Lrig1 and Wnt dependent niches dictate segregation of resident immune cells and melanocytes in murine tail epidermis

The barrier-forming, self-renewing mammalian epidermis comprises keratinocytes, pigment-producing melanocytes, and resident immune cells as first-line host defense. In murine tail skin, interfollicular epidermis patterns into pigmented ′scale′ and non-pigmented ′interscale′ epidermis. Why and how mature melanocytes confine to scale epidermis is unresolved. Here, we delineate a cellular hierarchy among epidermal cell types that determines skin patterning. Already during postnatal development, melanocytes co-segregate with newly forming scale compartments. Intriguingly, this process coincides with partitioning of both Langerhans cells and dendritic epidermal T-cells to interscale epidermis, suggesting functional segregation of pigmentation and immune surveillance. Analysis of non-pigmented mice and of mice lacking melanocytes or resident immune cells revealed that immunocyte patterning is melanocyte- and melanin-independent, and, vice versa, immune cells do not control melanocyte localization. Instead, genetically enforced progressive scale fusion upon Lrig1 deletion showed that melanocytes and immune cells dynamically follow epithelial scale:interscale patterns. Importantly, disrupting Wnt-Lef1 function in keratinocytes caused melanocyte mislocalization to interscale epidermis, implicating canonical Wnt signaling in organizing the pigmentation pattern. Together, this work uncovered cellular and molecular principles underlying the compartmentalization of tissue functions in skin.

EPCO-27. SINGLE-CELL ANALYSIS OF ETMR PATIENT SAMPLES LINKS TRANSCRIPTIONAL PHENOTYPES TO GENETIC DRIVER ALTERATIONS AND INFORMS NOVEL THERAPEUTIC STRATEGIES

Embryonal tumor with multilayered rosettes (ETMR) is a malignant brain tumor that typically occurs in children under the age of three. Most patients die within two years of diagnosis, and more effective, targeted therapies are urgently needed. To better characterize the oncogenic mechanisms of key driver alterations and identify novel therapeutic targets, we studied the cellular heterogeneity of ETMR using single-cell RNA sequencing. Analyses conducted on >3,000 high-quality cells collected from ten primary and relapse specimens revealed a common cellular hierarchy across all tumors: A highly proliferative neural stem cell-like population (SOX2+) gives rise to intermediate progenitors (ASCL1+) and more differentiated neuron-like cells (STMN2/4+). These malignant populations closely match histological patterns of ETMR (i.e. rosettes, neuropil), as observed by immunofluorescence microscopy. Comparison to single-cell datasets from human embryos indicates resemblance to cell populations of the developing brain, but also reveals key ETMR-specific differences, including expression of the chromosome 19 miRNA cluster (C19MC, the presumed genetic driver of most ETMRs), which is restricted to the stem cell-like population. We next investigated if targeting C19MC is a viable strategy to disrupt the cellular hierarchy of ETMR. Silencing with antisense oligonucleotides shows pronounced reduction of cell line growth for a specific subset of the 46 members of C19MC. These miRNAs share seed sequences with evolutionary conserved miRNAs that have been shown to regulate pluripotency and self-renewal of embryonic stem cells. We hypothesize that select C19MC members play similar roles in ETMR and represent bona fide targets for therapeutic targeting using antisense technology.

Accessibility Over Transposable Elements Reveals Genetic Determinants of Stemness Properties in Normal and Leukemic Hematopoiesis

