Β-catenin Mutations Research Articles

Craniopharyngiomas

Craniopharyngiomas are rare epithelial tumours situated primarily in the sellar/parasellar region, occurring along the path of the craniopharyngeal duct. Whilst classed as histologically benign tumours, their unpredictable growth pattern and proximity to vital structures including the optic chiasm, hypothalamus, and pituitary gland renders them a considerable threat, with significant associated morbidity and increase in mortality. Occurring both in child and adulthood, their clinical manifestations are broad, commonly with symptoms/signs secondary to hypothalamic-pituitary dysfunction, raised intracranial pressure and visual compromise. They have two distinct histological subtypes (adamantinomatous and papillary), with unique patterns of age distribution, and genetic and molecular make-up. With increasing understanding of their genetic pathogenesis including BRAF V600E mutations in the papillary subtype, and β-catenin mutations in the adamantinomatous, further research provides hope for the discovery of targeted medical therapy that can exploit molecular changes occurring as a result of such alterations. Until then, primary treatment consists of surgery with or without radiotherapy, with intracystic aspiration, chemotherapy or irradiation being alternative options in selected patients. Long term management by an experienced multidisciplinary team is essential, given the breadth of complications, including hypothalamic morbidity, visual compromise, cognitive and neuropsychological sequelae and impairment to quality of life.

Inhibition of Isorhamnetin on β-Catenin/Tcf Signaling and β-Catenin-Activated Melanogenesis

Activated β-catenin/T-cell factor (Tcf) signaling plays various roles in human cell mechanisms. We identified the inhibitory effect of isorhamnetin against β-catenin/Tcf signaling in β-catenin activated cells. Isorhamnetin inhibited the transcriptional activity of β-catenin/Tcf in HEK293 cells transiently transfected with a constitutively active mutant β-catenin gene. Also, isorhamnetin inhibited theβ-catenin-activated melanogenesis in B16F1 cells. Regarding the inhibitory mechanism, the shift assay showed that the binding of Tcf complexes with its specific DNA-binding sites was suppressed byisorhamnetin. Western blot analysis showed a decreased level of β-catenin in nucleus caused by isorhamnetin. These results suggest that the isorhamnetin acts as a negative regulator of β-catenin/Tcf signaling and its inhibitory mechanism is related to the decreased binding of β-catenin/Tcf complexes to consensus DNA.

Pan-Cancer Analysis of Glycolytic and Ketone Bodies Metabolic Genes: Implications for Response to Ketogenic Dietary Therapy.

BackgroundThe Warburg effect, also termed “aerobic glycolysis”, is one of the most remarkable and ubiquitous metabolic characteristics exhibited by cancer cells, representing a potential vulnerability that might be targeted for tumor therapy. Ketogenic diets (KDs), composed of high-fat, moderate-protein and low carbohydrates, are aimed at targeting the Warburg effect for cancer treatment, which have recently gained considerable attention. However, the efficiency of KDs was inconsistent, and the genotypic contribution is still largely unknown.MethodsThe bulk RNA-seq data from The Cancer Genome Atlas (TCGA), single cell RNA sequencing (scRNA-seq), and microarray data from Gene Expression Omnibus (GEO) and Cancer Cell Line Encyclopedia (CCLE) were collected. A joint analysis of glycolysis and ketone bodies metabolism (KBM) pathway was performed across over 10,000 tumor samples and nearly 1,000 cancer cell lines. A series of bioinformatic approaches were combined to identify a metabolic subtype that may predict the response to ketogenic dietary therapy (KDT). Mouse xenografts were established to validate the predictive utility of our subtypes in response to KDT.ResultsWe first provided a system-level view of the expression pattern and prognosis of the signature genes from glycolysis and KBM pathway across 33 cancer types. Analysis by joint stratification of glycolysis and KBM revealed four metabolic subtypes, which correlated extensively but diversely with clinical outcomes across cancers. The glycolytic subtypes may be driven by TP53 mutations, whereas the KB-metabolic subtypes may be mediated by CTNNB1 (β-catenin) mutations. The glycolytic subtypes may have a better response to KDs compared to the other three subtypes. We preliminarily confirmed the idea by literature review and further performed a proof-of-concept experiment to validate the predictive value of the metabolic subtype in liver cancer xenografts.ConclusionsOur findings identified a metabolic subtype based on glycolysis and KBM that may serve as a promising biomarker to predict the clinical outcomes and therapeutic responses to KDT.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

Nrf2 Mutation/Activation Is Dispensable for the Development of Chemically Induced Mouse HCC.

