193J Contemp Med Sci | Vol. 9, No. 3, May-June 2023: 193–196 Original Statins in Combination with Ibrutinib bypasses Resistance to Ibrutinib in Mantle Cell Lymphoma Aoula Al-Zebeeby1* , Ali Abbas2 1Department of pathology and poultry diseases, Faculty of veterinary medicine, University of Kufa, Najaf, Iraq. 2Department of microbiology, Faculty of veterinary medicine, University of Kufa, Najaf, Iraq. *Correspondence to: Aoula Al-Zebeeby (E-mail: aoulae.alzebeeby@uokufa.edu.iq) (Submitted: 20 March 2023 – Revised version received: 18 April 2023 – Accepted: 10 May 2023 – Published online: 26 June 2023) Abstract Objective: In this study, we report that a novel combination therapy of different statins with Ibrutinib can overcome such resistance. Methods: For this, we generated a cell line model, exhibiting resistance to Ibrutinib, by repeated exposure of mantle cell lymphoma cell line to Ibrutinib. Apoptosis was assessed by the extent of phosphatidylserine externalisation. Results: Our results indicated that resistance to Ibrutinib could be overcome by targeting a key enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the cholesterogenesis pathway. Conclusion: Reusing different statins in combination with Ibrutinib could improve therapy and enhance sensitivity to Ibrutinib mediated apoptosis. Keywords: Statins, Ibrutinib, chemoresistance, HMG-CoA reductase, cholecterogensis pathway ISSN 2413-0516 Introduction Mantle cell lymphoma (MCL) is an aggressive form of malig- nant B-lymphocytes of non-Hodgkin’s lymphoma, which arise from the outer edge of a lymph node follicle, also known as the mantle zone of lymph node. The majority of mantle cell lym- phoma cases are considered as incurable with multiple inci- dences of relapse. Several treatment strategies have been developed over the past few years with significant improve- ment in patient outcome.1–3 Signalling of B cell receptor (BCR) is crucial for the matu- ration and development of B cells. Bruton’s Tyrosine Kinase (BTK) is a key enzyme in B cell receptor cascade.4–6 Many studies have identified an important role of BTK in the activa- tion of BCR signalling pathway,7–10 which in turn activates MAP kinase and NF-κB pathways, thereby enhancing B cell activity, survival rate, proliferation and migration.3 Therefore, BTK is considered an important therapeutic target for MCL and other B cell malignancies.11 In the last few decades, various molecules have been used in clinical practice for relapsed/refractory (R/R) of MCL and other types of B cell malignancies.10 Among these pharmaco- logical molecules, Ibrutinib, commercially available under the name Imbruvica, is a BTK inhibitor and first line treatment for MCL.12 However, chemoresistance to anticancer agents, including Ibrutinib poses a critical challenge for the successful implementation of this therapy. Tackling this challenge becomes vital as the resistance affects patient outcome.13 In recent years, several studies indicated that modulation of some cancer metabolic pathways improved cancer therapy.14,15 Statins form an important classification of these metabolic inhibitors, and exhibit pleiotropic abilities, including anti-angiogenic, antioxidant, anti-inflammatory and anti- cancer activities.16 Therefore, our study was aimed at identi- fying whether a combination of statins with Ibrutinib could overcome resistance to Ibrutinib-mediated apoptosis in MCL. Methods Reagents Ibrutinib, Simvastatin and Atorvastatin were from Selleck (Syl- vanfield Drive, Houston, USA). Pitavastatin was from Tocris (Abingdon, UK). Filipin III stain solution was from SigmaAl- drich (Gillingham, UK). Generation of Chemoresistance Parental cells of MAVER-1 cells (labelled as I in Figure 1A) were exposed to Ibrutinib (10 µM) for 48h, and the extent of apop- tosis was assessed using flow cytometry. This was followed by two weeks (2W) of recovery period, during which the cells were cultured in drug-free media (RPMI media + 10% FBS) at 37°C and 5% CO2. The resulting cells were labelled as II, and this process was repeated three more times more, to generate cells labelled as III, IV and V. The extent of apoptosis was checked after