MYC gain (5/34, 15%), was associated with HCK≥5 (100% vs 36%, p=0,01) and trisomy 3 (80% vs 14%, p=0,008). Altogether, 26/34 patients (76%) had a MYC activation, translocation or gain, that were mutually exclusive. The median overall survival (OS) for the entire cohort was 126 months with a median follow-up time of 47 months [ 0.2-141]. We found MYC activation (translocation or gain) to be associated with a shorter OS (p=0.03). Regarding MYC and del17p, we identified 3 distinct cytogenetic prognostic groups, with significant differences in OS (p=0.0006) (Fig 2). The patients without MYC activation had the lower risk (n=8, median not reached). Patients with a MYC activation without del17p had an intermediate risk (n=18, 125 months). The highest risk group corresponded to patients with both MYC and TP53 aberrations (n=7, 11 months). We performed drug response profiling on primary B-PLL cells using the ATP-based CellTiter Glo kit (Promega) (n=5). We observed that after 48h of exposure to increased doses, response was heterogeneous, with a majority of samples resistant to fludarabine (n=3), ibrutinib (n=3), idelalisib (n=4), venetoclax (n=3) and OTX015 (n=4). Annexin/PI assays using flow cytometry showed that the induced cell death could be increased by combination of ibrutinib or venetoclax with OTX015 or JQ1, two BET protein inhibitors that target MYC signaling (n=1/2). In summary, B-PLL have complex and highly complex K, a high frequency of MYC activation by translocation or gain, frequent 17p deletion, and frequent mutations in MYC, TP53, BCOR, and MYD88 genes. We identified 3 prognostic subgroups according to MYC and 17p status. Patients with MYC activation + 17p deletion have the shorter OS, and should be considered as a high-risk "double-hit" subgroup. Our results show that cytogenetic analysis is a useful diagnostic tool in B-PLL that improves prognostic stratification. We recommend to perform K and FISH (MYC and TP53) analyses systematically when a B-PLL is suspected. Our in vitro data suggest that drugs targeting the BCR and BCL2 in combination with MYC inhibition may be a therapeutic option in some patients. Disclosures Baseggio: Takeda Oncology: Honoraria.

Abstract BCR-ABL1 inhibitors have revolutionized treatment of CML patients. However several drawbacks remain, including therapy resistance of T315I-mutated CML and incapability of current drugs to eliminate quiescent CML stem cells warranting development of novel therapies. In addition, drugs with the potential to enhance efficacy of BCR-ABL1 targeting agents could improve treatment of CML patients. Members of the Tyro3, Axl, Mer receptor (TAMR) tyrosine kinase family are abundantly expressed in physiological and malignant hematopoiesis and their ligand Gas6 can support hematopoietic (progenitor) cells. Evidence in the literature indicates that Axl is upregulated upon treatment with imatinib (IM). In this study, we investigated the relevance of the Gas6-Axl axis in CML patients and the therapeutic potential of the clinically applicable small molecule Axl inhibitor BGB324 (former designation R428) in primary CML (stem cell) samples, cell lines and preclinical models. In a first step we quantified Axl-expressing cells by flow cytometry in chronic phase (CP) CML bone marrow at primary diagnosis and healthy bone marrow donors. Here, we found higher numbers of Axl-positive cells in CML bone marrow compared to controls (11.42±0.42% (n=5) vs. 0.65±0.10% (n=6), respectively; p=0.0015). In addition, we determined Gas6 plasma levels by ELISA in healthy controls and CML patients in CP and blast crisis (BC). These analyses revealed that Gas6 plasma levels were upregulated in a stage specific manner (plasma levels of Gas6: healthy controls 1290±684 pg/ml (n=14), CP 3465±405 pg/ml (n=50), BC 10940±3868 pg/ml (n=7); p=0.0001). Thus, the Gas6-Axl axis represents a potential therapeutic target in CML patients. Based on this finding we analyzed efficacy of BGB324 in Axl-expressing BV173, KCL22, K562, BaF3_BCR-ABL1-wt and BaF3_BCR-ABL1-T315I cell lines in vitro. We found inhibition of proliferation in all analyzed cell lines with IC50 values ranging from 500-3000 nM. Combination experiments with the IC50 dose of BGB324 and IM revealed additive effects of both treatments in all cell lines. Dose finding experiments with BGB324 in sorted CD34+ primary CML cells grown in the presence of physiological growth factors yielded a mean IC50 of 1.1 ± 0.3 mM (n=3), which was similar to nilotinib. BGB324 did not accumulate CD34+CFSEmax (undivided) cells any more than nilotinib but enhanced apoptosis of CD34+ cells in combination with nilotinib. Notably, there was a consistent inhibitory effect of 3 mM BGB324 alone or in combination with nilotinib against colony forming cells (CFC) with the more primitive BFU-E and GEMM being most sensitive to inhibition (n=4). Interestingly, the