bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2022‒03‒13
eighteen papers selected by
Lucas B. Zeiger
Beatson Institute for Cancer Research
  1. Structures of RGL1 RAS-Association domain in complex with KRAS and the oncogenic G12V mutant.
  2. Iron out KRAS-driven cancer.
  3. An integrative gene expression signature analysis identifies CMS4 KRAS-mutated colorectal cancers sensitive to combined MEK and SRC targeted therapy.
  4. Triple targeting of HER receptors overcomes heregulin-mediated resistance to EGFR blockade in colorectal cancer.
  5. A mutation breaks its silence on mutant KRAS.
  6. mTOR-Mediated Regulation of Immune Responses in Cancer and Tumor Microenvironment.
  7. Ferrous iron-activatable drug conjugate achieves potent MAPK blockade in KRAS-driven tumors.
  8. PIK3CA-related overgrowth: silver bullets from the cancer arsenal?
  9. KRAS-G12C Mutation in One Real-Life and Three Population-Based Nordic Cohorts of Metastatic Colorectal Cancer.
  10. miRNA-Dependent Regulation of AKT1 Phosphorylation.
  11. Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors.
  12. BRAF Mutations Are Associated with Poor Survival Outcomes in Advanced-stage Mismatch Repair-deficient/Microsatellite High Colorectal Cancer.
  13. RASGRF1 Fusions Activate Oncogenic RAS Signaling and Confer Sensitivity to MEK Inhibition.
  14. Structural basis of phosphatidylinositol 3-kinase C2α function.
  15. PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment.
  16. The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling.
  17. Comprehensive analysis of R-spondin fusions and RNF43 mutations implicate novel therapeutic options in colorectal cancer.
  18. Molecular Landscape of Small Bowel Adenocarcinoma.
  1. J Mol Biol. 2022 Mar 04. pii: S0022-2836(22)00101-2. [Epub ahead of print] 167527
    Structures of RGL1 RAS-Association domain in complex with KRAS and the oncogenic G12V mutant.
    Ben J Eves, Teklab Gebregiworgis, Geneviève M C Gasmi-Seabrook, Douglas A Kuntz, Gilbert G Privé, Christopher B Marshall, Mitsuhiko Ikura.
      Ral Guanine Nucleotide Dissociation Stimulator Like 1 (RGL1) is a RAS effector protein that activates Ral GTPase by stimulating nucleotide exchange. Most structures of RAS-effector complexes are for the HRAS isoform; relatively few KRAS-effector structures have been solved, even though KRAS mutations are more frequent in human cancers. We determined crystal structures of KRAS/RGL1-RAS-association (RA) domain complexes and characterized the interaction in solution using nuclear magnetic resonance spectroscopy, size-exclusion chromatography combined with multi-angle light scattering and biolayer interferometry. We report structures of wild-type KRAS and the oncogenic G12V mutant in complex with the RA domain of RGL1 at <2 Å resolution. KRASWT/RGL1-RA crystallized as a 1:1 heterodimer, whilst KRASG12V/RGL1-RA crystallized as a heterotetrameric structure in which RGL1-RA dimerized via domain-swapping the C-terminal beta-strand. Solution data indicated that KRASWT and KRASG12V in complex with RGL1-RA both exist predominantly as 1:1 dimers, while tetramerization occurs through very slow association. Through detailed structural analyses, the distance and angle between RAS α1 helix and RBD/RA α1 helix were found to differ significantly among RAS and RBD/RA complexes. The KRAS/RGL1-RA structures possess some of the largest α1RAS/α1Effector distances (21.7-22.2 Å), whereas the corresponding distances in previously reported RAS/RAF complexes are significantly shorter (15.2-17.7 Å). Contact map analysis identified unique structural signatures involving contacts between the β1-β2 loop of RA and the α1 helix of RAS, clearly distinguishing the KRAS/RGL1-RA (and other RAS/RA complexes) from RAS/RBD complexes. These results demonstrate that RAS effectors employ an assortment of finely-tuned docking surfaces to achieve optimal interactions with RAS.
