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. 2022 Nov 2;12(11):2646-2665.
doi: 10.1158/2159-8290.CD-22-0010.

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

Clara Reglero #  1 , Chelsea L Dieck #  1 , Arie Zask  2 , Farhad Forouhar  3 , Anouchka P Laurent  1 , Wen-Hsuan W Lin  4 , Robert Albero  1 , Hannah I Miller  1 , Cindy Ma  1 , Julie M Gastier-Foster  5   6 , Mignon L Loh  7 , Liang Tong  8 , Brent R Stockwell  2 , Teresa Palomero  1   4 , Adolfo A Ferrando  1   4   9   10
Affiliations

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

Clara Reglero et al. Cancer Discov. .

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 multiagent chemotherapy protocols. Activating mutations in the 5'-nucleotidase cytosolic 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 Ser502 phosphorylation as a novel NT5C2-mediated mechanism of 6-MP resistance in this disease. These results uncover an unanticipated role of nongenetic 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.

Significance: Relapse-associated NT5C2 mutations directly contribute to relapse in ALL by driving resistance to chemotherapy with 6-MP. Pharmacologic inhibition of NT5C2 with CRCD2, a first-in-class nucleotidase inhibitor, enhances the cytotoxic effects of 6-MP and effectively reverses thiopurine resistance mediated by genetic and nongenetic mechanisms of NT5C2 activation in ALL. This article is highlighted in the In This Issue feature, p. 2483.

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Conflict of interest statement

Declaration of Interests

The authors declare no competing financial interests relevant for the work reported here. Financial disclosures for Adolfo Ferrando: Employment Regeneron Genetics Center, Consulting for VantAI, and Brystol Myers Squibb. B.R.S. is an inventor on patents and patent applications involving small molecule therapeutics, co-founded and serves as a consultant to Inzen Therapeutics, Nevrox Limited, Exarta Therapeutics, and ProJenX, Inc. and serves as a consultant to Weatherwax Biotechnologies Corporation and Akin Gump Strauss Hauer & Feld LLP. Arie Zask is a co-founder of and consultant to ProJenX, Inc. A provisional patent protecting composition of matter on lead compounds has been filed by Columbia University.

