Igor Chernukhin's research while affiliated with Cancer Research UK Cambridge Institute and other places

Background: Pre-clinical data suggest that combining anti-estrogen treatment with a progesterone receptor agonist leads to greater inhibition of tumor proliferation, due to molecular interactions between ER and PR [1]. A high dose of the PR agonist megestrol (160mg daily) is approved as monotherapy for the treatment of ER positive metastatic breast cancer. A lower dose of megestrol (20-40mg daily) can be an effective treatment for severe hot flashes associated with endocrine therapy [2] but whether this dose has anti-tumor activity is unknown. The PIONEER trial evaluated the potential anti-proliferative effect of low and high dose megestrol in combination with letrozole, relative to letrozole alone, using a short-term preoperative ‘window’ trial design assessing the direct effects of the trial treatment on tumor tissue before and after treatment. Methods: Eligible patients were post-menopausal women with histologically confirmed ER+ (Allred ≥ 3) HER2 negative breast cancer at least 10mm in size, with an ECOG performance status ≤ 2, planned for primary surgery or endocrine therapy. Enrolled patients were randomised 2:3:3 to Arm A: letrozole alone, Arm B: letrozole + lower-dose megestrol (40mg) or Arm C: letrozole + higher-dose megestrol (160mg). Treatment was given for 15 (13-19) days prior to surgery or end of treatment (EOT) core biopsy. The primary endpoint was change in tumor proliferation between baseline and EOT in Arm A vs (Arms B+C combined), measured by Ki67 immunohistochemistry (IHC). Secondary endpoints were comparison of Ki67 change in high versus low dose megestrol arms, absolute Ki67 at EOT, and change in tumor apoptosis (cleaved caspase 3 IHC), proliferation (Aurora Kinase A IHC), PR and androgen receptor expression. Exploratory analysis of ER chromatin binding (ChIP-Seq) was conducted on paired fresh-frozen samples from a subset of patients. Results: A total of 243 patients were randomised from July 2017 to October 2022 with recruitment paused for 3 months at onset of the COVID pandemic. 198 patients completed treatment and had evaluable tissue samples at baseline and EOT (Arm A: n = 51, Arm B: n = 74, Arm C: n = 73). Baseline mean Ki67 values were well balanced. Therapy was well tolerated and adverse events ≥ grade 3 were similarly rare across arms (A: 3.3%, B+C: 3.5%). The mean % reduction in Ki67 for each arm was: Arm A (letrozole): 71%, Arm B (letrozole + 40mg megestrol): 79%, Arm C (letrozole + 160mg megestrol): 80%. There was a statistically significantly greater reduction in Ki67 with megestrol combinations (B+C) versus letrozole alone (A) (P = 0.013). Analyses of secondary IHC endpoints and ER ChIP-Seq are ongoing and will be presented. Conclusion: Addition of the PR agonist megestrol enhanced the anti-proliferative effect of letrozole in this window-of opportunity trial. Megestrol combinations were well tolerated, and the anti-proliferative effect was observed in both low and high dose arms. These data support the potential use of low-dose megestrol as an inexpensive and well-tolerated means of improving aromatase inhibitor efficacy. Low dose megestrol can also ameliorate hot flashes and therefore might be a strategy to improve both treatment adherence and clinical outcomes for patients taking adjuvant endocrine therapy. References 1. Mohammed et al., Nature 523: 313–317 (2015) 2. Loprinzi et al., NEJM 331: 347-352 (1994) Citation Format: Rebecca Burrell, Sanjeev Kumar, Stuart McIntosh, Vassilis Pitsinis, Polly King, Beatrix Elsberger, Sasirekha Govindarajulu, Lucy Satherley, Sirwan Hadad, Peter Schmid, Jean Abraham, Amit Agrawal, John Benson, Danya Cheeseman, Igor Chernukhin, Parto Forouhi, Eleftheria Kleidi, Cleopatra Pike, Karen Pinilla, Elena Provenzano, Wendi Qian, Jason Carroll, Richard Baird. Results of the window-of-opportunity PIONEER trial evaluating addition of the progesterone receptor (PR) agonist megestrol to letrozole for early stage estrogen receptor (ER) positive breast cancer: exploiting ER-PR interaction [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS15-06.

Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late-stage disease and have limited treatment options. Therefore, there is an immediate need for the generation of new and innovative therapeutic targets. Little is known about the role of pioneer factors such as FOXA1 and their interaction partners in a PDAC context. Inspired by literature reports implicating FOXA1 and GATA5/GATA6 in regulating pancreatic cancer resistance and metastasis, our study hypothesised the possibility of an undiscovered nuclear receptor that works with FOXA1 (similar to Estrogen receptor in breast cancer). Using innovative ‘omic’ based approaches (RIME) developed in our laboratory 1 we discovered a nuclear receptor (NR) complex involving HNF4A and HNF4G in the classical sub-type of pancreatic cancer. Subsequently the interaction was independently validated in Whipple surgical biopsies from PDAC patients using ChIP-sequencing studies. Across multiple patient tumors (n=7) a binding overlap specifically between FOXA1 and HNF4G (3461 sites) was established. To investigate the therapeutic potential of HNF4G in the classical subtype of PDAC, we generated CRISPR deletions (KO) of HNF4G in the HPAF-II cells. HNF4G-KO cells were orthotopically implanted into the pancreas of NSG mice. A significant survival advantage of 12 days (<0.001) and reduced growth (<0.02) was observed in these mice compared to the controls. To better understand the impact of HNF4G-KO on gene expression, the orthotopic tumors were subjected to RNA-seq analyses. Gene set enrichment and pathway analysis revealed significantly down-regulated pathways to include EMT transition. TCGA data highlighted HNF4G amplification in 9% of PDAC patients with concomitant decreased progression free survival. These data point towards HNF4G being a therapeutically viable target for further exploration. Protein Arginine Methyl Transferase 1 (PRMT1) is a common interactor of both FOXA1 and HNF4G. Our study reveals a unique dependency of PRMT1 on the HNF4G-FOXA1 complex in PDAC biopsies. HNF4G-KO drastically reduces the chromatin binding of PRMT1, implicating them as functionally dependent. Treatment of HNF4G-KO cells with GSK3368715 (PRMT1 inhibitor) further sensitizes these cells and results in a significant survival advantage (10 days <0.02). We propose a model of HNF4G inhibition in combination with PRMT1 as a novel therapeutic opportunity to treat the classical sub-type of PDAC. Further, our study reveals a unique reliance of primary classical tumors on HNF4G. Although HNF4G appears to dominate FOXA1 functionality in the primary disease, FOXA1 remains instrumental in priming metastasis and enhancer reprograming. 1. Papachristou EK, Kishore K, Holding AN, Harvey K, Roumeliotis TI, Chilamakuri CSR, et al. A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes. Nature Communications. 2018;9(1):2311. Citation Format: Shalini V. Rao, Lisa Young, Danya Cheeseman, Stephanie Mack, Jill Temple, Chandra Shekar Reddy Chilamakuri, Evangelia Papachristou, Catherina Pelicano, Amy Smith, Dominique-Laurent Couturier, Michael Gill, Duncan Jodrell Jodrell, Alasdair Russell, Igor Chernukhin, Jason Carroll. New insights into the role of FOXA1- HNF4 axis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C076.

