Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies

Journal name:
Nature Medicine
Volume:
23,
Pages:
256–263
Year published:
DOI:
doi:10.1038/nm.4265
Received
Accepted
Published online

The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults1. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP)2, 3, 4, 5, which causes DNA damage through perturbation of DNA synthesis6. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment7, 8, 9. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR–Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.

At a glance

Figures

  1. SAMHD1 is an ara-CTPase that protects cells from lethal misincorporation of ara-CTP into DNA.
    Figure 1: SAMHD1 is an ara-CTPase that protects cells from lethal misincorporation of ara-CTP into DNA.

    (a) Substrate-velocity curves measuring released inorganic triphosphate (PPPi) produced from hydrolysis of dCTP or ara-CTP by SAMHD1 in the presence of GTP or a nonhydrolyzable dGTP analog (dGTPαS) in the enzyme-coupled malachite green assay. Error bars indicate s.e.m. of three independent experiments performed at least in triplicate. Kinetic parameters are provided in Supplementary Table 1. (b) Pearson correlation of SAMHD1 mRNA expression and ara-C sensitivity in a panel of 138 hematopoietic and lymphoid-tissue-derived cell lines. AUC, area under the curve. (c,d) Cell viability of THP-1 (c) and HuT-78 cells (d), which were treated with SIV-Vpx-containing (X) or control (dX) VLPs, or PBS (no VLPs) in the presence of ara-C at the indicated concentrations. EC50 values: HuT-78 dX, 161 nM; HuT-78 X, 28 nM, HuT-78 no VLPs, 225 nM. THP-1 dX, 3,537 nM, THP-1 X, 69 nM, THP-1 no VLPs, 3,159 nM. Error bars indicate s.d. of a representative experiment out of three independent experiments performed in triplicate. Statistical testing was performed by comparing the logEC50 values by means of an extra-sum-of-squares F test. F = 92.02; DFn = 2; DFd = 85 (c). F = 32.01; DFn = 2; DFd = 85 (d). ****P ≤ 0.0001. (e) Cell viability of THP-1 clones with genetic disruption of SAMHD1 using CRISPR–Cas9 (g3-2, g2-2) and control clones (ctrl gRNA, g2-3) in the presence of ara-C at the indicated concentrations. EC50 values: g3-2, 92 nM; g2-2, 82 nM; ctrl gRNA, 1,305 nM, g2-3, 2,170 nM. Error bars indicate s.d. of a representative experiment out of three independent experiments performed in triplicates. Extra-sum-of-squares F test: F = 22.43; DFn = 3; DFd = 128. ****P ≤ 0.0001. (f) Cell viability of HuT-78 bulk cell populations in which endogenous SAMHD1 expression was disrupted by using CRISPR–Cas9 (−/−) and the cells were transduced for stable expression of the indicated SAMHD1 variants in the presence of ara-C at the indicated concentrations. EC50 values: (−), 120 nM; HA-D137N, 220 nM; HA-H233A, 164 nM; HA-wt, 17,215 nM. Error bars indicate s.d. of a representative experiment out of three independent experiments performed in triplicate. Extra-sum-of-squares F test: F = 76.46; DFn = 3; DFd = 128. ****P ≤ 0.0001. (g) Soluble 3H-ara-CTP levels in THP-1 cell clones either expressing SAMHD1(+, g2-3) or lacking SAMHD1(−, g2-2) treated with the indicated concentrations of 3H-ara-C. Values were normalized to the lowest ara-C dose treatment in SAMHD1 (+, g2-3) cells (dashed line). Individual data points from three independent experiments, each performed in duplicate, are plotted; the horizontal line and error bars indicate the mean and s.d. Statistical significance between SAMHD1+ and SAMHD1 cells was determined by using multiple two-tailed t-tests: ***P = 0.0006, t = 4.98, df = 10; ****P < 0.0001, t = 10.16, df = 10. (h) 3H-ara-CTP levels in DNA prepared from the cells in g. Values were normalized to the lowest ara-C dose treatment in SAMHD1+ cells (dashed line). Individual data points from two independent experiments, each performed in triplicate, are plotted; the horizontal line and error bars indicate the mean and s.d. Multiple two-tailed t-tests: ***P = 0.002, t = 5.69, df = 10; ****P < 0.0001, t = 6.49, df = 10. (i) 3H-thymidine levels in DNA prepared from SAMHD1+ and SAMHD1 cells treated with the indicated dose of unlabeled ara-C and 149 μM 3H-thymidine. Values were normalized to that observed for the lowest ara-C dose treatment in SAMHD1+ cells (dashed line). Individual data points from two independent experiments, each performed in triplicate, are plotted; the horizontal line and error bars indicate the mean and s.d. Multiple two-tailed t-tests: ***P = 0.003, t = 5.39, df = 10; 0.25 μM, **P = 0.006, t = 3.46, df = 10; 0.5 μM, **P = 0.002, t = 4.09, df = 10. (j) Western blot analysis of the indicated proteins in SAMHD1+ and SAMHD1 cells treated with increasing doses of ara-C (0.1, 0.5 and 1 μM) for 24 h. A representative blot is shown (for full blots, see Supplementary Fig. 20a; for quantification of repeat experiments, see Supplementary Fig. 13). (k,l) Immunofluorescence-microscopy analysis of subnuclear γH2Ax in isogenic HuT-78 SAMHD1−/− cells stably expressing the indicated SAMHD1 variant, treated with the indicated doses of ara-C. Mean fluorescence intensity per nucleus is plotted from two independent experiments, each performed in duplicate; the population median is indicated by horizontal bars. Wilcoxon rank–sum test, two-tailed: n.s., not significant, P = 0.85; ****P < 0.0001. Representative images are shown; DNA counterstained by DAPI. Scale bar, 50 μm.

