bims-pimaco 2022-12-04 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–12–04
nine papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Discovery of GDC-0077 (Inavolisib), a Highly Selective Inhibitor and Degrader of Mutant PI3Kα.
Development of SOS1 Inhibitor-Based Degraders to Target KRAS-Mutant Colorectal Cancer.
Elevated FSP1 protects KRAS-mutated cells from ferroptosis during tumor initiation.
Ras drives malignancy through stem cell crosstalk with the microenvironment.
Clinical and functional characterization of germline PIK3CA variants in patients with PIK3CA-related overgrowth Spectrum disorders.
ARD1 stabilizes NRF2 through direct interaction and promotes colon cancer progression.
Serum and glucocorticoid-regulated kinase 1: Structure, biological functions, and its inhibitors.
Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS.
Mother cells control daughter cell proliferation in intestinal organoids to minimize proliferation fluctuations.

J Med Chem. 2022 Dec 01.

Discovery of GDC-0077 (Inavolisib), a Highly Selective Inhibitor and Degrader of Mutant PI3Kα.

Emily J Hanan, Marie-Gabrielle Braun, Robert A Heald, Calum MacLeod, Connie Chan, Saundra Clausen, Kyle A Edgar, Charles Eigenbrot, Richard Elliott, Nicholas Endres, Lori S Friedman, Emily Gogol, Xiao-Hui Gu, Rebecca Hong Thibodeau, Philip S Jackson, James R Kiefer, Jamie D Knight, Michelle Nannini, Raman Narukulla, Amanda Pace, Jodie Pang, Hans E Purkey, Laurent Salphati, Deepak Sampath, Stephen Schmidt, Steve Sideris, Kyung Song, Swathi Sujatha-Bhaskar, Mark Ultsch, Heidi Wallweber, Jianfeng Xin, SiewKuen Yeap, Amy Young, Yu Zhong, Steven T Staben.

Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib, 32), which is now under evaluation in a Phase III clinical trial as a treatment for patients with PIK3CA-mutant breast cancer.

DOI: https://doi.org/10.1021/acs.jmedchem.2c01422
J Med Chem. 2022 Dec 02.

Development of SOS1 Inhibitor-Based Degraders to Target KRAS-Mutant Colorectal Cancer.

Yujia Bian, Diego Alem, Francisca Beato, Tara L Hogenson, Xinrui Yang, Kun Jiang, Jianfeng Cai, Wen Wee Ma, Martin Fernandez-Zapico, Aik Choon Tan, Nicholas J Lawrence, Jason B Fleming, Yu Yuan, Hao Xie.

Direct blockade of KRAS driver mutations in colorectal cancer (CRC) has been challenging. Targeting SOS1, a guanine nucleotide exchange factor, has arisen as an attractive approach for KRAS-mutant CRC. Here, we describe the development of novel SOS1 degraders and their activity in patient-derived CRC organoids (PDO). The design of these degraders as proteolysis-targeting chimera was based on the crystal structures of cereblon and SOS1. The synthesis used the 6- and 7-OH groups of a quinazoline core as anchor points to connect lenalidomide. Fifteen compounds were screened for SOS1 degradation. P7 was found to have up to 92% SOS1 degradation in both CRC cell lines and PDOs with excellent specificity. SOS1 degrader P7 demonstrated superior activity in inhibiting CRC PDO growth with an IC50 5 times lower than that of SOS1 inhibitor BI3406. In summary, we developed new SOS1 degraders and demonstrated SOS1 degradation as a feasible therapeutic strategy for KRAS-mutant CRC.

DOI: https://doi.org/10.1021/acs.jmedchem.2c01300
Cell Death Differ. 2022 Nov 29.

Elevated FSP1 protects KRAS-mutated cells from ferroptosis during tumor initiation.

Fabienne Müller, Jonathan K M Lim, Christina M Bebber, Eric Seidel, Sofya Tishina, Alina Dahlhaus, Jenny Stroh, Julia Beck, Fatma Isil Yapici, Keiko Nakayama, Lucia Torres Fernández, Johannes Brägelmann, Gabriel Leprivier, Silvia von Karstedt.

Oncogenic KRAS is the key driver oncogene for several of the most aggressive human cancers. One key feature of oncogenic KRAS expression is an early increase in cellular reactive oxygen species (ROS) which promotes cellular transformation if cells manage to escape cell death, mechanisms of which remain incompletely understood. Here, we identify that expression of oncogenic as compared to WT KRAS in isogenic cellular systems renders cells more resistant to ferroptosis, a recently described type of regulated necrosis. Mechanistically, we find that cells with mutant KRAS show a specific lack of ferroptosis-induced lipid peroxidation. Interestingly, KRAS-mutant cells upregulate expression of ferroptosis suppressor protein 1 (FSP1). Indeed, elevated levels of FSP1 in KRAS-mutant cells are responsible for mediating ferroptosis resistance and FSP1 is upregulated as a consequence of MAPK and NRF2 pathway activation downstream of KRAS. Strikingly, FSP1 activity promotes cellular transformation in soft agar and its overexpression is sufficient to promote spheroid growth in 3D in KRAS WT cells. Moreover, FSP1 expression and its activity in ferroptosis inhibition accelerates tumor onset of KRAS WT cells in the absence of oncogenic KRAS in vivo. Consequently, we find that pharmacological induction of ferroptosis in pancreatic organoids derived from the LsL-KRASG12D expressing mouse model is only effective in combination with FSP1 inhibition. Lastly, FSP1 is upregulated in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) as compared to the respective normal tissue of origin and correlates with NRF2 expression in PDAC patient datasets. Based on these data, we propose that KRAS-mutant cells must navigate a ferroptosis checkpoint by upregulating FSP1 during tumor establishment. Consequently, ferroptosis-inducing therapy should be combined with FSP1 inhibitors for efficient therapy of KRAS-mutant cancers.

