Inducible Cre-Mediated Deletion of the B1-8f HC Allele Leads to Loss of Surface Ig from Immature B Cells

<div><p>(A) Flow cytometry showing surface phenotype of B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre BM after 5-d IL-7 culture.</p> <p>(B) Flow cytometric analysis of B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre BM culture cells incubated with IFNαβ (1,000 or 5,000 units/ml), in the absence of IL-7, for 1, 2, or 3 d. The cell populations shown are gated on lymphocytes by forward and side scatter, and then for B220. At the end of the 5-d IL-7 culture, more than 90% of the cells were viable and B220⁺. The numbers shown indicate the percentage of gated cells.</p></div

Genes Differentially Expressed between Ctrl-M^hi and Cre-M^lo Cell Populations

<p>Shown are representative genes that were generally either (A) up-regulated or (B) down-regulated in Cre-M^lo cells compared with Ctrl-M^hi cells. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030082#pbio-0030082-g002" target="_blank">Figure 2</a> legend for details.</p

Microarray Gene Expression Analysis Demonstrates Co-Clustering of Cre-Deleted IgM^lo Cells with IgM⁻ Cell Populations

<div><p>(A) FACS sorting strategy for Ctrl-M^hi, Cre-M^hi, and Cre-M^lo immature B cells incubated with IFNαβ (1,000 units/ml) for 2 d. The numbers shown indicate the percentage of gated cells.</p> <p>(B) Affymetrix microarrays were used to identify genes differentially expressed between IFN-treated Ctrl-M^hi and Cre-M^lo cells. Analysis identified 327 transcripts that met the following criteria: 2-fold or greater change in mean expression level, a more than 200-unit difference in mean expression values, and Student's t-test <i>p</i> < 0.01. Individual expression values for each gene were divided by the mean of expression levels for three control IgM⁺ cell populations: IFN-treated Ctrl-M^hi; IgM^hi cells sorted from 5-d IL-7 HEL-lg BM cultures (HEL-M^hi); and sorted from normal Balb/c BM (FxE). Other populations included Cre-M^hi, Hardy Fraction D pre-B cells (FxD) sorted from normal Balb/c BM, and lgM⁻ cells sorted from 5-d IL-7 cultures of control B6 BM (B6-M⁻). Data were transformed into log₂ space, and represent fold-differences relative to the IgM⁺ cell populations (see scale bar). Data from 293 transcripts (duplicates and all but four representative Ig HC and LC transcripts removed) were clustered and visualized using CLUSTER and TREEVIEW [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030082#pbio-0030082-b51" target="_blank">51</a>]. Red represents genes expressed at higher levels, while green represents genes expressed at lower levels, than the mean of IgM⁺ cells. Each column represents an individual sorted cell population.</p></div

Protein Expression Confirms Reversal of Development in Immature B Cells Losing Surface IgM

<p>B1-8f/3-83κ control and B1-8f/3-83κ/Mx-Cre cell populations were harvested at the end of a 3- or 4-d culture with IFNαβ (3,000 units/ml), stained with mAbs for cell surface proteins, and analyzed by flow cytometry. Shown are the expression levels of B220-gated Ctrl-M^hi (thick line) and Cre-M^lo (thin line) cells at the end of the culture period. Essentially identical results were observed when Cre-M^hi and Cre-M^lo cells were compared (data not shown).</p

Immature B Cells That Lose Basal Signaling Show Induction of LC Rearrangements

<div><p>(A) PCR analysis of endogenous Ig light chain rearrangements (V-Jλ3, V-Jλ1, RS, and V-Jκ1) in genomic DNA of FACS-sorted HEL-Ig/Rag2-GFP BM cells incubated with medium alone or with 300 ng/ml herbimycin A for 24 h. IgM^a+GFP⁺ cells were sorted from herbimycin A-treated cultures, and IgM^a+GFP⁻ were sorted from control cultures. Data are from three independent experiments. CD14 is a loading control. −, negative control (C57Bl/6J tail DNA); +, positive control (C57Bl/6J spleen DNA).</p> <p>(B) Genomic DNA from the same cell populations described in (A) was subjected to ligation-mediated PCR to detect double-strand signal end DNA breaks at Jκ1. Controls in right three lanes of blot: H₂O, control; −, negative control (S17 stroma); +, positive control (C57Bl/6 BM).</p> <p>(C) Genomic DNA was extracted from B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre immature B cells 3 d following incubation with IFNαβ. Quantitative PCR analysis was used to determine the fold-induction of LC rearrangements in B1-8f/3-83κ/Mx-Cre immature B cells treated with IFN compared to medium alone. Data represent the mean ± standard deviation of two (V-Jλ1), three (V-Jλ3), or four experiments (V-Jκ1, RS).</p></div

Exposure and target engagement of MRLB-11055 in the peripheral blood of C57BL/6 mice stimulated with darbepoetin.

<p>A. Effect of MRLB-11055 on phosphoSTAT5 levels at various times post-dose. B. Calculation of IC50 value for inhibition of phosphoSTAT5. C. PK of 3 doses of MRLB-11055 in mouse blood, with calculated IC50 superimposed (dashed line).</p

Effect of 2 Cycles of Intermittent Dosing (3 days on, 4 days off) of MRLB-11055 on V617F-Luc2 Mice (N = 10).

<p>Effect on A & B. Bioluminescence in spleen C. Hematocrit D. Multiple endpoints at end of study (Day 14).</p

Effect of MRLB-11055 in a Darbepoetin-Induced PV Efficacy Model.

<p>The ability of MRLB-11055 to prevent darbepoetin-induced increases in hematocrit (Hct) and spleen mass (SPL) over 7 days is shown, as is the impact of MRLB-11055 on white blood cells (WBC) and its concentration in blood (PK). Dashed line indicates <i>in vivo</i> IC50 value.</p

Effect of MRLB-11055 on major lymphoid populations in spleen of WT B6 mice.

<p>MRLB-11055 was given for either 3 or 6 days (on), followed up by either a 0 or 4 day holiday (off), for up to 4 cycles. Effects on NK, B and T cells were measured by flow cytometry.</p

Optimization of Orally Bioavailable Enhancer of Zeste Homolog 2 (EZH2) Inhibitors Using Ligand and Property-Based Design Strategies: Identification of Development Candidate (<i>R</i>)‑5,8-Dichloro-7-(methoxy(oxetan-3-yl)methyl)-2-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3,4-dihydro­isoquinolin-1(2<i>H</i>)‑one (PF-06821497)

A new series of lactam-derived EZH2 inhibitors was designed via ligand-based and physicochemical-property-based strategies to address metabolic stability and thermodynamic solubility issues associated with previous lead compound <b>1</b>. The new inhibitors incorporated an sp³ hybridized carbon atom at the 7-position of the lactam moiety present in lead compound <b>1</b> as a replacement for a dimethylisoxazole group. This transformation enabled optimization of the physicochemical properties and potency compared to compound <b>1</b>. Analysis of relationships between calculated log <i>D</i> (clogD) values and in vitro metabolic stability and permeability parameters identified a clogD range that afforded an increased probability of achieving favorable ADME data in a single molecule. Compound <b>23a</b> exhibited the best overlap of potency and pharmaceutical properties as well as robust tumor growth inhibition in vivo and was therefore advanced as a development candidate (PF-06821497). A crystal structure of <b>23a</b> in complex with the three-protein PRC2 complex enabled understanding of the key structural features required for optimal binding