Mechanistic impact of immunoproteasome function on cell competition in acute myeloid leukemia (AML)

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Chromosomal translocations found in acute myelogenous leukemia (AML) can generate oncogenic fusions with aberrant epigenetic and transcriptional functions. Fusion-oncogene driven leukemias remain a therapeutic challenge. While targeted therapies have improved the therapeutic landscape of acute myeloid leukemia (AML), direct targeting of oncogenic fusions has not been successful so far. Specific subtypes such as leukemias harboring rearrangements of the MLL-gene (MLLr) have a particularly poor prognosis due to residual disease persistence and a high relapse rate. Epigenetic inhibitors of MLL-complex proteins are available and effective but failed to eradicate disease in clinical trials.This prompted us to assess for secondary dependencies of fusion-oncogene driven leukemias. We applied in-depth proteome profiling on murine leukemias harboring MLL-AF9 or AML1-ETO oncogenic fusions. Here, we found enrichment of proteins involved in proteostasis, specifically in MLL-rearranged leukemia. Specifically, catalytic subunits of the immunoproteasome – a specialized version of the proteasome with relevance during infection and inflammation - were highly expressed in transcriptome analyses of human AML. In a CRISPR/Cas9 mediated genome editing and negative selection screen, immuno-proteasome subunit PSMB8/LMP7 scored high for a selective dependency. When using genetic inactivation or pharmacologic inhibition of PSMB8/LMP7, MLLr murine leukemias showed selective dependency, which was not detectable for other types of oncogenic fusions or normal hematopoietic cells. Transcriptome analysis revealed overlap of PSMB8/LMP7 dependent gene sets with those regulated by epigenetic MLL-complex inhibitors According to our proposed working program we will investigate the mechanistic consequences of PSMB8/LMP7-mediated immunoproteasome function on MLL-rearranged acute myeloid leukemia (AML). By using a global genome editing screen in human MLLr leukemia cells, we aim to identify relevant molecules that depend on LMP7/PSMB8 function. Moreover, we aim to identify PSMB8/LMP7-dependent proteins by mass-spectrometry based analaysis of the cellular degradome. We will assess for metabolic changes following immunoproteasome inactivation by flow cytometry and seahorse technology and test for the synergistic potential of a combined pharmacologic inhibition of MLL-complex and the immunoproteasome. Finally, we will assess for the functional relevance of immunoproteasome inhibitor treatment in human xenograft models of MLLr leukemia, specifically in regard to cell competition against normal hematopoietic cells, a therapeutically relevant setting in a minimal residual disease situation.Here, we aim to confirm the selective dependency of MLLr AML on immunoproteasome function followed by pre-clinical validation in human AML cells. Our experimental approach may therefore facilitate therapeutic targeting of frequently persisting leukemic cells, the source of relapse in MLLr AML.