bims-pimaco 2022-02-27 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–02–27
ten papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Effective drug combinations in breast, colon and pancreatic cancer cells.
The Ragulator complex serves as a substrate-specific mTORC1 scaffold in regulating the nuclear translocation of transcription factor EB.
YAP regulates an SGK1/mTORC1/SREBP-dependent lipogenic program to support proliferation and tissue growth.
Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop.
Insights into the Cross Talk between Effector and Allosteric Lobes of KRAS from Methyl Conformational Dynamics.
Albumin metabolism targeted peptide-drug conjugate strategy for targeting pan-KRAS mutant cancer.
Regulation of GTPase function by autophosphorylation.
Enhanced O-GlcNAc modification induced by the RAS/MAPK/CDK1 pathway is required for SOX2 protein expression and generation of cancer stem cells.
Immunofluorescence Detection of Plasma Membranous PTEN in Cultured Cells.
Discovery of MAP855, an Efficacious and Selective MEK1/2 Inhibitor with an ATP-Competitive Mode of Action.

Nature. 2022 Feb 23.

Effective drug combinations in breast, colon and pancreatic cancer cells.

Patricia Jaaks, Elizabeth A Coker, Daniel J Vis, Olivia Edwards, Emma F Carpenter, Simonetta M Leto, Lisa Dwane, Francesco Sassi, Howard Lightfoot, Syd Barthorpe, Dieudonne van der Meer, Wanjuan Yang, Alexandra Beck, Tatiana Mironenko, Caitlin Hall, James Hall, Iman Mali, Laura Richardson, Charlotte Tolley, James Morris, Frances Thomas, Ermira Lleshi, Nanne Aben, Cyril H Benes, Andrea Bertotti, Livio Trusolino, Lodewyk Wessels, Mathew J Garnett.

Combinations of anti-cancer drugs can overcome resistance and provide new treatments1,2. The number of possible drug combinations vastly exceeds what could be tested clinically. Efforts to systematically identify active combinations and the tissues and molecular contexts in which they are most effective could accelerate the development of combination treatments. Here we evaluate the potency and efficacy of 2,025 clinically relevant two-drug combinations, generating a dataset encompassing 125 molecularly characterized breast, colorectal and pancreatic cancer cell lines. We show that synergy between drugs is rare and highly context-dependent, and that combinations of targeted agents are most likely to be synergistic. We incorporate multi-omic molecular features to identify combination biomarkers and specify synergistic drug combinations and their active contexts, including in basal-like breast cancer, and microsatellite-stable or KRAS-mutant colon cancer. Our results show that irinotecan and CHEK1 inhibition have synergistic effects in microsatellite-stable or KRAS-TP53 double-mutant colon cancer cells, leading to apoptosis and suppression of tumour xenograft growth. This study identifies clinically relevant effective drug combinations in distinct molecular subpopulations and is a resource to guide rational efforts to develop combinatorial drug treatments.

DOI: https://doi.org/10.1038/s41586-022-04437-2
J Biol Chem. 2022 Feb 17. pii: S0021-9258(22)00184-3. [Epub ahead of print] 101744

The Ragulator complex serves as a substrate-specific mTORC1 scaffold in regulating the nuclear translocation of transcription factor EB.

Tetsuya Kimura, Yoshitomo Hayama, Daisuke Okuzaki, Shigeyuki Nada, Masato Okada.

  The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by ten-fold in the Ragulator-deficient clone compared to that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling.

Keywords:  Ragulator; lysosome; mammalian target of rapamycin (mTOR); nuclear translocation; scaffold protein; transcription factor EB

DOI:  https://doi.org/10.1016/j.jbc.2022.101744
Dev Cell. 2022 Feb 15. pii: S1534-5807(22)00070-3. [Epub ahead of print]

YAP regulates an SGK1/mTORC1/SREBP-dependent lipogenic program to support proliferation and tissue growth.

Srimayee Vaidyanathan, Talhah M Salmi, Rasan M Sathiqu, Malcolm J McConville, Andrew G Cox, Kristin K Brown.

