RMC-7977 a broad RAS inhibitor increases the sensitivity towards Bortezomib

RMC-7977 a broad RAS inhibitor increases the sensitivity towards Bortezomib | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article RMC-7977 a broad RAS inhibitor increases the sensitivity towards Bortezomib Didhiti Singha, Meghna Mondal, Pushkar Malakar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5295233/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The drug Bortezomib commonly treats patients with multiple myeloma. However, its use is more limited in other cancers, such as hepatocellular and cervical cancer. Additionally, reports have indicated cases of resistance to Bortezomib. In this context, studies have demonstrated that Ras signaling plays a significant role in influencing the sensitivity of cancer cells to chemotherapeutic drugs. A new experimental drug called RMC-7977 was made by Revolution Medicines. It targets the active, GTP-bound forms of RAS proteins, such as KRAS, NRAS, and HRAS. Since RMC-7977 is a broad Ras inhibitor recently discovered, we sought to evaluate its efficacy in enhancing the sensitivity of cancer cells to Bortezomib. Our study aimed to assess the effect of RMC-7977 on Bortezomib sensitivity. The results show that HeLa and HepG2 cells exhibit lower sensitivity to Bortezomib compared to RPMI-8266 cells. However, combination therapy with RMC-7977 and Bortezomib increases the sensitivity of HeLa and HepG2 cells to Bortezomib. Cycloheximide has a big effect on the levels of Ras protein in RPMI-8266 cells, but it doesn't change them at all in HeLa and HepG2 cells. These findings underscore the crucial role of Ras signaling in determining sensitivity to Bortezomib. The present study also highlights the potential of the RMC-7977 inhibitor in enhancing the response to Bortezomib. This work could broaden the scope of FDA-approved Bortezomib to include additional cancers beyond multiple myeloma and improve outcomes for patients with chemoresistant multiple myeloma. Furthermore, it may open up new therapeutic applications for the recently discovered RMC-7977. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Cancer Biological sciences/Drug discovery RMC-7977 Ras Inhibitor Bortezomib Ras Signaling and Drug Sensitivity Figures Figure 1 Figure 2 Figure 3 Introduction Bortezomib is a proteasome inhibitor used primarily in the treatment of multiple myeloma (MM) 1 . Some patients may develop resistance to bortezomib 2 . Research is ongoing to understand resistance mechanisms and develop strategies to overcome resistance 1 , 2 . Additionally, the goal is to expand the use of bortezomib to other cancers. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer 3 . Several clinical trials have explored the efficacy of bortezomib in HCC patients, often in combination with other therapies 4 . Results have been mixed, with some studies showing limited benefits from monotherapy 4 . Identifying biomarkers that predict response to bortezomib can help select patients who are most likely to benefit from this treatment. Furthermore, bortezomib is not commonly used in cervical cancer treatment 5 , 6 . Cervical Cancer is the fourth-most common cancer in women 7 . HeLa's sensitivity to Bortezomib is low compared to the multiple myeloma cell line RPMI-8226. Bortezomib has shown efficacy in treating multiple myeloma by inducing apoptosis and inhibiting cell proliferation 8 . RAS signaling, which is often activated in various cancers, plays a crucial role in cell survival, proliferation, and resistance to apoptosis 9 . Understanding the interplay between RAS signaling and bortezomib can provide insights into potential combination therapies and overcoming resistance in cancer treatment 9 – 11 . RAS signaling plays a significant role in the development of chemoresistance in a variety of cancers 10 . RMC-7977 is a novel investigational drug developed by Revolution Medicines, targeting the active, GTP-bound form of RAS proteins (such as KRAS, NRAS, and HRAS) 12 – 14 . This drug is part of a new class of RAS(ON) inhibitors designed to address cancers driven by mutations in these proteins 12 – 14 . The primary mechanism of action involves blocking the activation of downstream signaling pathways like MAPK and PI3K, which are crucial for cancer cell proliferation and survival 12 – 14 . Preclinical studies have demonstrated that RMC-7977 effectively inhibits both wild-type and mutant forms of RAS, showing promising results in various models of cancer, including pancreatic cancer 12 . The drug has shown significant efficacy in reducing tumor growth, particularly in models resistant to other treatments 12 – 14 . Furthermore, studies have shown that RMC-7977 selectively inhibits tumor cells, exhibiting minimal effects on normal cells, a crucial feature for mitigating potential side effects 12 – 14 . We wanted to check the effect of RMC-7977 treatment on sensitivity to Bortezomib. This will expand Bortezomib's usage range. Our results show that the sensitivity of HeLa and HepG2 to Bortezomib is low as compared to RPMI-8266. Combination therapy of RMC-7977 and Bortezomib increases HeLa and HepG2's sensitivity to Bortezomib. Furthermore, the cycloheximide chase experiment revealed that Ras levels in RPMI-8226 are highly sensitive to cycloheximide, whereas they are more stable in HeLa and HepG2. All of these findings clearly demonstrate the significance of Ras signaling in determining Bortezomib sensitivity. The present study also demonstrated the importance of the RMC-7977 inhibitor in increasing the response to Bortezomib. Materials and Method Cell Culture: We obtained HeLa, HepG2, and RPMI-8226 from NCCS, Pune, India. We grew HeLa and HepG2 cells in DMEM. RPMI-8226 cells were grown in RPMI. We supplemented both DMEM and RPMI-8226 with 10% FCS, 0.1 mg/mL penicillin, and 0.1 mg/mL streptomycin. We used Bortezomib at a concentration of 100 nM. RMC-7977 was purchased from Med Chem Express and used at a concentration of 2.5 µM. Immunoblotting: Laemmli buffer was used to lyse the cells. The total protein from each lysate, which was about 20µg, was separated by SDS-PAGE and then moved to the PVDF membrane. The main antibodies that were used were cleaved caspase-3 (1:1000; Cell Signaling Technology), GAPDH (1:1000; Santa Cruz Biotechnology), phospho-MEK1/2 (1:1000; Santa Cruz Biotechnology), Total MEK (1:1000; Cell Signaling Technology), phospho-S6K1 (1:1000; Santa Cruz Biotechnology), Total S6K1 (1:1000; Santa Cruz Biotechnology), Tubulin (1:1000; Santa Cruz Biotechnology), and Ras (1:1000; Cell Signaling Technology). Secondary antibodies used were HRP-conjugated goat anti-mouse and goat anti-rabbit (1:5000, Cell Signaling Technology). The blots were developed with the help of ECL (Cell Signaling Technology) in Chemi-Doc (Biorad). Results HeLa and HepG2 cells are not as sensitive to Bortezomib as RPMI-8226 is. To look at the effect of Bortezomib on different cell lines, we treated HeLa (a cervical cancer cell line), HepG2 (a liver cancer cell line), and RPMI-8226 (a multiple myeloma cell line) with Bortezomib for 24 hours ( Fig. 1 A ) . We prepared the cell lysate after 24 hours and measured the cytotoxic effect of Bortezomib by observing the cell's apoptosis using the apoptotic marker, cleaved caspase-3 ( Fig. 1 A ) . HeLa and HepG2 did not show much apoptosis, while RPMI-8226 showed increased apoptosis ( Fig. 1 A ) , as marked by enhanced expression of cleaved caspase-3 15 . These results clearly indicate that HeLa and HepG2 cells are not as sensitive to Bortezomib as RPMI-8226 is. Ras protein levels upon cycloheximide treatment in HeLa and HepG2 were quite stable in comparison to RPMI-8226. To understand the molecular mechanism of differential sensitivity to Bortezomib, we examined the status of Ras protein levels. This is due to the significant role Ras signaling plays in controlling the sensitivity to chemotherapeutic drugs 9 , 10 . To confirm the involvement of Ras signaling in the regulation of Bortezomib sensitivity, HeLa, HepG2, and RPMI-8226 cells were treated with cycloheximide for different time intervals ( Fig. 1 B- 1 D ) . In the cell lines, we found differences in the stability of the Ras protein upon exposure to cycloheximide ( Fig. 1 B- 1 D ) . RPMI-8226 cells Ras protein levels were highly sensitive to cycloheximide, indicating the role of Ras protein in determining Bortezomib sensitivity ( Fig. 1 B ) . Ras protein levels upon cycloheximide treatment in HeLa ( Fig. 1 C ) and HepG2 ( Fig. 1 D ) were quite stable in comparison to RPMI-8226 ( Fig. 1 B ) . More stability means higher protein levels, resulting in more functional protein activity 16 . Ras inhibitor RMC-7977 enhances Bortezomib's apoptotic activity. To investigate the role of Ras signaling in determining Bortezomib sensitivity, we used the Ras inhibitor RMC-7977 12,14 . As a single agent, 2.5 µM of RMC-7977 did not induce any cell death at 24 hours in HeLa ( Fig. 2 A & 2 C ) and HepG2 cells ( Fig. 2 B & 2 D ) . We further examined whether RMC-7977 augmented apoptosis, as evidenced by cleaved caspase-3, triggered by Bortezomib in HeLa ( Fig. 2 A & 2 C ) and HepG2 ( Fig. 2 B & 2 D ) . Bortezomib (100 nM) alone did not induce any detectable apoptosis, as evidenced by no detectable expression of cleaved caspase-3 in HeLa ( Fig. 2 A & 2 C ) and HepG2 ( Fig. 2 B & 2 D ) in 24 hours. Importantly, RMC-7977 with Bortezomib induced apoptosis with an increased expression of cleaved caspase-3 ( Fig. 2 ) . These results indicate that the Ras inhibitor RMC-7977 enhances Bortezomib's apoptotic activity, which in turn increases the expression of cleaved caspase-3. Microscopy images further substantiated these results, demonstrating that RMC-7977 and Bortezomib together induced cytotoxicity in HeLa ( Fig. 2 A ) and HepG2 ( Fig. 2 B ) , while neither compound alone significantly induced apoptosis. Taken together, these results clearly indicate the importance of Ras activity inhibition in increasing sensitivity to Bortezomib ( Fig. 2 ) . RMC-7977 and Bortezomib together result in reduced phosphorylation of MEK1/2. We first looked at how RMC-7977 made HeLa and HepG2 more sensitive to Bortezomib. Then we looked at what RMC-7977 did to the signaling pathway for HeLa ( Fig. 3 A ) and HepG2 ( Fig. 3 B ) with and without Bortezomib ( Fig. 3 ) . In HeLa, RMC-7977 did not change the levels of Ras in the presence or absence of Bortezomib ( Fig. 3 A ) . However, it did affect the signaling of MEK1/2 ( Fig. 3 A ) . We looked at MEK1/2 signaling, as it is one of the downstream targets of Ras signaling 17 . In HeLa, Bortezomib, or RMC-7977, treatment alone resulted in increased phosphorylation of MEK1/2 ( Fig. 3 A ) . However, adding both RMC-7977 and Bortezomib together results in reduced phosphorylation of MEK1/2 ( Fig. 3 A ) . We also checked the levels of phospho-S6K1 activation because Ras has been shown to turn on mTORC1 signaling through S6K1, which is one of its targets 17 , 18 . mTORC1 has been found to be activated in many cancers 19 – 21 . Bortezomib or RMC-7977 alone did not change the phosphorylated levels of S6K1 compared to the control alone, but RMC-7977 and Bortezomib together reduced the phosphorylated levels of S6K1 ( Fig. 3 A ) . In the case of HepG2 cells, RMC-7977 alone or in combination with Bortezomib did not change the expression levels of Ras ( Fig. 3 B ) . When RMC-7977 is used alone or with Bortezomib, it lowers the levels of phosphorylated MEK1/2 in HepG2 cells compared to control cells ( Fig. 3 B ) . However, unlike in HeLa, the combined treatment of RMC-7977 and Bortezomib did not result in a reduction of phosphorylated S6K1 ( Fig. 3 ) . Taken together, these results indicate that RMC-7977 reduces the activity of downstream Ras signaling, resulting in increased sensitivity to Bortezomib ( Fig. 3 ) . Discussion Our overall objective was to see the effect of the Ras inhibitor RMC-7977 on the sensitivity response to Bortezomib. The increased sensitivity of RPMI-8227, a multiple myeloma cell line, aligns with the clinical use of Bortezomib for treating multiple myeloma patients 22 . The fact that HeLa and HepG2 cells show no apoptosis indicates that they are less sensitive to Bortezomib ( Fig. 1 A ) . This probably explains restricted use of Bortezomib in clinics as a single drug for the treatment of hepatocellular and cervical cancer 4 , 6 . We looked at Ras signaling because it has a lot to do with chemoresistance 23 – 25 and wanted to figure out why HeLa and HepG2 cells were less sensitive to Bortezomib. Adding cycloheximide to RPMI-8227 cells results in a decrease in Ras protein stability levels compared to HeLa and HepG2 cells ( Fig. 1 B- 1 D ) . This suggests that the increased protein stability of Ras in HeLa and HepG2 may be the cause. HeLa and HepG2 cells become more sensitive to Bortezomib when RMC-7977 is present, as shown by more cell death. The results likely demonstrate the crucial role of Ras signaling in regulating the response to Bortezomib and highlight the significance of the recently identified Ras inhibitor RMC-7977 as a chemotherapy drug that can enhance the response to Bortezomib. RMC-7977 is a Ras activity inhibitor, which means it downregulates the downstream signaling without changing the expression levels 12 – 14 . Ras signaling has two downstream targets: MEK1/2 and mTORC1 17,26 . Both MEK1/2 and mTORC1 signaling have been implicated in cancer 10 , 27 . HeLa and HepG2 cells showed more apoptosis with RMC-7977 and Bortezomib than with either alone ( Fig. 2 ) . This suggests that RMC-7977 makes the cells more sensitive to Bortezomib. HeLa and HepG2 cells treated with RMC-7977 and Bortezomib had less phosphorylated MEK1/2 than cells treated with Bortezomib alone. This result signifies the MEK1/2 signaling role in protection against chemotherapeutic agents. Increased apoptosis correlates with reduced phosphorylation of MEK1/2. Furthermore, we observed a concurrent decrease in S6K1 phosphorylation in HeLa cells, indicating a decrease in mTORC1 activity. The role of mTORC1 signaling is crucial in determining chemosensitivity 28 . HepG2 did not exhibit this effect. The decrease in phosphorylation of MEK1/2 was very steep in HepG2, while in HeLa the decrease was very low ( Fig. 3 ) , suggesting the difference might be due to different cellular types resulting in different responses. This work has the potential to widen the scope of FDA-approved Bortezomib in various other cancers apart from multiple myeloma as well as improve the chemoresistant condition in multiple myeloma. Furthermore, this work could open up a new application for the recently discovered RMC-7977, which has garnered significant attention in recent times. These findings showed that Ras signaling affects how well Bortezomib works and that stopping Ras signaling with RMC-7977 makes Bortezomib work better. At the same time, these results would pave a path for the widening use of Bortezomib in other cancers with the help of RMC-7977. Conclusion In this study, we attempted to develop an alternative strategy to overcome Bortezomib resistance based on inhibiting molecular mechanisms that mediate drug resistance. First, we demonstrated that Bortezomib induces cell death in RPMI-8226, whereas HeLa and HepG2 were resistant to apoptotic cell death after 24 hours of Bortezomib treatment. Since HeLa and HepG2 cells showed increased Ras stability upon cycloheximide, upregulation of Ras signaling in HeLa and HepG2 may protect these cells against apoptosis. Studies have demonstrated an increasing role of Ras signaling in chemoresistance 10 . Conversely, our data demonstrate that RMC-7977 inhibits Ras signaling and overcomes Bortezomib resistance. So, our results show that RMC-7977 should be tested in clinical trials with Bortezomib in order to either make it more effective or get around the drug's resistance, which would lead to better outcomes for patients. Declarations Ethical Approval: Not Applicable Consent to participate: Not Applicable Consent to publish All the authors have carefully read the manuscript and given consent for publication. Availability of Data and Materials The data and materials will be available upon request to the corresponding author. Competing Interests The authors declare no competing interests Acknowledgments PM would like to thank SERB India for The Ramanujan Fellowship Funding This work was supported by SERB Ramanujan Fellowship (RJF/2021/000123), DST, Govt. of India. Authors' Contributions P. M. conceptualize the Project. D.S., M.M., and P.M. performed the experiments, analyzed the data, and wrote the manuscript. P.M. proofread and supervised during the writing. All authors reviewed the manuscript. References Bortezomib (Velcade™) in the treatment of multiple myeloma. Ther Clin Risk Manag 2 , 271–279 (2006). Balsas, P., Galán-Malo, P., Marzo, I. & Naval, J. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5295233","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":376831076,"identity":"dba747ba-6352-43da-9d83-832fca7cf69c","order_by":0,"name":"Didhiti Singha","email":"","orcid":"","institution":"Ramakrishna Mission Vivekananda Educational Research Institute (RKMVERI)","correspondingAuthor":false,"prefix":"","firstName":"Didhiti","middleName":"","lastName":"Singha","suffix":""},{"id":376831078,"identity":"aca885c2-ef82-4bfa-b718-a76f4d28823c","order_by":1,"name":"Meghna Mondal","email":"","orcid":"","institution":"Ramakrishna Mission Vivekananda Educational Research Institute (RKMVERI)","correspondingAuthor":false,"prefix":"","firstName":"Meghna","middleName":"","lastName":"Mondal","suffix":""},{"id":376831079,"identity":"4a4e574e-aa0f-4535-b990-4a89fdead930","order_by":2,"name":"Pushkar Malakar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYHACNsYGBoYEBvYGECcBJipBWAsPzwGStUgkoGjBDcwl0p89nFFzL89e8vnVDYx70qINbiQwfvjBYJGHS4vljBxzww3Hiot5pHPKbjA8y8ndcCOBWbKHQaIYlxaDGzlskg/YEhJ7pHPSbjAcqMidOSOBQRrol8QGnFrSn0k++AfUInkGroX5N34tCWaSG9uAWiTYjwG15OT2SySw4bXFsueNmeTMvoRinjM5bDcSDqTl9vM8bLPsMcCtxZwd6LCebwl57O3Hn934cCA5t409+fCNHxV1uB2GYPIYQGMFFE8GWFWja2F/gFvZKBgFo2AUjGgAANeTW5/o5E5MAAAAAElFTkSuQmCC","orcid":"","institution":"Ramakrishna Mission Vivekananda Educational Research Institute (RKMVERI)","correspondingAuthor":true,"prefix":"","firstName":"Pushkar","middleName":"","lastName":"Malakar","suffix":""}],"badges":[],"createdAt":"2024-10-19 15:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5295233/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5295233/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70379925,"identity":"25890621-a6fd-46de-a465-cec4680a1054","added_by":"auto","created_at":"2024-12-02 15:51:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":310210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe sensitivity of HeLa and HepG2 cells to Bortezomib is lower than that of RPMI-8226.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eThe Western Blot analysis examines the apoptosis of HeLa, HepG2, and RPMI-8226 cells that have been treated with Bortezomib for 24 hours. We used a concentration of Bortezomib of 100 nM. We used the expression of cleaved caspase-3 as a marker for apoptosis. GAPDH was used as loading control. \u003cstrong\u003e(B-D)\u003c/strong\u003eWestern blot analysis was performed on HeLa, HepG2, and RPMI-8226 to check the stability of Ras protein for the indicated time period after treatment with cycloheximide. The concentration of cycloheximide used is 1µg/ml. GAPDH was used as loading control.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5295233/v1/8f1404b78bce499b500695a3.png"},{"id":70379923,"identity":"1b81ee90-be09-4ef8-a044-f887a697ad0a","added_by":"auto","created_at":"2024-12-02 15:51:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2144979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSensitivity of HeLa and HepG2 to Bortezomib increases in the presence of RMC-7977.\u003c/strong\u003e \u003cstrong\u003e(A-B)\u003c/strong\u003eMicroscopy images of HeLa and HepG2 without any treatment, with Bortezomib alone, with RMC-7977 alone, or with both Bortezomib and RMC-7977 together are presented. The concentration of Bortezomib used was 100 nM, and RMC-7977 used was 2.5 µM. Microscopy images were taken at 10X. \u003cstrong\u003e(C-D)\u003c/strong\u003e Western blot analysis of HeLa and HepG2 for checking apoptosis for cells described above in A \u0026amp; B. The expression of cleaved caspase-3 was used as a marker for apoptosis. GAPDH was used as loading control. The red line indicates the scale bar.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5295233/v1/4887f60bcd92db98bfc3de6b.png"},{"id":70379924,"identity":"a0243d40-9efe-43a0-82ab-559b7b5e1df7","added_by":"auto","created_at":"2024-12-02 15:51:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":539762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRMC-7977 decreases the activity of downstream targets of Ras signaling.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Western blot analysis of the proteins shown in HeLa cells that had not been treated, were treated with Bortezomib alone, were treated with RMC-7977 alone, or were treated with both RMC-7977 and Bortezomib. We used 2.5 µM of RMC-7977 and 100nM of Bortezomib. Tubulin was used as loading control. \u003cstrong\u003e(B)\u003c/strong\u003e Western blot analysis of the proteins shown in HepG2 cells that were not treated, treated with Bortezomib alone, treated with RMC-7977 alone, or treated with both RMC-7977 and Bortezomib together. We used 2.5 µM \u0026nbsp;of RMC-7977 and 100nM of Bortezomib. GAPDH was used as loading control. The non-specific band of MEK1/2 is represented by the symbol NS.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5295233/v1/80353869cc76c09b78b3f088.png"},{"id":71222186,"identity":"db7846e4-8d37-4a01-9d57-6aa553017d17","added_by":"auto","created_at":"2024-12-12 09:24:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3282654,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5295233/v1/77d442e6-e1ea-40f3-b739-66573f42710b.pdf"},{"id":70379926,"identity":"4c411962-03f8-4557-9fdd-862c4c31b381","added_by":"auto","created_at":"2024-12-02 15:51:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1902262,"visible":true,"origin":"","legend":"","description":"","filename":"RMCRAWDATAWestern.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5295233/v1/80c6c063d458471632620d73.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"RMC-7977 a broad RAS inhibitor increases the sensitivity towards Bortezomib","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBortezomib is a proteasome inhibitor used primarily in the treatment of multiple myeloma (MM) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Some patients may develop resistance to bortezomib \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Research is ongoing to understand resistance mechanisms and develop strategies to overcome resistance \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Additionally, the goal is to expand the use of bortezomib to other cancers.\u003c/p\u003e \u003cp\u003eHepatocellular carcinoma (HCC) is the most common type of primary liver cancer \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Several clinical trials have explored the efficacy of bortezomib in HCC patients, often in combination with other therapies \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Results have been mixed, with some studies showing limited benefits from monotherapy \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Identifying biomarkers that predict response to bortezomib can help select patients who are most likely to benefit from this treatment. Furthermore, bortezomib is not commonly used in cervical cancer treatment \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Cervical Cancer is the fourth-most common cancer in women \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. HeLa's sensitivity to Bortezomib is low compared to the multiple myeloma cell line RPMI-8226.\u003c/p\u003e \u003cp\u003eBortezomib has shown efficacy in treating multiple myeloma by inducing apoptosis and inhibiting cell proliferation \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. RAS signaling, which is often activated in various cancers, plays a crucial role in cell survival, proliferation, and resistance to apoptosis \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Understanding the interplay between RAS signaling and bortezomib can provide insights into potential combination therapies and overcoming resistance in cancer treatment \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRAS signaling plays a significant role in the development of chemoresistance in a variety of cancers \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. RMC-7977 is a novel investigational drug developed by Revolution Medicines, targeting the active, GTP-bound form of RAS proteins (such as KRAS, NRAS, and HRAS)\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This drug is part of a new class of RAS(ON) inhibitors designed to address cancers driven by mutations in these proteins \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The primary mechanism of action involves blocking the activation of downstream signaling pathways like MAPK and PI3K, which are crucial for cancer cell proliferation and survival \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Preclinical studies have demonstrated that RMC-7977 effectively inhibits both wild-type and mutant forms of RAS, showing promising results in various models of cancer, including pancreatic cancer \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The drug has shown significant efficacy in reducing tumor growth, particularly in models resistant to other treatments \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Furthermore, studies have shown that RMC-7977 selectively inhibits tumor cells, exhibiting minimal effects on normal cells, a crucial feature for mitigating potential side effects \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe wanted to check the effect of RMC-7977 treatment on sensitivity to Bortezomib. This will expand Bortezomib's usage range.\u003c/p\u003e \u003cp\u003eOur results show that the sensitivity of HeLa and HepG2 to Bortezomib is low as compared to RPMI-8266. Combination therapy of RMC-7977 and Bortezomib increases HeLa and HepG2's sensitivity to Bortezomib. Furthermore, the cycloheximide chase experiment revealed that Ras levels in RPMI-8226 are highly sensitive to cycloheximide, whereas they are more stable in HeLa and HepG2. All of these findings clearly demonstrate the significance of Ras signaling in determining Bortezomib sensitivity. The present study also demonstrated the importance of the RMC-7977 inhibitor in increasing the response to Bortezomib.\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cp\u003eCell Culture:\u003c/p\u003e \u003cp\u003eWe obtained HeLa, HepG2, and RPMI-8226 from NCCS, Pune, India. We grew HeLa and HepG2 cells in DMEM. RPMI-8226 cells were grown in RPMI. We supplemented both DMEM and RPMI-8226 with 10% FCS, 0.1 mg/mL penicillin, and 0.1 mg/mL streptomycin. We used Bortezomib at a concentration of 100 nM. RMC-7977 was purchased from Med Chem Express and used at a concentration of 2.5 \u0026micro;M.