bims-pimaco 2023-02-26 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2023–02–26
eleven papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

With a little help from my T friends: T cells increase efficacy of KRAS (G12D) inhibitors.
Genome-wide CRISPR/Cas9 screens reveal shared and cell-specific mechanisms of resistance to SHP2 inhibition.
Binding and selectivity studies of phosphatidylinositol 3-kinase (PI3K) inhibitors.
AKT blocks SIK1-mediated repression of STAT3 to promote breast tumorigenesis.
The impact of RAS mutation on the treatment strategy of colorectal cancer.
Nuclear PTEN's Functions in Suppressing Tumorigenesis: Implications for Rare Cancers.
Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance.
Peptides That Block RAS-p21 Protein-Induced Cell Transformation.
Monobody inhibitor selective to the phosphatase domain of SHP2 and its use as a probe for quantifying SHP2 allosteric regulation.
High dose isoleucine stabilizes nuclear PTEN to suppress the proliferation of lung cancer.
The KRAS-Mutant Consensus Molecular Subtype 3 Reveals an Immunosuppressive Tumor Microenvironment in Colorectal Cancer.

Cell Rep Med. 2023 Feb 21. pii: S2666-3791(23)00042-3. [Epub ahead of print]4(2): 100950

With a little help from my T friends: T cells increase efficacy of KRAS (G12D) inhibitors.

Sara Mainardi.

Mutation-selective KRAS inhibitors are revolutionizing the treatment of RAS-mutant tumors but cannot achieve durable effects alone. Kemp and colleagues1 recently showed how the KRAS-G12D-specific inhibitor MRTX1133, while impairing cancer proliferation, stimulates T cell infiltration, which is crucial for sustained disease control.

DOI: https://doi.org/10.1016/j.xcrm.2023.100950
J Exp Med. 2023 May 01. pii: e20221563. [Epub ahead of print]220(5):

Genome-wide CRISPR/Cas9 screens reveal shared and cell-specific mechanisms of resistance to SHP2 inhibition.

Wei Wei, Mitchell J Geer, Xinyi Guo, Igor Dolgalev, Neville E Sanjana, Benjamin G Neel.

SHP2 (PTPN11) acts upstream of SOS1/2 to enable RAS activation. Allosteric SHP2 inhibitors (SHP2i) in the clinic prevent SHP2 activation, block proliferation of RTK- or cycling RAS mutant-driven cancers, and overcome "adaptive resistance." To identify SHP2i resistance mechanisms, we performed genome-wide CRISPR/Cas9 knockout screens on two SHP2i-sensitive cell lines, recovering genes expected to cause resistance (NF1, PTEN, CDKN1B, LZTR1, and RASA2) and novel targets (INPPL1, MAP4K5, epigenetic modifiers). We screened 14 additional lines with a focused CRISPR library targeting common "hits" from the genome-wide screens. LZTR1 deletion conferred resistance in 12/14 lines, followed by MAP4K5 (8/14), SPRED2/STK40 (6/14), and INPPL1 (5/14). INPPL1, MAP4K5, or LZTR1 deletion reactivated ERK signaling. INPPL1-mediated sensitization to SHP2i required its NPXY motif but not lipid phosphatase activity. MAP4K5 acted upstream of MEK through a kinase-dependent target(s); LZTR1 had cell-dependent effects on RIT and RAS stability. INPPL1, MAP4K5, or LZTR1 deletion also conferred SHP2i resistance in vivo. Defining the SHP2i resistance landscape could suggest effective combination approaches.

DOI: https://doi.org/10.1084/jem.20221563
J Mol Graph Model. 2023 Feb 14. pii: S1093-3263(23)00031-1. [Epub ahead of print]121 108433

Binding and selectivity studies of phosphatidylinositol 3-kinase (PI3K) inhibitors.

Mohammad Al Hasan, Matthew Sabirianov, Grace Redwine, Kaitlin Goettsch, Stephen X Yang, Haizhen A Zhong.

  Overexpression of the Phosphatidylinositol 3-kinase (PI3K) proteins have been observed in cancer cells. Targeting the phosphatidylinositol 3-kinase (PI3K) signaling transduction pathway by inhibition of the PI3K substrate recognition sites has been proved to be an effective approach to block cancer progression. Many PI3K inhibitors have been developed. Seven drugs have been approved by the US FDA with a mechanism of targeting the phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. In this study, we used docking tools to investigate selective binding of ligands toward four different subtypes of PI3Ks (PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ). The affinity predicted from both the Glide dock and the Movable-Type (MT)-based free energy calculations agreed well with the experimental data. The validation of our predicted methods with a large dataset of 147 ligands showed very small mean errors. We identified residues that may dictate the subtype-specific binding. Particularly, residues Asp964, Ser806, Lys890 and Thr886 of PI3Kγ might be utilized for PI3Kγ-selective inhibitor design. Residues Val828, Trp760, Glu826 and Tyr813 may be important for PI3Kδ-selective inhibitor binding.

