Loss of HLF expression accelerates G1/S transition and reduces the expression levels of HES1 and CDKN1C. (A) CFSE experiment performed with sgGFP and sgHLF cells harvested from secondary recipients. (Upper) Fractions of cells in generations 3 to 6, 4 days after incubation with CFSE. Shown are results of mice injected with 2 × 106 sgGFP (n = 2 mice, 3 cultures per sample) or 2 × 106 sgHLF cells (n = 3 mice, 3 cultures per sample). *P < .05. (Lower) Representative histograms showing distribution of generations 4 days after experiment start. Blue bar indicates starting CFSE intensity, green bar indicates background fluorescence intensity. Numbers indicate mouse IDs. Data from recipients of 105 cells available in supplemental Figure 7A-B. (B) Representative FACS plots of EdU experiment performed with sgGFP and sgHLF cells harvested from secondary recipients injected with 105 cells. Shown is distribution of cells in different cell cycle phases after 90-minute pulse with EdU (t0) and 14 hours later (t14h). Numbers indicate percentages of total. 4′,6-diamidino-2-phenylindole was used to determine DNA content, EdU was detected in fluorescein isothiocyanate channel. (C) EdU experiment performed with cells harvested from secondary recipients injected with either 2 × 106 cells (sgGFP, n = 2; sgHLF, n = 3) or 105 cells (sgGFP, n = 3; sgHLF, n = 3). Shown are mean percentages (and standard deviations) of cells in the indicated cell cycle phases after 90-minute EdU pulse (upper) and after 14 hours (lower) of all viable cells. *P < .05; **P < .005; ***P < .0005. (D) Compound sensitivity testing. Cells harvested from secondary recipients injected with either 2 × 106 cells (sgGFP n = 4 cultures from 2 different mice; sgHLF, n = 6 cultures derived from 3 different mice) or 105 cells (n = 6 cultures derived from 3 different mice per group) were exposed to 50 nM AraC, 50 nM daunorubicin, or 2 µM etoposide compared with DMSO (vehicle) for 5 days. Viable cell counts on day 5 were normalized to cell counts in DMSO. *P < .05; ***P < .0005. (E, left) Volcano plot showing log2-fold changes in mRNA expression (x-axis) and transformed P values (y-axis) for RNA-Seq data performed on triple-mutated cells after 1 round of in vivo expansion, followed by infection with shRNAs against HLF (shHLF.630, n = 3) or shLuc control (n = 3). Data points highlighted by colors represent genes with log2-fold change more than 1 (blue) or less than −1 (red) and FDR<10%. Because of space constraints, not all gene symbols are displayed. See also supplemental Table 10 for gene names. (Right) Mean RPKM values for HES1 (upper) and CDKN1C (lower), n = 3 per group. *P < .05. (F) Expression levels in percentage of GAPDH expression determined by quantitative PCR (qPCR) for HES1 and CDKN1C in sgHLF vs sgGFP cells from primary recipients (upper, sgGFP: #247, #246 vs sgHLF: #197, #198, #199) and secondary recipients (lower, sgGFP: #8013, #8014 vs sgHLF: #8248, #8249). *P < .05; **P < .005. (G, upper) HLF, CDKN1C, and HES1 expression in percentage of GAPDH expression determined by qPCR in the model leukemia MN1/ND13 after lentiviral transduction with shRNA against HLF (sh.630) vs shLuc control after 1 round of in vivo expansion (n = 3 recipients). (Lower) HLF and HES1 expression in healthy cord blood CD34+ cells after lentiviral transduction with 2 different shRNAs against HLF or shLuc control 5 days after infection normalized to shLuc. Numbers indicate mean fractions of shLuc. (H) Kaplan-Meier survival curves showing overall survival from time of diagnosis in patients from the Leucegene prognostic cohort with very high (4th quartile, red line) vs low HLF expression (quartiles 1-3, blue line). Log-rank test. (I) Model indicating the proposed functional role of HLF in triple-mutated AML. HLF induces upregulation of HES1 and CDKN1C, decelerates cell cycle progression, and maintains the CD34+GPR56+ compartment in vivo. Gen, generation; K, thousand; M, million.