The RAS genes, which include H, N, and KRAS, comprise the most frequently mutated family of oncogenes in cancer. Mutations in KRAS – such as the G12C mutation – are found in most pancreatic, half of ...colorectal and a third of lung cancer cases and is thus responsible for a substantial proportion of cancer deaths. Consequently, KRAS has been the subject of exhaustive drug‐targeting efforts over the past 3–4 decades. These efforts have included targeting the KRAS protein itself but also its posttranslational modifications, membrane localization, protein–protein interactions and downstream signalling pathways. Most of these strategies have failed and no KRAS‐specific drugs have yet been approved. However, for one specific mutation, KRASG12C, there is light on the horizon. MRTX849 was recently identified as a potent, selective and covalent KRASG12C inhibitor that possesses favourable drug‐like properties. MRTX849 selectively modifies the mutant cysteine residue in GDP‐bound KRASG12C and inhibits GTP‐loading and downstream KRAS‐dependent signalling. The drug inhibits the in vivo growth of multiple KRASG12C‐mutant cell line xenografts, causes tumour regression in patient‐derived xenograft models and shows striking responses in combination with other agents. It has also produced objective responses in patients with mutant‐specific lung and colorectal cancer. In this review, we discuss the history of RAS drug‐targeting efforts, the discovery of MRTX849, and how this drug provides an exciting and long‐awaited opportunity to selectively target mutant KRAS in patients.
Investigating therapeutic “outliers” that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine ...acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D “knockin” mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.
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•Increased oncogenic Kras expression promotes clonal outgrowth of Kras mutant AMLs•Subsequent loss of WT Kras both enhances fitness and confers MEK dependence•Mutant KRAS allelic imbalance modulates MAPK sensitivity in colorectal cancer cells•55% of advanced KRAS mutant cancers exhibit allelic imbalance at the KRAS locus
An imbalance in the dosage of mutant and wild-type KRAS allele shapes the trade-off between rapid cancer cell growth and resistance to MEK inhibitor therapy. This imbalance explains the challenges encountered during inhibitor trials.
Pancreatic ductal adenocarcinomas (PDA) are extremely aggressive cancers and currently available therapies are only minimally effective in treating this disease. Tackling this devastating cancer has ...been a major challenge to the scientific and medical communities, in part due to its intense therapeutic resistance. One of the aspects of this tumor that contributes to its aggressive behavior is its altered cellular metabolism. Indeed, PDA cells seem to possess the ability to adapt their metabolism to the particular environment to which they are exposed, including utilizing diverse fuel sources depending on their availability. Moreover, PDA tumors are efficient at recycling various metabolic substrates through activation of different salvage pathways such as autophagy and macropinocytosis. Together, these diverse metabolic adaptations allow PDA cells to survive and thrive in harsh environments that may lack nutrients and oxygen. Not surprisingly, given its central role in the pathogenesis of this tumor, oncogenic Kras plays a critical role in much of the metabolic reprogramming seen in PDA. In this review, we discuss the metabolic landscape of PDA tumors, including the molecular underpinnings of the key regulatory nodes, and describe how such pathways can be exploited for future diagnostic and therapeutic approaches.
A quarter of all solid tumors harbor KRAS oncogenes. Yet, no selective drugs have been approved to treat these malignancies. Genetic interrogation of the MAPK pathway revealed that systemic ablation ...of MEK or ERK kinases in adult mice prevent tumor development but are unacceptably toxic. Here, we demonstrate that ablation of c-RAF expression in advanced tumors driven by KrasG12V/Trp53 mutations leads to significant tumor regression with no detectable appearance of resistance mechanisms. Tumor regression results from massive apoptosis. Importantly, systemic abrogation of c-RAF expression does not inhibit canonical MAPK signaling, hence, resulting in limited toxicities. These results are of significant relevance for the design of therapeutic strategies to treat K-RAS mutant cancers.
