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

在急性髓细胞白血病(AML)中发现的染色体易位可以产生具有异常表观遗传和转录功能的致癌融合。融合癌基因驱动的白血病仍然是一个治疗挑战。虽然靶向治疗改善了急性髓系白血病(AML)的治疗格局，但直接靶向致癌融合迄今尚未成功。特殊的亚型，如携带MLL基因重排(MLLr)的白血病，由于残留疾病的持久性和高复发率，预后特别差。MLL复合蛋白的表观遗传抑制剂是可用的和有效的，但在临床试验中未能根除疾病。这促使我们评估融合癌基因驱动的白血病的二级依赖性。我们对携带MLL-AF9或AML1-ETO致癌融合基因的小鼠白血病进行了深入的蛋白质组分析。在这里，我们发现了参与蛋白平衡的蛋白质的丰富，特别是在MLL重排白血病中。具体地说，免疫蛋白酶体的催化亚基-在感染和炎症过程中相关的蛋白酶体的一个专门版本-在人类AML的转录组分析中高度表达。在CRISPR/Cas9介导的基因组编辑和阴性选择筛选中，免疫蛋白酶体亚单位PSMB8/LMP7因选择性依赖而得分很高。当使用PSMB8/LMP7的基因失活或药物抑制时，MLLr小鼠白血病表现出选择性依赖，而其他类型的致癌融合或正常造血细胞不能检测到这种依赖。转录组分析显示PSMB8/LMP7依赖的基因集与表观遗传的MLL-Complex抑制剂调控的基因集重叠。根据我们提出的工作计划，我们将研究PSMB8/LMP7介导的免疫蛋白酶体功能在MLL重排急性髓系白血病(AML)中的机制后果。通过在人类MLLr白血病细胞中使用全球基因组编辑屏幕，我们的目标是识别依赖LMP7/PSMB8功能的相关分子。此外，我们的目标是通过基于细胞降解组的质谱分析来鉴定依赖于PSMB8/LMP7的蛋白质。我们将通过流式细胞术和海马技术评估免疫蛋白酶体失活后的代谢变化，并测试MLL-复合体和免疫蛋白酶体联合药物抑制的协同潜力。最后，我们将评估免疫蛋白酶体抑制剂治疗在人类MLLr白血病异种移植模型中的功能相关性，特别是在与正常造血细胞的细胞竞争方面，这是在最小残留疾病情况下的治疗相关环境。在此，我们旨在确认MLLr AML对免疫蛋白酶体功能的选择性依赖，随后在人类AML细胞中进行临床前验证。因此，我们的实验方法可能有助于针对经常持续存在的白血病细胞的治疗靶向，这是MLLr AML复发的来源。

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.