Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer.

Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways.

The spliceosome is a therapeutic vulnerability in MYC-driven cancer.

MYC (also known as c-MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its pro-tumorigenic functions have been attributed to its ability to regulate gene expression programs. Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts. While such increases in RNA and protein production may endow cancer cells with pro-tumour hallmarks, this increase in synthesis may also generate new or heightened burden on MYC-driven cancer cells to process these macromolecules properly. Here we discover that the spliceosome is a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene in human mammary epithelial cells, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (such as SF3B1 and U2AF1) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total precursor messenger RNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Notably, genetic or pharmacological inhibition of the spliceosome in vivo impairs survival, tumorigenicity and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing, and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.

Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase.

INTRODUCTION: Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance. METHODS: Altered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy. RESULTS: Moderate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression. CONCLUSIONS: Moderate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.

ZFTA-RELA Dictates Oncogenic Transcriptional Programs to Drive Aggressive Supratentorial Ependymoma.

More than 60% of supratentorial ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. SIGNIFICANCE: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA-RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.This article is highlighted in the In This Issue feature, p. 2113.

Modulating Androgen Receptor-Driven Transcription in Prostate Cancer with Selective CDK9 Inhibitors.

Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.

Trisomy of a Down Syndrome Critical Region Globally Amplifies Transcription via HMGN1 Overexpression.

Down syndrome (DS, trisomy 21) is associated with developmental abnormalities and increased leukemia risk. To reconcile chromatin alterations with transcriptome changes, we performed paired exogenous spike-in normalized RNA and chromatin immunoprecipitation sequencing in DS models. Absolute normalization unmasks global amplification of gene expression associated with trisomy 21. Overexpression of the nucleosome binding protein HMGN1 (encoded on chr21q22) recapitulates transcriptional changes seen with triplication of a Down syndrome critical region on distal chromosome 21, and HMGN1 is necessary for B cell phenotypes in DS models. Absolute exogenous-normalized chromatin immunoprecipitation sequencing (ChIP-Rx) also reveals a global increase in histone H3K27 acetylation caused by HMGN1. Transcriptional amplification downstream of HMGN1 is enriched for stage-specific programs of B cells and B cell acute lymphoblastic leukemia, dependent on the developmental cellular context. These data offer a mechanistic explanation for DS transcriptional patterns and suggest that further study of HMGN1 and RNA amplification in diverse DS phenotypes is warranted.

Enhancer invasion shapes MYCN-dependent transcriptional amplification in neuroblastoma.

Amplification of the locus encoding the oncogenic transcription factor MYCN is a defining feature of high-risk neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of MYCN perturbation in neuroblastoma. At oncogenic levels, MYCN associates with E-box binding motifs in an affinity-dependent manner, binding to strong canonical E-boxes at promoters and invading abundant weaker non-canonical E-boxes clustered at enhancers. Loss of MYCN leads to a global reduction in transcription, which is most pronounced at MYCN target genes with the greatest enhancer occupancy. These highly occupied MYCN target genes show tissue-specific expression and are linked to poor patient survival. The activity of genes with MYCN-occupied enhancers is dependent on the tissue-specific transcription factor TWIST1, which co-occupies enhancers with MYCN and is required for MYCN-dependent proliferation. These data implicate tissue-specific enhancers in defining often highly tumor-specific 'MYC target gene signatures' and identify disruption of the MYCN enhancer regulatory axis as a promising therapeutic strategy in neuroblastoma.

Mutations in the transcriptional repressor REST predispose to Wilms tumor.

Wilms tumor is the most common childhood renal cancer. To identify mutations that predispose to Wilms tumor, we are conducting exome sequencing studies. Here we describe 11 different inactivating mutations in the REST gene (encoding RE1-silencing transcription factor) in four familial Wilms tumor pedigrees and nine non-familial cases. Notably, no similar mutations were identified in the ICR1000 control series (13/558 versus 0/993; P < 0.0001) or in the ExAC series (13/558 versus 0/61,312; P < 0.0001). We identified a second mutational event in two tumors, suggesting that REST may act as a tumor-suppressor gene in Wilms tumor pathogenesis. REST is a zinc-finger transcription factor that functions in cellular differentiation and embryonic development. Notably, ten of 11 mutations clustered within the portion of REST encoding the DNA-binding domain, and functional analyses showed that these mutations compromise REST transcriptional repression. These data establish REST as a Wilms tumor predisposition gene accounting for ∼2% of Wilms tumor.

Tcf3 promotes cell migration and wound repair through regulation of lipocalin 2.

Cell migration is an integral part of re-epithelialization during skin wound healing, a complex process involving molecular controls that are still largely unknown. Here we identify a novel role for Tcf3, an essential transcription factor regulating embryonic and adult skin stem cell functions, as a key effector of epidermal wound repair. We show that Tcf3 is upregulated in skin wounds and that Tcf3 overexpression accelerates keratinocyte migration and skin wound healing. We also identify Stat3 as an upstream regulator of Tcf3. We show that the promigration effects of Tcf3 are non-cell autonomous and occur independently of its ability to interact with ß-catenin. Finally, we identify lipocalin-2 as the key secreted factor downstream of Tcf3 that promotes cell migration in vitro and wound healing in vivo. Our findings provide new insights into the molecular controls of wound-associated cell migration and identify potential therapeutic targets for the treatment of defective wound repair.

The oncogenic STP axis promotes triple-negative breast cancer via degradation of the REST tumor suppressor.

Defining the molecular networks that drive breast cancer has led to therapeutic interventions and improved patient survival. However, the aggressive triple-negative breast cancer subtype (TNBC) remains recalcitrant to targeted therapies because its molecular etiology is poorly defined. In this study, we used a forward genetic screen to discover an oncogenic network driving human TNBC. SCYL1, TEX14, and PLK1 ("STP axis") cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event in human TNBC. The STP axis induces REST degradation by phosphorylating a conserved REST phospho-degron and bridging REST interaction with the ubiquitin-ligase ßTRCP. Inhibition of the STP axis leads to increased REST protein levels and impairs TNBC transformation, tumor progression, and metastasis. Expression of the STP axis correlates with low REST protein levels in human TNBCs and poor clinical outcome for TNBC patients. Our findings demonstrate that the STP-REST axis is a molecular driver of human TNBC.