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

Abstract

2 Small molecule inhibitors of purine nucleoside phosphorylase (PNP) have been explored as a treatment strategy for leukemia and lymphoma, however, the determinants of response to this class of drugs are incompletely understood. PNP inhibitors impair cell proliferation by preventing catabolism of the nucleoside deoxyguanosine (dG) which induces toxic imbalances amongst intracellular deoxyribonucleotide triphosphate (dNTP) pools following its phosphorylation and trapping by nucleoside kinases. We hypothesized that differential nucleoside uptake or catabolism defines cancer cell lines as either sensitive or resistant to PNP inhibition. Among cancer cell lines we found that T-cell acute lymphoblastic leukemia (T-ALL) cells are uniquely and acutely sensitive to PNP inhibition whereas the B-cell leukemia and solid tumor models are completely resistant. We determined that although the nucleoside scavenging kinase deoxycytidine kinase (dCK) was active in all cells tested, PNP inhibitors only induced dGTP pool increases in sensitive models. By evaluating the expression of key genes involved in nucleotide scavenging, biosynthesis, and phosphohydrolysis in a panel of sensitive and resistant cell lines we found that the dNTP phosphohydrolase SAM histidine aspartate containing protein 1 (SAMHD1) was exclusively expressed in resistant models. Using CRISPR/Cas9 SAMHD1 knockout cell lines, we verified that PNP inhibitor sensitivity is a function of SAMHD1 expression and determined that the pharmacological inhibition of dCK or genetic restoration of SAMHD1 conferred resistance to PNP inhibition. Importantly, we determined that low expression of SAMHD1 is not limited to T-ALL as subset of established and primary solid tumors models are SAMHD1-deficient. These solid tumor models were consistently acutely sensitive to PNP inhibitors which indicates that the utility of PNP inhibitors extends beyond hematological malignancies. Additionally, we found that deoxycytidine (dC) can limit the anti-proliferative effects of PNP inhibitors by competing with dG for phosphorylation by dCK but this effect can be overcome by expression of dC-catabolizing gene cytidine deaminase (CDA). Collectively, these results indicate that SAMHD1, dCK and CDA are critical biomarkers that must be used to stratify patients in clinical trials evaluating pharmacological PNP inhibition.

Nucleotide imbalance decouples cell growth from cell proliferation

“…Recent work has shown that the dNTP-degrading enzyme SAMHD1 protects against cytotoxic dGTP buildup upon deoxyguanosine supplementation. SAMHD1-deficient tumor cells are sensitive to dGTP accumulation caused by deoxyguanosine supplementation and purine nucleoside phosphorylase (PNP) inhibition 46,47 . Nucleotide imbalance is also implicated in non-cancer human disease settings.…”

Targeting the DNA damage response and repair in cancer through nucleotide metabolism

“…[ 154 ]). Notably, SAMHD1 has also been reported to protect cells from build‐up of cytotoxic dGTP, which could be exploited to target SAMHD1‐deficient cancer cells with PNP inhibitors [ 155 , 156 ]. Thus, there is a striking parallel in the role of SAMHD1 in protecting cells from excess dGTP and the triphosphate metabolite of nelarabine (ara‐GTP).…”

“…Thus, there is a striking parallel in the role of SAMHD1 in protecting cells from excess dGTP and the triphosphate metabolite of nelarabine (ara‐GTP). Given the apparent lack of SAMHD1 expression in T‐cell malignancy cell lines [ 151 , 156 ], it is tempting to speculate that SAMHD1 (or rather lack of) is responsible for the original observations of dG and ara‐G selective T‐cell toxicity together with subsequent experiments, which formed the basis of nelarabine being developed as a T‐cell‐specific drug. Another interesting point is that many of the triphosphate metabolites of these nucleoside analogues can also allosterically activate SAMHD1 at the AS2 site [ 14 , 143 , 144 , 145 , 148 , 149 ], but the biological relevance in cancer cells, if any, has been little explored/observed, perhaps owing to basal dNTPs already being sufficient for tetramerisation, which is known to be long‐lived [ 104 ].…”

Design, Synthesis, Biological Evaluation, and Crystallographic Study of Novel Purine Nucleoside Phosphorylase Inhibitors

“…T-Cell-targeted antiproliferative properties of PNPIs open various therapeutic uses in the treatment of autoimmune diseases (e.g., inflammatory bowel disorders, multiple sclerosis, rheumatoid arthitis, psoriasis, and organ transplant rejection) and T-cell malignancies (leukemias and lymphomas) . Recent research shed light on specific mutations responsible for sensitivity of cells to PNP inhibition. , Such genetic markers potentially open other opportunities in more targeted therapies and even in a treatment of solid tumors. Moreover, it has been demonstrated that accumulation of guanosine (Guo) by PNPIs activates various toll-like receptors (TLRs), which, paradoxically, leads to immune activation effects.…”

“… 9 Recent research shed light on specific mutations responsible for sensitivity of cells to PNP inhibition. 13 , 14 Such genetic markers potentially open other opportunities in more targeted therapies and even in a treatment of solid tumors. Moreover, it has been demonstrated that accumulation of guanosine (Guo) by PNPIs activates various toll-like receptors (TLRs), which, paradoxically, leads to immune activation effects.…”