Figure 6.
Loss-of-function screen identified synthetic lethal interaction between DUSP6-KO and nelarabine treatment. (A-B) Experimental design of genome-wide CRISPR/Cas9 screen for synthetic lethal partner of NEL treatment in NEL-resistant subline U937/R (A). Briefly, Cas9 expressing-U937/R cells were transduced with GeCKO gRNA library. After culturing for 7 days, cells were divided into 2 groups for treatment with vehicle or NEL (20 µM). After another 7 days of exposure, sgRNA representation was assessed by sequencing. The gene negative scores (B) were calculated using MAGeCK-MLE module. Reported ERK negative feedback regulators were labeled. The red dots represent preferential depletion of DUSP1 and DUSP6 sgRNAs following NEL treatment over vehicle control. (C) Relative DUSP6 expression levels in indicated AML cells treated with vehicle, NEL (10 µM for U937 and KG1A, 20 µM for MOLM13 and AML#1), Ara-C (0.5 µM), or dG (10 µM for U937 and KG1A, 15 µM for MOLM13 and AML#1) for 24 hours. Results represent mean ± SEM. **P < .01. (D) Western blot of DUSP6 proteins in U937/R-Cas9 cells transduced with lentiviral vectors expressing sgRNA against DUSP6 (sgDUSP6) or nontargeting control (sgCtrl). (E) Phospho-ERK levels in sgCtrl- or sgDUSP6-U937/R cells treated with vehicle or NEL (20 µM) for 24 hours. (F-G) CD11b expression levels (F) and apoptosis (G) of sgCtrl- or sgDUSP6-U937/R cells treated with vehicle or NEL (20 µM) for 96 hours. (H) Western blot of the indicated proteins in U937/R cells transduced with lentiviral vectors expressing shRNA against ERK2 (shERK2) or scramble control (shCtrl), followed by treatment with NEL (20 µM) alone or NEL (20 µM) plus BCI (1 µM) for 24 hours. (I) CD11b expression levels in shCtrl- or shERK2-U937/R cells treated with NEL (20 µM) alone or NEL (20 µM) plus BCI (1 µM) for 96 hours. (J) Relative phospho-ERK levels in primary AML CD34+ cells (n = 3) treated with vehicle, NEL (20 µM), BCI (1 µM), or combination for 24 hours. Data were normalized to vehicle-treated controls. Results represent mean ± SEM. **P < .01. (K) CD11b and CD15 expression levels in primary AML CD34+ cells from specimen AML#4 treated with vehicle, NEL (20 µM), BCI (1 µM), or a combination for 96 hours. (L-M) MOLM13-luciferase cells (1 × 106 cells per mouse) were injected into sublethally irradiated NSGS mice. Following engraftment, mice were treated with vehicle (PBS), NEL (217 mg/kg, IV, daily), BCI (10 mg/kg, intraperitoneally, daily), or a combination for 2 weeks (n = 7 per group), assessed for engraftment by in vivo bioluminescence imaging (L) and monitored for survival (M). (N) A schematic model showing that the response to RRM2 hyperactivation-mediated dNTP imbalance depends on reaching a lethal threshold of RAS/ERK activity, and DUSP6 activity may antagonize this lethal effect.

Loss-of-function screen identified synthetic lethal interaction between DUSP6-KO and nelarabine treatment. (A-B) Experimental design of genome-wide CRISPR/Cas9 screen for synthetic lethal partner of NEL treatment in NEL-resistant subline U937/R (A). Briefly, Cas9 expressing-U937/R cells were transduced with GeCKO gRNA library. After culturing for 7 days, cells were divided into 2 groups for treatment with vehicle or NEL (20 µM). After another 7 days of exposure, sgRNA representation was assessed by sequencing. The gene negative scores (B) were calculated using MAGeCK-MLE module. Reported ERK negative feedback regulators were labeled. The red dots represent preferential depletion of DUSP1 and DUSP6 sgRNAs following NEL treatment over vehicle control. (C) Relative DUSP6 expression levels in indicated AML cells treated with vehicle, NEL (10 µM for U937 and KG1A, 20 µM for MOLM13 and AML#1), Ara-C (0.5 µM), or dG (10 µM for U937 and KG1A, 15 µM for MOLM13 and AML#1) for 24 hours. Results represent mean ± SEM. **P < .01. (D) Western blot of DUSP6 proteins in U937/R-Cas9 cells transduced with lentiviral vectors expressing sgRNA against DUSP6 (sgDUSP6) or nontargeting control (sgCtrl). (E) Phospho-ERK levels in sgCtrl- or sgDUSP6-U937/R cells treated with vehicle or NEL (20 µM) for 24 hours. (F-G) CD11b expression levels (F) and apoptosis (G) of sgCtrl- or sgDUSP6-U937/R cells treated with vehicle or NEL (20 µM) for 96 hours. (H) Western blot of the indicated proteins in U937/R cells transduced with lentiviral vectors expressing shRNA against ERK2 (shERK2) or scramble control (shCtrl), followed by treatment with NEL (20 µM) alone or NEL (20 µM) plus BCI (1 µM) for 24 hours. (I) CD11b expression levels in shCtrl- or shERK2-U937/R cells treated with NEL (20 µM) alone or NEL (20 µM) plus BCI (1 µM) for 96 hours. (J) Relative phospho-ERK levels in primary AML CD34+ cells (n = 3) treated with vehicle, NEL (20 µM), BCI (1 µM), or combination for 24 hours. Data were normalized to vehicle-treated controls. Results represent mean ± SEM. **P < .01. (K) CD11b and CD15 expression levels in primary AML CD34+ cells from specimen AML#4 treated with vehicle, NEL (20 µM), BCI (1 µM), or a combination for 96 hours. (L-M) MOLM13-luciferase cells (1 × 106 cells per mouse) were injected into sublethally irradiated NSGS mice. Following engraftment, mice were treated with vehicle (PBS), NEL (217 mg/kg, IV, daily), BCI (10 mg/kg, intraperitoneally, daily), or a combination for 2 weeks (n = 7 per group), assessed for engraftment by in vivo bioluminescence imaging (L) and monitored for survival (M). (N) A schematic model showing that the response to RRM2 hyperactivation-mediated dNTP imbalance depends on reaching a lethal threshold of RAS/ERK activity, and DUSP6 activity may antagonize this lethal effect.

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