Shujun Shentu

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 4848: Pegylated arginine deiminase induces autophagy in canine melanoma and canine osteosarcoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Arginine is a nonessential amino acid in adult mice, men and dogs, which undergoes both synthesis and degradation in complex series of compartmentalized steps at the cellular and systemic level. The urea cycle enzymes are responsible for this axis and is dependent on the levels of are argininosuccinate synthetase (ASS1) and argininosuccinate lyase (ASL). It has been shown that many human tumor types are auxotrophic for L-arginine typically due to very low or no specific activities of ASS1 and/or ASL and are thus sensitive to induction of cell death as a result of the depravation of this single nutrient. Pegylated arginine-deiminase (PEGADI) is a molecule with this mechanism of action and has entered phase I and II clinical trials in humans where it has proven its clinical utility in the treatment of hepatocellular carcinoma and melanoma. The cancer-bearing dog serves an excellent spontaneous out-bred immune competent large animal model for numerous homologous neoplastic diseases in humans. The purpose of this study is to determine the status of arginine-synthetic enzymes and then evaluate the action of PEGADI in cultured canine cancer melanoma (12, 23, 36) and osteosarcoma (D17, 48-4, 29) cell lines. Using western blot analysis we found that each of these six canine cell lines expressed ASS1 and ASL. PEGADI depleted arginine in a time- and concentration-dependent manner in the cultured canine melanoma and osteosarcoma as measured by HPLC. Furthermore, the canine melanoma and osteosarcoma cells were very sensitive to this depletion. In particular, there was a dramatic loss of clonogenic survival in these cells treated with PEGADI (IC50 range: 0.03 uM at 96 h to 0.3 at 24 h). This sensitivity was apparently due to caspase independent cell killing as caspase 3, 8, or 9 were not activated. Changes in cell cycle distribution were quantitated with flow cytometry. Despite the lack of caspase activation, there was an increase in the number of cells in the Sub G1 population. To determine if PEGADI induced autophagy in these cells the conversion of microtubule-associated protein 1 light chain 3B (LC3B)-I to its lipidated form LC3B-II was assessed and showed there was a dose dependent induction of LC3B-II. Consistent with the induction of autophagy, results demonstrate an induction in the formation autophagolysosomes as determined by flow cytometery analysis of acridine orange staining of acidic vesicles, as well as fluorescent microscopy of treated cells stained with monodansylcadaverine. Our results support the notion that PEGADI may have clinical utility in the management of canine neoplasia. A clinical trial for this species is planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4848.

Chlorinated adenosine analogue induces AMPK and autophagy in chronic lymphocytic leukaemia cells during therapy

8‐chloro‐adenosine (8‐Cl‐Ado) is currently in phase‐I clinical trials for acute myeloid leukaemia and chronic lymphocytic leukaemia (CLL). Previously, we demonstrated that treatment with 8‐Cl‐Ado leads to diminished ATP levels. We hypothesized that AMP‐activated protein kinase (AMPK) signalling would be initiated in these cells, leading to induction of autophagy. AMPK activation and induction of autophagy were demonstrated during preclinical incubations in CLL cells with the analogues. Importantly, we extended similar observations in CLL lymphocytes during an 8‐Cl‐Ado phase‐I trial. In conclusion, 8‐Cl‐Ado treatment induces autophagy in CLL lymphocytes in vitro as well as in vivo during clinical trial.

The Ribonucleoside Analog, 8-Chloro-Adenosine, Inhibits the mTOR Pathway and Induces Autophagy.

