bims-pimaco 2022-09-11 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–09–11
twelve papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

mTORC2 Is the Major Second Layer Kinase Negatively Regulating FOXO3 Activity.
The Genomic Environment of BRAF Mutated and BRAF/PIK3CA Double Mutated Colorectal Cancers.
Genomic Landscapes and Hallmarks of Mutant RAS in Human Cancers.
Combinatorial approaches for mitigating resistance to KRAS-targeted therapies.
Computer-Aided Drug Design Boosts RAS Inhibitor Discovery.
Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach.
Long-term treatment with low dose rapamycin extends lifespan and delays tumours in cancer-prone germline PTEN mutant mice.
Pharmacologic inhibition of LAT1 predominantly suppresses transport of large neutral amino acids and downregulates global translation in cancer cells.
A comprehensive review of SHP2 and its role in cancer.
Phospholipid Scramblase 4 (PLSCR4) Regulates Adipocyte Differentiation via PIP3-Mediated AKT Activation.
Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging.
Modeling Colorectal Cancer Progression Reveals Niche-Dependent Clonal Selection.

Molecules. 2022 Aug 24. pii: 5414. [Epub ahead of print]27(17):

mTORC2 Is the Major Second Layer Kinase Negatively Regulating FOXO3 Activity.

Lucia Jimenez, Carlos Amenabar, Victor Mayoral-Varo, Thomas A Mackenzie, Maria C Ramos, Andreia Silva, Giampaolo Calissi, Inês Grenho, Carmen Blanco-Aparicio, Joaquin Pastor, Diego Megías, Bibiana I Ferreira, Wolfgang Link.

  Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.

Keywords:  FOXO; aging; cancer; chemical biology; high content screening; kinases; mTOR

DOI:  https://doi.org/10.3390/molecules27175414
J Clin Med. 2022 Aug 31. pii: 5132. [Epub ahead of print]11(17):

The Genomic Environment of BRAF Mutated and BRAF/PIK3CA Double Mutated Colorectal Cancers.

Ioannis A Voutsadakis.

   BACKGROUND: Colorectal cancer represents the most prevalent gastrointestinal malignancy. Prognosis of metastatic disease has improved in recent years with the introduction of effective systemic therapies, but mean survival remains in the range of two to three years. Targeted therapies based on specific molecular alterations in sub-sets of colorectal cancers have the potential of contributing to therapeutic progress. BRAF and PIK3CA are oncogenic kinases commonly mutated in colorectal cancers and can be targeted through small molecule kinase inhibitors.
METHODS: Clinical and genomic data from two extensive series of colorectal cancers were interrogated to define the molecular characteristics of cancers with BRAF mutations with and without concomitant mutations in PIK3CA.
RESULTS: Colorectal cancers that are BRAF and PIK3CA double mutants represent a small minority of about 5% of colorectal cancers in the two examined series of mostly localized disease. They also represent about one third of all BRAF mutated colorectal cancers. Most mutations in BRAF are classic V600E mutations. A high prevalence of MSI and CIMP is observed in BRAF mutated colorectal cancers with or without PIK3CA mutations. Mutations in tumor suppressors FBXW7 and ATM display a higher prevalence in BRAF mutated cancers. The prognosis of BRAF mutated colorectal cancers with or without PIK3CA mutations is not significantly different than counterparts with wild type BRAF. This contrasts with the known adverse prognostic effect of BRAF in metastatic disease and relates to the different prevalence of MSI in mutant BRAF localized versus metastatic colorectal cancers.
CONCLUSIONS: BRAF mutations are the defining molecular alterations in double mutant BRAF and PIK3CA colorectal cancers as determined by increased MSI and CIMP in BRAF subsets with and without PIK3CA mutations. Moreover, BRAF mutated cancers with and without PIK3CA mutations are characterized by the absence of KRAS mutations and a lower prevalence of APC mutations than BRAF wild type counterparts. Mismatch-repair-associated gene mutations display higher frequencies in BRAF mutated colorectal cancers. Despite the absence of prognosis implications of BRAF mutations in the studied cohorts of mostly localized cancers, such mutations could be prognostic in certain subsets. The presence of mutations in other genes, such as ATM and high MSI status present opportunities for combination therapies.

