A developmental constraint model of cancer cell states and tumor heterogeneity.

Cancer is a disease that stems from a fundamental liability inherent to multicellular life forms in which an individual cell is capable of reneging on the interests of the collective organism. Although cancer is commonly described as an evolutionary process, a less appreciated aspect of tumorigenesis may be the constraints imposed by the organism's developmental programs. Recent work from single-cell transcriptomic analyses across a range of cancer types has revealed the recurrence, plasticity, and co-option of distinct cellular states among cancer cell populations. Here, we note that across diverse cancer types, the observed cell states are proximate within the developmental hierarchy of the cell of origin. We thus posit a model by which cancer cell states are directly constrained by the organism's "developmental map." According to this model, a population of cancer cells traverses the developmental map, thereby generating a heterogeneous set of states whose interactions underpin emergent tumor behavior.

Cancer stem cells: The adventurous journey from hematopoietic to leukemic stem cells.

This year's Gairdner Foundation Award for Biomedical Research is awarded to John Dick for the discovery of leukemic stem cells and the hierarchical organization of acute myeloid leukemias. His work laid the foundation for the cancer stem cell model with numerous clinical implications for hematopoietic malignancies and solid tumors.

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

Glioblastomas acquire myeloid-affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.

Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B.

Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I → KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.

Adaptive Immune Resistance Emerges from Tumor-Initiating Stem Cells.

Our bodies are equipped with powerful immune surveillance to clear cancerous cells as they emerge. How tumor-initiating stem cells (tSCs) that form and propagate cancers equip themselves to overcome this barrier remains poorly understood. To tackle this problem, we designed a skin cancer model for squamous cell carcinoma (SCC) that can be effectively challenged by adoptive cytotoxic T cell transfer (ACT)-based immunotherapy. Using single-cell RNA sequencing (RNA-seq) and lineage tracing, we found that transforming growth factor ß (TGF-ß)-responding tSCs are superior at resisting ACT and form the root of tumor relapse. Probing mechanism, we discovered that during malignancy, tSCs selectively acquire CD80, a surface ligand previously identified on immune cells. Moreover, upon engaging cytotoxic T lymphocyte antigen-4 (CTLA4), CD80-expressing tSCs directly dampen cytotoxic T cell activity. Conversely, upon CTLA4- or TGF-ß-blocking immunotherapies or Cd80 ablation, tSCs become vulnerable, diminishing tumor relapse after ACT treatment. Our findings place tSCs at the crux of how immune checkpoint pathways are activated.

Rare Pulmonary Neuroendocrine Cells Are Stem Cells Regulated by Rb, p53, and Notch.

Pulmonary neuroendocrine (NE) cells are neurosensory cells sparsely distributed throughout the bronchial epithelium, many in innervated clusters of 20-30 cells. Following lung injury, NE cells proliferate and generate other cell types to promote epithelial repair. Here, we show that only rare NE cells, typically 2-4 per cluster, function as stem cells. These fully differentiated cells display features of classical stem cells. Most proliferate (self-renew) following injury, and some migrate into the injured area. A week later, individual cells, often just one per cluster, lose NE identity (deprogram), transit amplify, and reprogram to other fates, creating large clonal repair patches. Small cell lung cancer (SCLC) tumor suppressors regulate the stem cells: Rb and p53 suppress self-renewal, whereas Notch marks the stem cells and initiates deprogramming and transit amplification. We propose that NE stem cells give rise to SCLC, and transformation results from constitutive activation of stem cell renewal and inhibition of deprogramming.

CD10+GPR77+ Cancer-Associated Fibroblasts Promote Cancer Formation and Chemoresistance by Sustaining Cancer Stemness.

Carcinoma-associated fibroblasts (CAFs) are abundant and heterogeneous stromal cells in tumor microenvironment that are critically involved in cancer progression. Here, we demonstrate that two cell-surface molecules, CD10 and GPR77, specifically define a CAF subset correlated with chemoresistance and poor survival in multiple cohorts of breast and lung cancer patients. CD10+GPR77+ CAFs promote tumor formation and chemoresistance by providing a survival niche for cancer stem cells (CSCs). Mechanistically, CD10+GPR77+ CAFs are driven by persistent NF-κB activation via p65 phosphorylation and acetylation, which is maintained by complement signaling via GPR77, a C5a receptor. Furthermore, CD10+GPR77+ CAFs promote successful engraftment of patient-derived xenografts (PDXs), and targeting these CAFs with a neutralizing anti-GPR77 antibody abolishes tumor formation and restores tumor chemosensitivity. Our study reveals a functional CAF subset that can be defined and isolated by specific cell-surface markers and suggests that targeting the CD10+GPR77+ CAF subset could be an effective therapeutic strategy against CSC-driven solid tumors.

Toward Minimal Residual Disease-Directed Therapy in Melanoma.

Many patients with advanced cancers achieve dramatic responses to a panoply of therapeutics yet retain minimal residual disease (MRD), which ultimately results in relapse. To gain insights into the biology of MRD, we applied single-cell RNA sequencing to malignant cells isolated from BRAF mutant patient-derived xenograft melanoma cohorts exposed to concurrent RAF/MEK-inhibition. We identified distinct drug-tolerant transcriptional states, varying combinations of which co-occurred within MRDs from PDXs and biopsies of patients on treatment. One of these exhibited a neural crest stem cell (NCSC) transcriptional program largely driven by the nuclear receptor RXRG. An RXR antagonist mitigated accumulation of NCSCs in MRD and delayed the development of resistance. These data identify NCSCs as key drivers of resistance and illustrate the therapeutic potential of MRD-directed therapy. They also highlight how gene regulatory network architecture reprogramming may be therapeutically exploited to limit cellular heterogeneity, a key driver of disease progression and therapy resistance.

