Bruton tyrosine kinase is a therapeutic target in stem-like cells from multiple myeloma

Abstract

Ibrutinib (Imbruvica), a small-drug inhibitor of Bruton tyrosine kinase (BTK), is currently undergoing clinical testing in patients with multiple myeloma, yet important questions on the role of BTK in myeloma biology and treatment are outstanding. Using flow-sorted side population cells from human myeloma cell lines and multiple myeloma primary samples as surrogate for the elusive multiple myeloma stem cell, we found that elevated expression of BTK in myeloma cells leads to AKT/WNT/β-catenin-dependent upregulation of key stemness genes (OCT4, SOX2, NANOG, and MYC) and enhanced self-renewal. Enforced transgenic expression of BTK in myeloma cells increased features of cancer stemness, including clonogenicity and resistance to widely used myeloma drugs, whereas inducible knockdown of BTK abolished them. Furthermore, overexpression of BTK in myeloma cells promoted tumor growth in laboratory mice and rendered side population-derived tumors that contained high levels of BTK more sensitive to the selective, second-generation BTK inhibitor, CGI1746, than side population-derived tumors that harbored low levels of BTK. Taken together, these findings implicate BTK as a positive regulator of myeloma stemness and provide additional support for the clinical testing of BTK-targeted therapies in patients with myeloma.

Conflict of interest statement

Conflicts of interest: None

Disclosure of Potential Conflicts Interest

No potential conflicts of interest were disclosed.

©2014 American Association for Cancer Research.

Figures

Figure 1. BTK protein expression in myeloma patient bone marrow samples and inhibition of human and mouse myeloma cells using CGI1746

(A) Serial sections of a bone marrow biopsy specimen of a myeloma patient were immunolabeled with antibodies to BTK (left) or CD138 (syndecan 1, right) respectively. (B) CGI1746 is cytotoxic to human myeloma cells, ARP1 and OPM2. (C) Kaplan-Meier survival curve of CGI1746 treatment on C57BL/KaLwRij mice following 5TGM1 cells.

Figure 2. Upregulation of BTK is associated with features of stemness in myeloma

(A) qPCR data indicating ratios of mean BTK mRNA levels (horizontal columns) and standard deviations of the mean (horizontal error bars) of flow-sorted CD138− and CD138+ myeloma cells. For each cell line (n = 10), the mean BTK expression level seen in CD138+ cells was set to 1 and then used as benchmark to calculate the fold-increase in CD138− cells. (B) qPCR results indicating ratios of mean mRNA levels of CSC-associated genes (horizontal columns) in flow-sorted side population (SP) vs. main population (MP) myeloma cells. (C) qPCR data indicating ratios of BTK mRNA levels in immunoglobulin light-chain (IgL)-restricted (IgLR) SP cells vs. CD138+ MP cells from 4 patients with myeloma. (D) Flow histogram depicting the fluorescence intensity profile of ARP1 myeloma cells harboring a lentivirus-encoded green fluorescence protein (GFP) reporter gene driven by the human BTK core promoter. The top and bottom 10 percent of cells featuring high and low GFP expression, respectively, were flow sorted and designated GFPHigh and GFPLow, respectively. (E) qPCR result indicating that GFPHigh cells contain elevated levels of BTK message compared to GFPLow cells. (F) Colony growth data demonstrate that ARP1 GFPHigh cells have increased clonogenic potential relative to GFPLow cells. Cell clusters counted as colony are circled. Two independent colonies that begin to merge are indicated by light arrow. Small aggregates of cells not counted as colonies are indicated by dark arrows.

Figure 3. Enforced expression of BTK in myeloma cells confers features of stemness

(A) Immunoblot analysis of BTKOE and BTKWT ARP1 and OPM2 myeloma cells. (B) Bar diagram of qPCR measurements indicating elevated expression of 4 stemness genes in BTKOE compared to BTKWT cells. (C) Shown to the left are the percentages of SP (indicated in circle) in ARP1-BTK overexpressing cells gated based on verapamil treated cells. The bar graph to the right depicts the fold increase in the SP fraction in BTKOE vs. BTKWT samples. (D) Time course of tumor growth in NOD-SCID mice indicating that BTKOE cells are more aggressive than BTKWT cells.

