Page 75 - Haematologica-April 2018

Gfi1b in AML and MDS
(Figure 4G and data not shown). We did not observe a sig- nificant change in the number of overall myeloid cells or apoptosis level in the different settings (Figure 4H and Online Supplementary Figure S6A-D).
Loss of Gfi1b increases the number of LSCs
Loss of Gfi1b leads to an expansion in the number of functional HSCs;6 therefore we investigated whether the same applies to LSCs. We performed a limiting dilution assay by transplanting MLL-AF9 leukemic BM cells derived from poly(I:C)-treated Gfi1bfl/flMxCrewt or Gfi1bfl/flMxCretg leukemic mice into sublethally irradiated congenic mice (Figure 5A). Gfi1b-deficient MLL-AF9 BM cells had a LSC frequency of 1:3500 compared with an LSC frequency of 1:63000 cells in Gfi1b-expressing leukemic cells (Figure 5B). The increased number of func-
tional LSCs in Gfi1b-deficient leukemic cells could explain why loss of Gfi1b accelerated disease progression, as it has already been shown that a higher number of LSCs is asso- ciated with a poor prognosis of leukemia patients.37 However, this hypothesis needs to be confirmed in inde- pendent experiments.
Loss of Gfi1b induces gene expression changes supporting AML development
To further study the molecular function of Gfi1b in AML, we performed whole genome gene expression analysis using Gfi1b-expressing and Gfi1b-deficient NUP98/HOXD13tg leukemic mice (Figure 6A). This model was used since the difference between Gfi1b-deficient and Gfi1b-expressing leukemic cells was most striking in the NUP98/HOXD13tg mouse model. Using gene set enrich-
A
B
D
E
C
FG Giemsa staining of bone marrow (BM) cytospins from representative Gfi1bwt/wtMxCretg and Gfi1bfl/flMxCrewt leukemic mice transgeni- cally expressing Kras after poly(I:C) administra- tion (bar=20 mm). (D) Flow cytometric analysis of the BM from the leukemic mice shown in (B) with regard to Gr-1 and Mac-1 expression. (E) Isolation, transduction and transplantation of
H
Figure 4. Absence of Gfi1b accelerates the
progression of myeloproliferative disorder and
acute myeloid leukemia (AML). (A) Crossing of
the Gfi1bwt/wtMxCretg and Gfi1bfl/flMxCretg
mouse strains with the Kras+/fl mouse model. wt tg
(B) Survival of Gfi1b /wtMxCre and Gfi1bfl/flMxCretg mice transgenically expressing
Kras after poly (I:C)
****P<0.0015. Numbers indicate the num- ber of mice succumbing to AML. (C) Wright-
lineage-negative (Lin-)
Gfi1bfl/flMxCrewt and Gfi1bfl/flMxCretg mice with MLL-AF9-expressing retrovirus. After Cre-medi- ated deletion of the Gfi1b gene upon poly(I:C) administration, the mice were monitored for signs of leukemia. (F) Survival of the mice
transplanted with
Gfi1bfl/flMxCretg MLL-AF9 transduced cells; ***P=0.0002. Number of mice succumbing to AML is indicated. (G) Wright-Giemsa staining of BM cytospins from the leukemic mice described in (F) (bar=20 mm). (H) Flow cyto- metric analysis of the BM from the leukemic mice described in (F) with regard to Gr-1 and Mac-1 expression.
administration;
cells from Gfi1bfl/flMxCrewt and
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