Background & AimsActivation of the kelch-like ECH-associated protein 1 (Keap1)–nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway has been associated with metabolic reprogramming in many tumors, including hepatocellular carcinoma (HCC). However, the contribution of Nrf2 mutations in this process remains elusive. Here, we investigated the occurrence of Nrf2 mutations in distinct models of mouse hepatocarcinogenesis.MethodsHCCs were generated by experimental protocols consisting of the following: (1) a single dose of diethylnitrosamine (DEN), followed by repeated treatments with the nuclear-receptor agonist 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene; (2) repeated treatments with 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene alone; (3) a single dose of DEN followed by exposure to a choline-deficient L-amino acid–defined diet; and (4) a single dose of DEN with no further treatment. All of these protocols led to HCC development within 28–42 weeks. Activation of the Keap1-Nrf2 pathway was investigated by analyzing the presence of Nrf2 gene mutations, and the expression of Nrf2 target genes. Metabolic reprogramming was assessed by evaluating the expression of genes involved in glycolysis, the pentose phosphate pathway, and glutaminolysis.ResultsNo Nrf2 mutations were found in any of the models of hepatocarcinogenesis analyzed. Intriguingly, despite the described cooperation between β-catenin and the Nrf2 pathway, we found no evidence of Nrf2 activation in both early dysplastic nodules and HCCs, characterized by the presence of up to 80%–90% β-catenin mutations. No HCC metabolic reprogramming was observed either.ConclusionsThese results show that, unlike rat hepatocarcinogenesis, Nrf2 mutations do not occur in 4 distinct models of chemically induced mouse HCC. Interestingly, in the same models, metabolic reprogramming also was minimal or absent, supporting the concept that Nrf2 activation is critical for the switch from oxidative to glycolytic metabolism.

1537P Dissecting inflammation in patients with desmoid fibromatosis to identify prognostic biomarkers

Desmoid fibromatosis (DF) is a locally aggressive rare tumor with high recurrence rate after surgery and unpredictable clinical course. Standard of care for DF patients is active surveillance; 30% of cases undergo disease progression and shift to treatment. Biomarkers discriminating aggressive DF and predicting progressive disease are needed. DF harbors mutations in β-catenin and a transcriptional ‘inflammatory phenotype’ (PMID: 28627792). Cancer-associated inflammation is fostered by systemic factors and detectable in circulating immune cells.

Abstract P169: β-catenin C-terminal Domain/Sphingosine-1-Phosphate Receptor 1 Axis Drives Neointima Formation After Carotid Injury

Introduction: The β-catenin C-terminal domain (CTD) is required for artery formation during embryogenesis, but its role in vascular remodeling in adulthood is unknown. Inhibitors targeting specifically the CTD of β-catenin have been developed, but their use in obstructive vascular disease is not yet justified because of this gap of knowledge. Loss of β-catenin in mouse aortic smooth muscle cells (MASMCs) decreases the transcript levels of sphingosine-1-phosphate receptor 1 (S1PR1). However, no studies have evaluated the crosstalk between these proteins in vascular remodeling. Hypothesis: We hypothesized that the CTD of β-catenin promotes neointima formation after vascular injury, and that it acts by inducing S1PR1 expression. Methods: We studied mice bearing a knockin mutant β-catenin allele (ΔC) that lacks β-catenin CTD transcriptional activity. Mice expressing the SMC-specific SMA-CreERT2 and bearing one floxed and one mutant β-catenin allele were treated with tamoxifen to inactivate the floxed allele, leaving the mutant allele as the only source of β-catenin in SMC (SMβCΔC mice). Carotid artery ligation was performed to induce vascular injury. MASMCs were isolated from SMβCΔC mice (βCΔC cells). Results: We found reduced neointima formation after injury in SMβCΔC male (SMβCWT/-: 0.52±0.09 vs SMβCΔC: 0.16±0.05; n=10; P<0.05) and female (SMβCWT/-: 0.56±0.11 vs SMβCΔC: 0.17±0.04; n=10; P<0.05) mice. These findings were associated with reduced SMC proliferation, increased SMC apoptosis, and prevention of loss of SMC markers. In vitro, βCΔC MASMCs showed reduced growth compared to control cells (n=6; P<0.05). βCΔC MASMCs also showed reduced S1PR1 expression and decreased S1pr1 promoter activity, which were both rescued by the transfection of a constitutively active form of β-catenin. Interestingly, overexpression of S1PR1 restored βCΔC MASMC growth towards control levels (n=6; P<0.05). In vivo, we found reduced S1PR1 expression in SMCs of injured arteries from SMβCΔC mice. Conclusion: Our results define an essential SMC β-catenin CTD-S1PR1 axis that drives neointima formation after arterial injury, and point to multiple points for potential therapeutic intervention to control vascular remodeling in disease.

SMAD4 is critical in suppression of BRAF-V600E serrated tumorigenesis.

BRAF-driven colorectal cancer is among the poorest prognosis subtypes of colon cancer. Previous studies suggest that BRAF-mutant serrated cancers frequently exhibit Microsatellite Instability (MSI) and elevated levels of WNT signaling. Loss of tumor-suppressor Smad4 in oncogenic BRAF-V600E mouse models promotes rapid serrated tumor development and progression, and SMAD4 mutations co-occur in human patient tumors with BRAF-V600E mutations. This study assesses the role of SMAD4 in early-stage serrated tumorigenesis. SMAD4 loss promotes Microsatellite Stable (MSS) serrated tumors in an oncogenic BRAF-V600E context, providing a model for MSS serrated cancers. Inactivation of Msh2 in these mice accelerated tumor formation, and whole exome sequencing of both MSS and MSI serrated tumors derived from these mouse models revealed that all serrated tumors developed oncogenic WNT mutations, predominantly in the WNT-effector gene Ctnnb1 (β-catenin). Mouse models mimicking the oncogenic β-catenin mutation show that the combination three oncogenic mutations (Ctnnb1, Braf, and Smad4) are critical to drive rapid serrated dysplasia formation. Re-analysis of human tumor data reveals BRAF-V600E mutations co-occur with oncogenic mutations in both WNT and SMAD4/TGFβ pathways. These findings identify SMAD4 as a critical factor in early-stage serrated cancers and helps broaden the knowledge of this rare but aggressive subset of colorectal cancer.

Sustained expression of unc-4 homeobox gene and unc-37/Groucho in postmitotic neurons specifies the spatial organization of the cholinergic synapses in C. elegans.

Neuronal cell fate determinants establish the identities of neurons by controlling gene expression to regulate neuronal morphology and synaptic connectivity. However, it is not understood if neuronal cell fate determinants have postmitotic functions in synapse pattern formation. Here we identify a novel role for UNC-4 homeobox protein and its corepressor UNC-37/Groucho, in tiled synaptic patterning of the cholinergic motor neurons in Caenorhabditis elegans. We show that unc-4 is not required during neurogenesis but is required in the postmitotic neurons for proper synapse patterning. In contrast, unc-37 is required in both developing and postmitotic neurons. The synaptic tiling defects of unc-4 mutants are suppressed by bar-1/β-catenin mutation, which positively regulates the expression of ceh-12/HB9. Ectopic ceh-12 expression partly underlies the synaptic tiling defects of unc-4 and unc-37 mutants. Our results reveal a novel postmitotic role of neuronal cell fate determinants in synapse pattern formation through inhibiting the canonical Wnt signaling pathway.

Babao Dan is a robust anti-tumor agent via inhibiting wnt/β-catenin activation and cancer cell stemness

Ethnopharmacological relevanceTraditional Chinese Medicine (TCM) is being increasingly used worldwide due to its diverse efficacy and relatively low side effects. Babao Dan (BBD) is a well-known TCM formula that is currently used for the effective treatment of various cancers, however its underlying molecular mechanism remains unknown. Aim of the studyTumor growth and tumor recurrence are characterized by two distinct populations of cells, namely the well-differentiated cancer cells composing the majority of tumor bulk, and cancer stem cells (CSCs) involved in tumor relapse, which are both strongly associated with excessive activation of Wnt/β-catenin signaling. Our study aims to elucidate the underlying molecular mechanisms associated with the anti-tumor proliferative effects of Babao Dan (BBD). Materials and methodsWe used a hepatoblastoma cell line HepG2 with stem cell-like traits that harbors a constitutively active mutant of β-catenin in order to study the anti-tumor ability of BBD via targeting Wnt/β-catenin signaling. ResultsBBD robustly attenuated both the intrinsic and extrinsic activation of Wnt/β-catenin pathway in HepG2 hepatoblastoma cells, as well as Wnt target genes. Moreover, BBD significantly inhibited both the proliferation of well-differentiated cancer cells, as well as the stem-like property of CSCs as evidenced by EpCAM, a Wnt target gene and a novel marker of cancer cell stemness. In addition, mice administered with BBD using HepG2 cell line derived xenograft model had marked reductions in tumor size and weight, as well as significantly decreased expressions of Wnt target genes and cancer cell stemness. ConclusionOur findings elucidated the underlying molecular mechanisms associated with the robust anti-tumor effects of BBD via potent inhibition of Wnt/β-catenin signaling, and implicate its use in the clinical treatment of cancers.

