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FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

Hematologic malignancies are model diseases for understanding neoplastic transformation and serve as prototypes for developing effective therapies. Indeed, the concept of systemic cancer therapy originated in hematologic malignancies and has guided the development of chemotherapy, cellular therapies, immunotherapy and modern precision oncology. Despite significant advances in the treatment of leukemias, lymphomas and multiple myelomas, treatment resistance associated with molecular and clinical relapse remains very common. Therapy of relapsed and refractory disease remains extremely difficult, and failure of disease control at this stage remains the leading cause of mortality in patients with hematologic malignancies. In recent years, many efforts have been made to identify the genetic and epigenetic mechanisms that drive the development of hematologic malignancies to the stage of full-blown disease requiring clinical intervention. In contrast, the mechanisms responsible for treatment resistance in hematologic malignancies remain poorly understood. For example, the molecular characteristics of therapy-resistant persisting cells in minimal residual disease (MRD) remain rather elusive. In this mini-review we want to discuss that cellular heterogeneity and plasticity, together with adaptive genetic and epigenetic processes, lead to reduced sensitivity to various treatment regimens such as chemotherapy and pathway inhibitors such as tyrosine kinase inhibitors. However, resistance mechanisms may be conserved across biologically distinct cancer entities. Recent technological advances have made it possible to explore the underlying mechanisms of therapy resistance with unprecedented resolution and depth. These include novel multi-omics technologies with single cell resolution combined with advanced biocomputational approaches, along with artificial intelligence (AI) and sophisticated disease models for functional validation.

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

Disseminated cancer cells frequently lodge near vasculature in secondary organs. However, our understanding of the cellular crosstalk invoked at perivascular sites is still rudimentary. Here, we identify intercellular machinery governing formation of a pro-metastatic vascular niche during breast cancer colonization in the lung. We show that specific secreted factors, induced in metastasis-associated endothelial cells (ECs), promote metastasis in mice by enhancing stem cell properties and the viability of cancer cells. Perivascular macrophages, activated via tenascin C (TNC) stimulation of Toll-like receptor 4 (TLR4), were shown to be crucial in niche activation by secreting nitric oxide (NO) and tumor necrosis factor (TNF) to induce EC-mediated production of niche components. Notably, this mechanism was independent of vascular endothelial growth factor (VEGF), a key regulator of EC behavior and angiogenesis. However, targeting both macrophage-mediated vascular niche activation and VEGF-regulated angiogenesis resulted in added potency to curb lung metastasis in mice. Together, our findings provide mechanistic insights into the formation of vascular niches in metastasis.

Inter-patient variability and the similarity of healthy and leukemic stem cells (LSCs) have impeded the characterization of LSCs in acute myeloid leukemia (AML) and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. Although residual healthy and preleukemic cells dominated the dormant stem cell compartment, active LSCs resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors constituted a highly aberrant, disease-defining compartment: their gene expression and differentiation state affected both the chemotherapy response and leukemia's ability to differentiate into transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers misregulated specifically in leukemic cells. Taken together, CloneTracer reveals a differentiation landscape that mimics its healthy counterpart and may determine biology and therapy response in AML., Cell Stem Cell, 30 (5), ISSN:1934-5909, ISSN:1875-9777

To acquire a better understanding of clonal evolution of acute myeloid leukemia (AML) and to identify the clone(s) responsible for disease recurrence, we have comparatively studied leukemia-specific mutations by whole-exome-sequencing (WES) of both the leukemia and the nonleukemia compartments derived from the bone marrow of AML patients. The T-lymphocytes, B-lymphocytes and the functionally normal hematopoietic stem cells (HSC), that is, CD34+ /CD38- /ALDH+ cells for AML with rare-ALDH+ blasts (

Purpose As an independent, negative-prognostic biomarker for progression-free survival (PFS) and overall survival (OS), circulating tumor cells (CTCs) constitute a promising component for developing a liquid biopsy for patients with metastatic breast cancer (MBC). The effects of HER2-targeted therapy such as trastuzumab, pertuzumab, T-DM1, and lapatinib on CTC status and longitudinal enumeration were assessed in this trial. Methods CTC status of 264 patients with MBC was analyzed prior to and after 4 weeks of a new line of palliative systemic therapy. CTCs were assessed using CellSearch®. Three groups were compared: patients with HER2-positive MBC receiving ongoing HER2-targeted therapy (n = 28), patients with de novo HER2-positive MBC and no HER2-targeted therapy in the last 12 months prior to enrollment and start of HER2-targeted therapy (n = 15), and patients with HER2-nonamplified disease and no HER2-targeted therapy (n = 212). Results Positive CTC status (≥ 5 CTC/7.5 ml blood) at enrollment was observed in the 3 groups for 17.9, 46.7, and 46.2% (p = 0.02) of patients, respectively. At least one CTC/7.5 ml was seen in 28.6, 53.3, and 67.0% (p p p = 0.001) of patients had still a positive CTC status (≥ 5 CTC/7.5 ml blood). At least one CTC/7.5 ml was still observed in 25.0, 20.0, and 50.5% (p = 0.004) of the patients. Furthermore, 7.1, 0.0, and 1.9% (p = 0.187) had at least one HER2-positive CTC. After 3 months of therapy, 35.7, 20.0, and 28.3% (p = 0.536) showed disease progression. Conclusions HER2-targeted therapy seems to reduce the overall CTC count in patients with MBC. This should be taken into account when CTC status is used as an indicator for aggressive or indolent metastatic tumor disease.

Metastatic colonization relies on interactions between disseminated cancer cells and the microenvironment in secondary organs. Here, we show that disseminated breast cancer cells evoke phenotypic changes in lung fibroblasts, forming a supportive metastatic niche. Colonization of the lungs confers an inflammatory phenotype in metastasis-associated fibroblasts. Specifically, IL-1α and IL-1β secreted by breast cancer cells induce CXCL9 and CXCL10 production in lung fibroblasts via NF-κB signaling, fueling the growth of lung metastases. Notably, we find that the chemokine receptor CXCR3, that binds CXCL9/10, is specifically expressed in a small subset of breast cancer cells, which exhibits tumor-initiating ability when co-transplanted with fibroblasts and has high JNK signaling that drives IL-1α/β expression. Importantly, disruption of the intercellular JNK-IL-1-CXCL9/10-CXCR3 axis reduces metastatic colonization in xenograft and syngeneic mouse models. These data mechanistically demonstrate an essential role for the molecular crosstalk between breast cancer cells and their fibroblast niche in the progression of metastasis., How cancer cells engage the microenvironment to establish metastasis is poorly understood. Here, the authors show that CXCR3-expressing breast cancer cells secrete IL-1 to induce a paracrine crosstalk with fibroblasts in the lung, which involves CXCL9/10 production and results in colonization of the lung.

Estrogen receptor-positive breast cancer is a highly prevalent but heterogeneous disease among women. Advanced molecular stratification is required to enable individually most efficient treatments based on relevant prognostic and predictive biomarkers. First objective of our study was the hypothesis-driven discovery of biomarkers involved in tumor progression upon xenotransplantation of Luminal breast cancer into humanized mice. The second objective was the marker validation and correlation with the clinical outcome of Luminal breast cancer disease within the GeparTrio trial. An elevated mdm2 gene copy number was associated with enhanced tumor growth and lung metastasis in humanized tumor mice. The viability, proliferation and migration capacity of inherently mdm2 positive breast cancer cells in vitro were significantly reduced upon mdm2 knockdown or anti-mdm2 targeting. An mdm2 gain significantly correlated with a worse DFS and OS of Luminal breast cancer patients, albeit it was also associated with an enhanced preoperative pathological response rate. We provide evidence for an enhanced Luminal breast cancer stratification based on mdm2. Moreover, mdm2 can potentially be utilized as a therapeutic target in the Luminal subtype.

Metastasis is the primary cause of cancer-related mortality. Tumor cell interactions with cells of the vessel wall are decisive and potentially rate-limiting for metastasis. The molecular nature of this cross-talk is, beyond candidate gene approaches, hitherto poorly understood. Using endothelial cell (EC) bulk and single-cell transcriptomics in combination with serum proteomics, we traced the evolution of the metastatic vascular niche in surgical models of lung metastasis. Temporal multiomics revealed that primary tumors systemically reprogram the body’s vascular endothelium to perturb homeostasis and to precondition the vascular niche for metastatic growth. The vasculature with its enormous surface thereby serves as amplifier of tumor-induced instructive signals. Comparative analysis of lung EC gene expression and secretome identified the transforming growth factor–β (TGFβ) pathway specifier LRG1, leucine-rich alpha-2-glycoprotein 1, as an early instructor of metastasis. In the presence of a primary tumor, ECs systemically up-regulated LRG1 in a signal transducer and activator of transcription 3 (STAT3)–dependent manner. A meta-analysis of retrospective clinical studies revealed a corresponding up-regulation of LRG1 concentrations in the serum of patients with cancer. Functionally, systemic up-regulation of LRG1 promoted metastasis in mice by increasing the number of prometastatic neural/glial antigen 2 (NG2)

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D—a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6–9—are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo. Leukaemic stem cells in acute myeloid leukaemia are defined by a lack of expression of NKG2D ligands, which mediates their ability to evade surveillance by NK cells.

Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a 5-day course of granulocyte colony-stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor plerixafor is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was coadministered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization achieved by the VLA4 inhibitor and CXCR2 agonist combination in mice compared with currently approved HSPC mobilization methods, the combination represents an exciting potential strategy for clinical development in the future.

Somatic mutations in DNA Methyltransferase 3A (DNMT3A) are among the most frequent alterations in clonal hematopoiesis (CH) and Acute Myeloid Leukemia (AML), with a hotspot in exon 23 at arginine 882 (DNMT3A-R882). Here we demonstrate that DNMT3A-R882H-dependent CH- and AML cells are specifically susceptible to the hypomethylating agent azacytidine (AZA). Addition of AZA to chemotherapy prolonged AML survival solely in patients with DNMT3A-R882 mutation, suggesting its potential as a novel predictive marker for AZA response. AML and CH mouse models confirmed AZA susceptibility specifically in DNMT3A-R882H-expressing cells. Hematopoietic stem and progenitor cells expressing DNMT3A-R882H exhibited cell-autonomous viral mimicry response as a result of focal DNA hypomethylation at retrotransposon sequences. Administration of AZA boosted hypomethylation of retrotransposons specifically in DNMT3A-R882H expressing cells and maintained elevated levels of canonical interferon-stimulated genes (ISGs), thus leading to suppressed protein translation and increased apoptosis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, which challenges the molecular analyses of bulk tumor samples. Here we FACS-purified epithelial cells from human PDAC and normal pancreas and derived their genome-wide transcriptome and DNA methylome landscapes. Clustering based on DNA methylation revealed two distinct PDAC groups displaying different methylation patterns at regions encoding repeat elements. Methylationlow tumors are characterized by higher expression of endogenous retroviral transcripts and double-stranded RNA sensors, which lead to a cell-intrinsic activation of an interferon signature (IFNsign). This results in a protumorigenic microenvironment and poor patient outcome. Methylationlow/IFNsignhigh and Methylationhigh/IFNsignlow PDAC cells preserve lineage traits, respective of normal ductal or acinar pancreatic cells. Moreover, ductal-derived KrasG12D/Trp53−/− mouse PDACs show higher expression of IFNsign compared with acinar-derived counterparts. Collectively, our data point to two different origins and etiologies of human PDACs, with the aggressive Methylationlow/IFNsignhigh subtype potentially targetable by agents blocking intrinsic IFN signaling. Significance: The mutational landscapes of PDAC alone cannot explain the observed interpatient heterogeneity. We identified two PDAC subtypes characterized by differential DNA methylation, preserving traits from normal ductal/acinar cells associated with IFN signaling. Our work suggests that epigenetic traits and the cell of origin contribute to PDAC heterogeneity. This article is highlighted in the In This Issue feature, p. 521

Cancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells., Nature Communications, 12 (1), ISSN:2041-1723

Despite advances in the treatment of breast cancer, it is still the second leading cause of cancer-related death in women worldwide. A large number of patients develop recurrence and die of advanced metastatic disease. More than 70% of metastatic breast cancers (mBC) express androgen receptor (AR) representing a potential target for anti-hormone therapies. AR is suggested to directly interact with an associated transcription factor (ARaTF) in breast cancer, however the functional role of ARaTF and its interaction with AR remains to be elucidated. We find that AR expression highly correlates with ARaTF in patients. To study its functional role driving tumorigenesis and metastatic outgrowth, we utilize patient-derived xenograft cell lines derived from liquid biopsies of mBC patients which reflect the highly heterogenous disease. Using genetically manipulated PDX cell models, we demonstrate that repression of ARaTF significantly reduces tumor growth of AR positive (AR+) BC cells in vivo. Notably, we observe the opposite phenotype in an AR negative cell model suggesting a tumor suppressive function when AR is not present. Mechanistically, we find that ARaTF regulates key metabolic processes: (1) Pharmacological inhibition of AR or silencing of ARaTF restricts mitochondrial respiration activity resulting in decreased energy production. (2) We also find that ARaTF positively regulates genes encoding enzymes involved in de novo fatty acid biosynthesis in AR+ BC. We further observe upregulation of the CD36 fatty acid transporter facilitating fatty acid uptake as compensatory pathway upon downregulation of ARaTF. Finally, abolishing CD36 expression in AR+ BC cells significantly reduces cell viability upon ARaTF downregulation. These findings suggest that lipid and energy metabolism are transcriptionally regulated by ARaTF facilitating cell survival in nutrient-depleted environments and thus facilitating metastatic outgrowth. These findings provide knowledge about the functional role of ARaTF regulating the lipogenic metabolic profile of AR+ breast cancer, and might help to identify patients who benefit from an anti-androgen treatment. Citation Format: Sarah-Jane Neuberth, Corinna Schumacher, Marteinn Thor Snaebjörnsson, Roberto Würth, Franziska Maria Zickgraf, Ornella Kossi, Felix Geist, Andrea Geist, Vera Thiel, Jonas Schwickert, Mattia Falcone, Massimo Saini, Almut Schulze, Andreas Trumpp, Martin R. Sprick. An androgen receptor-associated transcription factor regulates fatty acid metabolism driving tumor growth in androgen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 652.

