scholarly journals Sos-mediated cross-activation of wild-type Ras by oncogenic Ras is essential for tumorigenesis

2012 ◽  
Vol 3 (1) ◽  
Author(s):  
Hao-Hsuan Jeng ◽  
Laura J Taylor ◽  
Dafna Bar-Sagi
Keyword(s):  
Wild Type ◽  
Oncogenic Ras

scholarly journals Bcor Mutations Cooperate with Tet2 Deficiency to Promote RAS Transformation of Mouse Hematopoietic Progenitors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 888-888
Author(s):  
Iman Fares ◽  
Rahul S. Vedula ◽  
Shabbir M. Vahanvaty ◽  
Christopher S Waters ◽  
Marlise R. Luskin ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Somatic Mutations ◽  
Genetic Alterations ◽  
Ras Signaling ◽  
Wild Type ◽  
Ras Pathway ◽  
Tp53 Mutations ◽  
Oncogenic Ras ◽  
Mdm2 Overexpression

Abstract Somatic mutations can have highly stereotyped positions in the myeloid clonal hierarchy and distinct patterns of co-occurring mutations. Gene mutations that cause aberrant activation of RAS/MAPK signaling are typically late events in myeloid disease progression and are closely associated with leukemic transformation. We hypothesized that the phenotypic output of oncogenic RAS signaling is dynamically reprogrammed during leukemogenesis based on evolving genetic and epigenetic context. To identify genetic alterations that may modulate RAS-mediated transformation, we evaluated 1273 adults with myelodysplastic syndrome, including 150 with mutations in NRAS, KRAS, PTPN11, CBL, RIT1, NF1, or FLT3. Somatic mutations in ASXL1 (q<0.0001), RUNX1 (q<0.0001), EZH2 (q<0.0001), BCOR (q=0.0002), and STAG2 (q=0.001) were most significantly associated with co-occurring RAS pathway mutations, compared to those without RAS pathway mutations, while TP53 mutations were less frequent (q=0.059). We validated these observations in an independent cohort of 6343 unselected patients, including 1081 patients harboring either RAS pathway mutations (n=651),TP53 mutations (n=494), or both (n=57). To define the effects of sequential acquisition of driver mutations, we developed a mouse serial transplantation model of somatic myeloid transformation. First, we used in vivo pI:pC treatment to induce biallelic inactivation of Tet2 in adult Mx1-Cre/Tet2flox/floxmice. After 12 weeks, we purified Tet2-/-or control hematopoietic stem and progenitor cells (HSPCs) and used CRISPR/Cas9 to separately introduce inactivating mutations in Ezh2, Asxl1-exon12, Stag2, or Bcor, then evaluated their functional effects using ex vivo serial replating or in vivo competitive transplantation. Tet2-/-HSPCs with control sgRNA showed a modest enhancement of serial replating compared to Tet2-wild type HSPCs, while Tet2-/-HSPCs Asxl1, Stag2, and Bcor, but not Ezh2 sgRNA had markedly enhanced serial replating capacity (>6 platings in all replicates). In primary transplantation, secondary mutations caused in vivo clonal advantage after 16 weeks, but never resulted in histologic transformation to acute leukemia. We next evaluated the impact of tertiary NRASG12Dmutations in each pairwise Tet2-/-CRISPR combination (Asxl1, Bcor, Ezh2, Stag2, control). We purified HSPCs from recipient mice 16 weeks after primary transplantation, transduced with a lentiviral NRASG12Dexpression vector and transplanted into secondary recipients. Recipients of Tet2/Bcor/NRAS, Tet2/Asxl1/NRAS, or Tet2/Ezh2/NRAS cells succumbed to CD11b+myeloid disease with variable latency in Bcor (14 days), Ezh2 (50 days), and Asxl1 (120 days) cells, suggesting that combined Tet2 and PRC1/2 alterations may modify the effects of oncogenic RAS signaling. To determine whether pre-existing epigenetic mutations cooperate to alter the transcriptional response to acute oncogenic stress compared to wild type cells, weperformed RNA-seq 12 and 24 hours after induced expression of NRASG12D in isogenic immortalized mouse progenitor cells deficient for Tet2, Bcor, or both Tet2 and Bcor. We observed rapid activation of inflammatory and cellular senescence programs in all conditions, suggesting a genotype-independent immediate early response to oncogenic signaling. However, we also identified genotype-specific regulation of tumor suppressor and cell cycle checkpoint pathways. While Cdnk1a expression was strongly induced in all conditions, Cdnk2a expression (and p16Ink4a and p19ARF protein levels) was preferentially upregulated in the context of Bcor deficiency. Moreover, expression of the p53 negative regulator Mdm2 was increased 11-fold in Tet2/Bcor-deficient cells, but only 4 to 5-fold in wild type, Tet2-, or Bcor-deficient cells. Tet2/Bcor-deficient cells were significantly more sensitive to treatment with the Mdm2 antogonist, Nutlin, upon induction of NRAS expression than were wild-type cells, suggesting that Mdm2 overexpression directly mediates acquired tolerance of oncogene stress. These human genetic data and mouse models suggest that epigenetic alterations occurring during early myeloid leukemogenesis may enable evasion of oncogene protection mechanism. Bcor mutations can pair with initiating Tet2 mutations to facilitate RAS mediated transformation while incurring a dependency on Mdm2 overexpression. Disclosures No relevant conflicts of interest to declare.

