Upregulation Of Wnt Pathway Research Articles

Abstract 3338: Development of a CK2α inhibitor for treatment of wnt-driven colorectal cancer

Abstract A novel, potent and highly selective orally bioavailable small molecule inhibitor of Casein Kinase 2α (CK2α) has been developed and validated in pre-clinical models of wnt-driven colorectal carcinoma; and is poised to enter clinical development.CK2α is considered as a positive regulator of wnt signaling and tumorigenesis. The wnt pathway is known to be sensitive to and amplified by CK2α activity at multiple nodes and can be inhibited by loss of CK2α function. The human genetics of colorectal cancer (CRC) have been well characterized and approximately 80% tumors are identified as being wnt pathway mutation driven. CRC tumors can be further stratified into consensus molecular subtypes (CMS), with iCMS2 representing 37% of CRC patients, being characterized by ongoing wnt pathway upregulation. Development of CK2α inhibitors as a targeted therapy for CRC is therefore mechanistically well aligned to the wnt signaling drive and may serve as a promising therapeutic strategy for this cancer. A novel small molecule binding site on CK2α has been exploited to design and develop an inhibitor with Ki of 95 pM and exquisite selectivity over the kinome. This inhibitor has been comprehensively characterized using in vitro, ex vivo and in vivo pharmacology studies. Cellular mechanism of action studies in CRC cell lines have quantified the potency of target engagement, inhibition of known CK2α phospho-sites, inhibition of tumorigenic wnt pathway activity, and induction of apoptosis. This CK2α inhibitor was also profiled in a high content screen of CRC patient-derived organoids established from various driver mutation backgrounds. Use of these ex vivo models has validated the potent cytotoxic activity of CK2α inhibition in tumor samples with wnt-driven characteristics and highlights the potential therapeutic efficacy of CK2α inhibition in this setting. In vivo CRC xenograft studies have confirmed anti-tumor efficacy and developed the understanding of pharmacokinetic pharmacodynamic relationship between free plasma concentration of drug and inhibition of CK2α-specific biomarker in the tumor. The preclinical data presented establishes the strong rationale for clinical development of a novel CK2α inhibitor for the treatment of locally advanced or metastatic solid tumors, with an emphasis on wnt-driven CRC. Citation Format: Darren Cawkill. Development of a CK2α inhibitor for treatment of wnt-driven colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3338.

The Functional and Molecular Alterations in Human Bone Marrow Mesenchymal Stem Cells in Chronic Myelomonocytic Leukemia

