Comparative biomarker analysis of PALOMA-2/3 trials for palbociclib.

While cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, including palbociclib, combined with endocrine therapy (ET), are becoming the standard-of-care for hormone receptor-positive/human epidermal growth factor receptor 2‒negative metastatic breast cancer, further mechanistic insights are needed to maximize benefit from the treatment regimen. Herein, we conducted a systematic comparative analysis of gene expression/progression-free survival relationship from two phase 3 trials (PALOMA-2 [first-line] and PALOMA-3 [≥second-line]). In the ET-only arm, there was no inter-therapy line correlation. However, adding palbociclib resulted in concordant biomarkers independent of initial ET responsiveness, with shared sensitivity genes enriched in estrogen response and resistance genes over-represented by mTORC1 signaling and G2/M checkpoint. Biomarker patterns from the combination arm resembled patterns observed in ET in advanced treatment-naive patients, especially patients likely to be endocrine-responsive. Our findings suggest palbociclib may recondition endocrine-resistant tumors to ET, and may guide optimal therapeutic sequencing by partnering CDK4/6 inhibitors with different ETs. Pfizer (NCT01740427; NCT01942135).

A DNA-Methylation-Driven Genes Based Prognostic Signature Reveals Immune Microenvironment in Pancreatic Cancer.

Pancreatic cancer (PACA), which is characterized by an immunosuppressive nature, remains one of the deadliest malignancies worldwide. Aberrant DNA methylation (DNAm) reportedly influences tumor immune microenvironment. Here, we evaluated the role of DNA methylation driven genes (MDGs) in PACA through integrative analyses of epigenomic, transcriptomic, genomic and clinicopathological data obtained from TCGA, ICGC, ArrayExpress and GEO databases. Thereafter, we established a four-MDG signature, comprising GPRC5A, SOWAHC, S100A14, and ARNTL2. High signature risk-scores were associated with poor histologic grades and late TNM stages. Survival analyses showed the signature had a significant predictive effect on OS. WGCNA revealed that the signature may be associated with immune system, while high risk-scores might reflect immune dysregulation. Furthermore, GSEA and GSVA revealed significant enrichment of p53 pathway and mismatch repair pathways in high risk-score subgroups. Immune infiltration analysis showed that CD8+ T cells were more abundant in low score subgroups, while M0 macrophages exhibited an opposite trend. Moreover, negative regulatory genes of cancer-immunity cycle (CIC) illustrated that immunosuppressors TGFB1, VEGFA, and CD274 (PDL1) were all positively correlated with risk-scores. Furthermore, the four signature genes were negatively correlated with CD8+ lymphocytes, but positively associated with myeloid derived suppressor cells (MDSC). Conversely, specimens with high risk-scores exhibited heavier tumor mutation burdens (TMB) and might show better responses to some chemotherapy and targeted drugs, which would benefit stratification of PACA patients. On the other hand, we investigated the corresponding proteins of the four MDGs using paraffin-embedded PACA samples collected from patients who underwent radical surgery in our center and found that all these four proteins were elevated in cancerous tissues and might serve as prognostic markers for PACA patients, high expression levels indicated poor prognosis. In conclusion, we successfully established a four-MDG-based prognostic signature for PACA patients. We envisage that this signature will help in evaluation of intratumoral immune texture and enable identification of novel stratification biomarkers for precision therapies.

Therapeutic Potential of CUDC-907 (Fimepinostat) for Hepatocarcinoma Treatment Revealed by Tumor Spheroids-Based Drug Screening.