\u003c/p\u003e \u003cp\u003eImmunoblotting:\u003c/p\u003e \u003cp\u003eLaemmli buffer was used to lyse the cells. The total protein from each lysate, which was about 20\u0026micro;g, was separated by SDS-PAGE and then moved to the PVDF membrane. The main antibodies that were used were cleaved caspase-3 (1:1000; Cell Signaling Technology), GAPDH (1:1000; Santa Cruz Biotechnology), phospho-MEK1/2 (1:1000; Santa Cruz Biotechnology), Total MEK (1:1000; Cell Signaling Technology), phospho-S6K1 (1:1000; Santa Cruz Biotechnology), Total S6K1 (1:1000; Santa Cruz Biotechnology), Tubulin (1:1000; Santa Cruz Biotechnology), and Ras (1:1000; Cell Signaling Technology). Secondary antibodies used were HRP-conjugated goat anti-mouse and goat anti-rabbit (1:5000, Cell Signaling Technology). The blots were developed with the help of ECL (Cell Signaling Technology) in Chemi-Doc (Biorad).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eHeLa and HepG2 cells are not as sensitive to Bortezomib as RPMI-8226 is.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo look at the effect of Bortezomib on different cell lines, we treated HeLa (a cervical cancer cell line), HepG2 (a liver cancer cell line), and RPMI-8226 (a multiple myeloma cell line) with Bortezomib for 24 hours \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. We prepared the cell lysate after 24 hours and measured the cytotoxic effect of Bortezomib by observing the cell's apoptosis using the apoptotic marker, cleaved caspase-3 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. HeLa and HepG2 did not show much apoptosis, while RPMI-8226 showed increased apoptosis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, as marked by enhanced expression of cleaved caspase-3 \u003csup\u003e15\u003c/sup\u003e. These results clearly indicate that HeLa and HepG2 cells are not as sensitive to Bortezomib as RPMI-8226 is.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRas protein levels upon cycloheximide treatment in HeLa and HepG2 were quite stable in comparison to RPMI-8226.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo understand the molecular mechanism of differential sensitivity to Bortezomib, we examined the status of Ras protein levels. This is due to the significant role Ras signaling plays in controlling the sensitivity to chemotherapeutic drugs \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To confirm the involvement of Ras signaling in the regulation of Bortezomib sensitivity, HeLa, HepG2, and RPMI-8226 cells were treated with cycloheximide for different time intervals \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. In the cell lines, we found differences in the stability of the Ras protein upon exposure to cycloheximide \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. RPMI-8226 cells Ras protein levels were highly sensitive to cycloheximide, indicating the role of Ras protein in determining Bortezomib sensitivity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Ras protein levels upon cycloheximide treatment in HeLa \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e and HepG2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e were quite stable in comparison to RPMI-8226 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. More stability means higher protein levels, resulting in more functional protein activity \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRas inhibitor RMC-7977 enhances Bortezomib's apoptotic activity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the role of Ras signaling in determining Bortezomib sensitivity, we used the Ras inhibitor RMC-7977 \u003csup\u003e12,14\u003c/sup\u003e. As a single agent, 2.5 \u0026micro;M of RMC-7977 did not induce any cell death at 24 hours in HeLa \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e and HepG2 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. We further examined whether RMC-7977 augmented apoptosis, as evidenced by cleaved caspase-3, triggered by Bortezomib in HeLa \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e and HepG2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Bortezomib (100 nM) alone did not induce any detectable apoptosis, as evidenced by no detectable expression of cleaved caspase-3 in HeLa \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e and HepG2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e in 24 hours. Importantly, RMC-7977 with Bortezomib induced apoptosis with an increased expression of cleaved caspase-3 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. These results indicate that the Ras inhibitor RMC-7977 enhances Bortezomib's apoptotic activity, which in turn increases the expression of cleaved caspase-3. Microscopy images further substantiated these results, demonstrating that RMC-7977 and Bortezomib together induced cytotoxicity in HeLa \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e and HepG2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, while neither compound alone significantly induced apoptosis. Taken together, these results clearly indicate the importance of Ras activity inhibition in increasing sensitivity to Bortezomib \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRMC-7977 and Bortezomib together result in reduced phosphorylation of MEK1/2.