Primary transplants
The same process outlined in Section 4.3.2 was used to generate primary Ezh2 conditional AML1- ETO9a leukaemias. Retroviral transduction was performed on c-kit selected bone marrow HSPCs taken from 2-3 Ezh2 fl/fl; Cre+ mice using retroviral supernatant derived from HEK 293T cells transfected with the MIGR1-AML1-ETO9a-GFP construct. This reliably yielded, on average, a higher transduction efficiency of 15-25% per experiment given the construct size is considerably smaller than MLL-AF9 (Figure 4.9). Given the lower potency of this particular oncogene, this also meant that more GFP positive cells were required per mouse for each transplant (in the order of 105 per mouse).
After transduction, as above, the cell dosage was adjusted based on total cell number and percentage of GFP positivity, and an equal number of GFP positive cells were injected into five primary lethally irradiated C57/Bl6 recipients. These animals were then tracked with serial bleeds and GFP assessments, and clinically for the development of leukaemia. The expectation was that these animals would have a long latency (close to one year) as seen in previous studies. 4 However disease features became prominent at approximately 3 months, with significantly raised GFP in peripheral blood sampling and all 5 animals succumbed to leukaemia between Days 96 and 138 post transplantation. Each displayed hepatosplenomegaly, marked anaemia and thrombocytopenia as seen in the induction experiments. Splenic tissue and bone marrow was harvested and frozen in liquid nitrogen. Spleen cells were c-kit positive but negative for other myeloid antigens such as Mac-1 and Gr-1 (also negative for B220/CD4 and Ter119). Given the latency for these primary leukaemias was slightly shorter than
FIGURE 4.9 Transduction efficiency of AML1-ETO9a into c-kit positive HSPCs
Live cells gated initially by forward and side-scatter, doublets removed and then gated for GFP positivity. Population positive for GFP, reflecting AML1-ETO9a transduced cells seen in Q4, right (here ~21%)
111 expected, genotyping to assess Ezh2 recombination was performed, as with the MLL-AF9 primary leukaemias. Unsurprisingly given the presumably pro-inflammatory nature of leukaemogenesis it appeared once again that spontaneous Cre-recombinase activation had occurred, demonstrating 60- 90% Ezh2 excision had occurred using the qPCR method across the five animals (from DNA extracted from splenic tissues). This disease latency tallied with the acceleration and shorter disease latency seen in the Ezh2fl/fl;Cre+ pIpC pre-treated arm in the induction in vivo experiments, suggesting again even partial Ezh2 loss through spontaneous Cre effects was powerful enough to facilitate accelerated leukaemogenesis. Nonetheless, having seen this already happen with the MLL-AF9 primary transplants as described above, we decided to proceed with secondary transplants using splenic tissue from the mouse with the least Ezh2 excision (approximately 60%).
Secondary transplants and Ezh2 functional assessment
Splenic tissue from one of the primary Ezh2fl/fl;Cre+ AML1-ETO9a leukaemic mice with the lowest level of spontaneous Ezh2 excision was thawed. This had an approximately 60% level of Ezh2 excision. After washing the cells, approximately 1x106 cells were injected via tail-vein into 20 sub-lethally irradiated wild-type C57/Bl6 mice (split thereafter into two arms). The cells were allowed 7 days for engraftment and the mice were then treated with intraperitoneal injections of either PBS (control) or pIpC (test arm), to induce complete Ezh2 excision in the latter. Five doses were given on alternate days. They were then tracked for aggressive, short latency secondary leukaemias, through peripheral blood sampling, GFP assessment and clinical monitoring. Ezh2 excision in the pIpC treated arm lead to a significant increase in survival compared to PBS treated (Figure 4.10):
112
SecondaryAML1-ETO9a - Survival proportions
0 100 200 300 400 0 50 100 P value 0.0011
PBS treated (n=10)
PIPC treated (n=10)
Days post transplantation
Pe
rc
ent
s
ur
vi
va
l
FIGURE 4.10 Kaplan-Meier survival outcomes for Ezh2fl/fl; Cre+ AML1-ETO9a secondary recipient mice treated with PBS or pIpC
Mice in the pIpC treated (i.e. Ezh2 excised) arm demonstrate a significant increase in survival compared to PBS treated
