Development Of Farnesyltransferase Inhibitors Research Articles

Abstract 1181: PTEN loss-of-function mutations prevalent in HRAS-mutant cancers results in resistance to targeted therapy

Abstract Background: The clinical development of farnesyltransferase inhibitors (FTIs) as targeted therapy for HRAS-mutant cancers has demonstrated mixed responses dependent on cancer type. Co-occurring mutations may affect tumor response, supported by previous studies demonstrating that NF1 mutations confer resistance to HRAS inhibition by the FTI tipifarnib in thyroid cancer mouse models. We aimed to determine if PI3K pathway activating mutations altered responses to targeted therapy in HRAS-mutant cancers. Methods: Targeted sequencing data from MSK-IMPACT cohort and DFCI-GENIE (Version 9.0) database was used to investigate co-mutations amongst HRAS-mutant cancers. Fisher’s exact test was used to determine co-altered mutations found predominantly in HRAS-mutant cancers relative to respective KRAS- and NRAS-mutant cancers. ‘RASless’ (KRASlox/HRASKO/NRASKO) mouse embryonic fibroblasts (MEFs) were obtained that in the presence of 600nM tamoxifen (4OHT) resulted in a KRAS knock-out. ‘Rasless’ MEFs were transfected with HRASG13R to create a system for testing sensitivity to FTIs in the presence or absence of WT KRAS, or with concurrent PTEN loss generated by CRISPR-Cas9 technology. Results: A greater proportion of HRAS-mutant cancers had co-altered mutations (48.8%) in genes encoding effectors in the MAPK, PI3K or RTK pathways compared to KRAS- and NRAS-mutant cancers (41.4% and 38.4%, respectively; p<0.05). PTEN mutations were more prevalent in HRAS-mutant NSCLC (21%) compared to KRAS- and NRAS-mutant NSCLC (1% and 2%, respectively; p<0.05). Non-transfected MEFs were sensitized to tipifarnib by introduction of a HRASG13R allele in non-4OHT (IC50: MEF= >3uM, HRASG13R = 324.7nM) and 4OHT (IC50: MEF= >3uM, HRASG13R= 0.62nM; p<0.001) conditions, indicating that WT KRAS confers a relative resistance to the inhibitory effects of the FTI on HRAS. PTEN loss-of-function mutations led to tipifarnib resistance in HRASG13R MEFs in the absence (IC50: >3uM; p<0.001) or presence of 4OHT (IC50: 213.6nM; p<0.001). Combined treatment of HRASG13R/PTEN MEFs with the PIK3CB-specific inhibitor AZD8186 and tipifarnib sensitized cells in non-4OHT (IC50- 100nM:100nM Tipifarnib:AZD8186) and 4OHT (IC50- 100nM:10nM Tipifarnib:AZD8186) conditions. Conclusions: Co-altered mutations of MAPK, PI3K or RTK effectors are found more commonly in HRAS than in KRAS or NRAS-mutant cancers. Co-alteration of PTEN preferentially associated with HRAS-mutations in NSCLC. Deletion of PTEN resulted in resistance to FTI targeted therapy in vitro. Co-altered mutations may predict sensitivity and resistance to FTIs and guide clinical trial design. Citation Format: Aradhya Nigam, Walid K. Chatila, Gnana P. Krishnamoorthy, Alan L. Ho, James A. Fagin, Nikolaus D. Schultz, Brian R. Untch. PTEN loss-of-function mutations prevalent in HRAS-mutant cancers results in resistance to targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1181.

Concurrent Inhibition of ERK and Farnesyltransferase Suppresses the Growth of HRAS Mutant Head and Neck Squamous Cell Carcinoma.