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe first looked at how RMC-7977 made HeLa and HepG2 more sensitive to Bortezomib. Then we looked at what RMC-7977 did to the signaling pathway for HeLa \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e and HepG2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e with and without Bortezomib \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. In HeLa, RMC-7977 did not change the levels of Ras in the presence or absence of Bortezomib \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. However, it did affect the signaling of MEK1/2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. We looked at MEK1/2 signaling, as it is one of the downstream targets of Ras signaling \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In HeLa, Bortezomib, or RMC-7977, treatment alone resulted in increased phosphorylation of MEK1/2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. However, adding both RMC-7977 and Bortezomib together results in reduced phosphorylation of MEK1/2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. We also checked the levels of phospho-S6K1 activation because Ras has been shown to turn on mTORC1 signaling through S6K1, which is one of its targets \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. mTORC1 has been found to be activated in many cancers \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Bortezomib or RMC-7977 alone did not change the phosphorylated levels of S6K1 compared to the control alone, but RMC-7977 and Bortezomib together reduced the phosphorylated levels of S6K1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. In the case of HepG2 cells, RMC-7977 alone or in combination with Bortezomib did not change the expression levels of Ras \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. When RMC-7977 is used alone or with Bortezomib, it lowers the levels of phosphorylated MEK1/2 in HepG2 cells compared to control cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. However, unlike in HeLa, the combined treatment of RMC-7977 and Bortezomib did not result in a reduction of phosphorylated S6K1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Taken together, these results indicate that RMC-7977 reduces the activity of downstream Ras signaling, resulting in increased sensitivity to Bortezomib \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur overall objective was to see the effect of the Ras inhibitor RMC-7977 on the sensitivity response to Bortezomib. The increased sensitivity of RPMI-8227, a multiple myeloma cell line, aligns with the clinical use of Bortezomib for treating multiple myeloma patients \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The fact that HeLa and HepG2 cells show no apoptosis indicates that they are less sensitive to Bortezomib \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. This probably explains restricted use of Bortezomib in clinics as a single drug for the treatment of hepatocellular and cervical cancer \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe looked at Ras signaling because it has a lot to do with chemoresistance \u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003eand wanted to figure out why HeLa and HepG2 cells were less sensitive to Bortezomib. Adding cycloheximide to RPMI-8227 cells results in a decrease in Ras protein stability levels compared to HeLa and HepG2 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. This suggests that the increased protein stability of Ras in HeLa and HepG2 may be the cause. HeLa and HepG2 cells become more sensitive to Bortezomib when RMC-7977 is present, as shown by more cell death. The results likely demonstrate the crucial role of Ras signaling in regulating the response to Bortezomib and highlight the significance of the recently identified Ras inhibitor RMC-7977 as a chemotherapy drug that can enhance the response to Bortezomib.\u003c/p\u003e \u003cp\u003eRMC-7977 is a Ras activity inhibitor, which means it downregulates the downstream signaling without changing the expression levels \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Ras signaling has two downstream targets: MEK1/2 and mTORC1 \u003csup\u003e17,26\u003c/sup\u003e. Both MEK1/2 and mTORC1 signaling have been implicated in cancer \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. HeLa and HepG2 cells showed more apoptosis with RMC-7977 and Bortezomib than with either alone \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. This suggests that RMC-7977 makes the cells more sensitive to Bortezomib. HeLa and HepG2 cells treated with RMC-7977 and Bortezomib had less phosphorylated MEK1/2 than cells treated with Bortezomib alone. This result signifies the MEK1/2 signaling role in protection against chemotherapeutic agents. Increased apoptosis correlates with reduced phosphorylation of MEK1/2. Furthermore, we observed a concurrent decrease in S6K1 phosphorylation in HeLa cells, indicating a decrease in mTORC1 activity. The role of mTORC1 signaling is crucial in determining chemosensitivity \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. HepG2 did not exhibit this effect. The decrease in phosphorylation of MEK1/2 was very steep in HepG2, while in HeLa the decrease was very low \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, suggesting the difference might be due to different cellular types resulting in different responses.\u003c/p\u003e \u003cp\u003eThis work has the potential to widen the scope of FDA-approved Bortezomib in various other cancers apart from multiple myeloma as well as improve the chemoresistant condition in multiple myeloma. Furthermore, this work could open up a new application for the recently discovered RMC-7977, which has garnered significant attention in recent times. These findings showed that Ras signaling affects how well Bortezomib works and that stopping Ras signaling with RMC-7977 makes Bortezomib work better. At the same time, these results would pave a path for the widening use of Bortezomib in other cancers with the help of RMC-7977.