Ph- lymphocytes (confirmed by FISH) sorted simultaneously with Ph+ CD34+ cells from the same CML patient were unaffected in terms of viability when treated with Axl inhibitor; thus the BGB324’s activity is cell context specific. Encouraged by these data we analyzed efficacy of BGB324 in an aggressive preclinical CML model in which bone marrow cells were retrovirally transduced with constructs containing T315I-mutated BCR-ABL1. Transduced cells were subsequently i.v. transplanted into sublethally irradiated recipient mice, who rapidly developed blast crisis CML. Mice were treated with 25 mg BGB324 or vehicle BID by oral gavage starting from day 3 after transplantation when homing of transduced cells to the bone marrow is completed. In this model we found a significant prolongation of survival upon treatment with BGB324 (Figure 1). Analyses of the leukemia phenotype by differential blood counts and flow cytometry revealed that BGB324 reduced leukemia cell burden (WBC 138±20x103/µl (n=14) vs. 60x103±21 k/µl WBC in the BGB324 treated group (n=13); p=0.0078). Taken together, these data suggest that the Gas6-Axl axis represents a therapeutic target and that BGB324 is a potent molecule effective against T315I-mutated and wt CML alone and in combination with TKI. Furthermore, BGB324 induces apoptosis of quiescent Ph+ CML stem/progenitor cells. Thus BGB324 might open up novel therapeutic avenues in CML patients. Disclosures: Loges: BerGenBio: research support Other.

Abstract Introduction: The mammalian target of rapamycin (mTOR) is a serine/threonine-specific protein kinase, downstream of the phoshatidylinositol 3-kinase (P13-K/AKT) pathway. Constitutive activation of the mTOR related upstream and downstream effectors including P13-K, AKT, P70S6K and 4E-BP1 have been found in numerous malignancies. Previous studies demonstrated that rapamycin has preclinical potential as therapy for multiple myeloma (MM), especially when associated with other drugs. Methods. We performed immunohistochemical analysis with p-AKT (Ser 473), p-mTOR (Ser2448), p-P70S6K (Thr389) and p-4E-BP1 (Thr37,Trh46) on bone marrow sections of 73 symptomatic MM patients. Mielomatous plasmacells were identified and counted by mouse monoclonal CD138 nd all cases were analyzed using a semiquantitative histologic score (HSCORE) method. Specifically, immunostaining intensity of each case was semiquantitatively scored as follow: 0, no staining; 1,weak staining; 2, moderate staining; and 3, strong staining. For each case, a value designed HSCORE was obtained multiplying each intensity with the corresponding percentage of positive cells [HSCORE =∑(1XPC), where 1 and PC represent intensity and percentage of cells, respectively]. Specimen with an HSCORE of ≥30 were classified as p-AKT, p-mTor, p-P706SK and p-4E-BP1 positive. Wilcoxon test was used to compare mTOR expression with clinical data of all patients (including age, presence of bone lesions, isotype, Beta2-microglobulin, haemoglobin, creatinine and albumin serum levels). Common cytogenetic abnormalities (t(11;14), t(4;14), del 13q14 and del p53) were also detected in 61 of 73 (83.5%) patients by FISH analysis on CD138 purified plasma cells. Results. Fouty-four (60.2%) and 46 of 73 (63%) patients stained positive for p –AKT and p-mTOR with a cytoplasmic staining pattern, respectively. P-mTOR immunoreactivity was strongly, moderately and weakly positive in 23.9 %, 34.8 % and 41.3 % of the 46 positive samples, respectively. P-P70S6K and p-4E-BP1 was detected in 53 (72.6%) and 40 (54.8%) patients with a predominantly nuclear staining pattern. The intensity of positivity was distributed as follows: p-P70S6K strongly, moderately and weakly positive, 54.7 (%), 26.4 (%) and 18.9 (%),respectively; p-4E-BP1 strongly, moderately and weakly positive, 62.5 (%), 17.5 (%) and 20 (%), respectively; Of the 46 myelomas stained positive for p-mTOR, 35 expressed p-AKT, 33 expressed p-4E-BP1 and 40 demonstrated p-P70S6K positivity. P-mTOR expression significantly correlated with p-AKT (p=0.003), p-P70S6K (p&lt;0.001) and p-4E-BP1 (p&lt; 0.001) staining consistent with the hypothesis that the AKT/mTOR/P70S6K/4E-BP1 pathway is activated in a subset of MM cases. A significant statistical correlation was found between mTOR expression and serum levels of Beta2-microglobulin ≥3 g/dl (P=0.039). No clearly correlation was found between mTOR pathway activation and the molecular cytogenetic anomalies. Conclusion: We identify a subgroup of MM patients with mTOR pathway activation and poor prognosis. Phosphoprotein staining of the mTOR pathway should be studied further as a means to select patients to receive mTOR inhibitors in combination of chemotherapy. These data also strongly suggest that the AKT/mTOR/P70S6K/4E-BP1 pathway activation may constitute a new biologic parameter regardless of molecular cytogenetic.

