Search

Background: About half of all breast cancers exhibit low HER2 expression. Despite lack of ERBB2 amplification, HER2-low tumors respond to trastuzumab deruxtecan (T-DXd), leading to the NCCN recommendation of T-DXd both for patients with HER2+ and HER2-low metastatic breast cancer (MBC). It remains however unclear if HER2-low represents a distinct molecular entity, as compared to HER2-0 MBC. Here, we compare the genomic landscape of HER2-low versus HER2-0 breast cancers in a large, single institution cohort. Methods: We identified consecutive patients with MBC seen at Dana-Farber Cancer Institute between 07/2013 and 12/2020. Patients were included if they had HER2-negative MBC per ASCO/CAP Guidelines and had undergone next generation sequencing (NGS) testing with a targeted, tumor-only platform (OncoPanel). Based on the HER2 status of the specimen tested by NGS, patients were divided into 2 groups: (i) HER2-low if immunohistochemistry (IHC) 1+ or 2+ non-amplified, or (ii) HER2-0 if IHC 0. Mutations of interest detected on NGS were classified as oncogenic using the OncoKB tool and additional annotation. Genomic profiles of HER2-low and HER2-0 tumors were compared using Chi-Square and Kruskal-Wallis tests. To determine genomic event enrichment between the two HER2 groups, logistic regression models were used, accounting for background rate and estrogen receptor (ER) expression. ERBB2 copy counts were calculated for tumors with recorded histology-estimated purities and copy-number segmentation using a simple model of allelic gain/loss. Results: Among 1847 patients with HER2-negative MBC, 1043 underwent NGS testing on a HER2-low (n=489, 47%) or HER2-0 sample (n=554, 53%). Most samples were metastatic (71%, n=743) while 29% (n=300) were from primary tumors. 73% had ductal histology, 13% were lobular and 14% had mixed or other histology. ER expression was enriched among HER2-low vs. HER2-0 tumors (76% vs. 60%; p< 0.001). Focusing on the most commonly occurring genetic mutations, no major differences were observed in HER2-low vs. HER2-0 tumors, after correcting for ER status (Table 1). Among all mutational events, any mutation in MPL, CYLD, and MAP3K and oncogenic mutations in TP53 and NF1 were more common in HER2-0, while any mutation in MTOR, RAD21, DNMT3A, and PDGFRA were enriched in HER2-low patients, when controlling for ER status and background mutational rate (p< 0.05). However, no mutation reached significance after accounting for multiple hypothesis testing. Similarly, no deep deletion or high amplification CNV events reached significance for either group. Analysis of tumor mutational burden in HER2-low vs. HER-0 tumors revealed no significant differences (median: 7.26 muts/Mb vs. 7.60 muts/Mb, p=1.00), including when accounting for ER status. Finally, among tumors with sufficient tumor purity for ERBB2 copy count analysis (n=374 and 419 for HER2-low and HER2-0, respectively), HER2-low tumors had a significantly higher number of ERBB2 alleles as compared to HER2-0 (< 2 copies, 15.0% vs. 30.9%, 2 copies 67.4% vs. 60.5%, and >2 copies, 17.6% vs. 8.6%; p< 0.001 by Kruskal-Wallis). Conclusions: To our knowledge, this is the largest comprehensive genomic analysis of HER2-low MBC to date. In our cohort of patients with HER2-negative MBC, the genomic landscape of HER2-low and HER2-0 tumors did not differ significantly, apart from a higher number of ERBB2 alleles. These data further support the notion that HER2-low, as currently defined, is not a distinct molecular subtype of breast cancer. Citation Format: Paolo Tarantino, Hersh V. Gupta, Melissa E. Hughes, Janet L. Files, Sarah Strauss, Gregory Kirkner, Anne-Marie Feeney, Yvonne Y. Li, Ana C. Garrido-Castro, Romualdo Barroso-Sousa, Brittany Bychkovsky, Laura MacConaill, Neal Lindeman, Bruce Johnson, Matthew Meyerson, Sheheryar Kabraji, Rinath Jeselsohn, Xintao Qiu, Rong Li, Henry W. Long, Eric Winer, Deborah A. Dillon, Giuseppe Curigliano, Andrew Cherniack, Sara Tolaney, Nancy U. Lin. HER2-05 Comprehensive genomic characterization of HER2-low breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-05.