An Unusual Cause of Recurrent Subepithelial Mass in the Stomach

Question: A 58-year-old woman presented with a subepithelial tumor, approximately 2 cm in size, incidentally detected in the posterior wall of upper body of the stomach during a screening endoscopy (Figure A). The sample from an endoscopic ultrasound-guided fine-needle biopsy revealed spindle-shaped cells arranged in a fascicular pattern. The spindle cells were immunopositive for c-Kit, cluster of differentiation 34, DOG-1, and smooth muscle actin. The patient was diagnosed with a gastrointestinal stromal tumor (GIST) and underwent laparoscopic wedge resection. The tumor was completely resected with clear margins. The maximal tumor size was 2.3 cm, and the mitotic count was 2 per 50 high-power fields; therefore, it was diagnosed as a GIST with a low risk of malignant potential. Follow-up abdominal computed tomography after 2 years showed a new low-density mass in the previous resection site (Figure B, arrow). Endoscopy showed no definite lesion at the resection site (Figure C), and endoscopic ultrasound revealed a 1.5-cm heterogeneous hypoechoic lesion originating from the muscularis propria (Figure D). Laparoscopic wedge resection was performed again for a definitive diagnosis and treatment. What is your diagnosis? See the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI. Histopathologic examination of the resected specimen revealed sweeping fascicles of bland and uniform spindle cells with collagenous stroma and compressed, thin-walled vessels (Figure E). The spindle cells were immunonegative for c-Kit (Figure F), cluster of differentiation 34 (Figure G), DOG-1 (Figure H), and S-100 protein, and were immunopositive for smooth muscle actin (Figure I), which suggested the exclusion of GIST. Therefore, additional immunohistochemistry was performed for the differential diagnosis. The tumor cells were negative for anaplastic lymphoma kinase but positive for nuclear β-catenin (Figure J). Based on morphologic and immunohistochemical findings, a diagnosis of a desmoid tumor occurring at the previous resection site of the gastric GIST was made. No further treatment was provided because the tumor was completely resected. No signs of recurrence or metastasis were found during 12 months of follow-up. Desmoid tumors are rare mesenchymal tumors occurring in patients with risk factors, such as a previous history of abdominal surgery, familial adenomatous polyposis, or pregnancy, and they can be easily mistaken for GIST because both tumors show similar clinical presentation and morphology.1Murshid A.A. Al-Maghraby H.Q. Subsequent development of desmoid tumor after a resected gastrointestinal stromal tumor.Case Rep Pathol. 2018; 2018: 1082956Google Scholar The desmoid tumor and GIST are both microscopically characterized by spindle-cell proliferation, and the differential diagnosis of these two tumors is performed mainly by immunohistochemistry. Because desmoid tumors usually have activating mutations in β-catenin, abnormal nuclear positivity of β-catenin is seen in most desmoid tumors.2Lu Q. Wang K. Liu D. et al.Stomach desmoid tumor: a case report and review of the literature.Int J Clin Exp Pathol. 2017; 10: 10531-10538Google Scholar Surgical resection with a negative margin is currently the treatment of choice. When local recurrence occurs or complete surgical resection is not feasible, radiotherapy, chemotherapy, and other comprehensive therapies, such as estrogen receptor antagonists and nonsteroidal anti-inflammatory drugs, should be provided.2Lu Q. Wang K. Liu D. et al.Stomach desmoid tumor: a case report and review of the literature.Int J Clin Exp Pathol. 2017; 10: 10531-10538Google Scholar Owing to the rarity of gastric desmoid tumors, information about their prognosis is very limited. According to a review of the literature on gastric desmoid tumors, no recurrence was reported during follow-up assessments,2Lu Q. Wang K. Liu D. et al.Stomach desmoid tumor: a case report and review of the literature.Int J Clin Exp Pathol. 2017; 10: 10531-10538Google Scholar indicating a good prognosis. However, considering the local recurrence rate of 13% for intra-abdominal sporadic desmoid tumors,3Wilkinson M.J. Fitzgerald J.E. Thomas J.M. et al.Surgical resection for nonfamilial adenomatous polyposis-related intraabdominal fibromatosis.Br J Surg. 2012; 99: 706-713Google Scholar regular follow-up is mandatory.

β-Catenin is required for optimal exercise- and contraction-stimulated skeletal muscle glucose uptake.

Loss of β-catenin impairs in vivo and isolated muscle exercise/contraction-stimulated glucose uptake. β-Catenin is required for exercise-induced skeletal muscle actin cytoskeleton remodelling. β-Catenin675 phosphorylation during exercise may be intensity dependent. The conserved structural protein β-catenin is an emerging regulator of vesicle trafficking in multiple tissues and supports insulin-stimulated glucose transporter 4 (GLUT4) translocation in skeletal muscle by facilitating cortical actin remodelling. Actin remodelling may be a convergence point between insulin and exercise/contraction-stimulated glucose uptake. Here we investigated whether β-catenin is involved in regulating exercise/contraction-stimulated glucose uptake. We report that the muscle-specific deletion of β-catenin induced in adult mice (BCAT-mKO) impairs both exercise- and contraction (isolated muscle)-induced glucose uptake without affecting running performance or canonical exercise signalling pathways. Furthermore, high intensity exercise in mice and contraction of myotubes and isolated muscles led to the phosphorylation of β-cateninS675 , and this was impaired by Rac1 inhibition. Moderate intensity exercise in control and Rac1muscle-specific knockout mice did not induce muscle β-cateninS675 phosphorylation, suggesting exercise intensity-dependent regulation of β-cateninS675 . Introduction of a non-phosphorylatable S675A mutant of β-catenin into myoblasts impaired GLUT4 translocation and actin remodelling stimulated by carbachol, a Rac1 and RhoA activator. Exercise-induced increases in cross-sectional phalloidin staining (F-actin marker) of gastrocnemius muscle was impaired in muscle from BCAT-mKO mice. Collectively our findings suggest that β-catenin is required for optimal glucose transport in muscle during exercise/contraction, potentially via facilitating actin cytoskeleton remodelling.