each exposure to assess the extent of resistance. Thus, group V cells exhibited the most resistance to Ibrutinib. Flow Cytometry Apoptosis was assessed by phosphatidylserine (PS) externali- sation. Following 48 hours exposure to Ibrutinib and 72 hours in case of statins, cells were gathered and completed using an Annexin V buffer and propidium iodide (PI) stain. Then, the extent of apoptosis was measured by flow cytometry (Flores- cence-Activated Cell Sorting (FACS)). Filipin Staining Suspension MAVER-1 cells (0.2 × 106) were collected and resus- pended with 50ml of Phosphate-buffered saline (PBS). Then, by using polysineTM adhesion slide (Menzel-Glaser, UK) the mix- ture was placed and left for 5 minutes. This was followed by a fixation step using paraformaldehyde (4%) from Thermo Fisher Scientific (Waltham, MA, USA) for 5 minutes and Triton X-100 (0.5%) from SigmaAldrich (Gillingham, UK) for 10 minutes. Subsequently, the slides were incubated with filipin for 2 hours, and washed three times gently with PBS, before imaging using fluorescence microscopy with a UV filter set (340–380 nm excitation, 40 nm dichroic, 430-nm long pass filter). Statistical Analysis Multiple comparisons one-way F test or analysis of variance (ANOVA) and the least significant difference (Fisher test) https://orcid.org/0000-0003-1656-1841 194 J Contemp Med Sci | Vol. 9, No. 3, May-June 2023: 193–196 Overcoming Chemoresistance to Ibrutinib Original A. Al-Zebeeby et al. (P ≤ 0.01) were conducted to compare sensitive (I) and resistant (V) cells. Statistics was performed using GraphPad Prism 6 software for windows (La Jolla, CA, USA). Results Mantle Cell Lymphoma Cells Acquired Rapid Resistance to Lbrutinib In order to mimic the resistance observed in clinic, we developed a chemoresistance model, in which the initial MAVER-1 cells (labelled as I) were repeatedly exposed to Ibrutinib 10mM for 48h, followed by recovery periods of two weeks (2W), until increasing resistance was reached in the different groups of cells, labelled II-V (Figure 1 A and B). Taken together, these results sug- gested that resistance to Ibrutinib was developed successfully. Targeting HMG-CoA Reductase Overcomes Resistance to Ibrutinib Mediated Apoptosis in Mantle cell Iymphoma Cells In order to identify a way to restore sensitivity to Ibrutinib- mediated apoptosis, we investigated whether MAVER-1 sensi- tive (labelled as I) and resistant (labelled as V) cells exhibited differences in cholesterol synthesis. Staining of cells with filipin indicated that cholesterol synthesis was enhanced in the resistant cells (V) compared to the sensitive cells (I) (Figure 2A). Therefore, we investigated whether targeting cholesterogenesis by three mostly used statins, namely Atorvastatin, Pitavastatin and Simvastatin could overcome resistance to Ibrutinib-medi- ated apoptosis in mantle cell lymphoma. Our results revealed that targeting HMG-CoA reductase by these three different statins in combination with Ibrutinib enhanced sensitivity for sensitive cells (I) and overcame resistance to Ibrutinib-mediated apoptosis in resistant cells (V) (Figure 2B). Taken together, these results suggested that resistance to Ibrutinib-mediated apop- tosis could be because of the enhanced cholesterol synthesis in the resistant cells. Therefore, using statins in combination with Ibrutinib enhanced sensitivity and overcame resistance to Ibru- tinib-mediated apoptosis. Discussion Development of resistance to cancer therapy is currently one of the main obstacle that challenges the successful anticancer therapy and is often responsible for regression and incurable cancer.1,2,13,17 In the current study, the resistance to Ibrutinib in mantle cell lymphoma was rapidly developed (Figure 1A and B) to mimic chemoresistance in patients. Chemotherapy often creates a stressful environment for cancer cells, which in turn respond to such unfavourable conditions by activation of several pathways that enable the cells to escape therapy.18,19 One example of this is the rewiring of cancer metabolism. This is achieved by increasing choles- terol and lipid synthesis, in addition to activation of several other metabolic