Keywords:  Anticancer; Free energy calculation; Molecular docking; Movable-type (MT); Phosphatidylinositol 3-kinase (PI3K); Selectivity

DOI:  https://doi.org/10.1016/j.jmgm.2023.108433
Cancer Res. 2023 Feb 20. pii: CAN-22-3407. [Epub ahead of print]

AKT blocks SIK1-mediated repression of STAT3 to promote breast tumorigenesis.

Zhicheng Sun, Qiwei Jiang, Bing Gao, Xiaomei Zhang, Lang Bu, Lei Wang, Ying Lin, Wei Xie, Jie Li, Jianping Guo.

The PI3K-AKT signaling pathway is frequently dysregulated in cancer, and it is hyperactivated in approximately 50% of breast cancers. While inhibitors directly targeting the PI3K-AKT axis have been developed, clinical efficacy has been limited to only a subset of patients. Identification of mechanisms underlying AKT-driven tumorigenesis could lead to alternative approaches to block pathway signaling and suppress breast tumor growth. Mass spectrometry-based analyses demonstrated that salt-inducible kinase 1 (SIK1) binds AKT and undergoes AKT-mediated phosphorylation, which compromises SIK1 tumor-suppressive functions. As a result, AKT relieved the binding and repression of STAT3 by SIK1 in a phosphorylation-dependent manner, resulting in breast cell tumorigenesis. Following AKT-mediated phosphorylation, SIK1 interacted with 14-3-3 and was translocated to the cytoplasm where the isomerase Pin1 facilitated SIK1 interaction with the E3 ligase ITCH to promote SIK1 ubiquitination and subsequent degradation. These findings indicate that SIK1 is a substrate of AKT that links AKT oncogenic function to STAT3 activation, highlighting targeting of the JAK2-STAT3 axis as a strategy to treat AKT-driven breast cancer.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-3407
Med Pharm Rep. 2023 Jan;96(1): 5-15

The impact of RAS mutation on the treatment strategy of colorectal cancer.

Edvina Elena Pirvu, Emilia Severin, Irina Niţă, Ştefania Andreea Toma.

  Kirsten rat sarcoma (KRAS) is the most frequently mutated oncogene in colorectal cancer, being present in 30% of patients with localized disease and in almost half of the patients that develop metastatic disease. While the development of chemotherapy doublets and targeted therapy have improved survival in recent years, KRAS mutation still has a controversial role regarding its prognostic and predictive value both in the adjuvant and in the metastatic setting. The impact of KRAS mutation on treatment strategy remains to be better defined. The development of new KRAS inhibitors promising new treatment options is on the horizon.

Keywords:  KRAS; adjuvant; colorectal cancer; metastatic

DOI:  https://doi.org/10.15386/mpr-2408
Biomolecules. 2023 Jan 30. pii: 259. [Epub ahead of print]13(2):

Nuclear PTEN's Functions in Suppressing Tumorigenesis: Implications for Rare Cancers.

Casey G Langdon.

  Phosphatase and tensin homolog (PTEN) encodes a tumor-suppressive phosphatase with both lipid and protein phosphatase activity. The tumor-suppressive functions of PTEN are lost through a variety of mechanisms across a wide spectrum of human malignancies, including several rare cancers that affect pediatric and adult populations. Originally discovered and characterized as a negative regulator of the cytoplasmic, pro-oncogenic phosphoinositide-3-kinase (PI3K) pathway, PTEN is also localized to the nucleus where it can exert tumor-suppressive functions in a PI3K pathway-independent manner. Cancers can usurp the tumor-suppressive functions of PTEN to promote oncogenesis by disrupting homeostatic subcellular PTEN localization. The objective of this review is to describe the changes seen in PTEN subcellular localization during tumorigenesis, how PTEN enters the nucleus, and the spectrum of impacts and consequences arising from disrupted PTEN nuclear localization on tumor promotion. This review will highlight the immediate need in understanding not only the cytoplasmic but also the nuclear functions of PTEN to gain more complete insights into how important PTEN is in preventing human cancers.

Keywords:  DNA damage; PTEN; PTEN hamartoma tumor syndrome; genetically engineered mouse models; nuclear PTEN; nuclear import; oncogenic transcriptional regulation; post-translational modifications; subcellular localization

DOI:  https://doi.org/10.3390/biom13020259
Nat Commun. 2023 Feb 18. 14(1): 923

Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance.