Background: Cancer is the second leading cause of death globally. However, most of the new anti-cancer agents screened by traditional drug screening methods fail in the clinic because of lack of efficacy. Choosing an appropriate in vitro tumor model is crucial for preclinical drug screening. In this study, we screened anti-hepatocarcinoma (HCC) drugs using a novel spheroid cell culture device. Methods: Four HCC cell lines were three-dimensionally (3D) cultured to screen 19 small molecular agents. 3D-cultured primary HCC cells and a tumor-bearing mouse model were used to verify the candidate anti-hepatocarcinoma agent. Cell function experiments and western blotting were conducted to explore the anti-hepatocarcinoma mechanism of the candidate agent. Results: We found that CUDC-907 can serve as a potent anti-hepatocarcinoma agent. The study data show that CUDC-907 (fimepinostat), a novel dual acting inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HDAC), has potent inhibitory effects on HCC cell lines and primary HCC cells in vitro, Animal studies have shown that CUDC-907 can also suppress HCC cells in vivo. Furthermore, we found that CUDC-907 inhibits the PI3K/AKT/mTOR pathway and downregulates the expression of c-Myc, leading to the suppression of HCC cells. Conclusion: Our results suggest that CUDC-907 can be a candidate anti-HCC drug, and the 3D in vitro drug screening method based on our novel spheroid culture device is promising for future drug screening efforts.

Gemtuzumab Ozogamicin (GO) Inclusion to Induction Chemotherapy Eliminates Leukemic Initiating Cells and Significantly Improves Survival in Mouse Models of Acute Myeloid Leukemia.

Gemtuzumab ozogamicin (GO) is an anti-CD33 antibody-drug conjugate for the treatment of acute myeloid leukemia (AML). Although GO shows a narrow therapeutic window in early clinical studies, recent reports detailing a modified dosing regimen of GO can be safely combined with induction chemotherapy, and the combination provides significant survival benefits in AML patients. Here we tested whether the survival benefits seen with the combination arise from the enhanced reduction of chemoresidual disease and leukemic initiating cells (LICs). Herein, we use cell line and patient-derived xenograft (PDX) AML models to evaluate the combination of GO with daunorubicin and cytarabine (DA) induction chemotherapy on AML blast growth and animal survival. DA chemotherapy and GO as separate treatments reduced AML burden but left significant chemoresidual disease in multiple AML models. The combination of GO and DA chemotherapy eliminated nearly all AML burden and extended overall survival. In two small subsets of AML models, chemoresidual disease following DA chemotherapy displayed hallmark markers of leukemic LICs (CLL1 and CD34). In vivo, the two chemoresistant subpopulations (CLL1+/CD117- and CD34+/CD38+) showed higher ability to self-renewal than their counterpart subpopulations, respectively. CD33 was coexpressed in these functional LIC subpopulations. We demonstrate that the GO and DA induction chemotherapy combination more effectively eliminates LICs in AML PDX models than either single agent alone. These data suggest that the survival benefit seen by the combination of GO and induction chemotherapy, nonclinically and clinically, may be attributed to the enhanced reduction of LICs.

A novel CXCR4 antagonist IgG1 antibody (PF-06747143) for the treatment of hematologic malignancies.

The chemokine receptor CXCR4 is highly expressed and associated with poor prognosis in multiple malignancies. Upon engagement by its ligand, CXCL12, CXCR4 triggers intracellular signaling pathways that control trafficking of cells to tissues where the ligand is expressed, such as the bone marrow (BM). In hematologic cancers, CXCR4-driven homing of malignant cells to the BM protective niche is a key mechanism driving disease and therapy resistance. We developed a humanized CXCR4 immunoglobulin G1 (IgG1) antibody (Ab), PF-06747143, which binds to CXCR4 and inhibits CXCL12-mediated signaling pathways, as well as cell migration. In in vivo preclinical studies, PF-06747143 monotherapy rapidly and transiently mobilized cells from the BM into the peripheral blood. In addition, PF-06747143 effectively induced tumor cell death via its Fc constant region-mediated effector function. This Fc-mediated cell killing mechanism not only enhanced antitumor efficacy, but also played a role in reducing the duration of cell mobilization, when compared with an IgG4 version of the Ab, which does not have Fc-effector function. PF-06747143 treatment showed strong antitumor effect in multiple hematologic tumor models including non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), and multiple myeloma (MM). Importantly, PF-06747143 synergized with standard-of-care agents in a chemoresistant AML patient-derived xenograft model and in an MM model. These findings suggest that PF-06747143 is a potential best-in-class anti-CXCR4 antagonist for the treatment of hematologic malignancies, including in the resistant setting. PF-06747143 is currently in phase 1 clinical trial evaluation (registered at www.clinicaltrials.gov as #NCT02954653).

