Laurence Cooke

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.

The c-Met receptor tyrosine kinase inhibitor MP470 radiosensitizes glioblastoma cells

PurposeGlioblastoma multiforme (GBM) is resistant to current cytotoxic therapies, in part because of enhanced DNA repair. Activation of the receptor tyrosine kinase c-Met has been shown to protect cancer cells from DNA damage. We hypothesized that inhibiting c-Met would decrease this protection and thus sensitize resistant tumor cells to the effects of radiation therapy.Materials and methodsEight human GBM cell lines were screened for radiosensitivity to the small-molecule c-Met inhibitor MP470 with colony-count assays. Double-strand (ds) DNA breaks was quantified by using antibodies to gamma H2AX. Western blotting demonstrate expression of RAD51, glycogen synthase kinase (GSK)-3β, and other proteins. A murine xenograft tumor flank model was used for in vivo radiosensitization studies.ResultsMP470 reduced c-Met phosphorylation and enhanced radiation-induced cell kill by 0.4 logs in SF767 cells. Cells pretreated with MP470 had more ds DNA damage than cells treated with radiation alone. Mechanistically, MP470 was shown to inhibit dsDNA break repair and increase apoptosis. MP470 influences various survival and DNA repair related proteins such as pAKT, RAD51 and GSK3β. In vivo, the addition of MP470 to radiation resulted in a tumor-growth-delay enhancement ratio of 2.9 over radiation alone and extended survival time.ConclusionsGBM is a disease site where radiation is often used to address both macroscopic and microscopic disease. Despite attempts at dose escalation outcomes remain poor. MP470, a potent small-molecule tyrosine kinase inhibitor of c-Met, radiosensitized several GBM cell lines both in vitro and in vivo, and may help to improve outcomes for patients with GBM.

Aurora inhibitor MLN8237 in combination with docetaxel enhances apoptosis and anti-tumor activity in mantle cell lymphoma.

Auroras (A and B) are oncogenic serine/threonine kinases that play key roles in the mitotic phase of the eukaryotic cell cycle. Analysis of the leukemia lymphoma molecular profiling project (LLMPP) database indicates Aurora over-expression correlates with poor prognosis. A tissue microarray (TMA) composed of 20 paired mantle cell lymphoma (MCL) patients demonstrated >75% of patients had high levels Aurora expression. Aurora A and B were also found elevated in 13 aggressive B-NHL cell lines. MLN8237, an Aurora inhibitor induced G2/M arrest with polyploidy and abrogated Aurora A and histone-H3 phosphorylation. MLN8237 inhibited aggressive B-NHL cell proliferation at an IC(50) of 10-50 nM and induced apoptosis in a dose- and time-dependent manner. Low dose combinations of MLN8237+docetaxel enhanced apoptosis by ~3-4-fold in cell culture compared to single agents respectively. A mouse xenograft model of MCL demonstrated that MLN8237 (10 or 30 mg/kg) or docetaxel (10mg/kg) alone had modest anti-tumor activity. However, MLN8237 plus docetaxel demonstrated a statistically significant tumor growth inhibition and enhanced survival compared to single agent therapy. Together, our results suggest that MLN8237 plus docetaxel may represent a novel therapeutic strategy that could be evaluated in early phase trials in relapsed/refractory aggressive B-cell NHL.

NSC348884, a nucleophosmin inhibitor disrupts oligomer formation and induces apoptosis in human cancer cells

