Mechanisms and therapeutic targeting of NT5C2 mutations in relapsed acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy that results from the unregulated growth of B-cell and T-cell lymphoid progenitors. Despite the implementation of risk-stratification and improved multi-agent therapeutic regimens, 20% of pediatric and 50% of adult patients fail to achieve remission and end up relapsing. NT5C2 (5’ cytosolic nucleotidase II) is the most frequently mutated gene specifically found in relapsed ALL. NT5C2 mutations are present in 20% of relapsed T-ALLs and 3-10% of relapsed B-ALLs and present as heterozygous gain of function alleles exhibiting increased nucleotidase activity. As NT5C2 can dephosphorylate and inactivate the cytotoxic metabolites generated by 6-mercaptopurine, a chemotherapy used in the treatment of ALL, these NT5C2 activating mutations can contribute to thiopurine chemotherapy resistance (Tzoneva, Perez-Garcia et al. 2013). Here we perform an extensive structure-function study to understand how relapse-associated NT5C2 mutations result in increased nucleotidase activity and contribute to chemotherapy resistance in ALL. Crystallization of 15 NT5C2 WT and mutant structures as well as enzymatic, structural modeling, and genetic screens identified three regulatory mechanisms of NT5C2, which are disrupted by these gain of function alleles. Class I NT5C2 mutations lock the protein in an active configuration through stabilization of the helixA region, which allows for substrate processing and catalysis. Class II NT5C2 mutations disrupt an intramolecular switch off domain involving the arm region and the intermonomeric positively charged pocket. And a single C-terminus truncating mutant creates a third class of mutations, which show increased nucleotidase activity due to the loss of the C-terminus blockade against allosteric activation. These studies provide new insight into the regulatory controls that mediate NT5C2 activity providing a framework for the development of targeted inhibitors for the treatment of relapsed ALL. In addition to looking at relapse associated NT5C2 mutations on a structural level, we also explored how NT5C2 mutations shape the clonal architecture and evolutionary dynamics during tumor initiation and disease progression in ALL. To formally address these questions, we developed a murine NOTCH1-driven T-ALL with conditional knock-in of the Nt5c2R367Q mutation, the most recurrent mutation found in relapsed ALL, from the endogenous locus. Using this model, we confirmed that Nt5c2+/R367Q lymphoblasts show increased resistance to 6-MP in vitro and in vivo. We also found that Nt5c2+/R367Q mutant lymphoblasts exhibit impaired cell fitness and decreased leukemia initiating cell capacity. Metabolomic profiling and guanosine rescue experiments show that this decrease in cell fitness is due to excess clearance of purine metabolites out of the cell as a result of deregulated Nt5c2 nucleotidase activity. However, in the context of 6-MP therapy, Nt5c2+/R367Q mutant cells are positively selected for in mixed population studies in vitro and in vivo. These results identify a clear selective advantage for NT5C2 mutant cells in the context of 6-MP chemotherapy. In addition, NT5C2 mutant chemoresistant cells show collateral sensitivity to inhibition of inosine monophosphate dehydrogenase (IMPDH) with mizoribine, which further disrupts guanosine production pointing to a potentially selective therapy against NT5C2 mutant cells. We also show here the initial development of a small molecule NT5C2 inhibitor for the treatment of relapsed ALL. Using a malachite green based NT5C2 nucleotidase assay, we performed a small molecule high throughput assay and identified HTP_2 as a lead compound with low micromolar inhibitory activity against NT5C2 R367Q mutant recombinant protein. HTP_2 can reverse 6-MP resistance in Nt5c2+/R367Q mouse lymphoblasts and NT5C2 R29Q mutant expressing human cell lines. Interestingly, HTP_2 treatment also results in increased sensitivity to 6-MP therapy in NT5C2 wild-type cells, suggesting a role for wild-type NT5C2 activity in the clearance of 6-MP and supporting a potential therapeutic use for NT5C2 inhibitors in potentiating the effects of 6-MP based chemotherapy in NT5C2 wild-type cells as well. NT5C2 knockdown cells and Nt5c2 knockout mice show no apparent toxicities suggesting that systemic inhibition of NT5C2 could be fairly well tolerated. In all, this work presents a framework for the development of a high affinity NT5C2 inhibitor for the reversal of 6-MP resistance in relapsed ALL patients. These studies presented here address the role of NT5C2 mutant proteins as drivers of resistance and as therapeutic targets in relapsed ALL. Improved understanding of the molecular mechanisms responsible for increased NT5C2 nucleotidase activity and on the process of clonal evolution during disease progression provide important insight into the mechanism driving ALL resistance and relapse. The identification of IMPDH inhibition as a collateral vulnerability in NT5C2 mutant ALL cells and the development of a first-in-class NT5C2 inhibitor serve as framework for the development of new combination therapies aimed at curtailing the emergence of these thiopurine-resistant relapse driving clones in ALL

