The Fetal Specific Gene LIN28B Is Essential for Human Fetal B-Lymphopoiesis and Initiation of KMT2A-AFF1+ Infant Acute Lymphoblastic Leukemia

Introduction

The fetal origin of KMT2A rearranged infant acute lymphoblastic leukemia (iALL) can provide clues about the properties of fetal hematopoietic stem and progenitor cells (HSPC) that promote leukemic transformation and confer its aggressive phenotype. Fetal HSPC have different gene expression profiles to their postnatal counterparts, and one such fetal specific oncogene, LIN28B, is aberrantly expressed in many cancers, including those with putative fetal origins. Functionally, it confers a proliferative phenotype and in murine models, specifies fetal-like lymphopoiesis. The exact role of this gene in promoting initiation or maintenance of ALL is not clear.

We hypothesise that LIN28B is key to the ability of fetal HSPC to undergo leukemia initiation and the aggressiveness of ensuing ALL.

While it is difficult to study leukemia initiation in utero, an iALL model utilizing CRISPR-Cas9 editing to induce KMT2A-AFF1 in human fetal liver (FL) CD34+ cells (CRISPR-KMT2A-AFF1+ALL), allows us to interrogate the role of LIN28B in initiating iALL.

Methods

Bulk and scRNAseq in HSPC across ontogeny and in CRISPR-KMT2A-AFF1+ALL were compared to published iALL and childhood ALL (chALL) data.

Primary human FL CD34+ cells, either unmanipulated; or transduced with lentivirus (LV) encoding YFP and (a) control/scrambled RNA or (b) LIN28B knockdown shRNA (KD) underwent CRISPR-Cas9 induced KMT2A-AFF1 translocation. Downstream analyses included digital droplet PCR (ddPCR), RNA sequencing, in vitro assays and in vivo xenotransplantation in NSG mice. RNA Immunoprecipitation sequencing (RIPseq) was performed in LIN28B+ KMT2A-AFF1+ SEM cells and the data intersected with RNAseq.

Results

A fetal-specific gene signature derived by comparing transcriptomes of human FL and fetal BM HSPC to their adult counterparts, included LIN28B and its downstream effectors IGF2BP1 and HMGA2 among the top hits. These genes were also overexpressed in iALL compared to chALL.

RNAseq analysis revealed that LIN28B KD human FL CD34+ cells were enriched for fetal myeloid gene signatures and depleted for lymphoid, stem cell and cell cycle gene signatures compared to controls. In keeping with this, LIN28B KD FL CD34+ cells were less proliferative, and their B- cell output (p= 0.04) was severely abrogated in vitro, with a concomitant increase in myeloid output (p=0.003) compared to controls (n=4).

CRISPR-Cas9 mediated KMT2A-AFF1 translocation was detected by ddPCR in both control and LIN28B KD human FL CD34+ cells 24 hours after electroporation (n=4). However, while all 4 control samples showed transformation in vitro, with a dramatic proliferation of CD19+ cells, none of the LIN28B KD samples transformed.

In CRISPR-KMT2A-AFF1+ALLs generated from unmanipulated human FL CD34+ cells, LIN28B expression was heterogeneous, analogous to KMT2A-AFF1+ iALL patients. CRISPR-KMT2A-AFF1+ALL with high LIN28B expression (n=4) showed an enrichment for cell cycle, MYC target and mTORC1 signalling genesets compared to those with no LIN28B expression (n=3). LIN28B-high CRISPR-KMT2A-AFF1+ALL displayed significantly greater proliferation in vitro (p=0.02) and reduced leukemia latency in vivo (p=0.01) compared to CRISPR-KMT2A-AFF1+ALL with no LIN28B expression. LV LIN28B KD in LIN28B-high CRISPR-KMT2A-AFF1+ ALL, reduced their proliferative capacity in vitro and led to increased survival in vivo, confirming LIN28B's importance in driving an aggressive leukemic phenotype.

To understand the mechanism by which LIN28B exerts its 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; key early B-lymphoid genes, EBF1, ETS1/2, IL7R; and cell cycle and apoptotic genes, CDK4 and BCL2.

Conclusion

Our data suggest that LIN28B is necessary for the initiation of CRISPR-KMT2A-AFF1+ALL in human fetal HSPC. LIN28B has an essential physiological role in human fetal B lymphopoiesis while promoting proliferation and “stemness”, thus providing the permissive context for KMT2A-AFF1 B-ALL initiation in fetal HSPC in the absence of additional co-operating mutations. LIN28B-high CRISPR-KMT2A-AFF1+ALL display a more aggressive phenotype. These findings make oncofetal genes such as LIN28B attractive therapeutic targets in leukemia as they are not expressed in healthy postnatal tissues.

Harman:Dark Blue Therapeutics: Current Employment, Other: Involvement in this project prior to move to Industry. Psaila:Alethiomics: Consultancy, Current equity holder in private company, Research Funding; University of Oxford: Patents & Royalties: 2203947.3; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy; Incyte: Consultancy, Research Funding. Milne:Dark Blue Therapeutics: Consultancy, Other: T.A.M. is a paid consultant for and shareholder in Dark Blue Therapeutics Ltd..