The t(8,21) translocation is the most prevalent chromosomal translocation in Acute Myeloid Leukaemia (AML), which leads to the expression of chimaeric RUNX1/ETO oncogene protein. Our previous work showed that RNAi-mediated RUNX1/ETO knockdown inhibits cell proliferation, reduces clonogenicity, causes G1 cell cycle arrest and impairs engraftment in immunocompromised host.
Acquired mutations in exon 2 of GATA1 -in the background of trisomy 21- produce an N-terminus truncated protein, named GATA1s driving transient abnormal myelopoiesis (TAM) in newborns with Down syndrome. Additional mutations however are required to transform the pre-leukaemic TAM clones into a full-blown acute leukaemia (ML-DS). Prior work revealed common secondary mutations in ML-DS patients belonging to epigenetic modifiers, signalling pathways and cohesin complex, which were shown causative for leukaemic transformation in a murine model. However, with the observed mutational repertoire open questions remained. Here, we are utilising virus-free CRISPR techniques to introduce GATA1s and additional mutations in primary human foetal haematopoietic stem and progenitor cells. Xenotransplantation assays of edited cells showed a marked production of immature CD117 + CD41+ megakaryocytic progenitors. Comprehensive in vitro and in vivo functional assays will provide insight into the genetic players of TAM and ML-DS oncogenesis.
Non-coding RNAs (ncRNAs) recently emerged as central regulators of chromatin and gene expression, posing a novel window for targeted therapies in pediatric acute megakaryoblastic leukemia (AMKL). In our ncRNA expression atlas of the human hematopoietic system, we uncovered specific and coordinated expression of the DLK1-DIO3 locus in megakaryocytes and AMKL, which is essential of for promoting megakaryocytic differentiation, but also leukemic growth. To determine how expression of the DLK1-DIO3 locus is controlled, we performed several techniques elucidating different regulatory layers. Using bisulphite sequencing, a significant correlation between MEG3 expression and methylation of a CpG island downstream of the first exon of MEG3 was found, which displayed enhancer/promoter activity. CUT&RUN for key megakaryocytic transcription factors revealed increased binding of oncogenic GATA1s on several genomic locations upstream of DLK1 and MEG3. Overall, our study is the first step towards understanding the regulation of the DLK1-DIO3 locus in AMKL and normal hematopoiesis.
The fusion protein RUNX1/ETO is generated by the chromosomal translocation t(8;21) and found in 15 % of all pediatric acute myeloid leukemia (AML). It causes leukemogenic transformation by blocking differentiation and promoting self-renewal. Direct targeting using RNA interference promises a leukemia-specific therapeutic approach, but remains associated with poor pharmacokinetics. Here, we aimed to explore lipid nanoparticles (LNP) for safe and efficacious siRNA delivery. LNPs were prepared using microfluidic mixing techniques. Efficacies were examined in cell lines, patient-derived xenografts (PDX) and primary material. RUNX1/ETO mRNA and protein levels decreased more than twofold in all cultures. This reduction was associated with reduced proliferation, loss off clonogenicity and G1 cell cycle arrest. In vivo colocalisation studies, using fluorescence and bioluminescence, demonstrated that the LNPs reached the leukemic cells. Pharmacodynamic analyses proved RUNX1/ETO knockdown in all animals, as well as accordingly changed target gene expression levels. To conclude, LNP-mediated siRNA delivery is a promising new approach for specific targeting of fusion gene dependent cancers.
RUNX1/ETO is required for leukaemic clonogenicity and proliferation and is traditionally thought of as a transcriptional repressor. Our RNAi experiment identifies target genes that are transcriptionally upregulated.
Leukeamias are often driven by the expression of leukaemic-specific fusion genes. Exclusive targeting using RNA interference is therefore an attractive therapeutic concept, but lacks so far suitable delivery systems. Here, we use targeted lipid nanoparticles (LNPs) containing siRNA (siRE) to reduce RUNX1/ETO protein expression in patient-derived AML cells.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)
