AZACITIDINE AND ASCORBATE INHIBIT COMPETITIVE OUTGROWTH OF HUMAN TET2-MUTANT HSPCS IN A XENOGRAFT MODEL OF PRE-LEUKEMIA
Author(s): ,
Yusuke Nakauchi
Affiliations:
Hematology,Stanford University,Stanford,United States
,
Daniel Thomas
Affiliations:
Hematology,Stanford University,Stanford,United States
,
Rajiv Sharma
Affiliations:
Hematology,Stanford University,Stanford,United States
,
Ryan Corces
Affiliations:
Hematology,Stanford University,Stanford,United States
,
Andreas Reinisch
Affiliations:
Hematology,Stanford University,Stanford,United States
,
David Cruz-Hernandez
Affiliations:
Hematology,Stanford University,Stanford,United States
,
Thomas Koehnke
Affiliations:
Hematology,Stanford University,Stanford,United States
,
Daiki Karigane
Affiliations:
Hematology,Stanford University,Stanford,United States
,
Amy Fan
Affiliations:
Hematology,Stanford University,Stanford,United States
,
John Cao
Affiliations:
Hematology/Oncology,University of Chicago,Chicago,United States
,
Lucy Godley
Affiliations:
Hematology/Oncology,University of Chicago,Chicago,United States
Ravindra Majeti
Affiliations:
Hematology,Stanford University,Stanford,United States
EHA Library. Nakauchi Y. Jun 15, 2019; 267419; S836
Yusuke Nakauchi
Yusuke Nakauchi
Contributions
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Abstract

Abstract: S836

Type: Oral Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 12:30 - 12:45

Location: Forum Hall

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 & 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<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.

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.

Session topic: 3. Acute myeloid leukemia - Biology & Translational Research

Keyword(s): AML, Clonal expansion, Hematopoietic stem and progenitor cells, Methylation

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