    Keywords:  GTPase KRAS (KRAS); cancer biology; nuclear magnetic resonance (NMR); structure; x-ray crystallography
    DOI:  https://doi.org/10.1016/j.jmb.2022.167527
  2. J Exp Med. 2022 Apr 04. pii: e20212166. [Epub ahead of print]219(4):
    Iron out KRAS-driven cancer.
    Guang Lei, Boyi Gan.
      How to specifically target oncogenic KRAS-driven cancers while sparing normal tissues remains an unmet need in cancer therapy. In this issue of JEM, Jiang et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20210739) leveraged KRAS-induced iron addiction in cancer cells to design a clever drug delivery approach to enable selective inhibition of KRAS signaling in mutant KRAS tumors but not in normal tissues, offering a new strategy for treating this largely incurable disease.
    DOI:  https://doi.org/10.1084/jem.20212166
  3. BMC Cancer. 2022 Mar 10. 22(1): 256
    An integrative gene expression signature analysis identifies CMS4 KRAS-mutated colorectal cancers sensitive to combined MEK and SRC targeted therapy.
    Mingli Yang, Thomas B Davis, Lance Pflieger, Michael V Nebozhyn, Andrey Loboda, Heiman Wang, Michael J Schell, Ramya Thota, W Jack Pledger, Timothy J Yeatman.
      BACKGROUND: Over half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s).METHODS: We classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi "bypass" resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154).
    RESULTS: Here we report a central role of SRC in mediating "bypass"-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P < 10-320), which is similarly associated with EMT (epithelial to mesenchymal transition), regional metastasis and disease recurrence with poor prognosis. Deeper analysis shows that both MEKi-resistance and SRC activation are preferentially associated with a mesenchymal CSC phenotype. This association is validated in additional independent CRC tumor and cell lines datasets. The CMS classification analysis demonstrates the strikingly-distinct associations of CMS1-4 subtypes with the MEKi-resistance and SRC activation. Importantly, MEKi + SRCi sensitivities are predicted to occur predominantly in the KRAS mutant, mesenchymal CSC-like CMS4 CRCs.
    CONCLUSIONS: Large human tumor gene expression datasets representing CRC heterogeneity can provide deep biological insights heretofore not possible with cell line models, suggesting novel repurposed drug combinations. We identified SRC as a common targetable node--an Achilles' heel--in MEKi-targeted therapy-associated resistance in mesenchymal stem-like CRCs, which may help development of a biomarker-driven drug combination (MEKi + SRCi) to treat problematic subpopulations of CRC.
    Keywords:  CMS; Cancer stem cell; Colorectal cancer; EMT; Gene expression signature; MEK inhibitor; SRC; Targeted therapy
    DOI:  https://doi.org/10.1186/s12885-022-09344-3
  4. Mol Cancer Ther. 2022 Feb 28. pii: molcanther.0818.2021. [Epub ahead of print]
    Triple targeting of HER receptors overcomes heregulin-mediated resistance to EGFR blockade in colorectal cancer.
    Alexander Rau, Nicole Janssen, Lennart Kühl, Thomas Sell, Svetlana Kalmykova, Thomas E Mürdter, Marc-H Dahlke, Christine Sers, Markus Morkel, Matthias Schwab, Roland E Kontermann, Monilola A Olayioye.
      Current treatment options for patients with advanced colorectal cancers (CRC) include anti-EGFR/HER1 therapy with the blocking antibody cetuximab. Although a subset of patients with KRAS wild-type disease initially respond to the treatment, resistance develops in almost all cases. Relapse has been associated with the production of the ligand heregulin (HRG) and/or compensatory signaling involving the receptor tyrosine kinases HER2 and HER3. Here we provide evidence that triple HER receptor blockade based on a newly developed bispecific EGFRxHER3-targeting antibody (scDb-Fc) together with the HER2 blocking antibody trastuzumab effectively inhibited HRG-induced HER receptor phosphorylation, downstream signaling, proliferation and stem cell expansion of DiFi and LIM1215 CRC cells. Comparative analyses revealed that the biological activity of scDb-Fc plus trastuzumab was sometimes even superior to that of the combination of the parental antibodies, with PI3K/Akt pathway inhibition correlating with improved therapeutic response and apoptosis induction as seen by single cell analysis. Importantly, growth suppression by triple HER targeting was recapitulated in primary KRAS wild-type patient-derived organoid (PDO) cultures exposed to HRG. Collectively, our results provide strong support for a pan-HER receptor blocking approach to combat anti-EGFR therapy resistance of KRAS wild-type CRC tumors mediated by the upregulation of HRG and/or HER2/HER3 signaling.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-21-0818
  5. Sci Signal. 2022 Mar 08. 15(724): eabp8972
    A mutation breaks its silence on mutant KRAS.