Pancreatic Ductal Adenocarcinoma (PDAC) frequently metastasizes into the peritoneum forming peritoneal carcinomatosis, which are so far not treatable effectively. Metastasis-initiating cells need to acquire beneficial traits including cell plasticity, immune evasion, dormancy state control and organ colonization. These characteristics can be summarized in broad terms into two main processes, epithelial-to-mesenchymal transition (EMT) and stemness. Hyaluronic acid (HA), an extracellular matrix component, is a crucial factor in regulating these processes in PDAC, but it is so far not successfully targetable. Analyzing publicly available databases by gene set enrichment analysis (GSEA), a signature related to HA binding was enriched in tumor samples compared to normal tissue. Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1) was the top contributor to the enrichment score, being the 8th most enriched gene overall. We found that higher HAPLN1 expression correlated with shorter overall survival and that HAPLN1high patients had both, basal subtype and EMT signatures enriched. Moreover, these patients had a signature for peritoneal metastasis significantly enriched, suggesting a higher risk for peritoneal carcinomatosis. To study the role of HAPLN1 on PDAC in vitro, we stably overexpressed HAPLN1 in the murine PDAC cell line KPC. KPC-HAPLN1 cells expressed more EMT markers, more stem-related genes and changed the proteoglycan production from Aggrecan to Versican, which is known to be pro-metastatic. We found that spheroid formation, a feature of stemness, was improved in KPC-HAPLN1 vs KPC. Additionally, embedding these spheroids into matrigel led to an increased invasion of KPC-HAPLN1 cells. KPC-HAPLN1 cells improved KPC cell invasion capacities when co-cultured, indicating a paracrine effect. In vivo, intraperitoneal injection of luciferase expressing KPC cells resulted in higher luciferase activity when tumor cells expressed HAPLN1. Analyzing the peritoneal lavage (PL) from these mice, we obtained significantly more tumor cells in KPC-HAPLN1 injected mice. RNAseq data of tumor cells isolated from tumor nodules and PL showed that KPC-HAPLN1 cells acquired an increased metastatic potential and a strong immunomodulatory phenotype. Thus, we evaluated the immune cell composition of the PL by flow cytometry. Neutrophil and monocyte percentages were drastically reduced in KPC-HAPLN1 bearing mice. On the contrary, these mice had a significant increase in macrophages, which showed a reduction in pro-inflammatory gene expression. We conclude that HAPLN1 expression in tumor cells promotes a hyperplastic phenotype that facilitates invasion and colonization of the peritoneum, among others by creation of a pro-tumoral immune microenvironment. Citation Format: Lena Wiedmann, Francesca De Angelis Rigotti, Nuria Vaquero-Siguero, Elisa Donato, Elisa Espinet, Andreas Trumpp, Andreas Fischer, Juan Rodriguez-Vita. HAPLN1 increases peritoneal carcinomatosis by inducing tumor cell hyperplasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 960.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor entity with dismal prognosis. PDAC is characterized by an extensive desmoplastic stroma and the tumor microenvironment (TME) has been recognized to play crucial roles in tumor development, therapy resistance, progression and metastasis. Although different cell types of the TME have been molecularly analyzed, tumor-infiltrating neurons could not be considered so far, because their cell bodies are located in adjacent ganglia of the peripheral nervous system and thus they are not part of the tumor mass (Demir, Thiel et al, Nature Cancer 2020; Demir, Thiel et al. Cancer Cell 2020). One of the unique features of PDAC is the strong infiltration by nerve fibers which correlates directly with tumor malignancy. In recent years it has been shown that either neurotransmitters secreted by neurons, or modulators of neuronal infiltration secreted by cancer cells can influence tumor growth. Here we use 3D light sheet fluorescence microscopy imaging (LSFM) of full tissue cleared PDAC specimen of tumors growing in genetically engineered mouse models or patient-derived xenograft models. Our data showthat these PDACs are innervated by a complex array of sensory and sympathetic neurons. To molecularly analyse neurons innervating the tumor mass, we have developed a novel approach we termed Trace’n’seq. This combines retrograde axonal tracing and FACS analysis with single cell RNA sequencing with the aim to identify, isolate and analyse hundreds of traced neurons infiltrating tumors or their respective healthy organs. Thereby, we have identified previously undescribed neuronal cell types and provide a landscape of sympathetic and sensory nerves innervating the healthy pancreas as well as PDAC. Our single cell transcriptomic analysis revealed signs of reprogramming of neuronal behaviour orchestrated by the tumor cells. These datasets combined with single cell profiling of PDAC cells and its associated stroma provide a first complete, unbiased insight into neurons infiltrating both healthy pancreas and PDAC. Based on a comprehensive interactome analysis of the interplay between tumor cells, stroma and the neurons, we have identified various candidate genes involved in tumor-nerve signalling. We are currently performing gain-and loss- of function approaches to dissect the role of individual factors. In summary, with Trace'n'seq, we have developed a novel technology pipeline to isolate tissue innervating neurons to investigate the role of neuronal infiltration in the development and progression of PDAC. Citation Format: Vera Thiel, Simon Renders, Jasper Panten, Julian Mochayedi, Rienk Offringa, Martin Sprick, Andreas Trumpp. Trace'n'Seq: Assessing neuronal infiltration and its impact in pancreatic ductal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3650.

