Gluing GAP to RAS Mutants: A New Approach to an Old Problem in Cancer Drug Development.

Mutated genes may lead to cancer development in numerous tissues. While more than 600 cancer-causing genes are known today, some of the most widespread mutations are connected to the RAS gene; RAS mutations are found in approximately 25% of all human tumors. Specifically, KRAS mutations are involved in the three most lethal cancers in the U.S., namely pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. These cancers are among the most difficult to treat, and they are frequently excluded from chemotherapeutic attacks as hopeless cases. The mutated KRAS proteins have specific three-dimensional conformations, which perturb functional interaction with the GAP protein on the GAP-RAS complex surface, leading to a signaling cascade and uncontrolled cell growth. Here, we describe a gluing docking method for finding small molecules that bind to both the GAP and the mutated KRAS molecules. These small molecules glue together the GAP and the mutated KRAS molecules and may serve as new cancer drugs for the most lethal, most difficult-to-treat, carcinomas. As a proof of concept, we identify two new, drug-like small molecules with the new method; these compounds specifically inhibit the growth of the PANC-1 cell line with KRAS mutation G12D in vitro and in vivo. Importantly, the two new compounds show significantly lower IC50 and higher specificity against the G12D KRAS mutant human pancreatic cancer cell line PANC-1, as compared to the recently described selective G12D KRAS inhibitor MRTX-1133.

Farnesyl-transferase inhibitors show synergistic anticancer effects in combination with novel KRAS-G12C inhibitors.

BACKGROUND: Inhibition of mutant KRAS challenged cancer research for decades. Recently, allele-specific inhibitors were approved for the treatment of KRAS-G12C mutant lung cancer. However, de novo and acquired resistance limit their efficacy and several combinations are in clinical development. Our study shows the potential of combining G12C inhibitors with farnesyl-transferase inhibitors. METHODS: Combinations of clinically approved farnesyl-transferase inhibitors and KRAS G12C inhibitors are tested on human lung, colorectal and pancreatic adenocarcinoma cells in vitro in 2D, 3D and subcutaneous xenograft models of lung adenocarcinoma. Treatment effects on migration, proliferation, apoptosis, farnesylation and RAS signaling were measured by histopathological analyses, videomicroscopy, cell cycle analyses, immunoblot, immunofluorescence and RAS pulldown. RESULTS: Combination of tipifarnib with sotorasib shows synergistic inhibitory effects on lung adenocarcinoma cells in vitro in 2D and 3D. Mechanistically, we present antiproliferative effect of the combination and interference with compensatory HRAS activation and RHEB and lamin farnesylation. Enhanced efficacy of sotorasib in combination with tipifarnib is recapitulated in the subcutaneous xenograft model of lung adenocarcinoma. Finally, combination of additional KRAS G1C and farnesyl-transferase inhibitors also shows synergism in lung, colorectal and pancreatic adenocarcinoma cellular models. DISCUSSION: Our findings warrant the clinical exploration of KRAS-G12C inhibitors in combination with farnesyl-transferase inhibitors.

Krebs von den Lungen 6 (KL-6) is a novel diagnostic and prognostic biomarker in pleural mesothelioma.

OBJECTIVES: Pleural mesothelioma (PM) is a rare disease with dismal outcome. Systemic treatment options include chemotherapy and immunotherapy, but biomarkers for treatment personalization are missing. The only FDA-approved diagnostic biomarker is the soluble mesothelin-related protein (SMRP). Krebs von den Lungen-6 (KL-6) is a human mucin 1 (MUC1) glycoprotein, which has shown diagnostic and prognostic value as a biomarker in other malignancies. The present study investigated whether KL-6 can serve as a diagnostic and/or prognostic biomarker in PM. MATERIALS AND METHODS: Using a fully-automated chemiluminescence enzyme immunoassay (CLEIA) for KL-6 and SMRP, pleural effusion samples from 87 consecutive patients with PM and 25 patients with non-malignant pleural disorders were studied. In addition, KL-6 and SMRP levels were determined in corresponding patient sera, and in an independent validation cohort (n = 122). MUC1 mRNA and protein expression, and KL-6 levels in cell line supernatants were investigated in PM primary cell lines in vitro. RESULTS: PM patients had significantly higher KL-6 levels in pleural effusion than non-malignant controls (AUC 0.78, p < 0.0001). Among PM patients, levels were highest in those with epithelioid or biphasic histologies. There was a strong positive correlation between pleural effusion levels of KL-6 and SMRP (p < 0.0001). KL-6 levels in sera similarly associated with diagnosis of PM, however, to a lesser extent (AUC 0.71, p = 0.008). PM patients with high pleural effusion KL-6 levels (≥303 IU/mL) had significantly better overall survival (OS) compared to those with low KL-6 levels (HR 0.51, p = 0.004). Congruently, high tumor cell MUC1 mRNA expression in primary cell lines associated with prolonged corresponding patient OS (HR 0.35, p = 0.004). These findings were confirmed in an independent validation cohort. CONCLUSION: This is the first study demonstrating KL-6 as a potential novel liquid-based diagnostic and prognostic biomarker in PM.

