Tumor cells chronically treated with a trastuzumab-maytansinoid antibody-drug conjugate develop varied resistance mechanisms but respond to alternate treatments.

Antibody-drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab-maytansinoid ADC (TM-ADC) at IC80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non-cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab-maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADC-resistant cell models retain sensitivity to other ADCs or standard-of-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance. Mol Cancer Ther; 14(4); 952-63. ©2015 AACR.

Epigenetic reprogramming by tumor-derived EZH2 gain-of-function mutations promotes aggressive 3D cell morphologies and enhances melanoma tumor growth.

In addition to genetic alterations, cancer cells are characterized by myriad epigenetic changes. EZH2 is a histone methyltransferase that is over-expressed and mutated in cancer. The EZH2 gain-of-function (GOF) mutations first identified in lymphomas have recently been reported in melanoma (~2%) but remain uncharacterized. We expressed multiple EZH2 GOF mutations in the A375 metastatic skin melanoma cell line and observed both increased H3K27me3 and dramatic changes in 3D culture morphology. In these cells, prominent morphological changes were accompanied by a decrease in cell contractility and an increase in collective cell migration. At the molecular level, we observed significant alteration of the axonal guidance pathway, a pathway intricately involved in the regulation of cell shape and motility. Furthermore, the aggressive 3D morphology of EZH2 GOF-expressing melanoma cells (both endogenous and ectopic) was attenuated by EZH2 catalytic inhibition. Finally, A375 cells expressing exogenous EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. This study not only demonstrates the first functional characterization of EZH2 GOF mutants in non-hematopoietic cells, but also provides a rationale for EZH2 catalytic inhibition in melanoma.

Osteogenic effects of a potent Src-over-Abl-selective kinase inhibitor in the mouse.

Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC(50) 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1-10 nM). Compound I (≥0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 µM compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, ß (ß-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by ß-CGRP.

Delineation of a cellular hierarchy in lung cancer reveals an oncofetal antigen expressed on tumor-initiating cells.

Poorly differentiated tumors in non-small cell lung cancer (NSCLC) have been associated with shorter patient survival and shorter time to recurrence following treatment. Here, we integrate multiple experimental models with clinicopathologic analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We show that the oncofetal protein 5T4 is expressed on tumor-initiating cells and associated with worse clinical outcome in NSCLC. Coexpression of 5T4 and factors involved in the epithelial-to-mesenchymal transition were observed in undifferentiated but not in differentiated tumor cells. Despite heterogeneous expression of 5T4 in NSCLC patient-derived xenografts, treatment with an anti-5T4 antibody-drug conjugate resulted in complete and sustained tumor regression. Thus, the aggressive growth of heterogeneous solid tumors can be blocked by therapeutic agents that target a subpopulation of cells near the top of the cellular hierarchy.

Design and synthesis of novel diaminoquinazolines with in vivo efficacy for beta-catenin/T-cell transcriptional factor 4 pathway inhibition.

We are introducing a novel series of 2,4-diaminoquinazolines as beta-catenin/Tcf4 inhibitors which were identified by ligand-based design. Here we elucidate the SAR of this series and explain how we were able to improve key molecular properties such as solubility and cLogP leading to compound 9. Analogue 9 exhibited better biological activity and improved physical and pharmacological properties relative to the HTS hit 49. Furthermore, 9 demonstrated good cell growth inhibition against several human colorectal cancer lines such as LoVo and HT29. In addition, treatment with compound 9 led to gene expression changes that overlapped significantly with the transcriptional profile resulting from the pathway inhibition by siRNA knockdown of beta-catenin or Tcf4. Subsequently, 9 was tested for efficacy in a beta-catenin/RKE-mouse xenograft, where it led to more then 50% decrease in tumor volume.

Efficacy and pharmacodynamic effects of bosutinib (SKI-606), a Src/Abl inhibitor, in freshly generated human pancreas cancer xenografts.

Recently, Src tyrosine kinase has emerged as an attractive target for anticancer therapy, and Src is overexpressed in pancreatic cancer. The purpose of the study was to investigate the in vivo efficacy and pharmacodynamic effects of bosutinib (SKI-606), a Src/Abl inhibitor, using a panel of human pancreatic tumor xenografts. Surgically resected human pancreatic tumors were implanted into female nude mice and randomized to bosutinib versus control. Src and other pathways were analyzed by Western Blot, IHC, and Affymetrix U133 Plus 2.0 gene arrays. Of 15 patient tumors, 3 patient tumors were found to be sensitive to bosutinib, defined as tumor growth of <45% than that of control tumors. There were no definite differences between sensitive and resistant tumors in the baseline Src kinase pathway protein expression assessed by Western Blot. Caveolin-1 expression, as assessed by reverse transcription-PCR and immunohistochemistry, was frequently higher in sensitive cases. In sensitive tumors, bosutinib resulted in increased apoptosis. Phosphorylation of key signaling molecules downstream of Src, signal transducers and activators of transcription 3, and signal transducers and activators of transcription 3, were significantly inhibited by bosutinib. K-Top Scoring Pairs analysis of gene arrays gave a six-gene classifier that predicted resistance versus sensitivity in six validation cases. These results may aid the clinical development of bosutinib and other Src inhibitors in pancreas cancer.

IkappaBalpha kinase inhibitor IKI-1 conferred tumor necrosis factor alpha sensitivity to pancreatic cancer cells and a xenograft tumor model.

Tumor necrosis factor alpha (TNFalpha) has been used to treat patients with certain tumor types. However, its antitumor activity has been undermined by the activation of IkappaBalpha kinase (IKK), which in turn activates nuclear factor-kappaB (NF-kappaB) to help cancer cells survive. Therefore, inhibition of TNFalpha-induced IKK activity with specific IKK inhibitor represents an attractive strategy to treat cancer patients. This study reveals IKI-1 as a potent small molecule inhibitor of IKKalpha and IKKbeta, which effectively blocked TNFalpha-mediated IKK activation and subsequent NF-kappaB activity. Using gene profiling analysis, we show that IKI-1 blocked most of the TNFalpha-mediated mRNA expression, including many genes that play important roles in cell survival. We further show that in vitro and in vivo combination of TNFalpha with IKI-1 had superior potency than either agent alone. This increased potency was due primarily to the increased apoptosis in the presence of both TNFalpha and IKI-1. Additionally, IKKbeta small interfering RNA transfected cells were more sensitive to the treatment of TNFalpha. The study suggests that the limited efficacy of TNFalpha in cancer treatment was due in part to the activation of NF-kappaB, allowing tumor cells to escape apoptosis. Therefore, the combination of IKI-1 with TNFalpha may improve the efficacy of TNFalpha for certain tumor types.

Prenatal choline availability modulates hippocampal and cerebral cortical gene expression.

An increased supply of the essential nutrient choline during fetal development [embryonic day (E) 11-17] in rats causes life-long improvements in memory performance, whereas choline deficiency during this time impairs certain aspects of memory. We analyzed mRNA expression in brains of prenatally choline-deficient, choline-supplemented, or control rats of various ages [postnatal days (P) 1 to 34 for hippocampus and E16 to P34 for cortex] using oligonucleotide microarrays and found alterations in gene expression levels evoked by prenatal choline intake that were, in most cases, transient occurring during the P15-P34 period. We selected a subset of genes, encoding signaling proteins, and verified the microarray data by reverse transcriptase-polymerase chain reaction analyses. Prenatally choline-supplemented rats had the highest expression of calcium/calmodulin (CaM)-dependent protein kinase (CaMK) I and insulin-like growth factor (IGF) II (Igf2) in the cortex and of the transcription factor Zif268/EGR1 in the cortex and hippocampus. Prenatally choline deficient rats had the highest expression of CaMKIIbeta, protein kinase Cbeta2, and GABA(B) receptor 1 isoforms c and d in the hippocampus. Similar changes in the expression of the proteins encoded by these genes were observed using immunoblot analyses. These data show that the prenatal supply of choline causes multiple modifications in the developmental patterns of expression of genes known to influence learning and memory and provide molecular correlates for the cognitive changes evoked by altered availability of choline in utero.

Bone morphogenetic protein 9 induces the transcriptome of basal forebrain cholinergic neurons.

Basal forebrain cholinergic neurons (BFCN) participate in processes of learning, memory, and attention. Little is known about the genes expressed by BFCN and the extracellular signals that control their expression. Previous studies showed that bone morphogenetic protein (BMP) 9 induces and maintains the cholinergic phenotype of embryonic BFCN. We measured gene expression patterns in septal cultures of embryonic day 14 mice and rats grown in the presence or absence of BMP9 by using species-specific microarrays and validated the RNA expression data of selected genes by immunoblot and immunocytochemistry analysis of their protein products. BMP9 enhanced the expression of multiple genes in a time-dependent and, in most cases, reversible manner. The set of BMP9-responsive genes was concordant between mouse and rat and included genes encoding cell-cycle/growth control proteins, transcription factors, signal transduction molecules, extracellular matrix, and adhesion molecules, enzymes, transporters, and chaperonins. BMP9 induced the p75 neurotrophin receptor (NGFR), a marker of BFCN, and Cntf and Serpinf1, two trophic factors for cholinergic neurons, suggesting that BMP9 creates a trophic environment for BFCN. To determine whether the genes induced by BMP9 in culture were constituents of the BFCN transcriptome, we purified BFCN from embryonic day 18 mouse septum by using fluorescence-activated cell sorting of NGFR(+) cells and profiled mRNA expression of these and NGFR(-) cells. Approximately 30% of genes induced by BMP9 in vitro were overexpressed in purified BFCN, indicating that they belong to the BFCN transcriptome in situ and suggesting that BMP signaling contributes to maturation of BFCN in vivo.

Bacterial LPS and CpG DNA differentially induce gene expression profiles in mouse macrophages.

Bacterial DNA containing unmethylated CpG dinucleotides (CpG DNA) is a potent immune stimulating agent that holds strong promise in the treatment of many disorders. Studies have established that CpG DNA triggers an immune response through activated expression of genes in immune cells including macrophages. To dissect further the molecular mechanism(s) by which CpG DNA activates the immune system, we studied macrophage gene expression profiles in response to CpG DNA using microarray technology. Since CpG DNA is reported to use the TLR9 receptor that shares homology with the TLR4 receptor used by bacterial lipopolysaccharide (LPS), we also evaluated gene expression profiles in macrophages stimulated by LPS versus CpG DNA. Both CpG DNA and LPS modulate expression of a large array of genes. However, LPS modulated the expression of a much greater number of genes than did CpG DNA and all genes induced or repressed by CpG DNA were also induced or repressed by LPS. These data indicate that the CpG DNA signaling pathway through TLR9 activates only a subset of genes induced by the LPS TLR4 signaling pathway.

Regulation of gene expression in mouse macrophages stimulated with bacterial CpG-DNA and lipopolysaccharide.

CpG-DNA is known as a potent immunostimulating agent and may contribute in therapeutic treatment of many immune disorders. CpG-DNA triggers innate and acquired immune responses through activated expression of various genes in immune cells, including macrophages. To define the molecular mechanism(s) by which CpG-DNA activates immune cells, we studied macrophage gene expression following CpG-DNA exposure using high-density oligonucleotide microarrays. As CpG-DNA receptor Toll-like receptor 9 (TLR9) shares homology with the lipopolysaccharide (LPS)-TLR4 receptor, we compared gene expression profiles in macrophages stimulated by LPS versus CpG-DNA. CpG-DNA and LPS modulate expression of many genes encoding cytokines, cell surface receptors, transcription factors, and proteins related to cell proliferation/differentiation. However, LPS modulated expression of significantly more genes than did CpG-DNA, and all genes induced or repressed by CpG-DNA were induced or repressed by LPS. We conclude that CpG-DNA signaling through TLR9 activates a subset of genes induced by LPS-TLR4 signaling.