Development of Mutant KRAS Molecuar Target Drugs and Future Prospects: Report
, by Drs. Fuyuhiko Tamanoi, Hideyuki Saya, Toshio Imai, and Kiyoko Kato
Fuyuhiko Tamanoi is a professor at Kyoto University and also holds a cross-appointment at UCLA where he studies signal transduction and cancer therapy. He has recently been involved in initiating new approaches to radiation therapy at Kyoto University. He completed his PhD at Nagoya University and postdoctoral research at Harvard Medical School. Before his professorship at UCLA, he worked as a senior staff investigator at Cold Spring Harbor Laboratory and then as an assistant/associate professor at the University of Chicago.
Hideyuki Saya received his MD from Kobe University School of Medicine and completed his residency in neurosurgery before conducting his PhD research at the Graduate School of Medical Sciences. He completed his postdoctoral fellowship at UCSF, after which he became an assistant professor of Neuro-oncology at the MD Anderson Cancer Center. He served as a Professor at Kumamoto University School of Medicine, and subsequently, he held a professorial position at Keio University School of Medicine. Upon retiring from Keio University, Dr. Saya assumed the role of Director of the Cancer Center at Fujita Health University.
Toshio Imai is a professor of Advanced Therapeutic Target Discovery at Kobe University Graduate School of Medicine. His research interests include the role of chemokines (i.e. fractalkine) in inflammatory diseases and their potential for therapeutic target. Recently, he has been conducting research on early diagnosis and prevention of pancreatic cancer utilizing patient-derived organoids.
Kiyoko Kato is an MD, PhD researcher specializing in obstetrics and gynecology. She is a professor of reproductive and developmental medicine at Kyushu University where her research focuses on endometrial cancer stem cells and therapies for hormone-dependent cancers.
On April 26, 2023, from 9:00 a.m. to 3:30 p.m., an online conference entitled "Development of Mutant KRAS Molecular Target Drugs and Future Prospects" was held by the Quantum Nano Medicine Research Center, Institute for Advanced Studies, Kyoto University, and co-organized by the Japanese Cancer Association. The conference was the first time that cancer researchers in Japan got together and discussed the recent development of anticancer drugs targeting mutant KRAS. A keynote lecture by Dr. Channing J. Der started the meeting followed by four sessions featuring talks by Japanese researchers working on related fields. 521 people registered for the conference, indicating the high interest of researchers from universities and companies in the research and development of mutant KRAS targeted drugs. Research and development of therapeutic drugs targeting KRAS mutant genes, which had been considered difficult to achieve, is now actively pursued in the world. In the opening remarks, Dr. Fuyuhiko Tamanoi explained the purpose of the meeting, which was to gain an accurate understanding of the current development, to share up-to-date information and to discuss future prospects.
In the keynote address, Dr. Channing J. Der (University of North Carolina at Chapel Hill), who was involved in the study of mutations in the human RAS gene that were first identified in early 1980s, gave a talk entitled "Recent progress on the preclinical and clinical development of mutant KRAS targeted drugs. " He talked about the discovery of the RAS gene, RAS mutations in cancer, RAS as a molecular switch, RAS protein structure and RAS signaling, and the recent development of direct inhibitors including G12C-specific inhibitors, G12D-selective inhibitors, and tri-complex inhibitors. The development of competitive inhibitors has been challenging due to the extremely high affinity of GTP for RAS (60 pM). In 2013, Kevan Shokat et al. reported a molecularly targeted drug that irreversibly binds to G12C and inhibits effector activation, marking the beginning of the development of KRAS molecular targeted drugs. In 2014, to accelerate progress in understanding and drugging RAS, the U.S. National Cancer Institute (NCI) launched the “RAS Initiative”, a program led by Frank McCormick (University of California, San Francisco). In 2021, the first KRAS inhibitor, sotorasib, was approved for the treatment of non-small cell lung cancer (NSCLC) that harbors one specific KRAS mutation, G12C, and a second, adagrasib, approved in 2022 for the same indication. While remarkable responses were seen in a subset of patients, essentially all response patients relapse, developing acquired resistance. Thus, a major challenge for the field is to better understand mechanisms of primary and acquired resistance, to then develop combination strategies to expand and extend the clinical effectiveness of G12C, and likely all RAS, inhibitors. One promising combination involves anti-EGFR inhibitor in combination with G12C inhibitors in colorectal cancer. Astellas' ASP3082 and Mirati’s MRTX133 have been developed as KRAS G12D selective inhibitors. In addition, one tri-complex inhibitor that target the activated GTP-bound RAS proteins are being developed to treat cancers with a variety of RAS mutations. RMC-6236 from Revolution Medicines binds non-covalently to cyclophilin A (CypA), an intracellular chaperone protein, and forms a selective three-way complex with GTP-bound RAS, sterically inhibiting RAS binding to downstream effector molecules such as RAF. RAS Multi(ON) inhibitors are being shown to be effective with minimal side effects.
