| Özet: | <p>The MLL rearrangement which causes the poor prognosis of infant acute lymphoblastic leukemia (iALL) occurs before birth. Fetal haematopoietic stem and progenitor cells (HSPC) have different gene expression profiles to their postnatal counterparts. One such fetal specific oncogene, LIN28B is aberrantly expressed in a wide range of cancers, including many with putative fetal origins and in murine models specifies fetal-like lymphopoiesis.</p>
<p>I have addressed the hypothesis that LIN28B is key to the susceptibility of fetal HSPC to undergo leukaemia initiation and the aggressiveness of the ensuing iALL.</p>
<p>LIN28B is highly expressed in early fetal HSPC, lentiviral LIN28B knockdown in human FL CD34+ cells reduced proliferation in vitro with severe abrogation of B cell output and concomitant increase in myeloid output. The corresponding RNAseq analysis found LIN28B KD was enriched for fetal myeloid gene signatures and depleted for lymphoid, stem and cell cycle gene signatures compared to controls.</p>
<p>To study MLL-AF4 induced leukaemia initiation I used an iALL model, where CRISPR-Cas9 editing induced MLL-AF4 in human FL CD34+ cells. The MLL-AF4 fusion was detected by digital droplet PCR in LIN28B KD and scrambled control human FL CD34+ cells. All the control samples underwent leukaemic transformation, evidenced by a dramatic proliferation of CD19+ cells, whereas none of the LIN28B KD FL CD34+ transformed.</p>
<p>Expression of LIN28B in CRISPR-MLL-AF4 leukaemia derived from unmanipulated FL CD34+ cells was heterogeneous, analogous to that seen in MLL-AF4 iALL patients. The CRISPR-MLL-AF4 leukaemia with high LIN28B expression were enriched for cell cycle and MYC target genesets compared to those with no LIN28B expression. Functionally, LIN28B-high CRISPR-MLL-AF4 leukaemia had significantly greater proliferation in vitro and reduced leukaemia latency in vivo. Similarly, lentiviral LIN28B KD in LIN28B-high CRISPR-MLL-AF4 leukaemia, reduced their proliferative capacity in vitro and led to increased survival in vivo, confirming LIN28B’s importance in driving an aggressive leukaemic phenotype.</p>
<p>Finally, to understand the mechanism by which LIN28B exerts these effects, RIPseq was performed in SEM cells to identify LIN28B bound mRNA. Intersection with differentially expressed genes after LIN28B KD, suggested that LIN28B stabilizes epigenetic regulators, BRD4 and CXXC1 and key early B-lymphoid genes, EBF1, ETS1/2, IL7R.</p>
<p>In summary, LIN28B has an essential physiological role in human fetal B lymphopoiesis. It is a necessary part of the permissive context of fetal HSPC for MLL-AF4 induced B-ALL initiation. In established leukaemia high expression of LIN28B in CRISPR-MLL-AF4ALL results in a more aggressive phenotype. LIN28B influences lineage specification, possibly through direct binding to B-lymphoid transcripts, and epigenetic regulators with known to have roles in lineage commitment.</p>
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