[Farnesyltransferase inhibitors have antitumoral effects in mutant KRAS containing cancer cells in preclinical models]. / Farnezil-transzferáz-inhibitorok (FTI) szinergista módon potencírozzák a mutáns KRAS inhibitorai daganatellenes hatásait preklinikai modellekben.

In silico studies raised the possibility that farnesyltransferase inhibitors (FTIs) may have antitumoral effects on KRAS mutant cancer cells. Accordingly, we have tested FTIs (tipifarnib and lonafarnib) in G12C mutant human cancer cell lines in vitro and in vivo. We have discovered that the combination of the two drugs has a synergistic antitumoral effect. Next, we have tested FTIs on G12D mutant human cancer cell lines and found that the combination has antitumoral effect in various preclinical cancer models. At last, we have also tested FTIs on G12V mutant human cancer cells and again we have detected antitumoral effects. We suggest that FTIs may have clinical relevance outside the HRAS mutant cancers.

The Prognostic Relevance of PMCA4 Expression in Melanoma: Gender Specificity and Implications for Immune Checkpoint Inhibition.

PMCA4 is a critical regulator of Ca2+ homeostasis in mammalian cells. While its biological and prognostic relevance in several cancer types has already been demonstrated, only preclinical investigations suggested a metastasis suppressor function in melanoma. Therefore, we studied the expression pattern of PMCA4 in human skin, nevus, as well as in primary and metastatic melanoma using immunohistochemistry. Furthermore, we analyzed the prognostic power of PMCA4 mRNA levels in cutaneous melanoma both at the non-metastatic stage as well as after PD-1 blockade in advanced disease. PMCA4 localizes to the plasma membrane in a differentiation dependent manner in human skin and mucosa, while nevus cells showed no plasma membrane staining. In contrast, primary cutaneous, choroidal and conjunctival melanoma cells showed specific plasma membrane localization of PMCA4 with a wide range of intensities. Analyzing the TCGA cohort, PMCA4 mRNA levels showed a gender specific prognostic impact in stage I-III melanoma. Female patients with high transcript levels had a significantly longer progression-free survival. Melanoma cell specific PMCA4 protein expression is associated with anaplasticity in melanoma lung metastasis but had no impact on survival after lung metastasectomy. Importantly, high PMCA4 transcript levels derived from RNA-seq of cutaneous melanoma are associated with significantly longer overall survival after PD-1 blockade. In summary, we demonstrated that human melanoma cells express PMCA4 and PMCA4 transcript levels carry prognostic information in a gender specific manner.

Allosteric and ATP-Competitive MEK-Inhibition in a Novel Spitzoid Melanoma Model with a RAF- and Phosphorylation-Independent Mutation.