Rigosertib Blocks RAS Signaling By Acting As a Small Molecule RAS Mimetic That Binds to the RAS-Binding Domains of RAS Effector Proteins

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5616-5616 ◽  
Author(s):  
E. Premkumar Reddy ◽  
Sai Krishna Divakar ◽  
Rodrigo Vasquez-Del Carpio ◽  
Kaushik Dutta ◽  
Stacey J Baker ◽  
...  
Keyword(s):  
Growth Factor ◽  
Chemical Shift ◽  
Research Funding ◽  
Kinase Activity ◽  
Effector Proteins ◽  
Phase Iii ◽  
Wild Type ◽  
Oncogenic Ras ◽  
Binding Domains ◽  
In Cells

Abstract Oncogenic activation of RAS via point mutations occurs in more than 30% of all human cancers, including hematopoietic malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Investigations to understand the critical biochemical and biological mechanisms of RAS function are at the forefront of cancer research. Studies have shown that RAS interacts with a large number of effector proteins by a highly conserved mechanism that involves the switch region of RAS and the RAS-binding domains (RBDs) of its effector proteins. Because these interactions play an essential role in oncogenic RAS function, inhibiting them constitutes an attractive and important therapeutic approach for myeloid neoplasias and other cancers. Rigosertib is a novel styryl benzyl sulfone, which is in a Phase III clinical trial (ONTIME) for MDS. Here, we delineate the way rigosertib interacts with the RBDs of several RAS effector proteins: RAF, the PI3K family of proteins and RalGDS. To identify residues in the B-RAF RBD that interact with rigosertib, we recorded a series of 15N-1H HSQC spectra of 15N-labeled B-RAF RBD with increasing concentration of rigosertib. Strikingly, the chemical shift perturbations caused by addition of rigosertib are localized to the very region of the B-RAF-RBD implicated in RAS binding, namely the beta1 and beta2 strands and alpha3 helix (Fig 1). Additionally, this cluster of residues with largest chemical shift perturbation contains many of the same residues involved in RAS binding, namely Ile156, Lys164, Arg166, Thr167, Val168, Ala184 and Met187. These key residues are conserved within RAF RBDs, suggesting that rigosertib would bind to similar regions of the A- and c-RAF RBDs. Next, we examined the binding of rigosertib and GTP-RAS to wild type and mutant forms of c-RAF RBD that harbor mutations in residues that mediate binding to rigosertib. Our studies show that all mutations that cause dissociation of GTP-RAS binding also inhibit rigosertib binding to these mutant proteins. Taken together, the chemical shift data and mutagenesis data provide powerful evidence that rigosertib binds the B-RAF RBD at the same location as the RAS switch I region. A consequence of inhibiting RAS binding to RAF appears to be a block in growth factor-induced activation of RAF kinase activity. We also show that a result of this block in RAS/RAF interactions is an inability of RAF proteins to form dimers and activate MEK and ERK. This block in the activation of MEK/ERK pathways can be seen in cells that express wild-type RAS and RAF proteins (HeLa), in cells that express a constitutively active form of oncogenic RAS (HeLa-N-RAS-G12D), and in cells that exhibit amplification of EGF receptors (A431). Rigosertib also inhibits the phosphorylation of c-RAF serine 338, which has been shown to be essential for the activation of its kinase activity and for its association with and activation of PLK-1. Our results showing rigosertib-mediated inhibition of the PLK-1/RAF interaction might help explain the ability of this compound to induce mitotic arrest of human tumor cells and the ability of rigosertib to reduce blast counts in MDS patients (Seetharam et al, Leuk Res 2012). We have also demonstrated the binding of rigosertib to the RBDs of the PI3K family of kinases and RalGDS, both of which constitute important effectors of RAS. A consequence of the interaction of rigosertib with the RBD domains of PI3Ks appears to be a block in growth factor-induced AKT activation. These studies suggest that the disruption of multiple RAS-driven signaling pathways by rigosertib is mediated via rigosertib’s binding to RBDs of RAS effector proteins, leading to their inactivation. Figure 1 Figure 1. Disclosures Reddy: Onconova Therapeutics Inc: Research Funding. Divakar:Onconova Therapeutics Inc: Research Funding. Vasquez-Del Carpio:Onconova Therapeutics Inc: Research Funding. Dutta:Onconova Therapeutics Inc: Research Funding. Baker:Onconova Therapeautics Inc: Consultancy. Reddy:Onconova Therapeutics Inc: Consultancy. Aggarwal:Onconova Therapeutics Inc: Research Funding.