Keywords:  gastrointestinal cancers; genomics; kinase; molecular alterations; signal transduction

DOI:  https://doi.org/10.3390/jcm11175132
Cancer Res. 2022 Sep 08. pii: CAN-22-1731. [Epub ahead of print]

Genomic Landscapes and Hallmarks of Mutant RAS in Human Cancers.

Robert B Scharpf, Archana Balan, Biagio Ricciuti, Jacob Fiksel, Christopher Cherry, Chenguang Wang, Michele L LeNoue-Newton, Hira A Rizvi, James R White, Alexander S Baras, Jordan Anaya, Blair V Landon, Marta Majcherska-Agrawal, Paola Ghanem, Jocelyn Lee, Leon Raskin, Andrew S Park, Huakang Tu, Hil Hsu, Kathryn C Arbour, Mark M Awad, Gregory J Riely, Christine M Lovly, Valsamo Anagnostou.

The RAS family of small GTPases represents the most commonly activated oncogenes in human cancers. To better understand the prevalence of somatic RAS mutations and the compendium of genes that are co-altered in RAS mutant tumors, we analyzed targeted next-generation sequence data of 607,863 mutations from 66,372 tumors in 51 cancer types in the AACR Project GENIE Registry. Bayesian hierarchical models were implemented to estimate the cancer-specific prevalence of RAS and non-RAS somatic mutations, to evaluate co-occurrence and mutual exclusivity, and to model the effects of tumor mutational burden and mutational signatures on co-mutation patterns. These analyses revealed differential RAS prevalence and co-mutations with non-RAS genes in a cancer lineage and context-dependent manner, with differences across age, sex and ethnic groups. Allele specific RAS co-mutational patterns included an enrichment in NTRK3 and chromatin-regulating gene mutations in KRAS G12C-mutant non-small cell lung cancer. Integrated multi-omic analyses of 10,217 tumors from TCGA revealed distinct genotype-driven gene expression programs pointing to differential recruitment of cancer hallmarks as well as phenotypic differences and immune surveillance states in the tumor microenvironment of RAS mutant tumors. The distinct genomic tracks discovered in RAS mutant tumors reflected differential clinical outcomes in the TCGA cohort and in an independent cohort of patients with KRAS G12C mutant non-small cell lung cancer that received immunotherapy containing regimens. The RAS genetic architecture points to cancer lineage-specific therapeutic vulnerabilities that can be leveraged for rationally combining RAS mutant allele-directed therapies with targeted therapies and immunotherapy.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-1731
Biochem J. 2022 Sep 06. pii: BCJ20220440. [Epub ahead of print]

Combinatorial approaches for mitigating resistance to KRAS-targeted therapies.

Hannah R Warren, Sarah Ross, Paul D Smith, Judy M Coulson, Ian A Prior.

  Approximately 15% of all cancer patients harbor mutated KRAS. Direct inhibitors of KRAS have now been generated and are beginning to make progress through clinical trials. These include a suite of inhibitors targeting the KRASG12C mutation commonly found in lung cancer. We investigated emergent resistance to representative examples of different classes of Ras targeted therapies. They all exhibited rapid reactivation of Ras signaling within days of exposure and adaptive responses continued to change over long-term treatment schedules. Whilst the gene signatures were distinct for each inhibitor, they commonly involved upregulation of upstream nodes promoting mutant and wild type Ras activation. Experiments to reverse resistance unfortunately revealed frequent desensitization to members of a panel of anti-cancer therapeutics, suggesting that salvage approaches are unlikely to be feasible. Instead, we identified triple inhibitor combinations that resulted in more durable responses to KRAS inhibitors and that may benefit from further pre-clinical evaluation.