Human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is a common cancer worldwide with an unmet need for more effective, less toxic treatments. Currently, both the disease and the treatment of HNSCC cause significant mortality and morbidity. Targeted therapies hold new promise for patients with HPV-negative status whose tumors harbor oncogenic HRAS mutations. Recent promising clinical results have renewed interest in the development of farnesyltransferase inhibitors (FTIs) as a therapeutic strategy for HRAS-mutant cancers. With the advent of clinical evaluation of the FTI tipifarnib for the treatment of HRAS-mutant HNSCC, we investigated the activity of tipifarnib and inhibitors of HRAS effector signaling in HRAS-mutant HNSCC cell lines. First, we validated that HRAS is a cancer driver in HRAS-mutant HNSCC lines. Second, we showed that treatment with the FTI tipifarnib largely phenocopied HRAS silencing, supporting HRAS as a key target of FTI antitumor activity. Third, we performed reverse-phase protein array analyses to profile FTI treatment-induced changes in global signaling, and conducted CRISPR/Cas9 genetic loss-of-function screens to identify previously unreported genes and pathways that modulate sensitivity to tipifarnib. Fourth, we determined that concurrent inhibition of HRAS effector signaling (ERK, PI3K, mTORC1) increased sensitivity to tipifarnib treatment, in part by overcoming tipifarnib-induced compensatory signaling. We also determined that ERK inhibition could block tipifarnib-induced epithelial-to-mesenchymal transition, providing a potential basis for the effectiveness of this combination. Our results support future investigations of these and other combination treatments for HRAS mutant HNSCC.

159 Poster - Antitumor activity of tipifarnib and PI3K pathway inhibitors in HRAS-associated head and neck squamous cell carcinoma

HRAS is an important driver oncogene in head and neck squamous cell carcinoma (HNSCC) and other squamous cell carcinomas. HRAS mutations are found in 4–10% of HNSCC cases and appear to represent a unique subset associated with low TP53 mutation rates, co-mutated caspase 8 and a characteristic methylation pattern. RAS proteins undergo several posttranslational modifications that facilitate their attachment to membranes, including the addition of a farnesyl isoprenoid moiety by the enzyme farnesyltransferase (FT), prompting the development of FT inhibitors (FTIs) for the treatment of RAS-driven cancers. Tipifarnib is a potent and selective FTI that has demonstrated compelling antitumor activity in a heavily pretreated cohort of recurrent/metastatic HNSCC with HRAS mutations. Based on these data, a pivotal study (NCT03719690) evaluating the efficacy of tipifarnib in HRAS mutant HNSCC (AIM-HN) is currently ongoing.

Is there a future for prenyltransferase inhibitors in cancer therapy?

It has been over 20 years since it was first recognized that the function of both normal and oncogenic Ras is dependent on the post-translational modification termed farnesylation. Since that time, intense effort has been expended on the development of farnesyltransferase inhibitors as novel anticancer agents. Over 70 clinical trials have now been conducted, with limited efficacy demonstrated. Here we provide an update of the most recently published clinical trials, discuss the use of the RASGRP1/APTX two-gene expression screen to select patients with acute myeloid leukemia for therapy, and report on the latest discoveries related to the targets of prenyltransferase inhibitors.

Enzyme Flexibility and the Catalytic Mechanism of Farnesyltransferase: Targeting the Relation

Farnesyltransferase enzyme (FTase) is an interesting target for anticancer therapy that has been the subject of particular attention over the past decade. However, despite of the thrilling achievements in the development of farnesyltransferase inhibitors (FTIs) over the past few years, the farnesylation mechanism remains, to some degree, a mystery. This work describes the application of molecular dynamics simulations to the study of enzyme flexibility in the 4 key intermediate states formed during the FTase catalytic mechanism--FTase resting state, binary complex (FTase-FPP), ternary complex (FTase-FPP-Peptide), and product complex (FTase-Product)--thereby covering the main states in the mechanistic pathway of this mysterious enzyme, while relating, dissecting, and exploring, in minute detail, the set of structural and dynamical changes taking place with FPP binding, peptide coordination and product formation. This study reveals the existence of a series of variational patterns involving the mechanistic events taking place at the active site of the enzyme, increasing in magnitude away from the active-site, demonstrating that relatively small-scale events such as substrate binding or product formation cause minor changes at the neighboring residues and corresponding helices, but ultimately induce much more dramatic effects on the more external regions of the enzyme.

