decreasing the levels of intracellular dNTPs 14,15 , which apparently compete with the 47 thymidine analog triphosphates for incorporation into HIV-1 cDNA during reverse 48 transcription 16 . We postulated that SAMHD1 could have a similar effect on nucleoside 49analog-based therapy in leukemia 6 . 50To investigate whether SAMHD1 expression enhances Ara-C cytotoxicity in AML 51 cells, we tested whether Ara-C sensitivity in 13 AML cell lines, determined by the half 52 maximal inhibitory concentration (IC 50 ), is correlated with SAMHD1 protein and mRNA 53 levels. Both SAMHD1 expression (Fig. 1a and Supplementary Fig. 1) and Ara-C sensitivity 54 (Supplementary Table 1) varied considerably among these cell lines. Unexpectedly, 55 SAMHD1 levels inversely correlated with Ara-C cytotoxicity (p=0.0037, Fig. 1b and 56 Supplementary Fig. 2a,b), as well as with the levels of early (Caspase 3 and 7 activity, 57 p=0.02, Supplementary Fig. 3a,b) and late (sub-G1 cells, apoptotic DNA fragmentation, 58 p=0.029, Supplementary Fig. 3c,d) markers of apoptosis. In contrast, no significant 59 correlation could be established between Ara-C IC 50 values and the expression of cellular 60 4 proteins previously implicated in Ara-C uptake or its conversion to Ara-CTP 1 , including 61 equilibrative nucleoside transporter (ENT1/SLC29A1), deoxycytidine kinase (DCK), cytidine 62 deaminase (CDA), deoxycytidilate deaminase (DCTD), or 5'-nucleotidase (NT5C2) (Fig. 63 1a,c-g). 64To further investigate its role in Ara-C resistance, we tested the effects of SAMHD1 65 deficiency by a number of approaches: (i) depletion of SAMHD1 in AML cell lines 66 expressing high endogenous SAMHD1 levels using either lentiviral vectors encoding 67 SAMHD1-specific shRNA or transfection with SAMHD1-specific siRNA; (ii) CRISPR/Cas9-68 mediated disruption of the SAMHD1 gene; and (iii) targeted degradation of SAMHD1 using 69 virus-like particles (VLPs) which shuttle the SAMHD1-interacting lentiviral Vpx protein 70 (Vpx-VLPs) into cells 7,8,17 (Fig. 2a and Supplementary Fig. 4). Vpx recruits SAMHD1 to a 71 cullin4A-RING E3 ubiquitin ligase (CRL4 DCAF1 ), which targets the enzyme for proteasomal 72 degradation 7,8 . 73SAMHD1 depletion in AML cell lines by RNA interference (OCI-AML3, THP-1), 74 SAMHD1 knockout (THP-1 -/-), or transduction with Vpx-VLPs (MonoMac6 cells, THP-1) 75 markedly sensitized AML cell lines to Ara-C toxicity relative to the respective controls (Fig. 76 2a,b and Supplementary Fig. 4). In contrast, SAMHD1 siRNA had only a marginal effect on 77 Ara-C toxicity in low SAMHD1-expressing HEL cells (Fig. 2a,b). Interestingly, we observed 78 SAMHD1 dependency, although less pronounced, for the purine analog fludarabine 79 ( Supplementary Fig. 5a); however, the IC 50 values for the topoisomerase II inhibitors 80 etoposide and daunorubicin, as well as for dFdC (2',2'-difluorodeoxycytidine; gemcitabine), 81were not consistently affected by SAMHD1 down-modulation ( Supplementary Fig. 5b-d), 82 indicating a certain degree of drug specificity. 83
5In HEL...