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•Ablation of c-RAF expression induces regression of Kras/Trp53 mutant lung tumors•Loss of c-RAF expression does not affect MAPK signaling•Systemic ablation of c-RAF expression in adult mice has limited toxic effects
Sanclemente et al. generate oncogenic Kras mice that allow inducible deletion of Raf1, encoding c-RAF, and/or Braf in established KrasG12V or KrasG12V;Trp53−/− lung tumors. They show that systemic c-RAF ablation has limited toxicity and leads to significant tumor regression without having an impact on MAPK activity.
Treatment resistance is observed in all advanced cancers. Colorectal cancer (CRC) presenting as colorectal adenocarcinoma (COAD) is the second leading cause of cancer deaths worldwide. Multimodality ...treatment includes surgery, chemotherapy, and targeted therapies with selective utilization of immunotherapy and radiation therapy. Despite the early success of anti-epidermal growth factor receptor (anti-EGFR) therapy, treatment resistance is common and often driven by mutations in APC, KRAS, RAF, and PI3K/mTOR and positive feedback between activated KRAS and WNT effectors. Challenges in the direct targeting of WNT regulators and KRAS have caused alternative actionable targets to gain recent attention. Utilizing an unbiased drug screen, we identified combinatorial targeting of DDR1/BCR-ABL signaling axis with small-molecule inhibitors of EGFR-ERBB2 to be potentially cytotoxic against multicellular spheroids obtained from WNT-activated and KRAS-mutant COAD lines (HCT116, DLD1, and SW480) independent of their KRAS mutation type. Based on the data-driven approach using available patient datasets (The Cancer Genome Atlas (TCGA)), we constructed transcriptomic correlations between gene DDR1, with an expression of genes for EGFR, ERBB2-4, mitogen-activated protein kinase (MAPK) pathway intermediates, BCR, and ABL and genes for cancer stem cell reactivation, cell polarity, and adhesion; we identified a positive association of DDR1 with EGFR, ERBB2, BRAF, SOX9, and VANGL2 in Pan-Cancer. The evaluation of the pathway network using the STRING database and Pathway Commons database revealed DDR1 protein to relay its signaling
adaptor proteins (SHC1, GRB2, and SOS1) and BCR axis to contribute to the KRAS-PI3K-AKT signaling cascade, which was confirmed by Western blotting. We further confirmed the cytotoxic potential of our lead combination involving EGFR/ERBB2 inhibitor (lapatinib) with DDR1/BCR-ABL inhibitor (nilotinib) in radioresistant spheroids of HCT116 (COAD) and, in an additional devastating primary cancer model, glioblastoma (GBM). GBMs overexpress DDR1 and share some common genomic features with COAD like EGFR amplification and WNT activation. Moreover, genetic alterations in genes like NF1 make GBMs have an intrinsically high KRAS activity. We show the combination of nilotinib plus lapatinib to exhibit more potent cytotoxic efficacy than either of the drugs administered alone in tumoroids of patient-derived recurrent GBMs. Collectively, our findings suggest that combinatorial targeting of DDR1/BCR-ABL with EGFR-ERBB2 signaling may offer a therapeutic strategy against stem-like KRAS-driven chemoradioresistant tumors of COAD and GBM, widening the window for its applications in mainstream cancer therapeutics.
The development of the mouse salivary gland involves a tip-driven process of branching morphogenesis that takes place in concert with differentiation into acinar, myoepithelial, and ductal (basal and ...luminal) sub-lineages. By combining clonal lineage tracing with a three-dimensional (3D) reconstruction of the branched epithelial network and single-cell RNA-seq analysis, we show that in tips, a heterogeneous population of renewing progenitors transition from a Krt14+ multipotent state to unipotent states via two transcriptionally distinct bipotent states, one restricted to the Krt14+ basal and myoepithelial lineage and the other to the Krt8+ acinar and luminal lineage. Using genetic perturbations, we show how the differential expression of Notch signaling correlates with spatial segregation, exits from multipotency, and promotes the Krt8+ lineage, whereas Kras activation promotes proacinar fate. These findings provide a mechanistic basis for how positional cues within growing tips regulate the process of lineage segregation and ductal patterning.