The Effects Of Farnesyltransferase Inhibitor Abt-100 On Murine Helper T-Cell Differentiation And Cytokine Secretion

Farnesyltransferase Inhibitors (FTIs) are a class of drugs known to prevent the farnesylation and subsequent membrane attachment of a number of intracellular proteins. In various studies, the administration of FTIs has been found to play a role in the activation and development of T-cells in the immune system. FTIs have also been found to act as immunomodulators in delaying MHC-II mismatched skin allografts in mice. This study focuses on the effect of the FTI, ABT-100, on the differentiation and cytokine secretion of Th1 and Th2 helper T-cells in BALB/C mice to better understand which immune responses are targeted by FTIs. Splenocytes were isolated from BALB/C mice, skewed towards either a Th1 or a Th2 phenotype with the addition of cytokines, and treated with various concentrations of ABT-100. Splenocytes were also isolated and immediately cultured in the presence of ABT-100 to observe differentiation trends of helper T-cells. Cytokine production was measured using intracytoplasmic flow cytometry analysis. I found that ABT-100 treatment does not block Th1 or Th2 cell differentiation. Instead, ABT-100 treatment appears to affect cytokine production from effector T-cells. I found that ABT-100 causes a decrease in IFN-¿ production in mature Th1 cells yet does not affect IL-4 production in mature Th2 cells. This decrease in cytokine production as a result of ABT-100 treatments provides a potential mechanism for how ABT-100 works to delay MHC-II mismatched allograft rejection

Clonal evolution mechanisms in NT5C2 mutant-relapsed acute lymphoblastic leukaemia.

Relapsed acute lymphoblastic leukaemia (ALL) is associated with resistance to chemotherapy and poor prognosis. Gain-of-function mutations in the 5'-nucleotidase, cytosolic II (NT5C2) gene induce resistance to 6-mercaptopurine and are selectively present in relapsed ALL. Yet, the mechanisms involved in NT5C2 mutation-driven clonal evolution during the initiation of leukaemia, disease progression and relapse remain unknown. Here we use a conditional-and-inducible leukaemia model to demonstrate that expression of NT5C2(R367Q), a highly prevalent relapsed-ALL NT5C2 mutation, induces resistance to chemotherapy with 6-mercaptopurine at the cost of impaired leukaemia cell growth and leukaemia-initiating cell activity. The loss-of-fitness phenotype of NT5C2+/R367Q mutant cells is associated with excess export of purines to the extracellular space and depletion of the intracellular purine-nucleotide pool. Consequently, blocking guanosine synthesis by inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) induced increased cytotoxicity against NT5C2-mutant leukaemia lymphoblasts. These results identify the fitness cost of NT5C2 mutation and resistance to chemotherapy as key evolutionary drivers that shape clonal evolution in relapsed ALL and support a role for IMPDH inhibition in the treatment of ALL

Clonal evolution mechanisms in NT5C2 mutant-relapsed acute lymphoblastic leukaemia

Relapsed acute lymphoblastic leukaemia (ALL) is associated with resistance to chemotherapy and poor prognosis. Gain-of-function mutations in the 5'-nucleotidase, cytosolic II (NT5C2) gene induce resistance to 6-mercaptopurine and are selectively present in relapsed ALL. Yet, the mechanisms involved in NT5C2 mutation-driven clonal evolution during the initiation of leukaemia, disease progression and relapse remain unknown. Here we use a conditional-and-inducible leukaemia model to demonstrate that expression of NT5C2(R367Q), a highly prevalent relapsed-ALL NT5C2 mutation, induces resistance to chemotherapy with 6-mercaptopurine at the cost of impaired leukaemia cell growth and leukaemia-initiating cell activity. The loss-of-fitness phenotype of NT5C2+/R367Q mutant cells is associated with excess export of purines to the extracellular space and depletion of the intracellular purine-nucleotide pool. Consequently, blocking guanosine synthesis by inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) induced increased cytotoxicity against NT5C2-mutant leukaemia lymphoblasts. These results identify the fitness cost of NT5C2 mutation and resistance to chemotherapy as key evolutionary drivers that shape clonal evolution in relapsed ALL and support a role for IMPDH inhibition in the treatment of ALL