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. 2017 Aug 3;130(5):619-624.
doi: 10.1182/blood-2016-09-738591. Epub 2017 Jun 14.

ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells

Brian S Garrison  1   2   3 , Adrian P Rybak  1   2   3 , Isabel Beerman  4 , Balthasar Heesters  5 , Francois E Mercier  2   6 , David T Scadden  2   6   7 , David Bryder  8 , Roland Baron  9   10   11 , Derrick J Rossi  1   2   3   7
Affiliations

ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells

Brian S Garrison et al. Blood. .

Abstract

The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521/Zfp521 as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521-deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
ZFP521 regulates murine HSC self-renewal and differentiation. (A) Schematic representation of serial competitive transplantation studies. (B-D) Peripheral blood analysis of primary transplant recipients (10 recipients per genotype) of WT (Zfp521+/+) and Zfp521 KO (Zfp521−/−) whole fetal liver cells showing (B) total donor chimerism; (C) donor contribution to myeloid, B, and T cells; and (D) granulocyte chimerism. (E-H) Bone marrow analysis at 16 weeks posttransplantation showing (E) total chimerism and frequencies of (F) HSCs, MPPs (MPP1 and MPP2), (G) CMPs, GMPs, megakaryocyte-erythroid progenitors (MEPs), and (H) CLPs. Peripheral blood analysis of secondary transplant recipients (10 recipients per genotype) showing (I) total donor chimerism; (J) donor contribution to myeloid, B, and T cells; and (K) granulocyte chimerism. (L-P) Bone marrow analysis at 16 weeks posttransplantation showing (L) total chimerism and frequencies of (M) HSCs, MPP1, and MPP2; (N) CMPs, GMPs, and MEPs, (O) megakaryocyte progenitors, and erythroid progenitors, and (P) CLPs. Unpaired t test: *P < .05, **P < .01, ***P < .001. BM, bone marrow; EP, erythroid progenitor; Gr, granulocyte; MkP, megakaryocyte progenitor; PB, peripheral blood; wBM, whole bone marrow.
Figure 2.
Figure 2.
ZFP521 regulates self-renewal ex vivo and facilitates MLL-AF9–mediated AML leukemia in mice. (A) Serial replating assays of HSCs transduced with lentiviruses encoding ZsGreen (control), ZFP521, or ZFP521(ΔNuRD). (B) Representative histograms (left) and quantitation (right) of DNA content per cell cycle from 3 independent experiments. The percentage of cells in S/G2/M phase of the cell cycle is presented as mean ± SEM. Unpaired t test: ***P < .001. (C) Wright-Giemsa staining (G, granulocyte; M, macrophage; PL, progenitor-like cell) of cultured HSC-derived cells ectopically expressing ZsGreen (control), ZFP521, or ZFP521(ΔNuRD). Scale bar represents 50 µm. (D) Heatmap showing relative expression (red [high expression] to blue [low expression]) of 26 conserved HSC-enriched genes, including the 6 indicated transcription factors within patient samples from 4 AML cytogenetic subtypes. See supplemental Table 4 for a complete gene list. (E-F) Kaplan-Meier survival curves following (E) primary and (F) secondary transplantation of MLL-AF9–transduced cells on a WT or Zfp521-deficient (KO) background, with the number (n) of mouse recipients indicated per group. Log-rank (Mantel-Cox) testing was conducted for statistical analyses; *P < .05, **P < .01. (G) Serial plating of bone marrow–derived MLL-AF9–transduced cells. Number of colonies at each passage is presented as mean ± SEM of 3 independent experiments. Unpaired t test: *P < .05, **P < .01. (H) Relative cell growth of MLL-AF9–positive (NOMO-1 and THP-1) or non–MLL-AF9 AML cells (OCI-AML3, HL-60, and U937) following doxycycline (+DOX)–induced shRNA-mediated ZNF521 knockdown (shZNF521) or scramble shRNA (shCONTROL) compared with the uninduced (−DOX) condition. Relative cell growth (+DOX/−DOX) values are presented as mean ± SEM of 3 independent experiments. Unpaired t test: *P < .05, **P < .01.
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