bims-pimaco 2022-01-30 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–01–30
ten papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Novel approaches for the development of direct KRAS inhibitors: structural insights and drug design.
The mixed blessing of AMPK signaling in Cancer treatments.
HIPK2 cooperates with KRAS signaling and associates with colorectal cancer progression.
Combined MEK/MDM2 inhibition demonstrates antitumor efficacy in TP53 wild-type thyroid and colorectal cancers with MAPK alterations.
Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding.
Impact of the evolution in RAS mutation analysis in Australian patients with metastatic colorectal cancer.
The impact of DAPK1 and mTORC1 signaling association on autophagy in cancer.
KRAS(G12D) can be targeted by potent inhibitors via formation of salt bridge.
Nuclear S6K1 regulates cAMP-responsive element-dependent gene transcription through activation of mTOR signal pathway.
Experiences of running a stratified medicine adaptive platform trial: Challenges and lessons learned from 10 years of the FOCUS4 trial in metastatic colorectal cancer.

Expert Opin Drug Discov. 2022 Jan 27. 1-11

Novel approaches for the development of direct KRAS inhibitors: structural insights and drug design.

Kashif Haider, Anku Sharma, M Shahar Yar, Prasanna Anjaneyulu Yakkala, Syed Shafi, Ahmed Kamal.

   INTRODUCTION: Hyperactivated RAS signaling is reported in 13% of all human cancers, in which ~80% resulted due to KRAS mutations alone. Direct inhibition of KRAS is an important aspect in treating KRAS-related tumors. Despite the efforts of more than four decades, not many KRAS inhibitors have been successful in obtaining clinical approval, except the very recent FDA approval for sotorasib. In recent years, the understanding of structural insights and allosteric pocket identification at catalytic sites of KRAS are likely to provide an excellent opportunity for the development of much more effective clinical candidates.
AREA COVERED: The presented review article mainly summarizes the developments of small molecule KRAS inhibitors as drug candidates and rational approaches that are being utilized for the selective targeting of KRAS signaling in the mutant cancer cells.
EXPERT OPINION: After the initial success in targeting the mutant KRAS G12C variants, the search has been shifted to address the challenges concerning the resistance and efficacy of small molecule KRAS inhibitors. However, the contribution of other KRAS mutations at G12V, G13C, and G13D variants causing cancers is much higher than the mutations at G12C. In view of this aspect, specific attention is required to target all other mutations as well. Accordingly, for the development of KRAS targeted therapies, the design of small molecule inhibitors that can inhibit KRAS signaling and as well as target inhibition of other signaling pathways like RAS-SOS and RAS-PI3K has to be explored extensively.

Keywords:  KRAS inhibitors; Kirsten rat sarcoma; SOS1 inhibitors: clinical trials; colorectal cancer; farnesyltransferase; non-small cell lung cancer

DOI:  https://doi.org/10.1080/17460441.2022.2029842
BMC Cancer. 2022 Jan 25. 22(1): 105

The mixed blessing of AMPK signaling in Cancer treatments.

Mehrshad Sadria, Deokhwa Seo, Anita T Layton.