Specific biological properties of those circulating cancer cells that are the origin of brain metastases (BM) are not well understood. Here, single circulating breast cancer cells were fate-tracked during all steps of the brain metastatic cascade in mice after intracardial injection over weeks. A novel in vivo two-photon microscopy methodology was developed that allowed to determine the specific cellular and molecular features of breast cancer cells that homed in the brain, extravasated, and successfully established a brain macrometastasis. Those BM-initiating breast cancer cells (BMIC) were mainly originating from a slow-cycling subpopulation that included only 16% to 20% of all circulating cancer cells. BMICs showed enrichment of various markers of cellular stemness. As a proof of principle for the principal usefulness of this approach, expression profiling of BMICs versus non-BMICs was performed, which revealed upregulation of NDRG1 in the slow-cycling BMIC subpopulation in one BM model. Here, BM development was completely suppressed when NDRG1 expression was downregulated. In accordance, in primary human breast cancer, NDRG1 expression was heterogeneous, and high NDRG1 expression was associated with shorter metastasis-free survival. In conclusion, our data identify temporary slow-cycling breast cancer cells as the dominant source of brain and other metastases and demonstrates that this can lead to better understanding of BMIC-relevant pathways, including potential new approaches to prevent BM in patients. Implications: Cancer cells responsible for successful brain metastasis outgrowth are slow cycling and harbor stemness features. The molecular characteristics of these metastasis-initiating cells can be studied using intravital microscopy technology.

Acute myeloid leukemia (AML) is a very heterogeneous and highly malignant blood cancer. Mutations of the DNA methyltransferase DNMT3A are among the most frequent recurrent genetic lesions in AML. The majority of DNMT3A-mutant AML patients shows fast relapse and poor survival, but also patients with long survival or long-term remission have been reported. Underlying molecular signatures and mechanisms that contribute to these survival differences are only poorly understood and have not been studied in detail so far. We applied hierarchical clustering to somatic gene mutation profiles of 51 DNMT3A-mutant patients from The Cancer Genome Atlas (TCGA) AML cohort revealing two robust patient subgroups with profound differences in survival. We further determined molecular signatures that distinguish both subgroups. Our results suggest that FLT3 and/or NPM1 mutations contribute to survival differences of DNMT3A-mutant patients. We observed an upregulation of genes of the p53, VEGF and DNA replication pathway and a downregulation of genes of the PI3K-Akt pathway in short- compared to long-lived patients. We identified that the majority of measured miRNAs was downregulated in the short-lived group and we found differentially expressed microRNAs between both subgroups that have not been reported for AML so far (miR-153-2, miR-3065, miR-95, miR-6718) suggesting that miRNAs could be important for prognosis. In addition, we learned gene regulatory networks to predict potential major regulators and found several genes and miRNAs with known roles in AML pathogenesis, but also interesting novel candidates involved in the regulation of hematopoiesis, cell cycle, cell differentiation, and immunity that may contribute to the observed survival differences of both subgroups and could therefore be important for prognosis. Moreover, the characteristic gene mutation and expression signatures that distinguished short- from long-lived patients were also predictive for independent DNMT3A-mutant AML patients from other cohorts and could also contribute to further improve the European LeukemiaNet (ELN) prognostic scoring system. Our study represents the first in-depth computational approach to identify molecular factors associated with survival differences of DNMT3A-mutant AML patients and could trigger additional studies to develop robust molecular markers for a better stratification of AML patients with DNMT3A mutations.

Large-scale genomic profiling of pancreatic cancer (PDAC) has revealed two distinct subtypes: ‘classical’ and ‘basal-like’. Their variable coexistence within the stromal immune microenvironment is linked to differential prognosis; however, the extent to which these neoplastic subtypes shape the stromal immune landscape and impact clinical outcome remains unclear. By combining preclinical models, patient-derived xenografts, as well as FACS-sorted PDAC patient biopsies, we show that the basal-like neoplastic state is sustained via BRD4-mediated cJUN/AP1 expression, which induces CCL2 to recruit tumor necrosis factor (TNF)-α-secreting macrophages. TNF-α+ macrophages force classical neoplastic cells into an aggressive phenotypic state via lineage reprogramming. Integration of ATAC-, ChIP- and RNA-seq data revealed distinct JUNB/AP1 (classical) and cJUN/AP1 (basal-like)-driven regulation of PDAC subtype identity. Pharmacological inhibition of BRD4 led to suppression of the BRD4–cJUN–CCL2–TNF-α axis, restoration of classical subtype identity and a favorable prognosis. Hence, patient-tailored therapy for a cJUNhigh/TNF-αhigh subtype is paramount in overcoming highly inflamed and aggressive PDAC states. Singh and colleagues show that PDAC phenotypic plasticity is regulated via AP1 enhancer remodeling and modulated by TNF-α+ macrophages and pharmacological inhibition of the BRD4–cJUN–CCL2–TNF-α axis restores favorable PDAC subtypes and prognosis.

Despite important advances in the treatment of breast cancer, the 5-year survival rate for patients with distant metastasis remains less than 30%. Metastasis is a complex, multi-step process beginning with local invasion and ending with the outgrowth of systemically disseminated cells into actively proliferating metastases that ultimately cause the destruction of vital organs. It is this last step that limits patient survival and, at the same time, remains the least understood mechanistically. Here, we focus on understanding determinants of metastatic outgrowth using metastatic effusion biopsies from stage IV breast cancer patients. By modelling metastatic outgrowth through xenograft transplantation, we show that tumour initiation potential of patient-derived metastatic breast cancer cells across breast cancer subtypes is strongly linked to high levels of EPCAM expression. Breast cancer cells with high EPCAM levels are highly plastic and, upon induction of epithelial-mesenchymal transition (EMT), readily adopt mesenchymal traits while maintaining epithelial identity. In contrast, low EPCAM levels are caused by the irreversible reprogramming to a mesenchymal state with concomitant suppression of metastatic outgrowth. The ability of breast cancer cells to retain epithelial traits is tied to a global epigenetic program that limits the actions of EMT-transcription factor ZEB1, a suppressor of epithelial genes. Our results provide direct evidence that maintenance of epithelial identity is required for metastatic outgrowth while concomitant expression of mesenchymal markers enables plasticity. In contrast, loss of epithelial traits is characteristic of an irreversible mesenchymal reprogramming associated to a deficiency for metastatic outgrowth. Collectively, our data provide a framework for the intricate intercalation of mesenchymal and epithelial traits in metastatic growth.

Acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis. We report a comprehensive proteogenomic analysis of bone marrow biopsies from 252 uniformly treated AML patients to elucidate the molecular pathophysiology of AML in order to inform future diagnostic and therapeutic approaches. In addition to in-depth quantitative proteomics, our analysis includes cytogenetic profiling and DNA/RNA sequencing. We identify five proteomic AML subtypes, each reflecting specific biological features spanning genomic boundaries. Two of these proteomic subtypes correlate with patient outcome, but none is exclusively associated with specific genomic aberrations. Remarkably, one subtype (Mito-AML), which is captured only in the proteome, is characterized by high expression of mitochondrial proteins and confers poor outcome, with reduced remission rate and shorter overall survival on treatment with intensive induction chemotherapy. Functional analyses reveal that Mito-AML is metabolically wired toward stronger complex I-dependent respiration and is more responsive to treatment with the BCL2 inhibitor venetoclax.

Background: Luminal, i.e., estrogen receptor (ER)-positive breast cancer (BC) is a heterogeneous disease in terms of tumor progression, therapy response, and relapse. Additional biomarkers with a prognostic and predictive impact would facilitate advanced patient stratification and can reveal advanced therapeutic options for individual patients suffering from luminal BC subtype. First objective of this study was the hypothesis driven validation and identification of biomarkers associated with successful engraftment, augmented tumor growth, and enhanced metastasis upon xenotransplantation into HTM and non-humanized tumor mice. Second objective was the retrospective validation and correlation of aforementioned markers with clinical outcomes (disease free-survival [DFS] and overall survival [OS]) of luminal BC patients after neoadjuvant chemotherapy within the GeparTrio trial. Methods: Immunodeficient NSG mice with and without immunological humanization were transplanted with primary, ER-positive BC tissues and cells. Engraftment rates, tumor progression, and metastasis were monitored as a function of marker expression and genomic alterations, considered to be associated with tumor cell stemness and tumor initiating capacity. Functional assays were applied to demonstrate the impact of identified markers on tumor cell viability, growth and migration in-vitro. Dual color fluorescence-in-situ-hybridization to monitor mdm2 gene and the cen12 region was applied to 502 pretherapeutic ER-positive tissue specimens of BC patients treated with anthracycline/taxane based neoadjuvant chemotherapy within the GeparTrio trial. Mdm2 gene expression was analyzed in the entire luminal BC cohort as well as in luminal-A and luminal-B samples classified based on a Ki-67 cut-off of 20% (St. Gallen guideline). Associations with survival outcomes were studied by Cox regression models. Results: We observed an elevated CD44 and c-MET expression in metastatic cells compared to the primarily growing xenotransplantants. Moreover, we found mdm2 gene amplification was associated with tumor growth and pronounced metastatic potential in NSG mice. Functional assays unveiled a reduced viability, proliferation, and migration capacity of inherently mdm2 positive breast cancer cells upon mdm2 knock-down or anti-mdm2 targeting. Validation of mdm2 gain in luminal BC cohort within the GeparTrio trial revealed a significant association of mdm2 amplification with worse DFS (HR=1.80 [95%CI 1.16-2.79], log rank p=0.008) and OS (HR=1.75 [95%CI 1.00-3.05], log-rank p=0.047). This association was even stronger in luminal-A BC: DFS (HR=2.56 [95%CI 1.40-4.71], log rank p=0.002 and OS (HR=3.27 [95%CI 1.51-7.09], log-rank p=0.001). Conclusions: Mdm2 gene amplification facilitates ER-positive BC engraftment and progression in a preclinical in-vivo xenograft humanized NSG mouse model. Targeting mdm2 in-vitro reduced malignant cell propagation and growth. In addition, an increased mdm2 gene dose is strongly associated with an unfavorable outcome of luminal BC. Prospective studies are required to verify the suitability of mdm2 for advanced luminal BC stratification and therapeutic targeting. Citation Format: Anja Kathrin Wege, Valentina Vladimirova, Christine Solbach, Eva-Maria Rom-Jurek, Jens-Uwe Blohmer, Paul Jank, Bruno Sinn, Andreas Trumpp, Elisabetta Marangoni, Knut Engels, Wilko Weichert, Nicole Pfarr, Christoph Irlbeck, Bernhard Polzer, Olaf Ortmann, Marion van Mackelenbergh, Carsten Denkert, Sibylle Loibl, Gero Brockhoff. Mdm2 gene amplification in estrogen receptor-positive breast cancer cells is associated with enhanced solid tumor growth and pronounced metastatic potential in humanized tumor mice (HTM) and a poor outcome of patients with luminal breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-07.

Serial longitudinal enumeration of circulating tumor cells (CTCs) has shown its prognostic value on progression-free survival and overall survival (OS) in patients with stage IV breast cancer. This study prospectively evaluated the role of CTCs as a prognostic marker during further progression of metastatic breast cancer (MBC). Among 476 MBC patients recruited between 2010 and 2015, the 103 patients with a known CTC status at baseline (CTCBL) and within 4 weeks of tumor progression (CTCPD) were included. Progressive disease (PD) was defined according to the Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1). Using the CellSearch method

Enrichment of leukemic blasts with a stem cell phenotype correlates with poor survival in acute myeloid leukemia (AML). In this context, measurement of the stem cell marker aldehyde-dehydrogenase (ALDH) activity can distinguish poor prognosis cases with increased fractions of ALDH-positive cells (ALDH-numerous AML) and favorable outcome cases with low percentages (ALDH-rare AML). It has been shown that ALDH-numerous AML favor leukemic engraftment in xenotransplantation assays which suggests increased leukemic stem cell (LSC) potential. To test if this reflects an immature cell of origin, comparative gene-expression studies of CD34+ leukemic blasts were performed. This analysis revealed increased expression of LSC and HSC signatures in ALDH-numerous AML, whereas ALDH-rare AML were enriched for a progenitor signature. The enrichment of stemness-associated transcriptional programs suggests that ALDH-numerous AML derive from immature hematopoietic progenitors and offers an explanation for the poor prognosis and therapy resistance of this subgroup which is likely caused by inherited stem cell properties.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is associated with a dismal prognosis and poor therapeutic response to current chemotherapy regimens in unselected patient populations. Recently, it has been shown that PDAC may be stratified into functionally and therapeutically relevant molecular subgroups and that some of these subtypes can be recapitulated by IHC for KRT81 [quasi-mesenchymal (QM)/squamous/basal-like] and HNF1A (non-QM, overlap with exocrine/ADEX subtype). Experimental Design: We validated the different outcome of the HNF1A/KRT81 PDAC subtypes in two independent cohorts of surgically treated patients and examined the treatment response to chemotherapy in a third cohort of unresectable patients. The first two cohorts included 262 and 130 patients, respectively, and the third independent cohort comprised advanced-stage PDAC patients who were treated with either FOLFIRINOX (64 patients) or gemcitabine (61 patients). Results: In both cohorts with resected PDAC, the HNF1A-positive subtype showed the best, the KRT81-positive subtype the worst, and the double-negative subtype an intermediate survival (P < 0.013 and P < 0.009, respectively). In the chemotherapy cohort, the survival difference between the double-negative and the HNF1A-positive subtype was lost, whereas the dismal prognosis of KRT81-positive PDAC patients was retained (P < 0.021). Patients with a KRT81-positive subtype did not benefit from FOLFIRINOX therapy, whereas those with HNF1A-positive tumors responded better compared with gemcitabine-based treatment (P < 0.038). Conclusions: IHC stratification recapitulating molecular subtypes of PDAC using HNF1A and KRT81 is associated with significantly different outcomes and responses to chemotherapy. These results may pave the way toward future pretherapeutic biomarker-based stratification of PDAC patients. Clin Cancer Res; 24(2); 351–9. ©2017 AACR.

