Figure 6.
Genetic and biochemical targeting of POLθ exerted antileukemia effect in HR-deficient and HR-proficient leukemias. (A) Representative anti-Flag flow cytometry profiles in Nalm6 (parental) and Nalm6(RAD54−/−) B-ALL cells infected with Flag-POLθ (D2230A+Y2231A) DNA polymerase–inactive mutant (orange), Flag- POLθ wild-type (blue), or GFP only (green). Geo mean ± SD of anti-Flag immunofluorescence is shown; ∗P < .001 when compared with wild-type using t test. (B) Results represent the percentage of colonies ± SD of GFP+ cells expressing POLθ (D2230A+Y2231A) mutant (orange) or POLθ wild-type (blue), when compared with cells expressing GFP only; ∗P < .01 using t test. (C) Results represent number of colonies ± SD from Lin-CD34+GFP+ BCR-ABL1–positive CML-CP and FLT3(ITD)-positive AML cells expressing POLθ (D2230A+Y2231A) mutant (orange) or POLθ wild-type (blue); ∗P < .01 using t test. (D) Representative anti-POLθ flow cytometry analyses showing expression of POLθ in GFP+ cells transduced with POLQ shRNA (orange) or scrambled RNA (blue). Results show Geo mean ± SD of anti-POLθ immunofluorescence; ∗P < .05 using t test. (E) Results represent the percentages of colonies ± SD from GFP+ cells transduced with POLQ shRNA (orange) and scrambled RNA (blue) when compared with cells expressing GFP only; ∗P < .01 using t test. (F) Results represent number of colonies ± SD from Lin-CD34+GFP+ BCR-ABL1–positive CML-CP and FLT3(ITD)-positive AML cells transduced with POLQ shRNA (orange) or scrambled RNA (blue); ∗P < .01 using t test. (G) Results represent the percentages of colonies ± SD from Lin-CD34+ cells isolated from FLT3(ITD)-positive AMLs (n = 3), FLT3(D835Y)-positive AML (n = 1), cKIT(D816V)-positive AML (n = 1), JAK2(V617F) MPN (n = 1), BCR-ABL1–positive CML-CP (n = 3), and normal healthy donors (n = 3) incubated with POLθi (ART558). (H-I) Cells from patients with Lin- AML were treated with ART558 for 6 days after SC-tDNAseq. (H) The fish plot reflects number of cells before (0 days) and 6 days after the treatment and the inferred clonal evolution pattern based on SC-tDNAseq data. (I) The phylogenic tree visualizes the predicted order of mutation acquisition and the proportion of subclones with a different combination of mutations in control and ART558-treated cells: ADO rate = 3.9%, FPR = 1.0%. ADO, allele dropout; FPR, false positive rate; SC-tDNAseq, single-cell targeted DNA sequencing; SD, standard deviation.

Genetic and biochemical targeting of POLθ exerted antileukemia effect in HR-deficient and HR-proficient leukemias. (A) Representative anti-Flag flow cytometry profiles in Nalm6 (parental) and Nalm6(RAD54−/−) B-ALL cells infected with Flag-POLθ (D2230A+Y2231A) DNA polymerase–inactive mutant (orange), Flag- POLθ wild-type (blue), or GFP only (green). Geo mean ± SD of anti-Flag immunofluorescence is shown; ∗P < .001 when compared with wild-type using t test. (B) Results represent the percentage of colonies ± SD of GFP+ cells expressing POLθ (D2230A+Y2231A) mutant (orange) or POLθ wild-type (blue), when compared with cells expressing GFP only; ∗P < .01 using t test. (C) Results represent number of colonies ± SD from Lin-CD34+GFP+ BCR-ABL1–positive CML-CP and FLT3(ITD)-positive AML cells expressing POLθ (D2230A+Y2231A) mutant (orange) or POLθ wild-type (blue); ∗P < .01 using t test. (D) Representative anti-POLθ flow cytometry analyses showing expression of POLθ in GFP+ cells transduced with POLQ shRNA (orange) or scrambled RNA (blue). Results show Geo mean ± SD of anti-POLθ immunofluorescence; ∗P < .05 using t test. (E) Results represent the percentages of colonies ± SD from GFP+ cells transduced with POLQ shRNA (orange) and scrambled RNA (blue) when compared with cells expressing GFP only; ∗P < .01 using t test. (F) Results represent number of colonies ± SD from Lin-CD34+GFP+ BCR-ABL1–positive CML-CP and FLT3(ITD)-positive AML cells transduced with POLQ shRNA (orange) or scrambled RNA (blue); ∗P < .01 using t test. (G) Results represent the percentages of colonies ± SD from Lin-CD34+ cells isolated from FLT3(ITD)-positive AMLs (n = 3), FLT3(D835Y)-positive AML (n = 1), cKIT(D816V)-positive AML (n = 1), JAK2(V617F) MPN (n = 1), BCR-ABL1–positive CML-CP (n = 3), and normal healthy donors (n = 3) incubated with POLθi (ART558). (H-I) Cells from patients with Lin- AML were treated with ART558 for 6 days after SC-tDNAseq. (H) The fish plot reflects number of cells before (0 days) and 6 days after the treatment and the inferred clonal evolution pattern based on SC-tDNAseq data. (I) The phylogenic tree visualizes the predicted order of mutation acquisition and the proportion of subclones with a different combination of mutations in control and ART558-treated cells: ADO rate = 3.9%, FPR = 1.0%. ADO, allele dropout; FPR, false positive rate; SC-tDNAseq, single-cell targeted DNA sequencing; SD, standard deviation.

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