   BACKGROUND: Nutrient acquisition and metabolism pathways are altered in cancer cells to meet bioenergetic and biosynthetic demands. A major regulator of cellular metabolism and energy homeostasis, in normal and cancer cells, is AMP-activated protein kinase (AMPK). AMPK influences cell growth via its modulation of the mechanistic target of Rapamycin (mTOR) pathway, specifically, by inhibiting mTOR complex mTORC1, which facilitates cell proliferation, and by activating mTORC2 and cell survival. Given its conflicting roles, the effects of AMPK activation in cancer can be counter intuitive. Prior to the establishment of cancer, AMPK acts as a tumor suppressor. However, following the onset of cancer, AMPK has been shown to either suppress or promote cancer, depending on cell type or state.
METHODS: To unravel the controversial roles of AMPK in cancer, we developed a computational model to simulate the effects of pharmacological maneuvers that target key metabolic signalling nodes, with a specific focus on AMPK, mTORC, and their modulators. Specifically, we constructed an ordinary differential equation-based mechanistic model of AMPK-mTORC signaling, and parametrized the model based on existing experimental data.
RESULTS: Model simulations were conducted to yield the following predictions: (i) increasing AMPK activity has opposite effects on mTORC depending on the nutrient availability; (ii) indirect inhibition of AMPK activity through inhibition of sirtuin 1 (SIRT1) only has an effect on mTORC activity under conditions of low nutrient availability; (iii) the balance between cell proliferation and survival exhibits an intricate dependence on DEP domain-containing mTOR-interacting protein (DEPTOR) abundance and AMPK activity; (iv) simultaneous direct inhibition of mTORC2 and activation of AMPK is a potential strategy for suppressing both cell survival and proliferation.
CONCLUSIONS: Taken together, model simulations clarify the competing effects and the roles of key metabolic signalling pathways in tumorigenesis, which may yield insights on innovative therapeutic strategies.

Keywords:  AMPK; Cancer; Dynamical system; Metabolism; mTORC

DOI:  https://doi.org/10.1186/s12885-022-09211-1
Mol Cancer Res. 2022 Jan 26. pii: molcanres.MCR-21-0628-A.2021. [Epub ahead of print]

HIPK2 cooperates with KRAS signaling and associates with colorectal cancer progression.

Micol Di Segni, Ilaria Virdia, Alessandra Verdina, Carla Azzurra Amoreo, Silvia Baldari, Gabriele Toietta, Maria Grazia Diodoro, Marcella Mottolese, Isabella Sperduti, Fabiola Moretti, Simonetta Buglioni, Silvia Soddu, Giuliana Di Rocco.

HIPK2 is an evolutionary conserved kinase that has gained attention as a fine tuner of multiple signaling pathways, among which those commonly altered in colorectal cancer (CRC). The aim of this study was to evaluate the relationship of HIPK2 expression with progression markers and mutational pattern and gain insights into the contribution of HIPK2 activity in CRC. We evaluated a retrospective cohort of CRC samples by immunohistochemistry for HIPK2 expression and by NGS for the detection of mutations of cancer associated genes. We show that the percentage of HIPK2 positive cells increases with tumor progression, significantly correlates with TNM staging and associates with a worse outcome. In addition, we observed that high HIPK2 expression significantly associates with KRAS mutations but not with other cancer related genes. Functional characterization of the link between HIPK2 and KRAS show that activation of the RAS pathway either due to KRAS mutation or via upstream receptor stimulation, increases HIPK2 expression at the protein level. Of note, HIPK2 physically participates in the active RAS complex while HIPK2 depletion impairs ERK phosphorylation and the growth of tumors derived from KRAS mutated CRC cells. Overall, this study identifies HIPK2 as a prognostic biomarker candidate in CRC patients and underscores a previously unknown functional link between HIPK2 and the KRAS signaling pathway. Implications: Our data indicate HIPK2 as a new player in the complex picture of the KRAS signaling network, providing rationales for future clinical studies and new treatment strategies for KRAS mutated CRC.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0628
Sci Rep. 2022 Jan 24. 12(1): 1248

Combined MEK/MDM2 inhibition demonstrates antitumor efficacy in TP53 wild-type thyroid and colorectal cancers with MAPK alterations.

Seyed Pairawan, Argun Akcakanat, Scott Kopetz, Coya Tapia, Xiaofeng Zheng, Huiqin Chen, Min Jin Ha, Yasmeen Rizvi, Vijaykumar Holla, Jing Wang, Kurt W Evans, Ming Zhao, Naifa Busaidy, Bingliang Fang, Jack A Roth, Ecaterina Ileana Dumbrava, Funda Meric-Bernstam.