Pancreatic cancer is set to become the 3rd deadliest cancer by 2025. Pancreatic ductal adenocarcinoma (PDAC) represents the majority of cases and is the most aggressive subtype, with a 5-year survival rate of around 10% and patients often diagnosed at a metastatic stage. Current treatments offer dismal survival benefit for the patients, highlighting the need for innovative therapeutic strategies. PDAC is characterized by genetic and epigenetic modifications and an abundant tumor microenvironment (TME) largely composed of cancer-associated fibroblasts (CAFs). Multiple populations of CAFs have been described and implicated in PDAC progression, however, little is known regarding the molecular crosstalk between them and the cancer cells. This project aims to investigate how the epigenetic landscape of PDAC is affected by recently described CAF populations. We have optimized a co-culture model consisting of cancer cells and genetically-locked inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs) to study the link between the TME and the gene regulatory machinery in PDAC through proteomic and genomic approaches. Firstly, a full proteome analysis revealed the two CAF populations drive expression of different subsets of proteins in PDAC cells, including transcription factors. We then interrogated chromatin accessibility via ATAC-seq, which showed that CAF subsets differentially affect chromatin dynamics in epithelial cells. Transcription factor interactomes were investigated using rapid immunoprecipitation of endogenous proteins (RIME), a method developed in our lab that allows the discovery of protein complexes in a robust manner. We leverage this technique to discover druggable transcription factor interactors. RIME data shows that upon co-culture with each CAF population, distinct interactors are recruited towards FOXA1, a pioneer factor that drives a transcriptional program associated with PDAC metastasis to the liver, and towards FOXA2, which has been shown to play distinct roles in different grades of PDAC. We are now assessing the potential of these interactors as drug candidates in each context. Chromatin immunoprecipitation assays have shown that the CAF populations also affect the binding profile of FOXA1 and FOXA2 to the chromatin. We are currently investigating how CAFs impact tumor progression in vivo using an orthotopic implantation model. Preliminary data suggests that CAF subsets affect the metastatic profile in PDAC, as tumors co-injected with iCAFs metastasize to different organs than those co-injected with myCAFs. Ongoing experiments aim to explore the differences between tumors and metastases at the epigenetic level. Subsequent studies will aim to employ distinct approaches that target tumor progression in each context. PDAC is a highly heterogeneous cancer with several layers of complexity; therefore, a more holistic approach is critical to tackle the disease. Understanding tumor-stroma interactions from an epigenetics standpoint may enable identification of druggable molecules much needed for PDAC patients. Citation Format: Catarina Pelicano, Igor Chernukhin, Lisa Young, Naomi Vranas, Yi Cheng, Joshua Kent, Kamal Kishore, Clive D'Santos, Alasdair Russell, Giulia Biffi, Shalivi V. Rao, Jason S. Carroll. Unravelling the epigenetic landscape of pancreatic cancer: The role of cancer-associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR07.

Inhibiting the androgen receptor (AR), a ligand-activated transcription factor, with androgen deprivation therapy is a standard-of-care treatment for metastatic prostate cancer. Paradoxically, activation of AR can also inhibit the growth of prostate cancer in some patients and experimental systems, but the mechanisms underlying this phenomenon are poorly understood. This study exploited a potent synthetic androgen, methyltestosterone (MeT), to investigate AR agonist-induced growth inhibition. MeT strongly inhibited growth of prostate cancer cells expressing AR, but not AR-negative models. Genes and pathways regulated by MeT were highly analogous to those regulated by DHT, although MeT induced a quantitatively greater androgenic response in prostate cancer cells. MeT potently downregulated DNA methyltransferases, leading to global DNA hypomethylation. These epigenomic changes were associated with dysregulation of transposable element expression, including upregulation of endogenous retrovirus (ERV) transcripts after sustained MeT treatment. Increased ERV expression led to accumulation of double-stranded RNA and a “viral mimicry” response characterized by activation of IFN signaling, upregulation of MHC class I molecules, and enhanced recognition of murine prostate cancer cells by CD8+ T cells. Positive associations between AR activity and ERVs/antiviral pathways were evident in patient transcriptomic data, supporting the clinical relevance of our findings. Collectively, our study reveals that the potent androgen MeT can increase the immunogenicity of prostate cancer cells via a viral mimicry response, a finding that has potential implications for the development of strategies to sensitize this cancer type to immunotherapies. Significance Our study demonstrates that potent androgen stimulation of prostate cancer cells can elicit a viral mimicry response, resulting in enhanced IFN signaling. This finding may have implications for the development of strategies to sensitize prostate cancer to immunotherapies.

