Oncogene Family Member Research Articles

Baohuoside I chemosensitises breast cancer to paclitaxel by suppressing extracellular vesicle/CXCL1 signal released from apoptotic cells.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and chemotherapy is the cornerstone treatment for TNBC. Regrettably, emerging findings suggest that chemotherapy facilitates pro-metastatic changes in the tumour microenvironment. Extracellular vesicles (EVs) have been highly implicated in cancer drug resistance and metastasis. However, the effects of the EVs released from dying cancer cells on TNBC prognosis and corresponding therapeutic strategies have been poorly investigated. This study demonstrated that paclitaxel chemotherapy elicited CXCL1-enriched EVs from apoptotic TNBC cells (EV-Apo). EV-Apo promoted the chemoresistance and invasion of co-cultured TNBC cells by polarizing M2 macrophages through activating PD-L1 signalling. However, baohuoside I (BHS) remarkably sensitized the co-cultured TNBC cells to paclitaxel chemotherapy via modulating EV-Apo signalling. Mechanistically, BHS remarkably decreased C-X-C motif chemokine ligand 1 (CXCL1) cargo within EV-Apo and therefore attenuated macrophage M2 polarization by suppressing PD-L1 activation. Additionally, BHS decreased EV-Apo release by diminishing the biogenesis of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs) of TNBC cells. Furthermore, BHS bound to the LEU104 residue of flotillin 2 (FLOT2) and interrupted its interaction with RAS oncogene family member 31 (RAB31), leading to the blockage of RAB31-FLOT2 complex-driven ILV biogenesis. Importantly, BHS remarkably chemosensitised paclitaxel to inhibit TNBC metastasis in vivo by suppressing EV-ApoCXCL1-induced PD-L1 activation and M2 polarization of tumour-associated macrophages (TAMs). This pioneering study sheds light on EV-ApoCXCL1 as a novel therapeutic target to chemosensitise TNBC, and presents BHS as a promising chemotherapy adjuvant to improve TNBC chemosensitivity and prognosis by disturbing EV-ApoCXCL1 biogenesis.

MODL-14. TARGETING GL1/GL2-DNMT1-UHRF1 EPIGENETIC COMPLEX DISRUPTION VIA BET INHIBITION IMPAIRS SHH MB DEVELOPMENT

Abstract BACKGROUND Sonic Hedgehog Medulloblastoma (SHH MB) shares 30% among all molecular subtypes of medulloblastomas (MBs). Constitutive activation of SHH signaling in the cerebellum’s granular cell precursors (GCPs) is critical for this MB development. When SHH receptor PATCHED-1 (PTCH1) is mutated, stimulation of this receptor by SHH ligand binding leads to derepression of heterotrimeric GTP-binding protein–coupled receptor Smoothened (SMO) eventually altering the activity, location, and stability of the three downstream glioma associated oncogene (GLI) family members: GLI1, GLI2, and GLI3. GLI1 and GLI2 mostly function as transcriptional activators, while GLI3 mainly plays a repressor role. The bromodomain and extra-terminal domain (BET) family of proteins comprises four conserved members (Brd2, Brd3, Brd4, and Brdt), which are critical regulators of GLI1 and GLI2-mediated transcription. METHODS In the present study, we pharmacologically inhibited BETs with a previously described BET inhibitor, BMS986158, and a novel BET inhibitor, UM002, discovered by our lab. We treated human SHH MB cells (ONS76 cells) in vitro and ex vivo in mouse SHH MB cells (from Ptch1 conditional knockout mice) with minimum effective concentrations of these inhibitors. We screened for GLI1/GLI2-DNMT1-UHRF1 epigenetic complex components expressions by immunoblotting and assessed the effectiveness of inhibiting their interactions by performing PLA assays. RESULTS In this study, we show that pharmacological inhibition of BETs leads to attenuated GLI1 and GLI2 levels. This GLI1 and GLI2 inhibition eventually disrupts the previously reported GL1/GL2, DNMT1-UHRF1 epigenetic complex, which is crucial for SHH MB development. A previously described BET inhibitor, BMS986158, and a novel BET inhibitor, UM002, synthesized and characterized by our lab can inhibit BETs. This could be a potential therapeutic approach targeting GLI-associated epigenetic complexes to treat SHH medulloblastoma.

