Page 109 - Haematologica June

Figure 2. High leukemia-initiating cell activity is associated with increased cell cycle activity. (A) Higher phosphorylated histone 3 (P-H3; Ser10)-positive cells in active mitosis in TTLshort (n=10) as compared to TTLlong leukemia samples (n=10), Mann-Whitney U-test; the line represents the median; P=statistical significance. (B and C) Higher bromodeoxyuridine (BrdU) uptake (B) and increased decline (C) after in vivo labeling as detected by flow cytometry of ALL cells in TTLshort/high LIC fre- quency compared to TTLlong/low LIC frequency ALL bearing recipients (n=3/time point; biological replicates). Percentages of huCD19+/BrdU+ cells in bone marrow (BM) and spleen of ALL bearing recipients (mean ±SD). Unpaired t-test with Welch correction (two-tailed); P= statistical significance; *≤0.05; n.s.: not significant. (D) Similar high leukemia load in recipients used for in vivo proliferation analysis; percentages of huCD19+ ALL cells in spleen and BM over time in recipients (n=3 per group; biological replicates) bearing a TTLshort or TTLlong leukemia (mean ± Standard Deviation).
haematologica | 2018; 103(6)
D
Leukemia initiating cells in ALL
activity upon transplantation into subsequent mice, irre- spective of the originating cell cycle compartment and TTL phenotype (Online Supplementary Table S3), leading to full-blown leukemia of the initial common-ALL immunophenotype. Interestingly, of the four cell cycle fractions transplanted, G1blow cells showed quickest engraftment and were associated with the shortest leukemia-free survival. This feature was observed in sam- ples with both short and long TTL phenotypes, suggesting that the high leukemia repopulating and initiating capaci- ty of G1blow cells is a general feature of this early cell cycle leukemia cell subfraction. Importantly, despite these dis- tinct repopulating activities of ALL cells from different cell cycle subgroups, the overall short or long leukemia engraftment of unfractionated leukemia cells was recapit- ulated in the sorted subfractions. Moreover, G2/M cells were always the last to engraft, leading to longer leukemia free survival of the recipient animals (Figure 3B). In addi- tion, no differences in the expression of lineage/stem cell markers huCD19, huCD38, huCD10 or huCD34 were observed on ALL cells of either G1blow or G2/M cell frac- tions (Online Supplementary Figure S2). Most interestingly, the distinct engraftment of G1blow versus G2/M cells was also retained upon secondary transplantation of G1blow- and G2/M-derived, unsorted bulk leukemia cells, indicat- ing maintenance of LIC-capacities in the bulk of G1blow- and G2/M-derived cells (Figure 3C and Online Supplementary Table S3).
AB
C
G1blow acute lymphoblastic leukemia cells are characterized by a stem cell signature
To further corroborate our observation of higher LIC activity in G1blow leukemia cells as suggested by our func- tional in vivo data, we investigated transcriptional signa- tures of sorted G1blow and G2/M leukemia cells (pairs of 4 samples; TTLshort= 1; TTLlong= 3). We identified 865 genes (1122 probe sets, false discovery rate q-value <0.05) as being differentially regulated between G1blow and G2/M subpopulations irrespective of their TTL phenotype, with 330 up- and 535 down-regulated genes in G1blow, which were mainly attributed to cell cycle regulation (Figure 4A and Online Supplementary Tables S4 and S5). Interestingly, gene set enrichment analysis identified a positive enrich- ment of 4 out of 16 gene sets previously associated with stem cell activity with the G1blow/high LIC-enriched cells (Figure 4B and Online Supplementary Table S6A), whereas genes sets from short-term HSC or mature cells were found to be positively enriched in G2/M/low LIC8,25-29 (Figure 4B and Online Supplementary Table S6B). Thus, these gene expression data further support the higher LIC activity of G1blow cells observed upon transplantation.
Different cell death sensitivities of cell cycle annotated acute lymphoblastic leukemia cells
Cancer initiating or stem cells have been described to be characterized by an increased resistance to anti-tumor therapies. To investigate this issue, we studied the intrin-
1011