Figure 1.
POLθ is required to resolve DPCs caused by formaldehyde generated via serine/1C cycle metabolism in OTKs-positive hematological malignancies. (A) Mean ± SD of DPCs in the indicated cell lines; ∗P < .05 using t test. (B) Mean ± SD of DPCs in: (left) 32Dcl3 parental cells (P) and cells expressing FLT3(ITD), JAK2(V617F), and BCR-ABL1, and (right) human Lin-CD34+ primary cells from leukemias expressing FLT3(ITD), JAK2(V617F), and BCR-ABL1 and from healthy donors. ∗P < .05 when compared with P/N using t test. (C) Mean formaldehyde (FA) levels ± SD in 32Dcl3 parental cells (P) and cells expressing FLT3(ITD), JAK2(V617F), and BCR-ABL1. ∗P < .05 when compared with P using t test. (D) Mean ± SD of FA levels in FLT3(ITD) 32Dcl3 cells cultured in standard medium (Reg) and in medium without glucose, serine, and glycine (no GSG); ∗P < .05 using t test when compared with corresponding Reg. (E) Mean ± SD of FA levels, DPCs, and % of γ-H2AX–positive cells in FLT3(ITD) 32Dcl3 cells cultured in glucose + 5× serine + 5× glycine high (high GSG) medium and in medium without glucose, serine, and glycine (no GSG). ∗P < .05 using t test. (F) Mean ± SD of FA levels and DPCs in FLT3(ITD) 32Dcl3 cells cultured in glucose-supplemented serine + glycine–free medium and treated or not with WQ-2101; ∗P < .05 using t test. (G) Mean ± SD of FA levels and DPCs from 3 experiments in FLT3(ITD) 32Dcl3 cells cultured in standard medium and treated or not with SHIN1. ∗P < .05 using t test. (H) Lin-cKit+ mBMCs from Polq+/+ and Polq−/− mice (blue and orange, respectively) were cultured in high GSG and/or no GSG medium. Results represent mean ± SD of FA levels and DPCs and proliferation rate; ∗P < .05 when compared with corresponding high GSG using t test. (I) Clonogenic activity of Lin-cKit+ BMCs from Polq+/+ and Polq−/− mice treated with the indicated concentrations of formaldehyde for 4 hours after plating in methylcellulose. Results represent the mean percentage of colonies ± SD when compared with untreated counterparts. (J) Lin-cKit+ mBMCs from Polq+/+ and Polq−/− mice were untreated (0) and treated with 400 nM formaldehyde for 24 and 48 hours. Results represent mean ± SD of DPCs and tail DNA percentage from the neutral comet assay. ∗P ≤ .001 when compared with Polq+/+ counterpart using t test. (K) Lin-cKit+ mBMCs from Flt3ITD;Polq−/− (orange) and Flt3ITD;Polq+/+ (blue) mice were cultured in high GSG and/or no GSG medium. Results represent mean ± SD of FA levels and DPCs and proliferation rate; ∗P < .05 when compared with corresponding high GSG using t test. (L) Sensitivity of FLT3(ITD) 32Dcl3 cells to 25 μM ART558 in high GSG and no GSG medium. Results represent the mean percentage of colonies ± SD when compared with untreated counterparts; ∗P < .05 using t test. (M) A scheme illustrating functional pathway where serine/1C cycle–produced formaldehyde induces DPCs, which are likely repaired by POLθ-mediated TMEJ. SD, standard deviation.

POLθ is required to resolve DPCs caused by formaldehyde generated via serine/1C cycle metabolism in OTKs-positive hematological malignancies. (A) Mean ± SD of DPCs in the indicated cell lines; ∗P < .05 using t test. (B) Mean ± SD of DPCs in: (left) 32Dcl3 parental cells (P) and cells expressing FLT3(ITD), JAK2(V617F), and BCR-ABL1, and (right) human Lin-CD34+ primary cells from leukemias expressing FLT3(ITD), JAK2(V617F), and BCR-ABL1 and from healthy donors. ∗P < .05 when compared with P/N using t test. (C) Mean formaldehyde (FA) levels ± SD in 32Dcl3 parental cells (P) and cells expressing FLT3(ITD), JAK2(V617F), and BCR-ABL1. ∗P < .05 when compared with P using t test. (D) Mean ± SD of FA levels in FLT3(ITD) 32Dcl3 cells cultured in standard medium (Reg) and in medium without glucose, serine, and glycine (no GSG); ∗P < .05 using t test when compared with corresponding Reg. (E) Mean ± SD of FA levels, DPCs, and % of γ-H2AX–positive cells in FLT3(ITD) 32Dcl3 cells cultured in glucose + 5× serine + 5× glycine high (high GSG) medium and in medium without glucose, serine, and glycine (no GSG). ∗P < .05 using t test. (F) Mean ± SD of FA levels and DPCs in FLT3(ITD) 32Dcl3 cells cultured in glucose-supplemented serine + glycine–free medium and treated or not with WQ-2101; ∗P < .05 using t test. (G) Mean ± SD of FA levels and DPCs from 3 experiments in FLT3(ITD) 32Dcl3 cells cultured in standard medium and treated or not with SHIN1. ∗P < .05 using t test. (H) Lin-cKit+ mBMCs from Polq+/+ and Polq−/− mice (blue and orange, respectively) were cultured in high GSG and/or no GSG medium. Results represent mean ± SD of FA levels and DPCs and proliferation rate; ∗P < .05 when compared with corresponding high GSG using t test. (I) Clonogenic activity of Lin-cKit+ BMCs from Polq+/+ and Polq−/− mice treated with the indicated concentrations of formaldehyde for 4 hours after plating in methylcellulose. Results represent the mean percentage of colonies ± SD when compared with untreated counterparts. (J) Lin-cKit+ mBMCs from Polq+/+ and Polq−/− mice were untreated (0) and treated with 400 nM formaldehyde for 24 and 48 hours. Results represent mean ± SD of DPCs and tail DNA percentage from the neutral comet assay. ∗P ≤ .001 when compared with Polq+/+ counterpart using t test. (K) Lin-cKit+ mBMCs from Flt3ITD;Polq−/− (orange) and Flt3ITD;Polq+/+ (blue) mice were cultured in high GSG and/or no GSG medium. Results represent mean ± SD of FA levels and DPCs and proliferation rate; ∗P < .05 when compared with corresponding high GSG using t test. (L) Sensitivity of FLT3(ITD) 32Dcl3 cells to 25 μM ART558 in high GSG and no GSG medium. Results represent the mean percentage of colonies ± SD when compared with untreated counterparts; ∗P < .05 using t test. (M) A scheme illustrating functional pathway where serine/1C cycle–produced formaldehyde induces DPCs, which are likely repaired by POLθ-mediated TMEJ. SD, standard deviation.

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