  2. Disruption of SAMHD1 expression sensitizes AML xenotransplants in mice and primary patient-derived AML blasts ex vivo to ara-C.
    Figure 2: Disruption of SAMHD1 expression sensitizes AML xenotransplants in mice and primary patient-derived AML blasts ex vivo to ara-C.

    (ac) NMRI nu/nu mice were subcutaneously xenotransplanted with either THP-1 SAMHD1 g2-2 cells (n = 8) or THP-1 SAMHD1+ g2-3 cells (n = 9). (a) Mouse body weight. Statistical significance was assessed using a two-tailed t-test of means. t = 1.62, df = 72; n.s., not significant (P = 0.11). (b) Tumor volumes during and after ara-C treatment. Thin lines represent calculated tumor volumes for individual mice; thick dotted lines represent a smoothened average for each group. Unpaired two-tailed t-test of means. t = 4.748, df = 54. ****P < 0.0001. (c) Kaplan–Meier survival analysis, based on a tumor-length endpoint of 20 mm. Statistical significance was assessed by using a Mantel–Cox log–rank test. χ2 = 17.47. df = 1. ****P < 0.0001. (d) Macroscopic images (top row), H&E staining (middle row) and anti-SAMHD1 immunohistochemistry (bottom row) of excised SAMHD1+ (right column) or SAMHD1 (left column) tumors. Scale bars, 25 μm. (e,f) NOD/SCID mice were injected i.v. with HL-60/iva SAMHD1 g2-2 cells (n = 12) or HL-60/iva SAMHD1+ g2-3 cells (n = 12) at day –6. Treatment with PBS or ara-C was started on day 0. (e) Cumulative incidence of clinical signs of systemic disease (see Online Methods, Supplementary Fig. 14 and Supplementary Table 3 for details). Mantel–Cox log–rank test. PBS treated: χ2 = 0.01852. df = 1. n.s., not significant (P = 0.8918). ara-C treated: χ2 = 8.268. df = 1; **P = 0.0040. (f) Kaplan–Meier survival analysis. Mantel–Cox log–rank test. PBS treated: χ2 = 2.502. df = 1. n.s, not significant (P = 0.1137). ara-C treated: χ2 = 7.037. df = 1; **P = 0.0080. (g) Cell viability of patient-derived AML blasts (patient P0; for patient characteristics see Supplementary Table 4) treated with VLPs containing SIV-Vpx (X), or control VLPs (dX) in the presence of ara-C at the indicated concentrations. EC50 values: dX, 460 nM; X, 52 nM. Extra-sum-of-squares F test: F = 161; DFn = 1; DFd = 68; P < 0.0001. (h) Representative (n = 14) western blot analysis of patient AML blasts stained with the indicated antibodies (for full blot, see Supplementary Fig. 20b).

  3. Low SAMHD1 expression levels are associated with a better response to ara-C therapy in patients with AML.
    Figure 3: Low SAMHD1 expression levels are associated with a better response to ara-C therapy in patients with AML.

    (a,d) Histogram of SAMHD1 mRNA expression levels in AML blasts at diagnosis from 147 TCGA (a) and 145 TARGET (d) patients with AML subsequently treated with ara-C-containing regimens. (b) Kaplan–Meier OS analysis of the 147 TCGA patients with AML after dichotomization for SAMHD1 mRNA levels below (red, n = 81) and above (black, n = 66) 105 RPKM. Log–rank test; χ2 = 1.0900; df = 1; P = 0.30. (c) Kaplan–Meier OS analysis in the 147 TCGA patients with AML for the first 18 months after diagnosis. Log–rank test; χ2 = 7.8854; df = 1; P = 0.005. (e) Kaplan–Meier OS analysis in the 145 TARGET patients with AML after dichotomization for SAMHD1 mRNA levels below (red, n = 109) and above (black, n = 36) 1,288,288 RPKM-UQ. Log–rank test; χ2 = 0.9571; df = 1; P = 0.33. (f) Kaplan–Meier OS analysis in the 145 TARGET patients with AML for the first 12 months after diagnosis. Log–rank test; χ2 = 10.3108; df = 1; P = 0.001. (g) Model for the role of SAMHD1 in ara-C metabolism. SAMHD1 limits ara-C cytotoxicity by hydrolyzing the active metabolite ara-CTP, thus preventing its lethal misincorporation into genomic DNA. Ara-C is a substrate of deoxycytidine kinase (DCK), the rate-limiting enzyme in the multistep process by which ara-C is phosphorylated and activated. Deoxycytidylate deaminase (DCTD) converts ara-CMP to ara-UMP, whereas cytosolic 5′-nucleotidase-II (NT5C2) can dephosphorylate Ara-CMP. Ara-C can also be converted to ara-U by cytidine deaminase (CDA).

