Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute up to 30% of human solid tumours including lung adenocarcinoma, pancreatic and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. Here, APEX-2 proximity labelling was used to profile the molecular environment of wild type and G12D, G13D and Q61H activating mutants of KRAS under both, starvation and stimulation conditions. We demonstrate by quantitative proteomics the presence of known interactors of KRAS including a-RAF and LZTR1, which varied in abundance with wildtype and KRAS mutants. Notably, the KRAS mutations G12D, G13D and Q61H abrogate association with LZTR1. Wildtype KRAS and LZTR1, as part of the CUL3 ubiquitin E3 ligase complex, affect each other's protein stability, revealing a direct feedback loop mechanism. KRAS mutations disconnect this regulatory circuit, thereby contributing to oncogenesis.

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Prevalence of low-penetrance KRAS (codons 12/13 and 61) and BRAF mutations in metastatic colorectal carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e14147 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e14147-e14147

Author(s):

Federico Rojo ◽

Trinidad Caldes ◽

Sandra Zazo ◽

Miguel de la Hoya ◽

Cristina Carames ◽

...

Keyword(s):

Wild Type ◽

Braf Mutations ◽

Kras Mutations ◽

Activating Mutations ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Intracellular Signal ◽

Polymerase Chain ◽

Current Guidelines ◽

Formalin Fixed

e14147 Background: In patients with metastatic colorectal cancer (mCRC), activating mutations within KRAS, which result in EGFR-independent intracellular signal transduction activation, are found in approximately 35-40% of patients with mCRC have been significantly associated with lack of response to cetuximab or panitumumab therapy. Although current guidelines recommend testing for frequent KRAS codons 12/13 mutations, emerging data indicate that additional KRAS and BRAF mutations are also predictive of non-responsiveness to anti-EGFR antibodies in mCRC. This study is aimed to analyze the prevalence of low-penetrance KRAS and BRAF V600 mutations in caucasian mCRC population. Methods: A two-institution retrospective cohort of 1,238 consecutive KRAS wild type mCRC patients previously studied for 7 mutations in codons 12/13 (G12D, G12A, G12V, G12S, G12R, G12C and G13D) by the CE-IVD marked ARMS-scorpion real-time polymerase chain reaction PCR (Therascreen, Qiagen) was assayed by the diagnostic TaqMelt PCR assay cobas KRAS mutation and cobas BRAF V600 mutation tests (Roche), which are designed to detect 19 mutations in KRAS codons 12, 13 and 61 (including G12F, G13C, G13R, G13S, G13A, G13V, G13I, Q61H, Q61K, Q61R, Q61L, Q61E and Q61P) and BRAF V600 (V600E, V600K and V600D) mutations. An additional cohort of 146 KRAS mutated patients by ARMS-scorpion PCR was studied. DNA was obtained by cobas DNA preparation kit from one single 5µm formalin-fixed paraffin-embedded tissue section. Results: In all samples, sufficient DNA was obtained for KRAS and BRAF mutational studies. Among 1238 KRAS codons 12/13 wild-type patients by ARMS-scorpion PCR,166 (13.4%) showed KRAS mutations, 117 (9.5%) in codons 12/13, and 49 (4%) in codon 61. BRAF V600 mutations were detected in 9% cases. In ARMS-scorpion PCR KRAS mutated patients, mutations were confirmed by cobas in all cases. Conclusions: The cobas mutation tests are robust and reproducible assays that, 1) detects a higher incidence (13.4%) of mutations in codons 12, 13, and 61 of the KRAS gene in wild-type mCRC population, 2) a relevant rate of BRAF mutations is present in the same population, and 3) requires a very small amount of tissue.

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Randomized phase II study of erlotinib (E) or intercalated E with carboplatin/paclitaxel (CP) in chemotherapy-naive advanced NSCLC: Correlation of biomarker status and clinical benefit

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.8026 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 8026-8026

Author(s):

F. R. Hirsch ◽

R. Dziadziuszko ◽

L. Varella-Garcia ◽

W. Franklin ◽

P. Bunn ◽

...

Keyword(s):

Phase Ii ◽

Kras Mutation ◽

Phase Ii Study ◽

Egfr Mutations ◽

Gene Copy ◽

Wild Type ◽

Kras Mutations ◽

Activating Mutations ◽

Randomized Phase Ii ◽

Evaluable Population

8026 Background: E plus chemotherapy showed no additive effects in NSCLC but preclinical studies suggested that intercalation of E and chemotherapy could give synergy. Clinical studies suggested that EGFR mutations could aid in pt selection. KRAS mutation status of tumors was also evaluated. We conducted a randomized phase II study of E and E intercalated with CP in pts with chemonaive NSCLC. Methods: Stage IIIB/IV EGFR+ NSCLC pts were randomized to E 150 mg/d or CP d1 plus E days 2–15 q21 days (ECP). After 4 cycles, E continued until PD. Tumor tissue was evaluated by IHC (EGFR), FISH (EGFR gene copy number), and PCR amplification followed by DNA sequencing (EGFR and KRAS mutations). Results: Among 143 pts randomized 53% EGFR FISH+; 13% activating EGFR mutations and 8% non-activating EGFR mutations (evaluable samples=114); and 22% KRAS mutations (evaluable samples=130). No pt had both EGFR and KRAS mutations. EGFR-activating mutations were higher among females (19% vs 6% males), adenocarcinoma histology (17% vs 0% others), Asians (45% vs 7% non-Asian), and never smokers (29% vs 7% former and 0% current); KRAS mutations were higher in current smokers (41% vs 27% former and 0% never) and adenocarcinoma histology (22% vs 18% squamous). In the E arm, 6-mo PFS probability for the efficacy evaluable population (n=69) was significantly higher in pts with EGFR activating mutations vs no mutations (89% vs 25%, HR=0.17, P=0.001), numerically higher in pts with EGFR FISH+ vs FISH- (40% vs 22%, HR=0.61, P=0.07), and with KRAS wild type vs mutation+ (38% vs 12%, HR=0.56, P=0.10). Response rates, PFS and OS by type of EGFR/KRAS mutation will be presented. In the ECP arm, 6-mo PFS probability for the efficacy evaluable population (n=68) was numerically higher in pts with EGFR activating mutations (42% vs 29%, HR=0.72, P=0.53), numerically higher in pts with wild type KRAS (32% vs 9%, HR=0.57, P=0.08), and numerically lower in pts with EGFR FISH+ vs FISH- (23% vs 30%, HR=0.93, P=0.78). Conclusions: Activating EGFR mutations correlate with increased 6 mo PFS probability in 1st line therapy with E. EGFR FISH + and absence of KRAS mutation trend towards increased 6 mo PFS rate with E. [Table: see text]

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The Q61H mutation decouples KRAS from upstream regulation and renders cancer cells resistant to SHP2 inhibitors

Nature Communications ◽

10.1038/s41467-021-26526-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Teklab Gebregiworgis ◽

Yoshihito Kano ◽

Jonathan St-Germain ◽

Nikolina Radulovich ◽

Molly L. Udaskin ◽

...

Keyword(s):

Molecular Dynamics ◽

Cancer Cells ◽

Wild Type ◽

Kras Mutations ◽

Gtp Hydrolysis ◽

High Affinity ◽

Erk Phosphorylation ◽

Variable Sensitivity ◽

Underlying Mechanisms ◽

Tyrosyl Phosphorylation

AbstractCancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.

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RAS Pathway Mutations Are Associated with Proliferative Features and Frequently Co-Occur with TET2 mutationsin Philadelphia Negative MPN Subtypes

Blood ◽

10.1182/blood.v128.22.4269.4269 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4269-4269 ◽

Cited By ~ 1

Author(s):

Bartlomiej M Getta ◽

Emily C Zabor ◽

Sean Devlin ◽

Kristina Marie Knapp ◽

Minal Patel ◽

...

