Early response in phosphorylation of ribosomal protein S6 is associated with sensitivity to trametinib in colorectal cancer cells

Hirashita, Yuka; Tsukamoto, Yoshiyuki; Kudo, Yoko; Kurogi, Shusaku; Hijiya, Naoki; Nakada, Chisato; Uchida, Tomohisa; Hirashita, Teijiro; Hiratsuka, Takahiro; Akagi, Tomonori; Ueda, Y.; Shiroshita, Hidefumi; Etoh, Tsuyoshi; Mizukami, Kazuhiro; Honda, Koichi; Kodama, Masaaki; Ito, Masafumi; Moriyama, Masatsugu; Murakami, Kazunari

doi:10.1038/s41374-021-00590-w

Cited by 11 publications

(10 citation statements)

References 36 publications

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“…As previously reported, a change in pERK was not significantly associated with trametinib sensitivity, only a reduction of phosphorylated ribosomal protein S6 (pS6) [248]. Trametinib resistance in CRC cell lines arises due to upregulation of pS6 [248]. In contrast, the proteomic results showed a reduction of pS6 in cells which are both, sensitive and resistant towards trametinib treatment (Figure 55).…”

Section: Bmp-pathway Activation Leads To Mek-inhibitor Resistancesupporting

confidence: 61%

“…None of the tested PDOs displayed changes in MEK and phosphorylated ERK (pERK) protein expression after 24 h of treatment (Figure 54), indicating that this signaling pathway is not involved. As previously reported, a change in pERK was not significantly associated with trametinib sensitivity, only a reduction of phosphorylated ribosomal protein S6 (pS6) [248]. Trametinib resistance in CRC cell lines arises due to upregulation of pS6 [248].…”

Section: Bmp-pathway Activation Leads To Mek-inhibitor Resistancementioning

confidence: 65%

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Identification of a gene signature as a predictive biomarker for successfull MEK inhibition in colorectal cancer

