Diverse, Potent, and Efficacious Inhibitors That Target the EED Subunit of the Polycomb Repressive Complex 2 Methyltransferase.

Aberrant activity of the histone methyltransferase polycomb repressive complex 2 (PRC2) has been linked to several cancers, with small-molecule inhibitors of the catalytic subunit of the PRC2 enhancer of zeste homologue 2 (EZH2) being recently approved for the treatment of epithelioid sarcoma (ES) and follicular lymphoma (FL). Compounds binding to the EED subunit of PRC2 have recently emerged as allosteric inhibitors of PRC2 methyltransferase activity. In contrast to orthosteric inhibitors that target EZH2, small molecules that bind to EED retain their efficacy in EZH2 inhibitor-resistant cell lines. In this paper we disclose the discovery of potent and orally bioavailable EED ligands with good solubilities. The solubility of the EED ligands was optimized through a variety of design tactics, with the resulting compounds exhibiting in vivo efficacy in EZH2-driven tumors.

Free energy perturbation in the design of EED ligands as inhibitors of polycomb repressive complex 2 (PRC2) methyltransferase.

Free Energy Perturbation (FEP) calculations can provide high-confidence predictions of the interaction strength between a ligand and its protein target. We sought to explore a series of triazolopyrimidines which bind to the EED subunit of the PRC2 complex as potential anticancer therapeutics, using FEP calculations to inform compound design. Combining FEP predictions with a late-stage functionalisation (LSF) inspired synthetic approach allowed us to rapidly evaluate structural modifications in a previously unexplored region of the EED binding site. This approach generated a series of novel triazolopyrimidine EED ligands with improved physicochemical properties and which inhibit PRC2 methyltransferase activity in a cancer-relevant G401 cell line.

EED-Targeted PROTACs Degrade EED, EZH2, and SUZ12 in the PRC2 Complex.

Deregulation of the PRC2 complex, comprised of the core subunits EZH2, SUZ12, and EED, drives aberrant hypermethylation of H3K27 and tumorigenicity of many cancers. Although inhibitors of EZH2 have shown promising clinical activity, preclinical data suggest that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (proteolysis-targeting chimera) to efficiently target EED for elimination. Our PROTACs bind to EED (pKD ∼ 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50 ∼ 8.1) and induce rapid degradation of not only EED but also EZH2 and SUZ12 within the PRC2 complex. Furthermore, the PROTACs selectively inhibit proliferation of PRC2-dependent cancer cells (half maximal growth inhibition [GI50] = 49-58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.

Discovery of a Series of 5-Azaquinazolines as Orally Efficacious IRAK4 Inhibitors Targeting MyD88L265P Mutant Diffuse Large B Cell Lymphoma.

In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline 4, we introduced a 5-aza substitution to mask the 4-NH hydrogen bond donor (HBD). This allowed us to substitute the core with a 2-aminopyrazole, which showed large gains in cellular potency despite the additional formal HBD. Further optimization led to 6-cyanomethyl-5-azaquinazoline 13, a selective IRAK4 inhibitor, which proved efficacious in combination with ibrutinib, while showing very little activity as a single agent up to 100 mg/kg. This contrasted to previously reported IRAK4 inhibitors that exhibited efficacy in the same model as single agents and was attributed to the enhanced specificity of 13 toward IRAK4.

An Unbiased Oncology Compound Screen to Identify Novel Combination Strategies.

Combination drug therapy is a widely used paradigm for managing numerous human malignancies. In cancer treatment, additive and/or synergistic drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. This can be explained in part through pathway interdependencies that are critical for cancer cell proliferation and survival. However, identification of the various interdependencies is difficult due to the complex molecular circuitry that underlies tumor development and progression. Here, we present a high-throughput platform that allows for an unbiased identification of synergistic and efficacious drug combinations. In a screen of 22,737 experiments of 583 doublet combinations in 39 diverse cancer cell lines using a 4 by 4 dosing regimen, both well-known and novel synergistic and efficacious combinations were identified. Here, we present an example of one such novel combination, a Wee1 inhibitor (AZD1775) and an mTOR inhibitor (ridaforolimus), and demonstrate that the combination potently and synergistically inhibits cancer cell growth in vitro and in vivo This approach has identified novel combinations that would be difficult to reliably predict based purely on our current understanding of cancer cell biology. Mol Cancer Ther; 15(6); 1155-62. ©2016 AACR.

Unique functions of CHK1 and WEE1 underlie synergistic anti-tumor activity upon pharmacologic inhibition.

