Pre-Treatment Whole Blood Gene Expression Is Associated with 14-Week Response Assessed by Dynamic Contrast Enhanced Magnetic Resonance Imaging in Infliximab-Treated Rheumatoid Arthritis Patients

<div><p>Approximately 30% of rheumatoid arthritis patients achieve inadequate response to anti-TNF biologics. Attempts to identify molecular biomarkers predicting response have met with mixed success. This may be attributable, in part, to the variable and subjective disease assessment endpoints with large placebo effects typically used to classify patient response. Sixty-one patients with active RA despite methotrexate treatment, and with MRI-documented synovitis, were randomized to receive infliximab or placebo. Blood was collected at baseline and genome-wide transcription in whole blood was measured using microarrays. The primary endpoint in this study was determined by measuring the transfer rate constant (K_trans) of a gadolinium-based contrast agent from plasma to synovium using MRI. Secondary endpoints included repeated clinical assessments with DAS28(CRP), and assessments of osteitis and synovitis by the RAMRIS method. Infliximab showed greater decrease from baseline in DCE-MRI K_trans of wrist and MCP at all visits compared with placebo (<i>P</i><0.001). Statistical analysis was performed to identify genes associated with treatment-specific 14-week change in K_trans. The 256 genes identified were used to derive a gene signature score by averaging their log expression within each patient. The resulting score correlated with improvement of K_trans in infliximab-treated patients and with deterioration of K_trans in placebo-treated subjects. Poor responders showed high expression of activated B-cell genes whereas good responders exhibited a gene expression pattern consistent with mobilization of neutrophils and monocytes and high levels of reticulated platelets. This gene signature was significantly associated with clinical response in two previously published whole blood gene expression studies using anti-TNF therapies. These data provide support for the hypothesis that anti-TNF inadequate responders comprise a distinct molecular subtype of RA characterized by differences in pre-treatment blood mRNA expression. They also highlight the importance of placebo controls and robust, objective endpoints in biomarker discovery.</p><p><b><i>Trial Registration:</i></b> ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/show/NCT01313520" target="_blank">NCT01313520</a></p></div

Gene signature score is higher in EULAR responders than non-responders in two independent studies.

<p>The predictive signature is associated with clinical response in two additional whole blood gene expression studies. The distribution of predictive signature scores was compared in responder and non-responder groups in the studies of Julia et al. and Toonen et al. In both cases, a one-tailed t-test identified statistically significant (p<0.05) differences.</p

Baseline characteristics.

<p>Baseline characteristics.</p

MRI and clinical endpoints.

<p>MRI and clinical endpoints.</p

Gene expression is associated with change in disease activity measured by DCE-MRI.

<p>(A) High signature score correlates with K_trans improvement in the treatment arm and K_trans deterioration in the placebo arm. Scatter plots show baseline and treatment adjusted 14-week change in log Ktrans vs. signature score in both the treatment and placebo arms at weeks 2, 4, and 14. Linear models including terms for baseline K_trans, treatment allocation, signature score, and the interaction between signature and treatment were fit to log K_trans change from baseline at each week. At both week 4 and 14, the signature score main effect and interaction with treatment were significant at p<0.05. (B) Whole blood gene expression improves prediction of week 14 change in K_trans. In ten repeated rounds of random subsampling, 40 patients were selected and their whole blood gene expression data was used to identify genes associated with treatment response measured by K_trans, DAS28(CRP), and RAMRIS. A linear model including terms for baseline disease activity, treatment allocation, signature score, and the interaction between signature and treatment was fit to week 14 data and used to predict week 14 changes for held out subjects. The distribution of mean squared prediction errors (MSE) minus the MSE achieved by a model excluding signature score terms is plotted for each endpoint. For K_trans, but not DAS28(CRP), or RAMRIS, incorporation of baseline blood gene expression consistently improved prediction performance (p = 0.015, t-test).</p

Correlation coefficient (p-value) of baseline signature score and 14-week change in log K_trans of wrist and DAS28(CRP) for previously reported blood gene signatures.

<p>Correlation coefficient (p-value) of baseline signature score and 14-week change in log K_trans of wrist and DAS28(CRP) for previously reported blood gene signatures.</p

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 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

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