Nicholas Kwiatkowski

Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity.

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.

Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function.

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, it is expected the development of a potent, selective small molecule inhibitor of Mps1 would greatly facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 co-crystal structures of novel, selective small molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit, and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells possessing extra centrosomes, an abnormality found in some cancers, Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

Chemical genetic strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell disease

The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a unique bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.

Structural Origin of Selectivity in Class II-Selective Histone Deacetylase Inhibitors

The development of class- and isoform-selective histone deacetylase (HDAC) inhibitors is highly desirable for the study of the complex interactions of these proteins central to transcription regulation as well as for the development of selective HDAC inhibitors as drugs in epigenetics. To provide a structural basis for the rational design of such inhibitors, a combined computational and experimental study of inhibition of three different histone deacetylase isoforms, HDAC1, -6, and -8, with three different hydroxamate inhibitors is reported. While SAHA was found to be unselective for the inhibition of class I and class II HDACs, the other inhibitors were found to be selective toward class II HDACs. Molecular dynamics simulations indicate that this selectivity is caused by both the overall shape of the protein surface leading to the active site and specific interactions of an aspartate residue in a polar loop and two phenylalanines and a methionine in a nonpolar loop. Monitoring the specific interactions as a function of the simulation time identifies a key sulfur-pi interaction. The implications of the structural motifs for the design of class II-selective HDAC inhibitors are discussed.

Release of Mps1 from kinetochores is crucial for timely anaphase onset

Mps1 regulates its own turnover at kinetochores to ensure mitotic checkpoint silencing in metaphase.

Effects of the Selective MPS1 Inhibitor MPS1-IN-3 on Glioblastoma Sensitivity to Antimitotic Drugs

BACKGROUND Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest. METHODS We used glioblastoma cells to determine the expression of MPS1 and to determine the effects of MPS1 inhibition on mitotic errors and cell viability in combination with vincristine and taxol. The effect of MPS1 inhibition was assessed in different orthotopic glioblastoma mouse models (n = 3-7 mice/group). MPS1 expression levels were examined in relation to patient survival. RESULTS Using publicly available gene expression data, we determined that MPS1 overexpression corresponds positively with tumor grade and negatively with patient survival (two-sided t test, P < .001). Patients with high MPS1 expression (n = 203) had a median and mean survival of 487 and 913 days (95% confidence intervals [CI] = 751 to 1075), respectively, and a 2-year survival rate of 35%, whereas patients with intermediate MPS1 expression (n = 140) had a median and mean survival of 858 and 1183 days (95% CI = 1177 to 1189), respectively, and a 2-year survival rate of 56%. We demonstrate that MPS1 inhibition by RNAi results in sensitization to antimitotic agents. We developed a selective small-molecule inhibitor of MPS1, MPS1-IN-3, which caused mitotic aberrancies in glioblastoma cells and, in combination with vincristine, induced mitotic checkpoint override, increased aneuploidy, and augmented cell death. MPS1-IN-3 sensitizes glioblastoma cells to vincristine in orthotopic mouse models (two-sided log-rank test, P < .01), resulting in prolonged survival without toxicity. CONCLUSIONS Our results collectively demonstrate that MPS1, a putative therapeutic target in glioblastoma, can be selectively inhibited by MPS1-IN-3 sensitizing glioblastoma cells to antimitotic drugs.

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

Discovery of selective aminothiazole aurora kinase inhibitors

Aurora family kinases regulate important events during mitosis including centrosome maturation and separation, mitotic spindle assembly, and chromosome segregation. Misregulation of Aurora kinases due to genetic amplification and protein overexpression results in aneuploidy and may contribute to tumorigenesis. Here we report the discovery of new small molecule aminothiazole inhibitors of Aurora kinases with exceptional kinase selectivity and report a 1.7 A cocrystal structure with the Aurora B:INCENP complex from Xenopus laevis. The compounds recapitulate the hallmarks of Aurora kinase inhibition, including decreased histone H3 serine 10 phosphorylation, failure to complete cytokinesis, and endoreduplication.

Tivantinib (ARQ 197) efficacy is independent of MET inhibition in non-small-cell lung cancer cell lines

MET targeted therapies are under clinical evaluation for non‐small‐cell lung cancer (NSCLC) patients. Tyrosine kinase inhibitors (TKI) against MET have varying degrees of specificity. Tivantinib (ARQ 197) is reported to be a non‐ATP competitive selective MET inhibitor. We aimed to compare the activity of tivantinib to established MET TKIs in a panel of NSCLC cell lines characterized by their MET dependency and by different relevant genotypes. A549, H3122, PC9 and HCC827, their respective resistant clones PC9 GR4 and HCC827 GR6 and the MET amplified cell lines H1993 and EBC‐1 were treated in vitro with tivantinib, crizotinib or PHA‐665752. Crizotinib and PHA‐665752 showed growth inhibition restricted to MET dependent cell lines. The pattern of activity was related to MET inhibition and downstream signaling inhibition of AKT and ERK1/2, resulting in G0/G1 cycle arrest and apoptosis. In contrast, tivantinib possessed more potent anti‐proliferative activity that was not restricted to only MET dependent cell lines. Tivantinib did not inhibit cellular MET activity or phosphorylation of downstream signaling proteins AKT or ERK1/2 in either MET dependent or independent cell lines. Cell cycle analysis demonstrated that tivantinib induced a G2/M arrest and induced apoptosis. Tivantinib but not crizotinib effected microtubule dynamics, disrupting mitotic spindles by a mechanism consistent with it functioning as a microtubule depolymerizer. Tivantinib activity is independent of MET signaling in NSCLC and suggests alternative mechanisms of action that should be considered when interpreting the results from on‐going clinical studies.

Cut homeobox 1 causes chromosomal instability by promoting bipolar division after cytokinesis failure

Cell populations able to generate a large repertoire of genetic variants have increased potential to generate tumor cells that survive through the multiple selection steps involved in tumor progression. A mechanism for the generation of aneuploid cancer cells involves passage through a tetraploid stage. Supernumerary centrosomes, however, can lead to multipolar mitosis and cell death. Using tissue culture and transgenic mouse models of breast cancer, we report that Cut homeobox 1 (CUX1) causes chromosomal instability by activating a transcriptional program that prevents multipolar divisions and enables the survival of tetraploid cells that evolve to become genetically unstable and tumorigenic. Transcriptional targets of CUX1 involved in DNA replication and bipolar mitosis defined a gene expression signature that, across 12 breast cancer gene expression datasets, was associated with poor clinical outcome. The signature not only was higher in breast tumor subtypes of worse prognosis, like the basal-like and HER2+ subtypes, but also identified poor outcome among estrogen receptor-positive/node-negative tumors, a subgroup considered to be at lower risk. The CUX1 signature therefore represents a unique criterion to stratify patients and provides insight into the molecular determinants of poor clinical outcome.