bims-pimaco 2021-08-01 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2021–08–01
eleven papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

A Covalent Calmodulin Inhibitor as a Tool to Study Cellular Mechanisms of K-Ras-Driven Stemness.
Stopping the beating heart of cancer: KRAS reviewed.
Leucyl-tRNA synthetase 1 is required for proliferation of TSC-null cells.
Targeting mTOR signaling overcomes acquired resistance to combined BRAF and MEK inhibition in BRAF-mutant melanoma.
The dynamic nature of the K-Ras/calmodulin complex can be altered by oncogenic mutations.
A new layer of phosphoinositide-mediated allosteric regulation uncovered for SHIP2.
ASTX029, a Novel Dual-Mechanism ERK Inhibitor, Modulates Both the Phosphorylation and Catalytic Activity of ERK.
Structural basis of the effect of activating mutations on the EGF receptor.
Sequential oncogenic mutations influence cell competition.
Statin-mediated inhibition of RAS prenylation activates ER stress to enhance the immunogenicity of KRAS mutant cancer.
Hydroxyphenyl Butanone Induces Cell Cycle Arrest through Inhibition of GSK3β in Colorectal Cancer.

Front Cell Dev Biol. 2021 ;9 665673

A Covalent Calmodulin Inhibitor as a Tool to Study Cellular Mechanisms of K-Ras-Driven Stemness.

Sunday Okutachi, Ganesh Babu Manoharan, Alexandros Kiriazis, Christina Laurini, Marie Catillon, Frank McCormick, Jari Yli-Kauhaluoma, Daniel Abankwa.

  Recently, the highly mutated oncoprotein K-Ras4B (hereafter K-Ras) was shown to drive cancer cell stemness in conjunction with calmodulin (CaM). We previously showed that the covalent CaM inhibitor ophiobolin A (OphA) can potently inhibit K-Ras stemness activity. However, OphA, a fungus-derived natural product, exhibits an unspecific, broad toxicity across all phyla. Here we identified a less toxic, functional analog of OphA that can efficiently inactivate CaM by covalent inhibition. We analyzed a small series of benzazulenones, which bear some structural similarity to OphA and can be synthesized in only six steps. We identified the formyl aminobenzazulenone 1, here named Calmirasone1, as a novel and potent covalent CaM inhibitor. Calmirasone1 has a 4-fold increased affinity for CaM as compared to OphA and was active against K-Ras in cells within minutes, as compared to hours required by OphA. Calmirasone1 displayed a 2.5-4.5-fold higher selectivity for KRAS over BRAF mutant 3D spheroid growth than OphA, suggesting improved relative on-target activity. Importantly, Calmirasone1 has a 40-260-fold lower unspecific toxic effect on HRAS mutant cells, while it reaches almost 50% of the activity of novel K-RasG12C specific inhibitors in 3D spheroid assays. Our results suggest that Calmirasone1 can serve as a new tool compound to further investigate the cancer cell biology of the K-Ras and CaM associated stemness activities.

Keywords:  BRET; K-Ras; calmodulin; cancer stem cell (CSC); covalent inhibitor

DOI:  https://doi.org/10.3389/fcell.2021.665673
Curr Opin Struct Biol. 2021 Jul 22. pii: S0959-440X(21)00097-X. [Epub ahead of print]71 136-147

Stopping the beating heart of cancer: KRAS reviewed.

Lorenz Herdeis, Daniel Gerlach, Darryl B McConnell, Dirk Kessler.

  It has taken four decades of research to see the first major breakthrough for KRAS-driven cancers. In particular, the last decade has seen a paradigm shift with the discovery of druggable pockets on KRAS and clinical efficacy with covalent KRASG12C inhibitors, culminating in the first approval of sotorasib monotherapy as second-line treatment in KRASG12C-driven non-small-cell lung cancer. Nevertheless, 85% of all KRAS-mutated cancers still lack novel agents. In this review, we will outline the structure, function, and post-translational modifications of KRAS and highlight the various approaches being adopted to drug KRAS, ranging from selective to pan concepts. The range of molecular modalities being explored, including PROTACs and glues, will also be described. Finally, an outlook toward the next wave of KRAS drugs and the challenges of resistance will be given.

