bims-pimaco 2022-04-03 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–04–03
twelve papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Targeting SHP2 phosphatase in breast cancer overcomes RTK-mediated resistance to PI3K inhibitors.
Functional diversity in the RAS subfamily of small GTPases.
Systematic review of randomised clinical trials and observational studies for patients with RAS wild-type or BRAFV600E-mutant metastatic and/or unresectable colorectal cancer.
The Influence of Oncogenic RAS on Chemotherapy and Radiotherapy Resistance Through DNA Repair Pathways.
Quantitative phosphoproteomic analyses identify STK11IP as a lysosome-specific substrate of mTORC1 that regulates lysosomal acidification.
Folliculin promotes substrate-selective mTORC1 activity by activating RagC to recruit TFE3.
Exploring Survival Outcomes in Metastatic Colorectal Cancer Harboring KRAS A146 Mutations: Important Distinction or Simple Distraction?
Targeting ligand-dependent wnt pathway dysregulation in gastrointestinal cancers through porcupine inhibition.
Targeted Therapy for Colorectal Cancer.
Structural basis of activation of the tumor suppressor protein neurofibromin.
Covalent Proximity Scanning of a Distal Cysteine to Target PI3Kα.
Design, synthesis and activity study of a novel PI3K degradation by hijacking VHL E3 ubiquitin ligase.

Breast Cancer Res. 2022 Apr 01. 24(1): 23

Targeting SHP2 phosphatase in breast cancer overcomes RTK-mediated resistance to PI3K inhibitors.

Guus J J E Heynen, Kamil Lisek, Regina Vogel, Annika Wulf-Goldenberg, Joshua Alcaniz, Elodie Montaudon, Elisabetta Marangoni, Walter Birchmeier.

   BACKGROUND: PI3K signaling is frequently activated in breast cancer and is targeted by PI3K inhibitors. However, resistance of tumor cells to PI3K inhibition, often mediated by activated receptor tyrosine kinases, is commonly observed and reduces the potency of PI3K inhibitors. Therefore, new treatment strategies to overcome resistance to PI3K inhibitors are urgently needed to boost their efficacy. The phosphatase SHP2, which plays a crucial role in mediating signal transduction between receptor tyrosine kinases and both the PI3K and MAPK pathways, is a potential target for combination treatment.
METHODS: We tested combinations of PI3K and SHP2 inhibitors in several experimental breast cancer models that are resistant to PI3K inhibition. Using cell culturing, biochemical and genetic approaches, we evaluated tumor cell proliferation and signaling output in cells treated with PI3K and SHP2 inhibitors.
RESULTS: Combination treatment with PI3K and SHP2 inhibitors counteracted both acquired and intrinsic breast cancer cell resistance to PI3K inhibition that is mediated by activated receptor tyrosine kinases. Dual PI3K and SHP2 inhibition blocked proliferation and led to sustained inactivation of PI3K and MAPK signaling, where resistant cells rapidly re-activated these pathways upon PI3K inhibitor monotreatment. In addition, we demonstrate that overexpression of SHP2 induced resistance to PI3K inhibition, and that SHP2 was frequently activated during the development of PI3K inhibitor resistance after prolonged treatment of sensitive cells.
CONCLUSIONS: Our results highlight the importance of SHP2 as a player in resistance to PI3K inhibitors. Combination treatment with PI3K and SHP2 inhibitors could pave the way for significant improvements in therapies for breast cancer.

Keywords:  Breast cancer; Drug resistance; PI3K and MAPK signaling; SHP2; Targeted therapy

DOI:  https://doi.org/10.1186/s13058-022-01521-3
Biochem Soc Trans. 2022 Mar 31. pii: BST20211166. [Epub ahead of print]

Functional diversity in the RAS subfamily of small GTPases.

Gabriela Bernal Astrain, Maya Nikolova, Matthew J Smith.

  RAS small GTPases regulate important signalling pathways and are notorious drivers of cancer development and progression. While most research to date has focused on understanding and addressing the oncogenic potential of three RAS oncogenes: HRAS, KRAS, and NRAS; the full RAS subfamily is composed of 35 related GTPases with diverse cellular functions. Most remain deeply understudied despite strong evolutionary conservation. Here, we highlight a group of 17 poorly characterized RAS GTPases that are frequently down-regulated in cancer and evidence suggests may function not as oncogenes, but as tumour suppressors. These GTPases remain largely enigmatic in terms of their cellular function, regulation, and interaction with effector proteins. They cluster within two families we designate as 'distal-RAS' (D-RAS; comprised of DIRAS, RASD, and RASL10) and 'CaaX-Less RAS' (CL-RAS; comprised of RGK, NKIRAS, RERG, and RASL11/12 GTPases). Evidence of a tumour suppressive role for many of these GTPases supports the premise that RAS subfamily proteins may collectively regulate cellular proliferation.