scholarly journals An engineered chimeric toxin that cleaves activated mutant and wild-type RAS inhibits tumor growth

2020 ◽  
Vol 117 (29) ◽  
pp. 16938-16948 ◽  
Author(s):  
Vania Vidimar ◽  
Greg L. Beilhartz ◽  
Minyoung Park ◽  
Marco Biancucci ◽  
Matthew B. Kieffer ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Protein Delivery ◽  
Tumor Burden ◽  
Target Engagement ◽  
Wild Type ◽  
Downstream Signaling ◽  
Oncogenic Ras ◽  
Broad Array ◽  
Xenograft Models

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1–specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutantRAS. Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.

Membrane association of cathepsin B can be induced by transfection of human breast epithelial cells with c-Ha-ras oncogene

1994 ◽  
Vol 107 (2) ◽  
pp. 373-384
Author(s):  
B.F. Sloane ◽  
K. Moin ◽  
M. Sameni ◽  
L.R. Tait ◽  
J. Rozhin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cathepsin B ◽  
Immunofluorescent Staining ◽  
Ras Oncogene ◽  
Human Breast ◽  
Wild Type ◽  
Breast Epithelial Cells ◽  
Oncogenic Ras ◽  
Breast Epithelial ◽  
Human Breast Epithelial Cells

Alterations in trafficking and increases in expression of the lysosomal proteases cathepsins B, D and L have been observed in transformed cells and malignant tumors, including human breast carcinoma. ras and the related rab proteins participate in the vesicular transport processes required for normal trafficking of lysosomal enzymes. In addition, transfection of murine fibroblasts with the ras oncogene has been shown to increase the expression of cathepsins L and B. As human cancers are primarily epithelial in origin, we have investigated whether there are alterations in the trafficking and expression of cathepsin B in MCF-10 human breast epithelial cells transfected with wild-type and mutated ras. In all cells examined, i.e. mortal MCF-10M cells, immortal MCF-10A or MCF-10F cells, and transfected MCF-10A cells (transfected with the neomycin resistance gene (MCF-10Aneo) or cotransfected with wild-type proto-oncogenic ras (MCF-10AneoN) or mutated oncogenic ras (MCF-10AneoT)), levels of mRNA transcripts for cathepsin B were similar. However, alterations in trafficking of cathepsin B were observed in the cells transfected with oncogenic ras. In these cells there was an increased association of cathepsin B activity and cathepsin B protein with plasma membrane/endosomal fractions and a more peripheral distribution of immunofluorescent staining for cathepsin B. At the electron microscopic level, immunogold labeling for cathepsin B was localized to the cell membrane as well as to vesicles in the microvilli and adjacent to the cell membrane. In the parental MCF-10A cells, in contrast, cathepsin B was localized to vesicles in the perinuclear region. The cathepsin B associated with plasma membrane/endosomal fractions in the cells transfected with oncogenic ras was mature cathepsin B as demonstrated by immunoblot analysis. This was confirmed further by showing an absence of peripheral immunofluorescent staining in these cells using an antibody specific for the propeptide of cathepsin B. Thus, we have demonstrated by multiple techniques that transfection of human breast epithelial cells with oncogenic ras results in alterations in the trafficking of cathepsin B similar to those observed previously in human and animal tumors of both epithelial and mesenchymal origin.