Comparison of dynamic contrast-enhanced MR, ultrasound and optical imaging modalities to evaluate the antiangiogenic effect of PF-03084014 and sunitinib.

Noninvasive imaging has been widely applied for monitoring antiangiogenesis therapy in cancer drug discovery. In this report, we used different imaging modalities including high-frequency ultrasound (HFUS), dynamic contrast enhanced-MR (DCE-MR), and fluorescence molecular tomography (FMT) imaging systems to monitor the changes in the tumor vascular properties after treatment with γ-secretase inhibitor PF-03084014. Sunitinib was tested in parallel for comparison. In the MDA-MB-231Luc model, we demonstrated that antiangiogenesis was one of the contributing mechanisms for the therapeutic effect of PF-03084014. By immunohistochemistry and FITC-lectin perfusion assays, we showed that the vascular defects upon treatment with PF-03084014 were associated with Notch pathway modulation, evidenced by a decrease in the HES1 protein and by the changes in VEGFR2 and HIF1α levels, which indicates down-stream effects. Using a 3D power Doppler scanning method, ultrasound imaging showed that the% vascularity in the MDA-MB-231Luc tumor decreased significantly at 4 and 7 days after the treatment with PF-03084014. A decrease in the tumor vessel function was also observed through contrast-enhanced ultrasound imaging with microbubble injection. These findings were consistent with the PF-03084014-induced functional vessel changes measured by suppressing the K(trans) values using DCE-MRI. In contrast, the FMT imaging with the AngioSence 680EX failed to detect any treatment-associated tumor vascular changes. Sunitinib demonstrated an outcome similar to PF-03084014 in the tested imaging modalities. In summary, ultrasound and DCE-MR imaging successfully provided longitudinal measurement of the phenotypic and functional changes in tumor vasculature after treatment with PF-03084014 and sunitinib.

Monitoring metabolic responses to chemotherapy in single cells and tumors using nanostructure-initiator mass spectrometry (NIMS) imaging.

BACKGROUND: Tissue imaging of treatment-induced metabolic changes is useful for optimizing cancer therapies, but commonly used methods require trade-offs between assay sensitivity and spatial resolution. Nanostructure-Initiator Mass Spectrometry imaging (NIMS) permits quantitative co-localization of drugs and treatment response biomarkers in cells and tissues with relatively high resolution. The present feasibility studies use NIMS to monitor phosphorylation of 3'-deoxy-3'-fluorothymidine (FLT) to FLT-MP in lymphoma cells and solid tumors as an indicator of drug exposure and pharmacodynamic responses. METHODS: NIMS analytical sensitivity and spatial resolution were examined in cultured Burkitt's lymphoma cells treated briefly with Rapamycin or FLT. Sample aliquots were dispersed on NIMS surfaces for single cell imaging and metabolic profiling, or extracted in parallel for LC-MS/MS analysis. Docetaxel-induced changes in FLT metabolism were also monitored in tissues and tissue extracts from mice bearing drug-sensitive tumor xenografts. To correct for variations in FLT disposition, the ratio of FLT-MP to FLT was used as a measure of TK1 thymidine kinase activity in NIMS images. TK1 and tumor-specific luciferase were measured in adjacent tissue sections using immuno-fluorescence microscopy. RESULTS: NIMS and LC-MS/MS yielded consistent results. FLT, FLT-MP, and Rapamycin were readily detected at the single cell level using NIMS. Rapid changes in endogenous metabolism were detected in drug-treated cells, and rapid accumulation of FLT-MP was seen in most, but not all imaged cells. FLT-MP accumulation in xenograft tumors was shown to be sensitive to Docetaxel treatment, and TK1 immunoreactivity co-localized with tumor-specific antigens in xenograft tumors, supporting a role for xenograft-derived TK1 activity in tumor FLT metabolism. CONCLUSIONS: NIMS is suitable for monitoring drug exposure and metabolite biotransformation with essentially single cell resolution, and provides new spatial and functional dimensions to studies of cancer metabolism without the need for radiotracers or tissue extraction. These findings should prove useful for in vitro and pre-clinical studies of cancer metabolism, and aid the optimization of metabolism-based cancer therapies and diagnostics.

