Midkine as a driver of age-related changes and increase in mammary tumorigenesis.

Aging is a pivotal risk factor for cancer, yet the underlying mechanisms remain poorly defined. Here, we explore age-related changes in the rat mammary gland by single-cell multiomics. Our findings include increased epithelial proliferation, loss of luminal identity, and decreased naive B and T cells with age. We discover a luminal progenitor population unique to old rats with profiles reflecting precancerous changes and identify midkine (Mdk) as a gene upregulated with age and a regulator of age-related luminal progenitors. Midkine treatment of young rats mimics age-related changes via activating PI3K-AKT-SREBF1 pathway and promotes nitroso-N-methylurea-induced mammary tumorigenesis. Midkine levels increase with age in human blood and mammary epithelium, and higher MDK in normal breast tissue is associated with higher breast cancer risk in younger women. Our findings reveal a link between aging and susceptibility to tumor initiation and identify midkine as a mediator of age-dependent increase in breast tumorigenesis.

Mammalian SWI/SNF complex activity regulates POU2F3 and constitutes a targetable dependency in small cell lung cancer.

Small cell lung cancers (SCLCs) are composed of heterogeneous subtypes marked by lineage-specific transcription factors, including ASCL1, NEUROD1, and POU2F3. POU2F3-positive SCLCs, ∼12% of all cases, are uniquely dependent on POU2F3 itself; as such, approaches to attenuate POU2F3 expression may represent new therapeutic opportunities. Here using genome-scale screens for regulators of POU2F3 expression and SCLC proliferation, we define mSWI/SNF complexes as top dependencies specific to POU2F3-positive SCLC. Notably, chemical disruption of mSWI/SNF ATPase activity attenuates proliferation of all POU2F3-positive SCLCs, while disruption of non-canonical BAF (ncBAF) via BRD9 degradation is effective in pure non-neuroendocrine POU2F3-SCLCs. mSWI/SNF targets to and maintains accessibility over gene loci central to POU2F3-mediated gene regulatory networks. Finally, clinical-grade pharmacologic disruption of SMARCA4/2 ATPases and BRD9 decreases POU2F3-SCLC tumor growth and increases survival in vivo. These results demonstrate mSWI/SNF-mediated governance of the POU2F3 oncogenic program and suggest mSWI/SNF inhibition as a therapeutic strategy for POU2F3-positive SCLCs.

Loss of Kmt2c or Kmt2d drives brain metastasis via KDM6A-dependent upregulation of MMP3.

KMT2C and KMT2D, encoding histone H3 lysine 4 methyltransferases, are among the most commonly mutated genes in triple-negative breast cancer (TNBC). However, how these mutations may shape epigenomic and transcriptomic landscapes to promote tumorigenesis is largely unknown. Here we describe that deletion of Kmt2c or Kmt2d in non-metastatic murine models of TNBC drives metastasis, especially to the brain. Global chromatin profiling and chromatin immunoprecipitation followed by sequencing revealed altered H3K4me1, H3K27ac and H3K27me3 chromatin marks in knockout cells and demonstrated enhanced binding of the H3K27me3 lysine demethylase KDM6A, which significantly correlated with gene expression. We identified Mmp3 as being commonly upregulated via epigenetic mechanisms in both knockout models. Consistent with these findings, samples from patients with KMT2C-mutant TNBC have higher MMP3 levels. Downregulation or pharmacological inhibition of KDM6A diminished Mmp3 upregulation induced by the loss of histone-lysine N-methyltransferase 2 (KMT2) and prevented brain metastasis similar to direct downregulation of Mmp3. Taken together, we identified the KDM6A-matrix metalloproteinase 3 axis as a key mediator of KMT2C/D loss-driven metastasis in TNBC.

Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation.