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. Preclinically, it has been also found to be cytotoxic to a number of epithelial and neuronal cell lines. Previously, we demonstrated that in the breast cancer cell lines, MCF-7 and BT-474, 8-Cl-Ado accumulates as a triphosphate reaching its plateau by 6 h of treatment with 10 μM 8-Cl-Ado. This accumulation was coupled with a parallel decrease in the level of the endogenous ATP pool by 60 and 40%. These changes in nucleoside levels then lead to ∼50% inhibition of RNA synthesis. The net effect of these metabolic changes results in a growth inhibition of ∼90% after 5 d treatment with 10 μM 8-Cl-Ado as measured by MTS assay. This growth inhibition was not due to a cell cycle arrest. Additionally, flow cytometry analysis of annexin V staining showed only a modest induction of apoptosis first detected within 24 h of treatment and reached ∼30% by 5 d. Because the low level of apoptosis can not account for high percent of growth inhibition suggests alternative mechanism(s) for the cytotoxic activity of 8-Cl-Ado. Depletion of the cellular ATP pool is often associated with the activation of AMP-activated protein kinase (AMPK) through its phosphorylation on Thr172. Correspondingly, we demonstrated that 8-Cl-Ado treatment of MCF-7 and BT-474 cells induced AMPK Thr172 phosphorylation within 7 h. Once activated, AMPK alters various energy regulating signaling cascades which includes the mTOR pathway. Inhibition of mTOR leads to a decrease of energy depletion by reducing protein translation and promotes nutrient restoration through the induction of autophagy. 8-Cl-Ado treatment of MCF-7 and BT-474 cells was associated with a decrease in the phosphorylation of the mTOR translation targets, 4E-BP1 and p70S6K within 8 h of treatment. Likewise, there was an increase in the conversion of microtubule-associated protein 1 light chain 3B (LC3B)-I to its lipidated form, LC3B-II, which is an autophagosome marker. Consistent with the immunoblot analysis, fluorescence microscopic analysis of 8-Cl-Ado-treated MCF-7 cells and BT-474 cells expressing a GFP-LC3 construct also demonstrated an increase in the formation of autophagosomes. Finally, acridine orange staining of 8-Cl-Ado treated MCF-7 and BT-474 cells demonstrated the formation autophagolysosomes. This was further confirmed in MCF-7 cells by monodansylcadaverine staining. In conclusion, our data indicates that 8-Cl-Ado treatment induces autophagy in breast cancer cells which in part elicits its growth inhibitory effects on these cells. Based on this biological activity, we are planning to test 8-Cl-Ado in the clinic for patients with breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6116.

Targeting the Pro-Survival Protein MET with Tivantinib (ARQ 197) Inhibits Growth of Multiple Myeloma Cells12

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.

Targeting the Pro-Survival Protein MET with Tivantinib (ARQ 197) Inhibits Growth of Multiple Myeloma Cells 1 2

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 5506: In vivo activity of the ribonucleoside analogue 8-chloro-adenosine in breast cancer mouse models.

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. Preclinically, it has been found to be cytotoxic to a number of malignant cell lines. Previously, we demonstrated in the breast cancer cell lines, MCF-7 and BT-474, 8-Cl-Ado accumulates as a triphosphate (8-Cl-ATP) reaching ∼125 and ∼400 μM, respectively, by 6 h of treatment with 10 μM 8-Cl-Ado [Breast Cancer Res Treat 121:355, 2010]. This accumulation was coupled with a parallel decrease in the endogenous cellular ATP pool levels. These changes in nucleotide levels lead to ∼50% inhibition of RNA synthesis. The net effect of these metabolic changes results in a 90% loss of clonogenic survival after a 3 d treatment with 10 μM 8-Cl-Ado. This loss in survival was associated with both apoptotic and autophagic cell death. Cellular pharmacology analyses on CD 2 F 1 mice after i.v. administration of 50 and 100 mg/kg 8-Cl-Ado, revealed 8-Cl-ATP accumulated in the circulating peripheral blood mononuclear cells; within 1 hr of infusion the levels reached ∼350 and ∼1150 μM, respectively [Cancer Chemother Pharmacol 50:85, 2002] which was comparable to the levels seen in BT-474 cells treated with 10 μM for 6 and 72 hr, respectively. To test the in vivo antitumor activity of 8-Cl-Ado in breast cancer, orthotopic xenograft mouse models using both MCF-7 and BT-474 cell lines were evaluated. 8-Cl-Ado was administered i.p. 3 d/wk for 3 wk with a dose range of 25, 50 and 100 mg/kg/d once tumors reached ∼0.4 cm diameter. The MCF-7 xenograft mice showed dose dependent tumor growth inhibition evident after the second dose. In mice with the BT-474 tumors, the 100 mg/kg dose was highly effective as tumor sizes decreased after the first dose of treatment. Furthermore, in several of the mice there was an eventual loss of a palpable tumor. Likewise, the 100 mg/kg 8-Cl-Ado dose had a profound effect on the final tumor volumes in the BT-474 xenograft mice assessed during necropsy and completely eliminated any visible tumors in 9/21 (42.8%) mice assessed during necropsy. Importantly, none of these tested doses (25, 50 and 100 mg/kg/d) resulted in any untoward toxicity which was assessed by infusing various 8-Cl-Ado doses in CD1 mice. The results of the toxicology assessment showed that only repeated high i.v. doses (250 and 350 mg/kg/d for 5 d) showed a persistent nephrotoxicity 32 d after drug administration, while no other pathologic or hematologic toxicities were observed. In conclusion, our data indicates tumoricidal levels of 8-Cl-ATP are readily achieved in vivo without deleterious toxic effects and establishes 8-Cl-Ado in vivo efficacy against 2 breast cancer tumor models. Based on these findings, we plan to test 8-Cl-Ado in the clinic for patients with breast cancer. Citation Format: Christine M. Stellrecht, Shujun Shentu, Mary L. Ayres, Varsha Gandhi. In vivo activity of the ribonucleoside analogue 8-chloro-adenosine in breast cancer mouse models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5506. doi:10.1158/1538-7445.AM2013-5506

