AT7519, A novel small molecule multi-cyclin-dependent kinase inhibitor, induces apoptosis in multiple myeloma via GSK-3beta activation and RNA polymerase II inhibition

Abstract

Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM.

FIG 1. AT7519 treatment decreases viability of MM cells in a dose dependent manner and overcomes proliferative advantage conferred by cytokines and the protective effect of BMSC

(A) Chemical structure of AT7519 (left panel). In vitro kinase inhibition (right panel). (B) MM cell lines (MM.1S, U266, OPM1, RPMI, LR5, DOX 40, MM.1R) and primary CD138+ MM cells from five different patients were cultured in the presence of increasing doses of AT7519 for 48 hours.. The effect of AT7519 was determined by MTT assay. IC₅₀ ranged from 0.5 to 2 μM. (C) AT7519 does not affect viability of peripheral blood mononuclear cells (PBMNCs) from healthy volunteers. (D) MM.1S cells were cultured with BMSCs, IL-6 (10 ng/ml), IGF-1 (50 ng/ml). AT7519 induced inhibition of DNA synthesis at 48 hours in dose dependent manner. The results represent an average of triplicate experiments ± SD.

FIG 2. AT7519 treatment induces apoptosis of MM cells in a time-dependent manner

(A) Cell cycle analysis by PI staining was performed on MM.1S. MM cells cultured with media alone or AT7519 (0.5 μM) for the indicated time points. AT7519 resulted in an increase G0/G1 phase and G2/M phase starting at 6 h. (B) Apoptosis was evaluated by Annexin/PI staining. The percentage of cells undergoing apoptosis was defined as the sum of early apoptosis (AnnexinV-positive cells) and late apoptosis (Annexin V-positive and PI-positive cells). Apoptosis induction was observed starting from 12 hours. (C) The apoptosis induced by AT7519 was further confirmed by western blotting. Whole cell lysates (30 μg/lane) were subjected to Western blotting using the specified antibodies.

FIG 3. AT7519 does not affect the expression of relevant cyclins and CDKs at early points but induces dephosphorylation of RNA pol II CTD

(A) MM.1S cells were incubated with AT7519 0.5 μM. At indicated time points, cells were harvested and whole lysates were subjected to Western blotting using the antibodies indicated. (B) MM.1S cells were incubated with AT7519 0.5 μM. At indicated time points cells were harvested and whole lysate were subjected to Western blotting using anti-RNA pol II, phospho RNA pol II serine 2, phospho RNA pol II serine 5, α-tubulin, α-actin antibodies. (C) MM.1S cells were incubated with AT7519 0.5 μM. At indicated time points cells were harvested and whole lysate were subjected to Western blotting using anti-Mcl-1, anti-XIAP and anti-α tubulin antibodies. Densitometry is demonstrated for each panel. (D) AT7519 inhibited RNA synthesis in MM.1S cells. MM.1S cells were incubated with media alone and AT7519 0.5 μM for 4, 6, 24 and 48 hours; then 3.5 hours prior of harvesting, [³H] Uridine was added to the cell culture and RNA synthesis measured. The results represent an average of triplicate experiments ± SD.

FIG 4. AT7519 induced cytotoxicity was associated with dephosphorylation of GSK-3β at serine 9

(A) MM.1S cells were incubated with media alone and AT7519 0.5 μM. At indicated time points cells were collected and whole lysates were subjected to Western blotting using anti-phospho GSK-3β serine 9, GSK-3β, glycogen synthase serine 641, α-tubulin, GAPDH antibodies. (B) MM.1S cells were incubated with AT7519 0.5 μM. At indicated time points, cells were collected and whole lysates were subjected to Western blotting using the antibodies indicated. Densitometry is demonstrated for each panel.

FIG 5. Inhibition of GSK-3β attenuates AT7519-induced apoptosis

(A) MM.1S cells were incubated with DMSO and AR-A014418 at indicated doses for 24 hours. Whole lysates were subjected to western blotting using anti-phospho GSK-3β serine 9, GSK-3β, glycogen synthase serine 641, GAPDH antibodies. These results are representative of at least three independent experiments. (B) MM.1S cells were cultured for 30 minutes with media alone and indicated doses of AR-A014418, and then incubated for 48 hours with AT7519 0.5 μM. The viability was determined by MTT assay. The results represent an average of triplicate experiments ± SD. (C) Silencing GSK-3β with shRNA lentiviral constructs in MM.1S was evaluated by western blotting. GSK-3β knock down was evident in clone no. 2 and observable in clone no.4. (D) MM.1S cells and transfected MM.1S were treated with increasing doses if AT7519 for 48 h. MM.1S cells with knocked-down GSK-3β, were more resistant to AT7519-induced cytotoxicity with respect to control shRNA-transfected cells. The effect of AT7519 was determined by MTT assay.

FIG 6. AT7519 dephosphorylates GSK-3β independent of inhibition of transcription

(A) MM.1S cells were incubated with media alone and with increasing doses of α-amanitin as indicated. At 24 hours, cells were collected and whole lysate were subjected to Western blotting using the specified antibodies. (B) The effect of alpha-amanitin on MM.1S cells viability was determined by MTT assays after 24 hours of treatment. The results represent mean (± SD) of triplicate experiment.

FIG 7. AT7519 inhibits MM cell growth in vivo

(A) Average ±SEM of tumor volume (mm³) from groups of mice (n=10/group) versus time (days) from first day of treatment after the development of measurable tumor. (B) Immunohistochemestry confirmed caspase 3 activation. (C) Survival was evaluated from the first day of treatment until death. Using Kaplan Meier and log-rank analysis, statistically significant prolongation in median OS was observed in treated mice compared with the control group (p = 0.0324 and p= 0.0411, respectively). The median overall survival (OS) in the group treated with 15 mg/kg once a day for five days for 2 weeks was 40 days versus 27.50 days in the control cohort. The median OS in the group treated with 15 mg/kg once a day three days week for four consecutive weeks was 39 days versus 27.50 days in the control group. (D) Average ±SEM of weight (gr) from groups of mice (n=10/group) versus time (days) from first day of treatment. AT7519 did not affect the body weight.