Christine M. Stellrecht

MET receptor tyrosine kinase as a therapeutic anticancer target

Tyrosine kinases are frequently deregulated in cancer either by constitutive activation, mutation, or over-expression. Though they are often associated with an aggressive phenotype they are also proving to be a druggable target. Activation of the MET receptor tyrosine kinase promotes cell proliferation, scattering, invasion, survival, and angiogenesis. Deregulation of MET promotes tumor formation, growth, progression, metastasis, and therapeutic resistance. Because MET is a player in so many aspects of cancer development and progression, it is a strong candidate for targeted therapy. Numerous agents have been developed that are able to target MET expression and/or function and are the focus of this review.

RNA-directed agent, cordycepin, induces cell death in multiple myeloma cells

Multiple myeloma (MM) is an incurable plasma cell malignancy that is slow‐growing, and thus traditional DNA‐replication directed chemotherapeutics are ineffective. We hypothesized that those agents that target RNA‐directed processes would be successful in MM. To test this postulate, cordycepin, a polyadenylation inhibitor was used as a proof‐of‐principle towards MM cell lines. Cordycepin accumulated in MM.1S cells as its triphosphate metabolite, 3′dATP and subsequently inhibits RNA synthesis and cell growth. Cell death was via apoptosis induction and over 50% of treated cells were annexin‐V positive after 48 h. As a consequence of RNA synthesis inhibition, we hypothesized that specific genes with short half‐lives may be downregulated, leading to a reduction in protein. Indeed, a reduction in the transcript levels for MET, a survival gene for MM, was detected as early as 4 h and transcripts were reduced to c. 10% of control after 48 h. Interestingly, no significant change in protein levels was observed for Bcl‐2, XIAP, Mcl‐1 or survivin. Stabilization of p53 was not observed, and caspases‐8, ‐9 and ‐3 showed activation following cordycepin treatment but were not required for cell death. Our results suggest that RNA‐directed agents may be a new group of agents for the treatment of MM.

Multiple Myeloma Cell Killing by Depletion of the MET Receptor Tyrosine Kinase

Multiple myeloma (MM) is an invariably fatal plasma cell malignancy, primarily due to the therapeutic resistance which ultimately arises. Much of the resistance results from the expression of various survival factors. Despite this, the ribonucleoside analogue, 8-chloro-adenosine (8-Cl-Ado), is cytotoxic to a number of MM cell lines. Previously, we established that the analogue incorporates into the RNA and inhibits mRNA synthesis. Because 8-Cl-Ado is able to overcome survival signals present in MM cells and inhibits mRNA synthesis, it is likely that the drug induces cytotoxicity by depleting the expression of critical MM survival genes. We investigated this question using gene array analysis, real-time reverse transcription-PCR, and immunoblot analysis on 8-Cl-Ado-treated MM.1S cells and found that the mRNA and protein levels of the receptor tyrosine kinase MET decrease prior to apoptosis. To determine METs role in 8-Cl-Ado cytotoxicity, we generated MM.1S clones stably expressing a MET ribozyme. None of the clones expressed <25% of the basal levels of MET mRNA, suggesting that a threshold level of MET is necessary for their survival. Additionally, the ribozyme knockdown lines were more sensitive to the cytotoxic actions of 8-Cl-Ado as caspase-3 activation and the induction of poly-ADP-ribose polymerase (PARP) cleavage were more pronounced and evident 12 h earlier than in the parental cells. We further established METs role in MM cell survival by demonstrating that a retroviral MET RNA interference construct induces PARP cleavage in MM.1S cells. These results show that MET provides a survival mechanism for MM cells. 8-Cl-Ado overcomes MM cell survival by a mechanism that involves the depletion of MET.

Modulation of PAX6 Homeodomain Function by the Paired Domain

PAX6 is required for proper development of the eye, central nervous system, and nose. PAX6 has two DNA binding domains, a glycine-rich region that links the two DNA binding domains, and a transactivation domain. There is evidence that the different DNA binding domains of PAX6 have different target genes. However, it is not clear if the two DNA binding domains function independently. We have studied the effect of structural changes in the paired domain on the function of PAX6 mediated through its homeodomain. The R26G and I87R mutations have been reported in different human patients with clinically different phenotypes and are in the N- and the C-terminal halves of the paired domain, respectively. Surprisingly, we found that the I87R mutant protein not only lost the transactivation function but also failed to bind DNA by either of its DNA binding domains. In contrast, the R26G mutant protein lost DNA binding through its paired domain but had greater DNA binding and transactivation than wild-type PAX6 on homeodomain binding sites. Like R26G, the 5a isoform showed higher DNA binding than wild-type PAX6. This study demonstrates that the two subdomains of the paired domain influence the function of the homeodomain differentially and also provides an explanation for the difference in phenotypes associated with these mutations.

