In Section 5.1 we showed that physiological expression of s-Prdm16 in HSCs shortens the latency of resulting MLL-AF9 leukemia. Prdm16 expression, however, was downregulated in the MLL-AF9- transformed cells. Nonetheless, PRDM16 is often expressed in leukemic cells. Our previous data did not investigate the effect of forced Prdm16 expression. We therefore retrovirally overexpressed individual
Prdm16 isoforms in MLL-AF9 cells using an MSCV-IRES-GFP construct (vector maps in Figure 5-7A). We
used the Prdm16fl/fl.Vav-Cre+ background which allowed for the forced expression of one Prdm16 isoform without a risk of endogenous expression of the other, to distinguish the individual biological effects of each isoform. Furthermore, to mitigate potential artifacts from retroviral insertion at different sites, we expressed Prdm16 isoforms after MLL-AF9 immortalization, thereby using the same pool of leukemic cells for transduction of either empty vector, f-Prdm16, or s-Prdm16. This is illustrated schematically in Figure
5-7B. Cells transduced with either of these three constructs had similar levels of GFP expression (Figure 5-7C) and there were also comparable levels of mRNA expression in the Prdm16-overexpressing samples
as measured by subtractive qPCR (Figure 5-7D), indicating that the two Prdm16 isoforms were overexpressed at similar levels.
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Figure 5-7: Forced expression of Prdm16 isoforms in Prdm16fl/fl.Vav-Cre+ MLL-AF9 cells. (A) Maps of retroviral Prdm16 expression vectors used in MLL-AF9 leukemia studies. (B) Schematic of MLL-AF9 transformation of HSCs followed by forced expression of either empty vector, f-Prdm16-GFP, s-
Prdm16-GFP, or s-Prdm16-GFP/f-Prdm16-RFP. (C) Quantification of GFP mean fluorescence intensity (MFI)
of Prdm16fl/fl.Vav-Cre MLL-AF9 cells expressing either empty vector, f-Prdm16-GFP or s-Prdm16-GFP (n = 3). (D) Quantitative PCR of MLL-AF9 leukemia expressing either empty vector, f-Prdm16, or s-Prdm16, using probes for total Prdm16 (exon 14/15 junction, left panel) or f-Prdm16-only (exons 2/3, right panel) demonstrating selective expression of the correct isoform and similar total expression levels. (n.s – P > 0.05, One-way ANOVA for multiple comparisons).
Forced expression of either f-Prdm16 or s-Prdm16 did not have an effect on in vitro growth of MLL-AF9 cells in colony forming assays (Figure 5-8A). This finding was consistent with the lack of an in vitro growth phenotype in MLL-AF9 cells with Prdm16 deletion. However, transplantation of recipient mice with these cells revealed that forced expression of s-Prdm16 shortened latency, to near the latency of WT leukemia, whereas overexpression of f-Prdm16 further increased leukemic latency beyond that observed with the empty vector Prdm16fl/fl.Vav-Cre+ MLL-AF9 cells (Figure 5-8B). Co-expression of both s-Prdm16 and
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differences in leukemic latency could be ascribed to defects in engraftment into recipient bone marrow, we measured engraftment of MLL-AF9 cells 24 hours after transplantation. We found that
f-Prdm16-overexpressing MLL-AF9 cells did not have an engraftment defect and in fact homed more
effectively to bone marrow than leukemic cells expressing s-Prdm16 (Figure 5-8C). Additionally,
s-Prdm16-overexpressing MLL-AF9 populations had fewer cycling cells than those expressing either f-Prdm16 or empty vector, eliminating defects in proliferation as a cause of extended latency in f-Prdm16-expressing leukemia (Figure 5-8D). These findings suggest an oncogenic role for s-Prdm16 and
a tumor suppressor role for f-Prdm16 after forced expression in MLL-AF9 that is unrelated to differences in engraftment potential or cycling.
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Figure 5-8: Forced expression of s-Prdm16 and f-Prdm16 have opposite effects on MLL-AF9 latency. (A)
Colony-forming assays of Prdm16-deficient Vav-Cre+/- Prdm16fl/fl (KO) MLL-AF9 cells expressing empty vector, f-Prdm16, or s-Prdm16. (n = 3 independent assays in duplicate). (B) Survival of lethally irradiated mice transplanted with the MLL-AF9 cells described in (A), as well as a KO MLL-AF9 line co-expressing both
Prdm16 isoforms. (n = 14-15 recipients from 3 independent experiments). (C) Percent of MLL-AF9 cells in
bone marrow of recipient mice 24-hours post-transplant. (n = 9 recipients, 3 independent transplants). (D) Percentage of KI-67+ ex vivo MLL-AF9 cells isolated ex vivo from moribund mice transplanted MLL-AF9 cells described in (A) (n = 4 recipients). (n.s = P > 0.05; * = P < 0.05; ** = P < 0.01; *** = P < 0.001, One- way ANOVA for multiple comparisons, Gehan-Breslow-Wilcoxon test for comparison of survival curves)
We performed bone marrow smears from moribund mice to check for visible differences between leukemic cells expressing either f-Prdm16 or s-Prdm16 compared to empty vector. Hematoxylin/eosin staining showed that in MLL-AF9 leukemias overexpressing s-Prdm16 in comparison to empty vector or
f-Prdm16, there was a significant increase in the fraction of cells with abnormal or fragmented nuclei
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showing dysplastic changes in AML with PRDM16 translocations in which the PR domain is absent362,363,365,366.
Figure 5-9: Dysplastic phenotype of MLL-AF9 cells expressing s-Prdm16. (A) Percent of cells with
abnormal (elongated or multi-lobed) nuclei in bone marrow of leukemic mice transplanted with
Prdm16fl/fl.Vav-Cre MLL-AF9 cells expressing either empty vector, f-Prdm16-GFP or s-Prdm16-GFP (n = 4 fields from 3 independent mice, each field containing at least 50 bone marrow cells, (** = P < 0.01, One- way ANOVA for multiple comparisons). (B) Representative images of the data presented in (A). White arrows indicate abnormal nuclei.
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