Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma.

CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC50 = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC50 values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.

EZH2 Inhibition by Tazemetostat Results in Altered Dependency on B-cell Activation Signaling in DLBCL.

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2 Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586-97. ©2017 AACR.

Selective Killing of SMARCA2- and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: In Vitro and In Vivo Preclinical Models.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850-60. ©2017 AACR.

Structural Insights into Ternary Complex Formation of Human CARM1 with Various Substrates.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine N-methyltransferase (PRMT) enzyme that has been implicated in a variety of cancers. CARM1 is known to methylate histone H3 and nonhistone substrates. To date, several crystal structures of CARM1 have been solved, including structures with small molecule inhibitors, but no ternary structures with nucleoside and peptide substrates have been reported. Here, the crystal structures of human CARM1 with the S-adenosylmethione (SAM) mimic sinefungin and three different peptide sequences from histone H3 and PABP1 are presented, with both nonmethylated and singly methylated arginine residues exemplified. This is the first example of multiple substrate sequences solved in a single PRMT enzyme and demonstrates how the CARM1 binding site is capable of accommodating a variety of peptide sequences while maintaining a core binding mode for the unmethylated and monomethylated substrates. Comparison of these with other PRMT enzyme-peptide structures shows hydrogen bonding patterns that may be thematic of these binding sites.

Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.

The discovery and optimization of a novel class of potent, selective, and orally bioavailable anaplastic lymphoma kinase (ALK) inhibitors with potential utility for the treatment of cancer.

A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure-activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.

Relationship between chest lateral width, tube current, image noise, and radiation exposure associated with coronary artery calcium scanning on 320-detector row CT.

BACKGROUND: The relationship between chest lateral width, tube current, image noise, and radiation exposure on 320-detector row CT has not been reported. OBJECTIVE: We investigated the relationships between chest lateral width, estimated radiation exposure (DLPe), and image noise in 300 patients undergoing clinical coronary calcium scanning. METHODS: Patients undergoing coronary calcium scanning with 320-detector row CT (prospective, volumetric mode, 120 kV of tube voltage, 100-550 mA of tube current, 0.5-mm detector width) were grouped by chest lateral width (small, medium, and large) from anteroposterior topograms and 100 consecutive patients were selected from each group (n = 300). Tube current, DLPe, and noise were compared among groups with Kruskal-Wallis or one-way ANOVA. Phantom experiments were performed to evaluate the accuracy of calcium quantification as a function of size and tube current. RESULTS: Median tube current in small, medium, and large patients was 130, 200, and 250 mA, respectively (P < 0.0001). Despite the use of higher tube current settings, noise levels also increased with size (20.2 ± 4.5 HU, 22.0 ± 3.9 HU, and 25.1 ± 4.9 HU, respectively; global P < 0.001). DLPe was significantly higher with increasing size (54, 83, and 104 mGy · cm, respectively; P < 0.0001). Phantom experiments showed that 50-100 mA, 150-200 mA, and approximately 300 mA in small, medium, and large phantoms were associated with stable estimate of calcium. CONCLUSIONS: Increasing chest lateral width is associated with increasing radiation exposure and image noise. The use of 50-100 mA in small and 150-200 mA in medium patients is associated with acceptable noise and stable estimate of coronary artery calcium. In large patients, precise identification of individual calcified lesions remains difficult despite increasing tube current and radiation exposure.

Comparison of 2 cell-based phosphoprotein assays to support screening and development of an ALK inhibitor.

Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY(1604) ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen(®)) and phosflow platforms were used to detect modulation of pY(1604) ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY(1604) ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen(®) assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen(®) and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY(1604) ALK.

Nicotinic receptor-mediated regulation of the dopamine transporter in rat prefrontocortical slices following chronic in vivo administration of nicotine.

Low levels of dopaminergic activity in prefrontal cortex are thought to contribute to negative symptoms of schizophrenia. Negative symptoms are associated with the prefrontocortical area of the brain. Schizophrenic patients have a high rate of smoking, which by subjective as well as objective measures produces a cognitive benefit. We have previously shown that agonists at nicotinic receptors containing alpha4 and beta2 subunits can enhance amphetamine-stimulated [3H]dopamine ([3H]DA) release via the dopamine transporter (DAT) from slices of rat prefrontal cortex. This effect is selective for prefrontal cortex; the enhancement does not occur in striatum or nucleus accumbens. The enhancement is dependent upon activation of protein kinase C (PKC). In the current study, we show that the enhancement of amphetamine-stimulated [3H]DA release is maintained after 10 days of chronic nicotine treatment, delivered subcutaneously twice daily. There are no significant changes in the ability of prefrontocortical brain slices to take up [3H]DA in tissue prepared from nicotine-treated vs. saline-treated rats. Nicotinic receptors mediating enhancement of amphetamine-stimulated [3H]DA release are at least partially localized to nerve terminals, as an enhancement in release is also observed in synaptosomal preparations. Finally, the sensitivity of the nicotine enhancement in release to the PKC inhibitor chelerythrine is also seen in synaptosomal preparations, suggesting that the signaling mechanism activated through alpha4beta2 receptors is intact.