P332: lin28b: an important onco-fetal gene in infant acute lymphoblastic leukaemia

Background: Infant acute lymphoblastic leukaemia (iALL) has a dismal prognosis, and its characteristic MLL gene rearrangement (MLLr) invariably arises before birth. MLLr alone appears sufficient to transform human fetal liver haematopoietic cells suggesting that fetal molecular programmes may co-operate with MLLr to produce the aggressive phenotype of iALL. One such oncofetal gene, LIN28B is normally expressed exclusively before birth but is known to be aberrantly expressed in a range of malignancies. LIN28B is an RNA binding protein and its canonic oncogenic mechanism is believed to be through repression of let-7 micro RNAs, which act as tumour suppressors for oncogenes such as the MYC and RAS families. The role of LIN28B expression/function in MLLr iALL initiation and maintenance is unknown. Aims: We hypothesise that the oncofetal gene, LIN28B, is required for the initiation and maintenance of iALL. Methods: Molecular: Bulk and scRNAseq datasets were interrogated for LIN28B expression in healthy human fetal liver (FL), fetal bone marrow (BM) and postnatal BM; publicly available bulk RNAseq datasets were analysed for infant (<1year age) and childhood (1-18 years) MLLr ALL. RNAseq, ATACseq and ChIPseq was performed for a human FL-derived MLL-AF4 iALL model (CRISPRMLL-AF4 ALL), SEM cell line and primary patient samples. Functional: SEM cells transduced with lentivirus encoding YFP and (a) control or (b) LIN28B knockdown shRNA(KD) were characterised using RNA sequencing, liquid culture proliferation, apoptosis assay, colony forming assays, and in vivo xenotransplantation in NSG mice. Results:LIN28B is selectively expressed in human fetal HSC, MPP, LMPP and myeloid and erythroid precursor cells, but not B-progenitors or B-cells. It is not expressed in any postnatal HSPC. A subset of iALL patients express LIN28B but there was no LIN28B expression in childhood MLLr ALL. Similarly, a subset of CRISPRMLL-AF4 ALLs expressed LIN28B; further analysis revealed that LIN28B was accessible and bound by MLL-AF4 in CRISPRMLL-AF4 ALLs. In the cell-lines screened: SEM (MLL-AF4+), RCH-ACV (TCF3-PBX1+), and KOPN-8 (MLL-ENL+), only SEM cells express LIN28B. Knockdown of LIN28B in MLL-AF4+ SEM cells (confirmed by qPCR and western blot), caused rapid cell death by apoptosis in liquid culture (Fig 1a&b) and a severe reduction in colony formation in semi-solid medium (n=5, p=0.0004) (Fig 1c). It also significantly prolonged survival in an in vivo ALL model produced by transplantation of control YFP+ or LIN28B KD YFP+ SEM cells, with median survival being 38 days (n= 9 mice) and 58 days (n=12 mice) respectively (p<0.0001) (Fig 1d). When culled LIN28B KD recipients had a predominantly YFP-ve LIN28B+ leukaemia, suggesting a LIN28B KD escape. By contrast the control SEM cells remained YFP+ve. Gene set enrichment analysis of bulk RNA sequencing for SEM control and LIN28B KD cells showed LIN28B KD was negatively associated with let-7 target genes, consistent with knockdown causing an increase in let-7 miRNAs and suppression of their targets. MYC targets, E2F and G2M gene sets are also negatively associated with LIN28B knockdown, which may be let 7 mediated or a direct LIN28B effect. Summary: Aberrant LIN28B expression is seen in a subset of iALL. LIN28B is essential in vitro and in vivo for SEM cells (MLL-AF4+ cell line). Transcriptomic data suggests let-7 axis may cause these changes. Further work is needed to elucidate the role of LIN28B in iALL. As LIN28B is not expressed in postnatal HSPC, targeting LIN28B or downstream effectors could offer an attractive option for therapy with minimal off target effects.Keywords: Infant, Children, Acute lymphoblastic leukemia