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.

Most invasive lobular breast cancers (ILC) are of the luminal A subtype and are strongly hormone receptor–positive. Yet, ILC is relatively resistant to tamoxifen and associated with inferior long-term outcomes compared with invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. A comprehensive analysis of the epigenome of ILC in preclinical models and clinical samples showed that, compared with IDC, ILC harbored a distinct chromatin state linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This resulted in an ILC-unique FOXA1–estrogen receptor (ER) axis that promoted the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1–ER axis led to retained ER chromatin binding after tamoxifen treatment, which facilitated tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the autoinduction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, ILC is characterized by a unique chromatin state and FOXA1–ER axis that is associated with tumor progression, offering a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC in order to optimize treatments in this breast cancer subtype. Significance: A unique FOXA1–ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease. See related commentary by Blawski and Toska, p. 3668

Figure S2

Supplementary Table S3

Supplementary Table from A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer

Supplementary Figure from A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer

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.

Most invasive lobular breast cancers (ILC) are of the luminal A subtype and are strongly hormone receptor–positive. Yet, ILC is relatively resistant to tamoxifen and associated with inferior long-term outcomes compared with invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. A comprehensive analysis of the epigenome of ILC in preclinical models and clinical samples showed that, compared with IDC, ILC harbored a distinct chromatin state linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This resulted in an ILC-unique FOXA1–estrogen receptor (ER) axis that promoted the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1–ER axis led to retained ER chromatin binding after tamoxifen treatment, which facilitated tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the autoinduction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, ILC is characterized by a unique chromatin state and FOXA1–ER axis that is associated with tumor progression, offering a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC in order to optimize treatments in this breast cancer subtype.Significance:A unique FOXA1–ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease.See related commentary by Blawski and Toska, p. 3668

Supplementary Figures

Supplementary Data from A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer

Purpose:Defects in genes in the DNA repair pathways significantly contribute to prostate cancer progression. We hypothesize that overexpression of DNA repair genes may also drive poorer outcomes in prostate cancer. The ribonucleotide reductase small subunit M2 (RRM2) is essential for DNA synthesis and DNA repair by producing dNTPs. It is frequently overexpressed in cancers, but very little is known about its function in prostate cancer.Experimental Design:The oncogenic activity of RRM2 in prostate cancer cells was assessed by inhibiting or overexpressing RRM2. The molecular mechanisms of RRM2 function were determined. The clinical significance of RRM2 overexpression was evaluated in 11 prostate cancer clinical cohorts. The efficacy of an RRM2 inhibitor (COH29) was assessed in vitro and in vivo. Finally, the mechanism underlying the transcriptional activation of RRM2 in prostate cancer tissue and cells was determined.Results:Knockdown of RRM2 inhibited its oncogenic function, whereas overexpression of RRM2 promoted epithelial mesenchymal transition in prostate cancer cells. The prognostic value of RRM2 RNA levels in prostate cancer was confirmed in 11 clinical cohorts. Integrating the transcriptomic and phosphoproteomic changes induced by RRM2 unraveled multiple oncogenic pathways downstream of RRM2. Targeting RRM2 with COH29 showed excellent efficacy. Thirteen putative RRM2-targeting transcription factors were bioinformatically identified, and FOXM1 was validated to transcriptionally activate RRM2 in prostate cancer.Conclusions:We propose that increased expression of RRM2 is a mechanism driving poor patient outcomes in prostate cancer and that its inhibition may be of significant therapeutic value.

Supplemental tables, figures, and figure captions.