Figure 4. Over-expression of BTK in myeloma cells promotes drug resistance

(A) Bar diagram depicting percent clonogenic growth of ARP1 myeloma cells that either over-express BTK (BTKOE) or contain normal levels (BTKWT). Shown to the right are representative photomicroscopic images of two soft-agar dishes that contain myeloma cell colonies derived from Bz-treated BTKOE cell (top) and BTKWT cells (bottom). (B) Diagrammatic representation of percent apoptotic cell death of BTKOE/BTKWT ARP1 cells treated with the same drugs used in panel A. Shown to the right is a typical set of flow histograms, using again treatment with 5 nM Bz as an example. (C) Gold eFluxx assay data demonstrating that over-expression of BTK results in heightened drug efflux in myeloma cells. The Western blot on the right demonstrates BTK-dependent up-regulation of ABCB1 and pBCL2 in ARP1 and OPM2 cells. (D) Bar diagram summarizing results of colony formation assays using ARP1 BTKOE/BTKWT ARP1 cells treated with 10 nM verapamil, 5 nM Bz or combination of both drugs. Cells left untreated were used as control. The BTKOE to BTKWT ratios are indicated, analogous to panels A and B.

Figure 5. Down regulation of BTK in myeloma cells mitigates growth, survival, and stemness

(A) Western blots of OCI-MY5 and H929 myeloma cells that either under-express BTK due to lentiviral transduction of a BTK–targeted shRNA “knock down” construct (BTKKD) or express BTK at normal levels due to transduction of a non-targeted or “scrambled” shRNA (BTKWT). PARP indicates poly (ADP-ribose) polymerase. Casp3, 8 and 9 denote three different members of the apoptosis-related cysteine peptidase family of caspase proteins. (B) Line graphs presenting changes in cell number (top) and cell viability (bottom) of BTKKD and BTKWT OCI-MY5 cells grown for 7 days in vitro. (C) Same as panel B except H929 cells were used. (D) Time course of tumor growth in NOD-SCID mice, showing that BTKKD cells expand less vigorously in vivo than BTKWT cells. (E) Evidence indicating that BTK and NANOG are co-regulated in myeloma, and that the BTK-NANOG axis promotes clonogenicity of myeloma cells.

Figure 6. Interrogation of the BTK signaling pathway in myeloma

(A) Western blot of a co-immunoprecipitation (Co-IP) experiment indicating physical interaction of BTK and CDC73 in BTK-overexpressing ARP1 and OPM2 cells (lanes 1–2). IgG isotype control (lanes 3–4) and whole cell lysates without Co-IP (lanes 5–6) were included for comparison. (B) Immunoblots demonstrating reciprocal changes in AKT and WNT pathways in BTKOE and BTKKD cells, respectively, relative to their respective BTKWT controls. (C) Flow cytometry histogram depicting eFluxx assay fluorescence intensity profiles of BTKOE ARP1 and OPM2 cells treated with the indicated AKT inhibitor (bottom panels) or left untreated (top panels). (D) Immunoblots indicating AKT inhibitor-dependent reductions in ABCB1, pBCL-2 and β-CATENIN levels in BTKOE ARP1 and OPM2 cells. β-ACTIN and HIS2B were used as loading controls. (E) Immunoblots indicating WNT inhibitor-dependent reductions in NANOG in BTKOE ARP1 and OPM2 cells.

Figure 7. Preclinical testing of CGI1746

(A) Flow histograms of eFluxx dye retention assays using BTKOE ARP1 and OPM2 cells treated with CGI1746. (B) Bar diagram of colony formation results indicating CGI1746-dependent reductions in clonogenicity of GFPHighBTKHigh vs. GFPLowBTKLow myeloma cells. (C) Kaplan-Meier survival curve (left) of C57BL/KaLwRij mice treated with IV injections of either SP or non-SP 5TGM1 cells, followed by treatment of mice with CGI1746 or solvent control and the right panel is a graph of mean serum IgG2b levels of tumor-bearing mice. (D) Column diagram presenting results of colony formation assays of hematopoietic stem cells (HSC) obtained from BTK-deficient Xid or normal mice. (E) Working model on the putative mechanisms by which BTK promotes stemness and drug resistance in myeloma.