    Amy E Baek.
      Leveraging silent mutations and alternative splicing enables targeting of a KRAS mutant.
    DOI:  https://doi.org/10.1126/scisignal.abp8972
  6. Front Immunol. 2021 ;12 774103
    mTOR-Mediated Regulation of Immune Responses in Cancer and Tumor Microenvironment.
    Sahar Mafi, Behzad Mansoori, Shahram Taeb, Hossein Sadeghi, Reza Abbasi, William C Cho, Davoud Rostamzadeh.
      The mechanistic/mammalian target of rapamycin (mTOR) is a downstream mediator in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways, which plays a pivotal role in regulating numerous cellular functions including cell growth, proliferation, survival, and metabolism by integrating a variety of extracellular and intracellular signals in the tumor microenvironment (TME). Dysregulation of the mTOR pathway is frequently reported in many types of human tumors, and targeting the PI3K/Akt/mTOR signaling pathway has been considered an attractive potential therapeutic target in cancer. The PI3K/Akt/mTOR signaling transduction pathway is important not only in the development and progression of cancers but also for its critical regulatory role in the tumor microenvironment. Immunologically, mTOR is emerging as a key regulator of immune responses. The mTOR signaling pathway plays an essential regulatory role in the differentiation and function of both innate and adaptive immune cells. Considering the central role of mTOR in metabolic and translational reprogramming, it can affect tumor-associated immune cells to undergo phenotypic and functional reprogramming in TME. The mTOR-mediated inflammatory response can also promote the recruitment of immune cells to TME, resulting in exerting the anti-tumor functions or promoting cancer cell growth, progression, and metastasis. Thus, deregulated mTOR signaling in cancer can modulate the TME, thereby affecting the tumor immune microenvironment. Here, we review the current knowledge regarding the crucial role of the PI3K/Akt/mTOR pathway in controlling and shaping the immune responses in TME.
    Keywords:  PI3K/Akt/mTOR signaling pathway; T cell; Tumor microenvironment; cancer; immune response; mTOR
    DOI:  https://doi.org/10.3389/fimmu.2021.774103
  7. J Exp Med. 2022 Apr 04. pii: e20210739. [Epub ahead of print]219(4):
    Ferrous iron-activatable drug conjugate achieves potent MAPK blockade in KRAS-driven tumors.
    Honglin Jiang, Ryan K Muir, Ryan L Gonciarz, Adam B Olshen, Iwei Yeh, Byron C Hann, Ning Zhao, Yung-Hua Wang, Spencer C Behr, James E Korkola, Michael J Evans, Eric A Collisson, Adam R Renslo.
      KRAS mutations drive a quarter of cancer mortality, and most are undruggable. Several inhibitors of the MAPK pathway are FDA approved but poorly tolerated at the doses needed to adequately extinguish RAS/RAF/MAPK signaling in the tumor cell. We found that oncogenic KRAS signaling induced ferrous iron (Fe2+) accumulation early in and throughout mutant KRAS-mediated transformation. We converted an FDA-approved MEK inhibitor into a ferrous iron-activatable drug conjugate (FeADC) and achieved potent MAPK blockade in tumor cells while sparing normal tissues. This innovation allowed sustainable, effective treatment of tumor-bearing animals, with tumor-selective drug activation, producing superior systemic tolerability. Ferrous iron accumulation is an exploitable feature of KRAS transformation, and FeADCs hold promise for improving the treatment of KRAS-driven solid tumors.
