Page 235 - 2021_03-Haematologica-web

Letters to the Editor
Epigenetic and functional changes imposed by NUP98-HOXA9 in a genetically engineered model of chronic myeloid leukemia progression
NUP98-HOXA9 (“NA9”) is a fusion gene commonly found, together with other mutations, in human acute myeloid leukemia cells, and occasionally in blast-phase (BP) cells of patients with chronic myeloid leukemia (CML) in whom disease progression has occurred.1 These findings underscore interest in elucidating the role of NA9 in contributing to an aggressive leukemic program. Previous studies addressing this issue reported biological and molecular consequences of NA9 alone in human cells, including normal human hematopoietic cells,2,3 and evidence of a co-operative role of NA9 with BCR-ABL1 in experimentally-induced models of leukemia in mouse cells.4-6 We now report the results of assessing the biolog- ical and molecular consequences of lentivirally-intro- duced expression of NA9 in freshly isolated CD34+ CML cells from patients with chronic phase (CP) disease (for a general outline and technical details, see Online Supplementary Figure S1A-E). Samples from patients with CP CML are known to contain variable ratios of normal and BCR-ABL1+/Philadelphia chromosome-positive cells,
particularly in the most primitive cell compartments.7 Therefore, for this study, we selected samples from three CP-CML patients for whom we had previously shown the content of any type of normal CD34+ cells to be very low based on assessments of their progeny genotypes produced for >30 weeks (Online Supplementary Table S1).8
Initial in vitro experiments with these three CML patients’ CP cells showed that NA9-transduced cells (compared to control-transduced cells) produced particu- larly large expansions of granulopoietic colony-forming cells in growth factor-supplemented liquid suspension cultures, with somewhat reduced effects on expanding the outputs of these progenitors in 6-week long-term cul- tures on human growth factor-producing stroma, or in serial replating assays of the transduced cells cultured in semi-solid medium (Online Supplementary Figure S1F-H). However, in all cases, the progeny of the NA9-transduced CML cells ultimately underwent terminal differentiation (Online Supplementary Figure S1I), thus recapitulating results reported previously for NA9-transduced normal human CD34+ hematopoietic cells.2,9
Outputs of NA9- and control-transduced CD34+ cells from the same three CP-CML patients’ samples were also assessed in sublethally irradiated NOD-PrkdcscidIL2Rgc-/- mice engineered to produce human interleukin 3, granu-
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Figure 1. NA9 promotes disease progression in xenografts of transduced CD34+ chronic phase chronic myeloid leukemia cells. (A) Experimental design: 8-week old NSG-3GS mice were sublethally irradiated with 3.15 Gy of 137Cs g-rays. 2 x 106 cells of a mixture containing equal proportions of chronic phase (CP) chronic myeloid leukemia (CML) CD34+ cells exposed to control and NA9 virus were injected intravenously into ten mice (3 with cells from patient #1, 3 from patient #2, and 4 from patient #3). (B) Kinetics of % NA9+ (GFP+) cells and control (YFP+) cells in the blood (at week 3) and bone marrow (all other time points) of NSG-3GS mice transplanted with transduced CP CML CD34+ cells (left, patient #1; middle, patient #2; right patient #3. (C) Differences in bone marrow chimerism (left; paired t-test, P=0.002) and the proportion of mature granulocyte-macrophage (CD14+CD15+) cells (not significant, paired t-test, P=0.18) between control (YFP+) and NA9+ (GFP+) human cells 28 weeks after transplantation. (D) Survival curves for recipients of different patients’ samples (n=10 mice). (E) Representative flow cytometric analysis of two mouse bone marrow samples assessed 16 weeks after transplantation. The results indicate a predom- inant population derived from NA9-transduced (GFP+) cells (left histograms) of either the granulocyte (upper panels) or monocyte lineages (lower panel). YFP: yellow fluorescent protein, GFP: green fluorescent protein; FC: flow cytometry; RNA-Seq: RNA-sequencing; Chip-Seq: chromatin immunoprecipitation sequencing; CFC: colony-forming cells; SC: suspension culture; LTC: long-term culture; Pt: patient.
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