Shadia Zaman

Targeting the Apoptosis Pathway in Hematologic Malignancies

Abstract Apoptosis is a cell death program that is well-orchestrated for normal tissue homeostasis and for removal of damaged, old or infected cells. It is regulated by intrinsic and extrinsic pathways. The intrinsic pathway responds to signals such as ultraviolet radiation or DNA damage and activates “executioner” caspases through a mitochondria-dependent pathway. The extrinsic pathway is activated by death signals induced, for example, by an infection that activates the immune system or receptor-mediated pathways. The extrinsic pathway signals also cascade down to executioner caspases that cleave target proteins and lead to cell death. Strict control of cellular apoptosis is important for the hematopoietic system as it has a high turnover rate. However, the apoptosis program is often deregulated in hematologic malignancies leading to the accumulation of malignant cells. Therefore, apoptosis pathways have been identified for the development of anticancer therapeutics. We review here the proteins that have been targeted for anticancer drug development in hematologic malignancies. These include BCL-2 family proteins, death ligands and receptors, inhibitor of apoptosis family proteins and caspases. Except for caspase activators, drugs that target each of these classes of proteins have advanced into clinical trials.

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

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 3328: Caspase activation as an approach to treat multiple myeloma.

Apoptosis is a cell death program that is essential for normal organ development. Defects in apoptosis lead to uncontrolled cell accumulation resulting in cancer such as multiple myeloma (MM), an incurable plasma cell disorder. MM patients have defective apoptosis machinery largely due to the overexpression of anti-apoptotic proteins such as BCL-2, MCL-1, cIAP1, cIAP2 and XIAP. We are investigating a novel class of procaspase-3 activators as a strategy to activate apoptosis in myeloma cells. The Procaspase Activating Compounds (PAC), PAC-1 and its analog L14R8, were synthesized by Dr. Hergenrother9s lab (U. of Illinois, Urbana, IL) and are being developed for clinical application by Vanquish Oncology. PACs convert the inactive zymogen procaspase-3 to caspase-3 which activates apoptosis. Mechanistically, PACs activate procaspase-3 by chelating inhibitory zinc ions thus allowing procaspase-3 to auto-activate to caspase-3. Our hypothesis is that targeting procaspase-3 with PAC agents will bypass inhibitory activity of the anti-apoptotic proteins and induce cell death in myeloma cells. In initial experiments, we examined procaspase-3 expression in a panel of 9 myeloma cell lines and observed that it was expressed in all the cell lines suggesting that PAC agents will be active in myeloma cells. Our investigations with PAC-1 and L14R8 demonstrated that L14R8, but not PAC-1, induced cell death in myeloma cells in a dose- and time-dependent manner. Treatment with 10 μM L14R8 for 24 hours induced >80% apoptosis in U266 and MM.1S cells. Furthermore, L14R8 activity was inhibited by exogenous addition of zinc confirming that the mechanism of L14R8 action is through zinc chelation. The activation of procaspase-3 resulted in the cleavage of its target PARP and decreased the levels of the anti-apoptotic proteins MCL-1 and XIAP. 10 μM L14R8 was also effective in inducing >80% cell death in myeloma cells grown in the presence of protective microenvironment as demonstrated by co-culture experiments with NKtert stromal cells. In addition, when myeloma cells were grown in the presence of exogenous growth factors such as hepatocyte growth factor (50 ng/ml) or interleukin-6 (10 or 50 ng/ml), L14R8 was still able to induce apoptosis (> 60% with 10 μM L14R8 treatment in the presence of either growth factor). Finally, L14R8 was effective in inducing apoptosis in cells resistant to lenalidomide, a standard treatment for myeloma patients. Our future plan is to examine the effectiveness of this drug in primary CD138+ plasma cells (representing malignant myeloma cells). These studies demonstrate that targeting procaspase-3 with L14R8 is an effective strategy to induce myeloma cell death. Citation Format: Shadia Zaman, Rui Wang, Varsha Gandhi. Caspase activation as an approach to treat multiple myeloma. [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 3328. doi:10.1158/1538-7445.AM2013-3328

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