Spitzoid melanoma is a rare malignancy with histological characteristics similar to Spitz nevus. It has a diverse genetic background and in adults, a similarly grim clinical outcome as conventional malignant melanoma. We established a spitzoid melanoma cell line (PF130) from the pleural effusion sample of a 37-year-old male patient. We found that the cell line carries a rare MEK1 mutation (pGlu102_Lys104delinsGln) that belongs to the RAF- and phosphorylation-independent subgroup of MEK1 alternations supposedly insensitive to allosteric MEK inhibitors. The in vivo tumorigenicity was tested in three different models by injecting the cells subcutaneously, intravenously or into the thoracic cavity of SCID mice. In the intrapleural model, macroscopic tumors formed in the chest cavity after two months, while subcutaneously and intravenously delivered cells showed limited growth. In vitro, trametinib-but not selumentinib-and the ATP-competitive MEK inhibitor MAP855 strongly decreased the viability of the cells and induced cell death. In vivo, trametinib but not MAP855 significantly reduced tumor growth in the intrapleural model. To the best of our knowledge, this is the first patient-derived melanoma model with RAF- and phosphorylation-independent MEK mutation and we demonstrated its sensitivity to trametinib.

Horizontal Combination of MEK and PI3K/mTOR Inhibition in BRAF Mutant Tumor Cells with or without Concomitant PI3K Pathway Mutations.

The RAS/RAF and PI3K/Akt pathways play a key regulatory role in cancer and are often hit by oncogenic mutations. Despite molecular targeting, the long-term success of monotherapy is often hampered by de novo or acquired resistance. In the case of concurrent mutations in both pathways, horizontal combination could be a reasonable approach. In our study, we investigated the MEK inhibitor selumetinib and PI3K/mTOR dual inhibitor BEZ235 alone and in combination in BRAF-only mutant and BRAF + PI3K/PTEN double mutant cancer cells using short- and long-term 2D viability assays, spheroid assays, and immunoblots. In the 2D assays, selumetinib was more effective on BRAF-only mutant lines when compared to BRAF + PI3K/PTEN double mutants. Furthermore, combination therapy had an additive effect in most of the lines while synergism was observed in two of the double mutants. Importantly, in the SW1417 BRAF + PI3K double mutant cells, synergism was also confirmed in the spheroid and in the in vivo model. Mechanistically, p-Akt level decreased only in the SW1417 cell line after combination treatment. In conclusion, the presence of concurrent mutations alone did not predict a stronger response to combination treatment. Therefore, additional investigations are warranted to identify predictive factors that can select patients who can benefit from the horizontal combinational inhibition of these two pathways.

K-Ras prenylation as a potential anticancer target.

KRAS is one of the most commonly mutated oncogene and a negative predictive factor for a number of targeted therapies. Therefore, the development of targeting strategies against mutant KRAS is urgently needed. One potential strategy involves disruption of K-Ras membrane localization, which is necessary for its proper function. In this review, we summarize the current data about the importance of membrane-anchorage of K-Ras and provide a critical evaluation of this targeting paradigm focusing mainly on prenylation inhibition. Additionally, we performed a RAS mutation-specific analysis of prenylation-related drug sensitivity data from a publicly available database ( https://depmap.org/repurposing/ ) of three classes of prenylation inhibitors: statins, N-bisphosphonates, and farnesyl-transferase inhibitors. We observed significant differences in sensitivity to N-bisphosphonates and farnesyl-transferase inhibitors depending on KRAS mutational status and tissue of origin. These observations emphasize the importance of factors affecting efficacy of prenylation inhibition, like distinct features of different KRAS mutations, tissue-specific mutational patterns, K-Ras turnover, and changes in regulation of prenylation process. Finally, we enlist the factors that might be responsible for the large discrepancy between the outcomes in preclinical and clinical studies including methodological pitfalls, the incomplete understanding of K-Ras protein turnover, and the variation of KRAS dependency in KRAS mutant tumors.

Next Generation Lipophilic Bisphosphonate Shows Antitumor Effect in Colorectal Cancer In Vitro and In Vivo.