New lead compounds identification against KRas mediated cancers through pharmacophore-based virtual screening and in vitro assays

Cancer remains the leading cause of mortality and morbidity in the world, with 19.3 million new diagnoses and 10.1 million deaths in 2020. Cancer is caused due to mutations in proto-oncogenes and tumor-suppressor genes. Genetic analyses found that Ras (Rat sarcoma) is one of the most deregulated oncogenes in human cancers. The Ras oncogene family members including NRas (Neuroblastoma ras viral oncogene homolog), HRas (Harvey rat sarcoma) and KRas are involved in different types of human cancers. The mutant KRas is considered as the most frequent oncogene implicated in the development of lung, pancreatic and colon cancers. However, there is no efficient clinical drug even though it has been identified as an oncogene for 30 years. Therefore there is an emerging need to develop potent, new anticancer drugs. In this study, computer-aided drug designing approaches as well as experimental methods were employed to find new and potential anti-cancer drugs. The pharmacophore model was developed from an already known FDA approved anti-cancer drug Bortezomib using the software MOE. The validated pharmacophore model was then used to screen the in-house and commercially available databases. The pharmacophore-based virtual screening resulted in 26 and 86 hits from in-house and commercial databases respectively. Finally, 6/13 (in-house database) and 24/64 hits (commercial databases) were selected with different scaffolds having good interactions with the significant active residues of KRasG12D protein that were predicted as potent lead compounds. Finally, the results of pharmacophore-based virtual screening were further validated by molecular dynamics simulation analysis. The 6 hits of the in-house database were further evaluated experimentally. The experimental results showed that these compounds have good anti-cancer activity which validate the protocol of our in silico studies. KRasG12D protein is a very important anti-cancer target and potent inhibitors for this target are still not available, so small lead compound inhibitors were identified to inhibit the activity of this protein by blocking the GTP-binding pocket. Communicated by Ramaswamy H. Sarma

Circ_KCNQ5 participates in the progression of childhood acute myeloid leukemia by enhancing the expression of RAB10 via binding to miR-622

ABSTRACT Background : Acute myeloid leukemia (AML) is regarded as a haematological malignancy and seriously threatens the public’s health. Circular RNA (circRNA) is gradually confirmed to be involved in the development of AML. The purpose of this study was to disclose the role of circRNA Potassium Voltage-Gated Channel Subfamily Q Member 5 (circ_KCNQ5) in AML. Methods : Quantitative real-time PCR (qPCR) and western blot were used for expression analysis. Colony formation assay, EdU assay and MTT assay were performed to determine cell proliferation. Flow cytometry assay was conducted to determine cell apoptosis. The predicted binding relationship between miR-622 and circ_KCNQ5 or RAS oncogene family member 10 (RAB10) was verified by dual-luciferase reporter assay. Results : The expression of circ_KCNQ5 was increased in bone marrow samples of childhood AML patients and AML cell lines. The knockdown of circ_KCNQ5 largely suppressed AML cell proliferation and promoted cell apoptosis. Circ_KCNQ5 directly bound to miR-622 and inhibited miR-622 expression. The cotransfection of miR-622 inhibitor reversed the effects of circ_KCNQ5 knockdown and thus recovered cell proliferation and depleted cell apoptosis. RAB10 was a target of miR-622, and circ_KCNQ5 bound to miR-622 to increase the expression of RAB10. MiR-622 restoration inhibited AML cell proliferation and induced cell apoptosis, while RAB10 overexpression abolished these effects. Conclusion : Circ_KCNQ5 high expression was associated with childhood AML malignant development, and circ_KCNQ5 participated in AML progression by regulating the miR-622/RAB10 pathway.

Deleterious Rare Mutations of GLI1 Dysregulate Sonic Hedgehog Signaling in Human Congenital Heart Disease.

The Glioma-associated oncogene (Gli) family members of zinc finger DNA-binding proteins are core effectors of Sonic hedgehog (SHH) signaling pathway. Studies in model organisms have identified that the Gli genes play critical roles during organ development, including the heart, brain, kidneys, etc. Deleterious mutations in GLI genes have previously been revealed in several human developmental disorders, but few in congenital heart disease (CHD). In this study, the mutations in GLI1-3 genes were captured by next generation sequencing in human cohorts composed of 412 individuals with CHD and 213 ethnically matched normal controls. A total of 20 patient-specific nonsynonymous rare mutations in coding regions of human GLI1-3 genes were identified. Functional analyses showed that GLI1 c.820G> T (p.G274C) is a gain-of-function mutation, while GLI1 c.878G>A (p.R293H) and c.1442T>A (p.L481X) are loss-of-function mutations. Our findings suggested that deleterious rare mutations in GLI1 gene broke the balance of the SHH signaling pathway regulation and may constitute a great contribution to human CHD, which shed new light on understanding genetic mechanism of embryo cardiogenesis regulated by SHH signaling.

