Pre-clinical anti-tumor activity of Bruton's Tyrosine Kinase inhibitor in Hodgkin's Lymphoma cellular and subcutaneous tumor model

Abstract

Bruton's Tyrosine Kinase (BTK) is a member of the TEC family and plays a central role in B-cell signaling, activation, proliferation and differentiation. Here we evaluated the impact of BTK inhibitor Ibrutinib on a panel of HL models in vitro and in vivo. Ibrutinib suppressed viability and induced apoptosis in 4 HL cell lines in a dose and time dependent manner. Molecular analysis showed induction of both apoptotic and autophagy markers. Ibrutinib treatment resulted in suppression of BTK and other downstream targets including PI3K, mTOR and RICTOR. Ibrutinib given at 50 mg/kg p.o daily for three weeks caused statistically significant inhibition of HL cell line derived subcutaneous xenografts (p < 0.01) in ICR-SCID mice. Molecular analysis of residual tumor tissue revealed down-regulation of BTK; its related markers and autophagy markers. Our studies are the first showing in vitro and in vivo action of BTK inhibition in classical HL. A phase II study examining the activity of ibrutinib in relapsed or refractory HL is currently enrolling (NCT02824029).

Keywords: BTK; BTK inhibitor; Bruton's Tyrosine Kinase; Bruton's Tyrosine Kinase inhibitor; Cancer research; Cell biology; Clinical toxicology; Hodgkin's Lymphoma; Molecular biology; Oncology; Toxicology.

Figures

Fig. 1

Ibrutinib (BTK inhibitor) suppresses the activity of Hodgkin's Lymphoma cell lines. 4 different HL cell lines were seeded in 24 well plates at a density of 200,000 cells/well in triplicate and exposed to indicated concentrations of ibrutinib (BTK inhibitor) for 24–72 hrs. At the end of each treatment period cells were counted using Trypan Blue viability assessment method as described previously. Graph representative of two independent experiments .

Fig. 2

Ibrutinib induces apoptosis in HL cell lines in a dose dependent manner. HL cells lines were grown in quadruplet in 24 well plates (200,000 cells per well) overnight. After 24 hrs the cells were exposed to indicated doses of ibrutinib for an additional 72 hrs. At the end of the treatment period, cells were centrifuged and re-suspended in 500 μL of Annexin binding buffer in Corning glass tubes. Each tube was mixed with 5 μL of Annexin and 5 μL of PI solution in dark and incubated for 5 minutes. The cells were sorted for apoptotic fraction using Becton Dickinson Flow cytometer at Karmanos Cancer Institute Flow cytometry core facility.

Fig. 3

Impact of Ibrutinib on downstream targets: L-428 cell lines were grown in 24 well plates at a density of 200,000 cells per well in quadruplet and exposed to indicated concentrations of Ibrutinib for 72 hrs. At the end of the treatment period cells were pooled and RNA was isolated and RT-PCR was performed using standard procedures (*p

Fig. 4

Protein expression analysis post ibrutinib treatment. L-428 cell lines were grown in T75 culture flask in the presence of indicated doses of Ibrutinib for 72 hrs. At the end of the treatment period cells protein was isolated and western blot was performed using standard protocols . Blots were probed for antibodies against PARP, cleaved Caspase 3, ATG12 and LC3B (see methods for antibody source). β-actin was used as a loading control. Blots are representative of two independent experiments. Original uncropped gel images can be viewed in Supplementary Figs. 2–5.

Fig. 5

Ibrutinib suppresses the growth of KM-H2 subcutaneous xenograft in a statistically significant manner. Initially, 5X106 KM-H2 100% viable cells were subcutaneously injected bilaterally in 5–6 ICR-SCID mice. After several attempts, and once tumor(s) develop, they were surgically removed, dissected into ∼100 mg tumor pieces, and transplanted into the flanks of 15 naïve, 4-5-week-old female ICR-SCID mice. Roughly two months later, 10 mice that developed sizable tumors were randomly divided into two equal cohorts; Control (received diluent) and Ibrutinib-treated (orally 50 mg/kg qd X5 X3–4 weeks). [A] Graph showing anti-tumor efficacy. [B] Gross tumor weight at the last dose treatment. [C] Images of tumors excised from mice after treatment. [D] One mice was sacrificed to check the expression of BTK and one mice was sacrificed to check the tumor quality. (Tumor weight calculated by vernier caliper by multiplying the length by witdth2/2) as described previously . Molecular analysis of residual tumor tissue using RT-PCR. Total RNAs from 2 tumor tissues were extracted by using the RNeasy Mini Kit (QIAGEN, Valencia, CA) following the protocol provided by the manufacturer. The RNA (1μg) from each sample was converted to cDNA by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The expression level of BTK, PI3K, Akt, RICTOR, and 4EBP1 in cells or tumors was analyzed by real-time qPCR using SYBR Green Master Mixture from Applied Biosystems. Data was analyzed according to the comparative Ct method and was normalized by GAPDH and 18S rRNA expression in each sample. Original uncropped gel images can be viewed in Supplementary Fig. 6.