113 As early as Day 35 post transplant, PBS treated animals became pre-terminal and were sacrificed. By Day 126, all PBS treated animals had been culled due to leukaemia development. In the pIpC arm only three out of ten animals actually developed secondary leukaemias, in general significantly later than the PBS treated animals. The remainder died of GFP-negative other causes (likely secondary to the effects of irradiation and aging), with several having massive thymic enlargement on necropsy (but GFP negative throughout all organs and blood counts not indicative of leukaemia) (Figure 4.11):
This suggested that maximal Ezh2 excision in primary AML1-ETO9a transplanted leukaemic tissues, rendered a significant delay in the development of secondary leukaemia allowing other effects such as aging/post-irradiation to come into play, and/or through inhibiting leukaemia development, was influencing the development of other disease processes (outside the scope of this study). Details of all mice in this experiment (where available) are provided in Table 4.2:
PBS
PIPC
FIGURE 4.11 Marked thymic enlargement seen in non-leukaemic mice transplanted with
Ezh2fl/fl;Cre+ AML1-ETO9a splenic tissues and treated with pIpC (right)
compared to PBS treated secondary leukaemic mouse (heart and lungs with minimal thymus)
114
GFP%
Mouse
# PBS/PIPC treated culled Day (x10WCC 9/L) (g/dl) Hb (x10Plts 9/L) Periph blood marrow Spleen Bone Spleen wt (g) wt (g) Excision Liver
4818 PBS 35 15.6 42 42 35.6 43.2 84.1 0.9 1.4 90.09% 4822 PBS 35 29.6 29 125 54.6 54.8 90.9 0.69 1.44 89.76% 4825 PBS 35 4.2 ** 103 42.8 54.7 93.8 0.62 1.22 86.99% 4830 PIPC 37 40.5 46 55 92.0 85.0 90.0 0.69 2.03 90.95% 4821 PBS 43 74.0 54 165 94.7 30.6 82.8 1.01 2.77 84.63% 4823 PBS 56 15.6 36 134 41.8 50.1 82.5 1.02 2.45 79.17% 4826 PBS 57 176.0 40 115 93.9 58.8 84.2 0.74 3.18 83.43% 4817 PBS 64 26.1 73 95 53.6 53.4 55.5 1.02 3.16 41.49% 4820 PBS 73 0.64 1.95 75.51% 4814 PIPC 75 220 40 135 97.7 49.6 88.3 1.09 3.55 89.96% 4819 PBS 126 37.1 84 91 90.1 0.84 2.64 86.08% 4813 PIPC 134 75 56 171 39.4 77.0 66.9 1.52 3.22 90.70% 4812* PIPC 170 <10% <10% <10% 4815* PIPC 170 <5% <5% <5% 4816* PIPC 175 13.8 102 209 <2% <2% <2% 0.83 5.46 4831* PIPC 186 4827* PIPC 203 4829* PIPC 219 <1% <1% <1% 0.46 2.77 4824 PBS 281 N N N <1% <1% <1% 4828 PIPC 290 7.7 158 172 <1% <1% 1.28 3.24
Ezh2 excision was assessed in splenic tissue for only leukaemic mice where tissue/DNA was available.
This is shown in the final column of Table 4.2. There were varying levels of excision seen (between 41- 90%) and did not follow a particular treatment arm. In view of the aggressive nature of these secondary leukaemias, we assumed that this level of excision was likely due to sustained Cre-activation caused by ongoing inflammation/interferon responses in the face of leukaemia progression in the PBS treated arm, though the phenotypic effects on survival clearly favour the pIpC treated arm (i.e. maximally Ezh2 deleted after engraftment), producing a profound survival advantage. This again suggests that Ezh2 appears to be a facilitator of the transformed state, with its complete removal at an early phase of leukaemia progression either blocking leukaemia completely (seven of ten pIpC treated mice did not develop GFP-positive leukaemia) or delaying it sufficiently to allow other disease processes to occur. Finally, there were no significant phenotypic effects on GFP distribution (though GFP percentage was marginally higher in bone marrow in the pIpC treated arm), peripheral blood counts or terminal organ size for all leukaemic mice between both arms (where available) (Figure 4.12):
TABLE 4.2 Terminal data for all secondary AML1-ETO9a leukaemia transplanted mice (Ezh2fl/fl;Cre+ treated with PBS or pIpC)
Mice identified in Bold indicate those with GFP negative disease at time of culling
*indicates mice with massive thymic enlargement at death. qPCR assessment of Ezh2 excision not performed in these latter cases as no evidence of leukaemia as determined by GFP expression
115
FIGURE 4.12 Spleen and liver weights, terminal white blood cell counts and GFP percentages for blood, bone marrow and spleen for secondary AML1-ETO9a recipients treated with PBS or pIpC
116
4.3.4 Loss of Ezh2 during maintenance of MOZ-TIF2 leukaemias in vivo does not significantly alter