Background The pro-neural transcription factor ASCL1 is a master regulator of neurogenesis and a key factor necessary for the reprogramming of permissive cell types to neurons. Endogenously, ASCL1 expression is often associated with neuroblast stem-ness. Moreover, ASCL1-mediated reprogramming of fibroblasts to differentiated neurons is commonly achieved using artificially high levels of ASCL1 protein, where ASCL1 acts as an “on-target” pioneer factor. However, the genome-wide effects of enhancing ASCL1 activity in a permissive neurogenic environment has not been thoroughly investigated. Here, we overexpressed ASCL1 in the neuronally-permissive context of neuroblastoma (NB) cells where modest endogenous ASCL1 supports the neuroblast programme. Results Increasing ASCL1 in neuroblastoma cells both enhances binding at existing ASCL1 sites and also leads to creation of numerous additional, lower affinity binding sites. These extensive genome-wide changes in ASCL1 binding result in significant reprogramming of the NB transcriptome, redirecting it from a proliferative neuroblastic state towards one favouring neuronal differentiation. Mechanistically, ASCL1-mediated cell cycle exit and differentiation can be increased further by preventing its multi-site phosphorylation, which is associated with additional changes in genome-wide binding and gene activation profiles. Conclusions Our findings show that enhancing ASCL1 activity in a neurogenic environment both increases binding at endogenous ASCL1 sites and also results in additional binding to new low affinity sites that favours neuronal differentiation over the proliferating neuroblast programme supported by the endogenous protein. These findings have important implications for controlling processes of neurogenesis in cancer and cellular reprogramming.

Triple Negative Breast Cancer (TNBC) accounts for 15–20% of all breast cancer cases, yet is responsible for a disproportionately high percentage of breast cancer mortalities. Thus, there is an urgent need to identify novel biomarkers and therapeutic targets based on the molecular events driving TNBC pathobiology. Estrogen receptor beta (ERβ) is known to elicit anti-cancer effects in TNBC, however its mechanisms of action remain elusive. Here, we report the expression profiles of ERβ and its association with clinicopathological features and patient outcomes in the largest cohort of TNBC to date. In this cohort, ERβ was expressed in approximately 18% of TNBCs, and expression of ERβ was associated with favorable clinicopathological features, but correlated with different overall survival outcomes according to menopausal status. Mechanistically, ERβ formed a co-repressor complex involving enhancer of zeste homologue 2/polycomb repressive complex 2 (EZH2/PRC2) that functioned to suppress oncogenic NFκB/RELA (p65) activity. Importantly, p65 was shown to be required for formation of this complex and for ERβ-mediated suppression of TNBC. Our findings indicate that ERβ+ tumors exhibit different characteristics compared to ERβ− tumors and demonstrate that ERβ functions as a molecular switch for EZH2, repurposing it for tumor suppressive activities and repression of oncogenic p65 signaling.

Despite their abundance, mid-sized RNAs (30–300 nt) have not been extensively studied by high-throughput sequencing, mostly due to selective loss in library preparation. The authors propose simple and inexpensive modifications to the Illumina TruSeq protocol (ncRNAseq), allowing the capture and sequencing of mid-sized non-coding RNAs without detriment to the coverage of coding mRNAs. This protocol is coupled with a two-step alignment: a pre-alignment to a curated non-coding genome, passing only the non-mapping reads to a standard genomic alignment. ncRNAseq correctly assigns the highest read-numbers to established abundant non-coding RNAs and correctly identifies cytosolic and nuclear enrichment of known non-coding RNAs in two cell lines.