Enteroendocrine cells (EECs) secrete serotonin (enterochromaffin [EC] cells) or specific peptide hormones (non-EC cells) that serve vital metabolic functions. The basis for terminal EEC diversity remains obscure. By forcing activity of the transcription factor (TF) NEUROG3 in 2D cultures of human intestinal stem cells, we replicated physiologic EEC differentiation and examined transcriptional and cis-regulatory dynamics that culminate in discrete cell types. Abundant EEC precursors expressed stage-specific genes and TFs. Before expressing pre-terminal NEUROD1, post-mitotic precursors oscillated between transcriptionally distinct ASCL1+ and HES6hi cell states. Loss of either factor accelerated EEC differentiation substantially and disrupted EEC individuality; ASCL1 or NEUROD1 deficiency had opposing consequences on EC and non-EC cell features. These TFs mainly bind cis-elements that are accessible in undifferentiated stem cells, and they tailor subsequent expression of TF combinations that underlie discrete EEC identities. Thus, early TF oscillations retard EEC maturation to enable accurate diversity within a medically important cell lineage.

Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies.

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.

Targetable leukaemia dependency on noncanonical PI3Kγ signalling.

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.

Mammalian SWI/SNF complex activity regulates POU2F3 and constitutes a targetable dependency in small cell lung cancer.

Small cell lung cancers (SCLC) are comprised of heterogeneous subtypes marked by lineage-specific transcription factors, including ASCL1, NEUROD1, and POU2F3. POU2F3-positive SCLC, ∼12% of all cases, are uniquely dependent on POU2F3 itself; as such, approaches to attenuate POU2F3 expression may represent new therapeutic opportunities. Here using genome-scale screens for regulators of POU2F3 expression and SCLC proliferation, we define mSWI/SNF complexes, including non-canonical BAF (ncBAF) complexes, as top dependencies specific to POU2F3-positive SCLC. Notably, clinical-grade pharmacologic mSWI/SNF inhibition attenuates proliferation of all POU2F3-positive SCLCs, while disruption of ncBAF via BRD9 degradation is uniquely effective in pure non-neuroendocrine POU2F3-SCLCs. mSWI/SNF maintains accessibility over gene loci central to POU2F3-mediated gene regulatory networks. Finally, chemical targeting of SMARCA4/2 mSWI/SNF ATPases and BRD9 decrease POU2F3-SCLC tumor growth and increase survival in vivo . Taken together, these results characterize mSWI/SNF-mediated global governance of the POU2F3 oncogenic program and suggest mSWI/SNF inhibition as a therapeutic strategy for SCLC.

Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.

Increased chromatin accessibility mediated by nuclear factor I drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer.

Androgen receptor (AR) splice variants, of which ARv7 is the most common, are increased in prostate cancer (PC) that develops resistance to androgen signaling inhibitor drugs, but the extent to which these variants drive AR activity, and whether they have novel functions or dependencies, remain to be determined. We generated a subline of VCaP PC cells (VCaP16) that is resistant to the AR inhibitor enzalutamide (ENZ) and found that AR activity was independent of the full-length AR (ARfl), despite its continued high-level expression, and was instead driven by ARv7. The ARv7 cistrome and transcriptome in VCaP16 cells mirrored that of the ARfl in VCaP cells, although ARv7 chromatin binding was weaker, and strong ARv7 binding sites correlated with higher affinity ARfl binding sites across multiple models and clinical samples. Notably, although ARv7 expression in VCaP cells increased rapidly in response to ENZ, there was a long lag before it gained chromatin binding and transcriptional activity. This lag was associated with an increase in chromatin accessibility, with the AR and nuclear factor I (NFI) motifs being most enriched at these more accessible sites. Moreover, the transcriptional effects of combined NFIB and NFIX knockdown versus ARv7 knockdown were highly correlated. These findings indicate that ARv7 can drive the AR program, but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.

Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation.

