bims-pimaco 2022-08-07 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–08–07
eight papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

AKT phosphorylation as a predictive biomarker for PI3K/mTOR dual inhibition-induced proteolytic cleavage of mTOR companion proteins in small cell lung cancer.
Specific KRAS-Mutant Cancers Are Sensitive to Inhibition of V-type ATPases.
Argonaute 2 modulates EGFR-RAS signaling to promote mutant HRAS and NRAS-driven malignancies.
Afadin couples RAS GTPases to the polarity rheostat Scribble.
Targeting RAS mutant colorectal cancer with dual inhibition of MEK and CDK4/6.
Nucleotide imbalance decouples cell growth from cell proliferation.
Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt.
DFNA5 inhibits colorectal cancer proliferation by suppressing the mTORC1/2 signaling pathways via upregulation of DEPTOR.

Cell Biosci. 2022 Aug 02. 12(1): 122

AKT phosphorylation as a predictive biomarker for PI3K/mTOR dual inhibition-induced proteolytic cleavage of mTOR companion proteins in small cell lung cancer.

Ming-Chun Hung, Wan-Ping Wang, Ya-Hui Chi.

   BACKGROUND: Constitutive activation of PI3K signaling has been well recognized in a subset of small cell lung cancer (SCLC), the cancer type which has the most aggressive clinical course amongst pulmonary tumors. Whereas cancers that acquire a mutation/copy gain in PIK3CA or loss of PTEN have been implicated in enhanced sensitivity to inhibitors targeting the PI3K/AKT/mTOR pathway, the complexities of the pathway and corresponding feedback loops hamper clear predictions as to the response of tumors presenting these genomic features.
METHODS: The correlation between the expression profile of proteins involved in the PI3K/AKT/mTOR signaling and cell viability in response to treatment with small molecule inhibitors targeting isoform-specific PI3Ks, AKT, and mTOR was assessed in 13 SCLC cancer cell lines. Athymic nude mice were used to determine the effect of PI3K/mTOR dual inhibition on the growth of xenograft SCLC tumors in vivo. The activation of caspase signaling and proteolytic cleavages of mTOR companion proteins were assessed using recombinant caspases assays and Western blot analyses.
RESULTS: Our results indicate that the sensitivity of these SCLC cell lines to GSK2126458, a dual PI3K/mTOR inhibitor, is positively correlated with the expression levels of phosphorylated AKT (p-AKT) at Thr308 and Ser473. Inhibition of pan-class I PI3Ks or PI3K/mTOR dual inhibition was shown to induce proteolytic cleavage of RICTOR and RPTOR, which were respectively dependent on Caspase-6 and Caspase-3. A combination of a clinically approved PI3Kα-selective inhibitor and an mTORC1 inhibitor was shown to have synergistic effects in inducing the death of SCLC cells with high p-AKT. We observed no clear correlation between PTEN levels and the survival of SCLCs in response to PI3K/mTOR dual inhibition; however, PTEN depletion was shown to increase the susceptibility of low p-AKT SCLC cells to dual PI3K/mTOR inhibitor-induced cell death as well as the proteolytic cleavage of RICTOR.
CONCLUSIONS: These results suggest the level of p-AKT can be a companion diagnostic biomarker for the treatment of SCLC involving the combinational use of clinically approved isoform-specific PI3K and mTOR inhibitors.

Keywords:  PI3K inhibitor; Small cell lung cancer; mTOR

DOI:  https://doi.org/10.1186/s13578-022-00862-y
Cancer Discov. 2022 Aug 05. OF1

Specific KRAS-Mutant Cancers Are Sensitive to Inhibition of V-type ATPases.

249C was identified as a selective Ras-mutant cytotoxic agent that binds to and inhibits V-ATPase.

DOI: https://doi.org/10.1158/2159-8290.CD-RW2022-142
PNAS Nexus. 2022 Jul;1(3): pgac084

Argonaute 2 modulates EGFR-RAS signaling to promote mutant HRAS and NRAS-driven malignancies.

Ronald F Siebenaler, Seema Chugh, Jessica J Waninger, Vijaya L Dommeti, Carson Kenum, Malay Mody, Anudeeta Gautam, Nidhi Patel, Alec Chu, Pushpinder Bawa, Jennifer Hon, Richard D Smith, Heather Carlson, Xuhong Cao, John J G Tesmer, Sunita Shankar, Arul M Chinnaiyan.

  Activating mutations in RAS GTPases drive nearly 30% of all human cancers. Our prior work described an essential role for Argonaute 2 (AGO2), of the RNA-induced silencing complex, in mutant KRAS-driven cancers. Here, we identified a novel endogenous interaction between AGO2 and RAS in both wild-type (WT) and mutant HRAS/NRAS cells. This interaction was regulated through EGFR-mediated phosphorylation of Y393-AGO2, and utilizing molecular dynamic simulation, we identified a conformational change in pY393-AGO2 protein structure leading to disruption of the RAS binding site. Knockdown of AGO2 led to a profound decrease in proliferation of mutant HRAS/NRAS-driven cell lines but not WT RAS cells. These cells demonstrated oncogene-induced senescence (OIS) as evidenced by β-galactosidase staining and induction of multiple downstream senescence effectors. Mechanistically, we discovered that the senescent phenotype was mediated via induction of reactive oxygen species. Intriguingly, we further identified that loss of AGO2 promoted a novel feed forward pathway leading to inhibition of the PTP1B phosphatase and activation of EGFR-MAPK signaling, consequently resulting in OIS. Taken together, our study demonstrates that the EGFR-AGO2-RAS signaling axis is essential for maintaining mutant HRAS and NRAS-driven malignancies.

Keywords:  Argonaute 2; EGFR; HRAS; NRAS; senescence

DOI:  https://doi.org/10.1093/pnasnexus/pgac084
Nat Commun. 2022 Aug 05. 13(1): 4562

Afadin couples RAS GTPases to the polarity rheostat Scribble.

Marilyn Goudreault, Valérie Gagné, Chang Hwa Jo, Swati Singh, Ryan C Killoran, Anne-Claude Gingras, Matthew J Smith.

AFDN/Afadin is required for establishment and maintenance of cell-cell contacts and is a unique effector of RAS GTPases. The biological consequences of RAS complex with AFDN are unknown. We used proximity-based proteomics to generate an interaction map for two isoforms of AFDN, identifying the polarity protein SCRIB/Scribble as the top hit. We reveal that the first PDZ domain of SCRIB and the AFDN FHA domain mediate a direct but non-canonical interaction between these important adhesion and polarity proteins. Further, the dual RA domains of AFDN have broad specificity for RAS and RAP GTPases, and KRAS co-localizes with AFDN and promotes AFDN-SCRIB complex formation. Knockout of AFDN or SCRIB in epithelial cells disrupts MAPK and PI3K activation kinetics and inhibits motility in a growth factor-dependent manner. These data have important implications for understanding why cells with activated RAS have reduced cell contacts and polarity defects and implicate AFDN as a genuine RAS effector.

DOI: https://doi.org/10.1038/s41467-022-32335-8
Cancer Res. 2022 Aug 01. pii: CAN-22-0198. [Epub ahead of print]

Targeting RAS mutant colorectal cancer with dual inhibition of MEK and CDK4/6.

Alexey V Sorokin, Preeti Kanikarla Marie, Lea Bitner, Muddassir Syed, Melanie Woods, Ganiraju Manyam, Lawrence N Kwong, Benny Johnson, Van K Morris, Philip Jones, David G Menter, Michael S Lee, Scott Kopetz.

KRAS and NRAS mutations occur in 45% of colorectal cancers (CRC), with combined MAPK pathway and CDK4/6 inhibition identified as a potential therapeutic strategy. In the current study, this combinatorial treatment approach was evaluated in a co-clinical trial in patient-derived xenografts (PDX), and safety was established in a clinical trial of binimetinib and palbociclib in metastatic CRC patients with RAS mutations. Across 18 PDX models undergoing dual inhibition of MEK and CDK4/6, 60% of tumors regressed, meeting the co-clinical trial primary endpoint. Prolonged duration of response occurred predominantly in TP53 wild-type models. Clinical evaluation of binimetinib and palbociclib in a safety lead-in confirmed safety and provided preliminary evidence of activity. Prolonged treatment in PDX models resulted in feedback activation of receptor tyrosine kinases and acquired resistance, which was reversed with a SHP2 inhibitor. These results highlight the clinical potential of this combination in CRC, along with the utility of PDX-based co-clinical trial platforms for drug development.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-0198
Nat Cell Biol. 2022 Aug 04.

Nucleotide imbalance decouples cell growth from cell proliferation.

Frances F Diehl, Teemu P Miettinen, Ryan Elbashir, Christopher S Nabel, Alicia M Darnell, Brian T Do, Scott R Manalis, Caroline A Lewis, Matthew G Vander Heiden.

Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division.

DOI: https://doi.org/10.1038/s41556-022-00965-1
J Biol Chem. 2022 Aug 01. pii: S0021-9258(22)00730-X. [Epub ahead of print] 102288

Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt.

Zanlin Yu, Junliang Chen, Enzo Takagi, Feng Wang, Bidisha Saha, Xi Liu, Lydia-Marie Joubert, Catherine E Gleason, Mingliang Jin, Chengmin Li, Carlos Nowotny, David Agard, Yifan Cheng, David Pearce.

Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously, however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.

DOI: https://doi.org/10.1016/j.jbc.2022.102288
Cell Cycle. 2022 Aug 03. 1-14

DFNA5 inhibits colorectal cancer proliferation by suppressing the mTORC1/2 signaling pathways via upregulation of DEPTOR.

Jing Guo, Junhui Yu, Mingchao Mu, Zilu Chen, Zhengshui Xu, Chenye Zhao, Kui Yang, Jianbao Zheng, Xiao Qin, Wei Zhao, Xuejun Sun.

  The human deafness, autosomal dominant 5 gene (DFNA5), a newly discovered executor of pyroptosis, has been strongly implicated in the tumorigenesis of several human cancers. However, an understanding of the functional role of DFNA5 in the development and progression of colorectal cancer (CRC) is limited. In this study, we demonstrated that DFNA5 was downregulated in CRC tissues. Ectopic expression of DFNA5 inhibited tumor cell growth in vitro, retarded tumor formation in vivo, and blocked a cell-cycle transition from the G0/G1 to the S phase, whereas a DFNA5 knockdown promoted cell proliferation. Western blotting showed that the levels of cell cycle-related proteins, including cyclin D1, cyclin E, CDK2, and p21, were accordingly altered upon DFNA5 overexpression or DFNA5 knockdown. Mechanistic studies indicated that DFNA5 exerted its tumor suppressor functions by antagonizing mTORC1/2 signaling via upregulation of DEPTOR. In addition, blockage of mTORC1/2 signaling by Torin-1 abolished the accelerative proliferation by DFNA5 knockdown. In conclusion, these results indicated that DFNA5 inhibits the proliferation and tumor formation of colon cancer cells by suppressing mTORC1/2 signaling.

Keywords:  DEPTOR; DFNA5; cell cycle; colorectal cancer; mTORC1/2; proliferation

DOI:  https://doi.org/10.1080/15384101.2022.2088570