Bailin Zhang

Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells

Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice. The role of DNMT1 in human cancer cells, however, remains highly controversial. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase–mediated disruption of this allele results in hemimethylation of ∼20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival.

Towards prediction of in vivo intestinal absorption using a 96-well Caco-2 assay.

We systematically validated a robust 96-well Caco-2 assay via an extended set of 93 marketed drugs with diverse transport mechanisms and quantified by LC/MS/MS, to investigate its predictive utility while dealing with challenging discovery compounds. Utilizing nonlinear fit, the validation led to a good correlation (R(2) = 0.76) between absorptive permeability, log P(app)(A-B), from in vitro Caco-2 assay and reported human fraction of dose absorbed. We observed that paracellular compounds could be flagged by log P(app)(A-B) (<-5.5 cm/s) and physicochemical property space (c log P < 1). Of 8000 Novartis discovery compounds examined 13% were subject to low recovery (<30%). Compound loss was investigated by comparing cell monolayer and artificial membrane, while 0.5% bovine serum albumin (in both donor and acceptor compartments) was utilized to improve recovery. The second focus of this study was to investigate the advantages and limitations of the current Caco-2 assay for predicting in vivo intestinal absorption. Caco-2 measurements for compounds with high aqueous solubility and low in vitro metabolic clearance were compared to 88 in vivo rat bioavailability studies. Despite the challenges posed by discovery compounds with suboptimal physicochemical properties, Caco-2 data successfully projected low intestinal absorption. This platform sets the stage for mechanistically evaluating compounds towards improving in vitro-in vivo correlations.

Evaluation of Fluorescence- and Mass Spectrometry–Based CYP Inhibition Assays for Use in Drug Discovery

The potential for metabolism-related drug-drug interactions by new chemical entities is assessed by monitoring the impact of these compounds on cytochrome P450 (CYP) activity using well-characterized CYP substrates. The conventional gold standard approach for in vitro evaluation of CYP inhibitory potential uses pooled human liver microsomes (HLM) in conjunction with prototypical drug substrates, often quantified by LC-MS/MS. However, fluorescent CYP inhibition assays, which use recombinantly expressed CYPs and fluorogenic probe substrates, have been employed in early drug discovery to provide low-cost, high-throughput assessment of new chemical entities. Despite its greatly enhanced throughput, this approach has been met with mixed success in predicting the data obtained with the conventional gold standard approach (HLM+LC-MS). The authors find that the predictivity of fluorogenic assays for the major CYP isoforms 3A4 and 2D6 may depend on the quality of the test compounds. Although the structurally more optimized marketed drugs yielded acceptable correlations between the fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9 (r 2 = 0.5-0.7; p < 0.005), preoptimization, early discovery compounds yielded poorer correlations (r 2 ≤ 0.2) for 2 of these major isoforms, CYPs 3A4 and 2D6. Potential reasons for the observed differences are discussed. (Journal of Biomolecular Screening 2008;343-353)

Selective blockade of the hydrolysis of the endocannabinoid 2-arachidonoylglycerol impairs learning and memory performance while producing antinociceptive activity in rodents

Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), 2-arachidonoyglycerol (2-AG). This study reports a potent covalent MAGL inhibitor, SAR127303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of 2-AG in mice. In vivo, SAR127303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR127303. Although inactive in acute seizure tests, repeated administration of SAR127303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that 2-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.

PIM inhibitors target CD25-positive AML cells through concomitant suppression of STAT5 activation and degradation of MYC oncogene

Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.

Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.

Evaluation of cancer dependence and druggability of PRP4 kinase using cellular, biochemical, and structural approaches

Background: Little is known about the cancer dependence and druggability of PRP4. Results: Significance of PRP4 catalytic activity is demonstrated, novel substrates are identified, and features of kinase domain structure are revealed. Conclusion: PRP4 is required for cancer cell survival, displays substrate specificity, and is amenable to pharmacological inhibition. Significance: Our results indicate that PRP4 is a potential drug target to pursue in cancer. PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.

Glutaminase is essential for the growth of triple-negative breast cancer cells with a deregulated glutamine metabolism pathway and its suppression synergizes with mTOR inhibition

Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition in vitro and in vivo. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.

Abstract 5468: SAR650984, a humanized anti-CD38 antibody potently modulates intracellular and extracellular nucleotide levels of cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA CD38 is a type II transmembrane glycoprotein with both ectozyme activity and receptor function that has been implicated in cancer cell adhesion, signal transduction and calcium signaling. CD38 is highly expressed at the surface of many hematological cancer cells. SAR650984 is a humanized IgG1 antibody targeting CD38 in early clinical development to treat patients with CD38+ hematological malignancies. Several potential mechanisms of action of SAR650984 have been identified including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct apoptosis induction (ASH 2008 Abstract #2756). In addition, SAR650984 displays potent inhibition of recombinant CD38 ADP-ribosyl cyclase activity in a biochemical assay (AACR 2013, Abstract #4735). To further explore the impact of anti-CD38 antibodies on CD38 ecto-enzymatic activities in cancer cells, we have developed a robust LC-MS-based cellular CD38 enzymatic assay that monitors the depletion of CD38 substrates nicotinamide adenosine dinucleotide (NAD) or nicotinamide mononucleotide (NMN), and the production of cyclic adenosine diphosphoribose (cADPR) and adenosine diphosphoribose (ADPR) from CD38 cyclase and CD38 NAD glycohydrolyase activities, respectively. Preliminary results demonstrate that in response to the treatment of SAR650984, CD38 cyclase activity in multiple myeloma and lymphoma cancer cells was significantly inhibited by more than 90% as shown by a rapid and dose-dependent decrease in the rate of both substrate NAD/NMN depletion as well as extracellular cADPR production. Interestingly, a significant reduction of intracellular cADPR (over 60%) was also observed after SAR650984 treatment. On the other hand, extracellular ADPR level was only moderately affected by SAR650984 treatment with a reduction about 20% which could be hypothetically attributed to the relatively different potency of SAR650984 toward CD38 cyclase and hydrolase activity, or to its potential binding preference to different CD38 oligomers. Emerging data suggest that nucleotides such as NAD and cADPR are important regulators of cell growth, survival and homeostasis. The altered levels of intracellular and extracellular nucleotides resulting from CD38 inhibition by SAR65084 in tumor microenvironment therefore could contribute to the anti-tumor efficacy of the antibody in addition to the previously identified mechanisms. Collectively, these results provide new insights toward understanding the multiple antitumor mechanisms of SAR650984. Additional studies to further understand these observations are currently ongoing. Citation Format: Bailin Zhang, Guang Yang, Shujia Dai, Tim He, Daniel Simard, Zhili Song, Stuart Licht, Francisco Adrian, Hong Cheng, Ti Cai. SAR650984, a humanized anti-CD38 antibody potently modulates intracellular and extracellular nucleotide levels of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5468. doi:10.1158/1538-7445.AM2014-5468

Abstract 4746: Selective inhibition of mutant IDH1 via small molecule binding to the dimer interface

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitric acid to α-ketoglutaric acid (αKG). Point mutations in IDH1 and IDH2 confer a neomorphic enzymatic activity: the reduction of αKG to D-2-hydroxyglutaric acid (2HG), which acts as an oncometabolite by inducing hypermethylation of histones and DNA. IDH1 mutations (predominantly R132H) have been found in 50-80% of grade II gliomas. To find potential IDH1-targeted therapeutics, we conducted a full small-molecule HTS and hit validation campaign using recombinant R132H IDH1. This campaign identified Compound 1, which belongs to a phenol-azole series, as a potent and selective inhibitor of mutant IDH1. We investigated the mode of inhibition of Compound 1 and a published IDH1 mutant inhibitor with a different chemical scaffold (Compound 2). Steady-state kinetics and biophysical studies showed that both inhibitors reversibly interact with both free and substrate-bound enzyme at an allosteric site. A crystal structure of Compound 1 complexed with R132H demonstrates binding of the inhibitor at the dimer interface. A competitive binding study showed that Compound 2 competes with Compound 1, suggesting that Compound 2 also binds at the dimer interface. This is contrary to a previously published statement that Compound 2 is a competitive inhibitor with respect to αKG. In cancer cell lines engineered to produce high levels of 2HG, both inhibitors penetrate cells efficiently and inhibit 2HG production with minimal cell toxicity. This study indicates that the dimer interface pocket is a druggable binding site for IDH1 inhibitors, and suggests that Compound 1 is a promising new starting point for future structure-based drug discovery efforts. Citation Format: Gejing Deng, Stuart Licht, Junqing Shen, Ming Yin, Jessica McManus, Patricia Gee, Tim He, Giang Gao, Bailin Zhang, Magali Mathieu, Alexey Rak, Olivier Bedel, Chaomei Shi, Stefan Gross, Dietmar Hoffmann, Eamonn Rooney, Aurelie Vassort, Walter Englaro, Yi Li, Dmitri Wiederschain, Vinod Patel, Francisco Adrian, Hong Cheng. Selective inhibition of mutant IDH1 via small molecule binding to the dimer interface. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4746. doi:10.1158/1538-7445.AM2014-4746