    DOI:  https://doi.org/10.1084/jem.20210739
  8. Trends Mol Med. 2022 Mar 07. pii: S1471-4914(22)00050-8. [Epub ahead of print]
    PIK3CA-related overgrowth: silver bullets from the cancer arsenal?
    Ralitsa R Madsen, Robert K Semple.
      Mutations that activate growth factor signaling often drive cancer growth. Many also arise in isolation, causing developmental growth disorders. PIK3CA, that encodes a catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is a cardinal example of this paradigm. Recent exciting progress towards the key goal of cancer drug repurposing for PIK3CA-driven overgrowth is discussed.
    Keywords:  CLOVES; PI3K; PIK3CA; PROS; alpelisib; overgrowth; phosphatidylinositol 3-kinase; sirolimus; trials
    DOI:  https://doi.org/10.1016/j.molmed.2022.02.009
  9. Front Oncol. 2022 ;12 826073
    KRAS-G12C Mutation in One Real-Life and Three Population-Based Nordic Cohorts of Metastatic Colorectal Cancer.
    Emerik Osterlund, Ari Ristimäki, Soili Kytölä, Teijo Kuopio, Eetu Heervä, Timo Muhonen, Päivi Halonen, Raija Kallio, Leena-Maija Soveri, Jari Sundström, Mauri Keinänen, Annika Ålgars, Raija Ristamäki, Halfdan Sorbye, Per Pfeiffer, Luís Nunes, Tapio Salminen, Annamarja Lamminmäki, Markus J Mäkinen, Tobias Sjöblom, Helena Isoniemi, Bengt Glimelius, Pia Osterlund.
      Background: KRAS mutations, present in over 40% of metastatic colorectal cancer (mCRC), are negative predictive factors for anti-EGFR therapy. Mutations in KRAS-G12C have a cysteine residue for which drugs have been developed. Published data on this specific mutation are conflicting; thus, we studied the frequency and clinical characteristics in a real-world and population-based setting.Methods: Patients from three Nordic population-based cohorts and the real-life RAXO-study were combined. RAS and BRAF tests were performed in routine healthcare, except for one cohort. The dataset consisted of 2,559 patients, of which 1,871 could be accurately classified as KRAS, NRAS, and BRAF-V600E. Demographics, treatments, and outcomes were compared using logistic regression. Overall survival (OS) was estimated with Kaplan-Meier, and differences were compared using Cox regression, adjusted for baseline factors.
    Results: The KRAS-G12C frequency was 2%-4% of all tested in the seven cohorts (mean 3%) and 4%-8% of KRAS mutated tumors in the cohorts (mean 7%). Metastasectomies and ablations were performed more often (38% vs. 28%, p = 0.040), and bevacizumab was added more often (any line 74% vs. 59%, p = 0.007) for patients with KRAS-G12C- vs. other KRAS-mutated tumors, whereas chemotherapy was given to similar proportions. OS did not differ according to KRAS mutation, neither overall (adjusted hazard ratio (HR) 1.03; 95% CI 0.74-1.42, reference KRAS-G12C) nor within treatment groups defined as "systemic chemotherapy, alone or with biologics", "metastasectomy and/or ablations", or "best supportive care", RAS and BRAF wild-type tumors (n = 548) differed similarly to KRAS-G12C, as to other KRAS- or NRAS-mutated (n = 66) tumors.
    Conclusions: In these real-life and population-based cohorts, there were no significant differences in patient characteristics and outcomes between patients with KRAS-G12C tumors and those with other KRAS mutations. This contrasts with the results of most previous studies claiming differences in many aspects, often with worse outcomes for those with a KRAS-G12C mutation, although not consistent. When specific drugs are developed, as for this mutation, differences in outcome will hopefully emerge.
    Keywords:  KRAS mutation; KRAS-G12C mutation; colorectal cancer; metastatic; population-based; real-world
    DOI:  https://doi.org/10.3389/fonc.2022.826073
  10. Cells. 2022 Feb 26. pii: 821. [Epub ahead of print]11(5):
    miRNA-Dependent Regulation of AKT1 Phosphorylation.
