WEE1 gene relevance and its inhibition effect on Myeloma Multiple cancer stem cell survival

• Grant number: 17/17101-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): December 01, 2017
• Effective date (End): May 31, 2018
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Gisele Wally Braga Colleoni
• Grantee: Taís Guimarães
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 10/17668-6 - Identification of biomarkers and possible therapeutical targets in B-cell lymphoproliferative disorders, AP.TEM

Abstract

Multiple myeloma (MM) is a malignant hematologic cancer characterized by infiltration of plasma tumor cells into the bone marrow (BM), synthesis and secretion of clonal immunoglobulins and tissue damage such as bone lesions, hypercalcemia, anemia and renal failure. Despite progress in treatment, the MM remains incurable due to high rate of relapse. The presence of cancer stem cells (CSCs) is indicated as the predominant cause in drug resistance and disease´s relapse, making them possible therapeutic targets. Although there is no universal antigen for these cell´s identification, it is possible to identify and isolate them based on their intrinsic characteristics. This study is based on the work of Boucher et al (2012), which established as expression pattern the surface antigens CD34 + / CD138- and the activity of an aldehyde dehydrogenase (ALDH) enzyme to identify MM-CSC. According to the results described by Dantonio et al (2016) in our group it was possible to isolate MM-CSC immunophenotypically (CD138-/CD34+), functionally (ALDH1-positive) and through gene expression profile. MM-CSCs gene expression profile showed differentially expressed genes in relation to tumor plasma cells, such as the WEE1 gene. The WEE1 protein is a negative modulator of mitosis, regulating the G2-M checkpoint through the phosphorylation of the CDC2 molecule. Inhibition of WEE1 drives the cell to early mitosis leading to apoptosis, a feature that has been explored as a therapeutic strategy in many other tumors. MK-1775 inhibits WEE1 kinase and acts by competing with ATP, avoiding DNA repair, promoting cell cycle arrest and antiproliferative effect. Tabayashi et al (2016) investigated the role of WEE1 in MM as a potential therapeutic target using MK-1775, showing inhibition of several MM cell line´s proliferation, inducing early and late apoptosis. Furthermore, in cells resistant to bortezomib (BTZ), MK-1775 overcomes such resistance and resensitizes the cells. Allied to our results in MM-CSCs obtained from MM patients, we found that WEE1 gene is a possible therapeutic target that can be explored. OBJECTIVES: To identify the presence of possible MM-CSCs in RPMI-8226 and U266 MM cell lines, evaluate WEE1 gene expression and evaluate the effects of WEE1 inhibitor (MK-1775) in MM-CSCs. METHODS: CD138- cells will be pre-selected by magnetic sorting from RPMI-8226 and U266 cell lines. CD138- cells will be submitted to sorting by flow cytometry on FACSAria II, labeled with anti-CD19 Pacific Blue, anti-CD34 PE Cy7 and anti-CD138 APC antibodies, in addition to Aldefluor" reagent. The obtained material will be placed in cell culture to evaluate spheres formation. The MM-CSCs will then be treated with WEE1 inhibitor (MK-1775). After 48 hours, late apoptosis will be assessed by flow cytometry in the FACSCalibur, using as parameters the annexin V+ and PI+. The RNeasy Mini kit and SuperScript III reverse transcriptase will be used for RNA extraction and cDNA synthesis, respectively. Finally, WEE1 gene expression will be assessed by real-time quantitative PCR (qPCR). (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

BARBOSA, REBECCA S. S.; DANTONIO, PAOLA M.; GUIMARAES, TAIS; DE OLIVEIRA, MARIANA B.; FOOK ALVES, VERUSKA L.; SANDES, ALEX FREIRE; FERNANDO, RODRIGO CARLINI; COLLEONI, GISELE W. B.. Sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in multiple myeloma cell lines. Biochemical and Biophysical Research Communications, v. 519, n. 3, p. 597-604, NOV 12 2019. (10/17668-6, 17/21801-2, 15/23983-5, 17/17101-5)