Keywords:  G-proteins; GTPase; RAS; cell proliferation; oncogenesis; tumour suppressors

DOI:  https://doi.org/10.1042/BST20211166
Crit Rev Oncol Hematol. 2022 Mar 25. pii: S1040-8428(22)00070-1. [Epub ahead of print]173 103646

Systematic review of randomised clinical trials and observational studies for patients with RAS wild-type or BRAFV600E-mutant metastatic and/or unresectable colorectal cancer.

Pilar García-Alfonso, Astrid Lièvre, Fotios Loupakis, Abir Tadmouri, Sadya Khan, Leticia Barcena, Sebastian Stintzing.

  Approximately 8-10% of metastatic colorectal cancer (mCRC) tumours harbour BRAFV600E mutations. Eleven randomised controlled trials (RCTs) and 24 non-RCTs were identified. Seven studies evaluated BRAF inhibitors. Single-agent BRAF inhibitors had minimal efficacy, whereas BRAF inhibitor plus anti-EGFR therapy improved outcomes. In BEACON CRC, overall survival (OS) was significantly longer for patients receiving encorafenib plus cetuximab ± binimetinib when compared with irinotecan/FOLFIRI plus cetuximab as second- and third-line therapy. Seven prospective non-RCTs reported worse OS and progression-free survival (PFS) for patients with BRAFV600E-mutant vs BRAF wild-type mCRC. Eight RCTs reported that PFS and OS were generally shorter for patients with BRAFV600E-mutant mCRC vs those with KRAS or RAS wild-type mCRC. Patients with BRAFV600E-mutant mCRC have worse outcomes with conventional therapy vs patients with BRAF wild-type tumours. BRAF inhibitors in conjunction with anti-EGFR therapy improves outcomes for patients with BRAFV600E-mutant mCRC vs conventional therapy or a BRAF inhibitor alone.

Keywords:  BRAF kinase inhibitor; BRAFV600E; Efficacy; Metastatic colorectal cancer

DOI:  https://doi.org/10.1016/j.critrevonc.2022.103646
Front Cell Dev Biol. 2022 ;10 751367

The Influence of Oncogenic RAS on Chemotherapy and Radiotherapy Resistance Through DNA Repair Pathways.

Rodrigo E Cáceres-Gutiérrez, Yair Alfaro-Mora, Marco A Andonegui, José Díaz-Chávez, Luis A Herrera.

  RAS oncogenes are chief tumorigenic drivers, and their mutation constitutes a universal predictor of poor outcome and treatment resistance. Despite more than 30 years of intensive research since the identification of the first RAS mutation, most attempts to therapeutically target RAS mutants have failed to reach the clinic. In fact, the first mutant RAS inhibitor, Sotorasib, was only approved by the FDA until 2021. However, since Sotorasib targets the KRAS G12C mutant with high specificity, relatively few patients will benefit from this therapy. On the other hand, indirect approaches to inhibit the RAS pathway have revealed very intricate cascades involving feedback loops impossible to overcome with currently available therapies. Some of these mechanisms play different roles along the multistep carcinogenic process. For instance, although mutant RAS increases replicative, metabolic and oxidative stress, adaptive responses alleviate these conditions to preserve cellular survival and avoid the onset of oncogene-induced senescence during tumorigenesis. The resulting rewiring of cellular mechanisms involves the DNA damage response and pathways associated with oxidative stress, which are co-opted by cancer cells to promote survival, proliferation, and chemo- and radioresistance. Nonetheless, these systems become so crucial to cancer cells that they can be exploited as specific tumor vulnerabilities. Here, we discuss key aspects of RAS biology and detail some of the mechanisms that mediate chemo- and radiotherapy resistance of mutant RAS cancers through the DNA repair pathways. We also discuss recent progress in therapeutic RAS targeting and propose future directions for the field.

Keywords:  DNA damage response; cancer; chemotherapy and radiotherapy resistance; double strand breaks; oncogene-induced senescence; ras; reactive oxygen species

DOI:  https://doi.org/10.3389/fcell.2022.751367
Nat Commun. 2022 Apr 01. 13(1): 1760

Quantitative phosphoproteomic analyses identify STK11IP as a lysosome-specific substrate of mTORC1 that regulates lysosomal acidification.

Zhenzhen Zi, Zhuzhen Zhang, Qiang Feng, Chiho Kim, Xu-Dong Wang, Philipp E Scherer, Jinming Gao, Beth Levine, Yonghao Yu.

