Pharmacologic Inhibition of NT5C2 Reverses Genetic and Nongenetic Drivers of 6-MP Resistance in Acute Lymphoblastic Leukemia

Abstract

Low intensity maintenance therapy with 6-mercaptopurine (6-MP) limits the occurrence of acute lymphoblastic leukemia (ALL) relapse and is central to the success of multi-agent chemotherapy protocols. Activating mutations in the cytosolic 5’ nucleotidase II (NT5C2) gene drive resistance to 6-MP in over 35% of early relapse ALL cases. Here we identify CRCD2 as a first-in-class small molecule NT5C2 nucleotidase inhibitor broadly active against leukemias bearing highly prevalent relapse-associated mutant forms of NT5C2 in vitro and in vivo. Importantly, CRCD2 treatment also enhanced the cytotoxic activity of 6-MP in NT5C2 wild type leukemias leading to the identification of NT5C2 S502 phosphorylation as a novel NT5C2-mediated mechanism of 6-MP resistance in this disease. These results uncover an unanticipated role of non-genetic NT5C2 activation as a driver of 6-MP resistance in ALL and demonstrate the potential of NT5C2 inhibitor therapy for enhancing the efficacy of thiopurine maintenance therapy and overcoming resistance at relapse.

Inosine enhances tumor mitochondrial respiration by inducing Rag GTPases and nascent protein synthesis under nutrient starvation

“…Up to date, a quantity of anti-cancer drugs, most purine nucleotide analogs, including 6-mercaptopurine, 6-thiocitrulline, and methotrexate which target the key enzymes in purine metabolism in clinical [ 56 ]. CRCD2, a first-in-class specific small molecule inhibitor of the cytosolic 5′ nucleotidase II (NT5C2), has been reported to be broadly active against NT5C2 in acute lymphoblastic leukemia (ALL) [ 57 ]. Whether any NT5C2 inhibitor, such as CRCD2 or PNP agonizts, can effectively block inosine accumulation is awaiting further investigation.…”

An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Thiopurine Resistance in Acute Lymphoblastic Leukemia

“…This was apparent in both cell-free nucleotidase assays (Figure 3c) and in vitro and in vivo 6-MP resistance assays performed with engineered B-ALL cell lines (Figure 3e,g, Supplemental Figure 1f,h, Supplemental Figure 2b). Not only could robust expression of the NT5C2ex6a isoform predict resistance to thiopurines, but may suggest additional therapeutic options such as treatment with the emerging class of direct NT5C2 small-molecule inhibitors such as CRCD2 (54) and also with the already FDA-approved drug mizoribine (55). Indeed, NT5C2ex6a-expressing REH and NALM6 cells were found to exhibit heightened sensitivity to mizoribine compared to the isogenic cell lines reconstituted with the canonical NT5C2 isform (Figure 3f, Supplemental Figure 1g).…”

“…Other ways to therapeutically target NT5C2 dysregulation are likely to exist. Our recently published work elucidated the role of NT5C2 post-transcriptional modification in chemoresistance, with phosphorylation of the Ser-502 residue by a yet-to-be-identified protein kinase increasing its activity against 6-MP (54). Our new data showing that substituting Ser at position 131 with a phosphomimetic Asp augments NT5C2ex6a-induced resistance to 6-MP (Supplemental Figure 1h) lend further credence to the model linking NT5C2 phosphorylation and chemoresistance function.…”

Computational structure prediction methods enable the systematic identification of oncogenic mutations

“…Interestingly, no mutations have been identified in these regions in ALL patients. However, it has been reported that nongenetic mechanisms such us post-translational modifications, affect NT5C2 activation and become drivers of therapy resistance 30 . In addition, our results demonstrate the effect of altering these regions in the response to 6-MP.…”