Background: Crosstalk between the bone marrow microenvironment and leukemia cells plays a pivotal role in pathogenesis. Mesenchymal stem cells (MSCs) acquire defects during natural ageing, with reduced regenerative potential and functional impairment. Since chronic myelomonocytic leukemia (CMML) mainly affects older adults, defective MSCs could play a critical and targetable role in disease initiation and progression. Identifying molecular abnormalities associated with CMML MSCs would reveal novel therapeutic targets for patient benefit. Methods: Phenotypes of age-matched healthy and CMML MSCs were compared by a range of functional approaches, including senescence, multilineage differentiation and hematopoietic stem cell maintenance assays. To identify altered epigenetic and transcriptional networks we performed RNA-Seq (n=18 CMML/10 healthy) and ATAC-Seq (n=12 CMML/5 healthy) on cultured MSCs (CD44+73+90+105+146+), integrating datasets to explore gene regulatory networks operational within CMML MSCs. To investigate direct crosstalk between MSCs and malignant cells we performed RNA-Seq on healthy MSCs following 7 days co-culture with CMML blood mononuclear cells (MNCs). Functional validation was performed through inhibitor studies using apoptosis, senescence, osteogenesis, and stem cell maintenance as readouts. Results: CMML MSCs displayed consistent and profound intrinsic growth defects, with higher senescence and apoptosis, and lower proliferation and clonogenic capacity. CMML MSCs also displayed a marked differentiation bias, with increased osteogenic lineage potential but reduced adipogenicity and chondrogenicity. Importantly, CMML MSCs demonstrated reduced stem cell maintenance capability in co-culture experiments, confirming a functional consequence on hematopoiesis. Transcriptomic analysis showed activation of novel pathways including mTOR, PI3K-Akt signaling, and oxidative phosphorylation in CMML MSCs, in addition to Wnt pathway upregulation as shown previously (Figure 1A). Consistent with functional readouts, CMML MSCs were transcriptionally primed towards the osteo-lineage, with high expression of osteogenic promoting factors (CORIN, LTF, TREM2, SMAD5, ALX5) and downregulation of osteogenic inhibitors (RBPJ, CLU). In line with transcriptomic analysis, ATAC-Seq of CMML MSCs highlighted aberrantly accessible chromatin regions linked to genes involved in mTOR, PI3K-Akt and Wnt signaling as well as promotion of osteogenesis (Figure 1A). Integration of datasets identified downregulated expression and activity of the transcription factor EBF1, where reduced expression is associated with self-renewal defects, decreased adipogenesis, cellular senescence and defective stem cell maintenance ability, compatible with our observations in CMML MSCs. Similarly, we identified downregulated activity of the transcription factor RBPJ, potentially mediating the preferential enhancement of osteogenesis observed in CMML MSCs. Following co-culture with CMML MNCs, healthy MSCs showed activation of mTOR, PI3K-Akt and osteogenesis, verifying a direct influence of leukemic cells on MSCs. Remarkably, integration of all datasets identified consistently upregulated genes (RGS4, TPI1) which positively regulate mTOR and PI3K-Akt signaling. Inhibiting these aberrantly active pathways in CMML MSCs using small molecule modulators significantly reversed the phenotypic abnormalities with a reduction in apoptosis, senescence and osteogenesis and restoration of their ability to support and maintain stem cells (Figure 1B). Discussion: Our integrated analysis identified altered gene regulatory networks that are linked to abnormal pathways active in CMML MSCs, which can be targeted to restore their normal function. These results highlight novel pathogenic roles for non-clonal MSCs in the CMML microenvironment, and potential adjunctive strategies for targeting microenvironmental alongside cell intrinsic vulnerabilities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 1097: Mechanisms of primary and acquired resistance to venetoclax in chronic lymphocytic leukemia (CLL)

Abstract Mechanisms of resistance to venetoclax, a highly selective oral BCL2 inhibitor approved for therapy of CLL, remain poorly understood and we sought to characterize the clonal evolution of resistance in patients developing progressive disease on venetoclax. We performed whole-exome sequencing (WES) on 7 relapsed/refractory CLL patients with disease progressing on venetoclax. We observed del17p in 43% (3/7), TP53 mutation in 86% (6/7) and unmutated IGHV in 71% (5/7) of patients. A median of four longitudinal tumor samples were sequenced per patient (total Nsamples= 25). We performed digital droplet PCR (ddPCR) analysis to look at the emergence of BCL2 G101V mutations that have been previously linked with venetoclax resistance after long times on therapy. G101V mutation was detected by ddPCR in two patients at low variant allelic fractions (VAF) of 0.03% and 4.68% respectively, and not detected by WES. We further detected one droplet positive for G101V mutation in two other patients in venetoclax progression bone marrow samples. Due to the very low VAF of the BCL2 G101V mutations, we suspected that other mechanisms of acquired resistance were more significant in this cohort of relatively early relapses. Analysis of WES data showed no somatic single nucleotide variants (sSNVs) selected in more than one patient with resistance. However, copy number analysis revealed acquired del8p in 3 patients, resulting in large subclonal expansions. Furthermore, del 8p co-occurred with amp 1q21.2-21.3 affecting the MCL1 gene in 2 patients, and with amp 8q, del 17p, del 18q22.1-23 and del 9p23-21.2 (containing the MYC, TP53, BCL2 and CDKN2A/B genes respectively) in a third. Two other patients showed expansion of clones harboring del 10q23.31-24.1, that includes PTEN, while a third contains an expanding clone with two IRF8 mutations (S283C and SFF416fs). Resistance in the final patient is likely associated with marked expansion of clones with TP53 and SF3B1 mutations. RNA-seq analysis comparing pre-venetoclax to post-venetoclax resistant samples from 8 CLL patients showed downregulation of the BCR, FCGR and MAPK signaling pathways, with upregulation of mitochondrial translation, oxidative phosphorylation and the TCA/citric acid cycle at the time of progression. RNA-seq analysis focusing on the patients with del 8p shows significant downregulation of the TNFRSF10A/10B genes (TRAIL-Rs), with gene set enrichment analysis (GSEA) showing positive enrichment for WNT5A/FZD4 signaling and CREB signaling via the PKC and MAPK pathways, concomitant with downregulation of the BCR and FCGR pathways at progression. Our data suggest several mechanisms of venetoclax resistance in CLL, including loss of TNFRSF10A/B, sometimes with MCL1 upregulation, as well as WNT pathway upregulation and BCR pathway downregulation, which we are now validating in primary patient PBMCs and relevant cell line models. Citation Format: Ishwarya Murali, Justin Cha, Ignaty Leshchiner, Yanan Kuang, Kevin Vasquez, Jasneet Khalsa, Stacey M. Fernandes, Filippo Utro, Kahn Rhrissorrakrai, Chaya Levovitz, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Cloud P. Paweletz, Laxmi Parida, Gad Getz, Jennifer R. Brown. Mechanisms of primary and acquired resistance to venetoclax in chronic lymphocytic leukemia (CLL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1097.