Enteroendocrine cells (EECs), which secrete serotonin (enterochromaffin cells, EC) or a dominant peptide hormone, serve vital physiologic functions. As with any adult human lineage, the basis for terminal cell diversity remains obscure. We replicated human EEC differentiation in vitro , mapped transcriptional and chromatin dynamics that culminate in discrete cell types, and studied abundant EEC precursors expressing selected transcription factors (TFs) and gene programs. Before expressing the pre-terminal factor NEUROD1, non-replicating precursors oscillated between epigenetically similar but transcriptionally distinct ASCL1 + and HES6 hi cell states. Loss of either factor substantially accelerated EEC differentiation and disrupted EEC individuality; ASCL1 or NEUROD1 deficiency had opposing consequences on EC and hormone-producing cell features. Expressed late in EEC differentiation, the latter TFs mainly bind cis -elements that are accessible in undifferentiated stem cells and tailor the subsequent expression of TF combinations that specify EEC types. Thus, TF oscillations retard EEC maturation to enable accurate EEC diversification.

scATAnno: Automated Cell Type Annotation for single-cell ATAC Sequencing Data.

Recent advances in single-cell epigenomic techniques have created a growing demand for scATAC-seq analysis. One key analysis task is to determine cell type identity based on the epigenetic data. We introduce scATAnno, a python package designed to automatically annotate scATAC-seq data using large-scale scATAC-seq reference atlases. This workflow generates the reference atlases from publicly available datasets enabling accurate cell type annotation by integrating query data with reference atlases, without the use of scRNA-seq data. To enhance annotation accuracy, we have incorporated KNN-based and weighted distance-based uncertainty scores to effectively detect cell populations within the query data that are distinct from all cell types in the reference data. We compare and benchmark scATAnno against 7 other published approaches for cell annotation and show superior performance in multiple data sets and metrics. We showcase the utility of scATAnno across multiple datasets, including peripheral blood mononuclear cell (PBMC), Triple Negative Breast Cancer (TNBC), and basal cell carcinoma (BCC), and demonstrate that scATAnno accurately annotates cell types across conditions. Overall, scATAnno is a useful tool for scATAC-seq reference building and cell type annotation in scATAC-seq data and can aid in the interpretation of new scATAC-seq datasets in complex biological systems.

Heterogeneity and transcriptional drivers of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with limited treatment options. To characterize TNBC heterogeneity, we defined transcriptional, epigenetic, and metabolic subtypes and subtype-driving super-enhancers and transcription factors by combining functional and molecular profiling with computational analyses. Single-cell RNA sequencing revealed relative homogeneity of the major transcriptional subtypes (luminal, basal, and mesenchymal) within samples. We found that mesenchymal TNBCs share features with mesenchymal neuroblastoma and rhabdoid tumors and that the PRRX1 transcription factor is a key driver of these tumors. PRRX1 is sufficient for inducing mesenchymal features in basal but not in luminal TNBC cells via reprogramming super-enhancer landscapes, but it is not required for mesenchymal state maintenance or for cellular viability. Our comprehensive, large-scale, multiplatform, multiomics study of both experimental and clinical TNBC is an important resource for the scientific and clinical research communities and opens venues for future investigation.

Combination Therapies to Improve the Efficacy of Immunotherapy in Triple-negative Breast Cancer.

Immune checkpoint inhibition combined with chemotherapy is currently approved as first-line treatment for patients with advanced PD-L1-positive triple-negative breast cancer (TNBC). However, a significant proportion of metastatic TNBC is PD-L1-negative and, in this population, chemotherapy alone largely remains the standard-of-care and novel therapeutic strategies are needed to improve clinical outcomes. Here, we describe a triple combination of anti-PD-L1 immune checkpoint blockade, epigenetic modulation thorough bromodomain and extra-terminal (BET) bromodomain inhibition (BBDI), and chemotherapy with paclitaxel that effectively inhibits both primary and metastatic tumor growth in two different syngeneic murine models of TNBC. Detailed cellular and molecular profiling of tumors from single and combination treatment arms revealed increased T- and B-cell infiltration and macrophage reprogramming from MHCIIlow to a MHCIIhigh phenotype in mice treated with triple combination. Triple combination also had a major impact on gene expression and chromatin profiles shifting cells to a more immunogenic and senescent state. Our results provide strong preclinical evidence to justify clinical testing of BBDI, paclitaxel, and immune checkpoint blockade combination.

