Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

Stratification of Tamoxifen Synergistic Combinations for the Treatment of ER+ Breast Cancer.

Breast cancer alone accounts for the majority of cancer deaths among women, with the most commonly diagnosed subtype being estrogen receptor positive (ER+). Survival has greatly improved for patients with ER+ breast cancer, due in part to the development of antiestrogen compounds, such as tamoxifen. While treatment of the primary disease is often successful, as many as 30% of patients will experience recurrence and metastasis, mainly due to developed endocrine therapy resistance. In this study, we discovered two tamoxifen combination therapies, with simeprevir and VX-680, that reduce the tumor burden in animal models of ER+ breast cancer more than either compound or tamoxifen alone. Additionally, these tamoxifen combinations reduced the expression of HER2, a hallmark of tamoxifen treatment, which can facilitate acquisition of a treatment-resistant phenotype. These combinations could provide clinical benefit by potentiating tamoxifen treatment in ER+ breast cancer.

Discovering Synergistic Compounds with BYL-719 in PI3K Overactivated Basal-like PDXs.

Basal-like triple-negative breast cancer (TNBC) tumor cells are difficult to eliminate due to resistance mechanisms that promote survival. While this breast cancer subtype has low PIK3CA mutation rates when compared to estrogen receptor-positive (ER+) breast cancers, most basal-like TNBCs have an overactive PI3K pathway due to gene amplification or high gene expression. BYL-719 is a PIK3CA inhibitor that has been found to have low drug-drug interactions, which increases the likelihood that it could be useful for combinatorial therapy. Alpelisib (BYL-719) with fulvestrant was recently approved for treating ER+ breast cancer patients whose cancer had developed resistance to ER-targeting therapy. In these studies, a set of basal-like patient-derived xenograft (PDX) models was transcriptionally defined with bulk and single-cell RNA-sequencing and clinically actionable mutation profiles defined with Oncomine mutational profiling. This information was overlaid onto therapeutic drug screening results. BYL-719-based, synergistic two-drug combinations were identified with 20 different compounds, including everolimus, afatinib, and dronedarone, which were also found to be effective at minimizing tumor growth. These data support the use of these drug combinations towards cancers with activating PIK3CA mutations/gene amplifications or PTEN deficient/PI3K overactive pathways.

Transcriptomic changes underlying EGFR inhibitor resistance in human and mouse models of basal-like breast cancer.

The goals of this study were to identify transcriptomic changes that arise in basal-like breast cancer cells during the development of resistance to epidermal growth factor receptor inhibitors (EGFRi) and to identify drugs that are cytotoxic once EGFRi resistance occurs. Human patient-derived xenografts (PDXs) were grown in immunodeficient mice and treated with a set of EGFRi; the EGFRi erlotinib was selected for more expansive in vivo studies. Single-cell RNA sequencing was performed on mammary tumors from the basal-like PDX WHIM2 that was treated with vehicle or erlotinib for 9 weeks. The PDX was then subjected to long-term erlotinib treatment in vivo. Through serial passaging, an erlotinib-resistant subline of WHIM2 was generated. Bulk RNA-sequencing was performed on parental and erlotinib-resistant tumors. In vitro high-throughput drug screening with > 500 clinically used compounds was performed on parental and erlotinib-resistant cells. Previously published bulk gene expression microarray data from MMTV-Wnt1 tumors were contrasted with the WHIM2 PDX data. Erlotinib effectively inhibited WHIM2 tumor growth for approximately 4 weeks. Compared to untreated cells, single-cell RNA sequencing revealed that a greater proportion of erlotinib-treated cells were in the G1 phase of the cell cycle. Comparison of WHIM2 and MMTV-Wnt1 gene expression data revealed a set of 38 overlapping genes that were differentially expressed in the erlotinib-resistant WHIM2 and MMTV-Wnt1 tumors. Comparison of all three data types revealed five genes that were upregulated across all erlotinib-resistant samples: IL19, KLK7, LCN2, SAA1, and SAA2. Of these five genes, LCN2 was most abundantly expressed in triple-negative breast cancers, and its knockdown restored erlotinib sensitivity in vitro. Despite transcriptomic differences, parental and erlotinib-resistant WHIM2 displayed similar responses to the majority of drugs assessed for cytotoxicity in vitro. This study identified transcriptomic changes arising in erlotinib-resistant basal-like breast cancer. These data could be used to identify a biomarker or develop a gene signature predictive of patient response to EGFRi. Future studies should explore the predictive capacity of these gene signatures as well as how LCN2 contributes to the development of EGFRi resistance.

Inhibition of Ca2+ entry by capsazepine analog CIDD-99 prevents oral squamous carcinoma cell proliferation.