    Mallory I Frederick, Tarana Siddika, Pengcheng Zhang, Nileeka Balasuriya, Matthew A Turk, Patrick O'Donoghue, Ilka U Heinemann.
      The phosphoinositide-3-kinase (PI3K)/AKT pathway regulates cell survival and is over-activated in most human cancers, including ovarian cancer. Following growth factor stimulation, AKT1 is activated by phosphorylation at T308 and S473. Disruption of the AKT1 signaling pathway is sufficient to inhibit the epithelial-mesenchymal transition in epithelial ovarian cancer (EOC) cells. In metastatic disease, adherent EOC cells transition to a dormant spheroid state, characterized previously by low S473 phosphorylation in AKT1. We confirmed this finding and observed that T308 phosphorylation was yet further reduced in EOC spheroids and that the transition from adherent to spheroid growth is accompanied by significantly increased levels of let-7 miRNAs. We then used mechanistic studies to investigate the impact of let-7 miRNAs on AKT1 phosphorylation status and activity in cells. In growth factor-stimulated HEK 293T cells supplemented with let-7a, we found increased phosphorylation of AKT1 at T308, decreased phosphorylation at S473, and enhanced downstream AKT1 substrate GSK-3β phosphorylation. Let-7b and let-7g also deregulated AKT signaling by rendering AKT1 insensitive to growth factor simulation. We uncovered let-7a-dependent deregulation of PI3K pathway components, including PI3KC2A, PDK1, and RICTOR, that govern AKT1 phosphorylation and activity. Together, our data show a new role for miRNAs in regulating AKT signaling.
    Keywords:  miRNA; oncogenic kinase; posttranslational modification; protein phosphorylation; signaling
    DOI:  https://doi.org/10.3390/cells11050821
  11. Cancers (Basel). 2022 Feb 25. pii: 1195. [Epub ahead of print]14(5):
    Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors.
    Jean Descarpentrie, Marcos J Araúzo-Bravo, Zongsheng He, Alexia François, Álvaro González, Patricia Garcia-Gallastegi, Iker Badiola, Serge Evrard, Simon Pernot, John W M Creemers, Abdel-Majid Khatib.
      Proprotein convertases or PCs are known to regulate the malignant phenotype of colon cancer cells by different mechanisms, but their effects on cancer stem cells (CSCs) have been less widely investigated. Here, we report that PCs expression is altered in colon CSCs, and the inhibition of their activity reduced colon CSCs growth, survival, and invasion in three-dimensional spheroid cultures. In vivo, repression of PCs activity by the general PC inhibitors α1-PDX, Spn4A, or decanoyl-RVKR-chloromethylketone (CMK) significantly reduced tumor expression levels of the stem cell markers LGR5 and NANOG that are associated with reduced tumor xenografts. Further analysis revealed that reduced tumor growth mediated by specific silencing of the convertase Furin in KRAS or BRAF mutated-induced colon tumors was associated with reduced expression of LGR5 and NANOG compared to wild-type KRAS and BRAF tumors. Analysis of various calcium regulator molecules revealed that while the calcium-transporting ATPase 4 (ATP2B4) is downregulated in all the Furin-silenced colon cancer cells, the Ca2+-mobilizing P2Y receptors, was specifically repressed in BRAF mutated cells and ORAI1 and CACNA1H in KRAS mutated cells. Taken together, our findings indicate that PCs play an important role in the malignant phenotype of colon CSCs and stem cell markers' expression and highlight PCs repression, particularly of Furin, to target colon tumors with KRAS or BRAF mutation.
    Keywords:  BRAF; KRAS; LGR5; NANOG; calcium; cancer stem cells; colon cancer
    DOI:  https://doi.org/10.3390/cancers14051195
  12. Oncologist. 2022 Mar 11. 27(3): 191-197
    BRAF Mutations Are Associated with Poor Survival Outcomes in Advanced-stage Mismatch Repair-deficient/Microsatellite High Colorectal Cancer.
    Elaine Tan, Junmin Whiting, Hao Xie, Iman Imanirad, Estrella Carballido, Seth Felder, Jessica Frakes, Quanxing Mo, Christine Walko, Jennifer B Permuth, Katelyn Sommerer, Richard Kim, Daniel A Anaya, Jason B Fleming, Ibrahim Halil Sahin.
