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. 2022 Jun 30;139(26):3752–3770. doi: 10.1182/blood.2021015108

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© 2022 by The American Society of Hematology

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Figure 3. — Replication stress signaling mediated RRM2 upregulation is responsible for myeloid differentiation. (A) Gene expression levels of RRM1, RRM2, and RRM2B were assessed by quantitative polymerase chain reaction analyses in indicated AML cells treated with vehicle or NEL for 12 hours (U937 and KG1A, 10 µM; AML#1, 20 µM). Data were first normalized to GAPDH levels and then vehicle-treated controls. Results represent mean ± SEM. **P < .01. (B) Western blot of the indicated proteins in U937 and primary AML CD34⁺ cells from specimen AML#1 treated with vehicle or NEL (U937, 10 µM; AML#1, 20 µM) for 12 hours. (C) Immunofluorescence analysis of RRM2 expression in U937 cells treated with vehicle or NEL (10 µM) for 12 hours. Scale bar, 5 μm. (D) Western blot of the indicated proteins in U937 cells treated with vehicle, NEL (10 µM), BAY1895344 (50 nM), or combination for 12 hours. (E) Western blot of the indicated proteins in U937 cells transduced with shRNA against E2F1 (shE2F1) or scramble control (shCtrl) after NEL treatment (10 µM) for 12 hours. (F) Pearson correlation of RRM2 and SAMHD1 mRNA expression levels with NEL sensitivity in a panel of 57 hematopoietic cell lines. Data were sourced from Cancer Therapeutics Response Portal (CTRP). (G) Western blot of the indicated proteins in U937 and THP1 cells treated with vehicle or Ara-C (0.5 μM) for 12 hours. (H) U937 cells were treated with vehicle, Ara-C (0.5 μM), COH29 (10 μM), or combination for 12 hours, and intracellular dNTP levels were quantified relative to their NTP counterparts by HPLC/MS. Numbers denote the fold changes of dNTP/NTP ratios relative to vehicle-treated controls. Results represent mean ± SEM. *P < .05; **P < .01. (I) CD11b expression levels in primary AML CD34⁺ cells (n = 5) treated with vehicle, Ara-C (0.5 μM), COH29 (10 μM), or combination for 96 hours. (J) CD11b expression levels of ishCtrl- and ishRRM2-U937 cells with or without Ara-C treatment (0.5 μM, 96 hours) after doxycycline induction. (K) Pearson correlation of RRM2 mRNA expression levels with Ara-C sensitivity in a panel of 67 hematopoietic cell lines. Data were sourced from CTRP portal. (L) Kaplan-Meier survival analysis of a cohort of patients with AML (GSE14468) after dichotomization for RRM2 mRNA levels below (blue, n = 70) and above (red, n = 192) 7.69 log₂ transformed intensity (P = .019). (M-N) Representative immunofluorescence images (M) and quantification of nuclear S9.6 intensity (N) in U937 cells transduced with empty vector (MOCK) or V5-tagged RNASEH1 construct after treatment with NEL (10 µM) or HU (20 µM) for 6 hours. Regions of interest for specific quantification of nuclear S9.6 staining were highlighted by dotted white lines. Scale bar, 10 μm. Box-whisker plots indicate median, 25th to 75th percentile, and maximum and minimum values by line, box, and whiskers, respectively. ns, nonsignificant; **P < .01. (O) Western blot of the indicated proteins in MOCK- or RNASEH1-transduced U937 cells after treatment with vehicle, NEL (10 µM), CPT (20 nM), or HU (20 µM) for 12 hours. (P-Q) CD11b expression levels (P) and relative cell viability (Q) of MOCK- or RNASEH1-transduced U937 cells after treatment with vehicle or NEL (10 µM) for 96 hours. Results represent mean ± SEM. **P < .01.

Figure 3. — Replication stress signaling mediated RRM2 upregulation is responsible for myeloid differentiation. (A) Gene expression levels of RRM1, RRM2, and RRM2B were assessed by quantitative polymerase chain reaction analyses in indicated AML cells treated with vehicle or NEL for 12 hours (U937 and KG1A, 10 µM; AML#1, 20 µM). Data were first normalized to GAPDH levels and then vehicle-treated controls. Results represent mean ± SEM. **P < .01. (B) Western blot of the indicated proteins in U937 and primary AML CD34⁺ cells from specimen AML#1 treated with vehicle or NEL (U937, 10 µM; AML#1, 20 µM) for 12 hours. (C) Immunofluorescence analysis of RRM2 expression in U937 cells treated with vehicle or NEL (10 µM) for 12 hours. Scale bar, 5 μm. (D) Western blot of the indicated proteins in U937 cells treated with vehicle, NEL (10 µM), BAY1895344 (50 nM), or combination for 12 hours. (E) Western blot of the indicated proteins in U937 cells transduced with shRNA against E2F1 (shE2F1) or scramble control (shCtrl) after NEL treatment (10 µM) for 12 hours. (F) Pearson correlation of RRM2 and SAMHD1 mRNA expression levels with NEL sensitivity in a panel of 57 hematopoietic cell lines. Data were sourced from Cancer Therapeutics Response Portal (CTRP). (G) Western blot of the indicated proteins in U937 and THP1 cells treated with vehicle or Ara-C (0.5 μM) for 12 hours. (H) U937 cells were treated with vehicle, Ara-C (0.5 μM), COH29 (10 μM), or combination for 12 hours, and intracellular dNTP levels were quantified relative to their NTP counterparts by HPLC/MS. Numbers denote the fold changes of dNTP/NTP ratios relative to vehicle-treated controls. Results represent mean ± SEM. *P < .05; **P < .01. (I) CD11b expression levels in primary AML CD34⁺ cells (n = 5) treated with vehicle, Ara-C (0.5 μM), COH29 (10 μM), or combination for 96 hours. (J) CD11b expression levels of ishCtrl- and ishRRM2-U937 cells with or without Ara-C treatment (0.5 μM, 96 hours) after doxycycline induction. (K) Pearson correlation of RRM2 mRNA expression levels with Ara-C sensitivity in a panel of 67 hematopoietic cell lines. Data were sourced from CTRP portal. (L) Kaplan-Meier survival analysis of a cohort of patients with AML (GSE14468) after dichotomization for RRM2 mRNA levels below (blue, n = 70) and above (red, n = 192) 7.69 log₂ transformed intensity (P = .019). (M-N) Representative immunofluorescence images (M) and quantification of nuclear S9.6 intensity (N) in U937 cells transduced with empty vector (MOCK) or V5-tagged RNASEH1 construct after treatment with NEL (10 µM) or HU (20 µM) for 6 hours. Regions of interest for specific quantification of nuclear S9.6 staining were highlighted by dotted white lines. Scale bar, 10 μm. Box-whisker plots indicate median, 25th to 75th percentile, and maximum and minimum values by line, box, and whiskers, respectively. ns, nonsignificant; **P < .01. (O) Western blot of the indicated proteins in MOCK- or RNASEH1-transduced U937 cells after treatment with vehicle, NEL (10 µM), CPT (20 nM), or HU (20 µM) for 12 hours. (P-Q) CD11b expression levels (P) and relative cell viability (Q) of MOCK- or RNASEH1-transduced U937 cells after treatment with vehicle or NEL (10 µM) for 96 hours. Results represent mean ± SEM. **P < .01.