Keywords:  G-proteins; drug resistance; signalling

DOI:  https://doi.org/10.1042/BCJ20220440
Molecules. 2022 Sep 05. pii: 5710. [Epub ahead of print]27(17):

Computer-Aided Drug Design Boosts RAS Inhibitor Discovery.

Ge Wang, Yuhao Bai, Jiarui Cui, Zirui Zong, Yuan Gao, Zhen Zheng.

  The Rat Sarcoma (RAS) family (NRAS, HRAS, and KRAS) is endowed with GTPase activity to regulate various signaling pathways in ubiquitous animal cells. As proto-oncogenes, RAS mutations can maintain activation, leading to the growth and proliferation of abnormal cells and the development of a variety of human cancers. For the fight against tumors, the discovery of RAS-targeted drugs is of high significance. On the one hand, the structural properties of the RAS protein make it difficult to find inhibitors specifically targeted to it. On the other hand, targeting other molecules in the RAS signaling pathway often leads to severe tissue toxicities due to the lack of disease specificity. However, computer-aided drug design (CADD) can help solve the above problems. As an interdisciplinary approach that combines computational biology with medicinal chemistry, CADD has brought a variety of advances and numerous benefits to drug design, such as the rapid identification of new targets and discovery of new drugs. Based on an overview of RAS features and the history of inhibitor discovery, this review provides insight into the application of mainstream CADD methods to RAS drug design.

Keywords:  RAS inhibitor; computer-aided drug design; molecular docking; molecular dynamics simulation; virtual screening

DOI:  https://doi.org/10.3390/molecules27175710
Anal Chem. 2022 Sep 09.

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach.

Lingyao Meng, Emily W Chan, Carl Ng, Junko Aimi, John C Tran, Angela J Oh, Mark Merchant, Hans E Purkey, Timothy P Heffron, Surinder Kaur, Keyang Xu, Zhen Shi, Jintang He.

KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C-positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/μg of total protein has been achieved for both free and GDC-6036-bound KRAS G12C with as little as 4 μg of total protein extracted from human tumor samples. This sub-fmol/μg level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.

DOI: https://doi.org/10.1021/acs.analchem.2c03146
J Pathol. 2022 Sep 08.

Long-term treatment with low dose rapamycin extends lifespan and delays tumours in cancer-prone germline PTEN mutant mice.

Priyanka Tibarewal, Victoria Rathbone, Georgia Constantinou, Wayne Pearce, Mahreen Adil, Zofia Varyova, Lisa Folkes, Alix Hampson, Gala Anastasia Electra Classen, Adriana Alves, Sara Carvalho, Cheryl L Scudamore, Bart Vanhaesebroeck.

  PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underly the PTEN Hamartoma Tumour Syndrome (PHTS), a rare human cancer-predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI-3-kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralize the impact of PTEN loss, rendering this a more druggable context compared to those of other tumour suppressor pathways such as loss of TP53. However, this has not been explored in cancer prevention in a model of germline cancer predisposition, such as PHTS. Clinical trials of short-term treatment with rapamycin have recently been initiated for PHTS, focusing on cognition and colon polyposis. Here, we administered a low dose of rapamycin from the age of 6-weeks onwards to mice with heterozygous germline PTEN loss, a mouse model that recapitulates most characteristics of human PHTS. Rapamycin was well-tolerated and led to a highly significant improvement of survival in both male and female mice. This was accompanied by a delay, but not full blockade of the development of a range of proliferative lesions, including gastro-intestinal and thyroid tumours and endometrial hyperplasia, with no impact on mammary and prostate tumours, and no effect on brain overgrowth. Our data indicate that rapamycin may have cancer prevention potential in human PHTS. This might also be the case for sporadic cancers in which genetic PI3K pathway activation is an early event in tumour development, such as endometrial cancer and some breast cancers. To the best of our knowledge, this is the first report of a long-term treatment of a germline cancer predisposition model with a PI3K/mTOR pathway inhibitor This article is protected by copyright. All rights reserved.

