Articulación y diseño de los programas universitarios para mayores.

Sorted populations were washed and RNA was extracted. Quantitative RT PCR was performed with primers specific for the candidate stemness genes BMI1, CD34, EZH2, HMGA2, MEIS1 and TERT. Expression was analysed by ΔCt using GAPDH as a reference gene. Using Wilcoxon matched-pairs signed rank analysis, only CD34 expression differed significantly between sorted leukaemic populations, p=0.03, as it did for sorted umbilical cord blood specimens.

Unexpectedly, the polycomb group genes BMI1 and EZH2 showed no consistent difference in expression between CD34+ and CD34- populations from either leukaemic or umbilical cord blood specimens. However, expression of TERT, MEIS1 and HMGA2 showed substantially higher expression in CD34+ umbilical cord blood stem cells than in the corresponding CD34- population, as would be expected (Figure 4-1B-D).

In contrast to sorted umbilical cord blood HSCs, expression of TERT was not different between sorted leukaemic populations and overall was higher than in either of the HSC populations, suggesting an important function across both populations of leukaemic blasts (Figure 4-1B). Expression of MEIS1 and

HMGA2 was more restricted. Neither of these genes showed higher expression in CD34+ cells, except for the expression of HMGA2 in the Philadelphia

chromosome positive ALL sample, L4951 (Figure 4-1C). However, the two fold higher expression seen in CD34+ leukaemic cells from this sample was

substantially lower than the 31-36 fold difference in expression seen in sorted HSC populations. MEIS1 was characteristically highly expressed in the one MLL rearranged sample tested (Figure 4-1D). Together, these findings argue against these candidate stemness genes having an important role in a CD34+ leukaemia stem cell population, consistent with the lack of a stem cell hierarchy in ALL.

Figure 4-1. Expression of candidate stemness genes in CD34+ and CD34- leukaemic blasts. A) CD34 expression is substantially higher in CD34+ cells (black bars) from both

umbilical cord blood (UCB) and leukaemic blasts. B) TERT expression is substantially higher in CD34+ UCB cells than in CD34- cells (hashed bars). In leukaemic blasts, no difference in

TERT expression is seen. The same pattern is seen in expression of both C) HMGA2 and D) MEIS1, although expression in leukaemic blasts is less consistent. Relative expression is 2-ΔCt, reference gene GAPDH

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4. Discussion

As leukaemia propagating cells in ALL are present in both CD34+ and CD34- sorted populations (Kong, Yoshida et al. 2008; le Viseur, Hotfilder et al. 2008), both populations would be predicted to express genes required for extensive cellular proliferation. This project aimed to assess the expression of a number of candidate stemness genes in sorted CD34+ and CD34- leukaemic blasts using qRT PCR. A normal haematopoietic control was provided by two umbilical cord blood specimens, also sorted for expression of CD34. The differential expression of the CD34 gene in all samples provided a control for the validity of this approach.

The first candidate showing differential expression in umbilical cord blood stem cells is telomerase reverse transcriptase (TERT). TERT encodes a key

component of the telomerase complex, which functions to maintain telomere length and prevent cellular senescence. As such it is an essential component of the self-renewal programme of normal and malignant stem cells. In

leukaemia, TERT expression is under the control of the leukaemic fusion genes MLL/AF4 and RUNX1/RUNX1T1 (Gessner, Thomas et al. 2010). Down-

regulation resulted in reduced clonogenicity and induction of replicative

senescence. Baseline expression is however, too low for TERT expression to be reliably represented in expression array analyses and therefore it is not surprising that it does not appear in the stemness signature of Eppert et al (Eppert, Takenaka et al. 2011).

MEIS1 is a homeobox protein implicated in the leukaemogenesis of MLL rearranged leukaemias and contained in the AML stemness signature defined by Eppert et al (Eppert, Takenaka et al. 2011). MEIS1 is up-regulated by MLL fusion proteins, whilst knockdown of MEIS1 in MLL rearranged leukaemic cell lines impairs engraftment and proliferation in murine xenograft models (Kumar, Li et al. 2009; Orlovsky, Kalinkovich et al. 2011). The present project

demonstrated higher expression of MEIS1 in CD34+ cord blood HSCs, compared with CD34- HSCs. Leukaemic expression was low in all samples other than L826, the only MLL rearranged leukaemia in the panel tested and

L4951, a Philadelphia chromosome positive ALL. In both of these samples, however, expression was higher in the CD34- population.

HMGA2 encodes the high mobility group protein A2 which is highly expressed during embryogenesis but is then down-regulated (Gattas, Quade et al. 1999). It functions to control growth and development, as evidenced by abnormal growth both in mutated mice (Zhou, Benson et al. 1995; Battista, Fidanza et al. 1999) and in a child with constitutional mutation of this gene (Ligon, Moore et al. 2005). HMGA2 expression has been identified in haematopoietic stem cells as well as blasts from both acute and chronic myeloid leukaemias (Meyer, Krisponeit et al. 2007). Expression in the present study was limited to four samples, although levels were substantially higher in sample L4951, positive for the Philadelphia chromosome. Overall there was no evidence for higher

expression in CD34+ leukaemic cells.

The polycomb group proteins BMI1 and EZH2 were not differentially expressed in either sorted HSCs or leukaemic blasts. This was a somewhat unexpected finding as both of these genes are essential to the maintenance of HSC self- renewal capacity by generating bivalent chromatin domains (Konuma, Oguro et al. 2010) and control the proliferative potential of leukaemia stem cells (Lessard and Sauvageau 2003). The reasons for the lack of differential expression in the present study are not clear. It is possible that the single marker sorting strategy was insufficiently effective in isolating HSCs resulting in analysis of a mixed population.

This project has shown that the candidate leukaemic stemness genes TERT, MEIS1 and HMGA2 are differentially expressed in sorted CD34+ and CD34- normal haematopoietic stem cells. In contrast, expression in sorted leukaemic blasts showed no bias towards higher expression in CD34+ cells consistent with the lack of a stem cell hierarchy in acute lymphoblastic leukaemia (Rehe,