DOI: https://doi.org/10.1038/s41418-022-01096-8
Nature. 2022 Nov 30.

Ras drives malignancy through stem cell crosstalk with the microenvironment.

Shaopeng Yuan, Katherine S Stewart, Yihao Yang, Merve Deniz Abdusselamoglu, S Martina Parigi, Tamar Y Feinberg, Karen Tumaneng, Hanseul Yang, John M Levorse, Lisa Polak, David Ng, Elaine Fuchs.

Squamous cell carcinomas are triggered by marked elevation of RAS-MAPK signalling and progression from benign papilloma to invasive malignancy1-4. At tumour-stromal interfaces, a subset of tumour-initiating progenitors, the cancer stem cells, obtain increased resistance to chemotherapy and immunotherapy along this pathway5,6. The distribution and changes in cancer stem cells during progression from a benign state to invasive squamous cell carcinoma remain unclear. Here we show in mice that, after oncogenic RAS activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signalling crosstalk with their tissue microenvironment that drives their malignant progression. The non-genetic, dynamic cascade of intercellular exchanges involves downstream pathways that are often mutated in advanced metastatic squamous cell carcinomas with high mutational burden7. Coupling our clonal skin HRASG12V mouse model with single-cell transcriptomics, chromatin landscaping, lentiviral reporters and lineage tracing, we show that aberrant crosstalk between cancer stem cells and their microenvironment triggers angiogenesis and TGFβ signalling, creating conditions that are conducive for hijacking leptin and leptin receptor signalling, which in turn launches downstream phosphoinositide 3-kinase (PI3K)-AKT-mTOR signalling during the benign-to-malignant transition. By functionally examining each step in this pathway, we reveal how dynamic temporal crosstalk with the microenvironment orchestrated by the stem cells profoundly fuels this path to malignancy. These insights suggest broad implications for cancer therapeutics.

DOI: https://doi.org/10.1038/s41586-022-05475-6
Hum Mol Genet. 2022 Dec 02. pii: ddac296. [Epub ahead of print]

Clinical and functional characterization of germline PIK3CA variants in patients with PIK3CA-related overgrowth Spectrum disorders.

Jessica A Cooley Coleman, Jennifer M Gass, Sujata Srikanth, Rini Pauly, Catherine A Ziats, David B Everman, Steven A Skinner, Shannon Bell, Raymond J Louie, Lauren Cascio, Wesley G Patterson, Julie R Jones, Nataliya Di Donato, Roger E Stevenson, Luigi Boccuto.

Mosaic variants in the PIK3CA gene, encoding the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), produce constitutive PI3K activation which causes PIK3CA-related overgrowth spectrum (PROS) disorders. To date, fewer than 20 patients have been described with germline alterations in PIK3CA. In this study, we describe three unrelated individuals with overgrowth and germline PIK3CA variants. These variants were discovered through whole-exome sequencing and confirmed as germline by testing multiple tissue types, when available. Functional analysis using Patient 1's fibroblast cell line and two previously reported patients' cell lines showed increased phosphorylation of AKT during cellular starvation revealing constitutive activation of the PI3K/AKT/mTOR pathway. Alternatively, stimulation of the cells by fetal bovine serum produced a reduced response, indicating an activated status of the PI3K complex reducing the pathway response to further external stimulation. Additional studies utilizing Biolog Phenotype Microarray technology indicated reduced energy production when cells were exposed to growth factors stimulating the PI3K/AKT/mTOR pathway, confirming the trend observed in the AKT phosphorylation test after stimulation. Furthermore, treatment with inhibitors of the PI3K/AKT/mTOR pathway rescued the normal energy response in the patients' cells. Collectively, these data demonstrate that disease-causing germline PIK3CA variants have a functional consequence, similar to mosaic variants in the PI3K/AKT/mTOR pathway.

DOI: https://doi.org/10.1093/hmg/ddac296
Life Sci. 2022 Nov 25. pii: S0024-3205(22)00917-1. [Epub ahead of print] 121217

ARD1 stabilizes NRF2 through direct interaction and promotes colon cancer progression.

Xizhu Fang, Yeon-Hwa Lee, Jeong-Hoon Jang, Su-Jung Kim, Seong Hoon Kim, Do-Hee Kim, Hye-Kyung Na, Kyung-Ok Kim, Jeong-Heum Baek, Young-Joon Surh.