Most tumors with activating MAPK (mitogen-activated protein kinase) pathway alterations respond poorly to MEK inhibitors alone. Here, we evaluated combination therapy with MEK inhibitor selumetinib and MDM2 inhibitor KRT-232 in TP53 wild-type and MAPK altered colon and thyroid cancer models. In vitro, we showed synergy between selumetinib and KRT-232 on cell proliferation and colony formation assays. Immunoblotting confirmed p53 upregulation and MEK pathway inhibition. The combination was tested in vivo in seven patient-derived xenograft (PDX) models (five colorectal carcinoma and two papillary thyroid carcinoma models) with different KRAS, BRAF, and NRAS mutations. Combination therapy significantly prolonged event-free survival compared with monotherapy in six of seven models tested. Reverse-phase protein arrays and immunohistochemistry, respectively, demonstrated upregulation of the p53 pathway and in two models cleaved caspase 3 with combination therapy. In summary, combined inhibition of MEK and MDM2 upregulated p53 expression, inhibited MAPK signaling and demonstrated greater antitumor efficacy than single drug therapy in both in vitro and in vivo settings. These findings support further clinical testing of the MEK/MDM2 inhibitor combination in tumors of epithelial origin with MAPK pathway alterations.

DOI: https://doi.org/10.1038/s41598-022-05193-z
Nat Commun. 2022 Jan 25. 13(1): 486

Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding.

Juliana A Martinez Fiesco, David E Durrant, Deborah K Morrison, Ping Zhang.

RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes, and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminal site and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.

DOI: https://doi.org/10.1038/s41467-022-28084-3
Asia Pac J Clin Oncol. 2022 Jan 24.

Impact of the evolution in RAS mutation analysis in Australian patients with metastatic colorectal cancer.

Chia Yuen Chong, Azim Jalali, Hui Li Wong, Matthew Loft, Rachel Wong, Margaret Lee, Lucy Gately, Wei Hong, Jeremy Shapiro, Suzanne Kosmider, Jeannie Tie, Sumitra Ananda, Justin M Yeung, Brigette Ma, Matthew Burge, Ross Jennens, Ben Tran, Belinda Lee, Lionel Lim, Andrew Dean, Louise Nott, Peter Gibbs.

   BACKGROUND: RAS mutation testing now routinely informs the optimal management of metastatic colorectal cancer (mCRC), specifically the finding of a RAS mutation defines patients who will not benefit from treatment with an epidermal growth factor receptor inhibitor. Over time more RAS genes have been tested and more sensitive techniques used.
AIMS: To review routine care RAS testing and results over time.
METHODS: A retrospective analysis of the molecular data collected prospectively in the multi-site Treatment of Recurrent and Advanced Colorectal Cancer (TRACC) registry from 2009 to 2018 was undertaken. Patients with RAS data were further analyzed. In parallel, the RAS mutation status of patients enrolled in the Test Tailor Treat (TTT) program was examined for 2011-2018.
RESULTS: Of 2908 patients in the TRACC registry, 1892 (65%) were tested, with 898 (47%) of tested patients found to be RAS mutant (RASmt). RAS data were available for 5935 TTT patients. Of the tested TRACC patients diagnosed in 2009 and 2010, 38% were RASmt. For each 2-year period from 2011/2012 through to 2017/2018, the prevalence of RASmt in TRACC and TTT was 42% and 40% (2011/2012), 52% and 40% (2013/2014), 47% and 49% (2015/2016), and 47% and 49% (2017/2018).
CONCLUSIONS: Based on both TRACC and TTT data, the proportion of patients reported to have a RAS mutation increased from 2009 to 2015 but has remained relatively stable in recent years. The increased proportion of RASmt patients observed over time is likely largely driven by the uptake of extended RAS testing.

Keywords:  RAS testing; colorectal cancer; metastatic

DOI:  https://doi.org/10.1111/ajco.13728
Mol Biol Rep. 2022 Jan 27.

The impact of DAPK1 and mTORC1 signaling association on autophagy in cancer.

Parvaneh Movahhed, Mohammadreza Saberiyan, Amir Safi, Zahra Arshadi, Faranak Kazerouni, Hossein Teimori.

