3114 – FROM MECHANISMS OF LEUKAEMOGENIC DYSREGULATION TO NEW CANDIDATE THERAPEUTICS

Abstract

Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities. Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities.