bims-pimaco 2021-10-31 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2021–10–31
nine papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Akt-mediated Ephexin1-Ras interaction promotes oncogenic Ras signaling and colorectal and lung cancer cell proliferation.
The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer.
Oncogenic KRAS G12D mutation promotes dimerization through a second, phosphatidylserine-dependent interface: a model for KRAS oligomerization.
KRAS Inhibitors- yes but what next? Direct targeting of KRAS- vaccines, adoptive T cell therapy and beyond.
Sin1-mediated mTOR signaling in cell growth, metabolism and immune response.
HER2 mediates clinical resistance to the KRASG12C inhibitor sotorasib, which is overcome by co-targeting SHP2.
Vacuolar localization via the N-terminal domain of Sch9 is required for TORC1-dependent phosphorylation and downstream signal transduction.
Unravelling the effect of the E545K mutation on PI3Kα kinase.
SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells.

Cell Death Dis. 2021 Oct 28. 12(11): 1013

Akt-mediated Ephexin1-Ras interaction promotes oncogenic Ras signaling and colorectal and lung cancer cell proliferation.

Jeeho Kim, Young Jin Jeon, Sung-Chul Lim, Joohyun Ryu, Jung-Hee Lee, In-Youb Chang, Ho Jin You.

ABSTRCT: Ephexin1 was reported to be highly upregulated by oncogenic Ras, but the functional consequences of this remain poorly understood. Here, we show that Ephexin1 is highly expressed in colorectal cancer (CRC) and lung cancer (LC) patient tissues. Knockdown of Ephexin1 markedly inhibited the cell growth of CRC and LC cells with oncogenic Ras mutations. Ephexin1 contributes to the positive regulation of Ras-mediated downstream target genes and promotes Ras-induced skin tumorigenesis. Mechanically, Akt phosphorylates Ephexin1 at Ser16 and Ser18 (pSer16/18) and pSer16/18 Ephexin1 then interacts with oncogenic K-Ras to promote downstream MAPK signaling, facilitating tumorigenesis. Furthermore, pSer16/18 Ephexin1 is associated with both an increased tumor grade and metastatic cases of CRC and LC, and those that highly express pSer16/18 exhibit poor overall survival rates. These data indicate that Ephexin1 plays a critical role in the Ras-mediated CRC and LC and pSer16/18 Ephexin1 might be an effective therapeutic target for CRC and LC.

DOI: https://doi.org/10.1038/s41419-021-04332-0
J Biol Chem. 2021 Oct 21. pii: S0021-9258(21)01141-8. [Epub ahead of print] 101335

The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer.

J Nathaniel Diehl, Jennifer E Klomp, Kayla R Snare, Priya S Hibshman, Devon R Blake, Zane D Kaiser, Thomas S K Gilbert, Elisa Baldelli, Mariaelena Pierobon, Björn Papke, Runying Yang, Richard G Hodge, Naim U Rashid, Emanuel F Petricoin, Laura E Herring, Lee M Graves, Adrienne D Cox, Channing J Der.

  Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines, and then applied Multiplexed Inhibitor Bead/Mass Spectrometry (MIB/MS) to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation, and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGFBR1, ILK), and pharmacologic inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacologic inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared to WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.

Keywords:  DDR1; RAS protein; WEE1; cancer biology; extracellular-signal-regulated kinase (ERK); kinome; mitogen-activated protein kinase (MAPK); pancreatic cancer; proteomics

DOI:  https://doi.org/10.1016/j.jbc.2021.101335
Chem Sci. 2021 Oct 06. 12(38): 12827-12837

Oncogenic KRAS G12D mutation promotes dimerization through a second, phosphatidylserine-dependent interface: a model for KRAS oligomerization.

Ki-Young Lee, Masahiro Enomoto, Teklab Gebregiworgis, Geneviève M C Gasmi-Seabrook, Mitsuhiko Ikura, Christopher B Marshall.

KRAS forms transient dimers and higher-order multimers (nanoclusters) on the plasma membrane, which drive MAPK signaling and cell proliferation. KRAS is a frequently mutated oncogene, and while it is well known that the most prevalent mutation, G12D, impairs GTP hydrolysis, thereby increasing KRAS activation, G12D has also been shown to enhance nanoclustering. Elucidating structures of dynamic KRAS assemblies on a membrane has been challenging, thus we have refined our NMR approach that uses nanodiscs to study KRAS associated with membranes. We incorporated paramagnetic relaxation enhancement (PRE) titrations and interface mutagenesis, which revealed that, in addition to the symmetric 'α-α' dimerization interface shared with wild-type KRAS, the G12D mutant also self-associates through an asymmetric 'α-β' interface. The 'α-β' association is dependent on the presence of phosphatidylserine lipids, consistent with previous reports that this lipid promotes KRAS self-assembly on the plasma membrane in cells. Experiments using engineered mutants to spoil each interface, together with PRE probes attached to the membrane or free in solvent, suggest that dimerization through the primary 'α-α' interface releases β interfaces from the membrane promoting formation of the secondary 'α-β' interaction, potentially initiating nanoclustering. In addition, the small molecule BI-2852 binds at a β-β interface, stabilizing a new dimer configuration that outcompetes native dimerization and blocks the effector-binding site. Our data indicate that KRAS self-association involves a delicately balanced conformational equilibrium between transient states, which is sensitive to disease-associated mutation and small molecule inhibitors. The methods developed here are applicable to biologically important transient interactions involving other membrane-associated proteins.