Nucleophosmin (NPM), a multifunctional nucleolar phosphoprotein is dysregulated in human malignancies leading to anti-apoptosis and inhibition of differentiation. We evaluated the precise three-dimensional structure of NPM based on the highly conserved structure of Xenopus NO38 and its requirement to form dimers and pentamers via its N-terminal domain (residues, 1–107). We hypothesized that a small molecular inhibitor (SMI) that could disrupt the formation of dimers would inhibit aberrant NPM function(s) in cancer cells. Molecular modeling, pharmacophore design, in silico screening and interactive docking identified NSC348884 as a putative NPM SMI that disrupts a defined hydrophobic pocket required for oligomerization. NSC348884 inhibited cell proliferation at an IC50 of 1.7–4.0 μM in distinct cancer cell lines and disrupted NPM oligomer formation by native polyacrylamide gel electrophoresis assay. Treatment of several different cancer cell types with NSC348884 upregulated p53 (increased Ser15 phosphorylation) and induced apoptosis in a dose-dependent manner that correlated with apoptotic markers: H2AX phosphorylation, poly(ADP-ribose) polymerase cleavage and Annexin V labeling. Further, NSC348884 synergized doxorubicin cytotoxicity on cancer cell viability. The data together show that NSC348884 is an SMI of NPM oligomer formation, upregulates p53, induces apoptosis and synergizes with chemotherapy. Hence, an SMI to NPM may be a useful approach to anticancer therapy.

Aurora A inhibitor (MLN8237) plus Vincristine plus Rituximab is synthetic lethal and a potential curative therapy in aggressive B-cell non-Hodgkin lymphoma

Purpose: Aurora A and B are oncogenic serine/threonine kinases that regulate mitosis. Overexpression of Auroras promotes resistance to microtubule-targeted agents. We investigated mechanistic synergy by inhibiting the mitotic spindle apparatus in the presence of MLN8237 [M], an Aurora A inhibitor with either vincristine [MV] or docetaxel [MD] in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The addition of rituximab [R] to MV or MD was evaluated for synthetic lethality. Experimental Design: Aggressive B-NHL cell subtypes were evaluated in vitro and in vivo for target modulation and anti-NHL activity with single agents, doublets, and triplets by analyzing cell proliferation, apoptosis, tumor growth, survival, and mechanisms of response/relapse by gene expression profiling with protein validation. Results: MV is synergistic whereas MD is additive for cell proliferation inhibition in B-NHL cell culture models. Addition of rituximab to MV is superior to MD, but both significantly induce apoptosis compared with doublet therapy. Mouse xenograft models of mantle cell lymphoma showed modest single-agent activity for MLN8237, rituximab, docetaxel, and vincristine with tumor growth inhibition (TGI) of approximately 10% to 15%. Of the doublets, MV caused tumor regression, whereas TGI was observed with MD (approximately 55%–60%) and MR (approximately 25%–50%), respectively. Although MV caused tumor regression, mice relapsed 20 days after stopping therapy. In contrast, MVR was curative, whereas MDR led to TGI of approximately 85%. Proliferation cell nuclear antigen, Aurora B, cyclin B1, cyclin D1, and Bcl-2 proteins of harvested tumors confirmed response and resistance to therapy. Conclusions: Addition of rituximab to MV is a novel therapeutic strategy for aggressive B-NHL and warrants clinical trial evaluation. Clin Cancer Res; 18(8); 2210–9. ©2012 AACR.

Alisertib (MLN8237) an investigational agent suppresses Aurora A and B activity, inhibits proliferation, promotes endo-reduplication and induces apoptosis in T-NHL cell lines supporting its importance in PTCL treatment

Peripheral T-cell lymphomas (PTCL) are a diverse group of rare non-Hodgkin lymphomas (NHL) that carry a poor prognosis and are in need of effective therapies. Alisertib (MLN8237) an investigational agent that inhibits Aurora A Ser/Thr kinase has shown activity in PTCL patients. Here we demonstrate that aurora A and B are highly expressed in T-cell lymphoma cell lines. In PTCL patient samples aurora A was positive in 3 of 24 samples and co-expressed with aurora B. Aurora B was positive in tumor cells in 22 of 32 samples. Of the subtypes of PTCL, aurora B was over-expressed in PTCL (NOS) [73%], T-NHL [100%], ALCL (Alk-Neg) [100%] and AITL [100%]. Treatment with MLN8237 inhibited PTCL cell proliferation in CRL-2396 and TIB-48 cells with an IC50 of 80-100nM. MLN8237 induced endo-reduplication in a dose and time dependent manner in PTCL cell lines leading to apoptosis demonstrated by flow cytometry and PARP-cleavage at concentrations achieved in early phase clinical trials. Moreover, inhibition of HisH3 and aurora A phosphorylation was dose dependent and strongly correlated with endo-reduplication. The data provide a sound rationale for aurora inhibition in PTCL as a therapeutic modality and warrants clinical trial evaluation.