BACKGROUND: Inhibition of kinases involved in the DNA damage response sensitizes cells to genotoxic agents by abrogating checkpoint-induced cell cycle arrest. CHK1 and WEE1 act in a pathway upstream of CDK1 to inhibit cell cycle progression in response to damaged DNA. Therapeutic targeting of either CHK1 or WEE1, in combination with chemotherapy, is under clinical evaluation. These studies examine the overlap and potential for synergy when CHK1 and WEE1 are inhibited in cancer cell models. METHODS: Small molecules MK-8776 and MK-1775 were used to selectively and potently inhibit CHK1 and WEE1, respectively. RESULTS: In vitro, the combination of MK-8776 and MK-1775 induces up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone at a fixed concentration. This requires aberrant cyclin-dependent kinase activity but does not appear to be dependent on p53 status alone. Furthermore, DNA damage takes place primarily in S-phase cells, implying disrupted DNA replication. When dosed together, the combination of MK-8776 and MK-1775 induced more intense and more durable DNA damage as well as anti-tumor efficacy than either MK-8776 or MK-1775 dosed alone. DNA damage induced by the combination was detected in up to 40% of cells in a treated xenograft tumor model. CONCLUSIONS: These results highlight the roles of WEE1 and CHK1 in maintaining genomic integrity. Importantly, the strong synergy observed upon inhibition of both kinases suggests unique yet complimentary anti-tumor effects of WEE1 and CHK1 inhibition. This demonstration of DNA double strand breaks in the absence of a DNA damaging chemotherapeutic provides preclinical rationale for combining WEE1 and CHK1 inhibitors as a cancer treatment regimen.

N-acetylation and ubiquitin-independent proteasomal degradation of p21(Cip1).

Expression of the CDK inhibitor p21(Cip1) is tightly regulated by signals that control cell division. p21 is an unstable protein that is degraded by the proteasome; however, the pathway that leads to proteasomal degradation of p21 has proven to be enigmatic. An important issue is whether proteasomal degradation of p21 occurs independently of ubiquitylation or, alternatively, whether ubiquitylation on its N terminus is crucial. We resolve this uncertainty by showing that endogenous cellular p21 is completely acetylated at its amino terminus and is therefore not a substrate for N-ubiquitylation. We further show that inactivation of essential components of the ubiquitylation machinery does not directly impact endogenous p21 degradation. Our results underscore the importance of N-acetylation in restricting N-ubiquitylation and show, in particular, that ubiquitylation of endogenous p21 either at internal lysines or on the N terminus is unlikely to control its degradation by the proteasome.

Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation.

Autophosphorylation-triggered ubiquitination has been proposed to be the major pathway regulating cyclin E protein abundance: phosphorylation of cyclin E on T380 by its associated CDK allows binding to the receptor subunit, Fbw7, of the SCFFbw7 ubiquitin ligase. We have tested this model in vivo and found it to be an inadequate representation of the pathways that regulate cyclin E degradation. We show that assembly of cyclin E into cyclin E-Cdk2 complexes is required in vivo for turnover by the Fbw7 pathway; that Cdk2 activity is required for cyclin E turnover in vivo because it phosphorylates S384; that phosphorylation of T380 in vivo does not require Cdk2 and is mediated primarily by GSK3; and that two additional phosphorylation sites, T62 and S372, are also required for turnover. Thus, cyclin E turnover is controlled by multiple biological inputs and cannot be understood in terms of autophosphorylation alone.

A Bni4-Glc7 phosphatase complex that recruits chitin synthase to the site of bud emergence.

Bni4 is a scaffold protein in the yeast Saccharomyces cerevisiae that tethers chitin synthase III to the bud neck by interacting with septin neck filaments and with Chs4, a regulatory subunit of chitin synthase III. We show herein that Bni4 is also a limiting determinant for the targeting of the type 1 serine/threonine phosphatase (Glc7) to the bud neck. Yeast cells containing a Bni4 variant that fails to associate with Glc7 fail to tether Chs4 to the neck, due in part to the failure of Bni4(V831A/F833A) to localize properly. Conversely, the Glc7-129 mutant protein fails to bind Bni4 properly and glc7-129 mutants exhibit reduced levels of Bni4 at the bud neck. Bni4 is phosphorylated in a cell cycle-dependent manner and Bni4(V831A/F833A) is both hyperphosphorylated and mislocalized in vivo. Yeast cells lacking the protein kinase Hsl1 exhibit increased levels of Bni4-GFP at the bud neck. GFP-Chs4 does not accumulate at the incipient bud site in either a bni4::TRP1 or a bni4(V831A/F833A) mutant but does mobilize to the neck at cytokinesis. Together, these results indicate that the formation of the Bni4-Glc7 complex is required for localization to the site of bud emergence and for subsequent targeting of chitin synthase.

Essential functions of Sds22p in chromosome stability and nuclear localization of PP1.

Sds22p is a conserved, leucine-rich repeat protein that interacts with the catalytic subunit of protein phosphatase 1 (PP1(C)) and which has been proposed to regulate one or more functions of PP1(C) during mitosis. Here we show that Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is present as a sTable 1:1 complex with yeast PP1(C) (Glc7p). Temperature-sensitive (Ts(-)) S. cerevisiae sds22 mutants show profound chromosome instability at elevated growth temperatures but do not confer a cell cycle stage-specific arrest. In the sds22-6 Ts(-) mutant, nuclear Glc7p is both reduced in level and aberrantly localized at 37 degrees C and the interaction between Glc7p and Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo Ts(-) growth defect. Like some glc7 mutations, sds22-6 can suppress the Ts(-) growth defect associated with ipl1-2, a loss of function mutation in a protein kinase that is known to work in opposition to PP1 on at least two nuclear substrates. This, together with reciprocal genetic interactions between GLC7 and SDS22, suggests that Sds22p functions positively with Glc7p to promote dephosphorylation of nuclear substrates required for faithful transmission of chromosomes during mitosis, and this role is at least partly mediated by effects of Sds22p on the nuclear distribution of Glc7p