KDM6A epigenetically regulates subtype plasticity in small cell lung cancer.

Small cell lung cancer (SCLC) exists broadly in four molecular subtypes: ASCL1, NEUROD1, POU2F3 and Inflammatory. Initially, SCLC subtypes were thought to be mutually exclusive, but recent evidence shows intra-tumoural subtype heterogeneity and plasticity between subtypes. Here, using a CRISPR-based autochthonous SCLC genetically engineered mouse model to study the consequences of KDM6A/UTX inactivation, we show that KDM6A inactivation induced plasticity from ASCL1 to NEUROD1 resulting in SCLC tumours that express both ASCL1 and NEUROD1. Mechanistically, KDM6A normally maintains an active chromatin state that favours the ASCL1 subtype with its loss decreasing H3K4me1 and increasing H3K27me3 at enhancers of neuroendocrine genes leading to a cell state that is primed for ASCL1-to-NEUROD1 subtype switching. This work identifies KDM6A as an epigenetic regulator that controls ASCL1 to NEUROD1 subtype plasticity and provides an autochthonous SCLC genetically engineered mouse model to model ASCL1 and NEUROD1 subtype heterogeneity and plasticity, which is found in 35-40% of human SCLCs.

Tutorial: integrative computational analysis of bulk RNA-sequencing data to characterize tumor immunity using RIMA.

RNA-sequencing (RNA-seq) has become an increasingly cost-effective technique for molecular profiling and immune characterization of tumors. In the past decade, many computational tools have been developed to characterize tumor immunity from gene expression data. However, the analysis of large-scale RNA-seq data requires bioinformatics proficiency, large computational resources and cancer genomics and immunology knowledge. In this tutorial, we provide an overview of computational analysis of bulk RNA-seq data for immune characterization of tumors and introduce commonly used computational tools with relevance to cancer immunology and immunotherapy. These tools have diverse functions such as evaluation of expression signatures, estimation of immune infiltration, inference of the immune repertoire, prediction of immunotherapy response, neoantigen detection and microbiome quantification. We describe the RNA-seq IMmune Analysis (RIMA) pipeline integrating many of these tools to streamline RNA-seq analysis. We also developed a comprehensive and user-friendly guide in the form of a GitBook with text and video demos to assist users in analyzing bulk RNA-seq data for immune characterization at both individual sample and cohort levels by using RIMA.

Landscape of prostate-specific membrane antigen heterogeneity and regulation in AR-positive and AR-negative metastatic prostate cancer.

Tumor expression of prostate-specific membrane antigen (PSMA) is lost in 15-20% of men with castration-resistant prostate cancer (CRPC), yet the underlying mechanisms remain poorly defined. In androgen receptor (AR)-positive CRPC, we observed lower PSMA expression in liver lesions versus other sites, suggesting a role of the microenvironment in modulating PSMA. PSMA suppression was associated with promoter histone 3 lysine 27 methylation and higher levels of neutral amino acid transporters, correlating with 18F-fluciclovine uptake on positron emission tomography imaging. While PSMA is regulated by AR, we identified a subset of AR-negative CRPC with high PSMA. HOXB13 and AR co-occupancy at the PSMA enhancer and knockout models point to HOXB13 as an upstream regulator of PSMA in AR-positive and AR-negative prostate cancer. These data demonstrate how PSMA expression is differentially regulated across metastatic lesions and in the context of the AR, which may inform selection for PSMA-targeted therapies and development of complementary biomarkers.

Universal NicE-Seq: A Simple and Quick Method for Accessible Chromatin Detection in Fixed Cells.

