Novel Spirocyclic Dimer, SpiD3, Targets Chronic Lymphocytic Leukemia Survival Pathways with Potent Preclinical Effects.

Chronic lymphocytic leukemia (CLL) cell survival and growth is fueled by the induction of B-cell receptor (BCR) signaling within the tumor microenvironment (TME) driving activation of NFκB signaling and the unfolded protein response (UPR). Malignant cells have higher basal levels of UPR posing a unique therapeutic window to combat CLL cell growth using pharmacologic agents that induce accumulation of misfolded proteins. Frontline CLL therapeutics that directly target BCR signaling such as Bruton tyrosine kinase (BTK) inhibitors (e.g., ibrutinib) have enhanced patient survival. However, resistance mechanisms wherein tumor cells bypass BTK inhibition through acquired BTK mutations, and/or activation of alternative survival mechanisms have rendered ibrutinib ineffective, imposing the need for novel therapeutics. We evaluated SpiD3, a novel spirocyclic dimer, in CLL cell lines, patient-derived CLL samples, ibrutinib-resistant CLL cells, and in the Eµ-TCL1 mouse model. Our integrated multi-omics and functional analyses revealed BCR signaling, NFκB signaling, and endoplasmic reticulum stress among the top pathways modulated by SpiD3. This was accompanied by marked upregulation of the UPR and inhibition of global protein synthesis in CLL cell lines and patient-derived CLL cells. In ibrutinib-resistant CLL cells, SpiD3 retained its antileukemic effects, mirrored in reduced activation of key proliferative pathways (e.g., PRAS, ERK, MYC). Translationally, we observed reduced tumor burden in SpiD3-treated Eµ-TCL1 mice. Our findings reveal that SpiD3 exploits critical vulnerabilities in CLL cells including NFκB signaling and the UPR, culminating in profound antitumor properties independent of TME stimuli. SIGNIFICANCE: SpiD3 demonstrates cytotoxicity in CLL partially through inhibition of NFκB signaling independent of tumor-supportive stimuli. By inducing the accumulation of unfolded proteins, SpiD3 activates the UPR and hinders protein synthesis in CLL cells. Overall, SpiD3 exploits critical CLL vulnerabilities (i.e., the NFκB pathway and UPR) highlighting its use in drug-resistant CLL.

HiCrayon reveals distinct layers of multi-state 3D chromatin organization.

The co-visualization of chromatin conformation with 1D 'omics data is key to the multi-omics driven data analysis of 3D genome organization. Chromatin contact maps are often shown as 2D heatmaps and visually compared to 1D genomic data by simple juxtaposition. While common, this strategy is imprecise, placing the onus on the reader to align features with each other. To remedy this, we developed HiCrayon, an interactive tool that facilitates the integration of 3D chromatin organization maps and 1D datasets. This visualization method integrates data from genomic assays directly into the chromatin contact map by coloring interactions according to 1D signal. HiCrayon is implemented using R shiny and python to create a graphical user interface (GUI) application, available in both web or containerized format to promote accessibility. HiCrayon is implemented in R, and includes a graphical user interface (GUI), as well as a slimmed-down web-based version that lets users quickly produce publication-ready images. We demonstrate the utility of HiCrayon in visualizing the effectiveness of compartment calling and the relationship between ChIP-seq and various features of chromatin organization. We also demonstrate the improved visualization of other 3D genomic phenomena, such as differences between loops associated with CTCF/cohesin vs. those associated with H3K27ac. We then demonstrate HiCrayon's visualization of organizational changes that occur during differentiation and use HiCrayon to detect compartment patterns that cannot be assigned to either A or B compartments, revealing a distinct 3rd chromatin compartment. Overall, we demonstrate the utility of co-visualizing 2D chromatin conformation with 1D genomic signals within the same matrix to reveal fundamental aspects of genome organization. Local version: https://github.com/JRowleyLab/HiCrayon Web version: https://jrowleylab.com/HiCrayon.

EHD1-dependent traffic of IGF-1 receptor to the cell surface is essential for Ewing sarcoma tumorigenesis and metastasis.

