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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 4. — Genetically elevating RRM2 levels impairs AML maintenance. (A-B) Western blot of RRM2 protein levels (A) and primer extension assay of intracellular dNTP levels (B) in THP1 cells engineered with empty vector (MOCK) or inducible RRM2 Rxl/AxA (iRxl/AxA) mutant, further treated with vehicle or NEL (20 µM, 12 hours) after doxycycline (DOX) induction. Rxl/AxA, CCNF binding-deficient RRM2 construct: RxI motif located at RRM2 residues aa49-aa51, reportedly binding to cyclin F, was mutated to AxA. Numbers denote the fold changes relative to vehicle-treated MOCK controls. For panel B, results represent mean ± SEM. *P < .05; **P < .01. (C) CD11b expression levels in MOCK- or iRxl/AxA-THP1 cells treated with vehicle, NEL (20 µM), or Ara-C (0.5 µM) for 96 hours after DOX induction. Results represent mean ± SEM. **P < .01. (D-E) Engineered THP1 cells (1 × 10⁶ cells per mouse) were injected into NSGS mice. Following engraftment, 2 groups of mice receiving either MOCK cells or iRxl/AxA cells were treated with DOX (10 mg/kg, daily). In MOCK or iRxl/AxA transplants, mice were divided into 2 treatment groups injected with either vehicle (PBS) or NEL (217 mg/kg, IV daily; n = 5 per group) and then assessed for engraftment by bioluminescence imaging (D) or monitored for survival (E). (F) Pearson correlation of RRM2 mRNA expression with CCNF gene dependency scores in 94 hematologic cancer cell lines, sourced from Depmap portal. The x axis denotes to RRM2 expression levels of cell lines presented as log2-transformed TPM values. (G) Western blot of the indicated proteins in THP1 cells transduced with lentiviral vectors expressing shRNA against CCNF (shCCNF#1 [chosen to be used further, namely shCCNF], shCCNF#2) or scramble control (shCtrl), followed by G₂/M enrichment through treatment with nocodazole (100 ng/mL, 16 hours). (H) Intracellular dNTP levels of shCtrl- or shCCNF-THP1 cells followed by G₂/M enrichment through treatment with nocodazole (100 ng/mL, 16 hours). Numbers denote the fold changes relative to shCtrl-THP1 cells. Results represent mean ± SEM. **P < .01. (I) CD11b expression levels in THP1 cells transduced with shCtrl, shCCNF#1, or shCCNF#2 lentivirus for 4 days. (J) Western blot of the indicated proteins in primary AML CD34⁺ cells (n = 6) transduced with shCtrl or shCCNF lentivirus, followed by G₂/M enrichment through treatment with nocodazole (100 ng/mL, 16 hours). (K) Primary AML CD34⁺ cells (n = 6) were transduced with shCtrl or shCCNF lentivirus before viability inhibition assay for 96 hours in the presence of Ara-C at the indicated concentrations. The IC₅₀ values between cells transduced with shCtrl lentivirus and their shCCNF counterparts were analyzed by means of extra-sum-of-squares F test. Results represent mean ± SEM. *P < .05; **P < .01. (L) Serial replating of Samhd1 WT/MOCK, Samhd1 KO/MOCK, Samhd1 WT/RRM2 OE, and Samhd1 KO/RRM2 OE cells engineered from MA9⁺ murine AML cells. Results represent mean ± SEM. ns, nonsignificant; *P < .05; **P < .01. (M) Percentages of CD45.2⁺ donor chimerism in BM of recipients (n = 6 per group) transplanted with engineered cells as indicated on day 30 after DOX induction. Results represent mean ± SEM. **P < .01. (N) Survival of CD45.1⁺ congenic recipients transplanted with engineered MA9⁺ murine AML cells as indicated. Doxycycline induction (10 mg/kg, daily) started 2 weeks after transplantation. (O) In vivo LDA assay showing LSC frequency changes in MA9⁺ primary murine AML cells after Samhd1 KO or RRM2 OE.

