S836 AZACITIDINE AND ASCORBATE INHIBIT COMPETITIVE OUTGROWTH OF HUMAN TET2‐MUTANT HSPCS IN A XENOGRAFT MODEL OF PRE‐LEUKEMIA
Authors: Y. Nakauchi, D. Thomas, R. Sharma, R. Corces, A. Reinisch, D. Cruz‐Hernandez, T. Koehnke, D. Karigane, A. Fan, J. Cao, L. Godley, R. Majeti
Journal: HemaSphere (2019)

Background: 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. Aims: 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. Methods: First, in “Hit &amp; 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. Results: 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 &lt; 0.02) or azacitidine (HDR, n = 4, p &lt; 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 &lt; 0.0001). In vivo , transplantation of TET2 ‐disrupted Hit &amp; 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. Summary/Conclusion: 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.