  The coordinated regulation of growth control and metabolic pathways is required to meet the energetic and biosynthetic demands associated with proliferation. Emerging evidence suggests that the Hippo pathway effector Yes-associated protein 1 (YAP) reprograms cellular metabolism to meet the anabolic demands of growth, although the mechanisms involved are poorly understood. Here, we demonstrate that YAP co-opts the sterol regulatory element-binding protein (SREBP)-dependent lipogenic program to facilitate proliferation and tissue growth. Mechanistically, YAP stimulates de novo lipogenesis via mechanistic target of rapamcyin (mTOR) complex 1 (mTORC1) signaling and subsequent activation of SREBP. Importantly, YAP-dependent regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1) is required to activate mTORC1/SREBP and stimulate de novo lipogenesis. We also find that the SREBP target genes fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) are conditionally required to support YAP-dependent proliferation and tissue growth. These studies reveal that de novo lipogenesis is a metabolic vulnerability that can be targeted to disrupt YAP-dependent proliferation and tissue growth.

Keywords:  SGK1; SREBP; YAP; cell metabolism; growth; lipogenesis; mTORC1; proliferation

DOI:  https://doi.org/10.1016/j.devcel.2022.02.004
Cell Death Dis. 2022 Feb 21. 13(2): 168

Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop.

Romina J Walter, Steffen J Sonnentag, Leonel Munoz-Sagredo, Melanie Merkel, Ludovic Richert, Felix Bunert, Yvonne M Heneka, Thomas Loustau, Michael Hodder, Rachel A Ridgway, Owen J Sansom, Yves Mely, Ulrich Rothbauer, Mark Schmitt, Véronique Orian-Rousseau.

Enhancement of Wnt signaling is fundamental for stem cell function during intestinal regeneration. Molecular modules control Wnt activity by regulating signal transduction. CD44 is such a positive regulator and a Wnt target gene. While highly expressed in intestinal crypts and used as a stem cell marker, its role during intestinal homeostasis and regeneration remains unknown. Here we propose a CD44 positive-feedback loop that boosts Wnt signal transduction, thus impacting intestinal regeneration. Excision of Cd44 in Cd44fl/fl;VillinCreERT2 mice reduced Wnt target gene expression in intestinal crypts and affected stem cell functionality in organoids. Although the integrity of the intestinal epithelium was conserved in mice lacking CD44, they were hypersensitive to dextran sulfate sodium, and showed more severe inflammation and delayed regeneration. We localized the molecular function of CD44 at the Wnt signalosome, and identified novel DVL/CD44 and AXIN/CD44 complexes. CD44 thus promotes optimal Wnt signaling during intestinal regeneration.

DOI: https://doi.org/10.1038/s41419-022-04607-0
J Am Chem Soc. 2022 Feb 25.

Insights into the Cross Talk between Effector and Allosteric Lobes of KRAS from Methyl Conformational Dynamics.

Fa-An Chao, Srisathiyanarayanan Dharmaiah, Troy Taylor, Simon Messing, William Gillette, Dominic Esposito, Dwight V Nissley, Frank McCormick, R Andrew Byrd, Dhirendra K Simanshu, Gabriel Cornilescu.

KRAS is the most frequently mutated RAS protein in cancer patients, and it is estimated that about 20% of the cancer patients in the United States carried mutant RAS proteins. To accelerate therapeutic development, structures and dynamics of RAS proteins had been extensively studied by various biophysical techniques for decades. Although 31P NMR studies revealed population equilibrium of the two major states in the active GMPPNP-bound form, more complex conformational dynamics in RAS proteins and oncogenic mutants subtly modulate the interactions with their downstream effectors. We established a set of customized NMR relaxation dispersion techniques to efficiently and systematically examine the ms-μs conformational dynamics of RAS proteins. This method allowed us to observe varying synchronized motions that connect the effector and allosteric lobes in KRAS. We demonstrated the role of conformational dynamics of KRAS in controlling its interaction with the Ras-binding domain of the downstream effector RAF1, the first kinase in the MAPK pathway. This allows one to explain, as well as to predict, the altered binding affinities of various KRAS mutants, which was neither previously reported nor apparent from the structural perspective.

DOI: https://doi.org/10.1021/jacs.2c00007
J Control Release. 2022 Feb 21. pii: S0168-3659(22)00100-6. [Epub ahead of print]

Albumin metabolism targeted peptide-drug conjugate strategy for targeting pan-KRAS mutant cancer.

