Evidence of mTOR Activation by an AKT-Independent Mechanism Provides Support for the Combined Treatment of PTEN-Deficient Prostate Tumors with mTOR and AKT Inhibitors

AbstractActivation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone

Splenectomy Normalizes Hematocrit in Murine Polycythemia Vera

Splenic enlargement (splenomegaly) develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV), an activating mutation in Janus kinase 2 (JAK2V617) induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH) or splenectomy (SPL) surgeries in a murine model of JAK2V617F-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2V617F transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2V617) is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2V617F-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen

A Quantitative Volumetric Micro-Computed Tomography Method to Analyze Lung Tumors in Genetically Engineered Mouse Models

Two genetically engineered, conditional mouse models of lung tumor formation, K-rasLSL-G12D and K-rasLSL-G12D/p53LSL-R270H, are commonly used to model human lung cancer. Developed by Tyler Jacks and colleagues, these models have been invaluable to study in vivo lung cancer initiation and progression in a genetically and physiologically relevant context. However, heterogeneity, multiplicity and complexity of tumor formation in these models make it challenging to monitor tumor growth in vivo and have limited the application of these models in oncology drug discovery. Here, we describe a novel analytical method to quantitatively measure total lung tumor burden in live animals using micro-computed tomography imaging. Applying this methodology, we studied the kinetics of tumor development and response to targeted therapy in vivo in K-ras and K-ras/p53 mice. Consistent with previous reports, lung tumors in both models developed in a time- and dose (Cre recombinase)-dependent manner. Furthermore, the compound K-rasLSL-G12D/p53LSL-R270H mice developed tumors faster and more robustly than mice harboring a single K-rasLSL-G12D oncogene, as expected. Erlotinib, a small molecule inhibitor of the epidermal growth factor receptor, significantly inhibited tumor growth in K-rasLSL-G12D/p53LSL-R270H mice. These results demonstrate that this novel imaging technique can be used to monitor both tumor progression and response to treatment and therefore supports a broader application of these genetically engineered mouse models in oncology drug discovery and development

MCL1 and BCL-xL Levels in Solid Tumors Are Predictive of Dinaciclib-Induced Apoptosis

<div><p>Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, <i>in vitro</i> and <i>in vivo</i> studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic <i>BCL2</i> family member <i>MCL1</i> and correlation of sensitivity with the <i>MCL1</i>-to-<i>BCL-xL</i> mRNA ratio or <i>MCL1</i> amplification in solid tumor models <i>in vitro</i> and <i>in vivo</i>. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating <i>MCL1</i> and <i>BCL-xL</i> as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.</p></div

Dinaciclib exhibits apoptotic-induction, tumor efficacy and antiangiogenic activity in human tumor xenograft models.

<p>(A) Immunoblot analysis of lysates prepared from NCI-H23 and COLO 320DM xenograft tumors resected at 1, 3, 6, 9, 18, 48 and 96 hr after a single 40 mg/kg, i.p. dinaciclib injection. Time 0 is a 1 hr vehicle treatment. Represented are three tumors from three animals per time-point. α-tubulin is included as a loading control. Quantification of the cleaved-PARP fragment in these lysates is shown in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108371#pone.0108371.s004" target="_blank">Figure S4</a>. (B) Quantification of cleaved-PARP fragment in lysates prepared from tumors resected 6 hr post-administration of vehicle or a single dose of dinaciclib at 40 mg/kg, i.p. Xenograft models designated as <i>MCL11:BCL-xL</i> high (filled bars) or low (open bars) mRNA ratio as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108371#pone.0108371.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108371#pone.0108371.s008" target="_blank">Table S1</a>. Fold change was determined from the mean of 3–5 dinaciclib-treated and 3–5 vehicle-treated tumors for each model. The mean and standard deviation are shown. (C) Efficacy of dinaciclib in 7 human xenograft models. Xenograft models designated as <i>MCL11:BCL-xL</i> high (filled bars) or low (open bars) mRNA ratio are indicated. Dinaciclib was given at 40 mg/kg, i.p., twice-weekly or 4qd. %TGI was measured at the end of the dosing period (n = 10 mice per group, except n = 8 mice in the JIMT-1 dinaciclib group). Tumor growth rates over the course of the study and endpoints are shown in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108371#pone.0108371.s006" target="_blank">Figure S6</a>. All dinaciclib-treated groups had mean tumor volumes that were significantly smaller than vehicle-treated groups at the end of study (p<0.05). (D) Antiangiogenic effect of dinaciclib relative to KDR inhibitor in A2780 xenograft tumors. Dinaciclib was given at 40 mg/kg, i.p., days 1, 4, 7 and KDR inhibitor was given at 10 mg/kg, po, days 1–7. Tumors were resected 2–4 hours after the last drug treatment. KDR-inhibitor compound B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108371#pone.0108371-Hardwick1" target="_blank">[19]</a> was utilized as a positive control in these studies. Mean vessel density by area  =  % of endothelial cells divided by the total tissue area of interest <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108371#pone.0108371-Shi1" target="_blank">[20]</a>.</p

Dinaciclib and navitoclax have an inverse cell-killing relationship and the combination is synergistic.