      BACKGROUND: Mismatch repair-deficient (MMR-D)/microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC) is a unique disease entity with growing interest given the rise of young-onset CRC. Given its heterogeneous behavior and potential for highly effective treatment outcomes, we sought to identify the clinical and molecular features that offer prognostic value for MMR-D CRC.MATERIALS/METHODS: This was a retrospective cohort study of patients with metastatic CRC with MMR-D or microsatellite instability in a real-world database. Overall survival (OS) was determined by the date of metastatic disease to date of death with stratification made based on factors including BRAF and RAS mutation status, age, and MMR protein loss type.
    RESULTS: There were 1101 patients in the study. Patients with BRAF mutations had worse OS compared with patients with wild-type BRAF with a median survival of 18.9 months versus 33.2 months (hazard ratio [HR] 1.52, 95% confidence interval [CI]: 1.25-1.86, P < .001). Patients with age >50 were found to have decreased OS versus age ≤50 with a median survival of 21.4 months versus 38.7 months (HR 1.66, 95% CI: 1.33-2.07, P < .001). BRAF mutations and age >50 remained significant predictors of OS in multivariate analysis.
    CONCLUSION: BRAF mutations and age >50 are associated with worse survival outcomes for patients with MMR-D mCRC. RAS mutations and specific MMR alterations are not associated with survival outcomes.
    Keywords:   BRAF V600E; KRAS; NRAS; colorectal cancer; late-onset disease; microsatellite instability high; mismatch repair deficiency; prognosis
    DOI:  https://doi.org/10.1093/oncolo/oyab055
  13. Clin Cancer Res. 2022 Mar 01. pii: clincanres.4291.2021. [Epub ahead of print]
    RASGRF1 Fusions Activate Oncogenic RAS Signaling and Confer Sensitivity to MEK Inhibition.
    Lisa Hunihan, Dejian Zhao, Heather Lazowski, Man Li, Yuping Qian, Laura Abriola, Yulia V Surovtseva, Viswanathan Muthusamy, Lynn T Tanoue, Bonnie E Gould Rothberg, Kurt A Schalper, Roy S Herbst, Frederick H Wilson.
      PURPOSE: The identification of actionable oncogenic alterations has enabled targeted therapeutic strategies for subsets of patients with advanced malignancies including lung adenocarcinoma (LUAD). We sought to assess the frequency of known drivers and identify new candidate drivers in a cohort of LUAD from patients with minimal smoking history.EXPERIMENTAL DESIGN: We performed genomic characterization of 103 LUADs from patients with {less than or equal to}10 pack-year smoking history. Tumors were subjected to targeted molecular profiling and/or whole-exome sequencing and RNA-seq in search of established and previously uncharacterized candidate drivers.
    RESULTS: We identified an established oncogenic driver in 98 of 103 tumors (95%). From one tumor lacking a known driver, we identified a novel gene rearrangement between OCLN and RASGRF1. The encoded OCLN-RASGRF1 chimera fuses the membrane-spanning portion of the tight junction protein occludin with the catalytic RAS-GEF domain of the RAS activator RASGRF1. We identified a similar SLC4A4-RASGRF1 fusion in a pancreatic ductal adenocarcinoma (PDAC) cell line lacking an activating KRAS mutation and an IQGAP1-RASGRF1 fusion from a sarcoma in The Cancer Genome Atlas. We demonstrate these fusions increase cellular levels of active GTP-RAS, induce cellular transformation, and promote in vivo tumorigenesis. Cells driven by RASGRF1 fusions are sensitive to targeting of the RAF-MEK-ERK pathway in vitro and in vivo.
    CONCLUSIONS: Our findings credential RASGRF1 fusions as a therapeutic target in multiple malignancies and implicate RAF-MEK-ERK inhibition as a potential treatment strategy for advanced tumors harboring these alterations.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-21-4291
  14. Nat Struct Mol Biol. 2022 Mar 07.
    Structural basis of phosphatidylinositol 3-kinase C2α function.
