De Novo Purine Biosynthesis in Drug Resistance and Tumor Relapse of Childhood ALL

Abstract

Background: Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Studies have shown that most ALL cases are polyclonal at diagnosis and that genetic changes in individual subclones influence sensitity to therapy and subsequent clonal evolution during therapy; but the molecular details remain to be worked out. Among different pathways enriched for mutations at relapse, purine metabolism is particularly interesting for two reasons: first, thiopurines are widely used in the ALL combination chemotherapy regimens, and are prodrugs that are converted by the purine salvage pathway to cytotoxic metabolites. Second, de novo nucleotide biosynthesis is often upregulated in cancer cells, and it is believed that sufficient nucleotide pools are required to maintain genomic stability, could bypass oncogene-induced senescence and promote tumor progression1. Therefore, we focus our current study on de novo purine biosynthesis in drug resistance and tumor relapse of childhood ALL.Methods and Results: Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. Targeted sequencing identified mutations in additional genes in de novo purine biosynthesis pathway, providing further genetic evidence for its importance in relapsed ALL. All individuals with PRPS1 mutation relapsed early on-treatment (P <0.001), having an inferior prognosis1.Using various functional assays, we demonstrated that rather than causing a simple gain-of-function effect, the mutations in PRPS1 resulted in the disruption of the normal feedback inhibition of purine synthesis, in which the enzyme remained active despite an increased concentration of nucleoside analogs. PRPS1 mutants increased synthesis of the nucleoside inosine monophosphate, its metabolite hypoxanthine (HX) and de novo purine biosynthesis intermediates (e.g. AICAR, SAICAR) in Reh cells. Increased intracellular HX can competively inhibit the conversion of thiopurines into their active metabolites. Furthermore, inhibition of de novo purine biosynthesis in vitro, either by CRISPR-Cas9 genome editing of de novo purine synthesis pathway genes (GART, ATIC etc.) or treatment with a pathway inhibitor lometrexol (GART inhibitor) alleviated the metabolic disturbance and drug resistance induced by PRPS1 mutations.Using ultra-deep sequencing of unique serial remission samples before clinical relapse, we noticed that the PRPS1 mutant allele fraction increased drastically before clinical relapse, suggesting rapid clonal expansion occurs after the acquisition of a PRPS1 mutation. Interestingly, we also noticed that PPRS1 mutation coexist with RAS mutation in many relapse cases and at single cell resolution. Functional analysis revealed that tumor cells which harbored RAS and PRPS1 double mutations are more drug resistant than those with RAS or PRPS1 mutation alone. Previous studies have shown that oncogenic RAS mutation can also induce various stress responses including oncogene-induced senensence and DNA damage response (DDR), which all could impede tumor cell proliferation during relapse. In vitro, we found PRPS1 mutation can release the replication and metabolic stress caused by RAS mutation, in addition to their role in thiopurine resistance. The PRPS1 mutants not only increase the nucleotide pools but also elevate purine biosynthesis intermediate AICAR, which can activate AMPK and reduce the RAS mutant-induced DDR. We are currently working on in vitro and in vivo models (including patient derived xenograft models) to further test the double mutant's effects on tumor-reinitiation and clonal evolution during ALL relapse.Conclusions: We demonstrated that negative feedback-defective PRPS1 mutants can drive de novo purine biosynthesis, which can exert drug resistance and reduce genomic instability during tumor relapse. Our study highlights the importance of de novo purine biosynthesis in the pathogenesis of relapse, and suggests a diagnostic approach to predicting early relapse and a therapeutic strategy to circumventing resistance in ALL.1 Li et al. Negative feedback-defective PRPS1 mutants drivee thiopurine resistance in relapsed childhood ALL. Nature Medicine,21(6): 563-571 (2015) DisclosuresNo relevant conflicts of interest to declare.