New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer.

Epithelial-mesenchymal transition (EMT) is a cellular programme that is known to be crucial for embryogenesis, wound healing and malignant progression. During EMT, cell-cell and cell-extracellular matrix interactions are remodelled, which leads to the detachment of epithelial cells from each other and the underlying basement membrane, and a new transcriptional programme is activated to promote the mesenchymal fate. In the context of neoplasias, EMT confers on cancer cells increased tumour-initiating and metastatic potential and a greater resistance to elimination by several therapeutic regimens. In this Review, we discuss recent findings on the mechanisms and roles of EMT in normal and neoplastic tissues, and the cell-intrinsic signals that sustain expression of this programme. We also highlight how EMT gives rise to a variety of intermediate cell states between the epithelial and the mesenchymal state, which could function as cancer stem cells. In addition, we describe the contributions of the tumour microenvironment in inducing EMT and the effects of EMT on the immunobiology of carcinomas.

Multi-organ Site Metastatic Reactivation Mediated by Non-canonical Discoidin Domain Receptor 1 Signaling.

Genetic screening identifies the atypical tetraspanin TM4SF1 as a strong mediator of metastatic reactivation of breast cancer. Intriguingly, TM4SF1 couples the collagen receptor tyrosine kinase DDR1 to the cortical adaptor syntenin 2 and, hence, to PKCα. The latter kinase phosphorylates and activates JAK2, leading to the activation of STAT3. This non-canonical mechanism of signaling induces the expression of SOX2 and NANOG; sustains the manifestation of cancer stem cell traits; and drives metastatic reactivation in the lung, bone, and brain. Bioinformatic analyses and pathological studies corroborate the clinical relevance of these findings. We conclude that non-canonical DDR1 signaling enables breast cancer cells to exploit the ubiquitous interstitial matrix component collagen I to undergo metastatic reactivation in multiple target organs.

Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML.

Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML.

WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis.

Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.

Persister cancer cells: Iron addiction and vulnerability to ferroptosis.

Ferroptosis is a unique type of non-apoptotic cell death resulting from the unrestrained occurrence of peroxidized phospholipids, which are subject to iron-mediated production of lethal oxygen radicals. This cell death modality has been detected across many organisms, including in mammals, where it can be used as a defense mechanism against pathogens or even harnessed by T cells to sensitize tumor cells toward effective killing. Conversely, ferroptosis is considered one of the main cell death mechanisms promoting degenerative diseases. Emerging evidence suggests that ferroptosis represents a vulnerability in certain cancers. Here, we critically review recent advances linking ferroptosis vulnerabilities of dedifferentiating and persister cancer cells to the dependency of these cells on iron, a potential Achilles heel for small-molecule intervention. We provide a perspective on the mechanisms reliant on iron that contribute to the persister cancer cell state and how this dependency may be exploited for therapeutic benefits.

Connect four with glioblastoma stem cell factors.

Hierarchical cell state models, wherein a few stem-like tumor-propagating cells repopulate the tumor after therapy, are often invoked in cancer. Suvà et al. demonstrate a plastic developmental hierarchy in glioma cell populations by characterizing the epigenetic states of phenotypically distinct cells and identifying four factors sufficient to reprogram differentiated cells into a tumorigenic stem-like state.

Reconstructing and reprogramming the tumor-propagating potential of glioblastoma stem-like cells.

Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, and OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements and are sufficient to fully reprogram differentiated GBM cells to "induced" TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies candidate therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies. PAPERCLIP:

Targeting KDM4A epigenetically activates tumor-cell-intrinsic immunity by inducing DNA replication stress.

Developing strategies to activate tumor-cell-intrinsic immune response is critical for improving tumor immunotherapy by exploiting tumor vulnerability. KDM4A, as a histone H3 lysine 9 trimethylation (H3K9me3) demethylase, has been found to play a critical role in squamous cell carcinoma (SCC) growth and metastasis. Here we report that KDM4A inhibition promoted heterochromatin compaction and induced DNA replication stress, which elicited antitumor immunity in SCC. Mechanistically, KDM4A inhibition promoted the formation of liquid-like HP1γ puncta on heterochromatin and stall DNA replication, which activated tumor-cell-intrinsic cGAS-STING signaling through replication-stress-induced cytosolic DNA accumulation. Moreover, KDM4A inhibition collaborated with PD1 blockade to inhibit SCC growth and metastasis by recruiting and activating CD8+ T cells. In vivo lineage tracing demonstrated that KDM4A inhibition plus PD1 blockade efficiently eliminated cancer stem cells. Altogether, our results demonstrate that targeting KDM4A can activate anti-tumor immunity and enable PD1 blockade immunotherapy by aggravating replication stress in SCC cells.

Definition of a small core transcriptional circuit regulated by AML1-ETO.

Transcription factors regulate gene networks controlling normal hematopoiesis and are frequently deregulated in acute myeloid leukemia (AML). Critical to our understanding of the mechanism of cellular transformation by oncogenic transcription factors is the ability to define their direct gene targets. However, gene network cascades can change within minutes to hours, making it difficult to distinguish direct from secondary or compensatory transcriptional changes by traditional methodologies. To overcome this limitation, we devised cell models in which the AML1-ETO protein could be quickly degraded upon addition of a small molecule. The rapid kinetics of AML1-ETO removal, when combined with analysis of transcriptional output by nascent transcript analysis and genome-wide AML1-ETO binding by CUT&RUN, enabled the identification of direct gene targets that constitute a core AML1-ETO regulatory network. Moreover, derepression of this gene network was associated with RUNX1 DNA binding and triggered a transcription cascade ultimately resulting in myeloid differentiation.

Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties.

Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.