Young Seok Cho, Gui Chul Kim, Hye Min Lee, Byoungmo Kim, Ha Rin Kim, Seung Woo Chung, Hyo Won Chang, Yoon Gun Ko, Yoon Se Lee, Seong Who Kim, Youngro Byun, Sang Yoon Kim.

  Despite recent breakthroughs in the development of direct KRAS inhibitors and modulators, no drugs targeting pan-KRAS mutant cancers are clinically available. Here, we report a novel strategy to treat pan-KRAS cancers using a caspase-3 cleavable peptide-drug conjugate that exploits enhanced albumin metabolism in KRAS altered cancers to deliver a cytotoxic agent that can induce a widespread bystander killing effect in tumor cells. Increased albumin metabolism in KRAS mutant cancer cells induced apoptosis via the intracellular uptake of albumin-bound MPD1. This allowed caspase-3 upregulation activated MPD1 to release the payload and exert the non-selective killing of neighboring cancer cells. MPD1 exhibited potent and durable antitumor efficacy in mouse xenograft models with different KRAS genotypes. An augmentation of anti-cancer efficacy was achieved by the bystander killing effect derived from the caspase-3 mediated activation of MPD1. In summary, albumin metabolism-induced apoptosis, together with the bystander killing effect of MPD1 boosted by caspase-3 mediated activation, intensified the efficacy of MPD1 in KRAS mutant cancers. These findings suggest that this novel peptide-drug conjugate could be a promising breakthrough for the treatment in the targeting of pan-KRAS mutant cancers.

Keywords:  Albumin metabolism; Bystander killing effect; Caspase-3; KRAS mutant cancer; Peptide-drug conjugate; Prodrug

DOI:  https://doi.org/10.1016/j.jconrel.2022.02.026
Mol Cell. 2022 Feb 18. pii: S1097-2765(22)00116-2. [Epub ahead of print]

Regulation of GTPase function by autophosphorylation.

Christian W Johnson, Hyuk-Soo Seo, Elizabeth M Terrell, Moon-Hee Yang, Fenneke KleinJan, Teklab Gebregiworgis, Genevieve M C Gasmi-Seabrook, Ezekiel A Geffken, Jimit Lakhani, Kijun Song, Puspalata Bashyal, Olesja Popow, Joao A Paulo, Andrea Liu, Carla Mattos, Christopher B Marshall, Mitsuhiko Ikura, Deborah K Morrison, Sirano Dhe-Paganon, Kevin M Haigis.

  A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.

Keywords:  GTPase; NMR; RAF; RAS; RASSF; autophosphorylation; kinase; molecular dynamics; phosphoryl transfer; protein crystallography

DOI:  https://doi.org/10.1016/j.molcel.2022.02.011
Sci Rep. 2022 02 21. 12(1): 2910

Enhanced O-GlcNAc modification induced by the RAS/MAPK/CDK1 pathway is required for SOX2 protein expression and generation of cancer stem cells.

Masahiro Shimizu, Hiroshi Shibuya, Nobuyuki Tanaka.

Cancer stem cells (CSCs) have tumour initiation, self-renewal, and long-term tumour repopulation properties, and it is postulated that differentiated somatic cells can be reprogrammed to CSCs by oncogenic signals. We previously showed that oncogenic HRASV12 conferred tumour initiation capacity in tumour suppressor p53-deficient (p53-/-) primary mouse embryonic fibroblasts (MEFs) through transcription factor NF-κB-mediated enhancement of glucose uptake; however, the underlying mechanisms of RAS oncogene-induced CSC reprogramming have not been elucidated. Here, we found that the expression of the reprogramming factor SOX2 was induced by HRASV12 in p53-/- MEFs. Moreover, gene knockout studies revealed that SOX2 is an essential factor for the generation of CSCs by HRASV12 in mouse and human fibroblasts. We demonstrated that HRASV12-induced cyclin-dependent kinase 1 (CDK1) activity and subsequent enhancement of protein O-GlcNAcylation were required for SOX2 induction and CSC generation in these fibroblasts and cancer cell lines containing RAS mutations. Moreover, the CDK inhibitor dinaciclib and O-GlcNAcylation inhibitor OSMI1 reduced the number of CSCs derived from these cells. Taken together, our results reveal a signalling pathway and mechanism for CSC generation by oncogenic RAS and suggest the possibility that this signalling pathway is a therapeutic target for CSCs.