Session 1 Radiotherapy Targeting the KRAS Gene
Chair: Fuyuhiko Tamanoi
Dr. Hiroshi Sugiyama (Kyoto University) presented a talk entitled, "DNA sequence-selective pyrrole-imidazole polyamides for cancer therapy". Using a sequence-selective DNA binding molecule, pyrrole-imidazole polyamide (PIP), he reported that PIP linked to an alkylating agent reacts selectively with KRAS mutant sequences, acts as an off-switch to suppress KRAS expression, resulting in tumor growth inhibition in tumor-bearing mice with KRAS mutations. This PIP off-switch approach could be used to inhibit transcription factor RUNX in various mouse tumor models and the enhancement of expression of specific gene clusters by HDAC inhibitor-linked PIP on-switch were also demonstrated, revealing the broad potential of PIP as an artificial gene switch.
Dr. Koichi Fukase (Osaka University) gave a presentation entitled, "Development of alpha-particle nuclear medicine therapy at Osaka University," in which he introduced that alpha-particle emitting radionuclide therapy (TAT) has two excellent features: strong cancer cell-killing effect due to the high energy of alpha-particles and minimally invasive effect on surrounding organs due to the short range of alpha-particles. Osaka University has facilities for 211At (Astatine) production using an accelerator, 211At drug development, preclinical and clinical research, and is planning to start a physician-initiated clinical trial using 211At for refractory thyroid cancer in 2021, and to develop a 211At-labeled TAT drug targeting prostate-specific membrane antigen (PSMA) for antitumor effects in carcinoma-bearing mice. Preparation of 211At-labeled pyrrole imidazole polyamide KR12, which selectively recognizes the KRAS G12V mutation sequence, is currently going on and 211At-labeled PIP drugs with a half-life of 7.2 hours are expected to rapidly accumulate in KRAS mutant tumors to achieve high therapeutic efficacy while minimizing side effects.
Dr. Hiroshi Harada (Kyoto University) discussed "Influence of the Oxygen Environment in Tumors on the Combined Effects of Mutant KRAS Molecular Targeted Drugs and Radiotherapy" from the perspective of radiation biology and hypoxia biology. He mentioned that the radiosensitizing effect can be enhanced by the inhibition of the EGF receptor-RAS-RAF-MEK-ERK pathway and that this is dependent on the tumor oxygen environment in the tissue and mutations in the EGFR and RAS genes. During radiotherapy, the DNA damage marker γH2AX is found around tumor blood vessels, but not in pimonidazole-positive hypoxic environment. On the other hand, EGF receptors are activated in a special type of lipid raft called caveolae in hypoxic cancer cells, and the effect of the anti-EGFR antibody drug cetuximab was strongly observed. Important basic findings leading to combination therapy with KRAS inhibitors and radiation therapy are expected in future studies.
