Ibrutinib (Imbruvica?) a small-drug inhibitor of Bruton tyrosine kinase (BTK) is currently undergoing clinical screening in patients with multiple myeloma (MM) yet important questions around the role of BTK in myeloma biology and treatment are outstanding. of key stemness genes (mRNA levels are elevated in myeloma cells compared to normal plasma cells. To complement these findings with BTK protein expression data we immunostained bone marrow biopsies of 34 patients with newly diagnosed myeloma using an antibody to BTK. Designating immunoreactivity in ≥25% of myeloma cells as cutoff for BTK expression we found 27 (~80%) cases to be positive and 7 (~20%) cases unfavorable. Semi-quantitative evaluation of tissue sections by a hematopathologist recognized 3 9 and 15 cases as BTKHigh BTKFair and BTKLow respectively. An example of moderate BTK expression is shown in Physique 1A. Examples of BTKHigh and BTKLow myelomas are depicted in Supplemental Physique 1. Next we asked whether CGI1746 inhibits HMCLs mRNA levels than seen in the CD138 assay: a ~150-fold increase in ARP1 and Cediranib (AZD2171) a ~35-fold increase in OPM2 Cediranib (AZD2171) (Physique 2B top rows). Be this as it may elevated BTK expression was associated with a marked up-regulation of 3 stem cell genes and and (Physique 2B). To translate this investigation to patient-derived myeloma samples we compared the expression of BTK in flow-sorted IgL-restricted (IgLR) SP cells with that in CD138+ MP cells: (26) BTK mRNA levels in the former were on average 2.5 times higher than in the latter (Figure 2C). Physique 2 Upregulation of is usually associated with features of stemness in myeloma To complement the results explained above with a method that yields larger samples of cells than possible using CD138? or SP fractionations we developed a reporter-based genetic method for circulation sorting of myeloma cells Cediranib (AZD2171) according to promoter activity. OCI-MY5 ARP1 and OPM2 cells were transduced with a lentivirus-encoded GFP reporter gene Cediranib (AZD2171) under transcriptional control of the BTK promoter. Cells were circulation sorted to collect the top and bottom deciles of GFP expressors (Physique 2D). RT-PCR analysis validated the method by demonstrating that GFPHigh cells harbored approximately 5 times more BTK message than GFPLow cells (Physique 2E). Next we performed serial colony formation assays using 3 consecutive passages of ARP1 cells to evaluate the possibility that BTK promotes clonogenicity. Compared to GFPLowBTKLow cells GFPHighBTKHigh cells not only exhibited significantly increased clonogenic potential upon initial plating (110 ± 23 vs. 58 ± 13 colonies < 0.05 student t test) but also greater capacity for further increase upon 2nd and 3rd re-plating (= 0.012 one-way ANOVA) (Figure 2F). Enforced expression of BTK enhances myeloma stemness To show BTK is usually a driver rather than a consequential phenomenon in keeping features of malignancy stemness in myeloma ARP1 and OPM2 cells were transfected with lentiviral particles that encoded a BTK cDNA gene. Western blotting showed that compared to cells infected with non-coding “vacant” computer virus (BTKWT used as control) cells over-expressing BTK (BTKOE) contained elevated amounts of (a) total and phosphorylated BTK (b) total and phosphorylated PLCγ2 a downstream substrate of BTK in the BCR signaling pathway and (c) NANOG a grasp regulator of stemness (Physique 3A). RT-PCR analysis of the iPS/ES genes Rabbit polyclonal to AFF3. and revealed 5-fold to 8-fold increases in mRNA levels in BTKOE cells compared to BTKWT cells (Physique 3B). Soft agar clonogenicity assays exhibited BTK-dependent elevations of colony figures in BTKOE vs. BTKWT cells: 12.9% vs. 8.63% in case of ARP1 and 13.7% vs. 9.94% for OPM2 (results not shown). Next using the circulation cytometric we found that over-expression of BTK in both ARP1 and OPM2 cells led to a ~3-fold increase in the large quantity of SP cells (Physique 3C). To determine whether enforced expression of BTK also increased tumorigenicity of myeloma cells we subcutaneously injected ARP1 cells into NOD-SCID mice. BTKOE cells generated tumors more effectively than their BTKWT counterparts (Physique 3D). Physique 3 Enforced expression of BTK in myeloma cells confers features of stemness BTK promotes drug resistance in myeloma Because CSCs have been implicated in acquisition of drug resistance in patients with malignancy we investigated whether enforced expression of BTK blunts the response of myeloma cells to widely used myeloma drugs. Clonogenic growth assays.