Detection of TET2 Mutation in Patients with De Novo Acute Myeloid Leukemia: A Mutation Analysis of 51 Iranian Patients.

Acute myeloid leukemia (AML) is a heterogeneous clonal disease that is considered to originate from hematopoietic stem cells, which are characterized by impaired myelopoiesis and blast proliferation. TET oncogene family member 2 (TET2) mutations are frequent in myeloid malignancies and several studies have assessed the clinical importance of TET2 mutations. However, its frequency ratio has not yet been fully clarified. Method:Hence, our study was aimed to analyze TET2mut in patients with de-novo AML and their association with clinical, molecular characteristics and Nucleophosmin 1 (NPM1), Fms-like tyrosine kinase 3 (FLT3), CCAAT Enhancer Binding Protein Alpha (CEBPA) and Wilms’ tumor protein (WT1) gene expression. Fifty-one Iranian patients were screened by polymerase chain reaction (PCR) and direct sequencing to evaluate TET2 mutations frequency. Results:Out of all patients, 10 mutations in 8 patients (15.6%) were detected and closely associated with higher age and higher hemoglobin levels (p-value <0.05). Although FLT3, NPM1 and CEBPA gene expression did not show any significant correlation with TET2mut, cytogenetically normal acute myeloid leukemia (CN-AML) patients appear to bear TET2mut more frequently with lower platelet counts. Monocyte-lineages leukemia has seemed to be more linked with TET2mut in these patients. Conclusion:Our study suggests the frequency of TET2mut in our study (15.6%) is in line with previous studies and reveals the critical role of TET2 in myeloid transformation, especially in leukemia with monocytic subtypes.

Identifying pathways regulating the oncogenic p53 family member \u0394Np63 provides therapeutic avenues for squamous cell carcinoma

BackgroundΔNp63 overexpression is a common event in squamous cell carcinoma (SCC) that contributes to tumorigenesis, making ΔNp63 a potential target for therapy.MethodsWe created inducible TP63-shRNA cells to study the effects of p63-depletion in SCC cell lines and non-malignant HaCaT keratinocytes. DNA damaging agents, growth factors, signaling pathway inhibitors, histone deacetylase inhibitors, and metabolism-modifying drugs were also investigated for their ability to influence ΔNp63 protein and mRNA levels.ResultsHaCaT keratinocytes, FaDu and SCC-25 cells express high levels of ΔNp63. HaCaT and FaDu inducible TP63-shRNA cells showed reduced proliferation after p63 depletion, with greater effects on FaDu than HaCaT cells, compatible with oncogene addiction in SCC. Genotoxic insults and histone deacetylase inhibitors variably reduced ΔNp63 levels in keratinocytes and SCC cells. Growth factors that regulate proliferation/survival of squamous cells (IGF-1, EGF, amphiregulin, KGF, and HGF) and PI3K, mTOR, MAPK/ERK or EGFR inhibitors showed lesser and inconsistent effects, with dual inhibition of PI3K and mTOR or EGFR inhibition selectively reducing ΔNp63 levels in HaCaT cells. In contrast, the antihyperlipidemic drug lovastatin selectively increased ΔNp63 in HaCaT cells.ConclusionsThese data confirm that ΔNp63-positive SCC cells require p63 for continued growth and provide proof of concept that p63 reduction is a therapeutic option for these tumors. Investigations of ΔNp63 regulation identified agent-specific and cell-specific pathways. In particular, dual inhibition of the PI3K and mTOR pathways reduced ΔNp63 more effectively than single pathway inhibition, and broad-spectrum histone deacetylase inhibitors showed a time-dependent biphasic response, with high level downregulation at the transcriptional level within 24 h. In addition to furthering our understanding of ΔNp63 regulation in squamous cells, these data identify novel drug combinations that may be useful for p63-based therapy of SCC.

Abstract 2420: Characterizing the oncogenic activity of ERBB4 mutations and their sensitivity to neratinib

