Search

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.

Includes Supplemental Figure S1 and Supplemental Tables S1-S7

Raw cell counts of FISH gene probes for (1) primary breast tumors, (2) metastatic sites, (3) pre- and post-treatment [Hungarian sample], and (4) TNBC cases.

Tumor cells that preferentially enter circulation include the precursors of metastatic cancer. Previously, we characterized circulating tumor cells (CTC) from patients with breast cancer and identified a signature of genomic regions with recurrent copy number gains. Through fluorescence in situ hybridization, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21-69%, median 55.5%, n=19). Furthermore, they are more prevalent in the metastases of patients who died from breast cancer after multiple rounds of treatment (70-100%, median 93%, n=41). Diversity indices revealed that higher spatial heterogeneity for these regions within primary tumors is associated with increased dissemination and metastasis. An identified subclone with multiple regions gained (MRG clone) was enriched in a post-treatment primary breast carcinoma as well as multiple metastatic tumors and local breast recurrences obtained at autopsy, indicative of a distinct early subclone with the capability to resist multiple lines of treatment and eventually cause death. Additionally, multiplex immunofluorescence revealed that tumor heterogeneity is significantly associated with the degree of infiltration of B lymphocytes in triple-negative breast cancer, a subtype with a large immune component. Collectively, these data reveal the functional potential of genetic subclones that comprise heterogeneous primary breast carcinomas and are selected for in CTCs and post-treatment breast cancer metastases. In addition, they uncover a relationship between tumor heterogeneity and host immune response in the tumor microenvironment.