scholarly journals Cellular Response to Oncogenic Ras Involves Induction of the Cdk4 and Cdk6 Inhibitor p15INK4b

2000 ◽  
Vol 20 (8) ◽  
pp. 2915-2925 ◽  
Author(s):  
Marcos Malumbres ◽  
Ignacio Pérez De Castro ◽  
María I. Hernández ◽  
María Jiménez ◽  
Teresa Corral ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cellular Response ◽  
Cellular Transformation ◽  
Dominant Negative ◽  
Erk Pathway ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
Oncogenic Ras ◽  
Cell Cycle Inhibitor

ABSTRACT The cell cycle inhibitor p15 INK4b is frequently inactivated by homozygous deletion together with p16 INK4a and p19 ARF in some types of tumors. Although the tumor suppressor capability of p15 INK4b is still questioned, it has been found to be specifically inactivated by hypermethylation in hematopoietic malignancies in the absence of p16 INK4a alterations. Here we show that, in vitro, p15 INK4b is a strong inhibitor of cellular transformation by Ras. Surprisingly, p15 INK4b is induced in cultured cells by oncogenic Ras to an extent similar to that of p16 INK4a , and their expression is associated with premature G1 arrest and senescence. Ras-dependent induction of these two INK4 genes is mediated mainly by the Raf-Mek-Erk pathway. Studies with activated and dominant negative forms of Ras effectors indicate that the Raf-Mek-Erk pathway is essential for induction of both the p15 INK4b and p16 INK4a promoters, although other Ras effector pathways can collaborate, giving rise to a stronger response. Our results indicate that p15 INK4b , by itself, is able to stop cell transformation by Ras and other oncogenes such as Rgr (a new oncogene member of the Ral-GDS family, whose action is mediated through Ras). In fact, embryonic fibroblasts isolated from p15 INK4b knockout mice are susceptible to transformation by the Ras or Rgr oncogene whereas wild-type embryonic fibroblasts are not. Similarly, p15 INK4b -deficient mouse embryo fibroblasts are more sensitive than wild-type cells to transformation by a combination of the Rgr and E1A oncogenes. The cell cycle inhibitor p15 INK4b is therefore involved, at least in some cell types, in the tumor suppressor activity triggered after inappropriate oncogenic Ras activation in the cell.

scholarly journals Activated Ras as a Therapeutic Target: Constraints on Directly Targeting Ras Isoforms and Wild-Type versus Mutated Proteins

ISRN Oncology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
Raymond R. Mattingly
Keyword(s):  
Mek Inhibitor ◽  
Ras Proteins ◽  
Wild Type ◽  
Farnesyl Transferase ◽  
Oncogenic Ras ◽  
Effective Inhibition ◽  
Ras Isoforms ◽  
And Function ◽  
Made In

The ability to selectively and directly target activated Ras would provide immense utility for treatment of the numerous cancers that are driven by oncogenic Ras mutations. Patients with disorders driven by overactivated wild-type Ras proteins, such as type 1 neurofibromatosis, might also benefit from progress made in that context. Activated Ras is an extremely challenging direct drug target due to the inherent difficulties in disrupting the protein:protein interactions that underlie its activation and function. Major investments have been made to target Ras through indirect routes. Inhibition of farnesyl transferase to block Ras maturation has failed in large clinical trials. Likely reasons for this disappointing outcome include the significant and underappreciated differences in the isoforms of Ras. It is still plausible that inhibition of farnesyl transferase will prove effective for disease that is driven by activated H-Ras. The principal current focus of drugs entering clinic trial is inhibition of pathways downstream of activated Ras, for example, trametinib, a first-in-class MEK inhibitor. The complexity of signaling that is driven by activated Ras indicates that effective inhibition of oncogenic transduction through this approach will be difficult, with resistance being likely to emerge through switch to parallel pathways. Durable disease responses will probably require combinatorial block of several downstream targets.