Abstract Hundreds of somatic ERBB4 mutations have been described in cancer tissues with very limited information about their functional significance. However, analyses of individual mutants have indicated that activating ERBB4 mutations, such as ERBB4 K935I, do exist. Understanding the functional consequences of ERBB4 mutations is needed in order to assess the relevance of targeting ERBB4 in human cancers with matched therapies such as neratinib. Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that binds to and potently inhibits ERBB4 kinases activity in-vitro, and is currently approved clinically for the treatment of early stage and metastatic HER2+ breast cancers. We set up to address the transforming potential of individual ERBB4 variants by selecting 18 mutations (Table 1) from cBioPortal data (www.cbioportal.org) using the following criteria: 1) the mutations were recurrent, 2) they were analogous to activating mutations described for other oncogenic ERBB family members and/or 3) their position at receptor dimerization interfaces suggested functional relevance. Retroviral pBABE-puro-gateway vector was used to achieve functional, but still near-physiological expression levels of ERBB4 in different cell backgrounds. A screen for optimal cellular background to assess the transforming potential of ERBB4 variants indicated that 1) IL-3 independent growth of mouse lymphoid Ba/F3 cells, 2) focus formation analysis of mouse NIH-3T3 fibroblasts, and 3) ligand-induced proliferation of human mammary epithelial MCF-10A provided read-outs with robust differences when the effects of the positive control mutant ERBB4 K935I was compared to wild-type ERBB4. These models are now being used to address the transforming potential of the 18 mutations (Table 1). Table 1.Somatic ERBB4 mutations chosen for functional analyses.R106CE452KR711CL798RG870RK1223TS303FR524CG741EV840IG907ES1289AR393WR544WS774GR847HR992CR1304W Citation Format: Peppi Suominen, Deepankar Chakroborty, Kari J. Kurppa, Irmina Diala, Lisa D. Eli, Alshad S. Lalani, Klaus Elenius. Characterizing the oncogenic activity of ERBB4 mutations and their sensitivity to neratinib [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 2420.

TET2 mutations in acute myeloid leukemia: a comprehensive study in patients of Sindh, Pakistan.

BackgroundThe tet oncogene family member 2 (TET2) gene has been reported to be involved in DNA methylation and epigenetic regulation in acute myeloid leukemia (AML). Various studies have proven functional role of TET2 mutations in AML. We herein studied the frequency and genotype-phenotype correlation of TET2 gene in AML patients in Sindh, Pakistan.Patients and methodsThe current study was carried out at Liaquat University of Medical & Health Sciences, Jamshoro, Pakistan, in collaboration with National Institute of Blood Disease & Bone Marrow Transplant, Karachi, Pakistan, during the period from June 2019 to June 2020. A total of 130 patients diagnosed with AML were screened for TET2 mutations. Whole exome sequencing of 14 individuals was carried out to find the genetic variants in TET2 gene. The pathogenicity of the variants was predicted by SIFT, PolyPhen2, Mutation Taster and CADD Phred scores. The allele frequency of the variants was compared with global population using 1000 genomes project and Exome Aggregation Consortium (ExAC). Furthermore, exon 3 and exon 5 of the TET2 gene were sequenced by using Sanger sequencing. The findings were correlated with subtypes of AML and corresponding karyotypes.ResultsThrough the exome sequencing, 17 genetic variants (13 SNPs and four indels) were identified in 14 individuals. Of these, four variants that is, one frameshift deletion, one frameshift insertion and two nonsense variants were novel and not present in dbSNP151 database. Three novel variants were found in exon 3 including two frameshift variants that is, p.T395fs and G494fs, predicted as deleterious by CADD Phred scores, and one stop-gain variant (p.G898X) predicted as deleterious by Mutation Taster and CADD Phred scores. One novel non sense variant (p.Q1191X) was found in the exon 5 predicted as deleterious by SIFT, Mutation Taster and CADD Phred scores. Sanger sequencing analysis revealed one novel deletion at g105233851: del.TAGATAGA, and one novel SNP g;105233861 T>G identified in the TET2 gene. Majority of the exon 3 mutations were seen in the patients diagnosed with AML with maturation, and had a normal karyotype.ConclusionTET2 mutations were identified in around 16% of the total patients of our study indicating other mechanisms being involved in pathophysiology of AML in this cohort. The TET2 mutations provide a prognostic value in determining AML classification.

Single-Cell Gene Network Analysis and Transcriptional Landscape of MYCN-Amplified Neuroblastoma Cell Lines.