Differential expression of BMP inhibitors gremlin and noggin in Hydra suggests distinct roles during budding and patterning of tentacles.

Mechanisms regulating BMP and Wnt pathways and their interactions are not well studied in Hydra. We report identification of BMP inhibitor gremlin, comparison of its expression with that of noggin and possible antagonism between Wnt and BMP signaling in Hydra. Gremlin is expressed in body column with high levels in budding region and in early buds. Noggin, on the other hand, is expressed in the hypostome, base of tentacles, lower body column, and basal disc. During budding, noggin is expressed at the sites of tentacle emergence. This was confirmed in ectopic tentacles in polyps treated with alsterpaullone (ALP), a GSK-3β inhibitor that leads to upregulation of Wnt pathway. RT-PCR data show that upregulation of Wnt is accompanied by downregulation of bmp 5-8b though noggin and gremlin remain unaltered till 24 hours. Different expression patterns of gremlin and noggin suggest their roles in budding and patterning of tentacles, respectively. Further, bmp 5-8b inhibition by activated Wnt signaling does not directly involve noggin and gremlin in Hydra. Our data suggest that Wnt/BMP antagonism may have evolved early for defining the oral-aboral axis, while the involvement of BMP antagonists during axial patterning is a recent evolutionary acquisition within the Bilateria lineage.

AB0070 LORECIVIVINT (SM04690), A POTENTIAL DISEASE-MODIFYING TREATMENT FOR KNEE OSTEOARTHRITIS, DEMONSTRATED CARTILAGE-PROTECTIVE EFFECTS ON HUMAN OSTEOARTHRITIC EXPLANTS

Background:Wnt pathway upregulation contributes to knee osteoarthritis (OA) through osteocyte differentiation, cartilage thinning, and inflammation. Lorecivivint (LOR; SM04690), a novel, small-molecule CLK/DYRK1A inhibitor that modulates the Wnt pathway, demonstrated disease-modifying potential for knee OA in preclinical studies.1However, the specific mechanisms by which LOR protects cartilage in knee OA are unclear.Objectives:To evaluate the cartilage-protective effects of LOR on human OA explants from total knee replacement (TKR) donors.Methods:Knee joint tissue from 22 TKR donors was obtained. IRB approval was obtained from Scripps Health. Cartilage was scored using the Outerbridge classification system based on gross appearance (grade 1=least-damaged tissue, grade 4=most-damaged tissue). Cartilage explants (4 mm in diameter) with Outerbridge grades 2–3 were harvested and cultured for 48 hours to reach metabolic stability. They were then treated with LOR (10 nM, 30 nM) or DMSO and stimulated with either IL-1β (10 ng/ml) or TNF-α (20 ng/ml)+oncostatin M (OM) (10 ng/ml) or left unstimulated. After 72 hours, supernatants and explants were collected. Gene expression of matrix metalloproteinases (MMPs) 1, 3, and 13 was measured by qPCR and protein levels of MMP-1, MMP-3, MMP-13, and thrombospondin-motif-containing disintegrins/metalloproteinases ADAMTS-4 and ADAMTS-5 were measured in supernatants by ELISA. Glycosaminoglycan (GAG) and nitric oxide (NO) levels were measured in supernatants using the dimethylmethylene blue assay and Griess assay, respectively. One-way ANOVA was used for multiple group comparisons.Results:Treatment with IL-1β or TNF-α+OM led to statistically significant increases in gene expression ofMMP1,MMP3, andMMP13and increased secretion of GAG, MMP-1, MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, and NO in supernatants compared to unstimulated control. Treatment with LOR decreased both IL-1β-stimulated and TNF-α+OM-stimulated gene expression ofMMP1,MMP3, andMMP13and secretion of GAG, MMP-1, MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, and NO in supernatants compared to treatment with DMSO.Conclusion:LOR demonstrated potent inhibition of cartilage catabolism enzyme production in human OA explants compared to controls. These cartilage-protective effects support the development of LOR as a potential disease-modifying treatment for knee OA. Human trials are ongoing.