Keywords:  PHTS; PI 3-kinase; PTEN; cancer prevention; drug; hamartoma; kinase inhibitor; mTORC1; rapamycin; rare disease; syndrome

DOI:  https://doi.org/10.1002/path.6009
J Cell Mol Med. 2022 Sep 07.

Pharmacologic inhibition of LAT1 predominantly suppresses transport of large neutral amino acids and downregulates global translation in cancer cells.

Kou Nishikubo, Ryuichi Ohgaki, Hiroki Okanishi, Suguru Okuda, Minhui Xu, Hitoshi Endou, Yoshikatsu Kanai.

  L-type amino acid transporter 1 (LAT1; SLC7A5), which preferentially transports large neutral amino acids, is highly upregulated in various cancers. LAT1 supplies cancer cells with amino acids as substrates for enhanced biosynthetic and bioenergetic reactions and stimulates signalling networks involved in the regulation of survival, growth and proliferation. LAT1 inhibitors show anti-cancer effects and a representative compound, JPH203, is under clinical evaluation. However, pharmacological impacts of LAT1 inhibition on the cellular amino acid transport and the translational activity in cancer cells that are conceptually pivotal for its anti-proliferative effect have not been elucidated yet. Here, we demonstrated that JPH203 drastically inhibits the transport of all the large neutral amino acids in pancreatic ductal adenocarcinoma cells. The inhibitory effects of JPH203 were observed even in competition with high concentrations of amino acids in a cell culture medium. The analyses of the nutrient-sensing mTORC1 and GAAC pathways and the protein synthesis activity revealed that JPH203 downregulates the global translation. This study demonstrates a predominant contribution of LAT1 to the transport of large neutral amino acids in cancer cells and the suppression of protein synthesis by JPH203 supposed to underly its broad anti-proliferative effects across various types of cancer cells.

Keywords:  LAT1; PDAC; SLC7A5; amino acid transporter; anti-cancer agent; global translation; inhibitor; large neutral amino acids

DOI:  https://doi.org/10.1111/jcmm.17553
Cell Oncol (Dordr). 2022 Sep 06.

A comprehensive review of SHP2 and its role in cancer.

Moges Dessale Asmamaw, Xiao-Jing Shi, Li-Rong Zhang, Hong-Min Liu.

  Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase ubiquitously expressed mainly in the cytoplasm of several tissues. SHP2 modulates diverse cell signaling events that control metabolism, cell growth, differentiation, cell migration, transcription and oncogenic transformation. It interacts with diverse molecules in the cell, and regulates key signaling events including RAS/ERK, PI3K/AKT, JAK/STAT and PD-1 pathways downstream of several receptor tyrosine kinases (RTKs) upon stimulation by growth factors and cytokines. SHP2 acts as both a phosphatase and a scaffold, and plays prominently oncogenic functions but can be tumor suppressor in a context-dependent manner. It typically acts as a positive regulator of RTKs signaling with some inhibitory functions reported as well. SHP2 expression and activity is regulated by such factors as allosteric autoinhibition, microRNAs, ubiquitination and SUMOylation. Dysregulation of SHP2 expression or activity causes many developmental diseases, and hematological and solid tumors. Moreover, upregulated SHP2 expression or activity also decreases sensitivity of cancer cells to anticancer drugs. SHP2 is now considered as a compelling anticancer drug target and several classes of SHP2 inhibitors with different mode of action are developed with some already in clinical trial phases. Moreover, novel SHP2 substrates and functions are rapidly growing both in cell and cancer. In view of this, we comprehensively and thoroughly reviewed literatures about SHP2 regulatory mechanisms, substrates and binding partners, biological functions, roles in human cancers, and different classes of small molecule inhibitors target this oncoprotein in cancer.