Neuroblastoma (NBL) is a pediatric cancer responsible for more than 15% of cancer deaths in children, with 800 new cases each year in the United States alone. Genomic amplification of the MYC oncogene family member MYCN characterizes a subset of high-risk pediatric neuroblastomas. Several cellular models have been implemented to study this disease over the years. Two of these, SK-N-BE-2-C (BE2C) and Kelly, are amongst the most used worldwide as models of MYCN-Amplified human NBL. Here, we provide a transcriptome-wide quantitative measurement of gene expression and transcriptional network activity in BE2C and Kelly cell lines at an unprecedented single-cell resolution. We obtained 1105 Kelly and 962 BE2C unsynchronized cells, with an average number of mapped reads/cell of roughly 38,000. The single-cell data recapitulate gene expression signatures previously generated from bulk RNA-Seq. We highlight low variance for commonly used housekeeping genes between different cells (ACTB, B2M and GAPDH), while showing higher than expected variance for metallothionein transcripts in Kelly cells. The high number of samples, despite the relatively low read coverage of single cells, allowed for robust pathway enrichment analysis and master regulator analysis (MRA), both of which highlight the more mesenchymal nature of BE2C cells as compared to Kelly cells, and the upregulation of TWIST1 and DNAJC1 transcriptional networks. We further defined master regulators at the single cell level and showed that MYCN is not constantly active or expressed within Kelly and BE2C cells, independently of cell cycle phase. The dataset, alongside a detailed and commented programming protocol to analyze it, is fully shared and reusable.

Mutation bias within oncogene families is related to proliferation-specific codon usage

It is well known that in cancer gene families some members are more frequently mutated in tumor samples than their family counterparts. A paradigmatic case of this phenomenon is KRAS from the RAS family. Different explanations have been proposed ranging from differential interaction with other proteins to preferential expression or localization. Interestingly, it has been described that despite the high amino acid identity between RAS family members, KRAS employs an intriguing differential codon usage. Here, we found that this phenomenon is not exclusive to the RAS family. Indeed, in the RAS family and other oncogene families with two or three members, the most prevalently mutated gene in tumor samples employs a differential codon usage that is characteristic of genes involved in proliferation. Prompted by these observations, we chose the RAS family to experimentally demonstrate that the translation efficiency of oncogenes that are preferentially mutated in tumor samples is increased in proliferative cells compared to quiescent cells. These results were further validated by assessing the translation efficiency of KRAS in cell lines that differ in their tRNA expression profile. These differences are related to the cell division rate of the studied cells and thus suggest an important role in context-specific oncogene expression regulation. Altogether, our study demonstrates that dynamic translation programs contribute to shaping the expression profiles of oncogenes. Therefore, we propose this codon bias as a regulatory layer to control cell context-specific expression and explain the differential prevalence of mutations in certain members of oncogene families.

Activation of STAT transcription factors by the Rho-family GTPases.

The Rho-family of small GTPases are biological molecular switches that are best known for their regulation of the actin cytoskeleton. Through their activation and stimulation of downstream effectors, the Rho-family control pathways involved in cellular morphology, which are commonly activated in cancer cell invasion and metastasis. While this makes them excellent potential therapeutic targets, a deeper understanding of the downstream signalling pathways they influence will be required for successful drug targeting. Signal transducers and activators of transcription (STATs) are a family of transcription factors that are hyper-activated in most cancer types and while STATs are widely understood to be activated by the JAK family of kinases, many additional activators have been discovered. A growing number of examples of Rho-family driven STAT activation, largely of the oncogenic family members, STAT3 and STAT5, are being identified. Cdc42, Rac1, RhoA, RhoC and RhoH have all been implicated in STAT activation, contributing to Rho GTPase-driven changes in cellular morphology that lead to cell proliferation, invasion and metastasis. This highlights the importance and therapeutic potential of the Rho-family as regulators of non-canonical activation of STAT signalling.

Breast Cancer and Major Deviations of Genetic and Gender-related Structures and Function.

We have searched the literature for information on the risk of breast cancer (BC) in relation to gender, breast development, and gonadal function in the following 8 populations: 1) females with the Turner syndrome (45, XO); 2) females and males with congenital hypogonadotropic hypogonadism and the Kallmann syndrome; 3) pure gonadal dysgenesis (PGD) in genotypic and phenotypic females and genotypic males (Swyer syndrome); 4) males with the Klinefelter syndrome (47, XXY); 5) male-to-female transgender individuals; 6) female-to-male transgender individuals; 7) genotypic males, but phenotypic females with the complete androgen insensitivity syndrome, and 8) females with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome (müllerian agenesis). Based on this search, we have drawn 3 major conclusions. First, the presence of a Y chromosome protects against the development of BC, even when female-size breasts and female-level estrogens are present. Second, without menstrual cycles, BC hardly occurs with an incidence comparable to males. There is a strong correlation between the lifetime number of menstrual cycles and the risk of BC. In our populations the BC risk in genetic females not exposed to progesterone (P4) is very low and comparable to males. Third, BC has been reported only once in genetic females with MRKH syndrome who have normal breasts and ovulating ovaries with normal levels of estrogens and P4. We hypothesize that the oncogenic glycoprotein WNT family member 4 is the link between the genetic cause of MRKH and the absence of BC women with MRKH syndrome.