    Wen-Ting Lo, Yingyi Zhang, Oscar Vadas, Yvette Roske, Federico Gulluni, Maria Chiara De Santis, Andreja Vujicic Zagar, Heike Stephanowitz, Emilio Hirsch, Fan Liu, Oliver Daumke, Misha Kudryashev, Volker Haucke.
      Phosphatidylinositol 3-kinase type 2α (PI3KC2α) is an essential member of the structurally unresolved class II PI3K family with crucial functions in lipid signaling, endocytosis, angiogenesis, viral replication, platelet formation and a role in mitosis. The molecular basis of these activities of PI3KC2α is poorly understood. Here, we report high-resolution crystal structures as well as a 4.4-Å cryogenic-electron microscopic (cryo-EM) structure of PI3KC2α in active and inactive conformations. We unravel a coincident mechanism of lipid-induced activation of PI3KC2α at membranes that involves large-scale repositioning of its Ras-binding and lipid-binding distal Phox-homology and C-C2 domains, and can serve as a model for the entire class II PI3K family. Moreover, we describe a PI3KC2α-specific helical bundle domain that underlies its scaffolding function at the mitotic spindle. Our results advance our understanding of PI3K biology and pave the way for the development of specific inhibitors of class II PI3K function with wide applications in biomedicine.
    DOI:  https://doi.org/10.1038/s41594-022-00730-w
  15. J Immunother Cancer. 2022 Mar;pii: e003402. [Epub ahead of print]10(3):
    PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment.
    Natalie B Collins, Rose Al Abosy, Brian C Miller, Kevin Bi, Qihong Zhao, Michael Quigley, Jeffrey J Ishizuka, Kathleen B Yates, Hans W Pope, Robert T Manguso, Yashaswi Shrestha, Marc Wadsworth, Travis Hughes, Alex K Shalek, Jesse S Boehm, William C Hahn, John G Doench, W Nicholas Haining.
      BACKGROUND: Oncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood.METHODS: We use an in vivo expression screen of known cancer-associated somatic mutations in mouse syngeneic tumor models treated with checkpoint blockade to identify oncogenes that promote immune evasion. We then validated candidates from this screen in vivo and analyzed the tumor immune microenvironment of tumors expressing mutant protein to identify mechanisms of immune evasion.
    RESULTS: We found that expression of a catalytically active mutation in phospho-inositol 3 kinase (PI3K), PIK3CA c.3140A>G (H1047R) confers a selective growth advantage to tumors treated with immunotherapy that is reversed by pharmacological PI3K inhibition. PIK3CA H1047R-expression in tumors decreased the number of CD8+ T cells but increased the number of inhibitory myeloid cells following immunotherapy. Inhibition of myeloid infiltration by pharmacological or genetic modulation of Ccl2 in PIK3CA H1047R tumors restored sensitivity to programmed cell death protein 1 (PD-1) checkpoint blockade.
    CONCLUSIONS: PI3K activation enables tumor immune evasion by promoting an inhibitory myeloid microenvironment. Activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy. Our data suggest that some oncogenes promote tumorigenesis by enabling tumor cells to avoid clearance by the immune system. Identification of those mechanisms can advance rational combination strategies to increase the efficacy of immunotherapy.
    Keywords:  biomarkers; immune evation; immunotherapy; tumor
    DOI:  https://doi.org/10.1136/jitc-2021-003402
  16. Front Pharmacol. 2022 ;13 816551
    The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling.
    Ruohan Zhang, Serra Ozgen, Hongke Luo, Judith Krigman, Yutong Zhao, Gang Xin, Nuo Sun.