<p>(A) Effects of 24 hr treatment of 1 µM navitoclax (open bars), 100 nM dinaciclib (filled bars) and the combination at the respective concentrations (hatched bars) on cell viability in 11 SCLC cell lines. (B) Bliss synergy analysis graph of the expected fractional cell viability response to the combination of dinaciclib and navitoclax at specified concentrations (grid intersections) compared to the observed (black balls) fractional cell viability response following 18 hr treatment using an 8×8 dose escalation matrix. The dotted lines correspond to the fractional viability of dinaciclib (left, rear) and navitoclax (right, rear) treatment alone at the specified concentrations. (C) Immunoblot analysis of total protein lysates from SW1573 cells treated for the indicated times with 1 µM navitoclax, 100 nM dinaciclib or the combination.</p

Sensitivity to short-term dinaciclib treatment correlates with <i>BCL-xL</i> and <i>MCL1</i> levels.

<p>(A) <i>BCL-xL</i> mRNA level positively correlated with viability of 254 solid tumor cell lines treated 24 hr with dinaciclib (100 nM). (B) The <i>MCL1:BCL-xL</i> mRNA ratio negatively correlated with viability in the same dinaciclib-treated cell line panel as (A). (C) Effect of 8 hr dinaciclib (100 nM) treatment on apoptosis induction as measured by cleaved PARP fragment (y axis) against the <i>MCL1:BCL-xL</i> mRNA ratio (x axis). Data were measured from 27 cell lines. The <i>MCL1:BCL-xL</i> mRNA ratios and relative cleaved PARP fragment values for these cell lines are listed in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108371#pone.0108371.s008" target="_blank">Table S1</a>. Relative cleaved PARP fragments values <1 were adjusted to 1. <i>MCL1</i> and <i>BCL-xL</i> expression values were obtained from the Broad Institute CCLE. (D) Percent change in activated caspase-3/7 levels after 12 hr (empty bars) and 24 hr (filled bars) of dinaciclib (100 nM) treatment in four high <i>MCL1:BCL-xL</i> mRNA ratio cell lines (COLO 320DM, 22Rv1, A2780, NCI-H23) and four low <i>MCL1:BCL-xL</i> mRNA ratio cell lines (MDA-MB-231, JIMT-1, SW480, PC3). (E) Effect of <i>MCL1</i> copy number on viability after 24 hr, 100 nM dinaciclib treatment in 18 cell lines. Insensitive cell lines (circles) exhibited ≥70% cell viability and sensitive cell lines (squares) exhibited <30% cell viability after dinaciclib treatment. (F) <i>MCL1</i> rescue of siRNA knockdown in NCI-H23 cells. Top, percent cell viability remaining in cells transfected with empty vector control or vector expressing <i>MCL1</i> lacking 3′ UTR, followed by 24 hr transfection with control siRNA (filled bar) or <i>MCL1</i> siRNA targeting 3′ UTR of endogenous <i>MCL1</i> (empty bar). Bottom, MCL1 immunoblot of NCI-H23 cells described in top panel. Cntrl  =  Control.</p

Summary of Bliss scores and sensitivity to dinaciclib, navitoclax and combination treatment.

1<p>Synergy analysis after 18 hr treatment. vBliss: synergy (>0.1), additive (0.1–0.05). neutral (<0.05-0), antagonistic (<0).</p>2<p>Percent viability after 18 hour treatment with 100 nM dinaciclib, 1 µM navitoclax or the combination.</p>3<p>Information source. Cancer Cell Line Encyclopedia converted from log 2 to log10.</p><p>Summary of Bliss scores and sensitivity to dinaciclib, navitoclax and combination treatment.</p

Dinaciclib functions as a transcriptional repressor requiring >2 hr exposure to induce apoptosis.

<p>(A) Effect of dinaciclib (100 nM) compared to transcriptional repressor triptolide (3 µM) in 33 ovarian cell lines after a 24 hr treatment. (B) Effect of dinaciclib (100 nM) compared to paclitaxel (3 µM) in 33 ovarian cell lines after a 24 hr treatment. (C) Dinaciclib (100 nM) downregulates <i>MCL1</i> mRNA expression levels in A2780 cells during a 5 hr treatment. Expression level was normalized to the geometric mean of α<i>-tubulin</i> and <i>GAPDH</i> mRNA levels. (D) Immunoblot analysis of A2780 cells during the 5 hr time-course in (C). (E) Immunoblot analysis of A2780 cells treated with dinaciclib (100 nM) for 0, 2 or 8 hr (lanes 1, 2 and 6). After 2 hr treatment, dinaciclib was washed-out and cells were analyzed at subsequent 2 hr intervals with the cumulative times from t = 0 as indicated (lanes 3, 4 and 5).</p