Effect of forkhead box M1 in multiple myeloma

Forkhead Box M1 (FOXM1) is a promising molecular target for high-risk multiple myeloma and relapse and refractory myeloma, but whether FOXM1 is essential in myeloma has not yet been established. To address this knowledge gap, we used Western blotting to measure FOXM1 protein levels in 11 human myeloma cell lines (HMCLs) and then chose the two lines expressing the largest amount of the transcription factor, OPM2 and Delta47, for gene editing using CRISPR-Cas9. In both cases, two independent FOXM1-deficient daughter lines, designated KO-1 and KO-2, were generated. They contained 2 different 10-bp deletions at the target site of their respective guide RNA in case of OPM2 and a 10-bp deletion and 1-bp insertion in case of Delta47. Western analysis confirmed the lack of FOXM1 in all knockout clones. FOXM1-deficient myeloma cells proliferated more slowly than their parental counterparts containing normal levels of FOXM1. Moreover, we added back FOXM1 to FOXM1-KO cells by transfection of a constitutively expressed FOXM1c cDNA gene. The reconstituted OPM2 and Delta47 cells, designated FOXM1 KO-R contained high amounts of FOXM1 protein. Here, we used these cell lines to investigate the role of FOXM1 in regulating the gene expression in multiple myeloma. Overall design: Three CRISPR-modified synthetic single guide RNAs (gRNAs) were purchased from Synthego to target FOXM1 exon 2. RNA sequences, 5' to 3', were as follows: UUG AGA AUC AGU GGC CGA CG, UGA GAA UCA GUG GCC GAC GG and UAA UGA AAA CUA GCC CCC GU. Guide RNA was complexed individually with Cas9 protein (a kind gift from Dr. Miles Pufall, Department of Biochemistry, University of Iowa) to make ribonucleoprotein (RNP). OPM2 and Delta47 cells were electroporated with RNP using a Lonza 4D-Nucleofector in CM-138 mode and individual knock out clones were selected by limited dilution and then verified using Western blotting and Sanger sequencing. For reconstituting of FOXM1, the FOXM1-KO cells were transfected with lentivirus containing a gene expression vector encoding FOXM1c by spininfection and then fractionated based on GFP expression using a cell sorter. Total RNA from those cells were extracted using the RNeasy Plus mini kit from Qiagen and then sent to BGI for RNA sequencing and bioinformatic analysis.