Figures

Figure 1.
Figure 1.. High-throughput compound screen for the identification of NT5C2 inhibitors.
(A) Malachite green NT5C2 enzyme assay reaction and colorimetric measurement. (B) Reaction progress curve of NT5C2 R367Q recombinant protein at five protein concentrations. (C) Initial velocity versus substrate concentration of 0.02 μM NT5C2 R367Q recombinant protein with IMP as substrate. Vmax= 610 μM IMP/min, Km = 308 μM IMP. (D) Malachite green assays showing dose-response curves of top six compounds from the screening library. Graphs show technical replicates from a single experiment performed in triplicate. logEC50 for each compound is shown. (E) Malachite green assays showing dose-response curves for top hits sourced from an independent synthesis batch sourced from Enamine as in (E). (F) Malachite green assay analysis of CRCD2 synthesized at the Columbia Probe Synthesis facility by two different synthesis routes (CU-1, CU-2) and two different lots provided by Enamine (Enamine-1, Enamine-2) incubated with recombinant NT5C2 R367Q protein. Graph shows mean ± SD of three independent experiments performed in triplicate. (G) Sensorgram for CRCD2 binding to NT5C2 D52N R367Q recombinant protein generated with Biacore surface plasmon resonance technology. Equilibrium dissociation constant (Kd) is shown.
Figure 2.
Figure 2.. Binding and inhibition of wild type, R367Q and K359Q recombinant protein by CRCD2.
(A) Chemical structure of CRCD2 compound. (B) Malachite green assay of wild type, R367Q and K359Q NT5C2 recombinant protein incubated with increasing concentrations of CRCD2. Graph shows average ± SD of three independent experiments performed in triplicate. P values were calculated using ordinary one-way ANOVA and Dunnett’s multiple comparisons test. (C) Michaelis-Menten curve showing NT5C2 enzyme kinetics in the absence and in the presence of 10μM CRCD2. Graph shows average ± SD of three independent experiments performed in triplicate. We calculated the P value using a Michaelis-Menten non-linear regression curve fit test. (D) Sensorgram for CRCD2 binding to NT5C2 D52N R367Q recombinant protein in the presence of 500μM IMP, generated with Biacore surface plasmon resonance technology. Equilibrium dissociation constant (Kd) is shown. (E) Malachite green assay of wild type, R367Q and K359Q recombinant protein incubated with 10 μM CRCD2 and increasing concentrations of IMP. Graph shows average ± SD of three independent experiments performed in triplicate. P values were calculated using ordinary one-way ANOVA and Dunnett’s multiple comparisons test. (F) Malachite green assay of wild type, R367Q and K359Q recombinant protein incubated with increasing concentrations of both CRCD2 and IMP. Graphs show average ± SD of three independent experiments performed in triplicate. We calculated P values using two-tailed Student’s t-test of the AUC of 500 mM vs 50 mM IMP.
Figure 3.
Figure 3.. CRCD2 sensitizes to 6-MP chemotherapy in human ALL cell lines harboring wild-type and mutant NT5C2 and in ALL patient-derived xenografts.
(A) Jurkat and CUTLL1 NT5C2 WT ALL cells treated with vehicle or 10 μM CRCD2 and increasing doses of 6-MP. Graph shows mean ± SD of three independent experiments performed in triplicate. (B) PEER and BE13 NT5C2 mutant ALL cells treated with vehicle or 10 μM CRCD2 and increasing doses of 6-MP. Graph shows mean ± SD of three independent experiments performed in triplicate. (C) REH (NT5C2 wild type) and 697 (NT5C2 R278W mutant) B-precursor ALL cells treated with vehicle or 10 μM CRCD2 and increasing doses of 6-MP. Graph shows mean ± SD of three independent experiments performed in triplicate. (D) Schematic representation of the NT5C2 mutants selected for the following assays. Mutants resulting in constitutive allosteric independent activation are shown in green Mutants with impaired intramolecular enzymatic switch off are shown in purple. (E) Immunoblot analysis of Jurkat cells expressing infected with empty vector or lentiviruses driving the expression of Flag-tagged mutants of NT5C2. Expression levels were verified in three independent experiments. (F) Viability assay of Jurkat cells infected with empty vector lentiviruses treated with vehicle or 10 μM CRCD2 and increasing doses of 6-MP. Graphs show mean ± SD of three independent experiments performed in triplicate. (G) Viability assay of Jurkat cells infected with mutant NT5C2 expressing lentiviruses treated with vehicle or 10 μM CRCD2 and increasing doses of 6-MP as in (F). (H) Patient-derived xenograft relapsed-T-ALL NT5C2-mutant lymphoblasts treated with vehicle or 10μM CRCD2 and increasing doses of 6-MP. Graph shows mean ± SD of two independent experiments performed in triplicate. All P values were calculated using IC50 values and two-tailed Student’s t-test over wild-type.
Figure 4.
Figure 4.. NT5C2 inhibitor CRCD2 sensitizes both NT5C2 WT (Nt5c2+/co-R367Q) and NT5C2 mutant (Nt5c2+/R367Q) mouse lymphoblasts to 6-MP.
(A) PCR amplification of Nt5c2 wild-type and R367Q mutant alleles after 4-OHT treatment. Isobologram analysis of NT5C2 WT (Nt5c2+/co-R367Q). (B) Cell viability of NT5C2 wild type (Nt5c2+/co-R367Q) and NT5C2 mutant (Nt5c2+/R367Q) mouse lymphoblasts treated with increasing doses of 6-MP and CRCD2. Graph shows one representative experiment with technical replicates. Two additional experiments showed similar results. (C) Cell viability of NT5C2 mutant (Nt5c2+/R367Q) mouse lymphoblasts treated with increasing doses of 6-MP and CRCD2. Graph shows one representative experiment with technical replicates. Two additional experiments showed similar results. (D) Cell viability of NT5C2 wild type (Nt5c2+/co-R367Q) and NT5C2 mutant (Nt5c2+/R367Q) mouse lymphoblasts treated with 2 μM 6-MP and increasing doses of CRCD2. Graph shows mean ± SD of three independent experiments performed in triplicate. P values were calculated using two-tailed Student’s t-test. (E) Isobologram analyses and cell viability of mouse wild type (Nt5c2+/co-R367Q) lymphoblasts treated with 6-MP, CRCD2, or a combination of 6-MP and CRCD2. Graphs show mean of three technical replicates. We repeated the experiment two additional times with similar results. (F) Isobologram analyses and cell viability of NT5C2 mutant (Nt5c2+/R367Q) mouse lymphoblasts treated with 6-MP, CRCD2, or a combination of 6-MP and CRCD2. Graphs show mean of three technical replicates. We repeated the experiment two additional times with similar results. (G) Combination Index of isogenic wild type and Nt5c2 R367Q mutant mouse ALL lymphoblasts treated with 6-MP and CRCD2. Dots represent mean values from three independent experiments performed in triplicate.