Synergistic effect of the γ-secretase inhibitor PF-03084014 and docetaxel in breast cancer models.

Notch signaling mediates breast cancer cell survival and chemoresistance. In this report, we aimed to evaluate the antitumor efficacy of PF-03084014 in combination with docetaxel in triple-negative breast cancer models. The mechanism of action was investigated. PF-03084014 significantly enhanced the antitumor activity of docetaxel in multiple xenograft models including HCC1599, MDA-MB-231Luc, and AA1077. Docetaxel activated the Notch pathway by increasing the cleaved Notch1 intracellular domain and suppressing the endogenous Notch inhibitor NUMB. PF-03084014 used in combination with docetaxel reversed these effects and demonstrated early-stage synergistic apoptosis. Docetaxel elicited chemoresistance by elevating cytokine release and expression of survivin and induced an endothelial mesenchymal transition (EMT) phenotype by increasing the expressions of Snail, Slug, and N-cadherin. When reimplanted, the docetaxel-residual cells not only became much more tumorigenic, as evidenced by a higher fraction of tumor-initiating cells (TICs), but also showed higher metastatic potential compared with nontreated cells, leading to significantly shortened survival. In contrast, PF-03084014 was able to suppress expression of survivin and MCL1, reduce ABCB1 and ABCC2, upregulate BIM, reverse the EMT phenotype, and diminish the TICs. Additionally, the changes to the ALDH(+) and CD133(+)/CD44(+) subpopulations following therapy corresponded with the TIC self-renewal assay outcome. In summary, PF-03084014 demonstrated synergistic effects with docetaxel through multiple mechanisms. This work provides a strong preclinical rationale for the clinical utility of PF-03084014 to improve taxane therapy.

Biomarker and pharmacologic evaluation of the γ-secretase inhibitor PF-03084014 in breast cancer models.

PURPOSE: We aimed to assess the biologic activity of PF-03084014 in breast xenograft models. The biomarkers for mechanism and patient stratification were also explored. EXPERIMENTAL DESIGN: The in vitro and in vivo properties of PF-03084014 were investigated. The mRNA expressions of 40 key Notch pathway genes at baseline or after treatment were analyzed to link with the antitumor efficacy of PF-03084014 in a panel of breast cancer xenograft models. RESULTS: In vitro, PF-03084014 exhibited activity against tumor cell migration, endothelial cell tube formation, and mammosphere formation. In vivo, we observed apoptosis, antiproliferation, reduced tumor cell self-renewal ability, impaired tumor vasculature, and decreased metastasis activity after the treatment of PF-03084014. PF-03084014 treatment displayed significant antitumor activity in 10 of the 18 breast xenograft models. However, the antitumor efficacy in most models did not correlate with the in vitro antiproliferation results in the corresponding cell lines, suggesting the critical involvement of tumor microenvironment during Notch activation. In the tested breast xenograft models, the baseline expressions of the Notch receptors, ligands, and the cleaved Notch1 failed to predict the antitumor response to PF-03084014, whereas several Notch pathway target genes, including HEY2, HES4, and HES3, strongly corresponded with the response with a P value less than 0.01. Many of the best molecular predictors of response were also significantly modulated following PF-03084014 treatment. CONCLUSIONS: PF-03084014 showed antitumor and antimetastatic properties via pleiotropic mechanisms. The Notch pathway downstream genes may be used to predict the antitumor activity of PF-03084014 and enrich for responders among breast cancer patients.

[(18)F]FLT-PET imaging does not always "light up" proliferating tumor cells.