Oral cancer patients have a poor prognosis, with approximately 66% of patients surviving 5-years after diagnosis. Treatments for oral cancer are limited and have many adverse side effects; thus, further studies are needed to develop drugs that are more efficacious. To achieve this objective, we developed CIDD-99, which produces cytotoxic effects in multiple oral squamous cell carcinoma (OSCC) cell lines. While we demonstrated that CIDD-99 induces ER stress and apoptosis in OSCC, the mechanism was unclear. Investigation of the Bcl-family of proteins showed that OSCC cells treated with CIDD-99 undergo downregulation of Bcl-XL and Bcl-2 anti-apoptotic proteins and upregulation of Bax (pro-apoptotic). Importantly, OSCC cells treated with CIDD-99 displayed decreased calcium signaling in a dose and time-dependent manner, suggesting that blockage of calcium signaling is the key mechanism that induces cell death in OSCC. Indeed, CIDD-99 anti-proliferative effects were reversed by the addition of exogenous calcium. Moreover, electrophysiological properties further established that calcium entry was via the non-selective TRPC1 channel and prolonged CIDD-99 incubation inhibited STIM1 expression. CIDD-99 inhibition of calcium signaling also led to ER stress and inhibited mitochondrial complexes II and V in vitro. Taken together, these findings suggest that inhibition of TRPC mediates induction of ER stress and mitochondrial dysfunction as a part of the cellular response to CIDD-99 in OSCC.

Dynamic Interactions of Transcription Factors and Enhancer Reprogramming in Cancer Progression.

While improved tumor treatment has significantly reduced the overall mortality rates, invasive progression including recurrence, therapy resistance and metastasis contributes to the majority of deaths caused by cancer. Enhancers are essential distal DNA regulatory elements that control temporal- or spatial-specific gene expression patterns during development and other biological processes. Genome-wide sequencing has revealed frequent alterations of enhancers in cancers and reprogramming of distal enhancers has emerged as one of the important features for tumors. In this review, we will discuss tumor progression-associated enhancer dynamics, its transcription factor (TF) drivers and how enhancer reprogramming modulates gene expression during cancer invasive progression. Additionally, we will explore recent advancements in contemporary technology including single-cell sequencing, spatial transcriptomics and CUT&RUN, which have permitted integrated studies of enhancer reprogramming in vivo. Given the essential roles of enhancer dynamics and its drivers in controlling cancer progression and treatment outcome, understanding these changes will be paramount in mitigating invasive events and discovering novel therapeutic targets.

Pontin Functions as A Transcriptional Co-activator for Retinoic Acid-induced HOX Gene Expression.

Pontin is a AAA+ ATPase protein that has functions in various biological contexts including gene transcription regulation, chromatin remodeling, DNA damage sensing and repair, as well as assembly of protein and ribonucleoprotein complexes. Pontin is known to regulate the transcription of several important signaling pathways, including Wnt signaling. However, its role in early embryonic signaling regulation remains unclear. Retinoic acid (RA) signaling plays a central role in vertebrate development. Using an in vivo biotin tagging technology, we mapped the genome-wide binding pattern of Pontin before and after RA-induced differentiation in the pluripotent embryo carcinoma cell line NTERA-2. Biotin ChIP-seq revealed significant changes in genome-wide Pontin binding sites upon RA stimulation. We also identified a substantial amount of overlapping binding peaks between Pontin and RARα, especially on all of the HOX gene loci (A-D clusters). Pontin knockdown experiments showed that its chromatin binding at the HOX gene clusters is required for RA-induced HOX gene expression. Furthermore, we performed Global Run-On sequencing (GRO-seq) to map de novo transcripts genome-wide and found that Pontin knockdown significantly diminished nascent HOX gene transcripts, indicating that Pontin regulates HOX gene expression at the transcriptional level. Finally, proteomic analysis demonstrated that Pontin associates with chromatin organization/remodeling complexes and various other functional complexes. Altogether, we have demonstrated that Pontin is a critical transcriptional co-activator for RA-induced HOX gene activation.

Loss of Ca2+ entry via Orai-TRPC1 induces ER stress, initiating immune activation in macrophages.

Activation of cellular stresses is associated with inflammation; however, the mechanisms are not well identified. Here, we provide evidence that loss of Ca2+ influx induces endoplasmic reticulum (ER) stress in primary macrophages and in murine macrophage cell line Raw 264.7, in which the unfolded protein response is initiated to modulate cytokine production, thereby activating the immune response. Stressors that initiate the ER stress response block store-dependent Ca2+ entry in macrophages prior to the activation of the unfolded protein response. The endogenous Ca2+ entry channel is dependent on the Orai1-TRPC1-STIM1 complex, and the presence of ER stressors decreased expression of TRPC1, Orai1 and STIM1. Additionally, blocking Ca2+ entry with SKF96365 also induced ER stress, promoted cytokine production, activation of autophagy, increased caspase activation and induced apoptosis. Furthermore, ER stress inducers inhibited cell cycle progression, promoted the inflammatory M1 phenotype, and increased phagocytosis. Mechanistically, restoration of Orai1-STIM1 expression inhibited the ER stress-mediated loss of Ca2+ entry that prevents ER stress and inhibits cytokine production, and thus induced cell survival. These results suggest an unequivocal role of Ca2+ entry in modulating ER stress and in the induction of inflammation.