Session 2 Discovery of Allosteric Inhibitors and Potential for Further Development of Molecularly Targeted Drugs
Chair: Toshio Imai
Dr. Hiroaki Suga (University of Tokyo) presented his findings in his talk "Specialty Peptide Drug Discovery by Targeting KRAS and Related Proteins". The development of genetic code reprogramming combined with an in vitro display system called the RaPID (Random Nonstandard Peptide Integrated Discovery) system can yield candidate molecules with high affinity (nM level) from 1 trillion (1012) specialty cyclic peptide libraries in about one month. There were reports of an interesting 15-year development process involving the evolution of individual elemental technologies and their combination, such as cell-free translation systems involving non-natural peptides using Flexizyme and cyclization of peptides by -Cl and -SH groups. For KRAS, cyclic peptides that bind to the activating GTP-binding RAS but not to the inactive GDP-binding RAS were obtained. The binding of the cyclic peptides will be confirmed by X-ray structural analysis to find interesting regions that alter the structure of KRAS. Targeting the membrane-permeable peptide library and RASMulti(ON) will lead to the development of drugs with superior pharmacological properties.
Dr. Ryo Honda (Gifu University) presented his attempt to develop KRAS molecular targeting drugs in his presentation entitled "Attempt to develop KRAS molecular-targeted drugs by using artificial chimeric proteins." He described the generation of about 300 artificial chimeric proteins with molecular weights exceeding 10,000 Da, which consist of two domains, a protein that interacts with KRAS intracellularly (Ras-binding domain, RBD) and a cell membrane-permeable peptide (Cell-permeable peptide, CPP), and showed strong antitumor effects in a mouse model. The chimeric protein functions as a KRAS Multi inhibitor, and its efficacy in an allogeneic tumor transplantation model but not in a xenograft model suggested the involvement of the immune system. In combination with gemcitabine, tumor regression has been observed. The possibility of drug discovery modality by cell membrane permeable protein with weak cell membrane disruption was demonstrated.
Session 3 Current Status and Future Prospects of Clinical Trials in Japan
Chair: Kiyoko Kato
Dr. Toshihiko Doi (National Cancer Center Hospital East) presented his talk entitled "RAS Inhibitors: From the Standpoint of Early Clinical Development", in which he raised the issue of delayed development of KRAS inhibitors in Japan. The FDA approval of Amgen's Sotrasib has provided a new therapeutic direction for patients with KRAS G12C-mutated non-small cell lung cancer and has also greatly stimulated RAS inhibitor research. In addition to small molecule inhibitor approaches, drug discovery based on new mechanisms that directly target KRAS is accelerating. Research and development are progressing to determine what combination drugs with KRAS G12C are optimum and what the next targets should be. KRAS inhibitors currently being developed primarily in the U.S. are being done by so-called biotech sectors. So-called emerging bio pharma (EBP) tends to aim for approval only in the U.S. and not pursue international expansion due to limited funding. Therefore, if clinical development is not possible in Japan, translational research and next-generation basic research will be delayed, resulting in drug loss. In addition, clinical development of EBPs is limited in Japan compared to the U.S. and China, and drug discovery will face a difficult situation in the future. It was pointed out that basic researchers want to build supercars, but clinical research requires the development of mass-market cars, and that it is necessary to link basic research and clinical research from the early stages of research.
Dr. Isamu Okamoto (Kyushu University) presented his talk entitled "Advances in Genomic Medicine in Non-Small Cell Lung Cancer: Clinical Introduction of Sotorasib, a KRAS Inhibitor." In advanced non-small cell lung cancer, there are nine driver gene abnormalities that serve as therapeutic targets and molecular targeted therapies that capture these therapeutic targets and exert anti-tumor effects have been introduced into clinical practice, and Sotorasib was the first KRAS inhibitor to be introduced into clinical practice. In a global Phase III trial in previously treated patients with KRASG12C-positive advanced non-small cell lung cancer, in which Dr. Okamoto participated as an investigator, Sotrasib was shown to be more effective than Docetaxel, the conventional standard of care, establishing a new standard of care for this patient group, which comprises approximately 5% of Japanese lung adenocarcinoma patients. In the early 2000s, the median survival rate was less than one year, but in recent years, the median survival rate has improved dramatically to more than five years.