PURPOSE: [(18)F]FLT (3'-Fluoro-3' deoxythymidine)-PET imaging was proposed as a tool for measuring in vivo tumor cell proliferation. The aim of this article was to validate the use of [(18)F]FLT-PET imaging for measuring xenograft proliferation and subsequent monitoring of targeted therapy. EXPERIMENTAL DESIGN: In exponentially growing xenografts, factors that could impact the outcome of [(18)F]FLT-PET imaging, such as nucleoside transporters, thymidine kinase 1, the relative contribution of DNA salvage pathway, and the ratio of FLT to thymidine, were evaluated. The [(18)F]FLT tracer avidity was compared with other proliferation markers. RESULTS: In a panel of proliferating xenografts, [(18)F]FLT or [(3)H]thymidine tracer avidity failed to reflect the tumor growth rate across different tumor types, despite the high expressions of Ki67 and TK1. When FLT was injected at the same dose level as used in the preclinical [(18)F]FLT-PET imaging, the plasma exposure ratio of FLT to thymidine was approximately 1:200. Thymidine levels in different tumor types seemed to be variable and exhibited an inverse relationship with the FLT tracer avidity. In contrast, high-dose administration of bromdeoxyuridine (BrdUrd; 50 mg/kg) yielded a plasma exposure of more than 4-fold higher than thymidine and leads to a strong correlation between the BrdUrd uptake and the tumor proliferation rate. In FLT tracer-avid models, [(18)F]FLT-PET imaging as a surrogate biomarker predicted the therapeutic response of CDK4/6 inhibitor PD-0332991. CONCLUSIONS: Tumor thymidine level is one of the factors that impact the correlation between [(18)F]FLT uptake and tumor cell proliferation. With careful validation, [(18)F]FLT-PET imaging can be used to monitor antiproliferative therapies in tracer-avid malignancies.

PF-03732010: a fully human monoclonal antibody against P-cadherin with antitumor and antimetastatic activity.

PURPOSE: P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion, proliferation, and invasiveness. We characterized the biological properties of PF-03732010, a human monoclonal antibody against P-cadherin, in cell-based assays and tumor models. EXPERIMENTAL DESIGN: The affinity, selectivity, and cellular inhibitory activity of PF-03732010 were tested in vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumor and antimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were also investigated. RESULTS: PF-03732010 selectively inhibits P-cadherin-mediated cell adhesion and aggregation in vitro. In the P-cadherin-overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-435HAL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth of primary tumors and metastatic progression, as determined by bioluminescence imaging. Computed tomography imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of PF-03732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the counterpart tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence, immunohistochemical analyses, and 3'-[(18)F]fluoro-3'-deoxythymidine-positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane ß-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3'-[(18)F]fluoro-3'-deoxythymidine tracer uptake also indicated antiproliferative activity and increased apoptosis in the tested xenografts. CONCLUSIONS: These findings suggest that interrupting the P-cadherin signaling pathway may be a novel therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clinical trials.

PF-00477736 mediates checkpoint kinase 1 signaling pathway and potentiates docetaxel-induced efficacy in xenografts.

PURPOSE: Checkpoint kinase 1 (Chk1) plays a critical role in the activation of mitotic spindle checkpoint and DNA damage checkpoint. We examined the preclinical use of the Chk1 inhibitor PF-00477736 as a docetaxel-sensitizing agent. Specifically, we investigated the correlation between PF-00477736-mediated modulation of biomarkers and the sensitization of docetaxel efficacy. EXPERIMENTAL DESIGN: In vitro and in vivo studies using COLO205 and other cell lines were done to assess PF-00477736-induced enhancement of docetaxel efficacy and effects on associated biomarkers. RESULTS: PF-00477736 significantly enhanced the docetaxel-induced efficacy in tumor cells and xenografts. Docetaxel induced dose- and time-dependent increase in the levels of phosphorylated Chk1 (Ser(345)), phosphorylated histone H3 (Ser(10)), and gammaH2AX foci and promoted the cytoplasmic localization of phosphorylated Cdc25C (Ser(216)). PF-00477736 cotreatment suppressed docetaxel-induced changes in phosphorylated histone H3 and cytoplasmic phosphorylated Cdc25C (Ser(216)) levels and concurrently sensitized the docetaxel-induced apoptosis. Docetaxel alone or in combination with PF-00477736 induced significant antiproliferative activity in xenografts, shown via [18F]FLT-PET imaging. However, changes in [18F]FLT uptake did not reflect the potentiation of docetaxel efficacy. In contrast, bioluminescence imaging showed that PF-00477736 sensitized docetaxel-induced suppression of tumor survival. CONCLUSIONS: Docetaxel triggers mitotic spindle checkpoint activation at low concentrations and activates both the DNA damage checkpoint and the spindle checkpoint at high concentrations. In combination with docetaxel, PF-00477736 abrogates the mitotic checkpoint, as well as the DNA damage checkpoint, and results in sensitization to docetaxel. Chk1 inhibitor PF-00477736 offers a therapeutic potential for the enhancement of taxane therapy.