Post-translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets

The involvement of the RAS superfamily of monomeric GTPases in carcinogenesis is increasingly being appreciated. A complex array of post-translational modifications and a highly sophisticated protein network regulate the spatio-temporal activation of these GTPases. Previous attempts to pharmacologically target this family have focused on the development of farnesyltransferase inhibitors, but the performance of such agents in cancer clinical trials has not been as good as hoped. Here, we review emerging druggable targets and novel therapeutic approaches targeting prenylation and post-prenylation modifications and the functional regulation of GDP/GTP exchange as exciting alternatives for anticancer therapy.

Effective tailor-made force field parameterization of the several Zn coordination environments in the puzzling FTase enzyme: opening the door to the full understanding of its elusive catalytic mechanism

Protein farnesyltransferase (FTase) is very promising anticancer drug target, with several drugs in advanced stages of clinical testing. However, in spite of the thrilling achievements in the development of farnesyltransferase inhibitors (FTIs) over the past few years, the farnesylation mechanism remains, to some degree, a mystery. This work reports the determination and validation of three sets of molecular mechanical parameters specifically tailored to accurately account for the very specific nature of the several Zn coordination spheres formed during the unclear catalytic pathway of this puzzling metalloenzyme, and built on the top of recent experimental and theoretical results that have dramatically changed the way how the farnesylation mechanism is perceived. Extensive validation studies with 14 FTase crystallographic structures, EXAFS data, DFT, and QM/MM theoretical calculations are presented.

Development of Farnesyl Transferase Inhibitors: A Review

Farnesyl transferase inhibitors are a new class of biologically active anticancer drugs. The exact mechanism of action of this class of agents is, however, currently unknown. The drugs inhibit farnesylation of a wide range of target proteins, including Ras. It is thought that these agents block Ras activation through inhibition of the enzyme farnesyl transferase, ultimately resulting in cell growth arrest. In preclinical models, the farnesyl transferase inhibitors showed great potency against tumor cells; yet in clinical studies, their activity was far less than anticipated. Reasons for this disappointing clinical outcome might be found in the drug-development process. In this paper, we outline an algorithm that is potentially useful for the development of biologically active anticancer drugs. The development of farnesyl transferase inhibitors, from discovery to clinical trials, is reviewed on the basis of this algorithm. We found that two important steps of this algorithm were underestimated. First, understanding of the molecular biology of the defective pathway has mainly been focused on H-Ras activation, whereas activation of K-Ras or other farnesylated proteins is probably more important in tumorigenesis. Inhibition of farnesylation is possibly not sufficient, because geranylgeranylation might activate K-Ras and suppress the effect of farnesyl transferase inhibitors. Furthermore, a well-defined proof of concept in preclinical and clinical studies has not been achieved. Integrating the proposed algorithm in future studies of newly developed biologically active anti-cancer drugs might increase the rate of success of these compounds in patients.

Inhibitors of Farnesyltransferase and Geranylgeranyltransferase-I for Antitumor Therapy: Substrate-Based Design, Conformational Constraint and Biological Activity

The development of farnesyltransferase inhibitors, a novel approach to non-cytotoxic anticancer therapy, has been an active area of research over the past decade. Compounds that have advanced to clinical trials were evolved both from substrate-based design efforts and from compound library screening hits. This review focuses on the effort at Merck to evolve inhibitors from the protein substrate of farnesyltransferase, which resulted in the identification of a non-peptide inhibitor for clinical evaluation. X-ray crystal structures of farnesyltransferase complexed with early peptidomimetic as well as later non-peptide inhibitors have validated this design approach. NMR spectroscopic methods for studying enzyme-bound inhibitor structure, in conjunction with the use of conformational constraints, were critical components of subsequent efforts to provide potent inhibitors with varying levels of farnesyltransferase and geranylgeranyltransferase-I inhibitory specificity. Several of these compounds were important tools for investigating the use of prenyltransferase inhibitors to target Ki-Ras-mediated tumor growth.