How cancer cells adapt to evade the therapeutic effects of drugs targeting oncogenic drivers is poorly understood. Here we report an epigenetic mechanism leading to the adaptive resistance of triple-negative breast cancer (TNBC) to fibroblast growth factor receptor (FGFR) inhibitors. Prolonged FGFR inhibition suppresses the function of BRG1-dependent chromatin remodelling, leading to an epigenetic state that derepresses YAP-associated enhancers. These chromatin changes induce the expression of several amino acid transporters, resulting in increased intracellular levels of specific amino acids that reactivate mTORC1. Consistent with this mechanism, addition of mTORC1 or YAP inhibitors to FGFR blockade synergistically attenuated the growth of TNBC patient-derived xenograft models. Collectively, these findings reveal a feedback loop involving an epigenetic state transition and metabolic reprogramming that leads to adaptive therapeutic resistance and provides potential therapeutic strategies to overcome this mechanism of resistance. Li et al. define an adaptive resistance mechanism against FGFR inhibitor treatment in breast cancer attributed to loss of BRG1 chromatin recruitment, reactivation of YAP-dependent enhancers and upregulation of amino acid-induced mTORC1 activity.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain (NTD) of the AR drives transcriptional activity in CRPC but is intrinsically disordered and remains a challenging therapeutic target. Therefore, inhibiting critical co-chaperones, such as BAG-1L, is an attractive alternative strategy. We performed druggability analyses demonstrating the BAG domain to be a challenging drug target. Thio-2, a tool compound, has been reported to bind the BAG domain of BAG-1L and inhibit BAG-1L-mediated AR transactivation. However, despite these data, the mechanism of action of Thio-2 is poorly understood and the BAG domain which is present in all BAG-1 isoforms has not been validated as a therapeutic target. Herein, we demonstrate growth inhibiting activity of Thio-2 in CRPC cell lines and patient derived models with decreased AR genomic binding and AR signaling independent of BAG-1 isoform function. Furthermore, genomic abrogation of BAG-1 isoforms did not recapitulate the described Thio-2 phenotype, and NMR studies suggest that Thio-2 may bind the AR NTD, uncovering a potential alternative mechanism of action, although in the context of low compound solubility. Furthermore, BAG-1 isoform knockout mice are viable and fertile, in contrast to previous studies, and when crossed with prostate cancer mouse models, BAG-1 deletion does not significantly impact prostate cancer development and growth. Overall, these data demonstrate that Thio-2 inhibits AR signaling and growth in CRPC independent of BAG-1 isoforms, and unlike previous studies of the activated AR, therapeutic targeting of the BAG domain requires further validation before being considered a therapeutic strategy for the treatment of CRPC.

Chromatin immunoprecipitation sequencing (ChIP-seq) and the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) have become essential technologies to effectively measure protein-DNA interactions and chromatin accessibility. However, there is a need for a scalable and reproducible pipeline that incorporates proper normalization between samples, correction of copy number variations, and integration of new downstream analysis tools. Here we present Containerized Bioinformatics workflow for Reproducible ChIP/ATAC-seq Analysis (CoBRA), a modularized computational workflow which quantifies ChIP-seq and ATAC-seq peak regions and performs unsupervised and supervised analyses. CoBRA provides a comprehensive state-of-the-art ChIP-seq and ATAC-seq analysis pipeline that can be used by scientists with limited computational experience. This enables researchers to gain rapid insight into protein-DNA interactions and chromatin accessibility through sample clustering, differential peak calling, motif enrichment, comparison of sites to a reference database, and pathway analysis. CoBRA is publicly available online at https://bitbucket.org/cfce/cobra.

Prostate cancers are considered to be immunologically ‘cold’ tumors given the very few patients who respond to checkpoint inhibitor (CPI) therapy. Recently, enrichment of interferon-stimulated genes (ISGs) predicted a favorable response to CPI across various disease sites. The enhancer of zeste homolog-2 (EZH2) is overexpressed in prostate cancer and known to negatively regulate ISGs. In the present study, we demonstrate that EZH2 inhibition in prostate cancer models activates a double-stranded RNA–STING–ISG stress response upregulating genes involved in antigen presentation, Th1 chemokine signaling and interferon response, including programmed cell death protein 1 (PD-L1) that is dependent on STING activation. EZH2 inhibition substantially increased intratumoral trafficking of activated CD8(+) T cells and increased M1 tumor-associated macrophages, overall reversing resistance to PD-1 CPI. Our study identifies EZH2 as a potent inhibitor of antitumor immunity and responsiveness to CPI. These data suggest EZH2 inhibition as a therapeutic direction to enhance prostate cancer response to PD-1 CPI.