Session 4 KRAS signaling, drug resistance, organoid medicine
Chair: Hideyuki Saya
Dr. Masanobu Oshima (Kanazawa University) presented a lecture entitled "Role of KrasG12D mutations in the malignant transformation of intestinal tumors". His research was aimed to elucidate the role of KRAS G12D mutations in the malignant transformation process of colorectal cancer, using various combinations of driver gene mutations such as APC, KRAS, SNAD4, TP53 and others in the intestinal using mouse models. He showed that the number of colonic polyps and tumors was significantly increased in ApcΔ716 KrasG12D double mutant mice compared to ApcΔ716 mutant mice. This suggests that activated KRAS induces COX-2 expression to form an inflammatory microenvironment that may support the survival and proliferation of early tumor cells. In addition, the ApcΔ716 KrasG12D double mutation did not cause submucosal invasion of intestinal tumors, but when the KrasG12D mutation was added to tumors with submucosal invasion by the ApcΔ716 Trp53R270H or ApcΔ716 Tgfbr2-/- double mutations, malignant traits such as EMT with fibrous microenvironment formation in submucosal tissues were enhanced and played an important role in metastasis formation. Dr. Ryohei Katayama (Cancer Research Institute, Japan) presented latest findings in his presentation entitled "Mechanisms of KRAS Inhibitor Resistance in KRAS-Positive Cancers." The recent development of mutant KRAS-specific inhibitors has revolutionized the therapeutic strategy for KRAS G12C mutant lung cancer. However, the therapeutic efficacy of KRAS G12C-specific inhibitors such as Sotorasib varies among cancer types, suggesting limited efficacy and early resistance in patients with KRAS G12C-mutated colorectal cancer. Relapse due to acquired resistance is also an issue in KRAS G12C mutant lung cancer. Indeed, most patient-derived KRAS-mutant colorectal cancer cell lines (PDCs) showed initial resistance to KRAS inhibition by KRAS inhibitors or KRAS siRNA. The mechanisms of early resistance can be classified into at least two groups, one of which involves reactivation of the EGFR-mediated MAPK pathway by KRAS inhibition.
Dr. Kazuhiro Togasaki (Keio University) presented his latest results in his talk entitled, "Organoids as an approach to the development of personalized medicine", focusing on lung cancer harboring KRAS mutations. They have established a genotype-phenotype association method through a new research approach that combines in vitro disease modeling using patient-derived organoids and genome editing. To understand the heterogeneity of lung cancer, a 43-line lung cancer organoid library was generated and revealed that lung adenocarcinomas can be divided into two subgroups, Wnt-dependent and Wnt-independent: the Wnt-dependent group harbors KRAS mutations and expresses HNF4A, while the Wnt-independent group harbors EGFR mutations and expresses NKX2-1. Deletion of the alveolar identity gene NKX2-1 resulted in Wnt-dependence regardless of EGFR mutation. Sensitivity to Wnt-targeted therapy can be stratified by NKX2-1 expression status, an important finding that suggests Wnt-targeted therapy for NKX2-1 negative lung adenocarcinomas, which account for about 10-20% of lung adenocarcinomas and are considered resistant to therapy. Thus, phenotype-driven screening and engineering of organoids are useful for the development of new cancer treatment strategies.
In the general discussion, the participants discussed the resistance mechanism of KRAS inhibitors, the possibility of developing new molecular-targeted drugs, and the possibility of combining KRAS inhibitors with other drugs. There was also a lively discussion on how Japan can contribute to the field of KRAS-targeted therapy. In order for Japan to win the competition, it is important to promote Japan's unique modality development capabilities and clinical development originating from biotech in Japan by linking translational research with next-generation basic research on KRAS. Regarding research funding, it was pointed out that it is an issue how to access not only public funds but also private funds in Japan. The development of drugs targeting KRAS mutant genes, which had been considered difficult to develop, has required long-term efforts. This symposium provided an opportunity to further strengthen ties among KRAS researchers in Japan. It was agreed that further gatherings should be held in the future.