Bisphosphonates, despite proven antitumor effect in vitro in many tumor types, are currently used only for treatment of osteoporosis and bone metastasis. Colorectal cancer is the third most commonly diagnosed type of cancer and lacks targeted therapy for RAS or RAF mutation carrying cases. A new lipophilic bisphosphonate showed promising results in lung cancer models, but their effect on colorectal cancer cells was not investigated excessively. Antitumor effects and impact on RAS-related signalization of zoledronic acid (ZA) and a lipophilic bisphosphonate (BPH1222) were investigated on 7 human colorectal cancer cell lines in vitro and in vivo. Furthermore, mutant KRAS dependent effect of prenylation inhibition was investigated using isogeneic cell lines. Both bisphosphonates reduced cell viability in vitro in a dose-dependent manner. Both compounds changed cell cycle distribution similarly by increasing the proportion of cells either in the S or in the subG1 phase or both. However, BPH1222 exerted higher inhibitory effect on spheroid growth than ZA. Interestingly, we found profound alterations in phosphorylation level of Erk and S6 proteins upon ZA or BPH1222 treatment. Furthermore, investigation of a mutant KRAS isogeneic model system suggests that the drugs interfere also with the mutant KRAS proteins. In vivo experiments with KRAS mutant xenograft model also revealed growth inhibitory potential of bisphosphonate treatment. Our results show that lipophilic bisphosphonates might extend the therapeutic spectrum of bisphosphonate drugs and could be considered as additional treatment approaches in colorectal cancer.

[Impact of prenylation inhibition on RAS mutant tumors in preclinical studies]. / Prenilációgátlás hatása RAS-mutáns daganatokra kísérleti rendszerekben.

Oncogenic mutation of RAS occurs in 20-25% of all malignancies. Our research group have examined inhibition of RAS prenylation on RAS wild type and RAS mutated melanoma, colorectal cancer and lung adenocarcinoma cell lines. Effects of clinically approved bisphosphonate (zoledronic acid) and its lipophilic derivate (BPH1222) on cell viability and cell signaling were determined. In models of melanoma and colorectal cancer we found no relevant difference in sensitivity to the drugs in light of RAS mutation presence. In case of lung adenocarcinoma bisphosphonate treatment inhibited both wild-type and mutated K-RAS, although tumor cells carrying mutated K-RAS seemed to be more sensitive to the bisphosphonate treatment. In summary, further investigations are warranted to identify tumor subgroups where bisphosphonates could have an effective therapeutic potential.

[Allele-specific inhibitors of mutant KRAS are in the focus of RASopathy consortium]. / Allélspecifikus inhibitorok nyomában: a RASopátia konzorcium célpontjában a KRAS fehérje onkogén mutációi.

The RASopathy consortium was built from research groups of the Budapest University of Technology and Economics, Eötvös Loránd University, Semmelweis University and two startups: KINETO Lab Ltd. and Fototronic Ltd. The goal was to design and test novel covalent and allele-specific KRAS small molecular inhibitors. KRAS is the most frequently mutated human oncogene which was unsuccessfully targeted until recently. The consortium established G12C-expressing bacterial and human cancer cell models (homo- and heterozygous variants) of lung, colorectal and pancreatic tumors. Using covalent fragment and acrylamide warhead libraries we were able to select novel candidates of small molecular G12C-specific inhibitors which were compared to published best-in-class drug candidates.

The Antitumor Effect of Lipophilic Bisphosphonate BPH1222 in Melanoma Models: The Role of the PI3K/Akt Pathway and the Small G Protein Rheb.

Malignant melanoma is one of the most metastatic cancer types, and despite recent success with novel treatment strategies, there is still a group of patients who do not respond to any therapies. Earlier, the prenylation inhibitor hydrophilic bisphosphonate zoledronic acid (ZA) was found to inhibit melanoma growth in vitro, but only a weaker effect was observed in vivo due to its hydrophilic properties. Recently, lipophilic bisphosphonates (such as BPH1222) were developed. Accordingly, for the first time, we compared the effect of BPH1222 to ZA in eight melanoma lines using viability, cell-cycle, clonogenic and spheroid assays, videomicroscopy, immunoblot, and xenograft experiments. Based on 2D and spheroid assays, the majority of cell lines were more sensitive to BPH. The activation of Akt and S6 proteins, but not Erk, was inhibited by BPH. Additionally, BPH had a stronger apoptotic effect than ZA, and the changes of Rheb showed a correlation with apoptosis. In vitro, only M24met cells were more sensitive to ZA than to BPH; however, in vivo growth of M24met was inhibited more strongly by BPH. Here, we present that lipophilic BPH is more effective on melanoma cells than ZA and identify the PI3K pathway, particularly Rheb as an important mediator of growth inhibition.