Advancing bioluminescence imaging technology for the evaluation of anticancer agents in the MDA-MB-435-HAL-Luc mammary fat pad and subrenal capsule tumor models.

PURPOSE: Tumors grafted s.c. or under the mammary fat pad (MFP) rarely develop efficient metastasis. By applying bioluminescence imaging (BLI) technology, the MDA-MB-435-HAL-Luc subrenal capsule (SRC) model was compared with the MFP model for disease progression, metastatic potential, and response to therapy. EXPERIMENTAL DESIGN: The luciferase-expressing MDA-MB-435-HAL-Luc cell line was used in both MFP and SRC models. BLI technology allowed longitudinal assessment of disease progression and the therapeutic response to PD-0332991, Avastin, and docetaxel. Immunohistochemical analysis of Ki67 and CD31 staining in the primary tumors was compared in these models. Caliper measurement was used in the MFP model to validate the BLI quantification of primary tumors. RESULTS: The primary tumors in MDA-MB-435-HAL-Luc MFP and SRC models displayed comparable growth rates and vascularity. However, tumor-bearing mice in the SRC model developed lung metastases much earlier (4 weeks) than in the MFP model (>7 weeks), and the metastatic progression contributed significantly to the survival time. In the MFP model, BLI and caliper measurements were comparable for quantifying palpable tumors, but BLI offered an advantage for detecting the primary tumors that fell below a palpable threshold and for visualizing metastases. In the SRC model, BLI allowed longitudinal assessment of the antitumor and antimetastatic effects of PD-0332991, Avastin, and docetaxel, and the results correlated with the survival benefits of these agents. CONCLUSIONS: The MDA-MB-435-HAL-Luc SRC model and the MFP model displayed differences in disease progression. BLI is an innovative approach for developing animal models and creates opportunities for improving preclinical evaluations of anticancer agents.

Pharmacologic properties of AG-012986, a pan-cyclin-dependent kinase inhibitor with antitumor efficacy.

AG-012986 is a multitargeted cyclin-dependent kinase (CDK) inhibitor active against CDK1, CDK2, CDK4/6, CDK5, and CDK9, with selectivity over a diverse panel of non-CDK kinases. Here, we report the potent antitumor efficacies of AG-012986 against multiple tumor lines in vitro and in vivo. AG-012986 showed antiproliferative activities in vitro with IC(50)s of <100 nmol/L in 14 of 18 tumor cell lines. In vivo, significant antitumor efficacy induced by AG-012986 was seen (tumor growth inhibition, >83.1%) in 10 of 11 human xenograft tumor models when administered at or near the maximum tolerated dose for 8 or 12 days. AG-012986 caused dose-dependent hypophosphorylation at Ser(795) of the retinoblastoma protein, cell cycle arrest, and apoptosis in vitro. Colony-forming assays indicated that the potency of AG-012986 substantially decreased with treatment time of <24 h. In vivo, AG-012986 also showed dose-dependent retinoblastoma Ser(795) hypophosphorylation, cell cycle arrest, decreased Ki-67 tumor staining, and apoptosis in conjunction with antitumor activity. Studies comparing i.p. bolus with s.c. implanted minipump dosing regimens revealed that in vivo efficacy correlated with the duration of minimally effective plasma levels rather than maximal drug plasma levels. Dosing optimization of AG-012986 provided guidance for selecting a treatment schedule to achieve the best antitumor efficacy while minimizing the risk of adverse side effects.