Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive. METHODS: To assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients. RESULTS: In the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis. CONCLUSIONS: BMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.

Temporal Analysis of Brd4 Displacement in the Control of B Cell Survival, Proliferation, and Differentiation.

JQ1 is a BET-bromodomain inhibitor that has immunomodulatory effects. However, the precise molecular mechanism that JQ1 targets to elicit changes in antibody production is not understood. Our results show that JQ1 induces apoptosis, reduces cell proliferation, and as a consequence, inhibits antibody-secreting cell differentiation. ChIP-sequencing reveals a selective displacement of Brd4 in response to acute JQ1 treatment (<2 h), resulting in specific transcriptional repression. After 8 h, subsequent alterations in gene expression arise as a result of the global loss of Brd4 occupancy. We demonstrate that apoptosis induced by JQ1 is solely attributed to the pro-apoptotic protein Bim (Bcl2l11). Conversely, cell-cycle regulation by JQ1 is associated with multiple Myc-associated gene targets. Our results demonstrate that JQ1 drives temporal changes in Brd4 displacement that results in a specific transcriptional profile that directly affects B cell survival and proliferation to modulate the humoral immune response.

Discovery of Acylsulfonohydrazide-Derived Inhibitors of the Lysine Acetyltransferase, KAT6A, as Potent Senescence-Inducing Anti-Cancer Agents.

A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (1), a unique aryl acylsulfonohydrazide with an IC50 of 1.0 µM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as 55 and 80. These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 (3) as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly potent KAT6A inhibitor (IC50 = 6.3 nM; KD = 0.002 µM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.

HBO1 is required for the maintenance of leukaemia stem cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

Fragment screening for a protein-protein interaction inhibitor to WDR5.

The WD40-repeat protein WDR5 scaffolds various epigenetic writers and is a critical component of the mammalian SET/MLL histone methyltransferase complex. Dysregulation of the MLL1 catalytic function is associated with mixed-lineage leukemia, and antagonism of the WDR5-MLL1 interaction by small molecules has been proposed as a therapeutic strategy for MLL-rearranged cancers. Small molecule binders of the "WIN" site of WDR5 that cause displacement from chromatin have been additionally implicated to be of broader use in cancer treatment. In this study, a fragment screen with Surface Plasmon Resonance (SPR) was used to identify a highly ligand-efficient imidazole-containing compound that is bound in the WIN site. The subsequent medicinal chemistry campaign-guided by a suite of high-resolution cocrystal structures with WDR5-progressed the initial hit to a low micromolar binder. One outcome from this study is a moiety that substitutes well for the side chain of arginine; a tripeptide containing one such substitution was resolved in a high resolution structure (1.5 Å) with a binding mode analogous to the native tripeptide. SPR furthermore indicates a similar residence time (k d = ∼0.06 s-1) for these two analogs. This novel scaffold therefore represents a possible means to overcome the potential permeability issues of WDR5 ligands that possess highly basic groups like guanidine. The series reported here furthers the understanding of the WDR5 WIN site and functions as a starting point for the development of more potent WDR5 inhibitors that may serve as cancer therapeutics.

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

De-novo designed library of benzoylureas as inhibitors of BCL-XL: synthesis, structural and biochemical characterization.

The prosurvival BCL-2 proteins are attractive yet challenging targets for medicinal chemists. Their involvement in the initiation and progression of many, if not all, tumors makes them prime targets for developing new anticancer therapies. We present our approach based on de novo structure-based drug design. Using known structural information from complexes engaging opposing members of the BCL-2 family of proteins, we designed peptidomimetic compounds using a benzoylurea scaffold to reproduce key interactions between these proteins. A library stemming from the initial de novo designed scaffold led to the discovery of ligands with low micromolar potency (KD = 4 µM) and selectivity for BCL-XL. These compounds bind in the canonical BH3 binding groove in a binding mode distinct from previously known BCL-2 inhibitors. The results of our study provide insight into the design of a new class of antagonists targeting a challenging class of protein-protein interactions.

Discovery of potent and selective benzothiazole hydrazone inhibitors of Bcl-XL.

Developing potent molecules that inhibit Bcl-2 family mediated apoptosis affords opportunities to treat cancers via reactivation of the cell death machinery. We describe the hit-to-lead development of selective Bcl-XL inhibitors originating from a high-throughput screening campaign. Small structural changes to the hit compound increased binding affinity more than 300-fold (to IC50 < 20 nM). This molecular series exhibits drug-like characteristics, low molecular weights (Mw < 450), and unprecedented selectivity for Bcl-XL. Surface plasmon resonance experiments afford strong evidence of binding affinity within the hydrophobic groove of Bcl-XL. Biological experiments using engineered Mcl-1 deficient mouse embryonic fibroblasts (MEFs, reliant only on Bcl-XL for survival) and Bax/Bak deficient MEFs (insensitive to selective activation of Bcl-2-driven apoptosis) support a mechanism-based induction of apoptosis. This manuscript describes the first series of selective small-molecule inhibitors of Bcl-XL and provides promising leads for the development of efficacious therapeutics against solid tumors and chemoresistant cancer cell lines.

Structure-guided design of a selective BCL-X(L) inhibitor.