Lorecivivint (SM04690), a potential disease-modifying treatment for knee osteoarthritis, demonstrated cartilage-protective effects on human osteoarthritic explants

Purpose: Wnt pathway upregulation contributes to knee osteoarthritis (OA) through osteocyte differentiation, cartilage thinning, and inflammation. Lorecivivint (LOR; SM04690), a novel, small-molecule CLK/DRYK1A inhibitor, demonstrated disease-modifying potential for knee OA via Wnt pathway modulation in preclinical studies. Further studies were performed to evaluate cartilage-protective effects of LOR on human OA explants from total knee replacement (TKR) donors.

Pathogenic WDFY3 variants cause neurodevelopmental disorders and opposing effects on brain size.

The underpinnings of mild to moderate neurodevelopmental delay remain elusive, often leading to late diagnosis and interventions. Here, we present data on exome and genome sequencing as well as array analysis of 13 individuals that point to pathogenic, heterozygous, mostly de novo variants in WDFY3 (significant de novo enrichment P = 0.003) as a monogenic cause of mild and non-specific neurodevelopmental delay. Nine variants were protein-truncating and four missense. Overlapping symptoms included neurodevelopmental delay, intellectual disability, macrocephaly, and psychiatric disorders (autism spectrum disorders/attention deficit hyperactivity disorder). One proband presented with an opposing phenotype of microcephaly and the only missense-variant located in the PH-domain of WDFY3. Findings of this case are supported by previously published data, demonstrating that pathogenic PH-domain variants can lead to microcephaly via canonical Wnt-pathway upregulation. In a separate study, we reported that the autophagy scaffolding protein WDFY3 is required for cerebral cortical size regulation in mice, by controlling proper division of neural progenitors. Here, we show that proliferating cortical neural progenitors of human embryonic brains highly express WDFY3, further supporting a role for this molecule in the regulation of prenatal neurogenesis. We present data on Wnt-pathway dysregulation in Wdfy3-haploinsufficient mice, which display macrocephaly and deficits in motor coordination and associative learning, recapitulating the human phenotype. Consequently, we propose that in humans WDFY3 loss-of-function variants lead to macrocephaly via downregulation of the Wnt pathway. In summary, we present WDFY3 as a novel gene linked to mild to moderate neurodevelopmental delay and intellectual disability and conclude that variants putatively causing haploinsufficiency lead to macrocephaly, while an opposing pathomechanism due to variants in the PH-domain of WDFY3 leads to microcephaly.

Modulation of the Wnt pathway through inhibition of CLK2 and DYRK1A by lorecivivint as a novel, potentially disease-modifying approach for knee osteoarthritis treatment