Molecular Characterization of the Rat Insulin Enhancer-binding Complex 3b2 CLONING OF A BINDING FACTOR WITH PUTATIVE HELICASE MOTIFS

Cell-specific expression of the rat insulin II gene is in part mediated through an element located in the 5′-flanking region. The element, termed RIPE3b (−126 to −101), confers β-cell-specific expression in conjunction with an adjacent element RIPE3a (−110 to −86). Here we report the characterization of one of the RIPE3b-binding complexes, 3b2. UV cross-linking analysis demonstrated that it is composed of at least three polypeptides: p58, p62, and p110. Furthermore, a cDNA was isolated via expression screening for binding to RIPE3b. Sequence analysis reveals that the encoded protein, designated Rip-1, possessed putative helicase motifs and a potential transcription activation domain. Overexpression of Rip-1 in cells greatly enhances the 3b2 binding complex, suggesting that Rip-1 is involved in the binding of 3b2.

A unique RNA-directed nucleoside analog is cytotoxic to breast cancer cells and depletes cyclin E levels

In contrast to deoxyribose or arabinose containing nucleoside analogs that are currently established for cancer therapeutics, 8-chloro-adenosine (8-Cl-Ado) possesses a ribose sugar. This unique nucleoside analog is RNA-directed and is in a phase I clinical trial for hematological malignancies. RNA-directed therapies are effective for the treatment of many malignancies as their activities are primarily aimed at short-lived transcripts, which are typically encoded by genes that promote the growth and survival of tumor cells such as cyclin E in breast cancer. Based on this, we hypothesized that 8-Cl-Ado, a transcription inhibitor, will be effective for the treatment of breast cancer cells. The metabolism of 8-Cl-Ado and the effect on ATP in the breast cancer cell lines MCF-7 and BT-474 were measured using HPLC analysis. In these cells, 8-Cl-Ado was effectively taken up, converted to its cytotoxic metabolite, 8-Cl-ATP, and depleted the endogenous ATP levels. This in turn led to an inhibition of RNA synthesis. The RNA synthesis inhibition was associated with a depletion of cyclin E expression, which is indicative of a diminished tumorigenic phenotype. The final outcome of 8-Cl-Ado treatment of the breast cancer cells was growth inhibition due to an induction of apoptosis and a loss of clonogenic survival. These results indicate that 8-Cl-Ado, which is currently in clinic for hematological malignancies, may be an effective agent for the treatment of breast cancer.

B cell receptor signaling regulates metabolism in Chronic Lymphocytic Leukemia.

Peripheral blood chronic lymphocytic leukemia (CLL) cells are quiescent but have active transcription and translation processes, suggesting that these lymphocytes are metabolically active. Based on this premise, the metabolic phenotype of CLL lymphocytes was investigated by evaluating the two intracellular ATP-generating pathways. Metabolic flux was assessed by measuring glycolysis as extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation as oxygen consumption rate (OCR) and then correlated with prognostic factors. Further, the impact of B-cell receptor signaling (BCR) on metabolism was determined by genetic ablation and pharmacological inhibitors. Compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in CLL cells. ECAR was consistently low, but OCR varied considerably in human patient samples (n = 45). Higher OCR was associated with poor prognostic factors such as ZAP 70 positivity, unmutated IGHV, high β2M levels, and higher Rai stage. Consistent with the association of ZAP 70 and IGHV unmutated status with active BCR signaling, genetic ablation of BCR mitigated OCR in malignant B cells. Similarly, knocking out PI3Kδ, a critical component of the BCR pathway, decreased OCR and ECAR. In concert, PI3K pathway inhibitors dramatically reduced OCR and ECAR. In harmony with a decline in metabolic activity, the ribonucleotide pools in CLL cells were reduced with duvelisib treatment. Collectively, these data demonstrate that CLL metabolism, especially OCR, is linked to prognostic factors and is curbed by BCR and PI3K pathway inhibition. Implications: This study identifies a relationship between oxidative phosphorylation in CLL and prognostic factors providing a rationale to therapeutically target these processes. Mol Cancer Res; 15(12); 1692–703. ©2017 AACR.

Myeloma antioxidant status: The good, the bad and the reactive

Reactive oxygen species (ROS), which include free radicals such as superoxide (O2 ) and hydroxyl radicals (HO), as well as non-radical molecules such as hydrogen peroxide (H2O2), are constantly being generated through a variety of pathways. Mitochondria are a major source of cellular ROS [1]. During oxidative phosphorylation, electrons are produced for ATP synthesis via electron transport chain (eTC) (Figure 1). Some of these electrons may escape and react with oxygen to form O2 7 which may be subsequently converted to H2O2 and HO . Additional sites of cellular ROS generation include the endoplasmic reticulum. Normally, ROS formation is a balanced byproduct of metabolism and plays important roles in cell signalling. Antioxidants aid in the control of this balance by decreasing ROS levels. Vitamin E (tocopherol), vitamin C (ascorbic acid) and thiol redox cycles, work synergistically as an antioxidant network [2]. Catalase and glutathione peroxidase (GPX) reduce peroxides whereas superoxide dismutase (SOD) catalyses the reaction of O2 7

