Modulation of p210BCR-ABL activity in transduced primary human hematopoietic cells controls lineage programming

Abstract

IntroductionThe hematopoietic system is composed of a hierarchy of progenitors with the most primitive stem cells uniquely characterized by their combined capacity for lymphomyeloid lineage differentiation, extensive self-renewal, and, on transplantation, lifelong maintenance of blood cell production. The mechanisms regulating the initial differentiation steps these cells undergo are still poorly understood, although considerable evidence now indicates that this occurs in a consistently ordered fashion to give a relatively simple picture of progressive restriction in differentiation ability to specific lineages. 1 Thus it has been possible to define a series of phenotypic changes that distinguish progenitors with different lineage options [2][3][4] and to identify a number of transcription factors whose presence (or absence) is important to the ability of early hematopoietic cells to undertake specific differentiation steps. 1,5 To date, dedifferentiation or transdifferentiation during normal hematopoiesis has not been described, although examples of such events have recently been reported to occur in neural cells 6,7 or in occasional examples of transformed hematopoietic cells. [8][9][10][11][12][13] The BCR-ABL oncogene is a fusion gene that characterizes the neoplastic clone of patients with chronic myeloid leukemia (CML). 14,15 It encodes a 210-kd oncoprotein (p210 BCR-ABL ) 16 with constitutively active tyrosine kinase activity 17 and an abnormal cytoplasmic location. 18 Both of these features are important to its transforming activity in model systems. [19][20][21][22][23][24] The BCR-ABL oncoprotein is also known to perturb many intracellular signaling pathways, including the RAS, mitogen-activated protein (MAP) kinase, phosphatidylinositol-3Ј kinase (PI3-K), janus-kinase/signal transducer and activator of transcription (JAK/STAT), and MYC pathways (for a review, see Deininger et al 25 ). The development of packaging cells that allow production of high titer preparations of p210 BCR-ABL complementary (cDNA)-containing retroviruses has greatly facilitated analysis of the effects of this gene in primary hematopoietic cells. These have shown that BCR-ABL-transduced mouse bone marrow cells produce a rapidly fatal CML-like myeloid leukemia on their transplantation into irradiated recipients. [26][27][28] However, the speed with which the leukemia develops is clearly not comparable to the approximately 7 year average duration of the latent phase of human CML. 29 This discrepancy in disease kinetics might be caused by species-specific effects of p210 BCR-ABL or, alternatively, by differences in BCR-ABL message levels determined by differences in the promoters active in the 2 systems (the endogenous BCR promoter in human CML cells and the long terminal repeat [LTR] For personal use only. on April 5, 2019. by guest www.bloodjournal.org From retrovirus. This resulted in the unexpected but consistent observation of a rapid outgrowth of an erythroid population. Additional experiments designed to investigate the bas...

Growth autonomy and lineage switching in BCR-ABL-transduced human cord blood cells depend on different functional domains of BCR-ABL

“…Interestingly, within the CD34 þ subset of CB cells, the % GFP þ cells 2 days post-transduction was the same for both vectors (772 and 871%), perhaps due to a selectively enhanced early rate of amplification of an initially lower fraction of P210-expressing CD34 þ cells. 31 The rapidity of the effect of p210 BCR-ABL on the growth rate of the transduced CB cells was seen also in the present experiments (Figure 2a), where a 50-fold greater net expansion of the P210-transduced (sorted GFP þ ) cells vs MIG-transduced (sorted GFP þ ) cells over a 14-day period can be seen (100 000-fold vs 2000-fold, Po0.05, n ¼ 3).…”

“…Colony-forming cell (CFC) assays were performed in SFM-containing methylcellulose cultures (Methocult H4100, StemCell Technologies) supplemented or not with human SF (50 ng/ml) and 20 ng/ml each of human IL-3, IL-6, G-CSF and GM-CSF73 U/ml human Epo (StemCell Technologies) as described previously. 31 …”

“…We have previously shown that this lineage switching response is due to an ability of forced expression of P210 in uncommitted primary human progenitors to favor selection of the erythroid lineage, and a re-activation of erythroid potential and suppression of granulopoietic potential in progenitors otherwise thought to be already restricted to the granulocyte-macrophage pathway. 31 Rapid induction of expression of selected growth factors in P210-transduced human CD34 þ CB cells…”

“…17 Forced expression of BCR-ABL in primary human hematopoietic cells offers a powerful strategy for creating new models of human CML to interrogate mechanisms of disease genesis and the effects of novel therapeutics. To date, the use of this approach has been limited, but perturbations in the adhesive properties of the transduced cells 30 and their lineage commitment 31 have been documented in transduced human cord blood (CB) cells. Here we show that these cells, like their murine counterparts, 27 rapidly acquire a growth factor-independent phenotype concomitant with the activation of IL-3, G-CSF and Epo expression.…”

Investigating human leukemogenesis: from cell lines to in vivo models of human leukemia

“…Expression of TEL-JAK2, BCR-ABL or FLT3-ITD all result in extensive erythropoietin-independent erythropoiesis in vitro, a finding that had not been noted in previous work with mouse models or patient samples. 79,72,76 Together, these reports suggest that activated tyrosine kinases, when expressed at high levels in human hematopoietic progenitor cells by viral vectors, drive EPO-independent erythropoiesis at the expense of myelopoiesis, an effect which appears to be mediated at least in part by the constitutive activation of STAT5 and the downregulation of C/EBPa. 85 …”