Keywords:  Gab1; PTPN11; SHP2; SHP2 inhibitors; Tyrosine phosphorylation

DOI:  https://doi.org/10.1007/s13402-022-00698-1
Int J Mol Sci. 2022 Aug 29. pii: 9787. [Epub ahead of print]23(17):

Phospholipid Scramblase 4 (PLSCR4) Regulates Adipocyte Differentiation via PIP3-Mediated AKT Activation.

Lisa A G Barth, Michèle Nebe, Hermann Kalwa, Akhil Velluva, Stephanie Kehr, Florentien Kolbig, Patricia Prabutzki, Wieland Kiess, Diana Le Duc, Antje Garten, Anna S Kirstein.

  Phospholipid scramblase 4 (PLSCR4) is a member of a conserved enzyme family with high relevance for the remodeling of phospholipid distribution in the plasma membrane and the regulation of cellular signaling. While PLSCR1 and -3 are involved in the regulation of adipose-tissue expansion, the role of PLSCR4 is so far unknown. PLSCR4 is significantly downregulated in an adipose-progenitor-cell model of deficiency for phosphatase and tensin homolog (PTEN). PTEN acts as a tumor suppressor and antagonist of the growth and survival signaling phosphoinositide 3-kinase (PI3K)/AKT cascade by dephosphorylating phosphatidylinositol-3,4,5-trisphosphate (PIP3). Patients with PTEN germline deletion frequently develop lipomas. The underlying mechanism for this aberrant adipose-tissue growth is incompletely understood. PLSCR4 is most highly expressed in human adipose tissue, compared with other phospholipid scramblases, suggesting a specific role of PLSCR4 in adipose-tissue biology. In cell and mouse models of lipid accumulation, we found PLSCR4 to be downregulated. We observed increased adipogenesis in PLSCR4-knockdown adipose progenitor cells, while PLSCR4 overexpression attenuated lipid accumulation. PLSCR4 knockdown was associated with increased PIP3 levels and the activation of AKT. Our results indicated that PLSCR4 is a regulator of PI3K/AKT signaling and adipogenesis and may play a role in PTEN-associated adipose-tissue overgrowth and lipoma formation.

Keywords:  PIP3; PLSCR4; PTEN; adipogenesis; lipoma; scramblase

DOI:  https://doi.org/10.3390/ijms23179787
Nat Commun. 2022 Sep 09. 13(1): 5317

Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging.

Eva C Freckmann, Emma Sandilands, Erin Cumming, Matthew Neilson, Alvaro Román-Fernández, Konstantina Nikolatou, Marisa Nacke, Tamsin R M Lannagan, Ann Hedley, David Strachan, Mark Salji, Jennifer P Morton, Lynn McGarry, Hing Y Leung, Owen J Sansom, Crispin J Miller, David M Bryant.

Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture.

DOI: https://doi.org/10.1038/s41467-022-32958-x
Cancers (Basel). 2022 Aug 31. pii: 4260. [Epub ahead of print]14(17):

Modeling Colorectal Cancer Progression Reveals Niche-Dependent Clonal Selection.

Nuria Vaquero-Siguero, Nikolai Schleussner, Julia Volk, Manuel Mastel, Jasmin Meier, Rene Jackstadt.

  Colorectal cancer (CRC) is among the deadliest cancers worldwide, with metastasis being the main cause of patient mortality. During CRC progression the complex tumor ecosystem changes in its composition at virtually every stage. However, clonal dynamics and associated niche-dependencies at these stages are unknown. Hence, it is of importance to utilize models that faithfully recapitulate human CRC to define its clonal dynamics. We used an optical barcoding approach in mouse-derived organoids (MDOs) that revealed niche-dependent clonal selection. Our findings highlight that clonal selection is controlled by a site-specific niche, which critically contributes to cancer heterogeneity and has implications for therapeutic intervention.

Keywords:  clonal selection; colorectal cancer; metastasis; mouse models; mouse-derived organoids; niche; orthotopic transplantation; tumor heterogeneity

DOI:  https://doi.org/10.3390/cancers14174260