Videos

  1. Orthopic AML mouse model with SAMHD1 wildtype or knockout HL-60/iva cells 30 days post ara-C treatment.
    Video 1: Orthopic AML mouse model with SAMHD1 wildtype or knockout HL-60/iva cells 30 days post ara-C treatment.

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Author information

  1. Present address: Andalusian Molecular Biology and Regenerative Medicine Centre CABIMER, University of Seville-CSIC, Seville, Spain (J.M.C.-M.) and AstraZeneca R&D, Mölndal, Sweden (T.L.).

    • José M Calderón-Montaño &
    • Thomas Lundbäck
  2. These authors contributed equally to this work.

    • Nikolas Herold &
    • Sean G Rudd

Affiliations

  1. Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, and Karolinska University Hospital, Stockholm, Sweden.

    • Nikolas Herold,
    • Linda Ljungblad,
    • Ida Hed Myrberg,
    • Bianca Tesi,
    • Julia Bladh,
    • Mats Heyman,
    • Per Kogner &
    • Jan-Inge Henter
  2. Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

    • Sean G Rudd,
    • Kumar Sanjiv,
    • Cynthia B J Paulin,
    • Anna Hagenkort,
    • Brent D G Page,
    • José M Calderón-Montaño,
    • Olga Loseva,
    • Ann-Sofie Jemth,
    • Hanna Axelsson,
    • Nicholas C K Valerie,
    • Andreas Höglund,
    • Elisée Wiita,
    • Ulrika Warpman-Berglund,
    • Thomas Lundbäck &
    • Thomas Helleday
  3. Department of Medicine, Center of Hematology and Regenerative Medicine, Karolinska Hospital and Karolinska Institutet, Stockholm, Sweden.

    • Yaser Heshmati,
    • Julian Walfridsson &
    • Sören Lehmann
  4. Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.

    • Juliane Kutzner,
    • Lorenzo Bulli &
    • Torsten Schaller
  5. Chemical Biology Consortium, Stockholm, Sweden.

    • Hanna Axelsson &
    • Thomas Lundbäck
  6. Division of Pediatrics, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden.

    • Mikael Sundin
  7. Paediatric Blood Disorders, Immunodeficiency and Stem Cell Transplantation, Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm, Sweden.

    • Mikael Sundin
  8. Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.

    • Michael Uhlin,
    • Georgios Rassidakis,
    • Katja Pokrovskaja Tamm &
    • Dan Grandér
  9. Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden.

    • Michael Uhlin
  10. Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

    • Sören Lehmann

Contributions

N.H. and T.S. conceived the study. N.H., S.G.R., J.-I.H., T.H. and T.S. wrote the manuscript, which was revised by all authors. N.H., J.K., L.B. and T.S. created CRISPR–Cas9 knockout cell lines and Vpx-VLPs. N.H., S.G.R., C.B.J.P, E.W., J.K., J.B. and T.S. designed and performed the proliferation inhibition assays for THP-1, HuT-78 and HL-60 cells. Experiments with primary AML blasts and hematopoietic stem cells were planned by N.H., J.W., S.L., M.U., M.S., S.G.R., N.C.K.V., G.R., K.P.T., M.H. and D.G., and proliferation inhibition and apoptosis assays for blasts treated with Vpx-VLPs were performed by N.H., Y.H. and H.A. N.H., L.L., P.K. and T.S. designed the animal experiments. A.H. and U.W.-B. established the orthotopic AML animal model. N.H., L.L. and K.S. performed the animal experiments. N.H., I.H.M., B.T. and J.I.H. analysed TCGA and TARGET data. A.S.J. produced the expression construct, and O.L. purified recombinant SAMHD1. A.-S.J., T.L., S.G.R. and C.B.J.P. established the in vitro SAMHD1 activity assay, and subsequent experiments were performed by S.G.R. and C.B.J.P. N.H., S.G.R., B.D.G.P. and A.H. conceived the ara-CTP pool and ara-C DNA incorporation assays, and the respective experiments were performed by A.H. and B.D.G.P. S.G.R. and J.M.C.-M. performed DNA damage-response experiments. S.G.R. performed DNA content analysis. N.H., J.K., L.B. and T.S. performed kinetic analysis on Vpx-mediated SAMHD1 degradation.

Competing financial interests

T.L. is presently employed with AstraZeneca.

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