Decreased Leukemia Initiating Cells in hdac1 Haploinsufficient T Cell Acute Lymphoblastic Leukemia
Authors: Pruitt Margaret M. , McConnell Sean C. , Restaino Anthony , Marin Wilfredo , de Jong Jill L. O. 
Published: October 2019

Abstract
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for 15% of pediatric and 25% of adult ALL cases. While initial treatment of T-ALL has improved, relapse is common and is associated with a poor prognosis. As with other tumors, T-ALL is genetically heterogeneous and relapse is driven at least in part by a subpopulation of cells called leukemia initiating cells (LICs). Capable of regrowing the entire tumor from a single cell, failure to eliminate these LICs is hypothesized to be the major determinant of relapse. Therefore understanding the genetic mechanisms that drive LICs may lead to new therapeutic targets that are likely to enhance rates of cure. The data presented here indicate that histone deacetylase 1 (hdac1) is an important regulator of LICs in T-ALL.
Histone deacetylases (hdacs) modify chromatin structure and regulate gene expression by removing acetyl groups from histones and other proteins. As hdacs are aberrantly expressed in hematopoietic malignancies, and hdac inhibitors are used to treat some cancers, we hypothesized that hdac1 might play a role in leukemogenesis. To explore this question, we generated T cell acute lymphoblastic leukemias by overexpressing the murine c-Myc oncogene and the fluorochrome mCherry under the rag2 promoter in hdac1 haploinsufficient zebrafish. Tumor incidence and latency were not significantly different for hdac1+/- and wild type (WT) tumors. Mean tumor latency was 42.3 days post fertilization (dpf) for hdac1+/- fish and 47.9 dpf for their WT siblings. Likewise overall survival was not different. Mean survival was 58.0 dpf for hdac1+/- fish, and 64.6 dpf for WT fish.
In contrast, when primary leukemia cells were transplanted into syngeneic recipient fish, the tumor cells from hdac1 haploinsufficient fish grew at a slower rate when compared to tumor cells from WT fish. After transplanting 1x105 primary WT or hdac1+/- tumor cells intraperitoneally into syngeneic zebrafish recipients, the transplanted WT tumors grew more aggressively compared to the hdac1+/- tumors. By 21 days post transplant, 92% of the WT tumors (n=22 of 24) had spread from the site of injection into the thymus and other organs, while most of the hdac1+/- tumors were only growing at the site of injection. Only 8.3% (n=1 of 12) of the hdac1+/- tumors had spread past the local injection site at this time point. We found that the slower rate of growth was not due to differences in proliferation, as determined by EdU incorporation. Hdac1+/- tumors had a mean percentage of EdU incorporation of 6.0 ± 4.0% (n=12), and WT tumors had 6.4 ± 4.4% (n=7); (p=0.8). In contrast, limit dilution transplant assays showed a higher frequency of leukemia initiating cells in the WT tumors (1 in 44 cells) compared with the hdac1+/- tumors (1 in 135 cells) (p<0.05). RNA-sequencing and qPCR analyses have been employed to determine gene expression differences between hdac1+/- and WT tumors. Transcriptome analysis has identified 1731 genes that are differentially expressed (p<1x10-5), 674 of which have at least a 5-fold difference in expression. Of these genes, 261 exhibited decreased expression in hdac1+/- tumors, and 413 exhibited increased expression in hdac1+/- tumors. Experiments are ongoing to elucidate the core molecular mechanisms that inhibit LICs in hdac1 haploinsufficient cells. These results provide a starting point to identify new therapeutic targets for T-ALL.

Disclosures
de Jong: National Medical Consultants, P.C.: Consultancy.