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Transforming activities of the NUP98-KMT2A fusion
Rosa26 locus allows doxycycline-regulated transgene expression (Figure 1A).19,22,23 We used primarily female mice to mitigate the effects of any potential sex-related differences in transgene expression.
Induction of iNUP98-KMT2A results
in an myelodysplastic syndrome-like disease in vivo
Primary-induced iNUP98-KMT2A mice developed signs of distress after a variable latency of 13-106 weeks (median latency=80 weeks, n=22) (Figure 1B). Blood values from non-induced iNUP98-KMT2A mice did not differ signifi- cantly from those of WT mice (Online Supplementary Figure S1A). We also explored disease induction by transplanting total BM from naïve (off doxycycline) iNUP98-KMT2A mice into lethally irradiated syngenic WT mice. In compar- ison to primary-induced mice, BM transplant recipient ani- mals developed the symptoms earlier (median latency=32 weeks, P=0.0007, log-rank test, n=5) (Figure 1B, Online Supplementary Table S3).
Most symptomatic primary-induced iNUP98-KMT2A mice had peripheral blood counts in the normal range with slightly increased numbers of white blood cells, and reticulocytes. Decreases were seen in hemoglobin levels and red blood cell numbers (P=0.049, unpaired t-test, n=15) (Figure 1C). Blood values from iNUP98-KMT2A BM transplant recipients revealed a similar trend to those of primary-induced samples in all parameters analyzed with significantly increased numbers of white blood cells (P=0.0006, unpaired t-test, n=5) and reduced cellular hemoglobin levels (P=0.03, unpaired t-test, n=5). Notably, the overall cellularity of the BM of primary-induced mice was reduced (P=0.0099, unpaired t-test, n=9) relative to controls. Further analysis of different BM subpopulations revealed decreased numbers of myeloid cells (Mac-1+/Gr- 1+), B cells (B220+), and T cells (CD3+) (Online Supplementary Figure S1B). Signs of intramedullary apop- totic cell death was found in some but not all mice as measured by combined PO-PRO-1 and 7-aminoactino- mycin D staining (Figure 1C) with non-significant increased apoptosis of CD71+Ter119+, but not of myeloid (Mac-1+, Gr-1+) or lymphoid (CD3+/CD8+/B220+) cells from primary-induced animals relative to WT controls (Online Supplementary Figure S1C). In primary induced mice we occasionally observed signs of dysplasia on blood smears, with the appearance of bi-lobed myeloid cells and polychromatophilic reticulocytes (Figure 1D) as well as signs of extramedullary hematopoiesis in the spleen and the liver (Figure 1E).
The BM of iNUP98-KMT2A mice that had been on doxy- cycline for several months displayed rather heterogeneous immunophenotypes: some showed an increase in Mac- 1+/Gr-1+ cells, whereas others showed a marked decrease in mature myeloid cells with concomitant increases in FcgRII/III+ and c-Kit- populations (Figure 1F, G).
To address potential reversibility, we transplanted iNUP98-KMT2A BM cells into lethally-irradiated WT recip- ients on doxycycline: upon development of symptoms in the first mouse, we reverted to non-doxycycline chow and followed the remaining mice for several months. Disease- free survival was significantly increased by the removal of doxycycline food (median latency=49 weeks, P=0.002, log- rank test, n=6) (Figure 1B) although analysis of peripheral blood at death revealed that many parameters remained similar to those of BM transplant recipients on doxycycline until death (Figure 1C).
Collectively, these data show that transgenic expression of iNUP98-KMT2A alters the hematopoietic system in vivo: there were increases in the numbers of white blood cells and apoptotic cells, and decreases in hemoglobin and the numbers of red blood cells and cells in the BM with signs of extramedullary hematopoiesis and morphological signs of dysplasia with some interindividual differences.
Expression of iNUP98-KMT2A leads to expansion and competitive advantage of hematopoietic stem and progenitor cells
We next studied the impact of iNUP98-KMT2A expres- sion on the cellular hierarchy of the BM. We found that prior to developing symptoms (mean time on doxycycline: 36 weeks), iNUP98-KMT2A mice displayed an expansion of Lin- Sca-1+ c-Kit+ (LSK) hematopoietic stem and progeni- tor cells (HSPC) (P=0.04, unpaired t-test, n=4) (Figure 2A, Online Supplementary Figure S1D). Further breakdown of the LSK compartment revealed no significant changes in the rel- ative distribution of CD34- long-term hematopoietic stem cells, and CD34+, CD48+, CD150- multipotent progenitors (Figure 2B). Interestingly, we found that iNUP98-KMT2A LSK, but not mature cells, from asymptomatic mice (mean time on doxycycline: 55 weeks) were cycling more than control cells as shown by a reduced fraction of quiescent cells in G0 phase (P=0.0098, unpaired t-test, n=3) and an increase in the G1 fraction (Figure 2C, Online Supplementary Figure S1E).
To address the functional consequence of the increased LSK number in iNUP98-KMT2A mice on doxycycline, we performed competitive repopulation assays. Naïve CD45.2+ iNUP98-KMT2A or WT (CTRL) total BM cells were trans- planted 1:1 with CD45.1+ WT total BM cells into lethally- irradiated CD45.1+ WT recipients (on doxycycline) and the cellular chimerism in the peripheral blood was determined 4, 8, 12, 18, and 25 weeks after transplantation. We observed that the proportion of CD45.2+ iNUP98-KMT2A cells in the peripheral blood steadily increased over time (Figure 2D) whereas the chimerism in mice that received CD45.2+ WT BM cells did not change significantly, remain- ing near to 50%. The percentage of iNUP98-KMT2A CD45.2+ cells was initially lower than that of CTRL (WT) (P=0.0153, t-test, n=5). After 18 weeks, CD45.2+ iNUP98- KMT2A cell numbers had increased by 1.6-fold compared to week 4 (P=0.034, t-test, n=5) and by 25 weeks CD45.2+ iNUP98-KMT2A cell numbers had further increased rela- tive to initial measurements (P=0.004, t-test, n=5) and were 20% greater than CD45.2+ WT cells at the same time-point [P=0.019, 2-way analysis of variance (ANOVA), n=4]. Interestingly, iNUP98-KMT2A CD45.2+ cells contributed to a greater extent to the myeloid lineage (Gr-1+) and the B lin- eage (B220+) but equally to the T lineage (CD3+), compared to WT controls (Figure 2E). Transplantation of total BM from primary recipients into lethally-irradiated CD45.1+ secondary recipients resulted in a heterogeneous outcome: in two mice (“M1” & “M2”) the BM was dominated by CD45.2+ cells, while two other mice (“M4” & “M5”) showed predominantly CD45.1+ cells. One mouse (“M3”) developed a CD45.2+ AML 23 weeks after transplantation (Figure 2F).
Some iNUP98-KMT2A mice develop transplantable acute myeloid leukemia
After a median latency of 62 weeks, six out of 22 iNUP98-KMT2A mice on doxycycline developed a
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