Abstract 3926: Abrogating tamoxifen resistance with the ribonucleoside analog, 8-amino adenosine

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Hormone therapy is a prevalent treatment of choice for patients with estrogen receptor alpha (ERα) positive breast cancers. Unfortunately, due to resistance, many women do not benefit from these forms of therapy. AIB1 is a transcriptional co-activator that plays an important role in resistance to hormone therapy. Depending on the cell/gene context, agents such as tamoxifen either stimulate or antagonize the transcriptional activities of ERα. Tamoxifen binding alters ERα to an intermediate conformation with affinity for both co-activators and co-repressors, causing the conformation to be dependent on co-regulator levels in the cell milieu. The stoichiometric balance between AIB1 and co-repressors is a key determinant of tamoxifen resistance. Additionally, cross-talk between ERα and growth factor (GF) signaling further manipulates ERα activity and responsiveness to hormone therapy, particularly in cells with high AIB1 levels. Of the receptor signaling transducers, AKT, ERK, and p38 are key players in resistance. We hypothesized that strategies to target the stoichiometric balance of AIB1 to co-repressors would be clinically beneficial for abrogating tamoxifen resistance. Furthermore, it has been demonstrated that agents that are able to interfere with the ERα/GF crosstalk are also highly advantageous for abrogating resistance to tamoxifen. We have shown that the ribonucleoside analog, 8-aminoadenosine (8-NH2-Ado) is a transcription inhibitor. As such, it is expected to primarily deplete the expression of the small percentage (∼5%) of gene transcripts with short-half lives, such as AIB1. We have demonstrated that treatment of MCF-7 and BT-474 cells with non-tumoricidal concentrations of 8-NH2-Ado (1 µM) decreased AIB1 protein levels. Furthermore, using in vitro kinase assays, we demonstrated the active metabolite of 8-NH2-Ado is a selective inhibitor of MEK1, 2, and 6. An initial analysis of 4-hydroxytamoxifen dose response in two different tamoxifen resistant breast cancer cell lines, MCF-7/HER-2:18 and BT-474, demonstrated non-tumoricidal analog concentrations were able to reduce the dose of 4-hydroxytamoxifen needed to inhibit proliferation in these cells. Additionally, 8-NH2-Ado inhibited AKT and ERK1/2 activity in these cells. In conclusion, AIB1 and GF signaling molecules are attractive targets for reversing resistance to hormone therapy. Our results suggest that treating ERα positive breast cancer with combinations of tamoxifen and 8-NH2-Ado would be an effective strategy to overcome tamoxifen resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3926. doi:1538-7445.AM2012-3926

Abstract 844: Targeting the pro-survival protein c-MET with ARQ 197 inhibits growth of multiple myeloma cells