Non-Redundant and Overlapping Oncogenic Readouts of Non-Canonical and Novel Colorectal Cancer KRAS and NRAS Mutants.

RAS oncogene family members are molecular switches of signaling pathways that control cell growth, proliferation, differentiation, and survival. In colorectal cancer, Kirsten-RAS (KRAS) and neuroblastoma-RAS (NRAS) are the commonly mutated isoforms. Activating mutations in RAS result in cellular transformation independent of upregulated epidermal growth factor receptor (EGFR)-initiated signaling. The present study characterized the functional consequences of non-canonical/novel KRAS and NRAS mutants identified in a targeted next-generation sequencing study of colorectal cancer specimens from Filipino patients. In vitro assays in NIH3T3 cells showed that similar to the canonical KRAS G12D mutant, overexpression of KRAS G12S, A59T, and Y137C, but not NRAS G12D and NRAS A11V, confer higher proliferation and migration rates. HCT116 cells transfected with the novel NRAS A11V and the canonical NRAS G12D, but not the KRAS mutants, display enhanced resistance to apoptosis. All four non-canonical/novel KRAS and NRAS mutants induce gross changes in F-actin cytoskeletal organization and cellular morphology of NIH3T3 cells. Only KRAS G12S and KRAS A59T appear to deregulate extracellular signal-regulated kinase (ERK) and its downstream target ETS transcription factor ELK1 (ELK1). Elucidation of differential effector engagement responsible for the variable phenotypic readouts of the mutants is warranted. If validated by mouse studies and clinical correlates, these can have wider implications in choosing treatment options.

Combination of the MEK Inhibitor Trametinib and Pyrvinium Pamoate Efficiently Targets RAS Pathway-Mutated Acute Myeloid Leukemia in Preclinical Models

While acute myeloid leukemia (AML) is still associated with a low cure rate, recent advances in understanding its molecular complexity have significantly improved therapy for subgroups of patients, including those harboring FLT3, IDH1 or IDH2 mutations1. However, more than half of AML cases still lack a druggable oncogenic target. Human cancers frequently harbor mutations in RAS oncogene family members, including NRAS, KRAS and HRAS, which drive oncogenesis by augmenting cellular proliferation and survival. These are small protein GTPases, regulated by a switch between active GTP-linked and inactive GDP-bound states governed by a complex network of guanine exchange factors (GEFs, favoring RAS-GTP) and GTPase activating factors (GAPs, favoring RAS-GDP). RAS activation - due to either extrinsic recruitment by transmembrane tyrosine kinase receptors or intrinsic mutations - propagates through the downstream RAF/MEK/ERK and PI3K/AKT signaling pathways. Besides RAS-activating mutations conferring independence from physiological regulators, human cancers harbor mutations in other RAS network genes such as NF1 (encoding neurofibromin, a RAS GAP), BRAF or PTPN11 (encoding the SHP2 tyrosine phosphatase involved in RAS activation). Somatic alterations of RAS pathway genes, notably NRAS, KRAS, PTPN11 (missense mutations) and NF1 (mutations and deletions), are reported in up to 20% of AML cases2. Generally arising as late driver events, RAS pathway mutations participate in leukemogenesis through mitogen activated protein kinase (MAPK) activation. The anti-tumor activity of MEK inhibitors in Nras-mutated AML in mice and in some NRAS or KRAS-mutated AML patients suggests that deregulated RAS pathway signaling may represent a bona fide therapeutic target. However, strategies to inhibit RAS - indirectly in most cases - have been hampered by signaling feedback, redundancy and tumor heterogeneity 3. We identified 127 cases of AML with unmet therapeutic need within a cohort from which we excluded those with European leukemia network (ELN) favorable prognosis or FLT3-ITD mutations. Targeted next-generation sequencing revealed RAS pathway alterations in 50 patients (39.3%) and NF1 mutations and deletions, mostly large cytogenetically detected deletions, in 17 (14.8%). NRAS, KRAS, PTPN11, CBL and BRAF variants were detected in 13 (10.4%), 10 (7.9%), 9 (7.2%), 5 (3.9%) and 2 (1.6%) cases, respectively. Mutations in RAF1, RASA1, SOS1 and MAP2K2 were observed in a single case each. RAS pathway alterations appeared in the putative main leukemic clone as well as in subclones inferred from variant allele frequencies. Concurrent RAS pathway mutations were observed in nine cases. Among 79 patients homogeneously treated with intensive induction chemotherapy, RAS pathway alterations correlated with higher clinical proliferation markers (elevated white blood cell count, blast cell percentage and LDH levels) and reduced survival probability, particularly within the ELN intermediate-risk subgroup. We established robust models of RAS/MAPK activation through genetic NF1 disruption or expression of NRASG12D or PTPN11D61Y in growth factor (GF)-dependent cell lines. We assessed oncogenic addiction to the RAS pathway in these cells through GF-independence, increased RAS activity, faster propagation in immunocompromised mice and an exquisite sensitivity to pharmacological MEK inhibition in vitro and in vivo. High-content pharmacological screens with FDA-approved molecules identified pyrvinium pamoate, an anti-helminthic agent, as preferentially active in RAS-activated cells. This compound significantly impaired cell viability and colony formation in primary AML samples with RAS pathway alterations. Moreover, the combination of trametinib and pyrvinium pamoate demonstrated synergy in cell line models and even primary samples. While pyrvinium pamoate strongly inhibited mitochondrial respiration and induced metabolic reprograming towards increased glycolysis, trametinib impaired glycolysis and mitochondrial respiratory capacity, suggesting a mechanistic basis for the synergy observed. These data highlight the translational opportunity in developing pyrvinium pamoate for RAS pathway mutated AML.