Overexpression of the EPS15 Homology Domain containing 1 (EHD1) protein has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD1 mRNA expression specifying shorter patient survival. ShRNA-knockdown and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting.

Chromatin alternates between A and B compartments at kilobase scale for subgenic organization.

Nuclear compartments are prominent features of 3D chromatin organization, but sequencing depth limitations have impeded investigation at ultra fine-scale. CTCF loops are generally studied at a finer scale, but the impact of looping on proximal interactions remains enigmatic. Here, we critically examine nuclear compartments and CTCF loop-proximal interactions using a combination of in situ Hi-C at unparalleled depth, algorithm development, and biophysical modeling. Producing a large Hi-C map with 33 billion contacts in conjunction with an algorithm for performing principal component analysis on sparse, super massive matrices (POSSUMM), we resolve compartments to 500 bp. Our results demonstrate that essentially all active promoters and distal enhancers localize in the A compartment, even when flanking sequences do not. Furthermore, we find that the TSS and TTS of paused genes are often segregated into separate compartments. We then identify diffuse interactions that radiate from CTCF loop anchors, which correlate with strong enhancer-promoter interactions and proximal transcription. We also find that these diffuse interactions depend on CTCF's RNA binding domains. In this work, we demonstrate features of fine-scale chromatin organization consistent with a revised model in which compartments are more precise than commonly thought while CTCF loops are more protracted.

Considerations and caveats for analyzing chromatin compartments.

Genomes are organized into nuclear compartments, separating active from inactive chromatin. Chromatin compartments are readily visible in a large number of species by experiments that map chromatin conformation genome-wide. When analyzing these maps, a common step is the identification of genomic intervals that interact within A (active) and B (inactive) compartments. It has also become increasingly common to identify and analyze subcompartments. We review different strategies to identify A/B and subcompartment intervals, including a discussion of various machine-learning approaches to predict these features. We then discuss the strengths and limitations of current strategies and examine how these aspects of analysis may have impacted our understanding of chromatin compartments.

EHD1-dependent traffic of IGF-1 receptor to the cell surface is essential for Ewing sarcoma tumorigenesis and metastasis.

Overexpression of EPS15 Homology Domain containing 1 (EHD1) has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD mRNA expression specifying shorter patient survival. ShRNA and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified the IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting.

Understanding the role of Pax5 in development of taxane-resistant neuroendocrine like prostate cancers.

Resistance to the current Androgen Receptor Signaling Inhibitor (ARSI) therapies has led to higher incidences of therapy-induced neuroendocrine-like prostate cancer (t-NEPC). This highly aggressive subtype with predominant small cell-like characteristics is resistant to taxane chemotherapies and has a dismal overall survival. t-NEPCs are mostly treated with platinum-based drugs with a combination of etoposide or taxane and have less selectivity and high systemic toxicity, which often limit their clinical potential. During t-NEPC transformation, adenocarcinomas lose their luminal features and adopt neuro-basal characteristics. Whether the adaptive neuronal characteristics of t-NEPC are responsible for such taxane resistance remains unknown. Pathway analysis from patient gene-expression databases indicates that t-NEPC upregulates various neuronal pathways associated with enhanced cellular networks. To identify transcription factor(s) (TF) that could be important for promoting the gene expression for neuronal characters in t-NEPC, we performed ATAC-Seq, acetylated-histone ChIP-seq, and RNA-seq in our NE-like cell line models and analyzed the promoters of transcriptionally active and significantly enriched neuroendocrine-like (NE-like) cancer-specific genes. Our results indicate that Pax5 could be an important transcription factor for neuronal gene expression and specific to t-NEPC. Pathway analysis revealed that Pax5 expression is involved in axonal guidance, neurotransmitter regulation, and neuronal adhesion, which are critical for strong cellular communications. Further results suggest that depletion of Pax5 disrupts cellular interaction in NE-like cells and reduces surface growth factor receptor activation, thereby, sensitizing them to taxane therapies. Moreover, t-NEPC specific hydroxymethylation of Pax5 promoter CpG islands favors Pbx1 binding to induce Pax5 expression. Based on our study, we concluded that continuous exposure to ARSI therapies leads to epigenetic modifications and Pax5 activation in t-NEPC, which promotes the expression of genes necessary to adopt taxane-resistant NE-like cancer. Thus, targeting the Pax5 axis can be beneficial for reverting their taxane sensitivity.