Keywords:  KRAS, Protein-Protein Interaction, Cancer, Oncology, Resistance

DOI:  https://doi.org/10.1016/j.sbi.2021.06.013
Biochem Biophys Res Commun. 2021 Jul 26. pii: S0006-291X(21)01119-0. [Epub ahead of print]571 159-166

Leucyl-tRNA synthetase 1 is required for proliferation of TSC-null cells.

Ji-Hyun Bae, Jong Hyun Kim.

  Uncontrolled cell proliferation associated with cancer depends on the functional abrogation of at least one of tumor suppressor. In response to nutrient cue, tuberous sclerosis complex (TSC) works as a tumor suppressor which inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). However, the regulation mechanism of nutrient-dependent cell proliferation in TSC-null cells remains unclear. Here, we demonstrate that leucine is required for cell proliferation through the activation of leucyl-tRNA synthetase (LARS1)-mTORC1 pathway in TSC-null cells. Cell proliferation and survival were attenuated by LARS1 knock-down or inhibitors in TSC-null cells. In addition, either rapamycin or LARS1 inhibitors significantly decreased colony formation ability while their combined treatment drastically attenuated it. Taken together, we suggest that LARS1 inhibitors might considered as novel tools for the regression of tumor growth and proliferation in TSC-null tumor cells which regrow upon discontinuation of the mTORC1 inhibition.

Keywords:  Amino acids; Cell proliferation; LARS1; TSC; mTORC1

DOI:  https://doi.org/10.1016/j.bbrc.2021.07.080
Oncogene. 2021 Jul 24.

Targeting mTOR signaling overcomes acquired resistance to combined BRAF and MEK inhibition in BRAF-mutant melanoma.

Beike Wang, Wei Zhang, Gao Zhang, Lawrence Kwong, Hezhe Lu, Jiufeng Tan, Norah Sadek, Min Xiao, Jie Zhang, Marilyne Labrie, Sergio Randell, Aurelie Beroard, Eric Sugarman, Vito W Rebecca, Zhi Wei, Yiling Lu, Gordon B Mills, Jeffrey Field, Jessie Villanueva, Xiaowei Xu, Meenhard Herlyn, Wei Guo.

Targeting MAPK pathway using a combination of BRAF and MEK inhibitors is an efficient strategy to treat melanoma harboring BRAF-mutation. The development of acquired resistance is inevitable due to the signaling pathway rewiring. Combining western blotting, immunohistochemistry, and reverse phase protein array (RPPA), we aim to understanding the role of the mTORC1 signaling pathway, a center node of intracellular signaling network, in mediating drug resistance of BRAF-mutant melanoma to the combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) therapy. The mTORC1 signaling pathway is initially suppressed by BRAFi and MEKi combination in melanoma but rebounds overtime after tumors acquire resistance to the combination therapy (CR) as assayed in cultured cells and PDX models. In vitro experiments showed that a subset of CR melanoma cells was sensitive to mTORC1 inhibition. The mTOR inhibitors, rapamycin and NVP-BEZ235, induced cell cycle arrest and apoptosis in CR cell lines. As a proof-of-principle, we demonstrated that rapamycin and NVP-BEZ235 treatment reduced tumor growth in CR xenograft models. Mechanistically, AKT or ERK contributes to the activation of mTORC1 in CR cells, depending on PTEN status of these cells. Our study reveals that mTOR activation is essential for drug resistance of melanoma to MAPK inhibitors, and provides insight into the rewiring of the signaling networks in CR melanoma.

DOI: https://doi.org/10.1038/s41388-021-01911-5
Curr Opin Struct Biol. 2021 Jul 23. pii: S0959-440X(21)00092-0. [Epub ahead of print]71 164-170

The dynamic nature of the K-Ras/calmodulin complex can be altered by oncogenic mutations.

Hazem Abdelkarim, Nicholas Leschinsky, Hyunbum Jang, Avik Banerjee, Ruth Nussinov, Vadim Gaponenko.

