Figure 2.
Targeting MARCH5 inhibits cell proliferation and prevents cell-cycle entry in PDX-AML cells in vivo. (A-C) PDX-AML cells were transduced with CTL or anti-MARCH5 shRNAs. (A-B) Gene expression was assessed by microarrays, and differential gene expression was analyzed by gene set enrichment analysis (n = 7 different PDX models). (A) Results are plotted as normalized enrichment score (NES) for hallmark signatures vs false discovery rate (FDR) q value. (B) Enrichment plots for quiescence or cycling signatures in AML.18 (C) PDX-AML cells were incubated with CFSE and then seeded in methylcellulose. Cells were cultured for 10 days, and CFSE was quantified by flow cytometry. Results are presented as CFSE mean fluorescence intensity (n = 3 different PDX models). (D-G) Leukemic cells from the PDXTUH06 model were transduced with mCherry-positive anti-MARCH5 shRNA. In the first experiment, cells were labeled ex vivo with CFSE and expanded in NSG mice for 7 days (n = 5). In the second experiment, PDX-AML cells transduced with anti-MARCH5 shRNA were expanded in vivo for 21 days and Ki-67/DAPI staining was performed (n = 5). (D) Schematic representation. (E) CSFE retention was measured in mCherry-positive (efficiently transduced) vs mCherry-negative (untransduced CLTs) cells. (F) Representative flow cytometry contour plots of Ki-67 vs DAPI in mCherry-positive and mCherry-negative cells. (G) Analysis of cell-cycle phases using Ki-67 and DRAQ7 staining in mCherry-positive and mCherry-negative cells. (H-J) Leukemic cells from the PDXTUH84 model were transduced with a GFP-tagged M5 OE vector and transplanted into NSG mice for 12 to 14 weeks (n = 8). (H) Schematic representation. (I) Representative flow cytometry contour plots of Ki-67 vs DRAQ7 in GFP+ and GFP– leukemic cells. (J) Analysis of cell-cycle phases using Ki-67 and DRAQ7 staining in GFP+ and GFP– cells. (K-N) PDX-AML cells were transduced with mCherry-tagged CTL or anti-MARCH5 shRNAs and transplanted into NSG mice for 12 to 14 weeks. (K) Summary diagram of the experiments. (L) Representative contour plots for mCherry vs side scatter-A (SSC-A) before transplantation (input) and after 12 to 14 weeks in vivo (output). (M) Representative confocal microscopy images at 20× original magnification of mouse bone marrow sections (scale bars, 50 μm). (N) Relative engraftment (output to input ratio) in shRNA shCTL vs shRNA targeting MARCH5 (shMARCH5) groups. Each dot represents the results observed in a single mouse (n = 2 different PDX models; n = 5-6 mice per experimental arm). IFNA, interferon alfa; IFNG, interferon gamma; L-CFU, leukemia colony-forming unit; ns, not significant; shCTL, sh control; shM5, shMARCH5; TNFA, tumor necrosis factor-α. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Targeting MARCH5 inhibits cell proliferation and prevents cell-cycle entry in PDX-AML cells in vivo. (A-C) PDX-AML cells were transduced with CTL or anti-MARCH5 shRNAs. (A-B) Gene expression was assessed by microarrays, and differential gene expression was analyzed by gene set enrichment analysis (n = 7 different PDX models). (A) Results are plotted as normalized enrichment score (NES) for hallmark signatures vs false discovery rate (FDR) q value. (B) Enrichment plots for quiescence or cycling signatures in AML.18 (C) PDX-AML cells were incubated with CFSE and then seeded in methylcellulose. Cells were cultured for 10 days, and CFSE was quantified by flow cytometry. Results are presented as CFSE mean fluorescence intensity (n = 3 different PDX models). (D-G) Leukemic cells from the PDXTUH06 model were transduced with mCherry-positive anti-MARCH5 shRNA. In the first experiment, cells were labeled ex vivo with CFSE and expanded in NSG mice for 7 days (n = 5). In the second experiment, PDX-AML cells transduced with anti-MARCH5 shRNA were expanded in vivo for 21 days and Ki-67/DAPI staining was performed (n = 5). (D) Schematic representation. (E) CSFE retention was measured in mCherry-positive (efficiently transduced) vs mCherry-negative (untransduced CLTs) cells. (F) Representative flow cytometry contour plots of Ki-67 vs DAPI in mCherry-positive and mCherry-negative cells. (G) Analysis of cell-cycle phases using Ki-67 and DRAQ7 staining in mCherry-positive and mCherry-negative cells. (H-J) Leukemic cells from the PDXTUH84 model were transduced with a GFP-tagged M5 OE vector and transplanted into NSG mice for 12 to 14 weeks (n = 8). (H) Schematic representation. (I) Representative flow cytometry contour plots of Ki-67 vs DRAQ7 in GFP+ and GFP– leukemic cells. (J) Analysis of cell-cycle phases using Ki-67 and DRAQ7 staining in GFP+ and GFP– cells. (K-N) PDX-AML cells were transduced with mCherry-tagged CTL or anti-MARCH5 shRNAs and transplanted into NSG mice for 12 to 14 weeks. (K) Summary diagram of the experiments. (L) Representative contour plots for mCherry vs side scatter-A (SSC-A) before transplantation (input) and after 12 to 14 weeks in vivo (output). (M) Representative confocal microscopy images at 20× original magnification of mouse bone marrow sections (scale bars, 50 μm). (N) Relative engraftment (output to input ratio) in shRNA shCTL vs shRNA targeting MARCH5 (shMARCH5) groups. Each dot represents the results observed in a single mouse (n = 2 different PDX models; n = 5-6 mice per experimental arm). IFNA, interferon alfa; IFNG, interferon gamma; L-CFU, leukemia colony-forming unit; ns, not significant; shCTL, sh control; shM5, shMARCH5; TNFA, tumor necrosis factor-α. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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