Abstract ND10: Discovery of RMC-5552: A selective bi-steric inhibitor of mTORC1 that suppresses 4EBP1 phosphorylation, for the treatment of mTORC1-activated tumors including RAS pathway escape

Abstract The PI3K/mTOR and RAS signaling pathways are hyperactivated in a wide range of human cancers. Several points of cross-talk and intersecting feedback mechanisms have been described and activation of mTOR signaling frequently mediates escape from RAS pathway inhibition. Revolution Medicines has developed a novel class of selective mTORC1 inhibitors, termed ‘bi-steric inhibitors', that interact with both the ATP- and FKBP12/FRB-binding sites of mTORC1. These compounds potently and selectively inhibit mTORC1 over mTORC2 and other lipid kinases. Unlike rapalogs, such as everolimus, these bi-steric inhibitors suppress 4EBP1 phosphorylation in vivo and induce growth suppression and apoptosis in various genetically-defined preclinical models of human cancer. We will describe our SAR investigations and findings for our preclinical tool compound, RMC-6272 (also known as RM-006) and for RMC-5552, our clinical candidate. We will demonstrate that mTORC1-selective bi-steric inhibitors show combinatorial activity with both KRASG12C(OFF) and KRASG12C(ON) inhibitors, in KRASG12C STK11deficient NSCLC models and can forestall resistance to KRASG12C inhibitor monotherapy. In addition, we will show single agent activity of bi-steric mTORC1 inhibitors in preclinical models of tumors bearing PI3K/mTOR pathway alterations as primary drivers, including PTEN-deficient glioblastoma multiforme, TSC1/2-deficient renal cystadenoma cancer, and hepatocellular cancers harboring mTOR-activating β-catenin mutations. RMC-5552, through reactivation of 4EBP1 and inhibition of cap-dependent translation, has the potential to benefit cancer patients with genetically-defined tumors with activating mutations in the RAS and PI3K/mTOR pathways both as a single agent or in combination with RAS inhibitors. Citation Format: G. Leslie Burnett. Discovery of RMC-5552: A selective bi-steric inhibitor of mTORC1 that suppresses 4EBP1 phosphorylation, for the treatment of mTORC1-activated tumors including RAS pathway escape [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 ND10.

Long-term outcomes following resection of hepatocellular adenomas with small foci of malignant transformation or malignant adenomas

Background & AimsMalignant transformation of hepatocellular adenoma (MT-HCA) may occur in up to 5% of tumours. However, the prognostic value of this event remains poorly described. In this study, we aimed to analyse the long-term outcomes of patients undergoing liver resection (LR) for MT-HCA compared to those of patients resected for hepatocellular carcinoma (HCC) occurring on normal liver parenchyma (NP-HCC).MethodsThis single-centre retrospective study included all patients who underwent LR for MT-HCA at Beaujon Hospital between 2001 and 2019. MT-HCAs were classified as small foci of malignant transformation HCA (SF-HCA) and as malignant HCA (M-HCA) in cases of predominant HCC foci. Recurrence-free survival (RFS) of MT-HCA was compared with that of NP-HCC after propensity score matching.ResultsForty patients (24 men, 16 women) underwent LR for MT-HCA, including 23 with SF-HCA and 17 with M-HCA. Of these cases, 16/40 (40%) had β-catenin mutations, 19/40 (47.5%) were inflammatory, 1 was HNF1α-mutated HCA and 4 (10%) were unclassified HCA. Microvascular invasion (12% vs. 0%, p = 0.091) and satellite nodules (25% vs. 4%, p = 0.028) were more frequently observed in M-HCA than in SF-HCA. After a median follow-up of 67 months, 10 (25%) patients with MT-HCA had tumour recurrence, including 9 with M-HCA and 1 with SF-HCA (p = 0.007). M-HCA was linked to significantly poorer 1-, 3-, 5- and 10-year RFS rates than SF-HCA (76%, 63%, 39%, 37% vs. 100%, 100%, 100%, 91%, p = 0.003). Multivariate analysis showed that SF-HCA was independently associated with improved RFS (hazard ratio 0.064; 95% CI 0.008–0.519; p = 0.01). After propensity score matching, NP-HCC was associated with significantly poorer 1-, 3-, 5- and 10-year RFS rates than MT-HCA (p = 0.01).ConclusionsHCA with malignant transformation yields a better long-term prognosis than NP-HCC. Among MT-HCA, SF-HCA is associated with a better prognosis than M-HCA.Lay summaryThe prognostic relevance of malignant transformation of hepatocellular adenoma (HCA) remains unknown. Thus, the aim of our study was to compare the outcomes of patients undergoing liver resection for malignant transformation to those of patients undergoing liver resection for hepatocellular carcinoma (HCC). The main long-term risk after resection for carcinoma is recurrence. In this study, 10/40 patients with malignant transformation of HCA relapsed after resection and we identified age >55 years, presence of satellite nodes, and microvascular invasion as risk factors for long-term recurrence. Compared to patients with HCC, patients who underwent liver resection for HCA with malignant transformation had better long-term survival.