The evolutionarily conserved serine/threonine kinase mTORC1 is a central regulator of cell growth and proliferation. mTORC1 is activated on the lysosome surface. However, once mTORC1 is activated, it is unclear whether mTORC1 phosphorylates local lysosomal proteins to regulate specific aspects of lysosomal biology. Through cross-reference analyses of the lysosome proteome with the mTORC1-regulated phosphoproteome, we identify STK11IP as a lysosome-specific substrate of mTORC1. mTORC1 phosphorylates STK11IP at Ser404. Knockout of STK11IP leads to a robust increase of autophagy flux. Dephosphorylation of STK11IP at Ser404 represses the role of STK11IP as an autophagy inhibitor. Mechanistically, STK11IP binds to V-ATPase, and regulates the activity of V-ATPase. Knockout of STK11IP protects mice from fasting or Methionine/Choline-Deficient Diet (MCD)-induced fatty liver. Thus, our study demonstrates that STK11IP phosphorylation represents a mechanism for mTORC1 to regulate lysosomal acidification and autophagy, and points to STK11IP as a promising therapeutic target for the amelioration of diseases with aberrant autophagy signaling.

DOI: https://doi.org/10.1038/s41467-022-29461-8
PLoS Biol. 2022 Mar 31. 20(3): e3001594

Folliculin promotes substrate-selective mTORC1 activity by activating RagC to recruit TFE3.

Kristina Li, Shogo Wada, Bridget S Gosis, Chelsea Thorsheim, Paige Loose, Zolt Arany.

Mechanistic target of rapamycin complex I (mTORC1) is central to cellular metabolic regulation. mTORC1 phosphorylates a myriad of substrates, but how different substrate specificity is conferred on mTORC1 by different conditions remains poorly defined. Here, we show how loss of the mTORC1 regulator folliculin (FLCN) renders mTORC1 specifically incompetent to phosphorylate TFE3, a master regulator of lysosome biogenesis, without affecting phosphorylation of other canonical mTORC1 substrates, such as S6 kinase. FLCN is a GTPase-activating protein (GAP) for RagC, a component of the mTORC1 amino acid (AA) sensing pathway, and we show that active RagC is necessary and sufficient to recruit TFE3 onto the lysosomal surface, allowing subsequent phosphorylation of TFE3 by mTORC1. Active mutants of RagC, but not of RagA, rescue both phosphorylation and lysosomal recruitment of TFE3 in the absence of FLCN. These data thus advance the paradigm that mTORC1 substrate specificity is in part conferred by direct recruitment of substrates to the subcellular compartments where mTORC1 resides and identify potential targets for specific modulation of specific branches of the mTOR pathway.

DOI: https://doi.org/10.1371/journal.pbio.3001594
JCO Precis Oncol. 2022 Mar;6 e2100564

Exploring Survival Outcomes in Metastatic Colorectal Cancer Harboring KRAS A146 Mutations: Important Distinction or Simple Distraction?

Richard Pham, Azim Jalali, Chia Chong, Vanessa Wong, Kate Dunn, Peter Gibbs.

DOI: https://doi.org/10.1200/PO.21.00564
Pharmacol Ther. 2022 Mar 28. pii: S0163-7258(22)00073-0. [Epub ahead of print] 108179

Targeting ligand-dependent wnt pathway dysregulation in gastrointestinal cancers through porcupine inhibition.

Dustin J Flanagan, Simon A Woodcock, Caroline Phillips, Catherine Eagle, Owen J Sansom.

  Gastrointestinal cancers are responsible for more cancer deaths than any other system of the body. This review summarises how Wnt pathway dysregulation contributes to the development of the most common gastrointestinal cancers, with a particular focus on the nature and frequency of upstream pathway aberrations. Tumors with upstream aberrations maintain a dependency on the presence of functional Wnt ligand, and are predicted to be tractable to inhibitors of Porcupine, an enzyme that plays a key role in Wnt secretion. We summarise available pre-clinical efficacy data from Porcupine inhibitors in vitro and in vivo, as well as potential toxicities and the data from early phase clinical trials. We appraise the rationale for biomarker-defined targeted approaches, as well as outlining future opportunities for combination with other therapeutics.

Keywords:  Porcupine inhibitor; RNF43; RSPO; Wnt; clinical trials; gastrointestinal cancer

DOI:  https://doi.org/10.1016/j.pharmthera.2022.108179
Surg Oncol Clin N Am. 2022 Apr;pii: S1055-3207(21)00098-3. [Epub ahead of print]31(2): 255-264

Targeted Therapy for Colorectal Cancer.

Shinichiro Sakata, David W Larson.

