ATR inhibition facilitates targeting of leukemia dependence on convergent nucleotide biosynthetic pathways

Abstract

Leukemia cells rely on two nucleotide biosynthetic pathways, de novo and salvage, to produce dNTPs for DNA replication. Here, using metabolomic, proteomic, and phosphoproteomic approaches, we show that inhibition of the replication stress sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) reduces the output of both de novo and salvage pathways by regulating the activity of their respective rate-limiting enzymes, ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), via distinct molecular mechanisms. Quantification of nucleotide biosynthesis in ATR-inhibited acute lymphoblastic leukemia (ALL) cells reveals substantial remaining de novo and salvage activities, and could not eliminate the disease in vivo. However, targeting these remaining activities with RNR and dCK inhibitors triggers lethal replication stress in vitro and long-term disease-free survival in mice with B-ALL, without detectable toxicity. Thus the functional interplay between alternative nucleotide biosynthetic routes and ATR provides therapeutic opportunities in leukemia and potentially other cancers.

Suppression of p16 induces mTORC1-mediated nucleotide metabolic reprogramming

“…While previous reports have linked ATR signaling to nucleotide metabolism through upregulation of RRM2 protein stability (Le et al, 2017), our model suggests that ATR also modulates nucleotide metabolism through increasing mTORC1 signaling (Fig. 3).…”

Defective Nucleotide Catabolism Defines a Subset of Cancers Sensitive to Purine Nucleoside Phosphorylase Inhibition

“…Furthermore, dCK inactivation has also been associated with resistance to dG-mediated toxicity in PNP-deficient T-lymphoma cells 15 . We found that the lethality of PNP inhibitors be completely prevented by supplementing with a small molecule dCK inhibitor indicating that phosphorylation of dG by dGK does not contribute to the observed lethality at these timepoints ( Figure 2D) 4 .…”

“…We found that SAMHD1 KO cells exhibited a 200-fold lower thymidine IC50 than SAMHD1 WT controls (Figure 5A). This selectivity was unique to thymidine and did not extend to other RNR inhibitors, including 3-AP, which function by preventing RNR tyrosyl radical regeneration 4 . Similar selectivity was observed using crystal violet proliferation analysis (Figure 5B).…”

“…A sufficient supply of deoxyribonucleotide triphosphates (dNTPs) is essential for cell growth, survival and proliferation 1,2 . Thus, the metabolic network that is responsible for the synthesis of dNTPs is tightly regulated by transcriptional, post-translational and allosteric control mechanisms 3,4 . Cancer cells are particularly reliant on dNTP biosynthesis and this non-oncogene addiction has been leveraged clinically by the use of small molecule inhibitors of rate-limiting metabolic enzymes including dihydroorotate dehydrogenase (DHODH), ribonucleotide reductase (RNR), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS) 5 .…”

“…Giblett and colleagues were among the first to identify that inactivating, inherited mutations in the nucleotide catabolic enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) drive the development of SCID 8,9 . ADA and PNP function sequentially in the catabolism of purine deoxyribonucleosides dA and dG and counter their intracellular accumulation mediated by deoxycytidine kinase dCK 4 . For reasons not well understood defective dG catabolism results in remarkably selective T-cell defects, and otherwise normal cellular development, in individuals with PNP-linked SCID 10 .…”