Figure 5.
AML1-ETO transformed hematopoietic stem cells depend on PLCG1. (A) Targeting strategy for the conditional Plcg1 knockout mouse model. Exons 3 to 5 are flanked with LoxP sites (red triangles) to facilitate tissue-specific deletion. FRT sites, green triangles. (B-F) GFP+Kit+ BM cells of Plcg1+/+ and Plcg1F/F AML1-ETO/KRAS (AE/K) or MLL-AF9 (MA9) primary recipients were sorted and retrovirally infected with a Cre-recombinase (MSCV-Cre-puro), followed by 24 hours of puromycin selection. (C) Serial replating in methylcellulose. Colony counts per plating over 6 weeks are depicted for AML1-ETO/KRAS. Representative pictures of colonies (second plating). n = 3 independent experiments, in duplicate; paired t test. (D) Kaplan-Meier survival curves of recipient animals of AE/K transformed Plcg1+/+ (n = 16 mice) vs Plcg1−/− (n = 7 mice) LSCs, Mantel-Cox test. (E) Histologic analysis of liver, lung, and spleen morphology in Plcg1+/+ or Plcg1−/− AML1-ETO9a/KRAS (AE/K) transformed secondary recipients. Representative images are shown. Scale bars, 100 μm. (F) Serial replating in methylcellulose. Colony counts per plating over 6 weeks are depicted for MLL-AF9. Representative pictures of colonies (second plating). n = 3 independent experiments, in duplicate; paired t test. (G) Heatmap of differentially expressed genes in AE/KRAS transformed Plcg1+/+ (n = 2) vs Plcg1−/− (n = 3) LSCs 48 hours after genetic deletion of Plcg1. Red zones represent higher gene expression (upregulation); blue zones represent lower gene expression (downregulation). (H) GSEA indicating loss of AML1-ETO (RUNX1-RUNX1T1) target genes (top) and negative enrichment of PLCG1 target genes (bottom) in the AML1-ETO knockdown signature of Kasumi-1 cells. AE, AML1-ETO; k/d, knockdown; MM, mismatch control; NES, normalized enrichment score; NT, nontargeting control. (I) Schematic representation of the experimental setup to study the effects of Plcg1 inactivation on AML1-ETO/KRAS (AE/K)-transformed LSCs in vivo. (J) Analysis of sublethally (7 Gy) irradiated 6- to 8-week-old primary recipients of AE/K-transformed Plcg1+/+ and Plcg1F/F LSK cells. pIpC injections were administered intraperitoneally as indicated by arrows. Immunophenotyping of (GFP+) leukemia cells in peripheral blood of primary recipient mice. Plcg1+/+ (n = 9 mice) vs Plcg1−/− (n = 9 mice). (K) Survival of primary recipient mice. Plcg1+/+ (n = 12 mice) vs Plcg1−/− (n = 12 mice). Mantel-Cox test. (L) Immunophenotyping of GFP+ bone marrow (BM) LSKs (Plcg1+/+ n = 8 mice, Plcg1−/− n = 9 mice; Mann-Whitney U test. (M) Cytospins (May-Grünwald/Giemsa staining) of GFP+ LSK cells following short-term (24-hour) culture ex vivo. (N) Cell-cycle analysis (Ki67/Hoechst staining) of GFP+ LSK cells from primary recipient mice following genetic inactivation of Plcg1 in vivo (Plcg1+/+, n = 6 mice vs Plcg1−/−, n = 6 mice; Mann-Whitney U test). (O) Kaplan-Meier survival curves of secondary recipients of 2 × 106 BM cells from primary Plcg1+/+ (n = 11) and Plcg1−/− (n = 10) recipient mice, Mantel-Cox test. (P) Colony formation of leukemic bone marrow cells derived from patients at primary diagnosis of t(8;21) positive AML (n = 6 individual patients). Colony number following PLCG1 depletion by RNAi (shPLCG1-1 and 1-2) compared with nontargeting control (shSCR).