Unraveling the mechanism of the farnesyltransferase enzyme

Farnesyltransferase enzyme (FTase) is currently one of the most fascinating targets in cancer research. Studies in other areas, namely in the fight against parasites and viruses, have also led to very promising results. However, in spite of the thrilling achievements in the development of farnesyltransferase inhibitors (FTIs) over the past few years, the farnesylation mechanism remains, to some degree, a mystery. This review tries to shed some light on this puzzling enzyme by analyzing seven key mechanistic dilemmas, based on recent studies that have dramatically changed the way this enzyme is currently perceived.

Perspectives on farnesyl transferase inhibitors in cancer therapy

The discovery that the transforming activity of oncogenic Ras depends upon its post-translational farnesylation has led to the development of farnesyl transferase inhibitors (FTIs). FTIs inhibit the growth of ras-transformed cells in vitro and induce tumor regression in Ras-dependent tumors. Currently, FTIs are undergoing clinical trials in various solid or hematological malignancies. In this review, we will summarize our current knowledge on cellular effect and molecular mechanism of FTIs. We will then describe recent clinical trials and propose some clues for their interpretation. Based on pre-clinical findings, we will emphasize on the optimal use of FTIs in anti-cancer strategy and lastly, we will insist on the interest of combining FTIs with chemotherapy, radiotherapy or other targeted agents.

Inhibitors of farnesyltransferase and geranylgeranyltransferase-I for antitumor therapy: substrate-based design, conformational constraint and biological activity.

The development of farnesyltransferase inhibitors, a novel approach to non-cytotoxic anticancer therapy, has been an active area of research over the past decade. Compounds that have advanced to clinical trials were evolved both from substrate-based design efforts and from compound library screening hits. This review focuses on the effort at Merck to evolve inhibitors from the protein substrate of farnesyltransferase, which resulted in the identification of a non-peptide inhibitor for clinical evaluation. X-ray crystal structures of farnesyltransferase complexed with early peptidomimetic as well as later non-peptide inhibitors have validated this design approach. NMR spectroscopic methods for studying enzyme-bound inhibitor structure, in conjunction with the use of conformational constraints, were critical components of subsequent efforts to provide potent inhibitors with varying levels of farnesyltransferase and geranylgeranyltransferase-I inhibitory specificity. Several of these compounds were important tools for investigating the use of prenyltransferase inhibitors to target Ki-Ras-mediated tumor growth.

Structure based design of benzophenone-based non-thiol farnesyltransferase inhibitors.

Farnesyltransferase catalyzes the transfer of a farnesyl residue from farnesylpyrophosphate to the thiol of a cysteine side chain of proteins which carry at the C-terminus the so called CAAX-sequence. Although the exact cellular events affected by farnesyltransferase inhibiton remain to be determined, farnesyltransferase has become a major target in the development of potential anti-cancer drugs. Numerous farnesyltransferase inhibitors have been described from which the majority are CAAX-peptidomimetics possessing a free thiol group which coordinates the enzyme-bound zinc ion. The development of farnesyltransferase inhibitors is clearly directed towards the so-called non-thiol farnesyltransferase inhibitors because of adverse drug effects connected to free thiols. This review mainly deals with the efforts of the authers group towards the design of non-thiol-farnesyltransferase inhibitors. Our first step on the way to non-thiol farnesyltransferase inhibitors was the development of an CAAX-peptidomimetic based on a pharmacophore model. On the basis of this benzophenone core, bisubstrate analogues were developed as one class of non-thiol farnesyltransferase inhibitors. In most non-thiol farnesyltransferase inhibitors known in literature nitrogene containing heterocycles are used as cysteine replacements supposedly coordinating the enzyme bound zinc. However, we and others have shown that nitrogen heterocycles can be replaced by aryl residues lacking the ability to coordinate metal atoms, an observation which let to the postulation of two hitherto unknown aryl binding sites. Using flexible docking of model compounds and GRID analysis we were able to locate these postulated aryl binding sites. Subsequently, we used one of this aryl binding sites for the structure based design of highly active non-thiol farnesyltransferase inhibitors.

