Fig 1: Clonal heterogeneity in SAMHD1 levels drives intrinsic resistance to CNDAC but not population doubling time in MV4-11 cells. (A) Schematic illustration of the establishment of MV4-11 single cell-derived clones by limiting dilution. (B) CNDAC concentrations that reduce viability of 12 single-cell-derived MV4-11 clones by 50% (IC50). Values represent means ± SD of three independent experiments. (C) Representative Western blots of SAMHD1, phosphorylated SAMHD1 (pSAMHD1), and DCK in single cellderived MV4-11 clones. GAPDH served as a loading control. (D) Analysis of SAMHD1 promoter methylation in MV4-11 clones through amplification of a single PCR product (993-bp) corresponding to the promoter sequence after HpaII digestion. (E) Correlation of the CNDAC IC50 values with cellular SAMHD1 or DCK protein levels, quantified using nearinfrared Western blot images to determine the ratio SAMHD1/ GAPDH or DCK/ GAPDH. Closed circles and error bars represent means ± SD of three independent experiments, each performed in three technical replicates. Linear regression analyses were performed using GraphPad Prism. (F) Western Blots and IC50 values for CNDAC and daunorubicin in MV4-11 clones 9, 11, and 12 after transfection with SAMHD1-siRNAs (siSAMHD1) or non-targeting control siRNAs (siCTRL). Each symbol represents the mean ± SD of three technical replicates of one representative experiment out of three. P-values were determined by two-tailed Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001). (G) Population doubling time (PDT) in MV4-11 single cell-derived clones and correlation of the PDT with cellular SAMHD1 or DCK protein levels. Closed circles and error bars represent means ± SD from the quantification of three Western Blots. Linear regression analyses were performed using GraphPad Prism
Fig 2: SAMHD1 and DCK regulate CNDAC cross-resistance of AML cell lines adapted to drugs from different classes. (A) Representative Western blots of SAMHD1, phosphorylated SAMHD1 (pSAMH1), DGK, and DCK in HL-60 sublines adapted to cytarabine (Ara-C), arabinosylguanine (Ara-G), 5-azacytidine (AZA), fludarabine (FLU), 6-mercaptopurine (6-MP), venetoclax (VENE), olaparib (OLA), and volasertib (VOLA). GAPDH served as loading control. (B) CNDAC concentrations that reduce cell viability by 50% (IC50s) in drug-adapted HL-60 sublines. Horizontal lines and error bars represent means ± SD of three independent experiments, each performed in three technical replicates. p-values were determined by two-tailed Student’s t-tests (*p < 0.05; **p < 0.01; ***p < 0.001). (C) Correlation of CNDAC IC50 values with cellular SAMHD1 or DCK protein levels, quantified using the near-infrared Western blot image shown in (A) to determine the ratio SAMHD1/GAPDH or DCK/GAPDH. (D) CNDAC dose-response curves in drug-adapted HL-60 sublines in the absence or presence of VPX virus-like particles (VPX-VLPs, cause SAMHD1 depletion) or VPR virus-like particles (VPR-VLPs, negative control). Each symbol represents the mean ± SD of three technical replicates of one representative experiment out of three. Concentrations that reduce AML cell viability by 50% (IC50s) ± SD and Western Blots showing SAMHD1 degradation by VPXVLPs are provided. (E) CNDAC or cytarabine (Ara-C) dose-response curve in cytarabineadapted MV4-11 or MOLM-13 cells (characterised by loss of DCK expression) stably transduced with either DCK (pWPI+DCK) or an empty vector (pWPI) in the absence or presence of VPX virus-like particles (VPX-VLPs), or VPR virus-like particles (VPR-VLPs). Each symbol represents the mean ± SD of three technical replicates of one representative experiment out of three. IC50s (mean ± SD) and Western Blots showing successful transduction with DCK and SAMHD1 degradation by VPX-VLPs are provided
Fig 3: SAMHD1 (but not DCK) levels determine sensitivity to CNDAC and inversely correlate with CNDAC-triphosphate (CNDAC-TP) in leukaemia cell lines. (A) Representative Western blots of SAMHD1, phosphorylated SAMHD1 (pSAMHD1), and DCK in 13 AML cell lines. GAPDH served as loading control. Uncropped Western blots are presented in Supplementary Figure 1. (B) CNDAC concentrations that reduce the viability of AML cell lines by 50% (IC50). Horizontal lines and error bars represent means ± SD of three independent experiments. (C) CNDAC triphosphate (CNDAC-TP) levels determined by LC–MS/MS. Horizontal lines and error bars show means ± SD of three independent experiments. (D, E) Correlation of the CNDAC IC50 values with cellular DCK (D) or SAMHD1 (E) protein levels, quantified using near-infrared Western blot images to determine the ratio DCK/ GAPDH or SAMHD1/GAPDH. Closed circles and error bars represent means ± SD of three independent experiments. Linear regression analyses were performed using GraphPad Prism. (F, G) Correlation of CNDAC-TP levels with cellular DCK (F) or SAMHD1 (G) protein levels in AML cell lines, quantified using near-infrared Western blot images to determine the ratio DCK/GAPDH or SAMHD1/GAPDH. Closed circles and error bars represent means ± SD of three independent experiments. Linear regression analyses were performed using GraphPad Prism. (H) Analysis of SAMHD1 promoter methylation in AML cell lines through amplification of a single PCR product (993-bp) corresponding to the promoter sequence after HpaII digestion. A 0.25-kb fragment of the GAPDH gene lacking HpaII sites was PCR-amplified using the same template DNA served as loading control. THP-1 served as control cell for an unmethylated SAMHD1 promotor, while JURKAT served as control cell for a methylated promotor. (I) Correlation of CNDAC IC50 values in 26 ALL cell lines (11 T-ALL, 15 B-ALL) with SAMHD1 protein levels, quantified using near-infrared Western blot images to determine the ratio SAMHD1/ GAPDH relative to the positive control THP-1. Closed circles and error bars represent means ± SD of three independent experiments. Linear regression analyses were performed using GraphPad Prism. (J-L) Comparison of SAMHD1 protein levels (J), CNDAC IC50 values (K) and CNDAC-TP levels determined by LC-MS/MS (L) in T-ALL and B-ALL cells. Each point represents the mean of three independent experiments. One-tailed Student’s t-tests were used to compare means in T-ALL and B-ALL cells (represented as horizontal lines ± SEM)
Fig 4: Acquired resistance to CNDAC is associated with decreased DCK levels and accompanied by cross-resistance to DCK-dependent nucleoside analogues. (A) Schematic illustrations of the establishment of CNDAC-resistant HL-60 and PL-21 cells by step-wise increasing drug concentrations during cell culture and of the establishment of single cellderived clones by limiting dilution. Moreover, representative Western blots indicating SAMHD1 and DCK levels in CNDAC-adapted HL-60 (HL-60rCNDACI-XII) and PL-21 (PL-21rCNDACI-XII) sublines and in single cell-derived clonal sublines of these cell lines. GAPDH and β-Actin served as loading controls. (B) Resistance profiles of CNDAC-adapted HL-60 and PL-21 sublines and single cell-derived clones of HL-60 and PL-21. Left spider webs show sensitivity to the cytotoxic drugs CNDAC, 6-Thioguanine (6-TG), Clofarabine (CLOF), Cladribine (CLAD), Fludarabine (FLU), Gemcitabine (GEM), Decitabine (DAC), 5-Azacytidine (AZA), Daunorubicin (DAU), Cytarabine (ARA-C), and Sapacitabine (SAP), while right spider webs display sensitivity to the targeted drugs Vismodegib (VISMO), Olaparib (OLA), Ganetespib (GANE), Gedatolisib (GEDA), Volasertib (VOLA), Molibresib (MOLI), and Venetoclax (VENE). Values are depicted as fold changes in drug concentrations that reduce cell viability by 50% (IC50s) between the respective parental AML cell line (shown in red) and the resistant cell lines or clones. Points closer to the centre than red lines indicate higher sensitivity to drugs in CNDAC-resistant sublines or clonal sublines than in parental cell lines, while points lying outside red lines indicate reduced sensitivity to the respective drug. IC50 fold changes are shown as means from three independent experiments. Numerical values are provided in Supplementary Table 6
Supplier Page from DNASU for DCK (Homo sapiens) in pDNR-Dual (Creator donor/master vector)