Activating mutations in the proto-oncogene KRAS are a hallmark of pancreatic ductal adenocarcinoma (PDAC), an aggressive malignancy with few effective therapeutic options. Despite efforts to develop KRAS-targeted drugs, the absolute dependence of PDAC cells on KRAS remains incompletely understood. Here we model complete KRAS inhibition using CRISPR/Cas-mediated genome editing and demonstrate that KRAS is dispensable in a subset of human and mouse PDAC cells. Remarkably, nearly all KRAS deficient cells exhibit phosphoinositide 3-kinase (PI3K)-dependent mitogen-activated protein kinase (MAPK) signaling and induced sensitivity to PI3K inhibitors. Furthermore, comparison of gene expression profiles of PDAC cells retaining or lacking KRAS reveal a role of KRAS in the suppression of metastasis-related genes. Collectively, these data underscore the potential for PDAC resistance to even the very best KRAS inhibitors and provide insights into mechanisms of response and resistance to KRAS inhibition., Pancreatic cancer cells may develop resistance to KRAS inhibitors due to activation of compensatory pathways. In this study, the authors demonstrate that KRAS is dispensable in a subset of pancreatic cancer and that PI3K signalling may have an important role in mediating tumor growth following KRAS inhibition.

Quiescent long-term hematopoietic stem cells (LT-HSCs) are efficiently activated by type I interferon (IFN-I). However, this effect remains poorly investigated in the context of IFN-I-inducing virus infections. Here we report that both vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection induce LT-HSC activation that substantially differs from the effects triggered upon injection of synthetic IFN-I-inducing agents. In both infections, inflammatory responses had to exceed local thresholds within the bone marrow to confer LT-HSC cell cycle entry, and IFN-I receptor triggering was not critical for this activation. After resolution of acute MCMV infection, LT-HSCs returned to phenotypic quiescence. However, non-acute MCMV infection induced a sustained inflammatory milieu within the bone marrow that was associated with long-lasting impairment of LT-HSC function. In conclusion, our results show that systemic virus infections fundamentally affect LT-HSCs and that also non-acute inflammatory stimuli in bone marrow donors can affect the reconstitution potential of bone marrow transplants.

Endothelial cells (ECs) provide angiocrine factors orchestrating tumor progression. Here, we show that activated Notch1 receptors (N1ICD) are frequently observed in ECs of human carcinomas and melanoma, and in ECs of the pre-metastatic niche in mice. EC N1ICD expression in melanoma correlated with shorter progression-free survival. Sustained N1ICD activity induced EC senescence, expression of chemokines and the adhesion molecule VCAM1. This promoted neutrophil infiltration, tumor cell (TC) adhesion to the endothelium, intravasation, lung colonization, and postsurgical metastasis. Thus, sustained vascular Notch signaling facilitates metastasis by generating a senescent, pro-inflammatory endothelium. Consequently, treatment with Notch1 or VCAM1-blocking antibodies prevented Notch-driven metastasis, and genetic ablation of EC Notch signaling inhibited peritoneal neutrophil infiltration in an ovarian carcinoma mouse model.

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3’-untranslated region (3’-UTR) composition. The relevance of APA in stem cell hierarchies in vivo remains elusive. Using extensive in vitro and in vivo analysis approaches we show that deregulation of the APA regulator Pabpn1 results in severe hematopoietic stem cell (HSC) defects. Further, we performed low input 3’-sequencing and established bioinformatic pipelines to uncover dynamic APA patterns in numerous genes of HSCs and multipotent progenitors determining the genome-wide APA landscape (APAome). This revealed transcriptome-wide dynamic APA patterns and an overall shortening of 3’-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation (Sommerkamp et al., Cell Stem Cell, 2020). Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching. This process is mediated by Nudt21 and is required for the proper stress response of HSCs. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Inhibition of this metabolic adjustment leads to impaired HSC function and a partial block in differentiation. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal and commitment.

Metastatic colonization relies on interactions between disseminated cancer cells and the microenvironment in secondary organs. Here, we show that disseminated breast cancer cells evoke major phenotypic changes in lung fibroblasts to form a metastatic niche that supports malignant growth. Colonization of the lungs by cancer cells confers an inflammatory phenotype in associated fibroblasts, where IL-1α and IL-1β, secreted by breast cancer cells, induceCXCL9andCXCL10production in metastasis-associated fibroblasts via NF-κB signaling. These paracrine interactions fuel the growth of lung metastases. Notably, we find that the chemokine receptor CXCR3, that binds CXCL9/10, is specifically expressed in a small subset of breast cancer cells with stem/progenitor cell properties and high tumor-initiating ability when co-transplanted with fibroblasts. CXCR3-expressing cancer cells show high JNK signaling that drives IL-1α/β expression. Thus, CXCR3 marks a population of breast cancer cells that induces CXCL9/10 production in fibroblast, but can also respond to and benefit from these chemokines. Importantly, disruption of this intercellular JNK-IL-1-CXCL9/10-CXCR3 axis significantly reduces metastatic colonization in xenograft and syngeneic mouse models. These data mechanistically demonstrate an essential role for this molecular crosstalk between breast cancer cells and their fibroblast niche in the progression of metastasis.

Metastasis is the main cause of death in breast cancer patients. The development of reliable and cost-effective biomarker to evaluate the prognosis of metastatic breast cancer (MBC) patients is of great importance. S100P is a member of S100 family and has been proved to be associated with metastasis establishment. Methods: We investigated the plasma S100P levels in 60 healthy controls, 48 primary and 273 metastatic breast cancer patients. The MBC patients were followed-up for disease progression and death up to 3.5 years after recruitment. Radiographic response of MBC patients were also analyzed for investigation on treatment monitoring value of plasma S100P level. We found a robust association between high plasma S100P level (>7 ng/mL) and poor prognosis of metastatic breast cancer (MBC) patients (median progression-free survival time: 5.0 vs. 8.7 months, log-rank test p

Conventional tumor markers have limited value for prognostication and treatment monitoring in metastatic breast cancer (MBC) patients and novel circulating tumor markers therefore need to be explored. Hyaluronic acid (HA) is a major macropolysaccharide in the extracellular matrix and is reported to be associated with tumor progression. In our study, we investigated plasma HA level with respect to progression free survival (PFS) and overall survival (OS), as well as the treatment monitoring value in MBC patients. The prognostic value of plasma HA level was investigated in a discovery cohort of 212 MBC patients with 2.5-year follow-up and validated in an independent validation cohort of 334 patients with 5-year follow-up. The treatment monitoring value of plasma HA level was investigated in 61 MBC patients from discovery cohort who had been radiographically examined after first complete cycle of chemo therapy. We found a robust association between high plasma HA level and poor prognosis of MBC patients in both discovery (pPFS = 7.92 × 10(-6) and pOS = 5.27 × 10(-5)) and validation studies (pPFS = 3.66 × 10(-4) and pOS = 1.43 × 10(-4)). In the discovery cohort, the plasma HA level displayed independent prognostic value after adjusted for age and clinicopathological factors, with respect to PFS and OS. Further, the decrease of plasma HA level displayed good concordance with treatment response evaluated by radiographic examination (AUC = 0.79). Plasma HA level displays prognostic value, as well as treatment monitoring value for MBC patients.