Estrogen receptor alpha (ER) is a ligand activated transcription factor and a driver of ER+ breast cancer. The discovery of activating mutations in ER (gene name ESR1), in patients with advanced endocrine-resistant ER+ breast cancer, has ignited a large pharmaceutical effort to develop next-generation ER antagonists that also target the mutant protein. Here, we profiled the potency and selectivity of many next-generation ER antagonists currently in clinical trials, including ER-targeted proteolysis-targeting chimeras (PROTACs), selective ER modulators (SERMs), and selective ER degraders (SERDs). To contextualize these antagonists, we evaluated their activity against the only FDA-approved SERD, Fulvestrant, at the level of chromatin, transcriptome, proteome, and cell viability, using cells expressing wild-type ER as well as activating mutant ER (ERY537S and ERD538G). Overall, we find that many of the novel oral SERDs are potent ER antagonists, including against mutant ER. To anticipate resistance mechanisms to these next-generation ER antagonists, we developed a strategy initially focused on ARV471 (the most clinically advanced ER PROTAC), with two approaches. First, we performed whole-genome CRISPR/Cas9-knockout screening in ER+ breast cancer cell lines treated with ARV471. Among the enriched gRNAs were PTEN, NF2, and CRBN. Loss of PTEN and NF2 results in activation of the PI3K and MAPK pathways, respectively, and CRBN is the ubiquitin E3 ligase required for ER protein degradation by ARV471. PTEN and NF2 are known mechanisms of endocrine resistance but it is not yet known if CRBN loss in response to PROTACs is a predominant clinical resistance mechanism. Therefore, in a second approach we generated ARV471 resistant breast cancer cell lines through long term exposure (LTE-ARV471). These cells developed an ER-independent resistance mechanism, with decreased ER expression and increased expression of pathways associated with Ras/MAPK and EMT. Therefore, as more potent ER degraders enter the clinic, such as PROTACs and oral SERDs, it may be important to monitor resistance mechanisms that involve loss of ER. Currently, we are following up our findings with LTE-ARV471 cells and performing CRISPR screening to identify targetable dependencies and combination therapies for endocrine resistant breast cancer. Citation Format: Delia Margaret Friel, Zachary Sandusky, Yijia Jiang, Xintao Qiu, Rong Li, Henry Long, Rinath Jeselsohn, Myles Brown. Evaluation of resistance mechanisms to ARV471, an ER-targeted PROTAC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3875.

The androgen receptor (AR) is a prostate master transcription factor. It binds to genetic enhancers, where it regulates gene activity and plays a fundamental role in prostate pathophysiology. Previous work has demonstrated AR-DNA binding is systematically and consistently reprogrammed during prostate tumorigenesis and disease progression. We charted these reprogrammed AR sites and identified genes proximal to them. We were able to devise gene lists based on AR status within specific histological contexts: normal prostate epithelium, primary prostate tumor, and metastatic prostate cancer. We evaluated expression of the genes in these gene sets in subjects from two distinct clinical cohorts – men treated with surgery for localized prostate cancer and men with metastatic prostate cancer. Among men with localized prostate cancer, expression of genes proximal to AR sites lost in the transition from normal prostate to prostate tumor was associated with clinical outcome. Among men with metastatic disease, expression of genes proximal to AR sites gained in metastatic tumors were associated with clinical outcome. The study demonstrates the power of incorporating context-dependent epigenetic data into genetic analyses.