DOI: https://doi.org/10.1039/d1sc03484g
Cancer Treat Rev. 2021 Oct 21. pii: S0305-7372(21)00157-2. [Epub ahead of print]101 102309

KRAS Inhibitors- yes but what next? Direct targeting of KRAS- vaccines, adoptive T cell therapy and beyond.

Misako Nagasaka, Bindu Potugari, Alexis Nguyen, Ammar Sukari, Asfar S Azmi, Sai-Hong Ignatius Ou.

  Kirsten rat sarcoma viral oncogene homolog (KRAS) is a proto-oncogene of the RAS-MAPK pathway. KRAS mutations are present in a variety of malignancies including lung, colorectal, and pancreatic cancer. Until the recent approval of sotorasib, a KRAS G12C inhibitor, lack of targeted therapy for KRAS has resulted in poor prognosis of patients with tumors harboring KRAS mutations. While the conditional approval of sotorasib was a major breakthrough for those patients harboring KRAS G12C mutations, G12C only accounts for a fraction of those with KRAS mutations and eventual resistance to G12C inhibitors are unavoidable. This comprehensive review on KRAS inhibitors covers accumulating evidence on not only the G12C inhibitors but also other therapeutic attempts to tackle KRAS including combination therapy as well as direct inhibition with vaccines, adoptive T cell therapy, proteolysis-targeted chimeras (PROTACs) and CRISPR/Cas9.

Keywords:  Adagrasib; G12C; G12D; KRAS inhibitors; Kirsten rat sarcoma viral oncogene homolog; PROTAC; Sotorasib; Vaccine

DOI:  https://doi.org/10.1016/j.ctrv.2021.102309
Natl Sci Rev. 2019 Nov;6(6): 1149-1162

Sin1-mediated mTOR signaling in cell growth, metabolism and immune response.

Chun Ruan, Xinxing Ouyang, Hongzhi Liu, Song Li, Jingsi Jin, Weiyi Tang, Yu Xia, Bing Su.

  The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr protein kinase with essential cellular function via processing various extracellular and intracellular inputs. Two distinct multi-protein mTOR complexes (mTORC), mTORC1 and mTORC2, have been identified and well characterized in eukaryotic cells from yeast to human. Sin1, which stands for Sty1/Spc1-interacting protein1, also known as mitogen-activated protein kinase (MAPK) associated protein (MAPKAP)1, is an evolutionarily conserved adaptor protein. Mammalian Sin1 interacts with many cellular proteins, but it has been widely studied as an essential component of mTORC2, and it is crucial not only for the assembly of mTORC2 but also for the regulation of its substrate specificity. In this review, we summarize our current knowledge of the structure and functions of Sin1, focusing specifically on its protein interaction network and its roles in the mTOR pathway that could account for various cellular functions of mTOR in growth, metabolism, immunity and cancer.

Keywords:  AGC kinases; Akt; Sin1; mTOR complex; metabolism and immune response

DOI:  https://doi.org/10.1093/nsr/nwz171
Eur J Cancer. 2021 Oct 26. pii: S0959-8049(21)01146-1. [Epub ahead of print]159 16-23

HER2 mediates clinical resistance to the KRASG12C inhibitor sotorasib, which is overcome by co-targeting SHP2.

Cassandra S L Ho, Alicia I Tüns, Hans-Ulrich Schildhaus, Marcel Wiesweg, Barbara M Grüner, Balazs Hegedus, Martin Schuler, Alexander Schramm, Sebastian Oeck.

