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.

A new ELISA assay demonstrates sex differences in the concentration of serum polysialic acid.

Male and female immune systems are strikingly different and yet little is known about sex differences in immune glycans, though glycans play central roles in regulating the immune response. Polysialic acid (polySia) occurs on the majority of leukocytes and is a potent immunomodulatory glycan which enables cell migration and serves as an immune checkpoint. Due to widespread influence of polySia on the immune system, we aimed to characterize its levels in serum, its presence on specific proteins, and differences in the amounts of polySia in male and female serum. However, polySia is difficult to quantify and detect on specific proteins, which makes it challenging to elucidate the molecular details of polySia function. We developed a sandwich ELISA that allows for the quantification of polySia as well as specific polysialylated proteins in complex mixtures without any pretreatment or harsh conditions. The assay is quick, linear, and robust under a wide variety of conditions and gave a limit of detection of approximately 0.2 ng polySia per mL of serum. We then quantified polySia and polysialylated CD56 in human and mouse serum. These studies strongly support our hypothesis of differences in glycosylation between the sexes as significantly less polySia was observed in female samples than in male samples.

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.

Tunability of liquid-infused silicone materials for biointerfaces.

The ability to control the properties of bio-inspired liquid-infused surfaces is of interest in a wide range of applications. Liquid layers created using oil-infused polydimethylsiloxane elastomers offer a potentially simple way of accomplishing this goal through the adjustment of parameters such as curing agent ratio and oil viscosity. In this work, the effect of tuning these compositional parameters on the properties of the infused polymer are investigated, including infusion dynamics, stiffness, longevity in the face of continuous liquid overlayer removal, and resistance to bacterial adhesion. It is found that that curing agent concentration appears to have the greatest impact on the functionality of the system, with a lower base-to-curing agent ratio resulting in both increased longevity and improved resistance to adhesion by Escherichia coli. A demonstration of how these findings may be implemented to introduce patterned wettability to the surface of the infused polymers is presented by controlling the spatial arrangement of bacteria. These results demonstrate a new degree of control over immobilized liquid layers and will facilitate their use in future applications.

Infused polymers for cell sheet release.

Tissue engineering using whole, intact cell sheets has shown promise in many cell-based therapies. However, current systems for the growth and release of these sheets can be expensive to purchase or difficult to fabricate, hindering their widespread use. Here, we describe a new approach to cell sheet release surfaces based on silicone oil-infused polydimethylsiloxane. By coating the surfaces with a layer of fibronectin (FN), we were able to grow mesenchymal stem cells to densities comparable to those of tissue culture polystyrene controls (TCPS). Simple introduction of oil underneath an edge of the sheet caused it to separate from the substrate. Characterization of sheets post-transfer showed that they retain their FN layer and morphology, remain highly viable, and are able to grow and proliferate normally after transfer. We expect that this method of cell sheet growth and detachment may be useful for low-cost, flexible, and customizable production of cellular layers for tissue engineering.