Oncogenic Kirsten RAt Sarcoma (KRAS) mutations are attractive targets in non-small-cell lung cancer (NSCLC). Thus, the objective of this work is to discover promising inhibitors that target this ...protein using in silico methods that have become increasingly cost-effective in research and development of drugs. In this study, 24 triterpenoid saponins were selected for designing the potent inhibitors using different methods: quantitative structure activity relationships (QSAR) analysis, homology modeling, as well as molecular docking, molecular dynamics (MD) simulation and in silico Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) screening. The selected model was presented high-squared cross-validation coefficient Q
2
= 0.85, and external validation R
2
pred = 0.75. In addition, the best comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models presented high values of Q
2
= 0.77 and 0.784 and values of R
2
pred = 0.87 and 0.83, respectively. After that, homology modeling was carried out for modeling the selected target and then validated by both Ramachandran plot and Qualitative Model Energy ANalysis (QMEAN) score of 0.83, indicating the best accuracy of the modeled protein with the experimental results. Furthermore, molecular docking study was conducted to better understanding the binding mechanism of homologous protein with triterpenoid saponins. In addition, MD simulations confirmed the stability of the selected complex systems during 10,000 femtosecondes (fs). According to these studies, three molecules were picked out as potential inhibitors. Indeed, the oral bioavailability and the toxicity of the predicted triterpenoid saponins have been found respecting the ADMET properties.
Communicated by Ramaswamy H Sarma
Synthesis of molecules containing all-carbon quaternary stereocenters has been a longstanding challenge in organic chemistry. In one of our discovery oncology programs, a key chiral building block ...bearing an all-carbon quaternary chiral center was of particular interest and was later identified as a core structure for a KRAS G12C inhibitor. Herein, the development of a safer and practical route to the key building block 1 is described. By replacing processes involving the use of an energetic reagent and extensive chromatographic purifications, a scalable process utilizing chemical resolution was developed to access the chiral building block in kilogram quantities, enabling timely delivery of API for preclinical and clinical studies.
Abstract
The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach ...for drug discovery. However, organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening (HTS)-based drug discovery due to technical difficulties. Using genetically engineered human colon organoids as a model system, here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS. We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library. We further miniaturized the 3D organoid culturing in a 1536-well ultra-HTS format and demonstrated its robust performance for large-scale primary compound screening. Our miniaturized organoid culturing method may be adapted to other types of organoids. By leveraging the power of 3D organoid culture in a high-density plate format, we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.
Clinical treatment of RAS mutant cancers is challenging because of the complexity of the Ras signaling pathway. SLC7A5 is a newly discovered downstream gene of the Ras signaling pathway, but the ...regulatory mechanism is unclear. We aimed to explore the molecular mechanism and role in KRAS mutant lung adenocarcinoma progression.
Key gene that regulated SLC7A5 in KRAS mutant lung adenocarcinoma was screened by RNA sequencing and bioinformatics analysis. The effect of this gene on the expression of SLC7A5 was studied by RNAi. The regulatory mechanism between the two genes was investigated by immunofluorescence, CoIP, pulldown and yeast two-hybrid assays. The location of the two genes was determined by inhibiting Ras and the downstream pathways PI3K-AKT and MEK-ERK. By
and
experiments, the effects of the key gene on the biological functions of KRAS mutant lung adenocarcinoma were explored.
We found a novel gene, ZNF24, which upregulated SLC7A5 protein expression rather than mRNA expression in KRAS mutant lung adenocarcinoma. Endogenous protein interactions occurred between ZNF24 and SLC7A5. Ras inhibition reduced the expression of ZNF24 and SLC7A5. ZNF24 and SLC7A5 are located downstream of the MEK-ERK and PI3K-AKT pathways.
and
functional experiments confirmed that the ZNF24-SLC7A5 signaling axis promoted the proliferation, invasion and migration of KRAS mutant lung adenocarcinoma.
ZNF24 promoted the growth of KRAS mutant lung adenocarcinoma by upregulating SLC7A5 protein expression, which suggested that ZNF24 is a new biomarker of KRAS mutant tumors and could be a new potential therapeutic target for Ras-driven tumors.
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