      Mitophagy is an intracellular mechanism to maintain mitochondrial health by removing dysfunctional mitochondria. The E3 ligase Parkin ubiquitinates the membrane proteins on targeted mitochondria to initiate mitophagy, whereas USP30 antagonizes Parkin-dependent mitophagy by removing ubiquitin from Parkin substrates. The AKT/mTOR signaling is a master regulator of cell proliferation, differentiation, apoptosis, and autophagy. Although mounting evidence suggests that perturbations in the AKT/mTOR signaling pathway may contribute to mitophagy regulation, the specific mechanisms between Parkin/USP30 and AKT/mTOR signaling have not been elucidated. In this study, we employ a set of genetic reagents to investigate the role of Parkin and USP30 in regulating the AKT/mTOR signaling during mitophagy. We demonstrated that, in the setting of mitochondrial stress, the AKT/mTOR signaling is regulated, at least in part, by the activity of Parkin and USP30. Parkin inhibits AKT/mTOR signaling following an in vitro mitochondrial stress, thereby promoting apoptosis. However, USP30 overexpression antagonizes the activity of Parkin to sustain AKT/mTOR activity and inhibit apoptosis. These findings provide new insights into Parkin and USP30's role in apoptosis and suggest that inhibiting USP30 might provide a specific strategy to synergize with AKT/mTOR inhibitors in cancer treatment.
    Keywords:  USP30; akt; cancer; leukemia; mTOR; mitophagy; parkin
    DOI:  https://doi.org/10.3389/fphar.2022.816551
  17. Clin Cancer Res. 2022 Mar 07. pii: clincanres.3018.2021. [Epub ahead of print]
    Comprehensive analysis of R-spondin fusions and RNF43 mutations implicate novel therapeutic options in colorectal cancer.
    Andreas Seeber, Francesca Battaglin, Kai Zimmer, Florian Kocher, Yasmine Baca, Joanne Xiu, Gilbert Spizzo, Veronica Novotny-Diermayr, Dietmar Rieder, Alberto Puccini, Jeffrey Swensen, Michelle Ellis, Richard M Goldberg, Axel Grothey, Anthony F Shields, John L Marshall, Benjamin A Weinberg, Paul E Sackstein, Kiat Hon Lim, Gek San Tan, Chadi Nabhan, W Michael Korn, Arno Amann, Zlatko Trajanoski, Martin D Berger, Emil Lou, Dominik Wolf, Heinz-Josef Lenz.
      PURPOSE: Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer (CRC). Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared to wildtype CRCs to gain insights into potential rationales for therapeutic strategies.EXPERIMENTAL DESIGN: A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and immunohistochemistry. An independent cohort was used to validate our findings.
    RESULTS: The discovery cohort consisted of 7,245 CRC samples. RSPOfp and RNF43 mutations were detected in 1.3% (n=94) and 6.1% (n=443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g. IFNGR1-RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison to wildtype CRCs, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared to wildtype cases (4.4 vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared to wildtype samples. While RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a TMB {greater than or equal to}10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings.
    CONCLUSIONS: This is the largest series of RSPOfp/RNF43-mut CRCs reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-21-3018
  18. Cancers (Basel). 2022 Mar 02. pii: 1287. [Epub ahead of print]14(5):
    Molecular Landscape of Small Bowel Adenocarcinoma.
    Karan Pandya, Michael J Overman, Pat Gulhati.
      Small bowel adenocarcinoma (SBA) is a rare malignancy, with lower incidence, later stage at diagnosis, and poor overall prognosis compared to other cancers of the gastrointestinal tract. Owing to the rarity of the disease along with the paucity of high-quality tissue samples and preclinical models, little is known about the molecular alterations characteristic of SBA. This is reflected by the fact that the clinical management of SBA is primarily extrapolated from colorectal cancer (CRC). Recent advances in genomic profiling have highlighted key differences between these tumors, establishing SBA as a molecularly unique intestinal cancer. Moreover, comprehensive molecular analysis has identified a relatively high incidence of potentially targetable genomic alterations in SBA, predictive of response to targeted and immunotherapies. Further advances in our knowledge of the mutational and transcriptomic landscape of SBA, guided by an increased understanding of the molecular drivers of SBA, will provide opportunities to develop novel diagnostic tools and personalized therapeutic strategies.
    Keywords:  BRAF alteration; ERBB2/HER2 alteration; colorectal cancer (CRC); gastric cancer (GC); microsatellite instability (MSI); small bowel adenocarcinoma; tumor mutational burden (TMB)
    DOI:  https://doi.org/10.3390/cancers14051287
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