<i>JAK2^V617F</i> Drives Mcl-1 Expression and Sensitizes Hematologic Cell Lines to Dual Inhibition of JAK2 and Bcl-xL

<div><p>Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major <i>JAK2</i> mutation observed in these diseases (<i>JAK2^V617F</i>) enforces Mcl-1 transcription via STAT3 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in <i>JAK2^V617F</i>-harboring myelodyssplastic syndrome cell lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-xL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the <i>JAK2^V617F</i> mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by activating <i>JAK2</i> mutations.</p></div

Regulation of Mcl-1 and Bcl-XL by <i>JAK2</i>^<i>V617F</i>.

<p>(A) JAKi-I was evaluated in a panel of 66 human protein kinases as detailed in the Methods section, and Ki values determined. Red, <0.01 μM; black, 0.01–1.67 μM, green, >1.67 μM. (B) UKE-1 (<i>JAK2</i>^<i>V617F</i>) AML cells were treated for 10 min with JAKi-I as indicated. Tyrosine phoshorylation of STAT3 and STAT5 was determined by immunoblotting. (C) The <i>JAK2</i>^<i>V617F</i>-positive AML cell lines, SET-2, UKE-1, and HEL, the chronic myelogenous leukemia line, K562 (<i>JAK2</i>^<i>V617F</i>-negative), and the AML cell line, MV4;11 (<i>JAK2</i>^<i>V617F</i>-negative), were cultured in the absence of serum for 2 hr, then treated with 1 μM JAKi-I for 1 hr. Constitutive tyrosine phosphorylation of STAT3 and STAT5 was determined by immunoblotting. (D and E) Cells were treated for 6 hr with JAKi-I, and the abundance of Mcl-1 and Bcl-XL mRNA was determined by qPCR. Data represent means +/- standard deviation for two independent determinations each performed in triplicate. (F) Cells were treated with JAKi-I or Ruxolitinib over a 24-hr time course, and Mcl-1 and Bcl-XL levels were determined by immunoblotting (similar results were observed for 2 separate immuoblots). (G) Quantification of the data shown in (F). Data are expressed as the ratio of intensity of Mcl-1/β-actin for each time point. (H and I) HEL or K562 cells were transfected with either non-targeting (siNT-1) or Mcl-1-specific (siMcl1–1–4) siRNAs, treated for 72 hr with ABT-263, then lysates were prepared, and cell viability was determined. Data are means of duplicate samples and are representative of two independent experiments. (J) Cells were treated for 6 hr with or without 1 μM JAKi-I then subjected chromatin immunoprecipitation assays using normal mouse IgG, anti-acetylated histone H3, or anti-STAT3. Mcl-1 promoter binding was determined by PCR on chromatin immunoprecipitates (for <u>immunoblots</u>, similar results were obtained twice).</p

Combination of JAK2 and Bcl-2 family inhibitors yields synergistic antiproliferative activity in <i>JAK2</i>^<i>V617F</i>-harboring AML cell lines.

<p>(A/B) HEL and K562 cells were treated for 6 hr with 1 μM JAKi-I followed by 3 hr with 0.15 μM ABT-263, then lysates or Bcl-XL immunoprecipitates were prepared and immunoblotted. (C) Cells were treated for 6 hr with 1 μM JAKi-I followed by 0.15 μM ABT-263 over a 3-hr time period. Caspase-3 activity was determined at each time point. Data are from duplicate samples and are representative of at least three independent experiments. (D-G) Cells were treated in combination as indicated, and cell viability was determined after 72 hr. Data are means of duplicate determinations, and are representative of at least three independent experiments. (H) Drug-drug interactions were determined using a matrix of pairwise combinations covering half-log dose responses from 0.03 to 1 μM for both JAKi-I and ABT-263. Drugs were added simultaneously, and cell viability was determined after 72 hr. The data were then analyzed using the drug-drug interaction model of Bliss additivity¹⁶ to define dose combinations that were synergistic (values >15; red), antagonistic (values <-15; blue), or without effect (-15V617F constitutively phosphorylates and activates STAT3/5, thus enforcing expression of the transcriptional target, Mcl-1. Mcl-1 collaborates with Bcl-XL to oppose apoptosis and support viability. Inhibition of JAK2 in this context silences JAK/STAT-driven transcription of Mcl-1, leaving survival largely dependent upon Bcl-XL. Neutralization of Bcl-XL with ABT-263 is then achieved at a lower dose and is sufficient to induce apoptosis.</p

Azaindole-Based Inhibitors of Cdc7 Kinase: Impact of the Pre-DFG Residue, Val 195

To investigate the role played by the unique pre-DFG residue Val 195 of Cdc7 kinase on the potency of azaindole-chloropyridines (<b>1</b>), a series of novel analogues with various chloro replacements were synthesized and evaluated for their inhibitory activity against Cdc7. X-ray cocrystallization using a surrogate protein, GSK3β, and modeling studies confirmed the azaindole motif as the hinge binder. Weaker hydrophobic interactions with Met 134 and Val 195 by certain chloro replacements (e.g., H, methyl) led to reduced Cdc7 inhibition. Meanwhile, data from other replacements (e.g., F, O) indicated that loss of such hydrophobic interaction could be compensated by enhanced hydrogen bonding to Lys 90. Our findings not only provide an in-depth understanding of the pre-DFG residue as another viable position impacting kinase inhibition, they also expand the existing knowledge of ligand-Cdc7 binding

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

Novel Antibacterial Class

We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S. pneumoniae. The chemical structure of the NRI class is related to antibacterial quinolones, but, interestingly, the differences in structure are sufficient to completely alter the biochemical and intracellular mechanisms of action. Expression array studies and analysis of NRI-resistant mutants confirm this difference in intracellular mechanism and provide evidence that the NRIs inhibit bacterial protein synthesis by inhibiting ribosomes. Furthermore, compounds in the NRI series appear to inhibit bacterial ribosomes by a new mechanism, because NRI-resistant strains are not cross-resistant to other ribosome inhibitors, such as macrolides, chloramphenicol, tetracycline, aminoglycosides, or oxazolidinones. The NRIs are a promising new antibacterial class with activity against all major drug-resistant respiratory pathogens

Potent and Selective Inhibitors of CDPK1 from <i>T. gondii</i> and <i>C. parvum</i> Based on a 5‑Aminopyrazole-4-carboxamide Scaffold

5-Aminopyrazole-4-carboxamide was used as an alternative scaffold to substitute for the pyrazolopyrimidine of a known “bumped kinase inhibitor” to create selective inhibitors of calcium-dependent protein kinase-1 from both <i>Toxoplasma gondii</i> and <i>Cryptosporidium parvum</i>. Compounds with low nanomolar inhibitory potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in <i>T. gondii</i> cell proliferation assays and were shown to be nontoxic to mammalian cells