Epigenetic Regulation of Non-canonical Menin Targets Modulates Menin Inhibitor Response in Acute Myeloid Leukemia.

Menin inhibitors that disrupt Menin-MLL interaction hold promise for treating specific acute myeloid leukemia subtypes, including KMT2A rearrangements (KMT2A-r), yet resistance remains a challenge. Here, through systematic chromatin-focused CRISPR screens, along with genetic, epigenetic, and pharmacologic studies in a variety of human and mouse KMT2A-r AML models, we uncover a potential resistance mechanism independent of canonical Menin-MLL targets. We show that a group of non-canonical Menin targets, which are bivalently co-occupied by active Menin and repressive H2AK119ub marks, are typically downregulated following Menin inhibition. The loss of Polycomb Repressive Complex 1.1 (PRC1.1) subunits, such as PCGF1 or BCOR, leads to Menin inhibitor resistance by epigenetic reactivation of these non-canonical targets, including MYC. Genetic and pharmacological inhibition of MYC can resensitize PRC1.1-deficent leukemia cells to Menin inhibition. Moreover, we demonstrate that leukemia cells with the loss of PRC1.1 subunits exhibit reduced monocytic gene signatures and are susceptible to the BCL2 inhibition, and combinational treatment of venetoclax overcomes the resistance to Menin inhibition in PRC1.1-deficient leukemia cells. These findings highlight the important roles of PRC1.1 and its regulated non-canonical Menin targets in modulating Menin inhibitor response and provide potential strategies to treat leukemias with compromised PRC1.1 function.

RNA binding protein-directed control of leukemic stem cell evolution and function.

Strict control over hematopoietic stem cell decision making is essential for healthy life-long blood production and underpins the origins of hematopoietic diseases. Acute myeloid leukemia (AML) in particular is a devastating hematopoietic malignancy that arises from the clonal evolution of disease-initiating primitive cells which acquire compounding genetic changes over time and culminate in the generation of leukemic stem cells (LSCs). Understanding the molecular underpinnings of these driver cells throughout their development will be instrumental in the interception of leukemia, the enabling of effective treatment of pre-leukemic conditions, as well as the development of strategies to target frank AML disease. To this point, a number of precancerous myeloid disorders and age-related alterations are proving as instructive models to gain insights into the initiation of LSCs. Here, we explore this myeloid dysregulation at the level of post-transcriptional control, where RNA-binding proteins (RBPs) function as core effectors. Through regulating the interplay of a myriad of RNA metabolic processes, RBPs orchestrate transcript fates to govern gene expression in health and disease. We describe the expanding appreciation of the role of RBPs and their post-transcriptional networks in sustaining healthy hematopoiesis and their dysregulation in the pathogenesis of clonal myeloid disorders and AML, with a particular emphasis on findings described in human stem cells. Lastly, we discuss key breakthroughs that highlight RBPs and post-transcriptional control as actionable targets for precision therapy of AML.

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

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) 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 versus 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. SIGNIFICANCE: LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171.

TCF/LEF regulation of the topologically associated domain ADI promotes mESCs to exit the pluripotent ground state.

Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a ß-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

Arhgef2 regulates mitotic spindle orientation in hematopoietic stem cells and is essential for productive hematopoiesis.

How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell-driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2-/- fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.

Diminished AHR Signaling Drives Human Acute Myeloid Leukemia Stem Cell Maintenance.

Eliminating leukemic stem cells (LSC) is a sought after therapeutic paradigm for the treatment of acute myeloid leukemia (AML). While repression of aryl hydrocarbon receptor (AHR) signaling has been shown to promote short-term maintenance of primitive AML cells in culture, no work to date has examined whether altered AHR signaling plays a pathologic role in human AML or whether it contributes at all to endogenous LSC function. Here, we show AHR signaling is repressed in human AML blasts and preferentially downregulated in LSC-enriched populations within leukemias. A core set of AHR targets are uniquely repressed in LSCs across diverse genetic AML subtypes. In vitro and in vivo administration of the specific AHR agonist FICZ significantly impaired leukemic growth, promoted differentiation, and repressed self-renewal. Furthermore, LSCs suppressed a set of FICZ-responsive AHR target genes that function as tumor suppressors and promoters of differentiation. FICZ stimulation did not impair normal hematopoietic stem and progenitor (HSPC) function, and failed to upregulate a prominent LSC-specific AHR target in HSPCs, suggesting that differential mechanisms govern FICZ-induced AHR signaling manifestations in HSCs versus LSCs. Altogether, this work highlights AHR signaling suppression as a key LSC-regulating control mechanism and provides proof of concept in a preclinical model that FICZ-mediated AHR pathway activation enacts unique transcriptional programs in AML that identify it as a novel chemotherapeutic approach to selectively target human LSCs. SIGNIFICANCE: The AHR pathway is suppressed in leukemic stem cells (LSC), therefore activating AHR signaling is a potential therapeutic option to target LSCs and to treat acute myeloid leukemia.

A Single TCF Transcription Factor, Regardless of Its Activation Capacity, Is Sufficient for Effective Trilineage Differentiation of ESCs.

Co-expression and cross-regulation of the four TCF/LEFs render their redundant and unique functions ambiguous. Here, we describe quadruple-knockout (QKO) mouse ESCs lacking all full-length TCF/LEFs and cell lines rescued with TCF7 or TCF7L1. QKO cells self-renew, despite gene expression patterns that differ significantly from WT, and display delayed, neurectoderm-biased, embryoid body (EB) differentiation. QKO EBs have no contracting cardiomyocytes and differentiate poorly into mesendoderm but readily generate neuronal cells. QKO cells and TCF7L1-rescued cells cannot efficiently activate TCF reporters, whereas TCF7-rescued cells exhibit significant reporter responsiveness. Surprisingly, despite dramatically different transactivation capacities, re-expression of TCF7L1 or TCF7 in QKO cells restores their tri-lineage differentiation ability, with similar lineage marker expression patterns and beating cardiomyocyte frequencies observed in EBs. Both factors also similarly affect the transcriptome of QKO cells. Our data reveal that a single TCF, regardless of its activation capacity, is sufficient for effective trilineage differentiation of ESCs.