In-silico gene essentiality analysis of polyamine biosynthesis reveals APRT as a potential target in cancer

Abstract

Constraint-based modeling for genome-scale metabolic networks has emerged in the last years as a promising approach to elucidate drug targets in cancer. Beyond the canonical biosynthetic routes to produce biomass, it is of key importance to focus on metabolic routes that sustain the proliferative capacity through the regulation of other biological means in order to improve in-silico gene essentiality analyses. Polyamines are polycations with central roles in cancer cell proliferation, through the regulation of transcription and translation among other things, but are typically neglected in in silico cancer metabolic models. In this study, we analysed essential genes for the biosynthesis of polyamines. Our analysis corroborates the importance of previously known regulators of the pathway, such as Adenosylmethionine Decarboxylase 1 (AMD1) and uncovers novel enzymes predicted to be relevant for polyamine homeostasis. We focused on Adenine Phosphoribosyltransferase (APRT) and demonstrated the detrimental consequence of APRT gene silencing on different leukaemia cell lines. Our results highlight the importance of revisiting the metabolic models used for in-silico gene essentiality analyses in order to maximize the potential for drug target identification in cancer.

Figure 1

Genes and reactions involved in the biosynthesis of polyamines. Putrescine and spermine appear in an empty box, while spermidine in an empty dashed box. For clarity, spermidine is represented twice. Abbreviations: AMP: adenosine monophosphate.

Figure 2

Gene silencing analysis of APRT and PNP in ALL derived CEMO-1 cell line. (A and B) mRNA expression of the APRT and PNP genes 48h after nucleofection with the siRNAs. Data are referred to GUS human gene and an experimental group nucleofected with mock siRNA. Data represent mean ± standard deviation of four pooled experiments with similar results. C) Proliferation of CEMO-1 cell line nucleofected with siRNAs targeted to the indicated genes was studied by MTS. *Indicate p-value < 0.001 in a repeated measures ANOVA test followed by a Bonferroni post-test between the PNP and APRT groups. Data represent mean ± standard deviation of four pooled experiments with similar results.

Figure 3

Gene silencing analysis of APRT in acute leukemias cell lines. (A) mRNA expression of APRT gene 48h after nucleofection with the siRNAs. Data are referred to GUS human gene and an experimental group nucleofected with Silencer Select Negative Control-1 siRNA (Mock siRNA). (B) Cell proliferation of CEMO-1, KG-1 and PEER cell lines nucleofected with APRT siRNAs studied by MTS. Data represent mean ± standard deviation of three different experiments.

Figure 4

Gene expression analysis of cells sensitive and resistant to APRT knockdown for genes involved in the polyamines biosynthesis pathway. (A) Log (base 2) fold change estimates of differentially expressed genes (adj. p-value ≤ 0.05) involved in polyamines biosynthesis pathway. A gene with a positive fold change is upregulated in cells sensitive to APRT knockdown; (B) Boxplot of Log (base 2) APRT/SRM ratio in cells sensitive and resistant to APRT knockdown.