Abstract Abstract 2879 Background: Immunomodulatory drugs (IMiDs) are highly active in the treatment of multiple myeloma (MM), but the mechanisms of action are still not completely understood. Recently, cereblon (CRBN) has been identified as the primary target of thalidomide teratogenicity (Ito K et al, 2010) and, moreover as an essential requirement for IMiD therapy (Zhu YX et al, 2011). We wanted to investigate, if expression levels of CRBN could serve as a predictor of response. Patients and Methods: We measured CRBN mRNA expression in bone marrow samples of 44 well characterized MM patients treated with lenalidomide containing regimens, myeloma cell lines, and normal bone marrow (BM), using real time PCR. The median age of patients was 65 years (range: 37–85 years). Nine patients had ISS-stage I, 9 stage II, and 26 had stage III. All patients, except 12, were newly diagnosed. None of the patients had been exposed to lenalidomide before study entry. Full data documentation for response evaluation (> 2 cycles) was available in 37 patients (84%). Of these, lenalidomide was given in combination with dexamethasone in 27 patients with a starting dose of 25 mg per day on days 1–21 in a 28 days cycle, in combination with melphalan and prednisone (MPR, starting dose of lenalidomide 10 mg per day on days 1–21) in 9 patients, and in combination with bendamustine in 1 patient. Results: Normal BM was used as a reference with an expression level of one. All multiple myeloma cell lines tested (U266, KMS-12-BM, OPM-2, NCL-H929, MM.1S, SK-MM-1, and RPMI8226), had a higher CRBN expression than normal BM. CRBN was detected in all 44 MM samples distributed over a range covering 3 orders of magnitude (0.31 to 462.08-fold relative to normal BM; median: 3.61). Lenalidomide-based therapy resulted in CR in 3 (8%), nCR in 2 (5%), VGPR in 4 (11%), PR in 17 (46%), and in MR in 4 patients (11%), respectively. Three patients (8%) had SD, and 4 (11%) had PD. Median CRBN expression was three times higher in responding (≥MR) patients compared to non-responders (3.65 vs. 0.99, p<0.01). In addition, a significant correlation between quality of response and CRBN expression (r=0.34) was observed. This correlation remained statistically significant after exclusion of previously treated patients (r=0.37, p=0.02). Interestingly, among 9 available patients who had been pretreated, the lowest level of CRBN expression (0.90) was noted in the patient who progressed during lenalidomide treatment, indicating a predictive potential of CRBN expression also in pre-treated patients. When the analysis was restricted to the 27 patients who had uniformly been treated with lenalidomide and dexamethasone, an even more pronounced association between myeloma response and CRBN expression was noted (r=0.45; p=0.008). In patients with SD or PD, median CRBN expression was lower than in normal BM, while a higher CRBN expression was found in all patients with CR, nCR, VGPR, PR or MR (Table 1), suggesting that CRBN was required for anti-myeloma activity in these patients. This applied also to patients with marked myeloma response (CR, nCR, VGPR) and patients with PD (r=0.82; p= 0.003) (Figure 1). Univariate analysis between established prognostic factors such as beta-2-microglobulin, albumin, ISS stage, Hb, FISH defined cytogenetic aberrations, and response revealed no significant correlation in this patient cohort. A highly significant correlation between expression of CRBN and IRF4, an important transcription factor required for myeloma survival (p=0.00007), and XBP1 (p=0.00004) was observed. For PAX5 and BLIMP no such association were noted. When primary myeloma cells of 5 patients and cell lines (U 266, KMS-12-M) were exposed to lenalidomide, a significant down regulation of IRF4, but not of CRBN was found. Conclusion: Our studies show a significant association between CRBN expression and myeloma response in patients treated with lenalidomide containing regimens, especially in those receiving lenalidomide and dexamethasone therapy. These findings should be confirmed in larger, prospective clinical trials. Disclosures: Jäger: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; AMGEN: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Research Funding.

