Biology subjects

Grossman, S. R.

Publications and source records attributed to Grossman, S. R..

6 recordsLinked to original sources

bioRxiv2025

Combined inhibition of NAD synthesis and C-terminal binding protein cooperatively induce cell death and inhibit growth of High Grade Serous Ovarian Carcinoma

The transcriptional scaffolds C-terminal Binding Proteins (CtBP) 1 and 2 are overexpressed and act as oncogenic dependencies in multiple cancers but importantly encode a chemically targetable dehydrogenase domain. CtBP promotes survival of high grade serous ovarian carcinoma (HGSOC) cells by repressing expression of Death Receptors (DR) 4 and 5, which activate caspase 8-dependent apoptosis. We have previously developed a series of substrate competitive CtBP dehydrogenase inhibitors active in multiple cell and preclinical solid tumor models. In the current study, we validated CtBP 1 and 2 overexpression in a longitudinal series of primary and metastatic/recurrent HGSOC cases. Furthermore, our lead CtBP dehydrogenase inhibitor, JW-98 induced apoptosis and exhibited variable single agent IC50 values in HGSOC cell lines, but depletion of nicotinamide adenine dinucleotide (NAD) using the NAD synthesis inhibitor GMX1778 strikingly sensitized tumor cells to JW-98 treatment. Mechanistically, the JW-98/GMX-1778 combination effectively disrupted CtBP dimerization that requires stoichiometric levels of intracellular NAD and is required for oncogenic transcriptional activities. Highlighting the translational potential of this combination, combined JW-98/GMX1778 treatment of OVCAR3 HGSOC xenografts in immunodeficient mice abrogated tumor growth without observable toxicity. CtBP/NAD combined inhibition represents a novel therapeutic strategy that could improve outcomes in chemoresistant HGSOC.

cancer biology↗

bioRxiv2024

Oncogenic Mutant p53 Sensitizes Non-Small Cell Lung Cancer Cells to Proteasome Inhibition via Oxidative Stress-Dependent Induction of Mitochondrial Apoptosis

Non-small cell lung cancer (NSCLC) cells with oncogenic mutant p53 alleles (Onc-p53) exhibit significantly higher levels of proteasome activity, indicating that Onc-p53 induces proteotoxic stress which may be leveraged as a therapeutic vulnerability. Proteasome inhibitors (PIs), such as bortezomib (BTZ), can induce toxic levels of oxidative stress in cancer cells and thus we investigated whether PIs exhibit preferential cytotoxicity in Onc-p53 NSCLC cells. Indeed, BTZ and other PIs exhibited the IC50 6-7-fold lower in Onc-p53 cells vs. wild-type (WT) p53 cells. BTZ cytotoxic effects in Onc-p53 cells were nearly completely rescued by antioxidants such as N-acetyl cysteine, indicating that oxidative stress is the critical driver of BTZ-dependent cytotoxic effects in Onc-p53 cells. Importantly, we observed oxidative stress-dependent transcriptional induction of the pro-apoptotic NOXA with downstream cleaved caspase-3, consistent with apoptotic cell death in Onc-p53 but not in WT p53 cells treated with BTZ, and BTZ-generated oxidative stress was linked to nuclear translocation of NRF2 and transcriptional activation of ATF3, which in turn was required for NOXA induction. Validating BTZs translational potential in Onc-p53 NSCLC, BTZ and carboplatin or the BH3-mimetic navitoclax were synergistically cytotoxic in Onc-p53 but not WT p53 cells in vitro, and BTZ effectively limited growth of Onc-p53 NSCLC xenografts when combined with either carboplatin or navitoclax in vivo. Our data therefore support further investigation of the therapeutic utility of PIs combined with carboplatin or BH3-mimetics in Onc-p53 human NSCLC as novel therapeutic strategies. SignificanceNon-small cell lung cancer (NSCLC) is the leading cause of cancer death due, in part, to a lack of active therapies in advanced disease. We demonstrate that proteasome inhibitor/BH3-mimetic combination therapy is an active precision therapy in NSCLC cells and tumors expressing oncogenic mutant p53 alleles (Onc-p53).

cancer biology↗

bioRxiv2023

Bcl-xL is translocated to the nucleus via CtBP2 to epigenetically promote metastasis

Besides its mitochondria-based anti-apoptotic role, Bcl-xL also travels to the nucleus to promote cancer metastasis by upregulating global histone H3 trimethyl Lys4 (H3K4me3) and TGF{beta} transcription. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates H3K4me3 modification are not understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced cell migration and metastasis in mouse models. Furthermore, knockout of CtBP2 suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGF{beta} mRNA upregulation as well as cell invasion. Moreover, cleavage under targets and release using nuclease (CUT&RUN) coupled with next generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor region of genes encoding TGF{beta} and its signaling pathway in the cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1.

cancer biology↗

bioRxiv2021

Compatibility logic of human enhancer and promoter sequences

Gene regulation in the human genome is controlled by distal enhancers that activate specific nearby promoters. One model for the specificity of enhancer-promoter regulation is that different promoters might have sequence-encoded preferences for distinct classes of enhancers, for example mediated by interacting sets of transcription factors or cofactors. This "biochemical compatibility" model has been supported by observations at individual human promoters and by genome-wide measurements in Drosophila. However, the degree to which human enhancers and promoters are intrinsically compatible or specific has not been systematically measured, and how their activities combine to control RNA expression remains unclear. To address these questions, we designed a high-throughput reporter assay called enhancer x promoter (ExP) STARR-seq and applied it to examine the combinatorial compatibilities of 1,000 enhancer and 1,000 promoter sequences in human K562 cells. We identify a simple logic for enhancer-promoter compatibility - virtually all enhancers activated all promoters by similar amounts, and intrinsic enhancer and promoter activities combine multiplicatively to determine RNA output (R2=0.82). In addition, two classes of enhancers and promoters showed subtle preferential effects. Promoters of housekeeping genes contained built-in activating sequences, corresponding to motifs for factors such as GABPA and YY1, that correlated with both stronger autonomous promoter activity and enhancer activity, and weaker responsiveness to distal enhancers. Promoters of context-specific genes lacked these motifs and showed stronger responsiveness to enhancers. Together, this systematic assessment of enhancer-promoter compatibility suggests a multiplicative model tuned by enhancer and promoter class to control gene transcription in the human genome.

genetics↗

bioRxiv2020

CryoEM Structure of CtBP2 Confirms Tetrameric Architecture

C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis and metastasis. CtBP proteins are activated under hypoxic conditions where NAD(H) levels tend to be higher. NADH-dependent activation of CtBP2 has direct implication in multiple types of cancers and poor patient prognosis. Previous studies have proposed dimeric CtBP as the relevant oligomeric state, however our studies with multi-angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the cryoEM structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is indeed tetrameric. The physiological relevance of tetrameric CtBP2 was tested in HCT116; CtBP2 -/- cells transfected with tetramer destabilizing_mutants. Mutants that inhibit tetramer formation show a decrease in expression of the CtBP transcriptional target TIAM1 and exhibit a decrease in the ability to promote cell migration. Together with our cryoEM studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.

biochemistry↗

bioRxiv2019

Activity-by-Contact model of enhancer specificity from thousands of CRISPR perturbations

Mammalian genomes harbor millions of noncoding elements called enhancers that quantitatively regulate gene expression, but it remains unclear which enhancers regulate which genes. Here we describe an experimental approach, based on CRISPR interference, RNA FISH, and flow cytometry (CRISPRi-FlowFISH), to perturb enhancers in the genome, and apply it to test >3,000 potential regulatory enhancer-gene connections across multiple genomic loci. A simple equation based on a mechanistic model for enhancer function performed remarkably well at predicting the complex patterns of regulatory connections we observe in our CRISPR dataset. This Activity-by-Contact (ABC) model involves multiplying measures of enhancer activity and enhancer-promoter 3D contacts, and can predict enhancer-gene connections in a given cell type based on chromatin state maps. Together, CRISPRi-FlowFISH and the ABC model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.

genetics↗