Sex-specific multi-level 3D genome dynamics in the mouse brain.

The female mammalian brain exhibits sex hormone-driven plasticity during the reproductive period. Recent evidence implicates chromatin dynamics in gene regulation underlying this plasticity. However, whether ovarian hormones impact higher-order chromatin organization in post-mitotic neurons in vivo is unknown. Here, we mapped the 3D genome of ventral hippocampal neurons across the oestrous cycle and by sex in mice. In females, we find cycle-driven dynamism in 3D chromatin organization, including in oestrogen response elements-enriched X chromosome compartments, autosomal CTCF loops, and enhancer-promoter interactions. With rising oestrogen levels, the female 3D genome becomes more similar to the male 3D genome. Cyclical enhancer-promoter interactions are partially associated with gene expression and enriched for brain disorder-relevant genes and pathways. Our study reveals unique 3D genome dynamics in the female brain relevant to female-specific gene regulation, neuroplasticity, and disease risk.

Elevating SOX2 Downregulates MYC through a SOX2:MYC Signaling Axis and Induces a Slowly Cycling Proliferative State in Human Tumor Cells.

Slowly cycling/infrequently proliferating tumor cells present a clinical challenge due to their ability to evade treatment. Previous studies established that high levels of SOX2 in both fetal and tumor cells restrict cell proliferation and induce a slowly cycling state. However, the mechanisms through which elevated SOX2 levels inhibit tumor cell proliferation have not been identified. To identify common mechanisms through which SOX2 elevation restricts tumor cell proliferation, we initially performed RNA-seq using two diverse tumor cell types. SOX2 elevation in both cell types downregulated MYC target genes. Consistent with these findings, elevating SOX2 in five cell lines representing three different human cancer types decreased MYC expression. Importantly, the expression of a dominant-negative MYC variant, omomyc, recapitulated many of the effects of SOX2 on proliferation, cell cycle, gene expression, and biosynthetic activity. We also demonstrated that rescuing MYC activity in the context of elevated SOX2 induces cell death, indicating that the downregulation of MYC is a critical mechanistic step necessary to maintain survival in the slowly cycling state induced by elevated SOX2. Altogether, our findings uncover a novel SOX2:MYC signaling axis and provide important insights into the molecular mechanisms through which SOX2 elevation induces a slowly cycling proliferative state.

Ecdysoneless Protein Regulates Viral and Cellular mRNA Splicing to Promote Cervical Oncogenesis.

High-risk human papillomaviruses (HPV), exemplified by HPV16/18, are causally linked to human cancers of the anogenital tract, skin, and upper aerodigestive tract. Previously, we identified Ecdysoneless (ECD) protein, the human homolog of the Drosophila ecdysoneless gene, as a novel HPV16 E6-interacting protein. Here, we show that ECD, through its C-terminal region, selectively binds to high-risk but not to low-risk HPV E6 proteins. We demonstrate that ECD is overexpressed in cervical and head and neck squamous cell carcinoma (HNSCC) cell lines as well as in tumor tissues. Using The Cancer Genome Atlas dataset, we show that ECD mRNA overexpression predicts shorter survival in patients with cervical and HNSCC. We demonstrate that ECD knockdown in cervical cancer cell lines led to impaired oncogenic behavior, and ECD co-overexpression with E7 immortalized primary human keratinocytes. RNA-sequencing analyses of SiHa cells upon ECD knockdown showed to aberrations in E6/E7 RNA splicing, as well as RNA splicing of several HPV oncogenesis-linked cellular genes, including splicing of components of mRNA splicing machinery itself. Taken together, our results support a novel role of ECD in viral and cellular mRNA splicing to support HPV-driven oncogenesis. IMPLICATIONS: This study links ECD overexpression to poor prognosis and shorter survival in HNSCC and cervical cancers and identifies a critical role of ECD in cervical oncogenesis through regulation of viral and cellular mRNA splicing.