   INTRODUCTION: Mutant RAS guanosine triphosphate hydrolases (GTPases) are key oncogenic drivers in many cancers. The KRASG12C variant has recently become targetable by a new drug class specifically locking KRASG12C in its inactive guanosine diphosphate (GDP)-bound state. Clinical activity was demonstrated in patients with advanced lung cancers harbouring KRASG12C mutations but was limited by the development of resistance.
METHODS: A biopsy from progressing lung cancer of a patient treated with the KRASG12C inhibitor sotorasib was obtained, and the underlying resistance factors were analysed. Mechanistic studies were performed in vitro and in vivo to uncover strategies to overcome resistance to KRASG12C inhibition.
RESULTS: We demonstrated acquisition of HER2 copy number gain and KRASG12C mutation retention in the post-progression biopsy. To explore HER2 gain as the relevant resistance mechanism, we generated KRASG12C lung cancer models overexpressing HER2. MAPK pathway signalling remained active despite KRASG12C inhibitor treatment. Combined pharmacological inhibition of KRASG12C and SHP2 synergistically overcame HER2-mediated resistance in vitro and in vivo.
CONCLUSIONS: These findings establish HER2 copy number gain as a clinically relevant mechanism of resistance to pharmacological KRASG12C inhibition that can be overcome by co-targeting SHP2.

Keywords:  Acquired resistance; KRAS(G12C) inhibition; Lung cancer; SHP2 inhibition

DOI:  https://doi.org/10.1016/j.ejca.2021.10.003
J Mol Biol. 2021 Oct 22. pii: S0022-2836(21)00563-5. [Epub ahead of print] 167326

Vacuolar localization via the N-terminal domain of Sch9 is required for TORC1-dependent phosphorylation and downstream signal transduction.

Daniele Novarina, Paolo Guerra, Andreas Milias-Argeitis.

  The budding yeast Sch9 kinase (functional orthologue of the mammalian S6 kinase) is a major effector of the Target of Rapamycin Complex 1 (TORC1) complex in the regulation of cell growth in response to nutrient availability and stress. Sch9 is partially localized at the vacuolar surface, where it is phosphorylated by TORC1. The recruitment of Sch9 on the vacuole is mediated by direct interaction between phospholipids of the vacuolar membrane and the region of Sch9 encompassing amino acid residues 1-390, which contains a C2 domain. Since many C2 domains mediate phospholipid binding, it had been suggested that the C2 domain of Sch9 mediates its vacuolar recruitment. However, the in vivo requirement of the C2 domain for Sch9 localization had not been demonstrated, and the phenotypic consequences of Sch9 delocalization remained unknown. Here, by examining cellular localization, phosphorylation state and growth phenotypes of Sch9 truncation mutants, we show that deletion of the N-terminal domain of Sch9 (aa 1-182), but not the C2 domain (aa 183-399), impairs vacuolar localization and TORC1-dependent phosphorylation of Sch9, while causing growth defects similar to those observed in sch9Δ cells. These defects can be reversed either via artificial tethering of the protein to the vacuole, or by introducing phosphomimetic mutations at the TORC1 target sites, suggesting that Sch9 localization on the vacuole is needed for the TORC1-dependent activation of the kinase. Our study uncovers a key role for the N-terminal domain of Sch9 and provides new mechanistic insight into the regulation of a major TORC1 signaling branch.

Keywords:  C2 domain; Cell growth; Saccharomyces cerevisiae; TOR signaling; kinase

DOI:  https://doi.org/10.1016/j.jmb.2021.167326
Chem Sci. 2020 Apr 07. 11(13): 3511-3515

Unravelling the effect of the E545K mutation on PI3Kα kinase.

Ioannis Galdadas, Francesco Luigi Gervasio, Zoe Cournia.

PI3Kα controls several cellular processes and its aberrant signalling is implicated in tumorigenesis. One of its hotspot mutations, E545K, increases PI3Kα lipid kinase activity, but its mode of action is only partially understood. Here, we perform biased and unbiased molecular dynamics simulations of PI3Kα and uncover, for the first time, the free energy landscape of the E545K PI3Kα mutant. We reveal the mechanism by which E545K leads to PI3Kα activation in atomic-level detail, which is considerably more complex than previously thought.

DOI: https://doi.org/10.1039/c9sc05903b
Nature. 2021 Oct 27.

SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells.

Ying Wang, Frederick S Yen, Xiphias Ge Zhu, Rebecca C Timson, Ross Weber, Changrui Xing, Yuyang Liu, Benjamin Allwein, Hanzhi Luo, Hsi-Wen Yeh, Søren Heissel, Gokhan Unlu, Eric R Gamazon, Michael G Kharas, Richard Hite, Kıvanç Birsoy.

Glutathione (GSH) is a small-molecule thiol that is abundant in all eukaryotes and has key roles in oxidative metabolism1. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions2. GSH is synthesized exclusively in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remains unknown. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, as a regulator of GSH transport into mitochondria. Loss of SLC25A39 reduces mitochondrial GSH import and abundance without affecting cellular GSH levels. Cells lacking both SLC25A39 and its paralogue SLC25A40 exhibit defects in the activity and stability of proteins containing iron-sulfur clusters. We find that mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enables mitochondrial GSH production and ameliorates the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH-import machinery.

DOI: https://doi.org/10.1038/s41586-021-04025-w