The androgen receptor (AR) is a prostate master transcription factor. It binds to genetic enhancers, where it regulates gene activity and plays a fundamental role in prostate pathophysiology. Previous work has demonstrated that AR-DNA binding is systematically and consistently reprogrammed during prostate tumorigenesis and disease progression. We charted these reprogrammed AR sites and identified genes proximal to them. We were able to devise gene lists based on AR status within specific histological contexts: normal prostate epithelium, primary prostate tumor, and metastatic prostate cancer. We evaluated expression of the genes in these gene sets in subjects from two distinct clinical cohorts—men treated with surgery for localized prostate cancer and men with metastatic prostate cancer. Among men with localized prostate cancer, expression of genes proximal to AR sites lost in the transition from normal prostate to prostate tumor was associated with clinical outcome. Among men with metastatic disease, expression of genes proximal to AR sites gained in metastatic tumors was associated with clinical outcome. These results are consistent with the notion that AR is fundamental to both maintaining differentiation in normal prostate tissue and driving de-differentiation in advanced prostate cancer. More broadly, the study demonstrates the power of incorporating context-dependent epigenetic data into genetic analyses.

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyze cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single-cell analysis of human clinical samples exhibits a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall, our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy., Neuroendocrine carcinomas (NECs) arise from different anatomic sites, but have similar histological and clinical features. Here, the authors show that the epigenetic landscape of a range of NECs converges towards a common epigenetic state, while distinct subtypes occur within neuroendocrine prostate cancer contributing to intratumor heterogeneity in clinical samples.

Triple-negative breast cancer (TNBC) is a subtype accounting for 20-30% all breast cancer cases and is a highly aggressive disease with inferior prognosis. The acquired resistance is a major obstacle for efficient therapy and discovery of novel clinically-actionable targets has become an urgent need. In this study, we carried out an in-depth functional characterization of histone demethylase KDM4C in TNBC and uncovered a novel mechanism underlying KDM4C-driven tumorigenesis. KDM4C is the second most frequently amplified histone demethylase in TNBC. Genetic knockdown or pharmacological inhibition of KDM4C in two amplified basal breast cancer cell lines, SUM149 and HCC1954, drastically suppressed primary tumor growth in vitro and in vivo. Transcriptomic analysis underlined oxidative phosphorylation impairment as functional consequences of KDM4C blockade. Surprisingly, integrating series of histone ChIP-seq and ATAC-seq showed that KDM4C suppression caused accessible chromatin remodeling without substantial changes of its canonical substrates H3K9me3 and H3K36me3. Rather KDM4C loss caused proteolytic cleavage at histone 3 N-terminus (Ala21 site) identified by histone mass spectrometry. Protease inhibitor array pointed out cathepsin L (CTSL) as the endopeptidase mediating this procedure. KDM4C blockade induced CTSL activation and promoted H3 tail clipping at around 30% CTSL binding sites, which was associated with restricted chromatin accessibility and transcriptomic reprogramming. Proteomic interactome profiling revealed that grainyhead like transcription factor 2 (GRHL2) tightly binds to CTSL. In addition, GRHL2 knockout induced nearly complete loss of CTSL chromatin binding, demonstrating its role as a recruiter of CTSL to the chromatin. KDM4C serves as either a direct or indirect suppressor for CTSL-mediated H3 cleavage. The latter one was largely mediated by redox imbalance. Metabolomic profiling showed that KDM4C inhibition strongly dampened intracellular glutathione (GSH) levels and hence produced more reactive oxygen species (ROS) and alleviated mitochondrial respiration. This procedure was grounded on the mechanism that KDM4C blockade decreased expression of GSH synthesis rate-limiting enzyme glutamate-cysteine ligase catalytic subunit (GCLC) via CTSL-mediated H3 tail clipping. Knockout of CTSL rescued KDM4C blockade-associated metabolic dysfunction, confirming KDM4C links redox homeostasis and chromatin remodeling. Finally, we found KDM4C-CTSL-GSH axis was associated with cisplatin resistance in TNBC patients. Co-targeting KDM4C and GSH production efficiently improved cisplatin response in TNBC cells. Our study provided detailed and unbiased evidence supporting a novel non-canonical role of KDM4C in preventing CTSL-mediated histone clipping and maintaining redox balance, opening up possibilities to improve chemotherapy through targeting this circus. Citation Format: Zheqi Li, Guillermo Peluffo, Laura E. Stevens, Xintao Qiu, Shawn B. Egri, Malvina Papanastasiou, Natalie Kingston, Clive S. D'Santos, Eva Papachristou, Kyle Evans, Ji-Heui Seo, Kendell Clement, Daniel Temko, Muhammad Ekram, Anton Simeonov, Stephen C. Kales, Ganesha Rai, Madhu Lal-Nag, David J. Maloney, Ajit Jadhav, Franziska Michor, Alex Meissner, Jason S. Carroll, Matthew L. Freedman, Henry W. Long, Jacob D. Jaffe, Kornelia Polyak. KDM4C histone demethylase connects redox balance to chromatin remodeling via histone H3 tail clipping. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A018.