AML1-ETO transformed hematopoietic stem cells depend on PLCG1. (A) Targeting strategy for the conditional Plcg1 knockout mouse model. Exons 3 to 5 are flanked with LoxP sites (red triangles) to facilitate tissue-specific deletion. FRT sites, green triangles. (B-F) GFP+Kit+ BM cells of Plcg1+/+ and Plcg1F/F AML1-ETO/KRAS (AE/K) or MLL-AF9 (MA9) primary recipients were sorted and retrovirally infected with a Cre-recombinase (MSCV-Cre-puro), followed by 24 hours of puromycin selection. (C) Serial replating in methylcellulose. Colony counts per plating over 6 weeks are depicted for AML1-ETO/KRAS. Representative pictures of colonies (second plating). n = 3 independent experiments, in duplicate; paired t test. (D) Kaplan-Meier survival curves of recipient animals of AE/K transformed Plcg1+/+ (n = 16 mice) vs Plcg1−/− (n = 7 mice) LSCs, Mantel-Cox test. (E) Histologic analysis of liver, lung, and spleen morphology in Plcg1+/+ or Plcg1−/− AML1-ETO9a/KRAS (AE/K) transformed secondary recipients. Representative images are shown. Scale bars, 100 μm. (F) Serial replating in methylcellulose. Colony counts per plating over 6 weeks are depicted for MLL-AF9. Representative pictures of colonies (second plating). n = 3 independent experiments, in duplicate; paired t test. (G) Heatmap of differentially expressed genes in AE/KRAS transformed Plcg1+/+ (n = 2) vs Plcg1−/− (n = 3) LSCs 48 hours after genetic deletion of Plcg1. Red zones represent higher gene expression (upregulation); blue zones represent lower gene expression (downregulation). (H) GSEA indicating loss of AML1-ETO (RUNX1-RUNX1T1) target genes (top) and negative enrichment of PLCG1 target genes (bottom) in the AML1-ETO knockdown signature of Kasumi-1 cells. AE, AML1-ETO; k/d, knockdown; MM, mismatch control; NES, normalized enrichment score; NT, nontargeting control. (I) Schematic representation of the experimental setup to study the effects of Plcg1 inactivation on AML1-ETO/KRAS (AE/K)-transformed LSCs in vivo. (J) Analysis of sublethally (7 Gy) irradiated 6- to 8-week-old primary recipients of AE/K-transformed Plcg1+/+ and Plcg1F/F LSK cells. pIpC injections were administered intraperitoneally as indicated by arrows. Immunophenotyping of (GFP+) leukemia cells in peripheral blood of primary recipient mice. Plcg1+/+ (n = 9 mice) vs Plcg1−/− (n = 9 mice). (K) Survival of primary recipient mice. Plcg1+/+ (n = 12 mice) vs Plcg1−/− (n = 12 mice). Mantel-Cox test. (L) Immunophenotyping of GFP+ bone marrow (BM) LSKs (Plcg1+/+ n = 8 mice, Plcg1−/− n = 9 mice; Mann-Whitney U test. (M) Cytospins (May-Grünwald/Giemsa staining) of GFP+ LSK cells following short-term (24-hour) culture ex vivo. (N) Cell-cycle analysis (Ki67/Hoechst staining) of GFP+ LSK cells from primary recipient mice following genetic inactivation of Plcg1 in vivo (Plcg1+/+, n = 6 mice vs Plcg1−/−, n = 6 mice; Mann-Whitney U test). (O) Kaplan-Meier survival curves of secondary recipients of 2 × 106 BM cells from primary Plcg1+/+ (n = 11) and Plcg1−/− (n = 10) recipient mice, Mantel-Cox test. (P) Colony formation of leukemic bone marrow cells derived from patients at primary diagnosis of t(8;21) positive AML (n = 6 individual patients). Colony number following PLCG1 depletion by RNAi (shPLCG1-1 and 1-2) compared with nontargeting control (shSCR).

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