Figure 5.
Figure 5.. Response of NT5C2 wild type and R367Q tumors to CRCD2 and 6-MP combinatorial chemotherapy in vivo.
(A) Schematic illustration of 6-MP and CRCD2 combination experimental therapeutic treatment in Nt5c2+/co-R367Q (wild type) and Nt5c2+/R367Q (mutant) NOTCH1- induced ALL mouse model. (B) Luciferase in vivo bioimaging indicative of tumor burden and quantitative analysis of tumor response (fold change in bioluminescence relative to the basal signal before treatment) in Nt5c2 wild-type ALL tumors treated with vehicle, single-drug or 6-MP and CRCD2 combination. (C) Femoral bone marrow leukemia burden (GFP+ cells) analyzed by flow cytometry following treatment of wild type leukemias as in (A). (D) Images of spleens and quantitation of spleen weight following treatment of wild type leukemias as in (A). (E) Spleen leukemia burden (GFP+ cells) analyzed by flow cytometry following treatment of wild type leukemias as in (A). (F) Luciferase in vivo bioimaging indicative of tumor burden and quantitative analysis of tumor response (fold change in bioluminescence relative to the basal signal before treatment) in Nt5c2 R367Q ALL tumors treated with vehicle, single-drug or 6-MP and CRCD2 combination as in (A). (G) Femoral bone marrow leukemia burden (GFP+ cells) analyzed by flow cytometry following treatment of Nt5c2 R367Q ALL bearing mice as in (A). (H) Images of spleens and quantitation of spleen weight following treatment of in Nt5c2 R367Q ALL tumors as in (A). (I) Spleen leukemia burden (GFP+ cells) analyzed by flow cytometry following treatment of wild type leukemias as in (A). N = 5 independent mice per treatment condition. Data are presented as mean values ± SD. P values were calculated applying two-sided Student’s t-test.
Figure 6.
Figure 6.. Molecular and structural characterization of NT5C2 phosphorylation at Ser502.
(A) Schematic illustration of Ser502 phosphorylation at the C-terminal domain of NT5C2. (B) Immunoblot analysis of Jurkat cells infected with empty vector or lentiviruses driving the expression of Flag-tagged Ser502 mutants of NT5C2. Expression levels were verified in three independent experiments. (C) Viability assay of Jurkat cells infected with mutant Ser502 NT5C2 expressing lentiviruses treated with increasing doses of 6-MP. (D) In vitro nucleotidase assays assessing the enzymatic activity of wild-type and S502D NT5C2 using increasing concentrations of ATP represented as specific activity. (E) Close-up view of the crystal structure of full-length wild-type NT5C2 (PDB id: 6DDO) showing residue S502 (green) from protomer A forms a hydrogen bond (red dash line) with D229 from protomer B (cyan). (F) Close-up view of the crystal structure of the full-length NT5C2 mutant S502D showing that mutation of S502 to D results in destabilization of C-terminal region (494–561). Protomers A and B are shown in magenta and yellow, respectively. (G) Immunoblot analysis of Jurkat cells infected with empty vector or lentiviruses driving the expression of Flag-tagged Asp-229 and Ser502 mutants of NT5C2. Expression levels were verified in three independent experiments. (H) Viability assay of Jurkat cells infected with mutant D229A NT5C2 expressing lentiviruses treated with increasing doses of 6-MP. (I) Viability assay of Jurkat cells infected with mutant S502D NT5C2 expressing lentiviruses treated with increasing doses of 6-MP. (J) Viability assay of Jurkat cells infected with double Asp-229 and Ser502 mutants expressing lentiviruses treated with increasing doses of 6-MP. All graphs show mean ± SD of three independent experiments performed in triplicate. P values were calculated using IC50 values and two-tailed Student’s t-test over wild-type.
Figure 7.
Figure 7.. Prevalence and treatment of NT5C2 Ser502 phosphorylation in relapse ALL patient-derived xenografts.
(A) Western-blot detection of NT5C2 pSer502 in CUTLL1 T-ALL cells. AP: alkaline phosphatase. (B) Western-blot detection of NT5C2 Ser502 phosphorylation after anti-Flag immunoprecipitation in Jurkat cells infected with empty vector or lentiviruses driving the expression of Flag-tagged wild-type, S502A and S502D mutants of NT5C2. Two additional experiments showed similar results. (C) NT5C2 Ser502 phosphorylation analysis in diagnostic and relapsed ALL patient derived xenografts. Numbers show normalized fold change of pSer502-NT5C2 and total levels of NT5C2 in relapse compared to the matched diagnosis xenograft. (D) Heatmap representation of normalized fold change of pSer502-NT5C2 and total levels of NT5C2 as in (C). (E) Western-blot detection of NT5C2 pSer502 levels in CUTLL1 cells treated with 10 μM CRCD2 or 1.5 μM 6-MP for 0, 6, 24 or 48 hours. A representative immuno-blot is shown. (F) Immunoblot analysis of Jurkat cells infected with empty vector or lentiviruses driving the expression of Flag-tagged wild-type, R367Q and S502D mutants of NT5C2. Expression levels were verified in three independent experiments. (G) Viability assay of Jurkat cells infected with wild-type or R367Q or S502D mutant NT5C2 expressing lentiviruses treated with vehicle or CRCD2 and increasing doses of 6-MP. Graphs show mean ± SD of three independent experiments performed in triplicate. P values were calculated using IC50 values and two-tailed Student’s t-test over wild-type.
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