   AIMS: Aberrant overactivation/overexpression of NRF2 is implicated as a driving event in tumor progression, which has been attributed to its mutation or inactivation of the inhibitory protein, KEAP1. However, alternative mechanisms responsible for sustained activation of NRF2 are less understood.
MAIN METHODS: Human colon cancer cell lines and tissues obtained from colorectal cancer patients were used. To examine the expression levels of ARD1 and NRF2, Western blot and immunofluorescence analyses were performed. To investigate the potential relevance of NRF2 and ARD1 to human colorectal cancer (CRC), NRF2 and ARD1 were individually silenced in human CRC cells (HCT-116) by transfection with their specific small interfering RNA (siRNA). To determine the functional role of ARD1 in NRF2 regulation, PLA, immunoprecipitation, nano-LC-ESI MS/MS, and in vitro acetylation assays were performed.
KEY FINDINGS: ARD1 knockdown in human colon cancer cell lines significantly reduced the protein levels of NRF2 without affecting its mRNA expression; however, silencing of NRF2 did not alter ARD1 protein expression. In addition, these two proteins were co-localized and physically interacted with each other both in human colon cancer cells (HCT-116) and human colon tumor tissues. Mechanistically, ARD1 overexpression increased the acetylation levels of NRF2. Moreover, an in vitro acetylation assay and mass spectrometric analysis demonstrated that ARD1 could directly acetylate NRF2. Ectopic expression of mutant forms of ARD1 with defective acetyltransferase activity reduced the stability of NRF2.
SIGNIFICANCE: In conclusion, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer by stabilizing this transcription factor.

Keywords:  ARD1; Acetylation; Colorectal cancer; NRF2; Posttranslational modification

DOI:  https://doi.org/10.1016/j.lfs.2022.121217
Front Pharmacol. 2022 ;13 1036844

Serum and glucocorticoid-regulated kinase 1: Structure, biological functions, and its inhibitors.

Hyunsoo Jang, Youngjun Park, Jaebong Jang.

  Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase belonging to the protein kinase A, G, and C (AGC) family. Upon initiation of the phosphoinositide 3-kinase (PI3K) signaling pathway, mammalian target of rapamycin complex 2 (mTORC2) and phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylate the hydrophobic motif and kinase domain of SGK1, respectively, inducing SGK1 activation. SGK1 modulates essential cellular processes such as proliferation, survival, and apoptosis. Hence, dysregulated SGK1 expression can result in multiple diseases, including hypertension, cancer, autoimmunity, and neurodegenerative disorders. This review provides a current understanding of SGK1, particularly in sodium transport, cancer progression, and autoimmunity. In addition, we summarize the developmental status of SGK1 inhibitors, their structures, and respective potencies evaluated in pre-clinical experimental settings. Collectively, this review highlights the significance of SGK1 and proposes SGK1 inhibitors as potential drugs for treatment of clinically relevant diseases.

Keywords:  SGK1; T cell modulation; cancer; ion channel; kinase inhibitor

DOI:  https://doi.org/10.3389/fphar.2022.1036844
Elife. 2022 Dec 02. pii: e79747. [Epub ahead of print]11

Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS.

Metehan Ilter, Ramazan Kaşmer, Farzaneh Jalalypour, Canan Atilgan, Ozan Topcu, Nihal Karakaş, Ozge Sensoy.

  Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.

Keywords:  molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.79747
Elife. 2022 Nov 29. pii: e80682. [Epub ahead of print]11

Mother cells control daughter cell proliferation in intestinal organoids to minimize proliferation fluctuations.

Guizela Huelsz-Prince, Rutger Nico Ulbe Kok, Yvonne Goos, Lotte Bruens, Xuan Zheng, Saskia Ellenbroek, Jacco Van Rheenen, Sander Tans, Jeroen S van Zon.

  During renewal of the intestine, cells are continuously generated by proliferation. Proliferation and differentiation must be tightly balanced, as any bias toward proliferation results in uncontrolled exponential growth. Yet, the inherently stochastic nature of cells raises the question how such fluctuations are limited. We used time-lapse microscopy to track all cells in crypts of growing mouse intestinal organoids for multiple generations, allowing full reconstruction of the underlying lineage dynamics in space and time. Proliferative behavior was highly symmetric between sister cells, with both sisters either jointly ceasing or continuing proliferation. Simulations revealed that such symmetric proliferative behavior minimizes cell number fluctuations, explaining our observation that proliferating cell number remained constant even as crypts increased in size considerably. Proliferative symmetry did not reflect positional symmetry but rather lineage control through the mother cell. Our results indicate a concrete mechanism to balance proliferation and differentiation with minimal fluctuations that may be broadly relevant for other tissues.

Keywords:  cell lineage; cell proliferation; developmental biology; fluctuations; intestinal epithelium; mouse; physics of living systems; stem cell dynamics; time-lapse microscopy

DOI:  https://doi.org/10.7554/eLife.80682