Abstract Background: The MYC proto-oncogene encodes a DNA-binding factor that can induce widespread changes in gene expression profiles (GEP). Activation of MYC is a hallmark of aggressive lymphomas and frequently observed in Richter transformation of CLL. In contrast, the role of MYC-related pathogenic networks is less clearly defined in untransformed CLL. Aims: We hypothesized that MYC activation in CLL could lead to specific GEP associated with aggressive disease. We combined the analysis of genomic copy number alterations (CNA) and GEP involved in MYC pathway activation on specimens from patients registered on the CLL8 trial (front line therapy FC vs. FCR). Methods: GEP were derived from CD19-enriched CLL samples (n=337, Human Exon 1.0 ST, Affymetrix) and analysis of CNA was performed based on availability of DNA (n=309, Human SNP Arrays 6.0, Affymetrix). Sample work-up upon trial registration included FISH and TP53 mutation analysis. Results: Genomic gains involving the MYC locus on 8q24.21 were observed in 4.5% of cases. To test the hypothesis of specific GEP associated with MYC activation, we explored the distribution of cases with MYC gain using an unsupervised approach on GEP. After consensus clustering (k=6 clusters) of variably expressed genes (SD>0.5), cases with MYC gain were non-randomly distributed and showed a characteristic pattern. Preferential enrichment was observed in one cluster ("MYC-CNA" group, comprising 40% of all cases) with 64% of MYC gains. Gene set enrichment analysis (GSEA) confirmed overrepresentation of MYC target genes (gene set: HALLMARK_MYC_TARGETS_V1, FDR <0.05) in MYC-CNA and a second cluster, denoted as MYC endogenous activation cluster ("MYC-EA" group, 16% of cases). Conversely, a large cluster, which was most distant to MYC-CNA, did not show significant enrichment in GSEA for MYC target genes or for CNA and was defined as "MYC-silent" reference cluster (comprising 30% of cases). Other potential elements contributing to the regulation of MYC networks, included enrichment of TP53 alterations in both MYC clusters compared to the MYC-silent cluster (17.5% vs. 6%, p=0.015, Fisher`s exact test). We also observed frequent gains of chromosome 2p, involving NMYC on 2p24.3, in both MYC clusters. Losses of the MYC repressors MNT on 17p13.3, MGA on 15q15.1 and PRDM1 on 6q21 also constituted frequent events in the MYC-CNA cluster. Overall, CNA affecting MYC, NMYC and the MYC repressors were more frequent in MYC-CNA (41 in 127 cases) compared to MYC-silent cluster (15 in 93 cases) (p=0.03, Mann Whitney). In addition, expression of the MYC repressor BCL6 was downregulated in MYC-CNA compared to the MYC-silent cluster (fold change 1.5, q<1e-07). MYC protein overexpression was observed by Western blot densitometry in cases without the described CNA, both in MYC-CNA (n=11 cases tested) and MYC-EA (n=7 cases tested), confirming independent activation. Activation of PI3K-AKT and RAS-ERK-signaling was a prominent feature in both MYC clusters. Strong discrepancy between both MYC clusters was observed for cell cycle regulation with changes implicating either increased proliferation in MYC-CNA or cell cycle arrest in MYC-EA. PFS was different when comparing both treatment arms in MYC-CNA (HR 0.55 (95%CI 0.37-0.82), p=0.003) and MYC-EA (HR 0.30 (95%CI 0.15-0.60), p<0.001) with PFS rates at 5 years of 15% (FC) vs. 38.6% (FCR) for MYC-CNA and 17.9% (FC) vs. 57.6% (FCR) for MYC-EA. In contrast, the MYC-silent cluster showed a better outcome compared to the MYC clusters when treated with FC and no benefit from the addition of rituximab with PFS rates at 5 years of 43.1% (FC) vs. 42.9% (FCR) (p=0.56). Median OS was significantly different for treatment arms in MYC-EA and, compared to all other clusters, showed shortest median OS for FC treatment with OS rates at 5 years of 47.9% and strongest benefit for the addition of rituximab with 80.5% (FCR) (HR 0.32 (95%CI 0.13-0.79), p=0.009). Conclusion: MYC pathway alterations were frequently observed in treatment-naive CLL and may involve various mechanism such as CNA affecting MYC and its repressors, TP53 defect or sole transcriptional changes. Cases with MYC activation may be segregated based on cell cycle checkpoint deregulation and consecutive proliferative capacity. Clusters with MYC activation had an inferior clinical course when treated with FC but, when adding rituximab, both MYC-CNA and MYC-EA showed a significant improvement for outcome. Disclosures Bahlo: Roche: Honoraria, Other: Travel Grants. Humphrey:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Wenger:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership, Other: Ownership interests PLC. Tausch:AbbVie: Consultancy, Other: Travel grants; Celgene: Consultancy, Other: Travel grants; Gilead: Consultancy, Other: Travel grants. Bullinger:Bayer Oncology: Research Funding; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Fischer:Roche: Other: Travel support. Hallek:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Boehringer Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Stilgenbauer:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