The prognostic impact of tet oncogene family member 2 mutations in patients with acute myeloid leukemia: a systematic-review and meta-analysis

BackgroundThe impact of Tet oncogene family member 2 (TET2) mutations on the prognosis of acute myeloid leukemia (AML) is still controversial. A meta analysis is needed in order to assess the prognostic significance of TET2 mutation in AML.MethodsFive databases including PubMed, Cochrane, EMBase, China National Knowledge Internet (CNKI) and Wanfang database were retrieved to search studies that investigated the correlation between TET2 mutations and outcomes of AML patients. Pooled hazard ratios (HRs) and odds ratios (ORs) were used to assess the effects of TET2 mutations.ResultsSixteen studies were included. TET2 mutation was an unfavorable prognostic factor for overall survival (OS: HR = 1.386; P < 0.001) and event-free survival (EFS: HR = 1.594; P = 0.002) in patients with AML. For patients under 65 years of age, TET2 mutation predicted an inferior OS (HR = 1.310, P = 0.051) and EFS (HR = 1.429, P = 0.027). For patients with intermediate-risk cytogenetics (IR-AML), mutant TET2 had a significant association with adverse OS (HR = 0.474; P < 0.001). For patients with normal cytogenetics (CN-AML), mutant TET2 also conferred adverse OS (HR = 1.425; P < 0.001) and EFS (HR = 1.450, P < 0.001). Further, among patients with CN-AML, mutant TET2 was associated with inferior OS (HR = 2.034, P < 0.001) and EFS (HR = 2.140, P < 0.001) in the ELN favorable-risk subgroup and an inferior EFS (HR = 1.487; P < 0.001) in the ELN intermediate-Isubgroup. With respect to treatment outcome, TET2 mutation predicted a significantly lower rate of complete remission (CR) in cases with ELN favorable-risk cytogenetics (OR = 0.460, P = 0.011).ConclusionsTET2 mutation had adverse impacts on survival and treatment response in AML patients and will contribute to risk-stratification, prognosis prediction and therapy guidance.

Oncogenic Wnt3a: A Candidate Specific Marker and Novel Molecular Target for Hepatocellular Carcinoma.

Background and aim: It is of the utmost importance for the specific diagnosis and effective therapy of hepatocellular carcinoma (HCC). Abnormality of oncogenic Wingless-type MMTV integration site family member 3a (Wnt3a) has been associated with progression of HCC. In this study, we aimed to evaluate Wnt3a as a novel biomarker and target for HCC.Methods: Circulating Wnt3a levels were quantitatively detected in a cohort of chronic liver diseases by an enzyme-linked immune-absorbent assay (ELISA). Hepatic Wnt3a expression in HCC and para-cancerous tissues was analyzed by immunohistochemistry. Prognostic value of Wnt3a for HCC was discovered in the cohort from the Cancer Genome Atlas (TCGA). Dynamic alterations of Wnt3a levels were detected in the hepatocarcinogenesis model induced by 2-acetylaminofluorene. Effects of Wnt3a on biological behaviors were evaluated in vitro and in vivo based on Crispr/Cas9.Results: Up-regulated Wnt3a levels were observed in serum of HCC patients with high specificity and sensitivity for HCC diagnosis. Combination of Wnt3a and AFP could improve sensitivity to 93.9% in serological detection. In addition, Wnt3a expression in HCC tissues was significantly higher than that in para-cancerous tissues. The cohort of TCGA demonstrated that high Wnt3a expression led to a poor survival of HCC patients, especially in cases at advanced stages. Furthermore, the hepatocarcinogenesis model showed that Wnt3a dynamically increased in the development of HCC. Functionally, silencing Wnt3a by Crispr/Cas9 suppressed the proliferation, colony formation, induced cell cycle arrest of HCC cells by de-activating Wnt/β-catenin pathway in vitro, and inhibited xenograft tumor growth in vivo.Conclusions: Oncogenic Wnt3a could be considered as a candidate biomarker and novel target for HCC.