Pfohl¹

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Despite all advancements in cancer research and clinical practice, cancer remains a life- threatening disease with an increasing incidence. According to a 2018 WHO forecast, cancer incidence will double to approximately 37 million new cancer cases by 2040. Today, clinical management of cancer is based on a "one-fits-all" strategy. Most cancers are still treated by surgical therapy followed by adjuvant or neoadjuvant chemotherapy based on rather strict guidelines (S3 guidelines in Europe) which are based on studies of large cohorts of patients with the same tumor entity. While this approach has led to substantial increases in progression-free survival and overall patient survival, most patients do not benefit from the administered treatment regimen. One reason for this is intra-tumor heterogeneity, which results from clonal evolution between cancer cells and their environment. This means that cancer patients may respond differently to a particular drug due to the different mutation patterns of their tumor cells. Therefore, patients should be screened in advance for reliable cancer biomarkers that definitively predict whether they will respond to a particular therapy. This would increase the probability of a successful treatment. Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer deaths worldwide. The main cause of death in CRC is a metastatic disease, which is presented in 20 % of patients and eventually develops in more than 30 % of early-stage patients. Despite the significant increase (to more than 30 months) in median survival with the development of cytotoxic agents and the introduction of targeted therapy, the progression-free survival in the first-line setting has remained largely unchanged over the past decade. The heterogeneity in CRC is characterized by alterations in multiple signaling pathways that affect cellular functions such as cell proliferation or apoptosis. Commonly affected signaling pathways include the mitogen-activated protein kinase (MAPK)- and the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP)-pathway. Alterations in the TGF-β/BMP pathway, due to mutations in the SMAD4 gene (mothers against decapentaplegic homolog 4), are associated with different drug response and promote resistance to chemotherapy. In addition, they are associated with a higher recurrence rate. SMAD4 is one of the most common cancer driver genes, and mutations occur in up to 15 % of CRC cases. Therefore, there is an urgent need for therapeutic agents that can specifically target SMAD4-mutated tumors. The aim of the present study was the identification of the clinical relevance of the SMAD4 gene and the investigation of its suitability as a potential biomarker in CRC. For this purpose, I investigated sibling patient-derived organoids (PDOs) derived from different regions of a chemo-naïve CRC tumor. PDOs are 3D cell cultures that reliably recapitulate the architecture of the tissue of origin, as well as preserve the genomic background and intra-tumor heterogeneity. The sibling PDOs (R1R361H and R4wt) shared the most common CRC mutations, such as KRASG12D (kirsten rat sarcoma), PIK3CAH1047R (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha), and TP53C242F (tumor protein 53), but differed in a SMAD4R361H mutation and showed a different drug response. The single nucleotide variant R361H of the SMAD4 gene is among the most common pathogenic alterations in various cancers, including CRC. The sibling PDOs showed significant differences in response to the MEK-inhibitors cobimetinib, trametinib, and selumetinib. MEK-inhibitors are antineoplastic agents that inhibit the function of MEK1 and MEK2, preventing phosphorylation of transcription factors, which leads to inhibition of tumor cell proliferation. MEK-inhibitors are approved for the treatment of malignant melanoma. Currently, they are in phase-III clinical trials for the treatment of patients with metastatic CRC. To investigate whether SMAD4R361H is responsible for sensitivity to MEK-inhibitors, Iestablished three syngeneic PDOs harboring a SMAD4R361H mutation using the CRISPR/Cas9 genome editing system. All CRISPR-PDOs were significantly more sensitive to the MEK-inhibitors, compared to R4wt. I have shown that the SMAD4R361H mutation is responsible for sensitivity to MEK inhibition in CRC models and may be a predictive biomarker. To test this hypothesis, I examined 62 CRC PDO models and treated them with the MEK-inhibitors cobimetinib, trametinib, and selumetinib. All models that had a pathogenic mutation or deletion in the SMAD4 gene (15 %) were sensitive to cobimetinib, 10 % of models were sensitive to trametinib, and 8 % were sensitive to selumetinib. I performed transcriptome (RNA sequencing) and proteome analyses using the DigiWest® method to investigate the mechanism underlying MEK-inhibitor sensitivity. DigiWest® is a Luminex® bead-based analysis that allows the simultaneous analysis of over 100 (phospho-)proteins. The transcriptome and proteome data support the observation that MEK inhibition primarily affects SMAD4R361H PDOs. Furthermore, I have shown that activation of the BMP signaling pathway in organoids with wild-type SMAD4 appears to be responsible for resistance to MEK-inhibitors. Thus, a genetic alteration in the BMP signaling pathway, beyond SMAD4, could lead to sensitivity to MEK-inhibitors. I identified four genes involved in the TGF-β/BMP signaling pathway that are frequently mutated in CRC and grouped them into the so-called SFAB-signature (SMAD4, FBXW7 (F-box/WD repeat-containing protein 7), ARID1A (AT-rich interactive domain-containing protein 1A), or BMPR2 (Bone morphogenetic protein receptor type II). Clinical data show that approximately 36 % of CRC patients have at least one pathogenic mutation in these genes. I tested all 62 CRC PDO models and found a significant positive prediction for sensitivity to cobimetinib (95 %) and selumetinib (70 %) for the SFAB-signature. Trametinib and the newly approved MEK-inhibitor binimetinib showed a similar trend. Therefore, the SFAB-signature has high predictive power for response to MEK-inhibitors and could be used as a predictive biomarker panel. The current clinically used biomarkers for CRC are based on the mutation status of driver genes KRAS and BRAF, which are present in up to 50 % and 10 % of CRC, respectively. Investigation of molecular alterations in CRC revealed that mutations in the KRAS gene, which is downstream of EGFR (epidermal growth factor receptor) in the MAPK-pathway, interfere with an anti-EGFR-antibody therapy (e.g., cetuximab). Therefore, cetuximab is only relevant for RAS wild-type tumors. However, approximately 40 % of patients with RAS wild-type status do not respond to this treatment. About 53 % of CRC PDO models carry a pathogenic RAS mutation, about 10 % harbor a pathogenic BRAF mutation. Both, the RAS and RAF status alone as well as the combination of RAS and RAF status with SFAB-signature did not provide a better prediction of sensitivity to MEK inhibition.