Daniel J Fazakerley, Julian van Gerwen, Kristen C Cooke, Xiaowen Duan, Elise J Needham, Alexis Díaz-Vegas, Søren Madsen, Dougall M Norris, Amber S Shun-Shion, James R Krycer, James G Burchfield, Pengyi Yang, Mark R Wade, Joseph T Brozinick, David E James, Sean J Humphrey.

The failure of metabolic tissues to appropriately respond to insulin ("insulin resistance") is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employ phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults causing insulin resistance, we observe a marked rewiring of the insulin signaling network. This includes both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying dysregulated phosphosites common to multiple insults reveals subnetworks containing non-canonical regulators of insulin action, such as MARK2/3, and causal drivers of insulin resistance. The presence of several bona fide GSK3 substrates among these phosphosites led us to establish a pipeline for identifying context-specific kinase substrates, revealing widespread dysregulation of GSK3 signaling. Pharmacological inhibition of GSK3 partially reverses insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that includes dysregulated MARK2/3 and GSK3 activity.

DOI: https://doi.org/10.1038/s41467-023-36549-2
Biomedicines. 2023 Feb 06. pii: 471. [Epub ahead of print]11(2):

Peptides That Block RAS-p21 Protein-Induced Cell Transformation.

Matthew R Pincus, Bo Lin, Purvi Patel, Elmer Gabutan, Nitzan Zohar, Wilbur B Bowne.

  This is a review of approaches to the design of peptides and small molecules that selectively block the oncogenic RAS-p21 protein in ras-induced cancers. Single amino acid substitutions in this protein, at critical positions such as at Gly 12 and Gln 61, cause the protein to become oncogenic. These mutant proteins cause over 90 percent of pancreatic cancers, 40-50 percent of colon cancers and about one third of non-small cell cancers of the lung (NSCCL). RAS-p21 is a G-protein that becomes activated when it exchanges GDP for GTP. Several promising approaches have been developed that target mutant (oncogenic) RAS-p21 proteins in these different cancers. These approaches comprise: molecular simulations of mutant and wild-type proteins to identify effector domains, for which peptides can be made that selectively inhibit the oncogenic protein that include PNC-1 (ras residues 115-126), PNC-2 (ras residues 96-110) and PNC7 (ras residues 35-47); the use of contiguous RAS-p21 peptide sequences that can block ras signaling; cyclic peptides from large peptide libraries and small molecule libraries that can be identified in high throughput assays that can selectively stabilize inactive forms of RAS-p21; informatic approaches to discover peptides and small molecules that dock to specific domains of RAS-p21 that can block mitogenic signal transduction by oncogenic RAS-p21; and the use of cell-penetrating peptides (CPPs) that are attached to the variable domains of the anti-RAS-p21 inactivating monoclonal antibody, Y13 259, that selectively enters oncogenic RAS-p21-containing cancer cells, causing these cells to undergo apoptosis. Several new anti-oncogenic RAS-p21 agents, i.e., Amgen's AMG510 and Mirati Therapeutics' MRTX849, polycyclic aromatic compounds, have recently been FDA-approved and are already being used clinically to treat RAS-p21-induced NSCCL and colorectal carcinomas. These new drugs target the inactive form of RAS-p21 bound to GDP with G12C substitution at the critical Gly 12 residue by binding to a groove bordered by specific domains in this mutant protein into which these compounds insert, resulting in the stabilization of the inactive GDP-bound form of RAS-p21. Other peptides and small molecules have been discovered that block the G12D-RAS-p21 oncogenic protein. These agents can treat specific mutant protein-induced cancers and are excellent examples of personalized medicine. However, many oncogenic RAS-p21-induced tumors are caused by other mutations at positions 12, 13 and 61, requiring other, more general anti-oncogenic agents that are being provided using alternate methods.

Keywords:  RAS-p21 protein; amino acid substitutions; blockade of oncogenic protein; cell transformation; mutant protein; oncogenic forms; peptides; small molecules

DOI:  https://doi.org/10.3390/biomedicines11020471
J Mol Biol. 2023 Feb 15. pii: S0022-2836(23)00066-9. [Epub ahead of print] 168010

Monobody inhibitor selective to the phosphatase domain of SHP2 and its use as a probe for quantifying SHP2 allosteric regulation.

Fern Sha, Kohei Kurosawa, Eliezra Glasser, Gayatri Ketavarapu, Samara Albazzaz, Akiko Koide, Shohei Koide.

  SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the "PTP-dead" C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.

Keywords:  allostery; drug discovery; protein engineering; synthetic binding protein

DOI:  https://doi.org/10.1016/j.jmb.2023.168010
Discov Oncol. 2023 Feb 23. 14(1): 25

High dose isoleucine stabilizes nuclear PTEN to suppress the proliferation of lung cancer.

