In Vivo Screening Unveils Pervasive RNA-Binding Protein Dependencies in Leukemic Stem Cells and Identifies ELAVL1 as a Therapeutic Target

Abstract

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSCs) whose determinants are challenging to discern from hematopoietic stem cells (HSCs) or uncover by approaches focused on general cell properties. We have identified a set of RNA binding proteins (RBPs) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution and selectively depleted primitive malignant vs. healthy cells. Integrative multiomics revealed differentiation, splicing and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34 being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML.

RNA decay defines the response to transcriptional perturbation in leukaemia

“…5A, S12. A-B), which has been reported to stabilize oncogenic transcripts in AML and promote Leukemic Stem Cell (LSC) self-renewal and survival ( 37 , 38 ). THP-1 cells with knockout of ELAVL1 (Fig.…”

“…C), thereby independently validating the results from the genome-wide screens. Given the proposed oncogenic role of ELAVL1 ( 37 , 38 ) it was unsurprising to observe that ELAVL1 knockout also resulted in a proliferative disadvantage in the absence of any CDK9i treatment (Fig. 5D).…”

“…To interrogate the therapeutic potential of ELAVL1 targeting in the context of CDK9i in leukaemia, THP-1 cells were treated with CDK9i in combination with the small molecule MS-444, a potent inhibitor of ELAVL1 ( 42 ). Importantly, MS-444 has been demonstrated to exhibit single agent efficacy in models of leukameia in vivo ( 38 ). Assessment of cell death revealed that the drug combination resulted in greater cytotoxicity than each inhibitor alone and was highly synergistic (Fig.…”

“…An alternative approach to alter transcript decay in cancer for therapeutic benefit has been to target RBPs that promote oncogenic mRNA stability. In the leukaemia setting, recent work has demonstrated that the ELAVL1 inhibitor MS-444 destabilises the mitochondrial import protein TOMM34 and hampers LSC-driven leukaemic reconstitution in vivo ( 38 ). In line with these findings, we observe that genetic knockout and therapeutic inhibition of ELAVL1 limits THP-1 cell line growth.…”

Multiple roles for AU-rich RNA binding proteins in the development of haematologic malignancies and their resistance to chemotherapy

“…HuR regulates a broad spectrum of targets implicated in various cellular functions. HuR activity affected alternative splicing and upregulated expression of proteins involved in processes related to metabolism, translation, and mitochondrial respiration (such as Ewsr1, Samd8, Nsun3, Pam16, and Mrtfl1, TOMM34), while downregulated – markers of myeloid differentiation ( Figure 4 ) [ 29 ]. The range of targets and their role in the regulation of cellular processes important for haematopoiesis implicates the significance of HuR in the view of leukaemia development and progression.…”