Genome-wide accessible chromatin sequencing and identification has enabled deciphering the epigenetic information encoded in chromatin, revealing accessible promoters, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding. The starting biological materials are often fixed using formaldehyde crosslinking. Here, we describe accessible chromatin library preparation from low numbers of formaldehyde-crosslinked cells using a modified nick translation method, where a nicking enzyme nicks one strand of DNA and DNA polymerase incorporates biotin-conjugated dATP, dCTP, and methyl-dCTP. Once the DNA is labeled, it can be isolated for NGS library preparation. We termed this method as universal NicE-seq (nicking enzyme-assisted sequencing). We also demonstrate a single tube method that enables direct NGS library preparation from low cell numbers without DNA purification. Furthermore, we demonstrated universal NicE-seq on FFPE tissue section sample.

JAK-STAT Signaling in Inflammatory Breast Cancer Enables Chemotherapy-Resistant Cell States.

Inflammatory breast cancer (IBC) is a difficult-to-treat disease with poor clinical outcomes due to high risk of metastasis and resistance to treatment. In breast cancer, CD44+CD24- cells possess stem cell-like features and contribute to disease progression, and we previously described a CD44+CD24-pSTAT3+ breast cancer cell subpopulation that is dependent on JAK2/STAT3 signaling. Here we report that CD44+CD24- cells are the most frequent cell type in IBC and are commonly pSTAT3+. Combination of JAK2/STAT3 inhibition with paclitaxel decreased IBC xenograft growth more than either agent alone. IBC cell lines resistant to paclitaxel and doxorubicin were developed and characterized to mimic therapeutic resistance in patients. Multi-omic profiling of parental and resistant cells revealed enrichment of genes associated with lineage identity and inflammation in chemotherapy-resistant derivatives. Integrated pSTAT3 chromatin immunoprecipitation sequencing and RNA sequencing (RNA-seq) analyses showed pSTAT3 regulates genes related to inflammation and epithelial-to-mesenchymal transition (EMT) in resistant cells, as well as PDE4A, a cAMP-specific phosphodiesterase. Metabolomic characterization identified elevated cAMP signaling and CREB as a candidate therapeutic target in IBC. Investigation of cellular dynamics and heterogeneity at the single cell level during chemotherapy and acquired resistance by CyTOF and single cell RNA-seq identified mechanisms of resistance including a shift from luminal to basal/mesenchymal cell states through selection for rare preexisting subpopulations or an acquired change. Finally, combination treatment with paclitaxel and JAK2/STAT3 inhibition prevented the emergence of the mesenchymal chemo-resistant subpopulation. These results provide mechanistic rational for combination of chemotherapy with inhibition of JAK2/STAT3 signaling as a more effective therapeutic strategy in IBC. SIGNIFICANCE: Chemotherapy resistance in inflammatory breast cancer is driven by the JAK2/STAT3 pathway, in part via cAMP/PKA signaling and a cell state switch, which can be overcome using paclitaxel combined with JAK2 inhibitors.

Targetable leukemia dependency on noncanonical PI3Kγ signaling.

Phosphoinositide 3-kinase gamma (PI3Kγ) is implicated as a target to repolarize tumor-associated macrophages and promote anti-tumor immune responses in solid cancers. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid, and dendritic lineages. This dependency is characterized by innate inflammatory signaling and activation of phosphoinositide 3-kinase regulatory subunit 5 ( PIK3R5 ), which encodes a regulatory subunit of PI3Kγ and stabilizes the active enzymatic complex. Mechanistically, we identify p21 (RAC1) activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency independently of Akt kinase. PI3Kγ inhibition dephosphorylates PAK1, activates a transcriptional network of NFκB-related tumor suppressor genes, and impairs mitochondrial oxidative phosphorylation. We find that treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukemias with activated PIK3R5 , either at baseline or by exogenous inflammatory stimulation. Notably, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukemia xenografts with low baseline PIK3R5 expression, as residual leukemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Taken together, our study reveals a targetable dependency on PI3Kγ/PAK1 signaling that is amenable to near-term evaluation in patients with acute leukemia.