Haiqing Wang, Sen Chen, Wenhui Kang, Bojiao Ding, Shulan Cui, Li Zhou, Na Zhang, Huiying Luo, Mingjuan Wang, Fan Zhang, Zezhou Zhao, Zihu Guo, Chao Wang, Liang Li, Zhengzhong Wang, Xuetong Chen, Yonghua Wang.

   PURPOSE: Cancer cells require a supply of amino acids, particularly essential amino acids such as branched-chain amino acids (BCAAs, i.e., valine, leucine, and isoleucine), to meet the increased nutrient demands of malignant tumors. The cell-autonomous and non-autonomous roles of altered BCAA supply have been implicated in cancer progression. The critical proteins involved in BCAA uptake, transport, metabolism, etc. serve as potential therapeutic biomarkers in human cancers. Here, we summarize the potential anti-tumor mechanism of BCAA by exploring the chain reaction triggered by increased BCAA supply in the tumor.
METHOD: A system-wide strategy was employed to provide a generic solution to establish the links between BCAA and cancer based on comprehensive omics, molecular experimentation, and data analysis.
RESULTS: BCAA over-supplementation (900 mg/kg) significantly inhibited tumor growth and reduced tumor burden, with isoleucine having the most pronounced effect. Surprisingly, isoleucine inhibited tumor growth independently of mTORC1 activation, a classical amino acid sensor. Exploratory transcriptome analysis revealed that Phosphatase and tensin homolog (PTEN) is the critical factor in the anti-tumor effect of isoleucine. By inhibiting PTEN ubiquitination, isoleucine can promote PTEN nuclear import and maintain PTEN nuclear stability. Interestingly, this process was regulated by isoleucine-tRNA ligase, cytoplasmic (IARS), a direct target of isoleucine. We demonstrated the enhanced interaction between IARS and PTEN in the presence of excess isoleucine. At the same time, IARS knockout leads to loss of isoleucine tumor suppressor ability.
CONCLUSION: Overall, our results provide insights into the regulation of the IARS-PTEN anti-tumor axis by isoleucine and reveal a unique therapeutic approach based on enhancing cellular isoleucine supply.

Keywords:  Branched-chain amino acids; IARS; Isoleucine; PTEN; Tumor therapy

DOI:  https://doi.org/10.1007/s12672-023-00634-1
Cancers (Basel). 2023 Feb 08. pii: 1098. [Epub ahead of print]15(4):

The KRAS-Mutant Consensus Molecular Subtype 3 Reveals an Immunosuppressive Tumor Microenvironment in Colorectal Cancer.

Pariyada Tanjak, Amphun Chaiboonchoe, Tharathorn Suwatthanarak, Onchira Acharayothin, Kullanist Thanormjit, Jantappapa Chanthercrob, Thanawat Suwatthanarak, Bundit Wannasuphaphol, Kemmapon Chumchuen, Bhoom Suktitipat, Somponnat Sampattavanich, Krittiya Korphaisarn, Ananya Pongpaibul, Naravat Poungvarin, Harald Grove, Woramin Riansuwan, Atthaphorn Trakarnsanga, Asada Methasate, Manop Pithukpakorn, Vitoon Chinswangwatanakul.

  Colorectal cancers (CRC) with KRAS mutations (KRASmut) are frequently included in consensus molecular subtype 3 (CMS3) with profound metabolic deregulation. We explored the transcriptomic impact of KRASmut, focusing on the tumor microenvironment (TME) and pathways beyond metabolic deregulation. The status of KRASmut in patients with CRC was investigated and overall survival (OS) was compared with wild-type KRAS (KRASwt). Next, we identified CMS, and further investigated differentially expressed genes (DEG) of KRASmut and distinctive pathways. Lastly, we used spatially resolved gene expression profiling to define the effect of KRASmut in the TME regions of CMS3-classified CRC tissues. CRC patients with KRASmut were mainly enriched in CMS3. Their specific enrichments of immune gene signatures in immunosuppressive TME were associated with worse OS. Activation of TGFβ signaling by KRASmut was related to reduced pro-inflammatory and cytokine gene signatures, leading to suppression of immune infiltration. Digital spatial profiling in TME regions of KRASmut CMS3-classified tissues suggested up-regulated genes, CD40, CTLA4, ARG1, STAT3, IDO, and CD274, that could be characteristic of immune suppression in TME. This study may help to depict the complex transcriptomic profile of KRASmut in immunosuppressive TME. Future studies and clinical trials in CRC patients with KRASmut should consider these transcriptional landscapes.

Keywords:  KRAS mutation; TGFβ signaling; colorectal cancer; immunosuppressive tumor microenvironment; tumor microenvironment

DOI:  https://doi.org/10.3390/cancers15041098