Despite most acute myeloid leukemia (AML) patients achieving complete remission after induction chemotherapy, two thirds of patients will relapse with fatal disease within 5 years. AML is organized as a cellular hierarchy sustained by leukemia stem cells (LSC) at the apex, with LSC properties directly linked to tumor progression, therapy failure and disease relapse 1–5. Despite the central role of LSC in poor patient outcomes, little is known of the genetic determinants of their stemness properties 6–8. Although much AML research focuses on mutational processes and their impact on gene expression programs, the genetic determinants of cell state properties including stemness expand beyond mutations, relying on the genetic architecture captured in the chromatin of each cell 9–11. As LSCs share many functional and molecular properties with normal hematopoietic stem cells (HSC), we identified genetic determinants of primitive populations enriched for LSCs and HSCs in comparison with their downstream mature progeny by investigating their chromatin accessibility. Our work reveals how distinct transposable element (TE) subfamilies are used in primitive versus mature populations, functioning as docking sites for stem cell-associated regulators of genome topology, including CTCF, or lineage-specific transcription regulators in primitive and mature populations, respectively. We further show how TE subfamilies accessible in LSCs define docking sites for several oncogenic drivers in AML, namely FLI1, LYL1 and MEIS1. Using chromatin accessibility profiles from a cohort of AML patients, we further show the clinical utility of our TE accessibility-based LSCTE121 scoring scheme to identify patients with high rates of relapse. Collectively, our work reveals how different accessible TE subfamilies serve as genetic determinants of stemness properties in normal and leukemic hematopoietic stem cells.

The Tumor Microenvironment as a Driving Force of Breast Cancer Stem Cell Plasticity

Tumor progression involves the co-evolution of transformed cells and the milieu in which they live and expand. Breast cancer stem cells (BCSCs) are a specialized subset of cells that sustain tumor growth and drive metastatic colonization. However, the cellular hierarchy in breast tumors is rather plastic, and the capacity to transition from one cell state to another depends not only on the intrinsic properties of transformed cells, but also on the interplay with their niches. It has become evident that the tumor microenvironment (TME) is a major player in regulating the BCSC phenotype and metastasis. The complexity of the TME is reflected in its number of players and in the interactions that they establish with each other. Multiple types of immune cells, stromal cells, and the extracellular matrix (ECM) form an intricate communication network with cancer cells, exert a highly selective pressure on the tumor, and provide supportive niches for BCSC expansion. A better understanding of the mechanisms regulating these interactions is crucial to develop strategies aimed at interfering with key BCSC niche factors, which may help reducing tumor heterogeneity and impair metastasis.

Variation in Stem Cell Driven Hierarchies Underlies Clinical Outcome and Drug Response in AML