A CK1δ/CK1ε-to-β-Catenin Circuit Is a Therapeutic Vulnerability for Primary and Drug Resistant Multiple Myeloma

Multiple Myeloma (MM) remains an incurable malignancy, despite the advent of several new therapeutic agents, including immunomodulatory drugs (IMiDs, e.g., Lenalidomide (Len)) and proteasome inhibitors (PIs, e.g ., Bortezomib (Btz)). Accordingly, there is an urgent need to identify new targetable vulnerabilities for MM patients. We developed an ex vivo 384-well assay that allows one to define drug sensitivities of primary patient CD138+ MM cells in the context of a reconstructed tumor microenvironment (TME), including allogeneic BMSC, extracellular matrix and MM patient serum. Using this platform and activity-based proteomic profiling (ABPP), we identified shared signaling pathways induced by the interactions of MM with the TME and integrated these data with screens performed using a bank of protein kinase inhibitors (PKI) and current anti-MM therapeutics. These analyses revealed that the serine/threonine kinases casein kinase-1δ (CK1δ) and CK1ε are high priority targets for MM. In support of this data, a highly selective and potent dual inhibitor of CK1δ/CK1ε coined SR-3029 is the most potent PKI versus MM. Further, our studies revealed SR-3029 has potent activity in 75/86 primary patient MM specimens tested thus far, including quad and penta-refractory MM.Previous studies have shown that SR-3029 targets CK1δ in triple negative breast cancer (TNBC) and that the therapeutic response relied on disabling the Wnt/β-catenin signaling. Aberrant activation of the Wnt/β-catenin pathway has well established roles in the development and progression of cancer. Indeed, gain-of-function somatic mutations in β-catenin and upstream receptors such as LRP5, and loss-of-function mutations in components of the β-catenin destruction complex, including the scaffold proteins APC and Axin, are hallmarks of several tumor types.Our analyses of whole exome sequencing (WES) of MM patients revealed that ~40% of tumors have functional protein-altering mutations in the Wnt/β-catenin pathway, and that this is manifest in both treatment-naïve and relapsed MM. Additionally, RNA-seq and gene expression profiling analysis of over 1,300 MCC MM patients specimens have show that components of the Wnt/β-catenin signaling pathway are elevated in some subsets of MM. Further, we have shown that SR-3029 treatment or inducible knockdown of CK1δ or CK1ε rapidly compromises the growth and survival of MM cell lines. Importantly, the anti-MM activity of SR-3029 is augmented in MM cell lines with selected resistance to Btz and Len relative to parental myeloma cells. Further, treatment of MM cells with this kinase inhibitor leads to marked reductions in total β-catenin protein, decreases in β-catenin target genes (e.g., MYC, CCND1 and WNT3), and to marked changes in the expression of components of the Wnt/β-catenin signaling pathway. Finally, using the established 5TGM1/Kal-Ridge (C57B6/KaLwRijHsd) syngeneic mouse model of multiple myeloma, we show that tumors derived from 5TGM1 MM cells, which rapidly die following exposure to SR-3029 ex vivo, are also sensitive to CK1δ/CK1ε inhibition in vivo, where the SR-3029 treated cohort of animals demonstrated decreased tumor burden as assessed by IgG2b levels and imaging, and by significantly improved survival relative tovehicle treated recipients. Collectively, these findings demonstrate that SR-3029 has potent activity in both naïve and therapy resistant multiple myeloma and establish CK1δ and/or CK1ε as attractive targets for anti-myeloma therapy. DisclosuresSiqueira Silva:AbbVie: Research Funding. Teer:Interpares Biomedicine: Consultancy. Shain:Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Consultancy; Takeda Pharmaceuticals: Consultancy.

NGS-Based Analysis of Atypical Deep Penetrating Nevi.

