Targeting the Purine Biosynthesis Pathway in Relapsed ALL

Relapsed acute lymphoblastic leukemia (ALL) remains a leading cause of childhood cancer death and presents a challenge, as well-established risk factors have been imperfect in predicting treatment failure. Recently, high-throughput genomic analyses of serial samples from diagnosis to remission and relapse have enhanced our understanding of disease evolution and mechanisms of drug resistance in childhood ALL.^1,2 

Utilizing this strategy, Dr. Benshang Li and colleagues performed whole-exome sequencing on matched diagnosis-remission-relapse samples from 15 cases of B-precursor ALL from Shanghai Medical Center and identified recurrent relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes an essential enzyme in the purine biosynthesis pathway, in two cases. Targeted sequencing in an independent Chinese cohort of 144 cases of relapsed ALL and a German cohort of 220 cases confirmed the presence of relapse-specific PRPS1 mutations in 24 individuals. Overall, 17 distinct mutations were identified, with a frequency of 13 percent in the Chinese cohort and 2.7 percent in the German cohort. Notably, mutations in NT5C2, another gene involved in thiopurine metabolism and among the most commonly observed relapse-specific mutation in childhood ALL,^2,3  were also found in the German cohort (6.1%) but were mutually exclusive of PRPS1 mutations. Although treatment regimens varied between the Chinese and German cohorts, both included prolonged daily administration of thiopurines (6-mercaptopurine or 6-thioguanine), and all relapses in individuals with PRPS1 mutations occurred early (<36 months from diagnosis).

To evaluate whether PRPS1 mutations were present in a subclone at diagnosis, as well as the time course of their acquisition, the authors analyzed serial bone marrow samples in four patients. While mutations were not present at diagnosis, they were acquired after exposure to chemotherapy, and mutant clones exponentially expanded prior to overt relapse (Figure). Functionally, these PRPS1 mutations were predicted to block binding to nucleotide inhibitors. The authors hypothesized that with exposure to thiopurines over the course of treatment, mutations in PRPS1 emerge from the selective pressure of these agents and confer resistance.

The authors next confirmed a gain-of-function mechanism of drug resistance showing that cells transfected with mutant PRPS1 demonstrated resistance to thiopurines, exclusively, and not to other classes of agents. To determine how PRPS1 mutations led to resistance, the authors examined the impact of the mutations on thiopurine prodrug conversion and showed that the production of active metabolites that cause DNA damage was markedly diminished in the presence of mutations. The authors next went on to show that mutations imparted resistance by reducing feedback inhibition of de novo purine biosynthesis, producing an abundance of substrates (purines) that competitively inhibited the normal conversion of thiopurine pro-drugs to active metabolites.⁴ 

Finally, to test therapeutic strategies targeting de novo purine biosynthesis, the authors inhibited enzymes in the purine biosynthetic pathway using CRISPR-Cas9 technology and treatment with a pathway inhibitor, and demonstrated reversal of the resistant phenotype in cell lines harboring PRPS1 mutations. These findings are important, as several small molecule inhibitors of de novo purine synthesis are presently in clinical development, and this mechanism of resistance may be relevant in other tumor types as well.