scholarly journals Suppression of the HBP Function Increases Pancreatic Cancer Cell Sensitivity to a Pan-RAS Inhibitor

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 431
Author(s):  
Francesca Ricciardiello ◽  
Laura Bergamaschi ◽  
Humberto De Vitto ◽  
Yang Gang ◽  
Taiping Zhang ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Pancreatic Cancer Cell ◽  
Combined Treatment ◽  
Cancer Development ◽  
Ductal Adenocarcinoma ◽  
Wild Type ◽  
Novel Therapy ◽  
Hexosamine Biosynthetic Pathway ◽  
Oncogenic Ras ◽  
Negative Effect

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death and the search for a resolutive therapy is still a challenge. Since KRAS is commonly mutated in PDAC and is one of the main drivers of PDAC progression, its inhibition should be a key strategy for treatment, especially considering the recent development of specific KRAS inhibitors. Nevertheless, the effects of KRAS inhibition can be increased through the co-inhibition of other nodes important for cancer development. One of them could be the hexosamine biosynthetic pathway (HBP), whose enhancement is considered fundamental for PDAC. Here, we demonstrate that PDAC cells expressing oncogenic KRAS, owing to an increase in the HBP flux, become strongly reliant on HBP for both proliferation and survival. In particular, upon treatment with two different compounds, 2-deoxyglucose and FR054, inhibiting both HBP and protein N-glycosylation, these cells undergo apoptosis significantly more than PDAC cells expressing wild-type KRAS. Importantly, we also show that the combined treatment between FR054 and the pan-RAS inhibitor BI-2852 has an additive negative effect on cell proliferation and survival by means of the suppression of both Akt activity and cyclin D1 expression. Thus, co-inhibition of HBP and oncogenic RAS may represent a novel therapy for PDAC patients.

scholarly journals Cooperation between oncogenic Ras and wild-type p53 stimulates STAT non-cell autonomously to promote tumor radioresistance

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yong-Li Dong ◽  
Gangadhara P. Vadla ◽  
Jin-Yu (Jim) Lu ◽  
Vakil Ahmad ◽  
Thomas J. Klein ◽  
...  
Keyword(s):  
Radiation Therapy ◽  
Cellular Response ◽  
Cell Communication ◽  
Genotoxic Stress ◽  
Tumor Resistance ◽  
Wild Type ◽  
Therapy Outcomes ◽  
Oncogenic Ras ◽  
Tumor Tissues ◽  
Cell Cell

AbstractOncogenic RAS mutations are associated with tumor resistance to radiation therapy. Cell-cell interactions in the tumor microenvironment (TME) profoundly influence therapy outcomes. However, the nature of these interactions and their role in Ras tumor radioresistance remain unclear. Here we use Drosophila oncogenic Ras tissues and human Ras cancer cell radiation models to address these questions. We discover that cellular response to genotoxic stress cooperates with oncogenic Ras to activate JAK/STAT non-cell autonomously in the TME. Specifically, p53 is heterogeneously activated in Ras tumor tissues in response to irradiation. This mosaicism allows high p53-expressing Ras clones to stimulate JAK/STAT cytokines, which activate JAK/STAT in the nearby low p53-expressing surviving Ras clones, leading to robust tumor re-establishment. Blocking any part of this cell-cell communication loop re-sensitizes Ras tumor cells to irradiation. These findings suggest that coupling STAT inhibitors to radiotherapy might improve clinical outcomes for Ras cancer patients.

scholarly journals Selective and noncovalent targeting of RAS mutants for inhibition and degradation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kai Wen Teng ◽  
Steven T. Tsai ◽  
Takamitsu Hattori ◽  
Carmine Fedele ◽  
Akiko Koide ◽  
...  
Keyword(s):  
Active State ◽  
Wild Type ◽  
Erk Activation ◽  
Mouse Xenograft ◽  
Oncogenic Ras ◽  
Starting Point ◽  
Selective Targeting ◽  
Covalent Inhibitors ◽  
Xenograft Models

AbstractActivating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.

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