: Numerous novel medicinal agents isolated from plant sources were used as indigenous remedies for the management and treatment of various types of cancer diseases. Naringenin is a naturally occurring flavanone glycoside and aglycone (genin) moiety of naringin, predominantly found in citrus and grapefruits, has emerged as a potential therapeutic agent for the management of a variety of diseases. A huge number of scientific papers have been published on naringenin describing its detailed studies and its therapeutic application in different diseases. The current study highlights, a comprehensive study on naringenin concerning its biosynthesis, molecular targets/pathways involved in carcinogenesis, mechanism of actions (MOAs), and structure-activity relationships (SARs), and patents granted have been highlighted. Naringenin and its derivatives has remarkable anti-cancer activity due to their inhibitory potential against diverse targets namely ABCG2/P-gp/BCRP, 5a-reductase, 17-bhydroxysteroid dehydrogenase, aromatase, proteasome, HDAC/Situin-1, VEGF, VEGFR-2 kinase, MMP-2/9, JAK/STAT signaling pathways, CDC25B, tubulin, topoisomerase-II, cathepsin-K, Wnt, NF-kB, B-Raf and mTOR, etc. With the huge knowledge of molecular targets, structural intuition, and SARs, the current study may be beneficial to design more potent, safe, effective, and economic anti-cancer naringenin. This is concluded that naringenin is a promising natural product for the management and therapy of cancer. Further evolution for pharmacological importance, clinical research, and trials are required to manifest its therapeutic action on metabolic syndrome in the human community.

Abstract Background The 28-joint disease activity score (DAS28) is a widely used measure to assess disease activity in rheumatoid arthritis (RA). The DAS28-P index, a derived proportion of the patient-reported components (joint tenderness and patient global assessment) within the DAS28, has been utilized as a discriminatory measure of non-inflammatory pain mechanisms in RA. This study aimed to evaluate the use of the DAS28-P index as a predictor of treatment response in early RA. Methods Patients with early RA enrolled in a supplemental fish oil clinical trial received a combination of disease-modifying anti-rheumatic drugs (DMARDs) according to a ‘treat-to-target’ protocol. First, consecutive measures of the DAS28-P index, derived from the DAS28-erythrocyte sedimentation rate (DAS28-ESR), at each visit over a 1-year period were estimated for each patient. Then, distinct subgroups of treatment responders based on the trajectories of the DAS28-P indices were identified using bivariate k-means cluster analysis. Data on baseline predictors as well as longitudinal outcomes of disease impact and DMARD use over a 1-year period and radiographic progression over a 3-year period were collected and analyzed using a random intercept, population-averaged generalized estimating equation model. Results 121 patients were included (74% female; mean age of 57; median of 16 weeks of active disease) and a 3-cluster model was identified—the ‘Responders’ group (n = 58; 48%), the ‘Partial Responders’ group (n = 32; 26%), and the ‘Non-Responders’ group (n = 31; 26%). The ‘Partial Responders’ group had consistently higher proportions of the DAS28-P index throughout the study period and had minimal radiographic progression over time, with the lowest joint erosion score of 0.9 [95% confidence interval (CI) 0.2, 1.6], observed at the 3-year follow-up. At 52 weeks, the methotrexate dose was higher for both ‘Partial Responders’ and ‘Non-Responders’ groups (18.5 mg [95% CI 15.5, 21.5] and 18.6 mg [95% CI 15.3, 21.8] respectively), when compared with the ‘Responders’ group (12.8 mg [95% CI 14.7, 20.9]). Conclusions Persistently high DAS28-P index scores are useful to distinguish poor patient global assessment and excessive treatment escalation in early RA, suggestive of underlying non-inflammatory pain contributing to higher disease activity score. Early identification of patients with discordant subjective and objective components of composite disease activity measures may allow better tailoring of treatment in RA.