Cell-to-Cell Variation in p53 Dynamics Leads to Fractional Killing.

Many chemotherapeutic drugs kill only a fraction of cancer cells, limiting their efficacy. We used live-cell imaging to investigate the role of p53 dynamics in fractional killing of colon cancer cells in response to chemotherapy. We found that both surviving and dying cells reach similar levels of p53, indicating that cell death is not determined by a fixed p53 threshold. Instead, a cell's probability of death depends on the time and levels of p53. Cells must reach a threshold level of p53 to execute apoptosis, and this threshold increases with time. The increase in p53 apoptotic threshold is due to drug-dependent induction of anti-apoptotic genes, predominantly in the inhibitors of apoptosis (IAP) family. Our study underlines the importance of measuring the dynamics of key players in response to chemotherapy to determine mechanisms of resistance and optimize the timing of combination therapy.

Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

Mammalian SWI/SNF complex activity regulates POU2F3 and constitutes a targetable dependency in small cell lung cancer.

Small cell lung cancers (SCLC) are comprised of heterogeneous subtypes marked by lineage-specific transcription factors, including ASCL1, NEUROD1, and POU2F3. POU2F3-positive SCLC, ∼12% of all cases, are uniquely dependent on POU2F3 itself; as such, approaches to attenuate POU2F3 expression may represent new therapeutic opportunities. Here using genome-scale screens for regulators of POU2F3 expression and SCLC proliferation, we define mSWI/SNF complexes, including non-canonical BAF (ncBAF) complexes, as top dependencies specific to POU2F3-positive SCLC. Notably, clinical-grade pharmacologic mSWI/SNF inhibition attenuates proliferation of all POU2F3-positive SCLCs, while disruption of ncBAF via BRD9 degradation is uniquely effective in pure non-neuroendocrine POU2F3-SCLCs. mSWI/SNF maintains accessibility over gene loci central to POU2F3-mediated gene regulatory networks. Finally, chemical targeting of SMARCA4/2 mSWI/SNF ATPases and BRD9 decrease POU2F3-SCLC tumor growth and increase survival in vivo . Taken together, these results characterize mSWI/SNF-mediated global governance of the POU2F3 oncogenic program and suggest mSWI/SNF inhibition as a therapeutic strategy for SCLC.

Mammalian SWI/SNF complex activity regulates POU2F3 and constitutes a targetable dependency in small cell lung cancer.

Small cell lung cancers (SCLCs) are composed of heterogeneous subtypes marked by lineage-specific transcription factors, including ASCL1, NEUROD1, and POU2F3. POU2F3-positive SCLCs, ∼12% of all cases, are uniquely dependent on POU2F3 itself; as such, approaches to attenuate POU2F3 expression may represent new therapeutic opportunities. Here using genome-scale screens for regulators of POU2F3 expression and SCLC proliferation, we define mSWI/SNF complexes as top dependencies specific to POU2F3-positive SCLC. Notably, chemical disruption of mSWI/SNF ATPase activity attenuates proliferation of all POU2F3-positive SCLCs, while disruption of non-canonical BAF (ncBAF) via BRD9 degradation is effective in pure non-neuroendocrine POU2F3-SCLCs. mSWI/SNF targets to and maintains accessibility over gene loci central to POU2F3-mediated gene regulatory networks. Finally, clinical-grade pharmacologic disruption of SMARCA4/2 ATPases and BRD9 decreases POU2F3-SCLC tumor growth and increases survival in vivo. These results demonstrate mSWI/SNF-mediated governance of the POU2F3 oncogenic program and suggest mSWI/SNF inhibition as a therapeutic strategy for POU2F3-positive SCLCs.

A strategy to assess the cellular activity of E3 ligase components against neo-substrates using electrophilic probes.

While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.

p53 dynamics vary between tissues and are linked with radiation sensitivity.

Radiation sensitivity varies greatly between tissues. The transcription factor p53 mediates the response to radiation; however, the abundance of p53 protein does not correlate well with the extent of radiosensitivity across tissues. Given recent studies showing that the temporal dynamics of p53 influence the fate of cultured cells in response to irradiation, we set out to determine the dynamic behavior of p53 and its impact on radiation sensitivity in vivo. We find that radiosensitive tissues show prolonged p53 signaling after radiation, while more resistant tissues show transient p53 activation. Sustaining p53 using a small molecule (NMI801) that inhibits Mdm2, a negative regulator of p53, reduced viability in cell culture and suppressed tumor growth. Our work proposes a mechanism for the control of radiation sensitivity and suggests tools to alter the dynamics of p53 to enhance tumor clearance. Similar approaches can be used to enhance killing of cancer cells or reduce toxicity in normal tissues following genotoxic therapies.

Chemoproteomics Enabled Discovery of Selective Probes for NuA4 Factor BRD8.

Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside a homology-model-guided design, to develop chemical biology approaches for the study of BRD8 inhibition and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.

CYP27A1-dependent anti-melanoma activity of limonoid natural products targets mitochondrial metabolism.

Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.

p53 pulses lead to distinct patterns of gene expression albeit similar DNA-binding dynamics.

The dynamics of transcription factors play important roles in a variety of biological systems. However, the mechanisms by which these dynamics are decoded into different transcriptional responses are not well understood. Here we focus on the dynamics of the tumor-suppressor protein p53, which exhibits a series of pulses in response to DNA damage. We performed time course RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) measurements to determine how p53 oscillations are linked with gene expression genome wide. We discovered multiple distinct patterns of gene expression in response to p53 pulses. Surprisingly, p53-binding dynamics were uniform across all genomic loci, even for genes that exhibited distinct mRNA dynamics. Using a mathematical model, supported by additional experimental measurements in response to sustained p53 input, we determined that p53 binds to and activates transcription of its target genes uniformly, whereas post-transcriptional mechanisms are responsible for the differences in gene expression dynamics.

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C.

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.

Pushing the envelope: chromatin boundaries at the nuclear pore.

In a novel genetic screen, the nuclear-cytoplasmic transport system was found to reposition DNA to the nuclear pore and establish a barrier to the spread of heterochromatin. These data provide a mechanism for movement and attachment of DNA to a functional nuclear compartment.

A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, and Drosophila. The discovery that synthetic double-stranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells.