ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia

In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3–internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC_(50) approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC_(50) = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G_(0)/G_1 phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)–FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC_(50) approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC_(50) = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML

The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia.

Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2) is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1), but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells

Pyrimidine-Based Tricyclic Molecules as Potent and Orally Efficacious Inhibitors of Wee1 Kinase

Aided by molecular modeling, compounds with a pyrimidine-based tricyclic scaffold were designed and confirmed to inhibit Wee1 kinase. Structure–activity studies identified key pharmacophores at the aminoaryl and halo-benzene regions responsible for binding affinity with sub-nM <i>K</i>_i values. The potent inhibitors demonstrated sub-μM activities in both functional and mechanism-based cellular assays and also possessed desirable pharmacokinetic profiles. The lead molecule, <b>31</b>, showed oral efficacy in potentiating the antiproliferative activity of irinotecan, a cytotoxic agent, in a NCI-H1299 mouse xenograft model

A-366 inhibits H3K9me2 similar to UNC0638, but does not inhibit cell growth of MOLT-16 and HT-1080 cells.

<p>(A) The T-cell ALL cell line MOLT-16 was treated for 5 days with A-366 or UNC0638 before assessing their proliferation. (B) MOLT-16 cells were treated for 3 days with compounds and lysed. Lysates were normalized and western blot analysis was performed for H3K9me2, total H3 and G9a. (C) HT-1080 fibrosarcoma cells were treated for 2 or 5 days with A-366 or UNC0638 before assessing their proliferation. (D) H3K9me2 AlphaLISA was performed on HT-1080 cells treated for 2 days with compounds and % inhibition of H3K9me2 was calculated compared to untreated control cells.</p

<i>In vivo</i> efficacy study of A-366 in MV4;11 flank xenografts.

<p>(A) MV4;11 cells were implanted subcutaneously in SCID/bg mice and allowed to establish tumors of ~200 mm³. A-366 was administered to tumor-bearing mice at 30 mg/kg/day by osmotic mini-pump for 14 days. Tumors were measured at the indicated time points and tumor growth was plotted as a function of time. (B) In a parallel PK/PD arm carried out in MV4;11 tumor-bearing animals, plasma and tissues were collected at the indicated time points and analyzed for A-366 levels. (C) A-366-treated tumors from the indicated time points were harvested and analyzed by AlphaLISA for reductions in H3K9me2 relative to vehicle.</p

A-366 does not impact the proliferation of MCF-7 cells despite inhibition of H3K9me2.

<p>(A) MCF-7 and MDA-MB-231 breast cancer cell lines were treated with the indicated concentrations of A-366 or UNC0638 for 14 days before assessing colony formation. (B) MCF-7 and MDA-MB-231 cells were treated with the indicated concentrations of A-366 or UNC0638 for 3 days and subjected to western blot analysis of global H3K9me2 and total histone H3.</p

A-366 has cellular activity comparable to known G9a/GLP inhibitors.

<p>(A) The chemical structure of A-366. (B) PC-3 prostate adenocarcinoma cells were incubated in triplicate with DMSO or the indicated concentrations of A-366 or UNC0638 for 72 hours. H3K9me2 levels were assessed by In-Cell Western assay. (C) In-Cell Western assay for total histone H3 levels from the same plate as shown in (B).</p