Summary Sustained treatment of estrogen receptor (ER)-positive breast cancer with ER-targeting drugs results in ER mutations and refractory unresponsive cancers. Androgen receptor (AR), which is expressed in 80%–95% of ER-positive breast cancers, could serve as an alternate therapeutic target. Although AR agonists were used in the past to treat breast cancer, their use is currently infrequent due to virilizing side effects. Discovery of tissue-selective AR modulators (SARMs) has renewed interest in using AR agonists to treat breast cancer. Using translational models, we show that AR agonist and SARM, but not antagonist, inhibit the proliferation and growth of ER-positive breast cancer cells, patient-derived tissues, and patient-derived xenografts (PDX). Ligand-activated AR inhibits wild-type and mutant ER activity by reprogramming the ER and FOXA1 cistrome and rendering tumor growth inhibition. These findings suggest that ligand-activated AR may function as a non-canonical inhibitor of ER and that AR agonists may offer a safe and effective treatment for ER-positive breast cancer., Graphical Abstract, Highlights • Androgen receptor (AR) agonists inhibit estrogen receptor (ER)-positive breast cancer • Activating AR reprograms ER and FOXA1 cistrome, resulting in ER inhibition • AR agonist alters the phosphoproteome signature consistent with growth inhibition, Molecular Biology; Molecular Mechanism of Gene Regulation; Cancer

c-MYC (MYC) is a major driver of prostate cancer tumorigenesis and progression. Although MYC is overexpressed in both early and metastatic disease and associated with poor survival, its impact on prostate transcriptional reprogramming remains elusive. We demonstrate that MYC overexpression significantly diminishes the androgen receptor (AR) transcriptional program (the set of genes directly targeted by the AR protein) in luminal prostate cells without altering AR expression. Importantly, analyses of clinical specimens revealed that concurrent low AR and high MYC transcriptional programs accelerate prostate cancer progression toward a metastatic, castration-resistant disease. Data integration of single-cell transcriptomics together with ChIP-seq revealed an increased RNA polymerase II (Pol II) promoter-proximal pausing at AR-dependent genes following MYC overexpression without an accompanying deactivation of AR-bound enhancers. Altogether, our findings suggest that MYC overexpression antagonizes the canonical AR transcriptional program and contributes to prostate tumor initiation and progression by disrupting transcriptional pause release at AR-regulated genes.STATEMENT OF SIGNIFICANCEAR and MYC are key to prostate cancer etiology but our current understanding of their interplay is scarce. Here we show that the oncogenic transcription factor MYC can pause the transcriptional program of the master transcription factor in prostate cancer, AR, while turning on its own, even more lethal program.

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. Here, we investigate the epigenomic basis of this resistance mechanism by profiling histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identify a vast network of cis-regulatory elements (N~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying cancer dependencies through epigenomic profiling., The molecular processes that lead to neuroendocrine prostate cancer after treating prostate adenocarcinoma (PRAD) are not well understood. Here the authors show that regulation by FOXA1 and changes in the epigenomic profile drive the transition from PRAD to a neuroendocrine phenotype.