Pharmacological inhibition of BCL-2 with the FDA-approved drug venetoclax impairs longitudinal bone growth

Abstract

Treatment-related skeletal complications are common in childhood cancer patients and survivors. Venetoclax is a BCL-2 inhibitor that has shown efficacy in hematological malignancies in adults and is being investigated in pediatric cancer clinical trials as a promising therapeutic modality. Venetoclax triggers cell death in cancer cells, but whether it exerts similar effects in normal bone cells, is unknown. Chondrogenic ATDC5 cells, E20 fetal rat metatarsal bones, and human growth plate biopsies were treated with different concentrations of venetoclax. Female NMRI nu/nu mice were treated with venetoclax or vehicle for 15 days. Mice were X-rayed at baseline and at the end of the experiment to assess longitudinal bone growth and body weight was monitored throughout the study. Histomorphometric and immunohistochemical analyses were performed to evaluate treatment effects on the growth plate cartilage. Venetoclax decreased the viability of chondrocytes and impaired the growth of ex vivo cultured metatarsals while reducing the height of the resting/proliferative zone and the hypertrophic cell size. When tested in vivo, venetoclax suppressed bone growth and reduced growth plate height. Our experimental data suggest that venetoclax directly targets growth plate chondrocytes suppressing bone growth and we, therefore, encourage careful monitoring of longitudinal bone growth if treating growing children with venetoclax.

Figure 1

Effects of Venetoclax (Ven) on ATDC5 cells and E20 metatarsal bones. (A) Venetoclax decreased the viability of chondrogenic ATDC5 cells after 48 or 72 h of treatment. Three independent experiments were performed. Mean values ± SEM are presented. ***p < 0.001. (B) Treatment with venetoclax impaired bone growth ex vivo. E20 fetal rat metatarsal bones were treated with increasing concentrations of venetoclax and their growth was monitored for 12 days in culture. The graph shows the increase (%) in bone length over time from day 0. Venetoclax treatment caused dose-dependent growth retardation compared to the control group. Mean values ± SEM from three independent experiments are presented. *p < 0.05, ***p < 0.001, n = 15/group. (C) Microscopic images of fetal rat metatarsals cultured ex vivo with venetoclax or DMSO (control) as captured in real-time conditions on day 0 (start of the experiment) and day 12 (termination). (D) Representative images of metatarsal sections stained with Alcian blue and Nuclear Fast Red. Venetoclax treatment affected the organization of chondrocytes in the different zones. Magnification 5x, scale bar = 500 μm. (E) Quantification of the resting and proliferative (R + P) zone height, and hypertrophic cell size. Venetoclax treatment significantly reduced R + P zone height and hypertrophic cell size. Mean values ± SD are presented. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3–7 bones/group.

Figure 2

Venetoclax impairs protein expression in fetal rat metatarsals. (A) Representative images of metatarsal sections stained for ColX, PCNA, and humanin. Magnification 20x, Scale bar: 100 μm. (B) Quantification of PCNA, ColX, and humanin protein expression in E20 metatarsal bones. Mean values ± SEM are presented. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3–7 bones/group.

Figure 3

Venetoclax (Ven) caused growth retardation in vivo and reduced growth plate height. (A) Schematic illustration of the experimental set-up. 7-week-old female NMRI nu/nu mice were treated with vehicle (control, n = 4), Ven 100 mg/kg (n = 5), or Ven 200 mg/kg (n = 5) for 15 days. X-rays were performed at baseline (day 0) and at the endpoint (day 15). (B) Weight gain of the treated mice over time. No difference among the different treatment groups was detected. Mean values ± SD are presented. (C) Venetoclax treatment impaired bone growth in vivo. Mean values ± SD are presented. *p < 0.05, n = 4–5/ group (D) Representative images of proximal tibia growth plates stained with Alcian blue/Nuclear Fast Red solution for analysis of growth plate histomorphometry. Growth plate height was reduced in venetoclax-treated mice. Magnification 20x, scale bar = 100 μm. (E,F) Quantification of the histomorphometric analyses of the mice growth plates. The total growth plate height (μm), the height of R + P zone and the size of the terminal hypertrophic cell (THC) were reduced in both venetoclax-treated groups, compared to the control group. Mean values ± SD are presented. *p < 0.05, **p < 0.01, n = 4–5/group. H Hypertrophic.

Figure 4

Effects of venetoclax on the human growth plate. (A) Illustration showing the procedure of human growth plate tissue collection. A biopsy needle is used during the epiphysiodesis surgery to collect growth plate tissue from the tibia or femur. The tissue is then cut into small pieces which were individually placed in 24-well plates in medium supplemented with the drug of interest and cultured ex vivo. After 48 h the culture is terminated, the growth plate pieces are fixed in 4% PFA, decalcified in EDTA, and embedded in paraffin blocks for sectioning. IHC stainings are performed to analyze the expression of different markers. IHC immunohistochemistry. (B) Representative images of human growth plate tissue biopsies treated with venetoclax (Ven) or DMSO (control) for 48 h. Alcian blue counterstain. Magnification 20x, scale bar = 100 μm. (C) Quantification of PCNA and humanin expression in the ex vivo treated human growth plate biopsies. Results of 3 patients are presented. Mean values ± SD are shown. *p < 0.05, **p < 0.01.