Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

A Fragment-Derived Clinical Candidate for Antagonism of X-Linked and Cellular Inhibitor of Apoptosis Proteins: 1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1 H,2 H,3 H-pyrrolo[3,2- b]pyridin-1-yl)-2-[(2 R,5 R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660).

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP-XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

ASTX660, a Novel Non-peptidomimetic Antagonist of cIAP1/2 and XIAP, Potently Induces TNFα-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth.

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I-II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381-91. ©2018 AACR.

RECQL5 Suppresses Oncogenic JAK2-Induced Replication Stress and Genomic Instability.

JAK2V617F is the most common oncogenic lesion in patients with myeloproliferative neoplasms (MPNs). Despite the ability of JAK2V617F to instigate DNA damage in vitro, MPNs are nevertheless characterized by genomic stability. In this study, we address this paradox by identifying the DNA helicase RECQL5 as a suppressor of genomic instability in MPNs. We report increased RECQL5 expression in JAK2V617F-expressing cells and demonstrate that RECQL5 is required to counteract JAK2V617F-induced replication stress. Moreover, RECQL5 depletion sensitizes JAK2V617F mutant cells to hydroxyurea (HU), a pharmacological inducer of replication stress and the most common treatment for MPNs. Using single-fiber chromosome combing, we show that RECQL5 depletion in JAK2V617F mutant cells impairs replication dynamics following HU treatment, resulting in increased double-stranded breaks and apoptosis. Cumulatively, these findings identify RECQL5 as a critical regulator of genome stability in MPNs and demonstrate that replication stress-associated cytotoxicity can be amplified specifically in JAK2V617F mutant cells through RECQL5-targeted synthetic lethality.

JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response.

Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)-marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.

JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia.

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F-positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2(V617F) mice develop reduced numbers of lineage(-)Sca-1(+)c-Kit(+) cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2(V617F) mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

PwRn1, a novel Ty3/gypsy-like retrotransposon of Paragonimus westermani: molecular characters and its differentially preserved mobile potential according to host chromosomal polyploidy.

BACKGROUND: Retrotransposons have been known to involve in the remodeling and evolution of host genome. These reverse transcribing elements, which show a complex evolutionary pathway with diverse intermediate forms, have been comprehensively analyzed from a wide range of host genomes, while the information remains limited to only a few species in the phylum Platyhelminthes. RESULTS: A LTR retrotransposon and its homologs with a strong phylogenetic affinity toward CsRn1 of Clonorchis sinensis were isolated from a trematode parasite Paragonimus westermani via a degenerate PCR method and from an insect species Anopheles gambiae by in silico analysis of the whole mosquito genome, respectively. These elements, designated PwRn1 and AgCR-1 - AgCR-14 conserved unique features including a t-RNATrp primer binding site and the unusual CHCC signature of Gag proteins. Their flanking LTRs displayed >97% nucleotide identities and thus, these elements were likely to have expanded recently in the trematode and insect genomes. They evolved heterogeneous expression strategies: a single fused ORF, two separate ORFs with an identical reading frame and two ORFs overlapped by -1 frameshifting. Phylogenetic analyses suggested that the elements with the separate ORFs had evolved from an ancestral form(s) with the overlapped ORFs. The mobile potential of PwRn1 was likely to be maintained differentially in association with the karyotype of host genomes, as was examined by the presence/absence of intergenomic polymorphism and mRNA transcripts. CONCLUSION: Our results on the structural diversity of CsRn1-like elements can provide a molecular tool to dissect a more detailed evolutionary episode of LTR retrotransposons. The PwRn1-associated genomic polymorphism, which is substantial in diploids, will also be informative in addressing genomic diversification following inter-/intra-specific hybridization in P. westermani populations.