Figure 5.
Inhibiting pyrimidine synthesis triggered compensatory increases in pyrimidine salvage. (A) Teriflunomide treatment increased aspartate substrate that is upstream, and decreased UMP product that is downstream of DHODH and UMPS, in both teriflunomide-naïve (parental) and teriflunomide-resistant AML cells (THP1); products even further downstream that are also salvaged by DCK and UCK2, dCMP and CMP, were partially recovered. Parental THP1 were treated with teriflunomide 20μM for 72 hours. Teriflunomide-resistant THP1 had exponentially expanded through teriflunomide 20μM, emerging within ∼40 days. HR-LCMS analyses of extracts from equal numbers of cells, harvested from parental THP1 treated independently in triplicate, and in independent harvests in quadruplicate from teriflunomide-resistant THP1. Values are normalized to taurine levels in the same cells (taurine internal control). Mean ± SD. ∗P < .05; ∗∗P < .01 (unpaired 2-sided t test). (B) DCK and UCK2, that rate limit salvage of deoxycytidine and cytidine into dCMP and CMP, respectively, were upregulated acutely by teriflunomide treatment, and remained upregulated in teriflunomide-resistant AML cells. Western blot. (C) Messenger RNA levels were also measured by quantitative reverse transcription polymerase chain reaction, using glyceraldehyde-3-phosphate dehydrogenase internal control, and analyzed by Livak-Schmittgen method. Fold change vs vehicle-treated parental THP1 cells. Data represent mean ± SD from 3 independent experiments. ∗∗P < .01; ∗∗∗P < .001 (unpaired 2-sided t test). (D) Teriflunomide-resistant AML cells were also relatively resistant to the HMA. Teriflunomide-naïve (parental) and teriflunomide-resistant THP1 were treated with gradually increasing concentrations of 5-azacytidine (0-100μM) or decitabine (0-50μM) to identify concentrations that produced GI50. Cell counts by automated counter. P value from extra sum-of-squares F test. (E) Inhibiting pyrimidine salvage with dipyridamole (DP) enhanced differentiation induction by teriflunomide. Parental THP1 and teriflunomide-resistant THP1 were treated with the SLC29A1 inhibitor DP 10μM with or without teriflunomide 20μM. The granulomonocyte differentiation marker CD11b was measured by flow cytometry at 96 hours (left). Data represent MFI as mean ± SD from 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 (unpaired 2-sided t test). Giemsa-stained cytospin preparations, imaged with a Leica Upright Microscope-Orion (right). Original magnification, ×400; scale bar, 20 μm. (F) AML cell numbers. Automated counter, methods/statistics per panel D. (G) Assay for apoptosis activation by flow cytometry for Annexin V staining at 24 hours. Camptothecin 0.25μM served as a positive control. Methods/statistics are described in panel D. CMP, cytidine monophosphate; CTPS, cytidine triphosphate synthetase; dCMP, deoxycytidine monophosphate; GI50, 50% growth inhibition; ns, nonsignificant; Res, resistant; Teri, teriflunomide; Veh, vehicle.

Inhibiting pyrimidine synthesis triggered compensatory increases in pyrimidine salvage. (A) Teriflunomide treatment increased aspartate substrate that is upstream, and decreased UMP product that is downstream of DHODH and UMPS, in both teriflunomide-naïve (parental) and teriflunomide-resistant AML cells (THP1); products even further downstream that are also salvaged by DCK and UCK2, dCMP and CMP, were partially recovered. Parental THP1 were treated with teriflunomide 20μM for 72 hours. Teriflunomide-resistant THP1 had exponentially expanded through teriflunomide 20μM, emerging within ∼40 days. HR-LCMS analyses of extracts from equal numbers of cells, harvested from parental THP1 treated independently in triplicate, and in independent harvests in quadruplicate from teriflunomide-resistant THP1. Values are normalized to taurine levels in the same cells (taurine internal control). Mean ± SD. ∗P < .05; ∗∗P < .01 (unpaired 2-sided t test). (B) DCK and UCK2, that rate limit salvage of deoxycytidine and cytidine into dCMP and CMP, respectively, were upregulated acutely by teriflunomide treatment, and remained upregulated in teriflunomide-resistant AML cells. Western blot. (C) Messenger RNA levels were also measured by quantitative reverse transcription polymerase chain reaction, using glyceraldehyde-3-phosphate dehydrogenase internal control, and analyzed by Livak-Schmittgen method. Fold change vs vehicle-treated parental THP1 cells. Data represent mean ± SD from 3 independent experiments. ∗∗P < .01; ∗∗∗P < .001 (unpaired 2-sided t test). (D) Teriflunomide-resistant AML cells were also relatively resistant to the HMA. Teriflunomide-naïve (parental) and teriflunomide-resistant THP1 were treated with gradually increasing concentrations of 5-azacytidine (0-100μM) or decitabine (0-50μM) to identify concentrations that produced GI50. Cell counts by automated counter. P value from extra sum-of-squares F test. (E) Inhibiting pyrimidine salvage with dipyridamole (DP) enhanced differentiation induction by teriflunomide. Parental THP1 and teriflunomide-resistant THP1 were treated with the SLC29A1 inhibitor DP 10μM with or without teriflunomide 20μM. The granulomonocyte differentiation marker CD11b was measured by flow cytometry at 96 hours (left). Data represent MFI as mean ± SD from 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 (unpaired 2-sided t test). Giemsa-stained cytospin preparations, imaged with a Leica Upright Microscope-Orion (right). Original magnification, ×400; scale bar, 20 μm. (F) AML cell numbers. Automated counter, methods/statistics per panel D. (G) Assay for apoptosis activation by flow cytometry for Annexin V staining at 24 hours. Camptothecin 0.25μM served as a positive control. Methods/statistics are described in panel D. CMP, cytidine monophosphate; CTPS, cytidine triphosphate synthetase; dCMP, deoxycytidine monophosphate; GI50, 50% growth inhibition; ns, nonsignificant; Res, resistant; Teri, teriflunomide; Veh, vehicle.

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