Multiple myeloma (MM) is a B-cell disorder characterized by the accumulation of mature plasma cells in the bone marrow. Earlier studies have established that patients with MM have high plasma concentrations of hepatocyte growth factor (HGF) which is correlated with poor prognosis and advanced disease stages. HGF is the ligand for the c-MET receptor tyrosine kinase. Our group has previously shown that inactivation of the c-MET receptor by siRNA and ribozyme approaches inhibited proliferation and induced apoptosis in MM cell lines. Hence, we hypothesized that the HGF/c-MET axis plays a critical role in myeloma cell survival and targeting this pathway would be an effective strategy to treat MM. To further test our hypothesis, we used MM1.S, U266 and OPM-2 myeloma cell lines and ARQ 197 (tivantinib), a small molecule non-ATP-competitive and selective c-MET inhibitor (K i =355 nM). This drug is orally bioavailable, achieving steady-state levels of median 6-7 µM in plasma of patients with solid tumors. Cell growth was inhibited by at least 50% and annexin V/propidium iodide positive cells increased by at least 60% within 48 hours of treatment with 1 µM (U266 and OPM-2) and 3 µM (MM.1S) ARQ 197. Consistent with this biological response, downstream effectors of c-MET signaling including phospho-GAB1 and phospho-ERK1/2 were decreased. MM patients develop resistance to the current drugs such as lenalidomide and bortezomib. We tested ARQ 197 in paired MM cell lines - RPMI 8226, ANBL-6 and KAS-6/1 - that are sensitive and resistant to lenalidomide or bortezomib. In general, all the tested cell lines expressed readily detectable levels of proHGF as well as detectable albeit low levels of c-MET. Interestingly, the resistant cell lines expressed significantly higher c-MET protein levels compared to drug-naive controls, but were equally sensitive to ARQ 197 indicating that this c-MET kinase inhibitor overcomes inherent drug resistance. The bone marrow microenvironment is also a source for acquired drug resistance, yet ARQ 197 was effective in inhibiting growth of MM cell lines grown on human stromal cells (NKtert) which mimic the bone marrow microenvironment. Consistent with these data, exogenous addition of the cytokines interleukin-6 (0.5 and 1.0 ng/ml) or HGF (50 ng/ml) did not abrogate ARQ 197-mediated cell death or growth inhibition. Importantly, plasma cells from healthy donors (n = 15) showed no difference in c-MET mRNA expression compared to plasma cells from MM patients (n = 147) but showed relatively lower levels of HGF expression compared to MM patients. These results suggest that the HGF/c-MET pathway is upregulated in MM plasma cells. In conclusion, inhibition of the c-MET receptor tyrosine kinase activity with ARQ 197 is a novel-target based strategy to impact on the pathobiology of MM. Based on these data, we are initiating a clinical trial of ARQ 197 in patients with relapsed/refractory MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 844. doi:1538-7445.AM2012-844

Abstract 3781: The ribonucleoside analog, 8-chloro-adenosine, induces autophagy in primary chronic lymphocytic leukemia cells in vitro and in vivo

The nucleoside analog, 8-chloro-adenosine (8-Cl-Ado) is in a phase I clinical trial for chronic lymphocytic leukemia (CLL). In contrast to deoxyribose or arabinose containing analogs that are currently established for cancer therapeutics, this unique nucleoside analog possesses a ribose sugar and thus is RNA directed. Previously, we demonstrated that in primary CLL cells, 8-Cl-Ado accumulates as a triphosphate reaching a plateau of ∼400 μM by 6 h of treatment with 10 μM 8-Cl-Ado [Blood 105:4455, 2005]. This accumulation was coupled with a parallel decrease in the level of the endogenous ATP pool by ∼60%. These changes in nucleoside levels elicit an inhibition of RNA synthesis by up to 70%. Because 8-Cl-Ado treatment leads cells to become bioenergetically compromised, we further investigated the effect of this phenomenon on primary CLL cells. Depletion of the cellular ATP pool is often associated with the activation of AMP-activated protein kinase (AMPK) through its phosphorylation at Thr172. Once activated, AMPK then stimulates various energy regulating signaling cascades, most notable of which is the induction of autophagy. Because 8-Cl-Ado readily depletes the ATP pool, we tested if AMPK activity was affected. Using immunoblot analysis, we demonstrated that while total AMPK protein levels were not perturbed by 8-Cl-Ado treatment, the phosphorylation of Thr172 AMPK was induced in a dose dependent manner (3-10 μM) and was associated with an increase in the phosphorylation of an AMPK target, acetyl-CoA carboxylase. To determine if 8-Cl-Ado induces autophagy in primary CLL cells, we examined the cells after 8-Cl-Ado treatment for changes in microtubule-associated protein 1 light chain 3B (LC3B) and showed there was an increase conversion to its lipidated form, which is a marker for autophagosome formation. Also consistent with the induction of autophagy, 8-Cl-Ado promoted the formation autophagolysosomes as shown by acridine orange staining of the CLL cells. To further extend our observations, we tested induction of autophagy in CLL lymphocytes obtained from patients accrued on ongoing 8-Cl-Ado clinical trial. Patients received a 1-hr infusion of 8-Cl-Ado at escalating doses (45, 67.5, or 100 mg/m2/day × 5 day) and peripheral blood samples were obtained both pre- and post infusion. Induction of LC3B lipidation was seen as early as 3 hr post infusion in circulating CLL cells. Additionally, acridine orange staining was increased in these lymphocytes. Finally, p62/SQSTM1 levels fluctuated during and between infusions with the levels decreasing during infusion and rebounded prior to infusion on consecutive days which would be indicative of autophagic flux. In conclusion, our data demonstrate that 8-Cl-Ado treatment induces autophagy in CLL lymphocytes during in vitro incubations as well as in vivo during clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3781. doi:10.1158/1538-7445.AM2011-3781