  Metastatic colorectal cancer (mCRC) is incurable in patients with unresectable disease. For most patients, the primary treatment is palliative systemic chemotherapy. Genomic profiling is used to detect specific genetic mutations that may offer selected patients a modest survival benefit with targeted therapy. Patients with mCRC with KRAS/NRAS/BRAF wild-type left-sided tumors may benefit from epidermal growth factor receptor (EGFR) inhibition with either cetuximab or panitumumab, in conjunction with chemotherapy. EGFR inhibitors can extend survival by 6 months compared with chemotherapy alone. The vascular endothelial growth factor (VEGF) inhibitor bevacizumab can serve as an alternative to EGFR inhibitors in right-sided tumors or second-line therapy. Many patients will have RAS mutations, and targeted therapies will not provide any benefit. The PRIME trial demonstrated that the addition of panitumumab to FOLFOX was associated with reduced overall survival. Patients with BRAF mutations do not benefit from targeted therapy unless a BRAF inhibitor supplements treatment. Triple combination therapy with cetuximab, the BRAF inhibitor encorafenib, and the MEK kinase inhibitor binimetinib has extended overall survival by about 3 months compared with chemotherapy alone. Finally, for the minority patients with microsatellite instability (MSI) high/mismatch repair (MMR) deficient tumors, either due to Lynch syndrome or sporadic mutations, immunotherapy is recommended as first-line treatment. The KEYNOTE-177 trial demonstrated that therapy with single-agent pembrolizumab improved progression-free survival by 8 months compared with FOLFOX or FOLFIRI and with or without EGFR inhibition. At this time, targeted therapy should only be used in patients with unresectable metastatic disease.

Keywords:  Bevacizumab; EGFR; Metastatic colorectal cancer; Targeted therapy

DOI:  https://doi.org/10.1016/j.soc.2021.11.006
Mol Cell. 2022 Mar 25. pii: S1097-2765(22)00220-9. [Epub ahead of print]

Structural basis of activation of the tumor suppressor protein neurofibromin.

Malik Chaker-Margot, Sebastiaan Werten, Theresia Dunzendorfer-Matt, Stefan Lechner, Angela Ruepp, Klaus Scheffzek, Timm Maier.

  Mutations in the NF1 gene cause the familial genetic disease neurofibromatosis type I, as well as predisposition to cancer. The NF1 gene product, neurofibromin, is a GTPase-activating protein and acts as a tumor suppressor by negatively regulating the small GTPase, Ras. However, structural insights into neurofibromin activation remain incompletely defined. Here, we provide cryoelectron microscopy (cryo-EM) structures that reveal an extended neurofibromin homodimer in two functional states: an auto-inhibited state with occluded Ras-binding site and an asymmetric open state with an exposed Ras-binding site. Mechanistically, the transition to the active conformation is stimulated by nucleotide binding, which releases a lock that tethers the catalytic domain to an extended helical repeat scaffold in the occluded state. Structure-guided mutational analysis supports functional relevance of allosteric control. Disease-causing mutations are mapped and primarily impact neurofibromin stability. Our findings suggest a role for nucleotides in neurofibromin regulation and may lead to therapeutic modulation of Ras signaling.

Keywords:  G protein; GAP activity; Ras signaling; cellular signaling; conformational change; cryo-EM; neurofibromatosis type I; neurofibromin; nucleotides; small GTPase

DOI:  https://doi.org/10.1016/j.molcel.2022.03.011
J Am Chem Soc. 2022 Mar 30.

Covalent Proximity Scanning of a Distal Cysteine to Target PI3Kα.

Chiara Borsari, Erhan Keles, Jacob A McPhail, Alexander Schaefer, Rohitha Sriramaratnam, Wojciech Goch, Thorsten Schaefer, Martina De Pascale, Wojciech Bal, Matthias Gstaiger, John E Burke, Matthias P Wymann.

Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5'-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (kchem), rate of covalent bond formation and proximity (kinact and reaction space volume Vr), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of kchem, kinact, Ki, and Vr, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes.

DOI: https://doi.org/10.1021/jacs.1c13568
Bioorg Med Chem. 2022 Mar 18. pii: S0968-0896(22)00099-2. [Epub ahead of print]61 116707

Design, synthesis and activity study of a novel PI3K degradation by hijacking VHL E3 ubiquitin ligase.

Haili Wang, Chuchu Li, Xiaoqing Liu, Mingliang Ma.

  PI3K kinase plays an important role in regulating key processes in cells, such as cell growth, metabolism, proliferation, and apoptosis. The overexpression of PI3K kinase exists in many cancers. The proteolytic target chimera (PROTAC) technology is a new technology that uses the ubiquitin-proteasome system to degrade a given target protein. It has been described that CRBN-based PROTAC targets the degradation of PI3K kinase. However, PROTAC based on VHL has not been reported yet. Here, we connected the previously obtained highly active PI3K inhibitor to the VHL ligand through different small molecules, and obtained a series of PROTAC molecules targeting PI3K kinase. Obtain the most active compound through screening. It provides evidence for the feasibility of PROTAC technology to recruit VHL E3 ligase in PI3K kinase.

Keywords:  Degradation; PI3K; PROTAC; VHL

DOI:  https://doi.org/10.1016/j.bmc.2022.116707