Preclinical activity of combined HDAC and KDM1A inhibition in glioblastoma

Abstract

Background: Glioblastoma (GBM) is the most common and aggressive form of brain cancer. Our previous studies demonstrated that combined inhibition of HDAC and KDM1A increases apoptotic cell death in vitro. However, whether this combination also increases death of the glioma stem cell (GSC) population or has an effect in vivo is yet to be determined. Therefore, we evaluated the translational potential of combined HDAC and KDM1A inhibition on patient-derived GSCs and xenograft GBM mouse models. We also investigated the changes in transcriptional programing induced by the combination in an effort to understand the induced molecular mechanisms of GBM cell death.

Methods: Patient-derived GSCs were treated with the combination of vorinostat, a pan-HDAC inhibitor, and tranylcypromine, a KDM1A inhibitor, and viability was measured. To characterize transcriptional profiles associated with cell death, we used RNA-Seq and validated gene changes by RT-qPCR and protein expression via Western blot. Apoptosis was measured using DNA fragmentation assays. Orthotopic xenograft studies were conducted to evaluate the effects of the combination on tumorigenesis and to validate gene changes in vivo.

Results: The combination of vorinostat and tranylcypromine reduced GSC viability and displayed efficacy in the U87 xenograft model. Additionally, the combination led to changes in apoptosis-related genes, particularly TP53 and TP73 in vitro and in vivo.

Conclusions: These data support targeting HDACs and KDM1A in combination as a strategy for GBM and identifies TP53 and TP73 as being altered in response to treatment.

Keywords: HDAC inhibitors; KDM1A; LSD1; epigenetics; glioblastoma; lysine demethylases; orthotopic GBM models; tranylcypromine.

Fig. 1.

Combined targeting of HDACs and KDM1A leads to decreased viability of glioma stem cells (GSCs). (A) Expression of KDM1A protein expression in GSCs compared with normal neural progenitor cells. Actin was used as a loading control. (B) Viability of GSCs was measured 5 days after single dosing with 5 uM vorinostat, 1 mM tranylcypromine or the combination. n = 3, mean ± SEM. ***P ≤ .001.

Fig. 2.

Inhibition of HDACs and KDM1A alter gene expression in glioblastoma cell lines. (A) Venn diagram depicting the total number of genes changed in LN-18 cells by vorinostat, KDM1A knockdown, and the combination. The table illustrates the number of genes up- and downregulated by each of the treatment groups. (B) LN-18 cells transfected with KDM1A shRNA were treated with 5 µM vorinostat for 24 hours, and gene expression was measured using a RT-qPCR array. A waterfall plot shows the expression profiles of genes that displayed >2-fold upregulation and (C) >2-fold downregulation.

Fig. 3.

p53 mRNA and protein expression is rapidly regulated by HDAC and KDM1A inhibition. LN-18 cells transfected with control or KDM1A shRNA were treated with 5 µM vorinostat for 24 hours, and (A) TP53 gene expression or (B) p53 protein expression was measured. (C) LN-18 cells were treated with 5 µM of the HDACi indicated. p53 protein expression was evaluated 24 hours after treatment. (D) LN-18 cells were treated with 5 µM vorinostat, 1 mM tranylcypromine, or the combination for 24 hours, and p53 mRNA expression was evaluated. (E) p53 mRNA was measured in LN-18 cells treated with 5 µM vorinostat at the time points indicated. (F) p53 protein was assessed after treatment with 5 µM vorinostat by Western blot at the indicated time points. (G) LNZ308 (p53-null) cells were transfected with empty vector or vector-expressing wild-type p53 (Flag-p53). DNA fragmentation was measured 72 hours after treatment with 5 µM vorinostat, 1 mM tranylcypromine, or the combination. (H) Western blots demonstrating lack of p53 protein in LNZ308 cells and ectopic expression of wild-type p53 protein under conditions stated in part G. All Western blots are representative of 3 independent experiments. Actin was used as a loading control. n = 3, mean ± SEM. *P ≤ .05, ** P ≤ .01, ***P ≤ .001.

Fig. 4.

HDAC and KDM1A inhibition alter TAp73 expression. LN-18 cells transfected with control or KDM1A shRNA were treated with 5 µM vorinostat for 24 hours: (A) TA, Δexon 2 (Δex2), Δexon 2 and 3 (Δex2/3), ΔN or ΔN′ isoforms of p73 were measured by RT-qPCR., or (B) p73 protein expression was evaluated by Western blot. (C) LN-18 cells were treated with 5 µM vorinostat, 1 mM tranylcypromine, or the combination and TAp73 mRNA expression was evaluated by qPCR. (D) LN-18 cells were treated with 5 µM vorinostat, and TAp73 mRNA expression was measured at the indicated time points. (E) TAp73 expression was measured in p53-null glioblastoma cells (LNZ308) were transfected with vector or Flag-tagged wild-type p53 followed by treatment with 5 µM vorinostat, 1 mM tranylcypromine, or the combination. (F) Viability was measured in wild-type (p73^+/+), p73-deficient (p73^−/−), or ΔN-p73-deficient (ΔN^−/−) mouse embryonic fibroblasts treated with 5 µM vorinostat, 1 mM tranylcypromine, or the combination for 24 hours. n = 3, mean ± SEM. *P ≤ .05, **P ≤ .01, ***P ≤ .001.

Fig. 5.

Combination of vorinostat and tranylcypromine extends survival of glioblastoma xenograft mouse model. (A) Schematic diagram illustrating the dosing schedule for animal experiments. (B) Mice were treated 7 days after glioma cell implantation with the following regimens: 50 mg/kg vorinostat, 5 days/week (n = 4); 1 mg/mouse tranylcypromine, 7 days/week (n = 7), combination of vorinostat and tranylcypromine (n = 5), or vehicle as a control (n = 2). Kaplan-Meyer curves depict time to moribund characteristics (hunched posture, significant weight loss, or hemiparesis). (C) Luciferase-labeled U87 was injected into athymic nu/nu mice and imaged 2 weeks after implantation (pretreatment) and at the conclusion of the 2-week combination regimen (post treatment). (D) Luminescence signal was quantified and presented graphically.

Fig. 6.

Vorinostat affects p53 and TAp73 expression in an in vivo mouse model for glioblastoma. RNA from brain tissue was isolated from xenograft mice, and qPCR performed as previously described for (A) p53 and (B) TAp73. n = 3 or 4 depending on RNA quality, mean ± SEM. *P ≤ .05.