   BACKGROUND: The autophagy pathway is used by eukaryotic cells to maintain metabolic homeostasis. Autophagy has two functions in cancerous cells which could inhibit tumorigenesis or lead to cancer progression by increasing cell survival and proliferation.
METHODS AND RESULTS: In this review article, Web of Science, PubMed, Scopus, and Google Scholar were searched and summarized published studies to explore the relationship between DAPK1 and mTORC1 signaling association on autophagy in cancer. Autophagy is managed through various proteins including the mTOR, which is two separated structural and functional complexes known as mTORC1 and mTORC2. MTORC1 is an important component of the regulatory pathway affecting numerous cellular functions including proliferation, migration, invasion, and survival. This protein plays a key role in human cancers. The activity level of mTORC1 is regulated by the death-associated protein kinases (DAPks) family, especially DAPK1. In many cancers, DAPK1 acts as a tumor suppressor which can be attributed to its ability to suppress cellular transformation and to inhibit metastasis.
CONCLUSIONS: A deep investigation not only will reveal more about the function of DAPK1 but also might provide insights into novel therapies aimed to modulate the autophagy pathway in cancer and to achieve better cancer therapy.

Keywords:  Autophagy; Cancer; DAPK1; mTORC1

DOI:  https://doi.org/10.1007/s11033-022-07154-1
Cell Discov. 2022 Jan 25. 8(1): 5

KRAS(G12D) can be targeted by potent inhibitors via formation of salt bridge.

Zhongwei Mao, Hongying Xiao, Panpan Shen, Yu Yang, Jing Xue, Yunyun Yang, Yanguo Shang, Lilan Zhang, Xin Li, Yuying Zhang, Yanan Du, Chun-Chi Chen, Rey-Ting Guo, Yonghui Zhang.

KRAS mutation occurs in nearly 30% of human cancers, yet the most prevalent and oncogenic KRAS(G12D) variant still lacks inhibitors. Herein, we designed a series of potent inhibitors that can form a salt bridge with KRAS's Asp12 residue. Our ITC results show that these inhibitors have similar binding affinity with both GDP-bound and GTP-bound KRAS(G12D), and our crystallographic studies reveal the structural basis of inhibitor binding-induced switch-II pocket in KRAS(G12D), experimentally confirming the formation of a salt bridge between the piperazine moiety of the inhibitors and the Asp12 residue of the mutant protein. Among KRAS family proteins and mutants, both ITC and enzymatic assays demonstrate the selectivity of the inhibitors for KRAS(G12D); and the inhibitors disrupt the KRAS-CRAF interaction. We also observed the inhibition of cancer cell proliferation as well as MAPK signaling by a representative inhibitor (TH-Z835). However, since the inhibition was not fully dependent on KRAS mutation status, it is possible that our inhibitors may have off-target effects via targeting non-KRAS small GTPases. Experiments with mouse xenograft models of pancreatic cancer showed that TH-Z835 significantly reduced tumor volume and synergized with an anti-PD-1 antibody. Collectively, our study demonstrates proof-of-concept for a strategy based on salt-bridge and induced-fit pocket formation for KRAS(G12D) targeting, which warrants future medicinal chemistry efforts for optimal efficacy and minimized off-target effects.

DOI: https://doi.org/10.1038/s41421-021-00368-w
Biochem Biophys Res Commun. 2022 Jan 10. pii: S0006-291X(22)00029-8. [Epub ahead of print]594 101-108

Nuclear S6K1 regulates cAMP-responsive element-dependent gene transcription through activation of mTOR signal pathway.

Ye Ji Jeon, Sang Ah Yi, Jaecheol Lee, Jeung-Whan Han.