Non-peptidic prenyltransferase inhibitors: diverse structural classes and surprising anti-cancer mechanisms.

The development of farnesyltransferase inhibitors (FTIs) has been one of the most active areas of anticancer drug development for the past ten years. This review presents a general overview of the developments in this area, along with a critical appraisal of the anticancer activity of FTIs. A historical survey of the protein prenylation field is given, in particular to emphasize the key role played by the Ras oncoprotein in driving the discovery of prenyltransferase enzymes. The different classes of prenylated proteins will be described along with the biochemical characteristics of the key drug target--farnesyltransferase (FTase). Numerous potent farnesyltransferase inhibitors have been developed. The FTIs developed can be separated into three different categories, based on their origin and/or mechanism of action: a) natural products; b) peptidomimetics and other CAAX-competitive inhibitors; c) farnesyl pyrophosphate (FPP) mimetics or analogs and other FPP-competitive inhibitors. Along with a survey of newer FTIs in each class, the development of several representative, potent compounds will be discussed in depth as we discuss the potential advantages and liabilities of each class. Particular emphasis is given to the discovery of new, more potent FPP-competitive FTIs of several diverse structural classes. Testing of different FTIs for their ability to block the growth of various cancer cell types in animal models will be discussed. There are a number of key differences between these compounds and traditional cytotoxic cancer chemotherapeutic agents, with surprising exceptions to their expected modes of action. As some FTIs have entered human clinical trials, answers may soon become available to key mechanistic questions concerning the extent and nature of their antitumor growth properties.

Pre-clinical development of farnesyltransferase inhibitors.

ras is the oncogene most frequently found in human cancers, being detected in 30% of most human cancers and at significantly higher rates in certain cancers including pancreatic (90%) and colon (50%) [1]. Almost 10 years ago it was shown that a C-terminal lipid modification of Ras, catalyzed by a specific farnesyl-protein transferase (FPTase), was required for the function of both normal and oncogenic Ras proteins. This finding spurred the development of FPTase inhibitors (FTIs) as a potential cancer therapy directed at the ras oncogene. FTIs have exhibited potent antiproliferative activity in cell culture and animal tumor models with a surprising lack of toxicity to normal tissues. However, while FTIs were originally conceptualized as Ras-specific agents, their mechanism of action is significantly more complicated than originally envisioned.

Advances in the development of farnesyltransferase inhibitors: Substrate recognition by protein farnesyltransferase

A variety of compounds that show promise in cancer chemotherapy and chemoprevention have been identified as farnesyltransferase inhibitors. These can be classified into mainly two different types of inhibitors, farnesyl diphosphate competitors and CAAX peptidomimetics. The former type acts by competitively inhibiting farnesyltransferase with respect to one of the substrates, farnesyl diphosphate, whereas the latter type acts by mimicking the other substrate, the C-terminal CAAX motif of Ras protein. One example of a farnesyl diphosphate competitor is manumycin, an antibiotic detected in the culture media of a Streptomyces strain. The CAAX peptidomimetics were developed based on the unique property of farnesyltransferase to recognize the CAAX motif at the C-terminus of the protein substrate. Our recent studies have focused on understanding the structural basis of this CAAX recognition. By using in vitro mutagenesis, residues of yeast farnesyltransferase important for the recognition of the CAAX motif have been identified. Two of these residues are closely located at the C-terminal region of the beta-subunit of farnesyltransferase. These and other results on the structural basis of the CAAX recognition may provide information valuable for structure-based design of farnesyltransferase inhibitors.

Advances in the development of farnesyltransferase inhibitors: Substrate recognition by protein farnesyltransferase

Advances in the development of farnesyltransferase inhibitors: Substrate recognition by protein farnesyltransferase