Christopher Riley

A novel tyrosine kinase switch is a mechanism of imatinib resistance in gastrointestinal stromal tumors

KIT or α-platelet-derived growth factor receptor (α-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase – AXL – in a ‘kinase switch’. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase–polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.

Transcript profiling in peripheral T-cell lymphoma, not otherwise specified, and diffuse large B-cell lymphoma identifies distinct tumor profile signatures

To glean biological differences and similarities of peripheral T-cell lymphoma–not otherwise specified [PTCL-NOS] to diffuse large B-cell lymphoma (DLBCL), a transcriptosome analysis was done on five PTCL-NOS and four DLBCL patients and validated by quantitative real-time reverse transcription-PCR on 10 selected genes. Normal peripheral blood T cells, peripheral blood B cells, and lymph node were used as controls. The resultant gene expression profile delineated distinct “tumor profile signatures” for PTCL-NOS and DLBCL. Several highly overexpressed genes in both PTCL-NOS and DLBCL involve the immune network, stroma, angiogenesis, and cell survival cascades that make important contributions to lymphomagenesis. Inflammatory chemokines and their receptors likely play a central role in these complex interrelated pathways: CCL2 and CXCR4 in PTCL-NOS and CCL5 and CCR1 in DLBCL. Highly overexpressed oncogenes unique to PTCL-NOS are SPI1, STK6, α-PDGFR, and SH2D1A, whereas in DLBCL they are PIM1, PIM2, LYN, BCL2A1, and RAB13. Oncogenes common to both lymphomas are MAFB, MET, NF-κB2, LCK, and LYN. Several tumor suppressors are also down-regulated (TPTE, MGC154, PTCH, ST5, and SUI1). This study illustrates the relevance of tumor-stroma immune trafficking and identified potential novel prognostic markers and targets for therapeutic intervention. [Mol Cancer Ther 2005;4(12):1867–79]

MP470, a novel receptor tyrosine kinase inhibitor, in combination with Erlotinib inhibits the HER family/PI3K/Akt pathway and tumor growth in prostate cancer

BackgroundProstate cancer is a common disease in men and at present there is no effective therapy available due to its recurrence despite androgen deprivation therapy. The epidermal growth factor receptor family (EGFR/HER1, HER2/neu and HER3)/PI3K/Akt signaling axis has been implicated in prostate cancer development and progression. However, Erlotinib, an EGFR tyrosine kinase inhibitor, has less effect on proliferation and apoptosis in prostate cancer cell lines. In this study, we evaluate whether MP470, a novel receptor tyrosine kinase inhibitor alone or in combination with Erlotinib has inhibitory effect on prostate cancer in vitro and in vivo.MethodsThe efficacy of MP470 or MP470 plus Erlotinib was evaluated in vitro using three prostate cancer cell lines by MTS and apoptosis assays. The molecular mechanism study was carried out by phosphorylation antibody array, immunoblotting and immunohistochemistry. A LNCaP mouse xenograft model was also used to determine the tumor growth inhibition by MP470, Erlotinib or the combination treatments.ResultsMP470 exhibits low μM IC50 in prostate cancer cell lines. Additive effects on both cytotoxicity and induction of apoptosis were observed when LNCaP were treated with MP470 in combination with Erlotinib. This combination treatment completely inhibited phosphorylation of the HER family members (HER1, 2, 3), binding of PI3K regulatory unit p85 to HER3 and downstream Akt activity even after androgen depletion. Furthermore, in a LNCaP mouse xenograft model, the MP470-Erlotinib combination produced 30–65% dose-dependent tumor growth inhibition (TGI).ConclusionWe propose that MP470-Erlotinib targets the HER family/PI3K/Akt pathway and may represent a novel therapeutic strategy for prostate cancer.

Apoptosis resistance in Barrett's esophagus: Ex vivo bioassay of live stressed tissues

BACKGROUND AND AIMS:Barretts esophagus (BE) is a premalignant lesion of the distal esophagus in which squamous epithelial cells are replaced by metaplastic intestinal-like columnar epithelium that contains goblet cells. The factors that contribute to the progression from normal squamous mucosa to BE, Barretts dysplasia, and adenocarcinoma are not well understood at the molecular level. Since reflux of bile acids is associated with BE development, we speculate that cells with an apoptosis-resistant phenotype are selected after long-term repeated exposure to pulses of bile acids. This will result in the survival of cells with unrepaired DNA damage, and a consequent increase in genomic instability leading to cancer progression. The major goal of this study is to compare sensitivity to apoptosis induced by the bile acid, deoxycholate (DOC), a known inducer of apoptosis, in normal esophageal squamous epithelium, normal colon epithelium, and BE.METHODS:Thirteen patients with a confirmed diagnosis of BE and four patients who had undergone clinically indicated colectomy were included in the present study. Freshly obtained biopsies were incubated with control medium or medium supplemented with 1 mM DOC for 3 h and then evaluated for apoptotic changes using transmission electron microscopy and immunohistochemical staining for two apoptotic markers, cleaved caspase 3 and cleaved cytokeratin 18.RESULTS:Our results indicate that BE is resistant to apoptosis induced by DOC compared to esophageal squamous epithelium and normal colon epithelium. In addition, electron micrographs revealed mitochondrial swelling in squamous epithelial cells treated ex vivo with DOC, which was absent in epithelial cells of BE. Formation of swollen mitochondria is an early marker of apoptotic cell death. Altogether, the data indicate that reduced apoptosis capability in BE tissue may contribute to progression to esophageal adenocarcinoma.

Design and Activity of a Murine and Humanized Anti-CEACAM6 Single-Chain Variable Fragment in the Treatment of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease, with surgery being the only curative modality for localized disease, and gemcitabine with or without erlotinib remains the standard of therapy for unresectable or metastatic disease. CEACAM6 is overexpressed in human PDA independent of stage or grade and causes anoikis resistance when dysregulated. Because murine monoclonal antibody 13-1 possesses target-specific cytotoxicity in human PDA cell lines, we designed a humanized anti-CEACAM6 single-chain variable fragment (scFv) based on monoclonal antibody 13-1. PEGylation of the glycine-serine linker was used to enhance plasma half-life. These scFvs bound CEACAM6 with high affinity, exhibited cytotoxic activity, and induced dose-dependent poly(ADP-ribose) polymerase cleavage. Murine PDA xenograft models treated with humanized scFv alone elicited tumor growth inhibition, which was enhanced in combination with gemcitabine. Immunohistochemistry showed significant apoptosis, with inhibition of angiogenesis and proliferation, and preservation of the target. Collectively, our results have important implications for the development of novel antibody-based therapies against CEACAM6 in PDA.

Gene Expression and Serum Cytokine Profiling of Low Stage CLL Identify WNT/PCP, Flt-3L/Flt-3 and CXCL9/CXCR3 as Regulators of Cell Proliferation, Survival and Migration.

Gene expression profiling (GEP) of 8 stage 0/I untreated Chronic Lymphocytic Leukemia (CLL) patients showed over-expression of Frizzled 3 (FZD3)/ROR-1 receptor tyrosine kinase (RTK), FLT-3 RTK and CXCR3 G-protein coupled receptor (GPCR). RT-PCR of 24 genes in 21 patients of the WNT pathway corroborated the GEP. Transforming growth factorβ, fibromodulin, TGFβRIII and SMAD2 are also over-expressed by GEP. Serum cytokine profiling of 26 low stage patients showed elevation of IFNγ, CSF3, Flt-3L and insulin-like growth factor binding protein 4. In order to ascertain why CLL cells grow poorly in culture, a GEP of 4 CLL patients cells at 0 hr and 24 hr in culture demonstrated over expression of CXCL5, CCL2 and CXCL3, that may recruit immune cells for survival. Treatment with thalidomide, an immunomodulatory agent, showed elevation of CCL5 by GEP but was not cytotoxic to CLL cells. Our data suggest an interplay of several oncogenic pathways, cytokines and immune cells that promote a survival program in CLL.

Monoclonal Antibody Therapies Targeting Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a poor prognosis where incidence mirrors mortality. Gemcitabine and gemcitabine plus erlotinib (epidermal growth factor receptor tyrosine kinase inhibitor) are the only FDA approved therapies for unresectable or metastatic PDA and are at best palliative. Hence, considerable efforts have been initiated to identify novel targets for monoclonal antibody (Mab) therapies that may safely and effectively be combined with gemcitabine. Mabs to cell surface receptors and/or their ligands have shown efficacy in pre-clinical and clinical studies in both solid and hematological malignancies and can safely be given with chemotherapy. A number of clinical trials have evaluated the safety and efficacy of Mabs targeting the tumor and/or tumor micro-environment and in combination with chemotherapy for PDA with very little success. Here we review the rationale for Mab therapies, targeted clinical trials, rational basis for target selection, pre-clinical models and promising novel cell surface targets and/or growth factor ligands that are amenable to ongoing and future Mab therapies that hold promise and hope for patients and their families with this devastating disease.

Genetic and cytokine changes associated with symptomatic stages of CLL.

The pathogenesis and drug resistance of symptomatic CLL patients involves genetic changes associated with the CLL clone as well as changes within the microenvironment. To further understand these processes, we compared early stage CLL to symptomatic late stage using gene expression and serum cytokine profiling to gain insight of the genetic and microenvironment changes associated with the most severe form of the disease. Patients were classified into low stage (Rai stage 0/I/II) and high stage (Rai stage III/IV). Gene expression profiles were obtained on pretreatment samples using the HG-U133A 2.0 Affymetrix platform. A comparison of low versus high stage CLL revealed a set of 21 genes differentially expressed genes. 15 genes were up regulated in the high stage compared to low stage while 6 genes were down regulated. Analysis of GO molecular function revealed 9 of 21 genes were involved in transcription factor activity. Serum cytokine profiles showed six cytokines to be significantly different in high stage patients. Two chemokines, SDF-1/CXCL12 and uPAR known to be involved in stem cell mobilization and homing were increased in serum of high stage patients. This study has identified therapeutic targets for symptomatic CLL patients.