Figure 3.
Clonal selection of Trp53-mutant HSPCs with lenalidomide therapy but not with pomalidomide therapy. Hoxb8 cells derived from the CrbnI391V Rosa26-Cas9 mouse and transduced with sgRNAs targeting the labeled genes were grown in the presence of serially diluted lenalidomide (A) or pomalidomide (B) for 72 hours and cell density measured using CellTiter-Glo. Data are normalized to the vehicle (DMSO) control and shown as the mean ± standard deviation (n = 3 replicates). Curves represent the logistic regression. (C) Schematic for the generation of pooled parallel CRISPR-Cas9 in vivo mouse screen. LSK cells were lentivirally transduced with individual guides, pooled at equal ratios, and then transplanted into lethally irradiated Bl6.SJL recipient mice. (D) PB was collected before and after treatment of mice from pooled CRISPR-Cas9 experiment, DNA was harvested, sgRNA sequence was PCR amplified, and then subjected to NGS. The proportions of read mapping to each sgRNA sequence was determined and the percentage change in read frequency for each sgRNA between pre- and posttreatment samples was determined. Shown is the mean ± standard deviation (n = 6 mice per group). P values are from unpaired single-sided t tests. (E) PB was collected weekly and chimerism was measured using fluorescence-assisted cell sorting. (F) BM was harvested and chimerism measured within each of the following cellular compartments: LSK, long-term HSCs (LT-HSC: LSK, CD150+, and CD48−), short-term–HSC (ST-HSC: LSK, CD150−, and CD48−), multipotent progenitor (MPP: LSK, CD150−, and CD48+), common myeloid progenitor (CMP: LK, CD16/32−, and CD34+), granulocyte-monocyte progenitor (GMP: LK, CD16/32+, and CD34+), megakaryocyte-erythroid progenitor (MEP: LK, CD16/32−, and CD34−). Shown is the mean ± the standard error of the mean (n = 9-10 mice per group). ∗∗∗∗P < .0001, one-way ANOVA. (G) After hematopoietic reconstitution, chimeric transplants containing 50% Tet2−/−, CrbnI391V (mutant, CD45.2), and 50% CrbnI391V (WT, CD45.1) were treated with vehicle (DMSO) or lenalidomide 50 mg/kg, twice daily (BID). PB was collected weekly and chimerism was measured using fluorescence-assisted cell sorting. Shown is the mean ± the standard error of the mean (n = 15 mice per group). Actb, cutting guide targeting intronic sequence within β-actin gene; NTG, nontargeting guide; ns, not significant; PO, postoperative; tx, treatment.

Clonal selection of Trp53-mutant HSPCs with lenalidomide therapy but not with pomalidomide therapy. Hoxb8 cells derived from the CrbnI391V Rosa26-Cas9 mouse and transduced with sgRNAs targeting the labeled genes were grown in the presence of serially diluted lenalidomide (A) or pomalidomide (B) for 72 hours and cell density measured using CellTiter-Glo. Data are normalized to the vehicle (DMSO) control and shown as the mean ± standard deviation (n = 3 replicates). Curves represent the logistic regression. (C) Schematic for the generation of pooled parallel CRISPR-Cas9 in vivo mouse screen. LSK cells were lentivirally transduced with individual guides, pooled at equal ratios, and then transplanted into lethally irradiated Bl6.SJL recipient mice. (D) PB was collected before and after treatment of mice from pooled CRISPR-Cas9 experiment, DNA was harvested, sgRNA sequence was PCR amplified, and then subjected to NGS. The proportions of read mapping to each sgRNA sequence was determined and the percentage change in read frequency for each sgRNA between pre- and posttreatment samples was determined. Shown is the mean ± standard deviation (n = 6 mice per group). P values are from unpaired single-sided t tests. (E) PB was collected weekly and chimerism was measured using fluorescence-assisted cell sorting. (F) BM was harvested and chimerism measured within each of the following cellular compartments: LSK, long-term HSCs (LT-HSC: LSK, CD150+, and CD48−), short-term–HSC (ST-HSC: LSK, CD150−, and CD48−), multipotent progenitor (MPP: LSK, CD150−, and CD48+), common myeloid progenitor (CMP: LK, CD16/32−, and CD34+), granulocyte-monocyte progenitor (GMP: LK, CD16/32+, and CD34+), megakaryocyte-erythroid progenitor (MEP: LK, CD16/32−, and CD34−). Shown is the mean ± the standard error of the mean (n = 9-10 mice per group). ∗∗∗∗P < .0001, one-way ANOVA. (G) After hematopoietic reconstitution, chimeric transplants containing 50% Tet2−/−, CrbnI391V (mutant, CD45.2), and 50% CrbnI391V (WT, CD45.1) were treated with vehicle (DMSO) or lenalidomide 50 mg/kg, twice daily (BID). PB was collected weekly and chimerism was measured using fluorescence-assisted cell sorting. Shown is the mean ± the standard error of the mean (n = 15 mice per group). Actb, cutting guide targeting intronic sequence within β-actin gene; NTG, nontargeting guide; ns, not significant; PO, postoperative; tx, treatment.

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