The prosurvival BCL-2 family protein BCL-X(L) is often overexpressed in solid tumors and renders malignant tumor cells resistant to anticancer therapeutics. Enhancing apoptotic responses by inhibiting BCL-X(L) will most likely have widespread utility in cancer treatment and, instead of inhibiting multiple prosurvival BCL-2 family members, a BCL-X(L)-selective inhibitor would be expected to minimize the toxicity to normal tissues. We describe the use of a high-throughput screen to discover a new series of small molecules targeting BCL-X(L) and their structure-guided development by medicinal chemistry. The optimized compound, WEHI-539 (7), has high affinity (subnanomolar) and selectivity for BCL-X(L) and potently kills cells by selectively antagonizing its prosurvival activity. WEHI-539 will be an invaluable tool for distinguishing the roles of BCL-X(L) from those of its prosurvival relatives, both in normal cells and notably in malignant tumor cells, many of which may prove to rely upon BCL-X(L) for their sustained growth.

An in vivo large-scale chemical screening platform using Drosophila for anti-cancer drug discovery.

Anti-cancer drug development involves enormous expenditure and risk. For rapid and economical identification of novel, bioavailable anti-tumour chemicals, the use of appropriate in vivo tumour models suitable for large-scale screening is key. Using a Drosophila Ras-driven tumour model, we demonstrate that tumour overgrowth can be curtailed by feeding larvae with chemicals that have the in vivo pharmacokinetics essential for drug development and known efficacy against human tumour cells. We then develop an in vivo 96-well plate chemical screening platform to carry out large-scale chemical screening with the tumour model. In a proof-of-principle pilot screen of 2000 compounds, we identify the glutamine analogue, acivicin, a chemical with known activity against human tumour cells, as a potent and specific inhibitor of Drosophila tumour formation. RNAi-mediated knockdown of candidate acivicin target genes implicates an enzyme involved in pyrimidine biosynthesis, CTP synthase, as a possible crucial target of acivicin-mediated inhibition. Thus, the pilot screen has revealed that Drosophila tumours are glutamine-dependent, which is an emerging feature of many human cancers, and has validated the platform as a powerful and economical tool for in vivo chemical screening. The platform can also be adapted for use with other disease models, thus offering widespread applications in drug development.

Synthesis of conformationally constrained benzoylureas as BH3-mimetics.

The design of small molecules that mimic the BH3 domain and bind to Bcl-2 proteins has emerged as a promising approach to discovering novel anti-cancer therapeutics. We reveal the design and synthesis of conformationally constrained benzoylurea scaffolds as conformational probes. Central to helix mimicry, the intramolecular hydrogen bond in the benzoylurea plays a key role in the pre-organisation of the acyclic substrates for cyclisation via ring closing metathesis, providing efficient access to the constrained mimetics.

An efficient high-throughput screening method for MYST family acetyltransferases, a new class of epigenetic drug targets.

Epigenetic aberrations are increasingly regarded as key factors in cancer progression. Recently, deregulation of histone acetyltransferases (HATs) has been linked to several types of cancer. Monocytic leukemia zinc finger protein (MOZ) is a member of the MYST family of HATs, which regulate gene expression in cell proliferation and differentiation. Deregulation of these processes through constitutively active MOZ fusion proteins gives rise to the formation of leukemic stem cells, rendering MOZ an excellent target for treating myeloid leukemia. The authors implemented a hit discovery campaign to identify small-molecule inhibitors of MOZ-HAT activity. They developed a robust, homogeneous assay measuring the acetylation of synthetic histone peptides. In a primary screening campaign testing 243 000 lead-like compounds, they identified inhibitors from several chemical classes. Secondary assays were used to eliminate assay-interfering compounds and prioritize confirmed hits. This study establishes a new high-throughput assay for HAT activity and could provide the foundation for the development of a new class of drugs for the treatment of leukemias.

Identification of 5,6-substituted 4-aminothieno[2,3-d]pyrimidines as LIMK1 inhibitors.

4-Aminobenzothieno[3,2-d]pyrimidines were previously identified in a high throughput screening campaign as LIMK1 inhibitors. Scaffold reversal led to the identification of a series of simple 5,6-substituted 4-aminothieno[2,3-d]pyrimidines with low micromolar inhibition of LIMK1.

Quinazoline sulfonamides as dual binders of the proteins B-cell lymphoma 2 and B-cell lymphoma extra long with potent proapoptotic cell-based activity.

ABT-737 and ABT-263 are potent inhibitors of the BH3 antiapoptotic proteins, Bcl-x(L) and Bcl-2. This class of putative anticancer agents invariantly contains an acylsulfonamide core. We have designed and synthesized a series of novel quinazoline-based inhibitors of Bcl-2 and Bcl-x(L) that contain a heterocyclic alternative to the acylsulfonamide. These compounds exhibit submicromolar, mechanism-based activity in human small-cell lung carcinoma cell lines in the presence of 10% human serum. This comprises the first successful demonstration of a quinazoline sulfonamide core serving as an effective benzoylsulfonamide bioisostere. Additionally, these novel quinazolines comprise only the second known class of Bcl-2 family protein inhibitors to induce mechanism-based cell death.