With an overall survival rate of about 8% after five years, ductal adenocarcinoma of the pancreas (PDAC) is one of the cancers with the worst prognosis (Cancer Research UK and American Cancer Society). A hallmark of PDAC is the immense contribution of desmoplastic stroma to the total tumour mass. Typically, epithelial PDAC cells represent only a minority of cells within the tumour (as low as 10%). The excessive stromal presence represents a major technical challenge when aiming to interrogate the specific molecular signals contributed by the neoplastic epithelial cell compartment from data generated using bulk tumour samples. To address this, we FACS-purified epithelial cells from PDAC and, as controls, healthy human pancreas and performed genome-wide transcriptome and DNA methylome analyses. Clustering based on DNA methylation revealed two distinct groups of PDAC with different methylation levels at genomic regions encoding repeat elements. Methylationlow tumours showed higher expression of endogenous retroviral (ERV) transcripts and a strong engagement of the dsRNA sensing machinery. This results in the cell intrinsic activation of an interferon response signature (IFNsign), leading to the reprogramming of stromal cells towards a pro-tumourigenic microenvironment and poor patient outcome. Methylationlow/IFNsignhigh and Methylationhigh/IFNsignlow PDAC cells harboured distinct lineage traits specific for normal ductal or acinar pancreatic epithelial cells at the methylation and transcriptional level. Moreover, ductal-cell-derived KrasG12D/Trp53-/- mutant mouse PDACs showed higher expression of IFNsign compared to tumors initiated by the same genetic drivers in acinar cells. Collectively, our data point to two distinct origins and etiology of human PDACs, with the aggressive Methylationhigh/IFNsignlow tumour subtype potentially targetable by agents blocking cell intrinsic IFN-signaling. Citation Format: Elisa Espinet, Andreas Trumpp. Viral Mimicry in Pancreatic Cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr IA05.

Although the survival of patients diagnosed with breast cancer has greatly improved over the last decades, the metastatic stage of the disease still remains incurable. Blood circulating tumor cells (CTCs) are assumed to be the source for metastatic onset and the enumeration of CTCs has already proven its prognostic value for survival in metastatic breast cancer (mBC) patients. However, the biological profiles of CTCs are so far not considered in therapy selection, but hold the potential to display the disease stage in a minimally invasive procedure. Here we serially collect liquid biopsies of mBC patients throughout their course of therapy with the CDK4/6 inhibitor palbociclib to monitor treatment response and drug resistance development. To this end, we combine transcriptomic, genomic and proteomic analyses of primary CTCs on bulk but also on the single cell level. Furthermore, we generated patient-specific, in vitro expanded CTC cell lines to intensively study mechanisms of drug resistance and identify new therapeutic targets by drug sensitivity assays. By analyzing mBC CTCs of serial liquid biopsies we monitored cell cycle arrest upon palbociclib treatment and drug resistance development. Drug sensitivity and successive drug resistance can be observed in primary and in vitro expanded, patient-specific CTCs. As revealed by RNA-seq of bulk and single CTCs, acquired resistance to palbociclib in mBC CTCs is associated with altered transcriptional programs including RB transcriptional corepressor 1 (RB1) function and can be linked to a loss-of-function mutation in the RB1 gene. Interestingly, apart from altered RB1 function, we can identify a global transcriptional transition from palbociclib sensitivity to resistance. Based on the transcriptomic information, pathways that are upregulated in resistant CTCs were determined and the patient-derived CTC cell lines were used to validate and specifically target tumor cells that are resistant to CDK4/6 inhibitors. Using drug screening assays on the CTC-derived cell lines, we identified potential drugs that in combination with palbociclib could circumvent the acquired resistance and help to prolong patient survival. Our study indicates that information about the biological state of serially collected CTCs can help to earlier detect the onset of resistance and should be included in clinical disease management. This will help to spare the patient severe side effects from ineffective drug treatment. Citation Format: Andreas Trumpp, Lisa Becker, Michael Fletcher, Roberto Würth, Anand Thippeswamy, Simon Haas, Lars Velten, Laura Michel, Frederik Marme, Sarah Jauch, Mirjam Becker, Lars Steinmetz, Andreas Schneeweiss, Marc Zapatka, Peter Lichter, Martin Sprick. Exploring CDK4/6 inhibitor therapy response and drug resistance development at the single cell level in metastatic breast cancer CTCs [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-01.

Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state., This work was supported by the FOR2033 and SFB873 funded by the Deutsche Forschungsgemeinschaft (DFG), the Dietmar Hopp Foundation (all to A.T.), and the Wellcome Trust (to A.S.).

Although c‐Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the Nras (Q61K) INK4a (−/−) mouse melanoma model to show that c‐Myc is essential for tumor initiation, maintenance, and metastasis. c‐Myc‐expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c‐Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c‐Myc‐positive melanoma cells activated and became dependent on the metabolic energy sensor AMP‐activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c‐Myc‐expressing melanoma cells, while AMPK activation protected against cell death of c‐Myc‐depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early‐stage human melanoma samples revealed an inverse correlation between C‐MYC and patient survival, suggesting that C‐MYC expression levels could serve as a prognostic marker for early‐stage disease.