Keyword(s):

Clinical Features ◽

Somatic Mutations ◽

Jak2 V617f ◽

Wild Type ◽

Kras Mutations ◽

Ras Mutations ◽

Activating Mutations ◽

Disease Biology ◽

Proliferative Disease ◽

Stat Pathway

Abstract Introduction: The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders, which include polycythemia vera, essential thrombocythemia and myelofibrosis. These disorders are characterized by activation of the JAK-STAT pathway via somatic mutations in JAK2, MPL, and CALR. However, a number of recurrent somatic mutations outside the JAK-STAT pathway have been described. Many of these mutations, such as ASXL1 and EZH2, are know to confer risk for disease progression. Other mutations, such as TET2 mutations, appear to alter the biology of disease in preclinical and clinical studies. Thus, further investigation of co-occurring mutation events is important to further the understanding of disease biology. Recurrent activating mutations in the RAS signaling pathway have been described in patients with MPNs. We have sought to determine the clinical impact of RAS mutations in patients with MPN, and to assess the pattern of co-mutational events in patients with RAS mutations. Methods : A targeted 28-gene, amplicon based next-generation sequencing assay was used to sequenced bone marrow aspirate or peripheral blood samples from 211 pts with Philadelphia negative MPN including: ET=61, PV=35, PMF=54, Post ET MF=16, Post PV MF=17, Post MPN AML=20, MPN unclassified=5 and systemic mastocytosis=3. Pts with MDS/MPN overlap syndromes were excluded. NRAS and KRAS mutations were grouped together. FisherÕs exact test and Wilcoxon rank-sum test were used to compare categorical and continuous variables, respectively. Results: N/KRAS mutations occurred at a frequency of 6 % in this cohort (Fig 1a). The median variant allele frequency (VAF) of RAS mutations was 15.6%. This was lower than the VAF of JAK2 V617F (44.6%) (p<0.001), TET2 (43.0%) (p=0.011) and ASXL1 (29.5%) (p=0.029) suggesting that RAS mutations are sub-clonal relative to other MPN associated mutations. By contrast, mutant RAS VAF did not significantly differ from VAFs of genes frequently enriched at the time of leukemic transformation, such as IDH (33.7%) (p=0.079) and TP53 (18.2%) (p=0.582) (Fig 1b). The presence of concurrent JAK-STAT activating mutations with RAS mutations was associated with distinct clinical features. RAS mutations occurred more frequently in patients with fibrotic MPN and post MPN AML and were less frequent in patients with ET and PV (table 1). RAS mutant patients had clinical features of more proliferative disease with higher total WBC, absolute monocytes, percentage circulating blasts, higher frequency of splenomegaly, and were associated with higher DIPSS score in pts with myelofibrosis (Table 1). RAS mutations were associated with mutations in TET2 with 4/32 (13%) with mut-TET2 vs 6/152 (4%) wild type TET2 having RAS mutations, though this result did not reach statistical significance (p=0.074) (Fig 1d). No other gene mutation was positively associated with mutations in RAS. 3/6 pts with TET2/RAS co-mutation had post MPN AML, while the remaining 3 had PMF or post ET MF (Fig 1c). The VAF of mut-RAS was always lower than mut-TET2 in patients where these were co-mutated, a pattern not seen for RAS VAF relative to JAK2 V617F VAF. This suggests that RAS mutations were acquired after TET2 in all cases assessed. Patients with RAS/TET2 co-mutation had the highest incidence of post-MPN AML. In those with wild type TET2 and mutant RAS proliferative disease features were noted including high WBC, monocyte count and splenomegaly. Finally, patients with myelofibrosis and RAS mutations were significantly more likely to have high DIPSS scores. Conclusions: RAS mutations occur in 6% of pts with Philadelphia negative MPN. Patients with co-occurring JAK-STAT pathway activating mutations and RAS mutations had more proliferative disease and a higher incidence of post MPN AML. RAS mutations were more frequent in patients with mutant TET2 and in this context they were associated with post MPN AML. The RAS VAF was always lower relative to TET2 and other MPN driver mutations suggesting they are acquired later in disease evolution. These data suggest the presence of RAS mutations may alter the disease biology of MPNs, and raise the question of the possible clinical efficacy of utilizing therapeutic agents, such as MEK inhibitors, in MPN patients with RAS mutations. Further preclinical and clinical evaluation of the role of this pathway in MPN pathobiology is warranted. Disclosures Levine: Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

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The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

Nature Communications ◽

10.1038/s41467-021-22861-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sabrina Dietz ◽

Miguel Vasconcelos Almeida ◽

Emily Nischwitz ◽

Jan Schreier ◽

Nikenza Viceconte ◽

...