Oncogenic mutant K-Ras promotes cancer cell proliferation, migration, invasion, and survival by assembling signaling complexes. To date, the functional and structural roles of K-Ras mutations within these complexes are incompletely understood despite their mechanistic and therapeutic significance. Here, we review recent advances in understanding specific binding between K-Ras and the calcium sensor calmodulin. This interaction positively and negatively regulates diverse functions of K-Ras in cancer, suggesting flexibility in K-Ras/calmodulin complex formation. Also, structural data suggest that oncogenic K-Ras likely samples several conformational states, influencing its distinct assemblies with calmodulin and with other proteins. Understanding how K-Ras interacts with calmodulin and with other partners is essential to discovering novel inhibitors of K-Ras in cancer.

DOI: https://doi.org/10.1016/j.sbi.2021.06.008
FASEB J. 2021 08;35(8): e21815

A new layer of phosphoinositide-mediated allosteric regulation uncovered for SHIP2.

Johanne Le Coq, Pilar López Navajas, Bárbara Rodrigo Martin, Carlos Alfonso, Daniel Lietha.

  The Src homology 2 containing inositol 5-phosphatase 2 (SHIP2) is a large multidomain enzyme that catalyzes the dephosphorylation of the phospholipid phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3 ) to form PI(3,4)P2 . PI(3,4,5)P3 is a key lipid second messenger controlling the recruitment of signaling proteins to the plasma membrane, thereby regulating a plethora of cellular events, including proliferation, growth, apoptosis, and cytoskeletal rearrangements. SHIP2, alongside PI3K and PTEN, regulates PI(3,4,5)P3 levels at the plasma membrane and has been heavily implicated in serious diseases such as cancer and type 2 diabetes; however, many aspects of its regulation mechanism remain elusive. We recently reported an activating effect of the SHIP2 C2 domain and here we describe an additional layer of regulation via the pleckstrin homology-related (PHR) domain. We show a phosphoinositide-induced transition to a high activity state of the enzyme that increases phosphatase activity up to 10-15 fold. We further show that PI(3,4)P2 directly interacts with the PHR domain to trigger this allosteric activation. Modeling of the PHR-phosphatase-C2 region of SHIP2 on the membrane suggests no major inter-domain interactions with the PHR domain, but close contacts between the two linkers offer a possible path of allosteric communication. Together, our data show that the PHR domain acts as an allosteric module regulating the catalytic activity of SHIP2 in response to specific phosphoinositide levels in the cell membrane.

Keywords:  SHIP2; allosteric regulation; enzyme kinetics; lipid phosphatase; phosphinositides

DOI:  https://doi.org/10.1096/fj.202100561R
Mol Cancer Ther. 2021 Jul 30. pii: molcanther.0909.2020. [Epub ahead of print]

ASTX029, a Novel Dual-Mechanism ERK Inhibitor, Modulates Both the Phosphorylation and Catalytic Activity of ERK.

Joanne M Munck, Valerio Berdini, Luke Bevan, Jessica L Brothwood, Juan Castro, Aurélie Courtin, Charlotte East, Roberta Ferraldeschi, Tom D Heightman, Christopher J Hindley, Justyna Kucia-Tran, John F Lyons, Vanessa Martins, Sandra Muench, Christopher W Murray, David Norton, Marc O'Reilly, Michael Reader, David C Rees, Sharna J Rich, Caroline J Richardson, Alpesh D Shah, Lukas Stanczuk, Neil T Thompson, Nicola E Wilsher, Alison Ja Woolford, Nicola G Wallis.

The MAPK signaling pathway is commonly upregulated in human cancers. As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers and for overcoming resistance to upstream inhibition. ASTX029 is a highly potent and selective dual-mechanism ERK inhibitor, discovered using fragment-based drug design. Due to its distinctive ERK binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK itself by MEK, despite not directly inhibiting MEK activity. This dual-mechanism was demonstrated in cell-free systems, as well as cell lines and xenograft tumor tissue, where the phosphorylation of both ERK and its substrate, RSK, were modulated on treatment with ASTX029. Markers of sensitivity were highlighted in a large cell panel, where ASTX029 preferentially inhibited the proliferation of MAPK-activated cell lines, including those with BRAF or RAS mutations. In vivo, significant anti-tumor activity was observed in MAPK-activated tumor xenograft models following oral treatment. ASTX029 also demonstrated activity in both in vitro and in vivo models of acquired resistance to MAPK pathway inhibitors. Overall, these findings highlight the therapeutic potential of a dual-mechanism ERK inhibitor such as ASTX029 for the treatment of MAPK-activated cancers, including those which have acquired resistance to inhibitors of upstream components of the MAPK pathway. ASTX029 is currently being evaluated in a first in human Phase I-II clinical trial in patients with advanced solid tumors (NCT03520075).