Wnt pathway upregulation contributes to knee osteoarthritis (OA) through osteoblast differentiation, increased catabolic enzymes, and inflammation. The small-molecule Wnt pathway inhibitor, lorecivivint (SM04690), which previously demonstrated chondrogenesis and cartilage protection in an animal OA model, was evaluated to elucidate its mechanism of action. Biochemical assays measured kinase activity. Western blots measured protein phosphorylation in human mesenchymal stem cells (hMSCs), chondrocytes, and synovial fibroblasts. siRNA knockdown effects in hMSCs and BEAS-2B cells on Wnt pathway, chondrogenic genes, and LPS-induced inflammatory cytokines was measured by qPCR. Invivo anti-inflammation, pain, and function were evaluated following single intra-articular (IA) lorecivivint or vehicle injection in the monosodium iodoacetate (MIA)-induced rat OA model. Lorecivivint inhibited intranuclear kinases CDC-like kinase 2 (CLK2) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Lorecivivint inhibited CLK2-mediated phosphorylation of serine/arginine-rich (SR) splicing factors and DYRK1A-mediated phosphorylation of SIRT1 and FOXO1. siRNA knockdowns identified a role for CLK2 and DYRK1A in Wnt pathway modulation without affecting β-catenin with CLK2 inhibition inducing early chondrogenesis and DYRK1A inhibition enhancing mature chondrocyte function. NF-κB and STAT3 inhibition by lorecivivint reduced inflammation. DYRK1A knockdown was sufficient for anti-inflammatory effects, while combined DYRK1A/CLK2 knockdown enhanced this effect. In the MIA model, lorecivivint inhibited production of inflammatory cytokines and cartilage degradative enzymes, resulting in increased joint cartilage, decreased pain, and improved weight-bearing function. Lorecivivint inhibition of CLK2 and DYRK1A suggested a novel mechanism for Wnt pathway inhibition, enhancing chondrogenesis, chondrocyte function, and anti-inflammation. Lorecivivint shows potential to modify structure and improve symptoms of knee OA.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Salvianolic Acid B Enhances Hepatic Differentiation of Human Embryonic Stem Cells Through Upregulation of WNT Pathway and Inhibition of Notch Pathway.

Hepatocytes differentiated from human embryonic stem cells (ESCs) could provide a powerful tool for enabling cell-based therapies, studying the mechanisms underlying human liver development and disease, and testing the efficacy and safety of pharmaceuticals. However, currently most in vitro protocols yield hepatocytes with low levels of liver function. In this study, we investigated the potential of Salvianolic acid B (Sal B), an active pharmaceutical compound present in Salvia miltiorrhiza, which has been shown to have an antifibrotic effect in previous studies, to enhance hepatocyte differentiation from human ESCs. After treatment with Sal B, albumin expression and secretion were consistently increased, indicating that Sal B could promote hepatocyte differentiation process. Expression of a large number of important phase 1 and 2 metabolizing enzymes and phase 3 transporters was also increased in treated cells, indicating an enhanced biotransformation function. Our investigations further revealed the activation of Wnt pathway in treated cells, as determined by upregulation of Wnts, which increased amounts of nuclear β-catenin. This increased nuclear β-catenin led in turn to the enhanced expression of T cell factor (TCF) 3 and lymphoid enhancer-binding factor (LEF) 1 which upregulated their downstream targets, cyclin D1 and c-Myc. Notch receptors (Notch1, Notch3), Notch ligand (Jagged2), and Notch receptor targets [hairy and enhancer of split (Hes) 1, 5] were downregulated in treated cells, suggesting that Notch pathway was inhibited. Consistent with the inhibition of Notch pathway, expression of cholangiocyte marker, CK7, was significantly reduced by treatment with Sal B. Numb, a direct transcriptional target of Wnt pathway and a negative regulator of Notch pathway, was upregulated, consistent with activation of Wnt signaling and suppression of Notch signaling. In conclusion, our study demonstrated that Sal B enhanced hepatocyte differentiation from human ESCs through activation of Wnt pathway and inhibition of Notch pathway. Therefore, this study suggests that Sal B can be used as a potential agent to generate more mature hepatocytes for cell-based therapeutics and pharmaceutical studies.

Abstract 1359: How does RAC1 GTP-ase signal Wnt-beta-catenin pathway-mediated integrin-directed metastasis-associated tumor cell phenotypes in TNBC

Abstract Introduction: We reported that Wnt Pathway (WP) upregulation, one of the salient genetic features of metastatic TNBC, controls the acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes of TNBC cells (Dey et al., 2013). RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. Aim: Here we tested the role of the mechanism of involvement of RAC1 in the regulation of WP-mediated ID-MA phenotypes in TNBC cells and examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Method: Oncoprints showed that the major alterations of RAC1 gene in TNBC were constituted by the amplification/gain of the gene. The collective percentage of alteration(s) in RAC1 in ER+ve BC was 35% as compared to 57% in ER-ve BC (brca/tcga/pub2015). Using pharmacological agents (sulindac sulfide), genetic tools (siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed migration and various motility parameters (actin dynamics, filopodia and lamellipodia by confocal microscopy) in the context of fibronectin-directed RAC1 and Cdc42 activation. Result: Breaking down by molecular subtypes of BC, RAC1 gene alterations are observed in 20% in PAM50 Luminal A, 35% in PAM50 Luminal B, 37% in PAM50 HER2 enriched and 40% in PAM50 Basal-like. For the samples set of TCGA, Cell 2015, RAC1 gene was altered in 42% total while it was altered 37% in PAM50 Luminal A, 57% in PAM50 Luminal B, 55% in PAM50 HER2 enriched and 60% in PAM50 basal-like. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) altered actin dynamics and (d) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report (De et al., 2016) that WP upregulation stimulates ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in an RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2. Citation Format: Nandini Dey, Jennifer H. Carlson, Casey Williams, Tyler Jepperson, Pradip De, Brian Leyland-Jones. How does RAC1 GTP-ase signal Wnt-beta-catenin pathway-mediated integrin-directed metastasis-associated tumor cell phenotypes in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1359. doi:10.1158/1538-7445.AM2017-1359