pathways.15,18,19 For instance, in case of B cell malignancies, resistance to Ibrutinib could be achieved through the elevation of fatty acids synthesis and oxida- tion.20,21 Similarly, resistance to anti-cancer agents in mantle cell lymphoma has been reported to occur via the activation of glutamine metabolism.22 Furthermore, targeting interme- diary metabolism enhances sensitivity and overcome chemoresistance.23 The best inhibitors of the HMG-CoA reductase are statins, such as Atorvastatin, Ptivastatin and Simvastatin, which decrease cholesterol levels in serum.24,25 Several studies indi- cated that statins have the power to control growth of tumour both in vitro and in vivo by blocking the progression of cell cycle.26–30 Furthermore, different clinical trials have been Fig.1 Mantle cell lymphoma rapidly developed resistance to Ibrutinib. (A) Scheme for developing resistance to Ibrutinib in mantle cell lymphoma cell lines (MAVER-1), as explained in the Methods section. Sensitive (I) and resistant (V) cells of MAVER-1 cell line were exposed for 48 h to Ibrutinib (10 mM), and apoptosis was detected. (B) Gradual increase of resistance was observed in each group of cells (I, II, III, IV and V). ***P-0.001, Error bars = mean ± standard error of mean (n = 3) PS: phosphatidylserine. 195J Contemp Med Sci | Vol. 9, No. 3, May-June 2023: 193–196 A. Al-Zebeeby et al. Original Overcoming Chemoresistance to Ibrutinib Fig. 2 Targeting HMG-CoA reductase overcame resistance to Ibrutinib. (A) Sensitive (labelled as I) and resistant (labelled as V) MAVER-1 cells were stained with flipin (cholesterol dye) Scale bar: 10 µm. (B) Sensitive (labelled as I) and resistant (labelled as V) MAVER-1 cells were exposed to Ibrutinib (10 mM) for 48 h alone or in combination with pharmacological inhibitors of HMGR including, Atorvastatin (10 mM), Pitavastatin (1 mM) or Simvastatin (250 nM) for 72 h. ***P-0.001, Error bars = mean ± standard error of mean (n = 3). PS: phosphatidylserine. Fig. 3 Scheme representing the development of resistance to Ibrutinib due to increased cholesterogenesis. Targeting the key enzyme HMGR by statins enhances sensitivity to Ibrutinib-mediated apoptosis. examined the anticancer activity of statins in many cancers, such as non-metastatic rectal cancer, head and neck cancer, advanced liver carcinoma, pediatric tumors, colon cancer and acute myeloid leukemia.31 –33 However, in the present study a group of statins was used to bypass resistance to Ibrutinib in mantle cell lymphoma cell line (Figures 2B and 3). In agreement with these results, inhibition of HMG-CoA reductase enhances sensitivity to venetoclax in different blood malignancies.16,34 Conclusion Resistance to Ibrutinib mediated apoptosis was developed rap- idly in mantle cell lymphoma cell line to mimic chemore- sistance in patients. Increased cholesterol synthesis could be the metabolic reprogramming that MAVER-1 used to escape from therapeutic pressure presented by Ibrutinib. Therefore, re-purposing some drugs, such as statins could improve therapy and bypass resistance to Ibrutinib-mediated apoptosis. Acknowledgments The authors thank Dr. Shankar Varadarajan (Institute of Sys- tems, Molecular and Integrative Biology/University of Liver- pool) for his help with proofreading the manuscript. Competing Interests The authors declare no competing interests.  196 J Contemp Med Sci | Vol. 9, No. 3, May-June 2023: 193–196 Overcoming Chemoresistance to Ibrutinib Original A. Al-Zebeeby et al. lymphomas. 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Ibrutinib Resistance Mechanisms and Treatment Strategies for B-Cell This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i3.1330 https://pubmed.ncbi.nlm.nih.gov/?term=Hermine+O&cauthor_id=22873532 https://pubmed.ncbi.nlm.nih.gov/?term=Hermine+O&cauthor_id=22873532 https://doi.org/10.1200/jco.2012.44.4281 https://pubmed.ncbi.nlm.nih.gov/22688757/ https://doi.org/10.1016/j.cellsig.2012.09.008 https://pubmed.ncbi.nlm.nih.gov/22262035/ https://pubmed.ncbi.nlm.nih.gov/22361516/ https://pubmed.ncbi.nlm.nih.gov/22361516/ https://pubmed.ncbi.nlm.nih.gov/29799080/ https://pubmed.ncbi.nlm.nih.gov/29799080/