AML is a stem cell disease wherein the properties of the disease-driving leukemia stem cells (LSCs) are reflected in the cellular hierarchies that they generate. We sought to understand how these hierarchies vary across patients and whether their characteristics are clinically relevant. To evaluate cellular hierarchies in AML, we re-analyzed the scRNA-seq data of 13,653 cells from 12 AML patients at diagnosis (van Galen, Cell 2019) and in particular the stem and progenitor blast populations. We identified three novel leukemia stem populations differing in their depth of quiescence, inflammatory signaling, and myeloid priming. Using the signatures of 7 malignant populations as well as 7 immune cell populations from this scRNA-seq data, we applied CIBERSORTx deconvolution (Newman, Nat Biotechnol 2019) on bulk RNA-seq data from multiple patient cohorts. This enabled determination of the relative abundance of each cell type in each patient, thereby capturing the "shape" of the leukemic hierarchies of hundreds of AML patients. AML patients within these cohorts clustered into 4 groups defined by different proportions of stem, progenitor, and mature blasts - each of which differed in their underlying genomic alterations and overall survival. Differences in chemotherapy response were mediated by specific cell types, notably a GMP-like blast population and a quiescent stem-like population (qLSPC). Dominance of the GMP-like population was associated with longer survival (HR -3.1, p=0.002), and these blasts were enriched among younger patients (<65 years) and those with favorable risk cytogenetics. In contrast, qLSPCs were associated with poor survival (HR 2.3, p=0.02; TCGA) and were more abundant in older patients (> 65 years) and patients with adverse cytogenetic alterations. Critically, this qLSPC population was enriched at relapse as well as within a subset of pediatric AML patients that failed to respond to induction chemotherapy. We confirmed that qLSPCs were also enriched among functionally validated leukemia engrafting (LSC+) sorted AML fractions. Accordingly, a high LSC17 score was strongly correlated with qLSPC abundance and anti-correlated with GMP-like abundance, suggesting that the score may reflect the underlying cellular hierarchy of each AML patient. Next, we generated drug sensitivity profiles for each cell type by correlating ex vivo drug sensitivity data for each of 112 inhibitors screened in the BEAT-AML trial with the relative abundance of each cell type across individual patients. In particular, Venetoclax sensitivity correlated with the abundance of primitive cell types and anti-correlated with mature cell types, suggesting that the composition of the leukemic hierarchy in AML patients is associated with drug response. We previously demonstrated that a high LSC17 score identifies patients who do not benefit from standard chemotherapy (Ng, Nature 2016). To develop a diagnostic tool for therapy selection amongst these poor prognosis patients, we retrained the LSC17 genes against cell type abundance and derived a subscore (LSC-7) to map patients along an axis of primitive vs mature leukemic hierarchies. We show that AML samples with a high LSC-7 score (more stem-like blasts) were more sensitive to Venetoclax whereas AML patients with a low LSC-7 score (more mature blasts) benefited from treatment with Gemtuzumab-Ozogamicin (GO). Used together, the LSC17 and LSC-7 scores enable risk stratification as well as subsequent drug selection for high-risk patients, and can both be measured by a single rapid NanoString assay. To apply this approach more broadly, we identified several published studies with drug screening data from primary patient samples and accompanying RNAseq data. By correlating cell type abundance with drug response, we were able to map the critical cell types that mediate sensitivity and resistance to inhibitors of mitochondrial metabolism, histone demethylation, and the CD47-SIRPa axis, among others. Our data establish that scRNA-seq informed deconvolution of bulk expression data permits characterization of the cellular hierarchy of individual AML patients. This framework can enhance our understanding of many aspects of biological, genomic, and clinical heterogeneity in AML, and represents a powerful tool to enable personalized therapeutic decision-making in AML. Figure Disclosures Wang: Trilium Therapeutics: Patents & Royalties. Dick:Bristol-Myers Squibb/Celgene: Research Funding.

STEM-04. PLATELETS DRIVE GLIOBLASTOMA ONCOGENESIS BY ENHANCING THE GLIOMA STEM CELL PHENOTYPE

Glioblastoma (GBM) is recognized as one of the deadliest forms of cancer, despite aggressive therapy consisting of maximal surgical resection followed by concurrent radiation and chemotherapy, the median survival remains ~12 months. Glioma stem cells (GSCs) possess potent tumor-initiating properties and comprise a cellular hierarchy that is responsible for treatment resistance and progression. Specifically targeting GSCs has been considered a promising therapeutic approach, however no clear method has been identified. Histologically, it is known that GSCs are found in perivascular and pseudsopalisading regions of GBM. Similarly, platelet aggregates are often found in pseudsopalisading necrotic regions, suggesting a potential interaction between platelets and GSCs due to their spatial locations. High platelet counts have been associated with poor clinical outcome in many cancers including ovarian and endometrial cancer. While platelets are known to affect progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Our work aimed to understand the crosstalk between GSCs and platelets within GBM solid tumors that work to enhance disease progression and treatment resistance. Our clinical studies suggest elevated platelet counts positively correlate with tumor growth and negatively correlate to overall patient survival. We found platelets and GSC co-localization in GBM solid tissue; platelet exposure to GSCs results in increased proliferation of GSCs specifically, by increasing the self-renewing capacity of GSCs in a dose dependent manner, and resulted in an increased “Stem-like” transcriptional pattern. Inhibiting the GSC-platelet interaction results in a decrease in GSC renewal and stemness. These results introduce a novel interaction between GSCs and platelets and elucidate a novel therapeutic approach specifically targeting GSCs by disrupting the GSC-platelet interaction.