Figure 4. — Genetically elevating RRM2 levels impairs AML maintenance. (A-B) Western blot of RRM2 protein levels (A) and primer extension assay of intracellular dNTP levels (B) in THP1 cells engineered with empty vector (MOCK) or inducible RRM2 Rxl/AxA (iRxl/AxA) mutant, further treated with vehicle or NEL (20 µM, 12 hours) after doxycycline (DOX) induction. Rxl/AxA, CCNF binding-deficient RRM2 construct: RxI motif located at RRM2 residues aa49-aa51, reportedly binding to cyclin F, was mutated to AxA. Numbers denote the fold changes relative to vehicle-treated MOCK controls. For panel B, results represent mean ± SEM. *P < .05; **P < .01. (C) CD11b expression levels in MOCK- or iRxl/AxA-THP1 cells treated with vehicle, NEL (20 µM), or Ara-C (0.5 µM) for 96 hours after DOX induction. Results represent mean ± SEM. **P < .01. (D-E) Engineered THP1 cells (1 × 10⁶ cells per mouse) were injected into NSGS mice. Following engraftment, 2 groups of mice receiving either MOCK cells or iRxl/AxA cells were treated with DOX (10 mg/kg, daily). In MOCK or iRxl/AxA transplants, mice were divided into 2 treatment groups injected with either vehicle (PBS) or NEL (217 mg/kg, IV daily; n = 5 per group) and then assessed for engraftment by bioluminescence imaging (D) or monitored for survival (E). (F) Pearson correlation of RRM2 mRNA expression with CCNF gene dependency scores in 94 hematologic cancer cell lines, sourced from Depmap portal. The x axis denotes to RRM2 expression levels of cell lines presented as log2-transformed TPM values. (G) Western blot of the indicated proteins in THP1 cells transduced with lentiviral vectors expressing shRNA against CCNF (shCCNF#1 [chosen to be used further, namely shCCNF], shCCNF#2) or scramble control (shCtrl), followed by G₂/M enrichment through treatment with nocodazole (100 ng/mL, 16 hours). (H) Intracellular dNTP levels of shCtrl- or shCCNF-THP1 cells followed by G₂/M enrichment through treatment with nocodazole (100 ng/mL, 16 hours). Numbers denote the fold changes relative to shCtrl-THP1 cells. Results represent mean ± SEM. **P < .01. (I) CD11b expression levels in THP1 cells transduced with shCtrl, shCCNF#1, or shCCNF#2 lentivirus for 4 days. (J) Western blot of the indicated proteins in primary AML CD34⁺ cells (n = 6) transduced with shCtrl or shCCNF lentivirus, followed by G₂/M enrichment through treatment with nocodazole (100 ng/mL, 16 hours). (K) Primary AML CD34⁺ cells (n = 6) were transduced with shCtrl or shCCNF lentivirus before viability inhibition assay for 96 hours in the presence of Ara-C at the indicated concentrations. The IC₅₀ values between cells transduced with shCtrl lentivirus and their shCCNF counterparts were analyzed by means of extra-sum-of-squares F test. Results represent mean ± SEM. *P < .05; **P < .01. (L) Serial replating of Samhd1 WT/MOCK, Samhd1 KO/MOCK, Samhd1 WT/RRM2 OE, and Samhd1 KO/RRM2 OE cells engineered from MA9⁺ murine AML cells. Results represent mean ± SEM. ns, nonsignificant; *P < .05; **P < .01. (M) Percentages of CD45.2⁺ donor chimerism in BM of recipients (n = 6 per group) transplanted with engineered cells as indicated on day 30 after DOX induction. Results represent mean ± SEM. **P < .01. (N) Survival of CD45.1⁺ congenic recipients transplanted with engineered MA9⁺ murine AML cells as indicated. Doxycycline induction (10 mg/kg, daily) started 2 weeks after transplantation. (O) In vivo LDA assay showing LSC frequency changes in MA9⁺ primary murine AML cells after Samhd1 KO or RRM2 OE.