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

Abstract

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 adults. 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), which causes DNA damage through perturbation of DNA synthesis. 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 treatment. 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.

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

“…Indirect evidence that SAMHD1 is functional even in cycling cells with high dNTP pools comes also from two recent reports that tested the sensitivity to some nucleoside analogs in cancer cells and in cells permissive to HIV-1 infection [34,35]. …”

SAMHD1 is a barrier to antimetabolite-based cancer therapies

“…Furthermore, VLP-delivery of Vpx to primary AML patient blasts, sampled either at diagnosis or relapse, successfully depleted SAMHD1 protein and increased ara-C toxicity. Finally, retrospective analyses of SAMHD1 expression and survival in two well-characterized cohorts of adult and pediatric AML patients revealed a ∼20% absolute difference in OS at 18 and 12 months after diagnosis, respectively 3 . Surprisingly, the mRNA expression levels of genes involved in ara-C metabolism that had been previously reported as potential biomarkers were not significantly correlated with survival in these patient cohorts 3 .…”

“…Finally, retrospective analyses of SAMHD1 expression and survival in two well-characterized cohorts of adult and pediatric AML patients revealed a ∼20% absolute difference in OS at 18 and 12 months after diagnosis, respectively 3 . Surprisingly, the mRNA expression levels of genes involved in ara-C metabolism that had been previously reported as potential biomarkers were not significantly correlated with survival in these patient cohorts 3 . Taken together, our findings demonstrate that the therapeutic response of AML to ara-C is controlled by SAMHD1 (Fig.…”

“… 7 provided proof-of-concept that artificial dNTP analogs like the triphosphate of the anti-cancer drug clofarabine, Cl-F-ara-ATP, can be substrates for SAMHD1. This prompted us to hypothesize that other nucleotide analogs could be SAMHD1 substrates, such as ara-CTP 3 (Fig. 1), which differs only in the hydroxyl group configuration at the 2′-C atom from dCTP.…”

“…We subsequently demonstrated in AML and lymphoma cell lines that the removal of SAMHD1 could dramatically increase ara-C cytotoxicity. Firstly, we exploited virus-like particle (VLP)-delivery of the (lenti)viral protein X (Vpx), that is known to recruit SAMHD1 for degradation, 3 and observed a dose-dependent sensitization of AML cells to ara-C up to ∼130-fold, with respect to cells treated with control VLPs lacking Vpx. Secondly, we used clustered regularly interspaced short palindromic repeats (CRISPR) /Cas9 (CRISPR associated protein 9)-mediated disruption of the SAMHD1 gene, which resulted in a substantial increase in ara-C toxicity that could be abrogated by ectopic expression of wild-type, but not catalytically inactive SAMHD1.…”