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16(INK4a), p21(CIP1) and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eμ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of abundant desmoplastic stroma primarily composed of cancer-associated fibroblasts (CAFs). It is generally accepted that CAFs stimulate tumor progression and might be implicated in drug resistance and immunosuppression. Here, we have compared the transcriptional profile of PDGFRα + CAFs isolated from genetically engineered mouse PDAC tumors with that of normal pancreatic fibroblasts to identify genes potentially implicated in their protumorigenic properties. We report that the most differentially expressed gene, Saa3, a member of the serum amyloid A (SAA) apolipoprotein family, is a key mediator of the protumorigenic activity of PDGFRα + CAFs. Whereas Saa3-competent CAFs stimulate the growth of tumor cells in an orthotopic model, Saa3-null CAFs inhibit tumor growth. Saa3 also plays a role in the cross talk between CAFs and tumor cells. Ablation of Saa3 in pancreatic tumor cells makes them insensitive to the inhibitory effect of Saa3-null CAFs. As a consequence, germline ablation of Saa3 does not prevent PDAC development in mice. The protumorigenic activity of Saa3 in CAFs is mediated by Mpp6, a member of the palmitoylated membrane protein subfamily of the peripheral membrane-associated guanylate kinases (MAGUK). Finally, we interrogated whether these observations could be translated to a human scenario. Indeed, SAA1, the ortholog of murine Saa3, is overexpressed in human CAFs. Moreover, high levels of SAA1 in the stromal component correlate with worse survival. These findings support the concept that selective inhibition of SAA1 in CAFs may provide potential therapeutic benefit to PDAC patients., This work was supported by European Research Council Grants ERC-AG/250297-RAS AHEAD and ERC-AG/695566-THERACAN, Spanish Ministry of Economy and Competitiveness Grant SAF2014-59864-R, and Asociación Española contra el Cáncer Grant GC16173694BARB (to M. Barbacid). M.D. was supported by a fellowship from La Caixa International Fellowship Program. M. Barbacid is the recipient of an Endowed Chair from the AXA Research Fund

Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid–induced 2 (RAI2) expression to be significantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells. Significance: We identified downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specified the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer. Cancer Discov; 5(5); 506–19. ©2015 AACR. See related commentary by Esposito and Kang, p. 466 This article is highlighted in the In This Issue feature, p. 453

Background Clinical decisions in the primary and advanced situation of breast cancer are based on immunohistochemical staining (IHC) and semiquantitative assessment of ER, PgR, HER2 and Ki67. However, IHC carries an up to 20% risk of erroneous results. Moreover, metaanalysis revealed variable discordance rates between primary and metastatic sites (P/M) which may have implications for patient management (Aurilio, Eu J Cancer 2013). Herein, we undertook a pilot study aiming to investigate whether the reported discordance between primary lesion and paired distant metastatic sites is due to technical limitations of the IHC technique and could be improved by mRNA analysis using the MammaTyper® in vitro diagnostic assay. Materials and Methods One 10μm-thick sections from clinical routine FFPE tissues of 28 tumor samples were reexamined by RNA quantitation of ESR1, PGR, HER2 and KI67 using the RNXtract® kit for RNA extraction and MammaTyper® kit for objective assessment of receptor status. RNA levels were normalized using a synthetic in-vitro transcript for normalization according to the 40-DDCT method. Receptor status was reported based on predefined cut-offs according to the instruction for use. mRNA and IHC results were compared between paired primary and metastatic sites. Results Concordance per tumor sample between the two methods was 100% for HER2, 81.9% for ESR1 and 81.5% for PR. Among discrepant samples (N=8), 6 were primary and among these, half (N=3) were due to negative ER by IHC. With the exception of HER2, where no differences were observed for either method, the P/M concordance was superior by MammaTyper® kit (ESR1: 92.85 vs. 76.82, PR: 71.43 vs. 69.23). Importantly, several discordant cases by IHC were ER negative in the primary site, while being ER positive in the metastatic lesion, while being consistently ESR1 positive in both sites, when determined by the more sensitive PCR method. The subtype discordant cases were situated in the lung (2), the liver (1) and the bone marrow (1). Conclusion Our data indicate that receptor status shifts less frequently when determined by PCR methods compared to IHC. While some shifts between primary site and metastases may arise from true progression-related biologic alterations (Prat et al Nat Med 2009), others seem to be related to technical limitations of semiquantitative and subjective IHC, with several cases being conspicuous for ESR1. This finding is of great clinical significance and is consistent with previous reports implicating technical confounders of IHC for large part of the observed P/M deviations (Pusztai, The Oncologist 2010). The implied superiority of RT-qPCR by MammaTyper® vs. routine IHC in detecting true P/M tumor receptor status reversals and the possible clinical impact is clearly worth pursuing in larger datasets. Citation Format: Markus Wallwiener, Andreas Hartkopf, Thomas Deutsch, Lakis Sotiris, Florin-Andrei Taran, Andreas Trumpp, Ralph Wirtz, Andreas Schneeweiss. Concordance/discordance rates of HER2, ER, PR, and Ki67 in matched pair samples of primary (PBC) and metastatic breast cancer (MBC) tissues when comparing IHC with MammaTyper® RT-PCR kit [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-45.

Haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood cells. The accumulation of DNA damage in HSCs is a hallmark of ageing and is probably a major contributing factor in age-related tissue degeneration and malignant transformation. A number of accelerated ageing syndromes are associated with defective DNA repair and genomic instability, including the most common inherited bone marrow failure syndrome, Fanconi anaemia. However, the physiological source of DNA damage in HSCs from both normal and diseased individuals remains unclear. Here we show in mice that DNA damage is a direct consequence of inducing HSCs to exit their homeostatic quiescent state in response to conditions that model physiological stress, such as infection or chronic blood loss. Repeated activation of HSCs out of their dormant state provoked the attrition of normal HSCs and, in the case of mice with a non-functional Fanconi anaemia DNA repair pathway, led to a complete collapse of the haematopoietic system, which phenocopied the highly penetrant bone marrow failure seen in Fanconi anaemia patients. Our findings establish a novel link between physiological stress and DNA damage in normal HSCs and provide a mechanistic explanation for the universal accumulation of DNA damage in HSCs during ageing and the accelerated failure of the haematopoietic system in Fanconi anaemia patients.

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH+ cells, What's new? To understand the precise disease‐driving mechanisms in acute myeloid leukemia (AML), comparison of patient‐matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. This study demonstrates the relevance of aldehyde dehydrogenase (ALDH) for the prospective identification of AML cases in which separation of functionally normal HSC from LSC is possible. Increased activity of this biomarker also characterizes a subgroup of patients with adverse outcome, which might be helpful in risk stratification prior to therapy. Overall, this study demonstrates functional heterogeneity of leukemia cells and suggests divergent roles for ALDH activity in normal HSC versus leukemia‐initiating cells.

Summary To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance., Graphical Abstract, Highlights • Clinical outcome in AML correlates with LSC-associated miRNA expression • miR-126 targets multiple components of the PI3K/AKT/MTOR signaling pathway • miR-126 promotes chemotherapy resistance by preserving LSC in a quiescent state • miR-126 governs opposing self-renewal outcomes in normal and malignant stem cells, Lechman et al. show that miR-126 targets the PI3K/AKT/MTOR signaling pathway to preserve quiescence, increase self-renewal, and promote chemotherapy resistance of acute myeloid leukemia stem cells (LSC). Reducing the miR-126 level impairs LSC maintenance in contrast to expanding normal hematopoietic stem cells.