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Section: Bmp-pathway Activation Leads To Mek-inhibitor Resistancesupporting

confidence: 61%

Section: Bmp-pathway Activation Leads To Mek-inhibitor Resistancementioning

confidence: 65%

Identification of a gene signature as a predictive biomarker for successfull MEK inhibition in colorectal cancer

Pfohl¹

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“…Drug screening assays generally measure the efficacy of drugs by detecting organoid size or enzymatic activity. Hirashita et al established a simple method to evaluate the efficacy of an MEK inhibitor which involved immunostaining-based detection of a change in phosphorylation of ribosomal protein S6 [ 119 ]. We believe that such a method can improve the reproducibility and simplicity of organoid-based drug screening.…”

Section: Challenges and Prospects For The Clinical Application Of Pat...mentioning

confidence: 99%

Patient-Derived Organoids of Colorectal Cancer: A Useful Tool for Personalized Medicine

Kiwaki

Kataoka

2022

JPM

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Colorectal cancer is one of the most important malignancies worldwide, with high incidence and mortality rates. Several studies have been conducted using two-dimensional cultured cell lines; however, these cells do not represent a study model of patient tumors very well. In recent years, advancements in three-dimensional culture methods have facilitated the establishment of patient-derived organoids, which have become indispensable for molecular biology-related studies of colorectal cancer. Patient-derived organoids are useful in both basic science and clinical practice; they can help predict the sensitivity of patients with cancer to chemotherapy and radiotherapy and provide the right treatment to the right patient. Regarding precision medicine, combining gene panel testing and organoid-based screening can increase the effectiveness of medical care. In this study, we review the development of three-dimensional culture methods and present the most recent information on the clinical application of patient-derived organoids. Moreover, we discuss the problems and future prospects of organoid-based personalized medicine.

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“… [ 89 ] CRC MEK MTS assay The ribosomal pS6 has great value of predicting the drug response to trametinib (a MEK inhibitor) in RAS/BRAF mutant CRC PDOs. [ 90 ] CRC mTOR, MNK Cell-Titer Blue assay PDOs with KRAS mutation sustain expression of c-MYC via the MNK/eIF4E pathway in CRC. Patients with activation of h mTORC1 and MNKs may benefit from a c-MYC-dependent co-targeting strategy in clinic.…”

Section: Gic Pdos As An Advantageous Preclinical Model For Targeted T...mentioning

confidence: 99%

“…MEK inhibition could lead to increased LGR5 levels, Wnt activity and stemness- and cancer relapse-related gene expression in CRC PDOs, revealing a side effect of MEK inhibition via inducing stem cell plasticity [ 83 ]. The ribosomal protein S6 (pS6) had great value in predicting the treatment effects of trametinib (a MEK inhibitor) in RAS/BRAF mutant patients with CRC [ 90 ]. Crenolanib, targeting tyrosine kinase receptors, including PDGFRA, PDGFRB, and FLT3, suppressed the growth of both KRAS/BRAF mutation PDOs and KRAS/BRAF wild-type PDO, suggesting that crenolanib may be applied for CRC patients [ 89 ].…”

Section: Gic Pdos As An Advantageous Preclinical Model For Targeted T...mentioning

confidence: 99%

The pivotal application of patient-derived organoid biobanks for personalized treatment of gastrointestinal cancers

Zhu²,

Xiao³

et al. 2022

Biomark Res

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Gastrointestinal cancers (GICs) occupy more than 30% of the cancer-related incidence and mortality around the world. Despite advances in the treatment strategies, the long-term overall survival has not been improved for patients with GICs. Recently, the novel patient-derived organoid (PDO) culture technology has become a powerful tool for GICs in a manner that recapitulates the morphology, pathology, genetic, phenotypic, and behavior traits of the original tumors. Excitingly, a number of evidences suggest that the versatile technology has great potential for personalized treatment, suppling the clinical application of molecularly guided personalized treatment. In the paper, we summarize the literature on the topics of establishing organoid biobanks of PDOs, and their application in the personalized treatment allowing for radiotherapy, chemotherapy, targeted therapy, and immunotherapy selection for GICs. Despite the limitations of current organoid models, high-throughput drug screening of GIC PDO combined with next-generation sequencing technology represents a novel and pivotal preclinical model for precision medicine of tumors and has a great value in promoting the transformation from basic cancer research to clinical application.

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Early response in phosphorylation of ribosomal protein S6 is associated with sensitivity to trametinib in colorectal cancer cells

Cited by 11 publications

References 36 publications

Identification of a gene signature as a predictive biomarker for successfull MEK inhibition in colorectal cancer

Identification of a gene signature as a predictive biomarker for successfull MEK inhibition in colorectal cancer

Patient-Derived Organoids of Colorectal Cancer: A Useful Tool for Personalized Medicine

The pivotal application of patient-derived organoid biobanks for personalized treatment of gastrointestinal cancers

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