DOI: https://doi.org/10.1038/s41598-022-06916-y
J Histochem Cytochem. 2022 Feb 24. 221554221082539

Immunofluorescence Detection of Plasma Membranous PTEN in Cultured Cells.

Takashi Kato.

  PTEN is a well-known tumor suppressor with various functions that depend on its intracellular localization. Green fluorescent protein (GFP)-tagged live-cell images clarified the crucial amino acids needed to regulate the localization of PTEN in cells. However, it currently remains unknown whether GFP itself affects the intracellular localization of PTEN and its mutants, and the establishment of fixed-cell imaging is important for identifying the exact location of PTEN in cells. I herein investigated a number of immunofluorescence strategies for cell fixation, membrane permeabilization, and antigen retrieval. Permeabilization by detergents was necessary to observe nuclear and cytosolic PTEN in paraformaldehyde (PFA)-fixed cells; however, this permeabilization was not always valid. On the other hand, antigen retrieval by the pre-boiled EDTA treatment was useful for detecting plasma membranous PTEN in PFA-fixed cells in the same manner as in in vivo studies. Furthermore, methanol-fixed images of PTEN were consistent with GFP-tagged live-cell images. Two immunofluorescence methods (the PFA-fixed/pre-boiled EDTA treatment and methanol fixation) are applicable to investigations of the intracellular localization of PTEN without a GFP tag in cultured cells. In conclusion, live-cell imaging and appropriate immunofluorescence including a novel antigen retrieval treatment were both useful for detecting the cellular localization of PTEN, particularly at the plasma membrane.

Keywords:  PTEN; immunofluorescence; nucleus; plasma membrane

DOI:  https://doi.org/10.1369/00221554221082539
J Med Chem. 2022 Feb 23.

Discovery of MAP855, an Efficacious and Selective MEK1/2 Inhibitor with an ATP-Competitive Mode of Action.

Ramulu Poddutoori, Kimberly Aardalen, Kiran Aithal, Sanjeev Surendranath Barahagar, Charamanna Belliappa, Mark Bock, Shekar Chelur, Andrea Gerken, Sreevalsam Gopinath, Bjoern Gruenenfelder, Michael Kiffe, Maithreyi Krishnaswami, John Langowski, Sudharshan Madapa, Kishore Narayanan, Chetan Pandit, Sunil Kumar Panigrahi, Mark Perrone, Ravi Kumar Potakamuri, Murali Ramachandra, Anuradha Ramanathan, Rita Ramos, Emine Sager, Susanta Samajdar, Hosahalli S Subramanya, Devaraja Seethappa Thimmasandra, Eleni Venetsanakos, Henrik Möbitz.

Mutations in MEK1/2 have been described as a resistance mechanism to BRAF/MEK inhibitor treatment. We report the discovery of a novel ATP-competitive MEK1/2 inhibitor with efficacy in wildtype (WT) and mutant MEK12 models. Starting from a HTS hit, we obtained selective, cellularly active compounds that showed equipotent inhibition of WT MEK1/2 and a panel of MEK1/2 mutant cell lines. Using a structure-based approach, the optimization addressed the liabilities by systematic analysis of molecular matched pairs (MMPs) and ligand conformation. Addition of only three heavy atoms to early tool compound 6 removed Cyp3A4 liabilities and increased the cellular potency by 100-fold, while reducing log P by 5 units. Profiling of MAP855, compound 30, in pharmacokinetic-pharmacodynamic and efficacy studies in BRAF-mutant models showed comparable efficacy to clinical MEK1/2 inhibitors. Compound 30 is a novel highly potent and selective MEK1/2 kinase inhibitor with equipotent inhibition of WT and mutant MEK1/2, whose drug-like properties allow further investigation in the mutant MEK setting upon BRAF/MEK therapy.

DOI: https://doi.org/10.1021/acs.jmedchem.1c02192