Long-Term Vemurafenib Exposure Induced Alterations of Cell Phenotypes in Melanoma: Increased Cell Migration and Its Association with EGFR Expression.

Acquired resistance during BRAF inhibitor therapy remains a major challenge for melanoma treatment. Accordingly, we evaluated the phenotypical and molecular changes of isogeneic human V600E BRAF-mutant melanoma cell line pairs pre- and post-treatment with vemurafenib. Three treatment naïve lines were subjected to in vitro long-term vemurafenib treatment while three pairs were pre- and post-treatment patient-derived lines. Molecular and phenotypical changes were assessed by Sulforhodamine-B (SRB) assay, quantitative RT-PCR (q-RT-PCR), immunoblot, and time-lapse microscopy. We found that five out of six post-treatment cells had higher migration activity than pretreatment cells. However, no unequivocal correlation between increased migration and classic epithelial-mesenchymal transition (EMT) markers could be identified. In fast migrating cells, the microphthalmia-associated transcription factor (MITF) and epidermal growth factor receptor (EGFR) mRNA levels were considerably lower and significantly higher, respectively. Interestingly, high EGFR expression was associated with elevated migration but not with proliferation. Cells with high EGFR expression showed significantly decreased sensitivity to vemurafenib treatment, and had higher Erk activation and FRA-1 expression. Importantly, melanoma cells with higher EGFR expression were more resistant to the EGFR inhibitor erlotinib treatment than cells with lower expression, with respect to both proliferation and migration inhibition. Finally, EGFR-high melanoma cells were characterized by higher PD-L1 expression, which might in turn indicate that immunotherapy may be an effective approach in these cases.

Pan-RAF and MEK vertical inhibition enhances therapeutic response in non-V600 BRAF mutant cells.

BACKGROUND: Currently, there are no available targeted therapy options for non-V600 BRAF mutated tumors. The aim of this study was to investigate the effects of RAF and MEK concurrent inhibition on tumor growth, migration, signaling and apoptosis induction in preclinical models of non-V600 BRAF mutant tumor cell lines. METHODS: Six BRAF mutated human tumor cell lines CRL5885 (G466 V), WM3629 (D594G), WM3670 (G469E), MDAMB231 (G464 V), CRL5922 (L597 V) and A375 (V600E as control) were investigated. Pan-RAF inhibitor (sorafenib or AZ628) and MEK inhibitor (selumetinib) or their combination were used in in vitro viability, video microscopy, immunoblot, cell cycle and TUNEL assays. The in vivo effects of the drugs were assessed in an orthotopic NSG mouse breast cancer model. RESULTS: All cell lines showed a significant growth inhibition with synergism in the sorafenib/AZ628 and selumetinib combination. Combination treatment resulted in higher Erk1/2 inhibition and in increased induction of apoptosis when compared to single agent treatments. However, single selumetinib treatment could cause adverse therapeutic effects, like increased cell migration in certain cells, selumetinib and sorafenib combination treatment lowered migratory capacity in all the cell lines. Importantly, combination resulted in significantly increased tumor growth inhibition in orthotropic xenografts of MDAMB231 cells when compared to sorafenib - but not to selumetinib - treatment. CONCLUSIONS: Our data suggests that combined blocking of RAF and MEK may achieve increased therapeutic response in non-V600 BRAF mutant tumors.

[Role of the stroma in the initiation and progression of tumors]. / A stroma szerepe a tumorok kialakulásában és progressziójában.

In the last decade, growing attention was paid to the observation that tumors did not only consist of cancer cells, they are rather a complex tissue-like mixture of tumor and stromal cells, which are playing an important role in the course of the malignant disease. Their contribution is so essential that without them, tumors are not even able to grow. This short review summarizes how stromal cells can promote the cancerous transformation and early development of tumors, how chronic inflammation contributes to the progression of cancer and how the stroma takes part in the induction of angiogenesis. The main mechanisms by which tumors can escape the immune surveillance will be demonstrated as well as the complex contributions of stroma to the invasion, intravasation and metastasis of cancer cells. Finally, possible and promising therapies will be presented that aim at the stroma and its main effects on the progression of tumors.