Keyword(s):

Quantitative Proteomics ◽

Wild Type ◽

C Elegans ◽

Double Stranded Dna ◽

Telomere Sequence ◽

Proteomics Approach ◽

Telomeric Protein ◽

Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

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The effects of lestaurtinib (CEP701) and PKC412 on primary AML blasts: the induction of cytotoxicity varies with dependence on FLT3 signaling in both FLT3-mutated and wild-type cases

Blood ◽

10.1182/blood-2006-04-015487 ◽

2006 ◽

Vol 108 (10) ◽

pp. 3494-3503 ◽

Cited By ~ 88

Author(s):

Steven Knapper ◽

Kenneth I. Mills ◽

Amanda F. Gilkes ◽

Steve J. Austin ◽

Val Walsh ◽

...

Keyword(s):

Myeloid Leukemia ◽

Wild Type ◽

Mutation Status ◽

Activating Mutations ◽

Drug Concentrations ◽

Flt3 Inhibitors ◽

Show Evidence ◽

Flt3 Expression ◽

Molecular Therapeutic ◽

Cytotoxic Responses

Abstract The receptor tyrosine kinase FLT3 is a promising molecular therapeutic target in acute myeloid leukemia (AML). Activating mutations of FLT3 are present in approximately one-third of patients, while many nonmutants show evidence of FLT3 activation, which appears to play a significant role in leukemogenesis. We studied the effects of lestaurtinib (CEP701) and PKC412, 2 small molecule inhibitors of FLT3, on 65 diagnostic AML blast samples. Both agents induced concentration-dependent cytotoxicity in most cases, although responses to PKC412 required higher drug concentrations. Cytotoxic responses were highly heterogeneous and were only weakly associated with FLT3 mutation status and FLT3 expression. Importantly, lestaurtinib induced cytotoxicity in a synergistic fashion with cytarabine, particularly in FLT3 mutant samples. Both lestaurtinib and PKC412 caused inhibition of FLT3 phosphorylation in all samples. Translation of FLT3 inhibition into cytotoxicity was influenced by the degree of residual FLT3 phosphorylation remaining and correlated with deactivation of STAT5 and MAP kinase. FLT3 mutant and wild-type cases both varied considerably in their dependence on FLT3 signaling for survival. These findings support the continued clinical assessment of FLT3 inhibitors in combination with cytotoxic chemotherapy: Entry to future clinical trials should include FLT3 wild-type patients and should remain unrestricted by FLT3 expression level.

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RASAL3 preferentially stimulates GTP hydrolysis of the Rho family small GTPase Rac2

Biomedical Reports ◽

10.3892/br.2018.1119 ◽

2018 ◽

Author(s):

Yoonjae Shin ◽

Yong Kim ◽

Hyemin Kim ◽

Nakyoung Shin ◽

Tae Kim ◽

...

Keyword(s):

Small Gtpase ◽

Gtp Hydrolysis ◽

Rho Family ◽

Hydrolysis Of

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N-Acetylcysteine Reduces the Pro-Oxidant and Inflammatory Responses during Pancreatitis and Pancreas Tumorigenesis

Antioxidants ◽

10.3390/antiox10071107 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1107

Author(s):

Marie-Albane Minati ◽

Maxime Libert ◽

Hajar Dahou ◽

Patrick Jacquemin ◽

Mohamad Assi

Keyword(s):

Nuclear Translocation ◽

Inflammatory Responses ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Glutathione Metabolism ◽

Protective Effects ◽

Wild Type ◽

Kras Mutations ◽

Oxidative Damages ◽

Factor Α

Pancreatitis, an inflammation of the pancreas, appears to be a main driver of pancreatic cancer when combined with Kras mutations. In this context, the exact redox mechanisms are not clearly elucidated. Herein, we treated mice expressing a KrasG12D mutation in pancreatic acinar cells with cerulein to induce acute pancreatitis. In the presence of KrasG12D, pancreatitis triggered significantly greater redox unbalance and oxidative damages compared to control mice expressing wild-type Kras alleles. Further analyses identified the disruption in glutathione metabolism as the main redox event occurring during pancreatitis. Compared to the wild-type background, KrasG12D-bearing mice showed a greater responsiveness to treatment with a thiol-containing compound, N-acetylcysteine (NAC). Notably, NAC treatment increased the pancreatic glutathione pool, reduced systemic markers related to pancreatic and liver damages, limited the extent of pancreatic edema and fibrosis as well as reduced systemic and pancreatic oxidative damages. The protective effects of NAC were, at least, partly due to a decrease in the production of tumor necrosis factor-α (TNF-α) by acinar cells, which was concomitant with the inhibition of NF-κB(p65) nuclear translocation. Our data provide a rationale to use thiol-containing compounds as an adjuvant therapy to alleviate the severity of inflammation during pancreatitis and pancreatic tumorigenesis.

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MDM2 Suppresses p73 Function without Promoting p73 Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.19.5.3257 ◽

1999 ◽

Vol 19 (5) ◽

pp. 3257-3266 ◽

Cited By ~ 211

Author(s):

Xiaoya Zeng ◽

Lihong Chen ◽

Christine A. Jost ◽

Ruth Maya ◽

David Keller ◽

...

Keyword(s):

Transcriptional Activation ◽

Feedback Loop ◽

Human Lung ◽

Activation Function ◽

Wild Type ◽

Mdm2 Protein ◽

Induced Apoptosis ◽

P53 Inactivation

ABSTRACT The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73α and p73β stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.

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Loss of MCU prevents mitochondrial fusion in G1-S phase and blocks cell cycle progression and proliferation

Science Signaling ◽

10.1126/scisignal.aav1439 ◽

2019 ◽

Vol 12 (579) ◽

pp. eaav1439 ◽

Cited By ~ 19

Author(s):

Olha M. Koval ◽

Emily K. Nguyen ◽

Velarchana Santhana ◽

Trevor P. Fidler ◽

Sara C. Sebag ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Cycle Progression ◽

Mitochondrial Fission ◽

Mitochondrial Fusion ◽

Cycle Phase ◽

Wild Type ◽

Mitochondrial Fragmentation ◽

Cycle Progression ◽

Regulatory Circuit

The role of the mitochondrial Ca2+uniporter (MCU) in physiologic cell proliferation remains to be defined. Here, we demonstrated that the MCU was required to match mitochondrial function to metabolic demands during the cell cycle. During the G1-S transition (the cycle phase with the highest mitochondrial ATP output), mitochondrial fusion, oxygen consumption, and Ca2+uptake increased in wild-type cells but not in cells lacking MCU. In proliferating wild-type control cells, the addition of the growth factors promoted the activation of the Ca2+/calmodulin-dependent kinase II (CaMKII) and the phosphorylation of the mitochondrial fission factor Drp1 at Ser616. The lack of the MCU was associated with baseline activation of CaMKII, mitochondrial fragmentation due to increased Drp1 phosphorylation, and impaired mitochondrial respiration and glycolysis. The mitochondrial fission/fusion ratio and proliferation in MCU-deficient cells recovered after MCU restoration or inhibition of mitochondrial fragmentation or of CaMKII in the cytosol. Our data highlight a key function for the MCU in mitochondrial adaptation to the metabolic demands during cell cycle progression. Cytosolic CaMKII and the MCU participate in a regulatory circuit, whereby mitochondrial Ca2+uptake affects cell proliferation through Drp1.

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