Targeting the Pro-Survival Protein MET with Tivantinib (ARQ 197) Inhibits Growth of Multiple Myeloma Cells

The hepatocyte growth factor (HGF)/MNNG HOS transforming gene (MET) pathway regulates cell growth, survival, and migration. MET is mutated or amplified in several malignancies. In myeloma, MET is not mutated, but patients have high plasma concentrations of HGF, high levels of MET expression, and gene copy number, which are associated with poor prognosis and advanced disease. Our previous studies demonstrated that MET is critical for myeloma cell survival and its knockdown induces apoptosis. In our current study, we tested tivantinib (ARQ 197), a small-molecule pharmacological MET inhibitor. At clinically achievable concentrations, tivantinib induced apoptosis by > 50% in all 12 human myeloma cell lines tested. This biologic response was associated with down-regulation of MET signaling and inhibition of the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways, which are downstream of the HGF/MET axis. Tivantinib was equally effective in inducing apoptosis in myeloma cell lines resistant to standard chemotherapy (melphalan, dexamethasone, bortezomib, and lenalidomide) as well as in cells that were co-cultured with a protective bone marrow microenvironment or with exogenous cytokines. Tivantinib induced apoptosis in CD138 + plasma cells from patients and demonstrated efficacy in a myeloma xenograft mouse model. On the basis of these data, we initiated a clinical trial for relapsed/refractory multiple myeloma (MM). In conclusion, MET inhibitors may be an attractive target-based strategy for the treatment of MM.

Abstract 1710: Bortezomib and lenalidomide resistant myeloma cells overexpress the hepatocyte growth factor/MET signaling axis and respond to MET kinase inhibitors

Multiple Myeloma (MM) is a plasma cell neoplasm that responds well to therapeutic agents such as bortezomib, a proteasome inhibitor, and lenalidomide, an immunomodulatory drug. However, myeloma patients acquire resistance to these agents and disease relapses. Previously, it has been established that levels of hepatocyte growth factor (HGF) are high in serum of myeloma patients. In concert, our studies in myeloma indicated that HGF gene expression is also high in CD138+ myeloma plasma cells. High HGF levels in serum and in myeloma cells are correlated with poor prognosis and advanced disease. HGF is the ligand for the MET receptor tyrosine kinase that controls proliferation, survival and migration. Our earlier studies demonstrated that MET acts as a survival factor in myeloma cells. We hypothesized that MET/HGF axis may serve as a resistant mechanism in myeloma. To test this, we examined the HGF/MET signaling pathway in bortezomib and lenalidomide resistant myeloma cell lines. These lines were developed by continuous exposure of cells to increasing concentrations of the respective drug. We used three paired cell lines; KAS-6/1 wild type (WT) and bortezomib resistant (KAS-6/V10R) as well as lenalidomide resistant (KAS-6/R10R); WT MM1.S and its lenalidomide resistant counterpart (MM1.R10R), and WT ANBL-6 and its bortezomib resistant (ANBL-6/V10R) pair. Compared to WT cells (KAS-6/1, MM.1S), resistant cell lines overexpress MET at both the protein and mRNA levels. MET is four-fold higher in KAS-6/V10R and KAS-6/R10R compared to KAS-6/1 at both the protein and mRNA level while it is three-fold higher in MM.1/R10R compared to MM.1S at the protein level. On the other hand, compared to WT ANBL-6 cells, the bortezomib resistant cell line ANBL-6/V10R does not overexpress MET but secretes higher (2.6x) levels of HGF into the medium. The overexpression of MET in the resistant cell lines is associated with increased signaling of the HGF/MET pathway at both basal conditions and upon HGF-stimulated activation of the HGF/MET pathway as we observed higher levels of phospho-AKT(S473), phospho-GSK3β(S9) and phospho-ERK1/2 in lenalidomide resistant cell lines compared to WT controls. To test utility of MET kinase inhibitors in bortezomib and lenalidomide resistant cell lines, we used crizotinib, SU11274, ARQ 197 and SGX523, all small-molecule MET kinase inhibitors. Our studies demonstrate that targeted MET kinase inhibition induced apoptosis with concomitant inhibition of growth in these resistant myeloma cell lines. Depending on the potency of the compound, we observed 1 - 90% apoptosis and 15 - 70 % growth inhibition with these inhibitors at 3 μM level. Taken together, out study implies that bortezomib and lenalidomide resistant myeloma may have active HGF/MET axis and MET inhibitors may have therapeutic efficacy for these patients. Citation Format: Shadia Zaman, Christine M. Stellrecht, Robert Z. Orlowski, Varsha Gandhi. Bortezomib and lenalidomide resistant myeloma cells overexpress the hepatocyte growth factor/MET signaling axis and respond to MET kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1710. doi:10.1158/1538-7445.AM2014-1710