LDB1 overexpression is a negative prognostic factor in colorectal cancer.

BackgroundColorectal cancer (CRC) is the third most common cancer in western countries and is driven by the Wnt signaling pathway. LIM-domain-binding protein 1 (LDB1) interacts with the Wnt signaling pathway and has been connected to malignant diseases. We therefore aimed to evaluate the role of LDB1 in CRC.ResultsOverexpression of LDB1 in CRC is associated with strikingly reduced overall and metastasis free survival in all three independent patient cohorts. The expression of LDB1 positively correlates with genes involved in the Wnt signaling pathway (CTNNB1, AXIN2, MYC and CCND1). Overexpression of LDB1 in CRC cell lines induced Wnt pathway upregulation as well as increased invasivity and proliferation. Upon separate analysis, the role of LDB1 proved to be more prominent in proximal CRC, whereas distal CRC seems to be less influenced by LDB1.Materials and MethodsThe expression of LDB1 was measured via RT-qPCR in 59 clinical tumor and normal mucosa samples and correlated to clinical end-points. The role of LDB1 was examined in two additional large patient cohorts from publicly available microarray and RNAseq datasets. Functional characterization was done by lentiviral overexpression of LDB1 in CRC cell lines and TOP/FOP, proliferation and scratch assays.ConclusionsLDB1 has a strong role in CRC progression, confirmed in three large, independent patient cohorts. The in vitro data confirm an influence of LDB1 on the Wnt signaling pathway and tumor cell proliferation. LDB1 seems to have a more prominent role in proximal CRC, which confirms the different biology of proximal and distal CRC.

Targeting Wnt pathway in mantle cell lymphoma-initiating cells.

BackgroundMantle cell lymphoma (MCL) is an aggressive and incurable form of non-Hodgkin’s lymphoma. Despite initial intense chemotherapy, up to 50 % of cases of MCL relapse often in a chemoresistant form. We hypothesized that the recently identified MCL-initiating cells (MCL-ICs) are the main reason for relapse and chemoresistance of MCL. Cancer stem cell-related pathways such as Wnt could be responsible for their maintenance and survival.MethodsWe isolated MCL-ICs from primary MCL cells on the basis of a defined marker expression pattern (CD34-CD3-CD45+CD19-) and investigated Wnt pathway expression. We also tested the potential of Wnt pathway inhibitors in elimination of MCL-ICs.ResultsWe showed that MCL-ICs are resistant to genotoxic agents vincristine, doxorubicin, and the newly approved Burton tyrosine kinase (BTK) inhibitor ibrutinib. We confirmed the differential up-regulation of Wnt pathway in MCL-ICs. Indeed, MCL-ICs were particularly sensitive to Wnt pathway inhibitors. Targeting β-catenin-TCF4 interaction with CCT036477, iCRT14, or PKF118-310 preferentially eliminated the MCL-ICs.ConclusionsOur results suggest that Wnt signaling is critical for the maintenance and survival of MCL-ICs, and effective MCL therapy should aim to eliminate MCL-ICs through Wnt signaling inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0161-1) contains supplementary material, which is available to authorized users.

Non-Darwinian dynamics in therapy-induced cancer drug resistance.

