Marie Classon

An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method

Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.

Lead optimization of a pyrazolo[1,5-a]pyrimidin-7(4H)-one scaffold to identify potent, selective and orally bioavailable KDM5 inhibitors suitable for in vivo biological studies.

Starting with a lead [1,5-a]pyrimidin-7(4H)-one-containing molecule (1), we generated potent, selective and orally bioavailable KDM5 inhibitors. Using structure- and property-based approaches, we designed 48 with improved cell potency (PC9 H3K4Me3 EC50=0.34μM). Furthermore, 48 maintained suitable physiochemical properties and displayed an excellent pharmacokinetic (PK) profile in mice. When dosed orally in mice at 50mg/kg twice a day (BID), 48 showed an unbound maximal plasma concentration (Cmax) >15-fold over its cell EC50, thereby providing a robust chemical probe for studying KDM5 biological functions in vivo.

Proline Starvation Induces Unresolved ER Stress and Hinders mTORC1-Dependent Tumorigenesis

The role of essential amino acids in metabolic reprogramming of cancer cells is now well established, whereas the role of non-essential amino acids (NEAAs) in malignancy remains less clear. Here, we have identified an important role for the NEAA proline in the tumorigenic potential of a subset of cancer cells. By profiling a large panel of cancer cell lines, we observed that proline consumption and expression of proline biosynthesis enzymes were well correlated with clonogenic and tumorigenic potential. Moreover, proline starvation or inhibition of proline biosynthesis enzymes impaired clonogenic/tumorigenic potential. Cancer cells exhibiting dependency on exogenous proline displayed hyperactivation of the mTORC1-mediated 4EBP1 signaling axis, as well as unresolved ER stress. Exogenous proline alleviated ER stress and promoted cellular homeostasis and clonogenicity. Increased dependence on proline may therefore define a specific vulnerability in some cancers that can be exploited by proline depletion.

Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug Exposure

Maintenance of phenotypic heterogeneity within cell populations is an evolutionarily conserved mechanism that underlies population survival upon stressful exposures. We show that the genomes of a cancer cell subpopulation that survives treatment with otherwise lethal drugs, the drug-tolerant persisters (DTPs), exhibit a repressed chromatin state characterized by increased methylation of histone H3 lysines 9 and 27 (H3K9 and H3K27). We also show that survival of DTPs is, in part, maintained by regulators of H3K9me3-mediated heterochromatin formation and that the observed increase in H3K9me3 in DTPs is most prominent over long interspersed repeat element 1 (LINE-1). Disruption of the repressive chromatin over LINE-1 elements in DTPs results in DTP ablation, which is partially rescued by reducing LINE-1 expression or function.

An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition

Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.

Silencing of retrotransposons by SETDB1 inhibits the interferon response in acute myeloid leukemia

A propensity for rewiring genetic and epigenetic regulatory networks, thus enabling sustained cell proliferation, suppression of apoptosis, and the ability to evade the immune system, is vital to cancer cell propagation. An increased understanding of how this is achieved is critical for identifying or improving therapeutic interventions. In this study, using acute myeloid leukemia (AML) human cell lines and a custom CRISPR/Cas9 screening platform, we identify the H3K9 methyltransferase SETDB1 as a novel, negative regulator of innate immunity. SETDB1 is overexpressed in many cancers, and loss of this gene in AML cells triggers desilencing of retrotransposable elements that leads to the production of double-stranded RNAs (dsRNAs). This is coincident with induction of a type I interferon response and apoptosis through the dsRNA-sensing pathway. Collectively, our findings establish a unique gene regulatory axis that cancer cells can exploit to circumvent the immune system.

From a novel HTS hit to potent, selective, and orally bioavailable KDM5 inhibitors.

A high-throughput screening (HTS) of the Genentech/Roche library identified a novel, uncharged scaffold as a KDM5A inhibitor. Lacking insight into the binding mode, initial attempts to improve inhibitor potency failed to improve potency, and synthesis of analogs was further hampered by the presence of a C-C bond between the pyrrolidine and pyridine. Replacing this with a C-N bond significantly simplified synthesis, yielding pyrazole analog 35, of which we obtained a co-crystal structure with KDM5A. Using structure-based design approach, we identified 50 with improved biochemical, cell potency and reduced MW and lower lipophilicity (LogD) compared with the original hit. Furthermore, 50 showed lower clearance than 9 in mice. In combination with its remarkably low plasma protein binding (PPB) in mice (40%), oral dosing of 50 at 5mg/kg resulted in unbound Cmax ∼2-fold of its cell potency (PC9 H3K4Me3 0.96μM), meeting our criteria for an in vivo tool compound from a new scaffold.

SUV420H2 is an epigenetic regulator of epithelial/mesenchymal states in pancreatic cancer

Epithelial-to-mesenchymal transition is implicated in metastasis, where carcinoma cells lose sessile epithelial traits and acquire mesenchymal migratory potential. The mesenchymal state is also associated with cancer stem cells and resistance to chemotherapy. It might therefore be therapeutically beneficial to promote epithelial identity in cancer. Because large-scale cell identity shifts are often orchestrated on an epigenetic level, we screened for candidate epigenetic factors and identified the histone methyltransferase SUV420H2 (KMT5C) as favoring the mesenchymal identity in pancreatic cancer cell lines. Through its repressive mark H4K20me3, SUV420H2 silences several key drivers of the epithelial state. Its knockdown elicited mesenchymal-to-epithelial transition on a molecular and functional level, and cells displayed decreased stemness and increased drug sensitivity. An analysis of human pancreatic cancer biopsies was concordant with these findings, because high levels of SUV420H2 correlated with a loss of epithelial characteristics in progressively invasive cancer. Together, these data indicate that SUV420H2 is an upstream epigenetic regulator of epithelial/mesenchymal state control.