bims-pimaco 2022-07-03 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–07–03
eight papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Prognostic and Predictive Value of PIK3CA Mutations in Metastatic Colorectal Cancer.
Exploring CRD mobility during RAS/RAF engagement at the membrane.
The mTORC1-SLC4A7 axis stimulates bicarbonate import to enhance de novo nucleotide synthesis.
Structural basis for SHOC2 modulation of RAS signalling.
Conformational Dynamics Allows Sampling of an "Active-like" State by Oncogenic K-Ras-GDP.
Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation.
Mutational landscape of pan-cancer patients with PIK3CA alterations in Chinese population.
Molecular mechanism of S-adenosylmethionine sensing by SAMTOR in mTORC1 signaling.

Target Oncol. 2022 Jun 29.

Prognostic and Predictive Value of PIK3CA Mutations in Metastatic Colorectal Cancer.

Elaine S Tan, Wenyi Fan, Todd C Knepper, Michael J Schell, Ibrahim H Sahin, Jason B Fleming, Hao Xie.

BACKGROUND: Comprehensive genomic profiling is used to guide the management of metastatic colorectal cancer (mCRC); however, the role of PIK3CA mutations, present in up to 20% of mCRCs, is unclear.
OBJECTIVE: This study aimed to evaluate the association of PIK3CA mutations with other common mutations in mCRC and determine the prognostic and predictive value of PIK3CA mutations.
PATIENTS AND METHODS: A retrospective chart review was performed on patients in the Moffitt Clinical Genomic Database with mCRC. A meta-analysis was performed to further evaluate the predictive value of PIK3CA mutations to the response to anti-epidermal growth factor receptor (EGFR) therapy.
RESULTS: Among 639 patients, PIK3CA was positively correlated with KRAS mutation (r = 0.11, p = 0.006) and negatively correlated with TP53 mutation (r = - 0.18, p ≤ 0.001) and ERBB2 amplification (r = - 0.08, p = 0.046). The median overall survival (OS) of patients with PIK3CA-mutant mCRC (n = 49) was 35.5 (95% confidence interval [CI] 18.7-48.1) months vs. 55.3 (95% CI 47.5-65.6) months for PIK3CA wild-type mCRC (n = 286) [p = 0.003]. This OS difference remained significant with exon 9 and exon 20 subset analyses. There was no significant difference in response rate between patients with PIK3CA wild-type (n = 97) versus mutant (n = 9) mCRC who received anti-EGFR therapy (43% vs. 56%, p = 0.61) and no significant difference in median progression-free survival (PFS) of 10.3 versus 7.2 months (p = 0.60). However, our meta-analysis of 12 studies, including ours, using a common effect model identified that PIK3CA mutations are associated with reduced response to anti-EGFR therapy, with a relative risk of 0.56 (95% CI 0.38-0.82).
CONCLUSION: Our study identified PIK3CA mutations as a poor prognostic factor, and our meta-analysis identified PIK3CA mutations as predictive of decreased response to anti-EGFR therapy in patients with mCRC.

DOI: https://doi.org/10.1007/s11523-022-00898-7
Biophys J. 2022 Jul 01. pii: S0006-3495(22)00545-8. [Epub ahead of print]

Exploring CRD mobility during RAS/RAF engagement at the membrane.

Kien Nguyen, Cesar A López, Chris Neale, Que N Van, Timothy S Carpenter, Francesco Di Natale, Timothy Travers, Timothy H Tran, Albert H Chan, Harsh Bhatia, Peter H Frank, Marco Tonelli, Xiaohua Zhang, Gulcin Gulten, Tyler Reddy, Violetta Burns, Tomas Oppelstrup, Nick Hengartner, Dhirendra K Simanshu, Peer-Timo Bremer, De Chen, James N Glosli, Rebika Shrestha, Thomas Turbyville, Frederick H Streitz, Dwight V Nissley, Helgi I Ingólfsson, Andrew G Stephen, Felice C Lightstone, S Gnanakaran.

During the activation of MAPK signaling, the RBD and CRD domains of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane-orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3-5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane which correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.

DOI: https://doi.org/10.1016/j.bpj.2022.06.035
Mol Cell. 2022 Jun 24. pii: S1097-2765(22)00544-5. [Epub ahead of print]

The mTORC1-SLC4A7 axis stimulates bicarbonate import to enhance de novo nucleotide synthesis.

