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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

Table S5 shows the log2 fold change of differentially expressed transcripts from the ELAVL1 knockout RN2c RNA-sequencing data set.

Table S7 shows (a) reproducible peaks and (b) GO analysis of ELAVL1-bound transcripts identified in the eCLIP-sequencing data set. The negatively enriched (c) and positively-enriched (d) gene sets identified by GSEA of ELAVL1-bound and differentially expressed transcripts from the RN2c RNA-sequencing data set are shown.

Table S1 shows expression of RBPs in an RNA-sequencing data set of human bone marrow hematopoietic stem and progenitor subpopulations.

Supplemental Figure S4 shows the validation of ELAVL1 knockdown by western blot and effects of ELAVL1 loss on human primary AML cells in vitro and in vivo.

Table S8 shows transcripts from the ELAVL1 knockout RNA-sequencing data set that are (a) alternatively spliced and (b) alternatively spliced as well as bound by ELAVL1 as identified by eCLIP-sequencing.

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

Supplemental Figure S5 shows the effects of DHTS treatment on THP-1 and human primary AML cells in vitro, the effects of MS-444 treatment on human umbilical cord blood in vivo, and validation of ELAVL1 overexpression by western blot.

Supplemental Figure S2 shows validation of sgRNA -mediated knockout of hit RBPs, comparison of hit RBP depletion in RN2c in vitro versus in vivo, effects of ELAVL1 loss on human AML cell lines, characteristics of the MLL-AF9 AML mouse model, and ELAVL1 expression in normal hematopoietic cells.

Table S9 shows the (a) differentially expressed transcripts and (b) altered pathways as identified by GSEA from the ELAVL1 knockdown human primary AML RNA-sequencing data set.

Table S6 shows altered gene sets identified by GSEA of the ELAVL1 knockout RN2c RNA-sequencing data set.

Supplemental Figure S1 shows expression of RBPs in healthy HSPCs and RN2c cells, sgRNA drop out dynamics in primary and secondary transplants, the molecular functions and biological processes associated with the hit RBPs and their gene essentialities.

Supplemental Figure S6 shows the GSEA of ELAVL1 depletion in mouse RN2c and human primary AML cells, ELAVL1 subcellular localization in mouse and human AML, the qRT-PCR of ELAVL1 targets in MS-444-treated RN2c cells, and the integrative analysis of the mouse RNA-seq and eCLIP-seq.

Table S2 shows sequences of (a) sgRNAs from the RBP screen, (b) sequencing primers, (c) shRNAs and human-targeting sgRNAs, (d) eCLIP oligos.

Table S3 shows clinical information for all AML patient specimens used in this study.

Supplemental Figure S3 shows the generation of the bcCML mouse model and the effects of ELAVL1 on normal bone marrow in vivo.

SUMMARYAcute myeloid leukemia (AML) progression and relapse is fueled by self-renewing leukemic stem cells (LSCs) whose molecular determinants have been difficult to discern from normal hematopoietic stem cells (HSCs) or to uncover in screening approaches focused on general AML cell properties. We have identified a unique set of RNA binding proteins (RBPs) that are enriched in human AML LSCs but repressed in HSCs. Using an in vivo two step CRISPR-Cas9-mediated screening approach to specifically score for cancer stem cell functionality, we found 32 RBPs essential for LSC propagation and self-renewal in MLL-AF9 translocated AML. Using knockdown or small molecule approaches we show that targeting key hit RBP ELAVL1 impaired LSC-driven in vivo leukemic reconstitution and selectively depleted primitive AML cells vs. normal hematopoietic stem and progenitors. Importantly, knockdown of Elavl1 spared HSCs while significantly reducing LSC numbers across genetically diverse leukemias. Integrative RNA-seq and eCLIP-seq profiling revealed hematopoietic differentiation, RNA splicing and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with the mitochondrial import protein TOMM34 being a direct ELAVL1-stabilized target whose inhibition impairs AML propagation. Altogether, through the use of a stem cell-adapted in vivo CRISPR dropout screening strategy, this work demonstrates that a wide variety of post-transcriptionally acting RBPs are important regulators of LSC-survival and self-renewal and, as exemplified by ELAVL1, highlights their potential as therapeutic targets in AML.

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.

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.

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.