Development of drug resistance, the prime cause of failure in cancer therapy, is commonly explained by the selection of resistant mutant cancer cells. However, dynamic non-genetic heterogeneity of clonal cell populations continuously produces meta-stable phenotypic variants (persisters), some of which represent stem-like states that confer resistance. Even without genetic mutations, Darwinian selection can expand these resistant variants, which would explain the invariably rapid emergence of stem-like resistant cells. Here, using quantitative measurements and modeling we show that appearance of multi-drug resistance in HL60 leukemic cells following treatment with vincristine is not explained by Darwinian selection but by Lamarckian induction. Single-cell longitudinal monitoring confirms the induction of multi-drug resistance in individual cells. Associated transcriptome changes indicate a lasting stress-response consistent with a drug-induced switch between high-dimensional cancer attractors. Resistance-induction correlates with Wnt-pathway up-regulation and is suppressed by β-catenin knock-down, revealing a new opportunity for early therapeutic intervention against resistance development.

Down-Regulation of Canonical and Up-Regulation of Non-Canonical Wnt Signalling in the Carcinogenic Process of Squamous Cell Lung Carcinoma

The majority of lung cancers (LC) belong to the non-small cell lung carcinoma (NSCLC) type. The two main NSCLC sub-types, namely adenocarcinoma (AC) and squamous cell carcinoma (SCC), respond differently to therapy. Whereas the link between cigarette smoke and lung cancer risk is well established, the relevance of non-canonical Wnt pathway up-regulation detected in SCC remains poorly understood. The present study was undertaken to investigate further the molecular events in canonical and non-canonical Wnt signalling during SCC development. A total of 20 SCC and AC samples with matched non-cancerous controls were obtained after surgery. TaqMan array analysis confirmed up-regulation of non-canonical Wnt5a and Wnt11 and identified down-regulation of canonical Wnt signalling in SCC samples. The molecular changes were tested in primary small airway epithelial cells (SAEC) and various lung cancer cell lines (e.g. A549, H157, etc). Our studies identified Wnt11 and Wnt5a as regulators of cadherin expression and potentiated relocation of β-catenin to the nucleus as an important step in decreased cellular adhesion. The presented data identifies additional details in the regulation of SCC that can aid identification of therapeutic drug targets in the future.

Epigenetic Downregulation of Secreted Frizzled-Related Proteins In Philadelphia Positive and Philadelphia Negative Myeloproliferative Neoplasms

AbstractAbstract 4647 Introduction:Hypermethylation of CpG islands in promoter regions is associated with transcriptional inactivation and represents an important mechanism of tumor suppressor gene silencing and plays a role in the pathogenesis of hematopoietic malignancies. Myeloproliferative neoplasms (MPN) constitute a heterogenous group of hematopoietic malignancies and comprise chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). BCR/ABL-negative MPN (Ph-MPN) are often characterized by an autoactivating mutation in the JAK2 tyrosine kinase (JAK2V617F) as well as additional mutations in the JAK-STAT pathway. Secreted Frizzled-related proteins (SFRPs) are antagonists of the Wnt signaling pathway. Consistent with the central role of the Wnt pathway for stem cell maintenance and differentiation of hematopoietic progenitors. Upregulation of Wnt-pathway has been described in haematopoietic malignancies and epigenetic downregulation of SFRPs has been shown in acute leukemia. Methods:We determined the promoter methylation status of SFRP1, -2, -4 and -5 in human cell lines and in 60 samples from MPN patients by methylation-specific PCR (MSP). The JAK2V617F mutation was assessed by allele-specific PCR. Results:Aberrant methylation among primary MPN samples was 5 % (3/60) for SFRP-1, 24 % (14/60) for SFRP-2, 2% (1/60) for SFRP-4 and 0% (0/60) for SFRP-5. The SFRP-2 promoter was methylated in 0% of CML (0/12), 27% of PV (3/11), 30% of ET (3/10) and 26% of PMF patient samples (7/27). There was no correlation between SFRP-2 hypermethylation and MPN subtype. We detected a trend for a correlation between methylation of the SFRP-2 promoter and presence of JAK2V617F mutation (p = 0,1). Discussion:Our data indicate that epigenetic dysregulation of the Wnt signaling pathway is a common event in Ph-MPN with aberrant methylation of at least one SFRP being detected in 30% of the primary patient samples with the most predominant member of the family, SFRP2 being methylated in about one third of the cases. Aberrant methylation of SFRPs may contribute to the pathogenesis of MPN by influencing stem cell maintenance and proliferation of hematopoietic progenitors. Disclosures:No relevant conflicts of interest to declare.