  S6K1 serves as an important signaling regulator of cell proliferation and growth in the mTOR signaling pathway. Excessive activation of the mTOR/S6K1 signaling pathway promotes abnormal cell growth and survival, thereby resulting in tumorigenesis. The roles of S6K1 in protein synthesis and metabolism are well known, but an additional role of S6K1 as a gene transcription regulator has not been much understood. Here, we demonstrated that S6K1 is dynamically distributed in the cytoplasm and nuclei of human cervical cancer cells. S6K1 nuclear localization was serum dependent and serum deprivation or rapamycin treatment inhibited S6K1 Thr389 phosphorylation and, thereby, S6K1 was retained in the cytoplasm. Furthermore, we found that endogenous S6K1 interacted with CREB in the cervical cancer cells. Additionally, S6K1 upregulated the CRE-driven promoter luciferase activity. The proto-oncogene c-JUN, which has several CREs, was attenuated in the S6K1 knockdown cervical cancer cells. The binding of CREB/S6K1 to the c-JUN promoter, altered by serum restimulation, was associated with active epigenetic markers. In HeLa cell, 891 promoter regions, to which S6K1 directly binds, were detected. Our findings suggested that active S6K1, which is dynamically translocated into the nucleus, directly binds to chromatin and could play a role in epigenetic mechanisms or transcription factor recruitment.

Keywords:  CREB; Epigenetics; S6K1; c-JUN

DOI:  https://doi.org/10.1016/j.bbrc.2022.01.015
Clin Trials. 2022 Jan 27. 17407745211069879

Experiences of running a stratified medicine adaptive platform trial: Challenges and lessons learned from 10 years of the FOCUS4 trial in metastatic colorectal cancer.

FOCUS4 Trial Investigators

   BACKGROUND: Complex innovative design trials are becoming increasingly common and offer potential for improving patient outcomes in a faster time frame. FOCUS4 was the first molecularly stratified trial in metastatic colorectal cancer and it remains one of the first umbrella trial designs to be launched globally. Here, we aim to describe lessons learned from delivery of the trial over the last 10 years.
METHODS: FOCUS4 was a Phase II/III molecularly stratified umbrella trial testing the safety and efficacy of targeted therapies in metastatic colorectal cancer. It used adaptive statistical methodology to decide which sub-trial should close early, and new therapies were added as protocol amendments. Patients with newly diagnosed metastatic colorectal cancer were registered, and central laboratory testing was used to stratify their tumour into molecular subtypes. Following 16 weeks of first-line therapy, patients with stable or responding disease were eligible for randomisation into either a molecularly stratified sub-trial (FOCUS4-B, C or D) or non-stratified FOCUS4-N. The primary outcome for all studies was progression-free survival comparing the intervention with active monitoring/placebo. At the close of the trial, feedback was elicited from all investigators through surveys and interviews and consolidated into a series of recommendations and lessons learned for the delivery of similar future trials.
RESULTS: Between January 2014 and October 2020, 1434 patients were registered from 88 UK hospitals. Of the 20 drug combinations that were explored for inclusion in the platform trial, three molecularly targeted sub-trials were activated: FOCUS4-D (February 2014-March 2016) evaluated AZD8931 in the BRAF-PIK3CA-RAS wildtype subgroup; FOCUS4-B (February 2016-July 2018) evaluated aspirin in the PIK3CA mutant subgroup and FOCUS4-C (June 2017-October 2020) evaluated adavosertib in the RAS+TP53 double mutant subgroup. FOCUS4-N was active throughout and evaluated capecitabine monotherapy versus a treatment break. A total of 361 (25%) registered patients were randomised into a sub-trial. Feedback on the experiences of delivery of FOCUS4 could be grouped into three main areas of challenge: funding/infrastructure, biomarker testing procedures and trial design efficiencies within which 20 recommendations are summarised.
CONCLUSION: Adaptive stratified medicine platform studies are feasible in common cancers but present challenges. Our stakeholder feedback has helped to inform how these trial designs can succeed and answer multiple questions efficiently, providing resource is adequate.

Keywords:  Metastatic; adaptive; biomarker; clinical trial; colorectal cancer; complex innovative design; multi-arm multi-stage; stratified

DOI:  https://doi.org/10.1177/17407745211069879