American Society of Hematology

Figure 3.

$Figure 3. INKA1-OE in 8227 AML LSC model recapitulates relative enrichment of primitive cells while INKA1-KD reduces them. (A) Representative CD34/CD38 FACS plot of the phenotypic 8227 AML hierarchy. (B) Engraftment data of injected femurs at 12 weeks (human CD45+CD33+) of 8227 AML cells that had been sorted into 4 fractions according to CD34 and CD38 expression and transplanted into nonobese diabetic-severe combined immunodeficiency-SGM3 mice and analyzed after 12 weeks. Unsorted: 1.5 × 106; CD34+ fractions: 0.3 × 106, CD34− fractions: 0.5 × 106 cells/mouse. (C) Spearman’s correlation ρ values generated from gene expression data (NanoString, n = 4) from the four 8227 AML fractions of 104 genes that are differentially regulated in LSC+ vs LSC− with their expression in the LSC104 data set. (D) INKA1 expression across 8227 AML hierarchy, according to NanoString analysis (n = 4). (E) CD34+CD38− 8227 AML cells were transduced with CTRL and INKA1-OE vectors, and %BFP+ was assessed over the course of 43 days (n = 3). (F) Cell viability of BFP+ CD34− and CD34+CD38− was analyzed according to AnnexinV− and 7AAD− staining. (G) Relative gene-marking (BFP+) in CD34+ and CD34− fractions was assessed over time (n = 3). (H) Sorted CD34+CD38−BFP+ cells were cultured to assess the capability to regrow the hierarchy. Shown are representative FACS plots at 2 weeks (n = 3) and 6 weeks (n = 2) after sorting. Absolute (I) and relative (J) cell counts are shown over 3 weeks of follow-up and CD34+CD38− maintenance (K), and differentiation to CD38+ (L) cells was monitored over the course of 6 weeks. (M) Long-term in vitro follow-up of %CD34+ cells within BFP+ population upon INKA1-KD (shINKA1) vs control (shCTRL, n = 4). (N) Sorted CD34+CD38−BFP+ cells (shCTRL, shINKA1) were cultured to assess the capability to regrow the hierarchy. Shown are representative FACS plots at 2 weeks (n = 3) after sorting and absolute cell counts of CD34+CD38− cells are shown over the course of 3 weeks follow-up in panel O. Student t test *P < .05; **P < .01; ***P < .001; n.s., nonsignificant.$

INKA1-OE in 8227 AML LSC model recapitulates relative enrichment of primitive cells while INKA1-KD reduces them. (A) Representative CD34/CD38 FACS plot of the phenotypic 8227 AML hierarchy. (B) Engraftment data of injected femurs at 12 weeks (human CD45⁺CD33⁺) of 8227 AML cells that had been sorted into 4 fractions according to CD34 and CD38 expression and transplanted into nonobese diabetic-severe combined immunodeficiency-SGM3 mice and analyzed after 12 weeks. Unsorted: 1.5 × 10⁶; CD34⁺ fractions: 0.3 × 10⁶, CD34⁻ fractions: 0.5 × 10⁶ cells/mouse. (C) Spearman’s correlation ρ values generated from gene expression data (NanoString, n = 4) from the four 8227 AML fractions of 104 genes that are differentially regulated in LSC⁺ vs LSC⁻ with their expression in the LSC104 data set. (D) INKA1 expression across 8227 AML hierarchy, according to NanoString analysis (n = 4). (E) CD34⁺CD38⁻ 8227 AML cells were transduced with CTRL and INKA1-OE vectors, and %BFP⁺ was assessed over the course of 43 days (n = 3). (F) Cell viability of BFP⁺ CD34⁻ and CD34⁺CD38⁻ was analyzed according to AnnexinV⁻ and 7AAD⁻ staining. (G) Relative gene-marking (BFP⁺) in CD34⁺ and CD34⁻ fractions was assessed over time (n = 3). (H) Sorted CD34⁺CD38⁻BFP⁺ cells were cultured to assess the capability to regrow the hierarchy. Shown are representative FACS plots at 2 weeks (n = 3) and 6 weeks (n = 2) after sorting. Absolute (I) and relative (J) cell counts are shown over 3 weeks of follow-up and CD34⁺CD38⁻ maintenance (K), and differentiation to CD38⁺ (L) cells was monitored over the course of 6 weeks. (M) Long-term in vitro follow-up of %CD34⁺ cells within BFP⁺ population upon INKA1-KD (shINKA1) vs control (shCTRL, n = 4). (N) Sorted CD34⁺CD38⁻BFP⁺ cells (shCTRL, shINKA1) were cultured to assess the capability to regrow the hierarchy. Shown are representative FACS plots at 2 weeks (n = 3) after sorting and absolute cell counts of CD34⁺CD38⁻ cells are shown over the course of 3 weeks follow-up in panel O. Student t test *P < .05; **P < .01; ***P < .001; n.s., nonsignificant.

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