DOI: https://doi.org/10.1158/1535-7163.MCT-20-0909
Elife. 2021 Jul 28. pii: e65824. [Epub ahead of print]10

Structural basis of the effect of activating mutations on the EGF receptor.

Ioannis Galdadas, Luca Carlino, Richard A Ward, Samantha J Hughes, Shozeb Haider, Francesco Luigi Gervasio.

  Mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are common oncogenic driver events in non-small cell lung cancer. Although the activation of EGFR in normal cells is primarily driven by growth-factor-binding-induced dimerization, mutations on different exons of the kinase domain of the receptor have been found to affect the equilibrium between its active and inactive conformations giving rise to growth-factor-independent kinase activation. Using molecular dynamics simulations combined with enhanced sampling techniques, we compare here the conformational landscape of the monomers and homodimers of the wild-type and mutated forms of EGFR ΔELREA and L858R, as well as of two exon 20 insertions, D770-N771insNPG, and A763-Y764insFQEA. The differences in the conformational energy landscapes are consistent with multiple mechanisms of action including the regulation of the hinge motion, the stabilization of the dimeric interface, and local unfolding transitions. Overall, a combination of different effects is caused by the mutations and leads to the observed aberrant signaling.

Keywords:  cancer biology; computational; computational biology; human; in silico; molecular dynamics; mutations; none; simulations; systems biology

DOI:  https://doi.org/10.7554/eLife.65824
Curr Biol. 2021 Jul 16. pii: S0960-9822(21)00893-9. [Epub ahead of print]

Sequential oncogenic mutations influence cell competition.

Koki Kohashi, Yusuke Mori, Rika Narumi, Kei Kozawa, Tomoko Kamasaki, Susumu Ishikawa, Mihoko Kajita, Rei Kobayashi, Yoichiro Tamori, Yasuyuki Fujita.

  At the initial stage of carcinogenesis, newly emerging transformed cells are often eliminated from epithelial layers via cell competition with the surrounding normal cells. For instance, when surrounded by normal cells, oncoprotein RasV12-transformed cells are extruded into the apical lumen of epithelia. During cancer development, multiple oncogenic mutations accumulate within epithelial tissues. However, it remains elusive whether and how cell competition is also involved in this process. In this study, using a mammalian cell culture model system, we have investigated what happens upon the consecutive mutations of Ras and tumor suppressor protein Scribble. When Ras mutation occurs under the Scribble-knockdown background, apical extrusion of Scribble/Ras double-mutant cells is strongly diminished. In addition, at the boundary with Scribble/Ras cells, Scribble-knockdown cells frequently undergo apoptosis and are actively engulfed by the neighboring Scribble/Ras cells. The comparable apoptosis and engulfment phenotypes are also observed in Drosophila epithelial tissues between Scribble/Ras double-mutant and Scribble single-mutant cells. Furthermore, mitochondrial membrane potential is enhanced in Scribble/Ras cells, causing the increased mitochondrial reactive oxygen species (ROS). Suppression of mitochondrial membrane potential or ROS production diminishes apoptosis and engulfment of the surrounding Scribble-knockdown cells, indicating that mitochondrial metabolism plays a key role in the competitive interaction between double- and single-mutant cells. Moreover, mTOR (mechanistic target of rapamycin kinase) acts downstream of these processes. These results imply that sequential oncogenic mutations can profoundly influence cell competition, a transition from loser to winner. Further studies would open new avenues for cell competition-based cancer treatment, thereby blocking clonal expansion of more malignant populations within tumors.

Keywords:  ROS; RasV12; Scribble; apoptosis; cell competition; engulfment; entosis; mTOR; mitochondria; sequential mutations

DOI:  https://doi.org/10.1016/j.cub.2021.06.064
J Immunother Cancer. 2021 Jul;pii: e002474. [Epub ahead of print]9(7):

Statin-mediated inhibition of RAS prenylation activates ER stress to enhance the immunogenicity of KRAS mutant cancer.