Simple SummaryThe recent WHO classification of melanocytic tumors requires the implementation of combined phenotypic–genotypic diagnostics. For rare tumors, such as atypical deep penetrating nevi, there is insufficient information regarding genetic status, and it is not yet clear whether the observed unusual morphological cyto-architectures reflect a distinct genomic profile or are associated with an increased metastatic potential and aggressive clinical behavior. We report herein a comprehensive next-generation sequencing (NGS) analysis of a series of atypical DPNs, showing their mutational profile with some specific signatures for these rare and diagnostically challenging tumors.Deep penetrating nevi (DPNs) are rare melanocytic neoplasms consisting of pigmented spindled or epithelioid melanocytes with a distinctive wedge-shaped configuration showing activation of the WNT pathway, with unusual cyto-architectural features. It is unclear whether they show a distinct genomic profile associated with a diverse metastatic potential. We describe herein a cohort of 21 atypical DPNs analyzed by next-generation sequencing using the Ion AmpliSeq™ Comprehensive Cancer Panel. We found that β-catenin exon 3 was mutated in 95% and MAP kinase pathway genes in 71% of the cases. Less frequent mutations were observed in HRAS (19%) and MAP2K1 (24%). Isocitrate dehydrogenases 1 (IDH1) mutations, including R132C, V178I, and S278L, were identified in 38% of cases and co-existed with BRAF/HRAS mutations. The only case with progressive nodal disease carried alterations in the β-catenin pathway and mutations in IDH1 and NRAS (codon 61). By a comprehensive mutation analysis, we found low genetic heterogeneity and a lack of significant associations between specific gene mutations and histopathological features, despite atypical features. Whether the acquisition of an NRAS or IDH1 mutation in an atypical DPN may represent a molecular evolution implying a pathway to melanoma progression should be confirmed in a larger series.

Targeting Wnt Signaling in Endometrial Cancer

Simple SummaryWnt has diverse regulatory roles at multiple cellular levels and numerous targeting points, and aberrant Wnt signaling has crucial roles in carcinogenesis, metastasis, cancer recurrence, and chemotherapy resistance; based on these facts, Wnt represents an appealing therapeutic target for cancer treatment. Although preclinical data supports a role for the Wnt signaling pathway in uterine carcinogenesis, this area remains understudied. In this review, we identify the functions of several oncogenes of the Wnt/β-catenin signaling pathway in tumorigenesis and address the translation approach with potent Wnt inhibitors that have already been established or are being investigated to target key components of the pathway. Further research is likely to expand the potential for both biomarker and cancer drug development. There is a scarcity of treatment choices for advanced and recurrent endometrial cancer; investigating the sophisticated connections of Wnt signaling networks in endometrial cancer could address the unmet need for new therapeutic targets.This review presents new findings on Wnt signaling in endometrial carcinoma and implications for possible future treatments. The Wnt proteins are essential mediators in cell signaling during vertebrate embryo development. Recent biochemical and genetic studies have provided significant insight into Wnt signaling, in particular in cell cycle regulation, inflammation, and cancer. The role of Wnt signaling is well established in gastrointestinal and breast cancers, but its function in gynecologic cancers, especially in endometrial cancers, has not been well elucidated. Development of a subset of endometrial carcinomas has been attributed to activation of the APC/β-catenin signaling pathway (due to β-catenin mutations) and downregulation of Wnt antagonists by epigenetic silencing. The Wnt pathway also appears to be linked to estrogen and progesterone, and new findings implicate it in mTOR and Hedgehog signaling. Therapeutic interference of Wnt signaling remains a significant challenge. Herein, we discuss the Wnt-activating mechanisms in endometrial cancer and review the current advances and challenges in drug discovery.

Development of a novel biomarker for the early detection of colorectal cancer

Colorectal cancer (CRC) is one of the most common cancer and a leading cause of cancer related deaths both in men and in women worldwide including US. A positive family history is strongly correlated with an increased risk of CRC diagnosis during lifetime. However, CRC is an indolent disease in its early stages and usually becomes symptomatic when it progresses to more advanced stages. Conventional screening methods have several limitations and currently, there is no screening procedure available to detect CRC at a very early stage. Under normal physiological condition, the epithelial lining of the gut gets breached by various factors including food, that creates local wound. The surviving epithelial cells around the wound lose their differentiated structure and undergoes epithelial mesenchymal transition (EMT) and migrate and proliferate to cover the injured area. Once the wound is covered, the cells start differentiating back into their normal cell type and become a part of the epithelium. If the cells fail to stop proliferating following wound closure by overriding the switch to differentiating that can lead to colonic polyp, or tumor, and if not detected in a timely manner than, it leads ultimately to CRC development. Previously, we reported that β-catenin; an adherence junction protein gets phosphorylated by Janus kinase 3 (Jak3), a non-receptor tyrosine kinase, where Jak3 directly phosphorylates three tyrosine residues, viz. Tyr30, Tyr64, and Tyr86 in the N-terminal domain (NTD) of β-catenin. Now, our data show that human biopsies from colonic polyps not only associate with EMT, but also with only those mutation in β-catenin where Jak3, fail to phosphorylate β-catenin thereby making the cells override the switch and undergo uncontrolled proliferation and polyp formation. Based on this mechanistic understanding, the present study demonstrates the development of probe that detects the transition of colonic cells from proliferative stage to a differentiated (non-proliferative) stage that can diagnose CRC at the pre-polyp stage. The probe can not only play an important role in the early detection and/or treatment of patients with colorectal cancer but also increase life expectancy post detection. For this study, we collectedde-identified and discarded specimen from normal and/or polyps (n=40 cases; 20 normal and 20 polyps) through our collaboration with clinicians from local hospitals. Briefly, snap-frozen tissues were used to isolate DNA with the QIAamp DNA mini kit (Qiagen) as reported before by our group. All DNA samples were quantified using UV spectrophotometer and analyzed through agarose gel electrophoresis. The DNA sample was used as template in PCR reactions using the β-catenin-probes to amplify the regions encompassing Y30, Y64, and Y86 followed by sequencing of PCR samples. This was followed by histological analysis of colonic polyp sections from each patient stained with H&E and images were taken with phase contrast microscope. Our results show strongly correlation between mutations and histological analysis indicating the aforementioned detection of intactness of EMT switch as potential novel biomarker for the early detection of colorectal cancer.