Eunus S Ali, Anna Lipońska, Brendan P O'Hara, David R Amici, Michael D Torno, Peng Gao, John M Asara, Mee-Ngan F Yap, Marc L Mendillo, Issam Ben-Sahra.

  Bicarbonate (HCO3-) ions maintain pH homeostasis in eukaryotic cells and serve as a carbonyl donor to support cellular metabolism. However, whether the abundance of HCO3- is regulated or harnessed to promote cell growth is unknown. The mechanistic target of rapamycin complex 1 (mTORC1) adjusts cellular metabolism to support biomass production and cell growth. We find that mTORC1 stimulates the intracellular transport of HCO3- to promote nucleotide synthesis through the selective translational regulation of the sodium bicarbonate cotransporter SLC4A7. Downstream of mTORC1, SLC4A7 mRNA translation required the S6K-dependent phosphorylation of the translation factor eIF4B. In mTORC1-driven cells, loss of SLC4A7 resulted in reduced cell and tumor growth and decreased flux through de novo purine and pyrimidine synthesis in human cells and tumors without altering the intracellular pH. Thus, mTORC1 signaling, through the control of SLC4A7 expression, harnesses environmental bicarbonate to promote anabolic metabolism, cell biomass, and growth.

Keywords:  SLC4A7/NBCn1; bicarbonate metabolism; mTOR signaling; purine metabolism; pyrimidine metabolism

DOI:  https://doi.org/10.1016/j.molcel.2022.06.008
Nature. 2022 Jun 29.

Structural basis for SHOC2 modulation of RAS signalling.

Nicholas P D Liau, Matthew C Johnson, Saeed Izadi, Luca Gerosa, Michal Hammel, John M Bruning, Timothy J Wendorff, Wilson Phung, Sarah G Hymowitz, Jawahar Sudhamsu.

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers1-3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5-8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11-13, but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

DOI: https://doi.org/10.1038/s41586-022-04838-3
J Mol Biol. 2022 Jun 22. pii: S0022-2836(22)00287-X. [Epub ahead of print] 167695

Conformational Dynamics Allows Sampling of an "Active-like" State by Oncogenic K-Ras-GDP.

Patrick Grudzien, Hyunbum Jang, Nicholas Leschinsky, Ruth Nussinov, Vadim Gaponenko.

  Mutations in K-Ras GTPase replacing Gly12 with either Asp or Val are common in cancer. These mutations decelerate intrinsic and catalyzed GTP hydrolysis, leading to accumulation of K-Ras-GTP in cells. Signaling cascades initiated by K-Ras-GTP promote cell proliferation, survival, and invasion. Despite functional differences between the most frequent G12D mutation and the most aggressive and chemotherapy resistant G12V mutation, their long-suspected distinct structural features remain elusive. Using NMR, X-ray structures, and computational methods, we found that oncogenic mutants of K-Ras4B, the predominant splice variant of K-Ras, exhibit distinct conformational dynamics when GDP-bound, visiting the "active-like" conformational state similar to the one observed in GTP-bound K-Ras. This behavior distinguishes G12V from wild type and G12D K-Ras4B-GDP. The likely reason is hydrophobic interactions between the aliphatic sidechain of V12 and the Switch II region of K-Ras4BG12V-GDP, which are distinct in K-Ras4BG12D-GDP. In the X-ray structures, crystal contacts reduce the dynamics of the sidechain at position 12 by stabilizing the Switch I region of the protein. This explains why structural differences between G12V and G12D K-Ras have yet not been reported. Together, our results suggest a previously unknown mechanism of K-Ras activation. This mechanism relies on conformational dynamics caused by specific oncogenic mutations in the GDP-bound state. Our findings also imply that the therapeutic strategies decreasing the level of K-Ras-GTP by interfering with nucleotide exchange or by expediting GTP hydrolysis may work differently in different oncogenic mutants.

Keywords:  Chemical shift; Hydrolysis; NMR; Oncogenic mutations; Ras; molecular dynamics

DOI:  https://doi.org/10.1016/j.jmb.2022.167695
Elife. 2022 06 27. pii: e71929. [Epub ahead of print]11

Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation.

Haoran Zhu, Keefe T Chan, Xinran Huang, Carmelo Cerra, Shaun Blake, Anna S Trigos, Dovile Anderson, Darren J Creek, David P De Souza, Xi Wang, Caiyun Fu, Metta Jana, Elaine Sanij, Richard B Pearson, Jian Kang.

  Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-β-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (H2S) and glutathione (GSH) production, consequently protecting senescent cells from oxidative stress-induced cell death. CBS depletion allows AIS cells to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production by reducing mitochondrial localized CBS while retaining antioxidant capacity of transsulfuration pathway. These findings implicate a potential tumor-suppressive role for CBS in cells with aberrant PI3K/AKT pathway activation. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

Keywords:  PI3K/AKT signaling; cancer biology; cell biology; cystathionine-β-synthase; gastric cancer; glutathione; human; mouse; oxidative stress; senescence

DOI:  https://doi.org/10.7554/eLife.71929
BMC Med Genomics. 2022 Jul 01. 15(1): 146

Mutational landscape of pan-cancer patients with PIK3CA alterations in Chinese population.

Qingfeng Huang, Yang Zhou, Bowen Wang, Yi Zhao, Fengxia Zhang, Bowen Ding.

   PURPOSE: To analyze the mutational landscape of pan-cancer patients with PIK3CA mutations in Chinese population in real-world.
METHODS: We analyzed PIK3CA mutation status in sequencing data of cell-free DNA from plasma and genomic DNA from matched peripheral blood lymphocyte in 11,904 Chinese pan-cancer patients, and compared them with genomic data from the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Besides, concomitant genomic aberrations in PIK3CA-mutated samples were detected to investigate cancer driver genes, as well as their enriched pathways. Meanwhile, the mutations of Alpelisib targeting genes were screened and their co-alterations were analyzed according to OncoKB definition to identify the potential actionable ones.
RESULTS: The proportion of patients with PIK3CA mutations varied among 21 types of cancer, with the top being BRCA, CESC, SCL, and UCEC. The most common PIK3CA mutation hotspots were found to be E545K, E542K and H1047R. The Chinese cohort had significantly lower frequencies of PIK3CA mutations in breast and stomach cancers, but markedly higher PIK3CA mutation frequencies in large intestine, kidney and lung cancers than the COSMIC cohort. Compared with COSMIC cohort, the mutation frequencies of Alpelisib-targeted genes in breast cancer were significantly reduced in the Chinese cohort. All PIK3CA-mutated patients had concomitant genomic aberrations. While the most common concomitant genomic alterations occurred in TP53, EGFR and FAT1, these co-mutated genes were mainly enriched in RTK/RAS pathway, PI3K pathway and P53 pathway. Moreover, 83.6% of patients carrying mutations in Alpelisib-targeted genes had at least one actionable concomitant alteration. Level 1 actionable alteration was identified in LUAD, BRCA, COAD, LUSC, READ, and STAD.
CONCLUSION: Compared with the Western cohort, the mutation frequency of PIK3CA in breast cancer was reduced in the Chinese cohort. RTK/RAS pathway, PI3K pathway and P53 pathway were identified as the most common co-mutation pathways, suggesting that they may potentially serve as targets for possible Alpelisib-based combination therapy.

Keywords:  Alpelisib; Chinese population; Concomitant genomic aberrations; PIK3CA mutation

DOI:  https://doi.org/10.1186/s12920-022-01297-7
Sci Adv. 2022 Jul;8(26): eabn3868

Molecular mechanism of S-adenosylmethionine sensing by SAMTOR in mTORC1 signaling.

Xin Tang, Yifan Zhang, Guanchao Wang, Chunxiao Zhang, Fang Wang, Jiawen Shi, Tianlong Zhang, Jianping Ding.

The mechanistic target of rapamycin-mLST8-raptor complex (mTORC1) functions as a central regulator of cell growth and metabolism in response to changes in nutrient signals such as amino acids. SAMTOR is an S-adenosylmethionine (SAM) sensor, which regulates the mTORC1 activity through its interaction with the GTPase-activating protein activity toward Rags-1 (GATOR1)-KPTN, ITFG2, C12orf66 and SZT2-containing regulator (KICSTOR) complex. In this work, we report the crystal structures of Drosophila melanogaster SAMTOR in apo form and in complex with SAM. SAMTOR comprises an N-terminal helical domain and a C-terminal SAM-dependent methyltransferase (MTase) domain. The MTase domain contains the SAM-binding site and the potential GATOR1-KICSTOR-binding site. The helical domain functions as a molecular switch, which undergoes conformational change upon SAM binding and thereby modulates the interaction of SAMTOR with GATOR1-KICSTOR. The functional roles of the key residues and the helical domain are validated by functional assays. Our structural and functional data together reveal the molecular mechanism of the SAM sensing of SAMTOR and its functional role in mTORC1 signaling.

DOI: https://doi.org/10.1126/sciadv.abn3868