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Nakauchi, Y.1; Thomas, D.1; Sharma, R.1; Corces, R.1; Reinisch, A.1; Cruz-Hernandez, D.1; Koehnke, T.1; Karigane, D.1; Fan, A.1; Cao, J.2; Godley, L.2; Majeti, R.1

doi: 10.1097/01.HS9.0000561624.96745.6b
Simultaneous Sessions II: Acute myeloid leukemia - Biology & translational research - Towards molecular therapies

1Hematology, Stanford University, Stanford

2Hematology/Oncology, University of Chicago, Chicago, United States

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The TET2 gene is frequently mutated in pre-leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), a key intermediate for DNA demethylation. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5-methylcytosine can be blocked with azacitidine.

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To understand the mechanisms of TET2 mutation-driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ stem and progenitor cells.

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First, in “Hit & Run”, we use Cas9 with two single-guide RNAs to delete an internal region and disrupt TET2. Second, we use homology-directed repair (HDR) of Cas9-mediated double-strand breaks to disrupt TET2 by inserting GFP and/or mCherry expression cassettes to generate in vivo traceable cells. Global 5-hmC profiling revealed time-dependent loss of hydroxymethylation after 6 weeks of in vitro culture at predominantly genes and promoters, validating loss of TET2 enzymatic function. Thus, we have developed a tractable and cell-traceable model that recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis.

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First, we examined the biological effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both methods of TET2 disruption decreased CD71+CD235+ erythroblasts compared to control safe-harbor disrupted cells. Notably, exposure to ascorbate (HDR, n = 6, p < 0.02) or azacitidine (HDR, n = 4, p < 0.05) partially rescued the erythroid differentiation block. TET2 disruption consistently abolished erythroid colonies in methylcellulose and increased the numbers of granulocyte-macrophage colonies upon serial re-plating (HDR, n = 4 independent experiments, p < 0.0001).

In vivo, transplantation of TET2-disrupted Hit & Run CD34+ cells into NSG mice showed gradual expansion of TET2-disrupted cells in the CD33+ myeloid population. At 36 weeks after secondary transplantation, we detected a marked expansion of human myeloid lineage cells in keeping with a time-dependent myeloid skewing induced by TET2 mutation (lymphoid = 22.1%, myeloid = 73.0%, Mann-Whitney U, p = 0.0485).

We therefore performed in vivo competition studies to determine if TET2-disrupted HSPCs could be selectively targeted by azacitidine or ascorbate treatment. In PBS control treated mice, the percentage of TET2-disrupted cells increased from 29.3 to 71.6 over 4 weeks. Intriguingly, azacitidine slowed the expansion of TET2-disrupted cells in evaluable mice (delta increase of 42% in PBS vs 5% in azacitidine, p = 0.036), but did not eradicate established TET2 pre-leukemia. Similarly, high dose ascorbate treatment slowed the rate of expansion to a lesser degree (delta increase of 42% in PBS vs 18.3% in ascorbate, p = 0.14). Combination studies of both drugs in a larger cohort are ongoing coupled with global 5-hmC profiling.

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Our results indicate that disruption of TET2 in human hematopoietic stem and progenitor cells is sufficient to induce global changes in 5-hmC and suggest azacitidine or ascorbate treatment can slow the expansion of TET2-mutant human pre-leukemic clones suggesting an approach to preventing CHIP progression to de novo AML.

Copyright © 2019 The Authors. Published by Wolters Kluwer Health Inc., on behalf of the European Hematology Association.