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we attempted to develop an alternative strategy to overcome Bortezomib resistance based on inhibiting molecular mechanisms that mediate drug resistance. First, we demonstrated that Bortezomib induces cell death in RPMI-8226, whereas HeLa and HepG2 were resistant to apoptotic cell death after 24 hours of Bortezomib treatment. Since HeLa and HepG2 cells showed increased Ras stability upon cycloheximide, upregulation of Ras signaling in HeLa and HepG2 may protect these cells against apoptosis. Studies have demonstrated an increasing role of Ras signaling in chemoresistance \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Conversely, our data demonstrate that RMC-7977 inhibits Ras signaling and overcomes Bortezomib resistance.\u003c/p\u003e \u003cp\u003eSo, our results show that RMC-7977 should be tested in clinical trials with Bortezomib in order to either make it more effective or get around the drug's resistance, which would lead to better outcomes for patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have carefully read the manuscript and given consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and materials will be available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePM would like to thank SERB India for The Ramanujan Fellowship\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by SERB Ramanujan Fellowship (RJF/2021/000123), DST, Govt. of India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP. M. conceptualize the Project. D.S., M.M., and P.M. performed the experiments, analyzed the data, and wrote the manuscript. P.M. proofread and supervised during the writing. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBortezomib (Velcade\u0026amp;trade;) in the treatment of multiple myeloma. \u003cem\u003eTher Clin Risk Manag\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 271\u0026ndash;279 (2006).\u003c/li\u003e\n\u003cli\u003eBalsas, P., Gal\u0026aacute;n-Malo, P., Marzo, I. \u0026amp; Naval, J. Bortezomib resistance in a myeloma cell line is associated to PSM\u0026beta;5 overexpression and polyploidy. \u003cem\u003eLeuk Res\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 212\u0026ndash;218 (2012).\u003c/li\u003e\n\u003cli\u003eLlovet, J. 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N. mTOR Signaling Confers Resistance to Targeted Cancer Drugs. \u003cem\u003eTrends Cancer\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 688\u0026ndash;697 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"RMC-7977, Ras Inhibitor, Bortezomib, Ras Signaling, and Drug Sensitivity","lastPublishedDoi":"10.21203/rs.3.rs-5295233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5295233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe drug Bortezomib commonly treats patients with multiple myeloma. However, its use is more limited in other cancers, such as hepatocellular and cervical cancer. Additionally, reports have indicated cases of resistance to Bortezomib. In this context, studies have demonstrated that Ras signaling plays a significant role in influencing the sensitivity of cancer cells to chemotherapeutic drugs. A new experimental drug called RMC-7977 was made by Revolution Medicines. It targets the active, GTP-bound forms of RAS proteins, such as KRAS, NRAS, and HRAS. Since RMC-7977 is a broad Ras inhibitor recently discovered, we sought to evaluate its efficacy in enhancing the sensitivity of cancer cells to Bortezomib.\u003c/p\u003e \u003cp\u003eOur study aimed to assess the effect of RMC-7977 on Bortezomib sensitivity. The results show that HeLa and HepG2 cells exhibit lower sensitivity to Bortezomib compared to RPMI-8266 cells. However, combination therapy with RMC-7977 and Bortezomib increases the sensitivity of HeLa and HepG2 cells to Bortezomib. Cycloheximide has a big effect on the levels of Ras protein in RPMI-8266 cells, but it doesn't change them at all in HeLa and HepG2 cells.\u003c/p\u003e \u003cp\u003eThese findings underscore the crucial role of Ras signaling in determining sensitivity to Bortezomib. The present study also highlights the potential of the RMC-7977 inhibitor in enhancing the response to Bortezomib. This work could broaden the scope of FDA-approved Bortezomib to include additional cancers beyond multiple myeloma and improve outcomes for patients with chemoresistant multiple myeloma. Furthermore, it may open up new therapeutic applications for the recently discovered RMC-7977.\u003c/p\u003e","manuscriptTitle":"RMC-7977 a broad RAS inhibitor increases the sensitivity towards Bortezomib","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-02 15:51:20","doi":"10.21203/rs.3.rs-5295233/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a87d904c-6665-403f-8644-c4f2cccdfc44","owner":[],"postedDate":"December 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40115023,"name":"Biological sciences/Biochemistry"},{"id":40115024,"name":"Biological sciences/Biotechnology"},{"id":40115025,"name":"Biological sciences/Cancer"},{"id":40115026,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2024-12-12T09:24:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-02 15:51:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5295233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5295233","identity":"rs-5295233","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}