Expression of TCF3-ZNF384 in Human Hematopoietic Cells Induces Lineage Disruption in Vitro and Acute Leukemia in Vivo
Authors: Dickerson Kirsten , Yoshihara Hiroki , Janke Laura , Mullighan Charles G. 
Published: October 2019

Abstract
Introduction: Rearrangement of ZNF384, a transcription factor of poorly characterized function, defines a subtype of acute leukemia that may manifest as either B-ALL with aberrant myeloid marker expression or B/myeloid mixed phenotype acute leukemia (MPAL). Such leukemias are characterized by chromosomal rearrangements that result in the fusion of a diverse group of partners, often transcription factors or epigenetic modifiers, to ZNF384. Our prior studies have shown that ZNF384 B-ALL and B/myeloid MPAL are genomically indistinguishable, and that the fusion may be identified in a subset of hematopoietic stem cells, suggesting that the acquisition of a ZNF384 fusion in a primitive progenitor directly perturbs hematopoietic differentiation. The goals of this study were to determine the effect of expression of ZNF384 rearrangements on human hematopoietic stem and progenitor cell differentiation in vitro and in vivo, using TCF3-ZNF384 as a commonly observed exemplar of this form of leukemia.
Methods: For in vitro experiments, human CD34+ cord blood cells were sorted into stem and progenitor populations (hematopoietic stem cell (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP)) and lentivirally infected with wild type ZNF384, TCF3-ZNF384, or vector control. Single cells were sorted onto an MS-5 stromal layer and the immunophenotype of colonies was determined 15 days later by flow cytometry. In vivo studies were performed by sorting human CD34+ cord blood cells into stem-enriched (CD34+CD38-) or progenitor-enriched (CD34+CD38+) populations and lentivirally infecting with wild type ZNF384, TCF3-ZNF384, or vector control. Cells were transplanted into sub-lethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3) mice.
Results: Single-cell MS-5 stromal experiments revealed that expression of the fusion protein perturbed hematopoietic differentiation. In all stem and progenitor populations, cells expressing TCF3-ZNF384 lost the ability to differentiate into erythroid colonies. HSC, MPP, and CMP cells expressing the fusion most commonly form undifferentiated, CD45+, CD33+ colonies. Additionally, GMP and MEP cells expressing the fusion lost their ability to form colonies. Human CD34+ cells expressing TCF3-ZNF384 successfully initiate leukemia in NSG-SGM3 mice with a median latency of 123 days. Mice presented with anemia and pathological analysis using hematoxylin and eosin staining showed infiltration of leukemic cells into the bone marrow, spleen, liver, central nervous system, and ovary. Additionally, CD33, myeloperoxidase, and major basic protein staining confirmed myeloid leukemia with a subset of eosinophil differentiation.
Conclusion: Our results demonstrate that hematopoietic lineage determination is altered by the expression of TCF3-ZNF384 in human stem and progenitor cell populations. Additionally, we have created the first model of TCF3-ZNF384 leukemia which mimics the complexity of lineage deregulation in ZNF384-rearranged leukemia.

Disclosures
Mullighan: Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding.