Chromosomal rearrangements of the MLL gene (MLL-r) are strong drivers of acute leukemia with poor prognosis. During the last years, the first MLL-r targeting compounds have been developed. However, resistance has been described already. Here, we identified a highly upregulated core gene set and a secondary driver mutation in human long-term cultured CD34+ progenitors expressing the t(11;19) MLL-ENL (ME) fusion, with the intention to uncover druggable vulnerabilities in ME driven transformation.

ME was introduced into human CD34+ progenitors from healthy donors via retroviral transduction. Upon ex vivo expansion and outgrowth of ME monocytic progenitors, gene expression profiling was performed via the Nanostring technology, bulk and single cell (sc) RNA sequencing. We compiled a top 30 list of ME upregulated genes, including CEBPα and its target gene BCL2. To investigate cell biological consequences of CEBPα and BCL2 disruption, genetic downregulation via lentiviral shRNA and pharmacological inhibition with small molecule compounds were explored. For genomic survey whole exome sequencing was performed revealing mutated PTPN11, an activator of CEBPα gene expression, as additional driver for ME triggered cell growth.

Gene arrays identified high CEBPα levels in ME progenitors. Of note, CEBPα shRNA blocked proliferation indicating cooperativity with ME. Indeed, combination of the MLL inhibitor MI-503 and the CEBPα complex inhibitor OICR-9429 strongly reduced proliferation. Moreover, BCL2 was downregulated upon CEBPα knockdown. Hence, treatment with Venetoclax (VEN) significantly reduced growth. Combining VEN with either MI-503 or OICR-9429 further increased growth-inhibition. Additionally, exome sequencing revealed a monoclonal PTPN11(Y62S) mutation, indicating a pro-proliferative role of PTPN11. Accordingly, scRNA-seq discovered co-expression of PTPN11, CEBPA and BCL2 in ME blasts. Of note, a clinical PTPN11 inhibitor dose dependently blocked growth.

Our proposed t(11;19) pre-leukemia model proves the usefulness of human CD34+ progenitors derived from healthy donors for disease modeling and drug target validation. The study sheds light into the oncogenic mechanisms of ME fusion protein activity in human CD34+ progenitors suggesting oncogenic cooperativity of ME with the PTPN11/CEBPα/BCL2 network in AML transformation. The here identified vulnerabilities might be attractive drug targets to inhibit the aggressive tumor promoting effects of ME.

The authors declare no conflict of interest.