AT7519, a Cyclin-Dependent Kinase Inhibitor, Exerts Its Effects by Transcriptional Inhibition in Leukemia Cell Lines and Patient Samples

AT7519 is a potent inhibitor of several cyclin-dependent kinases and is currently in early phase clinical development. Recently, cyclin-dependent kinases 7, 8, and 9 have been shown to regulate transcription through phosphorylation of RNA polymerase II. B-cell lymphoproliferative disorders, including chronic lymphocytic leukemia, rely on the expression of transcripts with a short half-life, such as Mcl-1, Bcl-2, and XIAP, for survival. Here, we describe the characterization of AT7519 in leukemia cell lines, and compare and contrast the response in cell lines derived from solid tumors. Finally, we use these mechanistic insights to show activity in peripheral blood mononuclear cells isolated from 16 chronic lymphocytic leukemia patients. AT7519 induced apoptosis at concentrations of 100 to 700 nmol/L and was equally effective regardless of Rai stage or known prognostic markers. Short-term treatments (4-6 hours) resulted in inhibition of phosphorylation of the transcriptional marker RNA polymerase II and downregulation of the antiapoptotic protein Mcl-1, with no effect on either XIAP or Bcl-2 levels. The reduction in Mcl-1 protein level was associated with an increase in cleaved poly(ADP-ribose) polymerase. Together the data suggest AT7519 offers a promising treatment for patients with advanced B-cell leukemia. Mol Cancer Ther; 9(4); 920–8. ©2010 AACR.

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.

AT9283, a novel aurora kinase inhibitor, suppresses tumor growth in aggressive B-cell lymphomas†

Aurora kinases are oncogenic serine/threonine kinases that play key roles in regulating the mitotic phase of the eukaryotic cell cycle. Auroras are overexpressed in numerous tumors including B‐cell non‐Hodgkins lymphomas and are validated oncology targets. AT9283, a pan‐aurora inhibitor inhibited growth and survival of multiple solid tumors in vitro and in vivo. In this study, we demonstrated that AT9283 had potent activity against Aurora B in a variety of aggressive B‐(non‐Hodgkin lymphoma) B‐NHL cell lines. Cells treated with AT9283 exhibited endoreduplication confirming the mechanism of action of an Aurora B inhibitor. Also, treatment of B‐NHL cell lines with AT9283 induced apoptosis in a dose and time dependent manner and inhibited cell proliferation with an IC50 < 1 μM. It is well known that inhibition of auroras (A or B) synergistically enhances the effects of microtubule targeting agents such as taxanes and vinca alkaloids to induce antiproliferation and apoptosis. We evaluated whether AT9283 in combination with docetaxel is more efficient in inducing apoptosis than AT9283 or docetaxel alone. At very low doses (5 nM) apoptosis was doubled in the combination (23%) compared to AT9283 or docetaxel alone (10%). A mouse xenograft model of mantle cell lymphoma demonstrated that AT9283 at 15 mg/kg and docetaxel (10 mg/kg) alone had modest anti‐tumor activity. However, AT9283 at 20 mg/kg and AT9283 (15 or 20 mg/kg) plus docetaxel (10 mg/kg) demonstrated a statistically significant tumor growth inhibition and enhanced survival. Together, our results suggest that AT9283 plus docetaxel may represent a novel therapeutic strategy in B‐cell NHL and warrant early phase clinical trial evaluation.

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.