Codon usage regulates human KRAS expression at both transcriptional and translational levels

KRAS and HRAS are highly homologous oncogenic Ras GTPase family members that are mutated in a wide spectrum of human cancers. Despite having high amino acid identity, KRAS and HRAS have very different codon usage biases: the HRAS gene contains many common codons, and KRAS is enriched for rare codons. Rare codons in KRAS suppress its protein expression, which has been shown to affect both normal and cancer biology in mammals. Here, using HRAS or KRAS expression in different human cell lines and in vitro transcription and translation assays, we show that KRAS rare codons inhibit both translation efficiency and transcription and that the contribution of these two processes varies among different cell lines. We observed that codon usage regulates mRNA translation efficiency such that WT KRAS mRNA is poorly translated. On the other hand, common codons increased transcriptional rates by promoting activating histone modifications and recruitment of transcriptional coactivators. Finally, we found that codon usage also influences KRAS protein conformation, likely because of its effect on co-translational protein folding. Together, our results reveal that codon usage has multidimensional effects on protein expression, ranging from effects on transcription to protein folding in human cells.

MTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β

Glycogen synthase kinase 3β (GSK3β) phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3β substrates, such as c-Myc and Snail, are nuclear transcription factors, suggesting the possibility that GSK3β function is controlled through its nuclear localization. Here, using ARPE-19 and MDA-MB-231 human cell lines, we found that inhibition of mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3β from the cytosol to the nucleus and to a GSK3β-dependent reduction of the levels of both c-Myc and Snail. mTORC1 is known to be controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance, and we observed here that AMPK activation or amino acid deprivation promotes GSK3β nuclear localization in an mTORC1-dependent manner. GSK3β was detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through a perturbation of RAS oncogene family member 7 (Rab7) resulted in loss of GSK3β from lysosomes and in enhanced GSK3β nuclear localization as well as GSK3β-dependent reduction of c-Myc levels. These findings indicate that the nuclear localization and function of GSK3β is suppressed by mTORC1 and suggest a link between metabolic conditions sensed by mTORC1 and GSK3β-dependent regulation of transcriptional networks controlling cellular biomass production.

Clinical Importance of Myc Family Oncogene Aberrations in Epithelial Ovarian Cancer.

BackgroundThe Myc oncogene family has been implicated in many human malignancies and is often associated with particularly aggressive disease, suggesting Myc as an attractive prognostic marker and therapeutic target. However, for epithelial ovarian cancer (EOC), there is little consensus on the incidence and clinical relevance of Myc aberrations. Here we comprehensively investigated alterations in gene copy number, expression, and activity for Myc and evaluated their clinical significance in EOC.MethodsTo address inconsistencies in the literature regarding the definition of copy number variations, we developed a novel approach using quantitative polymerase chain reaction (qPCR) coupled with a statistical algorithm to estimate objective thresholds for detecting Myc gain/amplification in large cohorts of serous (n = 150) and endometrioid (n = 80) EOC. MYC, MYCN, and MYCL1 mRNA expression and Myc activity score for each case were examined by qPCR. Kaplan–Meier and Cox-regression analyses were conducted to assess clinical significance of Myc aberrations.ResultsUsing a large panel of cancer cell lines (n = 34), we validated the statistical algorithm for determining clear thresholds for Myc gain/amplification. MYC was the most predominantly amplified of the Myc oncogene family members, and high MYC mRNA expression levels were associated with amplification in EOC. However, there was no association between prognosis and increased copy number or gene expression of MYC/MYCN/MYCL1 or with a pan-Myc transcriptional activity score, in EOC, although MYC amplification was associated with late stage and high grade in endometrioid EOC.ConclusionA systematic and comprehensive analysis of Myc genes, transcripts, and activity levels using qPCR revealed that although such aberrations commonly occur in EOC, overall they have limited impact on outcome, suggesting that the biological relevance of Myc oncogene family members is limited to certain subsets of this disease.