Gi-Hoon Nam, Minsu Kwon, Hanul Jung, Eunbyeol Ko, Seong A Kim, Yoonjeong Choi, Su Jeong Song, Seohyun Kim, Yeji Lee, Gi Beom Kim, Jihoon Han, Jiwan Woo, Yakdol Cho, Cherlhyun Jeong, Seung-Yoon Park, Thomas M Roberts, Yong Beom Cho, In-San Kim.

   BACKGROUND: Statins preferentially promote tumor-specific apoptosis by depleting isoprenoid such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. However, statins have not yet been approved for clinical cancer treatment due, in part, to poor understanding of molecular determinants on statin sensitivity. Here, we investigated the potential of statins to elicit enhanced immunogenicity of KRAS-mutant (KRAS mut) tumors.
METHODS: The immunogenicity of treated cancer cells was determined by western blot, flow cytometry and confocal microscopy. The immunotherapeutic efficacy of mono or combination therapy using statin was assessed in KRASmut tumor models, including syngeneic colorectal cancer and genetically engineered lung and pancreatic tumors. Using NanoString analysis, we analyzed how statin influenced the gene signatures associated with the antigen presentation of dendritic cells in vivo and evaluated whether statin could induce CD8+ T-cell immunity. Multiplex immunohistochemistry was performed to better understand the complicated tumor-immune microenvironment.
RESULTS: Statin-mediated inhibition of KRAS prenylation provoked severe endoplasmic reticulum (ER) stress by attenuating the anti-ER stress effect of KRAS mutation, thereby resulting in the immunogenic cell death (ICD) of KRAS mut cancer cells. Moreover, statin-mediated ICD enhanced the cross-priming ability of dendritic cells, thereby provoking CD8+ T-cell immune responses against KRAS mut tumors. Combination therapy using statin and oxaliplatin, an ICD inducer, significantly enhanced the immunogenicity of KRAS mut tumors and promoted tumor-specific immunity in syngeneic and genetically engineered KRAS mut tumor models. Along with immune-checkpoint inhibitors, the abovementioned combination therapy overcame resistance to PD-1 blockade therapies, improving the survival rate of KRAS mut tumor models.
CONCLUSIONS: Our findings suggest that KRAS mutation could be a molecular target for statins to elicit potent tumor-specific immunity.

Keywords:  CD8-positive T-lymphocytes; antigen presentation; combination; dendritic cells; drug therapy; immunotherapy

DOI:  https://doi.org/10.1136/jitc-2021-002474
Biomed Res Int. 2021 ;2021 9981815

Hydroxyphenyl Butanone Induces Cell Cycle Arrest through Inhibition of GSK3β in Colorectal Cancer.

Songyan Zhang, Yunfeng Wang, Haopeng Zhang, Chengming Sun, Shuwei Dang, Ming Liu.

Background: Colorectal cancer (CRC) is among the top three gastrointestinal malignancy in morbidity and mortality. The abnormal activation of Wnt/β-catenin pathway is considered to be a key factor in the occurrence and development of CRC. Novel inhibitor discovery against key factor in WNT pathway is important for CRC treatment and prevention.
Methods: Cell proliferation was detected after hydroxyphenyl butanone treatment in human colorectal cancer HCT116, LOVO, and normal colonic epithelial NCM460 cells. Colony formation, cell invasion ability, and cell cycle were detected with and without GSK-3β knockdown.
Results: Hydroxyphenyl butanone induces cycle arresting on G1-S phase of colorectal cancer cell line through GSK3β in Wnt/β-catenin pathway and inhibits malignant biological manifestations of cell proliferation, colony formation, and invasion. The inhibition in the high concentration group is stronger than that in the low concentration group, and the antitumor effect is different for different tumor cells. Under the same concentration of natural hydroxyphenyl butanone, the inhibition on normal colonic epithelial cells is significantly lower than that on tumor cells. The natural hydroxyphenyl butanone with medium and low concentration could promote the proliferation of normal colonic epithelial cells.
Conclusion: This study illustrated natural hydroxyphenyl butanone as new inhibitor of GSK3β and revealed the mechanisms underlying the inhibitory effects in colorectal cancer.

DOI: https://doi.org/10.1155/2021/9981815