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. Author manuscript; available in PMC: 2023 Jan 6.
Published in final edited form as: Cancer Discov. 2022 Jul 6;12(7):1760–1781. doi: 10.1158/2159-8290.CD-21-0956

Figure 4. Knock-down of IRF2BP2 in PDX cells reduces leukemia burden and prolongs survival.

Figure 4.

A, Western blot analysis for IRF2BP2 in hCD34+ bone marrow cells nucleofected with synthetic IRF2BP2-targeting guides or a non-targeting control guide. Vinculin was used as a loading control.

B, Quantification of colony numbers of hCD34+ cells nucleofected with synthetic IRF2BP2-targeting guides, a non-targeting control guide or a positive control guide targeting an essential gene (RPA3) on day 10 post seeding. One-way ANOVA, Dunnett’s multiple comparison test (per week), **** p < 0.0001.

C, Quantification of colony numbers of six PDX models (for molecular characteristics see Table 1) nucleofected with synthetic IRF2BP2-targeting guides, a non-targeting control guide or a positive control guide targeting an essential gene (RPA3) on day 10 post seeding. One-way ANOVA, Dunnett’s multiple comparison test (per week), **** p < 0.0001.

D, GSEA for human monocytic lineage gene markers (35) run per individual PDX sample on the genome-wide genes ranked by log2(TPM+1) expression. NES >/= 1.3, p-value </= 0.05, FDR </= 0.25.

E, PDX cells from PDX model 16-01 (left) and 17-14 (right) were infected with mAmetrine-positive CRISPR guides targeting IRF2BP2 or a non-targeting control guide (sgNT). Flow-cytometry for mAmetrine-positive cells was performed 72 hours post infection, and then weekly. The fraction of mAmetrine-positive cells relative to week 0 was assessed at one and two weeks. One-way ANOVA, Dunnett’s multiple comparison test (per week), ** p < 0.01, *** p < 0.001, **** p < 0.0001.

F, Flow cytometry analysis for GFP-positive cells on bone marrow aspirates from sub-lethally irradiated NSG mice transplanted with PDX 16-01 cells infected with doxycycline-inducible CRISPR guides against IRF2BP2 or a non-targeting control guide after a two-week doxycycline diet (n = 6 to 8 mice per group, each dot represents one animal). One-way ANOVA, Dunnett’s multiple comparison test, * p < 0.05.

G, Flow cytometry analysis for GFP-positive cells on bone marrow aspirates from sub-lethally irradiated NSG mice transplanted with PDX 17-14 cells infected with doxycycline-inducible CRISPR guides against IRF2BP2 or a non-targeting control guide (sgNT) after a two-week doxycycline diet (n = 6 to 8 mice per group, each dot represents one animal). One-way ANOVA, Dunnett’s multiple comparison test, ** p < 0.01.

H, Kaplan-Meier curves depicting survival of NSG mice transplanted with PDX 16-01 cells infected with doxycycline-inducible CRISPR guides against IRF2BP2 or a non-targeting control guide under continuous doxycycline diet. The median survival of the mice transplanted with PDX 17-14 sgIRF2BP2_1 and sgIRF2BP2_3 cells was 49 and 52 days, respectively, as compared to 34 days for mice who received sgNT PDX cells. Log-rank Mantel-Cox test, **** p < 0.0001.

I, Kaplan-Meier curves depicting survival of NSG mice transplanted with PDX 17-14 cells infected with doxycycline-inducible CRISPR guides against IRF2BP2 or a non-targeting control guide under continuous doxycycline diet. The median survival of the mice transplanted with PDX 17-14 sgIRF2BP2_1 and sgIRF2BP2_3 was 63.5 and 60 days respectively, as compared to 38 days for mice who received sgNT PDX cells. Log-rank Mantel-Cox test, **** p < 0.0001.