Podocytes Specific Deletion of PKM2 Ameliorates LPS‐induced Podocyte Injury through Regulation of Beta‐Catenin Activity

Background Acute kidney injury (AKI) is associated with a severe decline in kidney function caused by abnormalities within the podocytes' glomerular matrix. Recently, AKI has been linked to alterations in glycolysis and the activity of glycolytic enzymes, including pyruvate kinase M2 (PKM2). However, the contribution of this enzyme to AKI remains largely unexplored. Methods Cre-loxP technology was used to examine the effects of PKM2 specific deletion in podocytes on the activation status of key signaling pathways involved in the pathophysiology of AKI by lipopolysaccharides (LPS). In addition, we used lentiviral shRNA to generate murine podocytes deficient in PKM2 and investigated the molecular mechanisms mediating PKM2 actions in vitro. Results Specific PKM2 deletion in podocytes ameliorated LPS-induced protein excretion and alleviated LPS-induced alterations in blood urea nitrogen and serum albumin levels. In addition, PKM2 deletion in podocytes alleviated LPS-induced renal injury and protected mice from LPS-induced pro-inflammatory cytokine expression. At the molecular level, PKM2 deficiency in podocytes alleviated LPS-induced inflammation, endoplasmic reticulum stress, and apoptosis, but exacerbated LPS-induced autophagy. In vitro, PKM2 knockdown in murine podocytes suppressed LPS-induced apoptosis. These effects were concomitant with a reduction in LPS-induced activation of β-catenin and the loss of Wilms’ Tumor 1 (WT1) and nephrin. Notably, the overexpression of a constitutively active mutant of β-catenin abolished the protective effect of PKM2 knockdown. Conversely, Knockdown cells reconstituted with the phosphotyrosine binding–deficient PKM2 mutant (K433E) recapitulated the effect of PKM2 depletion on LPS-induced apoptosis, β-catenin activation, and reduction in WT1 expression. Conclusion Our data demonstrate that PKM2 plays a key role in podocytes injury and suggest that targetting PKM2 in podocytes could serve as a promising therapeutic strategy for AKI.

Reconciling the Biological and Transcriptional Variability of Hepatoblastoma with Its Mutational Uniformity.

Simple SummaryHepatoblastoma (HB), the most common form of childhood liver cancer, is associated with dual mutation and/or dysregulation of the Wnt/β-catenin and Hippo pathways in ~50% of cases. However, this mutational simplicity cannot explain HB’s biological and histologic diversity. This discussion focuses upon recent work showing that specific β-catenin mutants are key determinants of this HB variability as well as their metabolic and transcriptional signatures. Dysregulation of the anti-oxidant NFE2L2 pathway also contributes to tumorigenesis by being directly transforming in association with either of the other two factors. The transcriptional overlap of tumors generated by pairs of factors identifies crucial targets that likely mediate HB tumorigenesis, behavior and appearance.Hepatoblastoma (HB), the most common childhood liver cancer, is associated with seven distinct histologic subtypes and variable degrees of clinical aggressiveness and presentation. Yet it is among the least genomically altered tumors known, with about half of HBs showing mutation and/or dysregulation of the Wnt/β-catenin and Hippo pathways. This raises the question of how this mutational simplicity can generate such biological and histologic complexity. Recent work shows that the identity of the underlying β-catenin mutation is a major contributor. Mutation or over-expression of the NFE2L2/NRF2 transcription factor, previously thought only to promote anti-oxidant responses, has also recently been shown to accelerate the growth of HBs generated by mutations in the Wnt/β-catenin and Hippo pathways while imparting novel features such as the tumor-associated cysts and necrosis. Moreover, patient-associated NFE2L2 mutations are overtly transforming when co-expressed with either mutant β-catenin or a Hippo pathway effector. The finding that tumorigenesis can be driven by any two arms of the β-catenin/Hippo/NFE2L2 axis has permitted the identification of a small subset of coordinately regulated tumor-specific transcripts, some of whose levels correlate with inferior long-term outcomes in HB and other cancers. Collectively, these findings begin to provide for more refined and molecularly based classification, survival algorithms and design of chemotherapeutic regimens.