Bruton tyrosine kinase inhibition is a novel therapeutic strategy targeting tumor in the bone marrow microenvironment in multiple myeloma

Abstract

Bruton tyrosine kinase (Btk) has a well-defined role in B-cell development, whereas its expression in osteoclasts (OCs) further suggests a role in osteoclastogenesis. Here we investigated effects of PCI-32765, an oral and selective Btk inhibitor, on osteoclastogenesis as well as on multiple myeloma (MM) growth within the BM microenvironment. PCI-32765 blocked RANKL/M-CSF-induced phosphorylation of Btk and downstream PLC-γ2 in OCs, resulting in diminished TRAP5b (ED50 = 17 nM) and bone resorption activity. PCI-32765 also inhibited secretion of multiple cytokines and chemokines from OC and BM stromal cell cultures from both normal donors (ED50 = 0.5 nM) and MM patients. It decreased SDF-1-induced migration of MM cells, and down-regulated MIP1-α/CCL3 in MM cells. It also blocked MM cell growth and survival triggered by IL-6 or coculture with BM stromal cells or OCs in vitro. Importantly, PCI-32765 treatment significantly inhibits in vivo MM cell growth (P < .03) and MM cell-induced osteolysis of implanted human bone chips in SCID mice. Moreover, PCI-32765 prevents in vitro colony formation by stem-like cells from MM patients. Together, these results delineate functional sequelae of Btk activation mediating osteolysis and growth of MM cells, supporting evaluation of PCI-32765 as a novel therapeutic in MM.

Figure 1

PCI-32765 blocked Btk-mediating osteoclastogenic signaling pathway and impacted osteoclastogenesis. (A) CD14⁺ OC precursor cells (OCPs) from normal human donor or mouse raw267.4 cells were stimulated with RANKL/M-CSF for the indicated time intervals. Cell lysates were subjected to immunoblotting with antiphosphotyrosine antibodies. Anti–α-tubulin and -Btk mAbs served as loading controls. (B) OCPs were pretreated with (+) or without (−) PCI-32765 (100nM) for 2 hours before stimulation with RANKL/M-CSF. (C) TRAP staining was performed at 10 days of OC culture to identify mature OCs (> 3 nuclei, > 50 μm per cell; original magnification ×40 and ×100). (D) TRAP-positive multinucleated OCs were quantified (P < .01) and assayed by MTT (E).

Figure 2

PCI-32765 diminished bone resorption activity. (A) Human OCPs from normal donors were stimulated with RANKL/M-CSF and cultured on glass cover slips for 15 to 17 days, followed by immunofluorescence staining to observe OC morphology using Alexa-568–conjugated phalloidin (red) for actin and 4,6-diamidino-2-phenylindole (blue) for nuclei. (B) Abnormal OCs observed in panel A were further quantitated for extended spreading area per multinucleated OC (> 3 nuclei) and number of nuclei per OC (C). (D) OCPs were cultured on the dentine slice for 2 weeks, in the presence or absence of PCI-32765, alone or with dexamethasone (Dex), and analyzed for pit formation to determine percentage of bone erosion area. Images of representative bone resorption on dentine slices, with or without PCI-32765 treatment, are shown on the right (10× lens).

Figure 3

PCI-32765 potently down-regulates cytokines/chemokine in supernatants from human OC and BMSC cultures from normal donors and MM patients. Supernatants collected at day 14 of human OC cultures treated with PCI-32765 starting on day 1 and day 5 were subjected to ELISA and Multiplex Luminex Assay (A-E). Many MM- and OC-related cytokines and chemokines were decreased (ED₅₀ = 0.1-0.48nM). TRAP5b, a mature OC-specific marker, was also inhibited (ED₅₀ = 17nM; F). (G) Survival of CD138-negative BMSCs from MM patients with active disease was measured by MTT assay, and supernatants assayed for secretion of cytokine/chemokines (H). (I) TRAP staining was performed at day 21 culture, visualized, and further measured using a spectrophotometer.

Figure 4

PCI-32765 induces cytotoxicity against Btk-expressing MM cells stimulated by IL-6 or coculture with BMSCs or OCs. (A) Immunoblotting analysis showed Btk expression in CD138⁺ MM cell lines and patient MM cells (n = 5). (B) INA6 cells, with or without IL-6 (B) or BMSCs (C) or OC (D), prepared from MM patients were cultured with PCI-32765. (E) INA6-GFP cells were cocultured with (+) or without (−) BMSCs overnight, sorted by FACS, and subjected to mRNA and protein extraction for Btk levels by quantitative RT-PCR and immunoblotting. (F) PCI-32765 was added for 3 days to CD138⁺ cells from 2 representative MM patients, alone or with BMSCs, followed by a luminescent cell viability assay.

Figure 5

SDF-1–induced adhesion and migration in MM cells were inhibited by PCI-32765, and Btk directly regulates MM cell survival. (A) MM cells were preincubated with PCI-32765 (100nM, +) or control media; the drug was then washed out before stimulation with SDF-1 for 2 and 5 minutes. Lysates were immunoprecipitated (IP) with anti-Btk, and the IPs probed with anti-pBtk. Total cell lysates were further probed with indicated phopho-specific Abs and anti–α-tubulin as a loading control. (B) PCI-32765 was added to patient MM cells in overnight coculture with BMSCs. Images were taken using a Leica DFC300FX and Leica IM50 Image Manager (original magnification ×100). (C) CD138⁺ patient MM cells were treated with PCI-32765 and allowed to migrate in the absence (−) or presence of SDF-1 (200 ng/mL) in transwells coated with 1 μg/mL sVCAM-1. Bars represent the mean ± SD of triplicates. (D) MM1R cells were transduced with control GFP or shBtk lentiviruses; cell lysates were then prepared for immunoblotting. MM1R cells pretreated with (PCI) or without (−) PCI-32765 served as controls. (E) Three days after lentivirus infection, MM1R cells were labeled with calcein-AM and adherence to BMSCs was assayed in 2 hours. Four days after lentiviral transduction, cell images (F, original magnification ×400) were taken, and cell viability as well as caspase 3/caspase 7 activity (G) were determined.

Figure 6

PCI-32765 significantly altered gene expression in MM cells and OC lineage cells stimulated with M-CSF/RANKL. (A) MM (ANBL6, INA-6) were treated with PCI-32765 (1μM) for an hour, and cell lysates were subjected to immunoblotting using specific Abs to determine the effects of the drug on Btk signaling cascade. (B) Patient MM cells (MM1, MM2) and MM1R cells were treated with PCI-32765. (C) INA6 cells, with or without IL-6, were treated with PCI-32765 overnight followed by real-time quantitative RT-PCR for MIP-1α/CCL3, which was normalized to HPRT as an internal control. Fold changes triggered by the drug were further normalized relative to the medium control. (D) Human donor OCPs stimulated with M-CSF/RANKL were treated with PCI-32765 for 7 days, and mRNA changes of these target genes were assayed by real-time quantitative RT-PCR using specific primers.

Figure 7

PCI-32765 inhibits MM cell growth and MM-induced bone lysis in a murine model of human MM. (A) SCID-hu mice were injected with INA-6 MM cells into the implanted human bone and continuously treated with PCI-32765 (12 mg/kg, n = 6) or vehicle control (n = 5) beginning after first detection of tumor by monitoring shuIL-6R in mouse serum samples weekly. (B) Bone chips were retrieved from SCID-hu mice, decalcified, and sectioned. Tissue slides were stained with H&E and immunohistochemically analyzed for CD138 (MM), TRAP (OC), and ALP (OB). Original magnification ×200, except for ALP (original magnification ×400). (C) Representative cross-sectional images by 3-dimensional reconstruction of the harvested human bones obtained after performing high-resolution micro-CT scan are shown and quantified (D). *P < .04. (E) Osteogenic activity per bone surface (ALP⁺/BS), indicating bone formation activity. **P < .01. The PCI-32765-treated group displayed significantly reduced osteolysis induced by MM cells and enhanced osteogenic activity, compared with vehicle control group. Effects of PCI-32765 were also quantitated in the left (mouse L) and right (mouse R) normal mouse extremities (F).

Figure 8

PCI-32765 inhibited MM stem-like cells. (A) Stem-like PRMI8226 MM cells (RPMI SP) sorted by flow cytometry were incubated with PCI-32765 in the presence or absence of BMSCs for 3 days, followed by a luminescent cell viability assay. (B) Clonogenic stem-like cells, purified from MM patient (n = 5) BMMCs by immunomagnetic depletion of CD138⁺ plasma cells and CD34⁺ normal hematopoietic progenitors, were subjected to colony formation assays in methylcellulose in the presence of PCI-32765. Btk expression in such clonogenic populations from 3 MM patients was shown by immunoblotting.