Cindy Marando

Identification of ADAM10 as a major source of HER2 ectodomain sheddase activity in HER2 overexpressing breast cancer cells

ALL AUTHORS: Phillip C.C. Liu, Xiangdong Liu, Yanlong Li, Maryanne Covington, Richard Wynn, Reid Huber, Milton Hillman, Gengjie Yang, Dawn Ellis, Cindy Marando, Kamna Katiyar, Jodi Bradley, Kenneth Abremski, Mark Stow, Mark Rupar, Jincong Zhuo, Yun-Long Li, Qiyan Lin, David Burns, Meizhong Xu, Colin Zhang, Ding-Quan Qian, Chunhong He, Vaqar Sharief, Lingkai Weng, Costas Agrios, Eric Shi, Brian Metcalf, Robert Newton, Steven Friedman, Wenqing Yaol, Peggy Scherlel, Gregory Hollis, Timothy C. Burn Overexpression and activating mutations of ErbB family members have been implicated in the development and progression of a variety of tumor types. Cleavage of the HER2 receptor by an as yet unidentified ectodomain sheddase has been shown to liberate the HER2 extracellular domain (ECD) leaving a fragment with constitutive kinase activity that can provide ligand-independent growth and survival signals to the cell. This process is clinically relevant since HER2 ECD serum levels in metastatic breast cancer patients are associated with a poorer prognosis. Thus, inhibition of the HER2 sheddase may provide a novel therapeutic approach for breast cancer. We describe the use of transcriptional profiling, pharmacological and in vitro approaches to identify the major source of HER2 sheddase activity. Real-time PCR was used to identify those ADAM family members which were expressed in HER2 shedding cell lines. siRNAs that selectively inhibited ADAM10 expression reduced HER2 shedding. In addition, we profiled over 1000 small molecules for in vitro inhibition of a panel of ADAM and MMP proteins; a positive correlation was observed only between ADAM10 inhibition and reduction of HER2 ECD shedding in a cell based assay. Finally, in vitro studies demonstrate that in combination with low doses of Herceptin, selective ADAM10 inhibitors decrease proliferation in HER2 overexpressing cell lines while inhibitors, that do not inhibit ADAM10, have no impact. These results are consistent with ADAM10 being a major determinant of HER2 shedding, the inhibition of which, may provide a novel therapeutic approach for treating a variety of cancers with active HER2 signaling.

A Novel Kinase Inhibitor, INCB28060, Blocks c-MET–Dependent Signaling, Neoplastic Activities, and Cross-Talk with EGFR and HER-3

Purpose: The c-MET receptor tyrosine kinase plays important roles in the formation, progression, and dissemination of human cancer and presents an attractive therapeutic target. This study describes the preclinical characterization of INCB28060, a novel inhibitor of c-MET kinase. Experimental Design: Studies were conducted using a series of in vitro and in vivo biochemical and biological experiments. Results: INCB28060 exhibits picomolar enzymatic potency and is highly specific for c-MET with more than 10,000-fold selectivity over a large panel of human kinases. This inhibitor potently blocks c-MET phosphorylation and activation of its key downstream effectors in c-MET–dependent tumor cell lines. As a result, INCB28060 potently inhibits c-MET–dependent tumor cell proliferation and migration and effectively induces apoptosis in vitro. Oral dosing of INCB28060 results in time- and dose-dependent inhibition of c-MET phosphorylation and tumor growth in c-MET–driven mouse tumor models, and the inhibitor is well tolerated at doses that achieve complete tumor inhibition. In a further exploration of potential interactions between c-MET and other signaling pathways, we found that activated c-MET positively regulates the activity of epidermal growth factor receptors (EGFR) and HER-3, as well as expression of their ligands. These effects are reversed with INCB28060 treatment. Finally, we confirmed that circulating hepatocyte growth factor levels are significantly elevated in patients with various cancers. Conclusions: Activated c-MET has pleiotropic effects on multiple cancer-promoting signaling pathways and may play a critical role in driving tumor cell growth and survival. INCB28060 is a potent and selective c-MET kinase inhibitor that may have therapeutic potential in cancer treatment. Clin Cancer Res; 17(22); 7127–38. ©2011 AACR.

Selective inhibition of ADAM metalloproteases blocks HER-2 extracellular domain (ECD) cleavage and potentiates the anti-tumor effects of trastuzumab

The HER-2 receptor tyrosine kinase is an important regulator of cell proliferation and survival, and it is a clinically validated target of therapeutic intervention for HER-2 positive breast cancer patients. Its extracellular domain (ECD) is frequently cleaved by protease(s) in HER-2 overexpressing breast cancer patients, rendering the remaining membrane-bound portion (p95) a constitutively activated kinase. The presence of both serum ECD and cellular p95 protein has been linked to poor clinical outcome as well as reduced effectiveness of some therapeutic treatments. We have identified a series of potent, selective small molecule inhibitors of ADAM proteases, exemplified here by INCB003619, and demonstrate that these inhibitors effectively block HER-2 cleavage in HER-2 overexpressing human breast cancer cell lines. Intriguingly, when used in combination, INCB003619 dramatically enhances the antiproliferative activity of suboptimal doses of the anti-HER-2 antibody, trastuzumab, in HER-2 overexpressing/shedding breast cancer cell lines, accompanied by reduced ERK and AKT phosphorylation. Furthermore, INCB003619, in combination with trastuzumab, augments the pro-apoptotic and antiproliferative effects of the chemotherapeutic agent paclitaxel. Consistent with these in vitro data, INCB003619 reduces serum ECD levels and enhances the antitumor effect of trastuzumab in a xenograft tumor model derived from the HER-2 overexpressing BT-474 breast cancer cell line. Collectively, these findings suggest that blocking HER-2 cleavage with selective ADAM inhibitors may represent a novel therapeutic approach for treating HER-2 overexpressing breast cancer patients.

Compelling P1 substituent affect on metalloprotease binding profile enables the design of a novel cyclohexyl core scaffold with excellent MMP selectivity and HER-2 sheddase inhibition.

A serendipitous discovery that the metalloprotease binding profile of a novel class of 2-carboxamide-3-hydroxamic acid piperidines could be significantly attenuated by the modification of the unexplored P1 substituent enabled the design and synthesis of a novel 2-carboxamide-1-hydroxamic acid cyclohexyl scaffold core that exhibited excellent HER-2 potency and unprecedented MMP-selectivity that we believe would not have been possible via conventional P1 perturbations.

Abstract 5071: Preclinical characterization of the potent and selective BET inhibitor INCB057643 in models of hematologic malignancies

Inhibitors of the Bromodomain and Extra-Terminal (BET) family of bromodomain containing proteins regulate expression of key cell fate, cell cycle, and survival genes including c-myc. In preclinical models, BET inhibitors have demonstrated significant efficacy in a variety of different oncology indications, including hematological malignancies. Here we describe the preclinical profile of the novel, orally bioavailable BET inhibitor INCB057643 in preclinical models of hematologic malignancies. INCB057643 inhibited binding of BRD2/BRD3/BRD4 to an acetylated histone H4 peptide in the low nM range, and was selective against other bromodomain containing proteins. In vitro analyses showed that INCB057643 inhibited proliferation of human AML, DLBCL, and multiple myeloma cell lines, with a corresponding decrease in MYC protein levels. Cell cycle analyses indicated that G1 arrest and a concentration-dependent increase in apoptosis were seen within 48 hours of treatment with INCB057643. BRD proteins also regulate the expression of many pro-inflammatory genes. Production of several cytokines, including IL-6, IL-10 and MIP-1α, was repressed by INCB057643 in human and mouse whole blood stimulated ex vivo with LPS. Consistent with these effects, analyses of gene expression in cells treated with INCB057643 revealed that pathways involved in cell cycle progression, apoptosis, and IL-6 were among the most significantly altered in vitro. Oral administration of INCB057643 resulted in significant anti-tumor efficacy in xenograft models of AML, myeloma, and DLBCL. Additionally, combining INCB057643 with standard of care agents used for the treatment of DLBCL including rituximab and bendamustine resulted in enhanced anti-tumor efficacy relative to that achieved with single agent therapies at doses that were well tolerated. In addition, many B cell malignancies are reliant on the PI3Kδ pathway for proliferation and survival, suggesting that the combination of INCB057643 with the clinical stage PI3Kδ specific inhibitor INCB050465 may be a rational therapeutic strategy for DLBCL. Compared with single agent BETi or PI3Kδi therapy, the combination significantly potentiated tumor growth inhibition in DLBCL models representative of the ABC subtype (HBL-1), and the double hit GCB subtype (WILL2). These data suggest that clinical exploration of INCB057643 as a monotherapy or in combination in hematologic malignancies is warranted. Citation Format: Matthew C. Stubbs, Thomas Maduskuie, Timothy Burn, Sharon Diamond-Fosbenner, Nikoo Falahatpisheh, Alla Volgina, Nina Zolotarjova, Xiaoming Wen, Patricia Feldman, Mark Rupar, Robert Collins, Cindy Marando, Bruce Ruggeri, Maryanne Covington, Xuesong Mike Liu, Richard Wynn, Swamy Yeleswaram, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Andrew P. Combs, Phillip C. Liu. Preclinical characterization of the potent and selective BET inhibitor INCB057643 in models of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5071. doi:10.1158/1538-7445.AM2017-5071

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies

The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine protein kinases are overexpressed in many hematologic and solid tumor malignancies and play central roles in intracellular signaling networks important in tumorigenesis, including the Janus kinase–signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. The three PIM kinase isozymes (PIM1, PIM2, and PIM3) share similar downstream substrates with other key oncogenic kinases and have differing but mutually compensatory functions across tumors. This supports the therapeutic potential of pan-PIM kinase inhibitors, especially in combination with other anticancer agents chosen based on their role in overlapping signaling networks. Reported here is a preclinical characterization of INCB053914, a novel, potent, and selective adenosine triphosphate-competitive pan-PIM kinase inhibitor. In vitro, INCB053914 inhibited proliferation and the phosphorylation of downstream substrates in cell lines from multiple hematologic malignancies. Effects were confirmed in primary bone marrow blasts from patients with acute myeloid leukemia treated ex vivo and in blood samples from patients receiving INCB053914 in an ongoing phase 1 dose-escalation study. In vivo, single-agent INCB053914 inhibited Bcl-2–associated death promoter protein phosphorylation and dose-dependently inhibited tumor growth in acute myeloid leukemia and multiple myeloma xenografts. Additive or synergistic inhibition of tumor growth was observed when INCB053914 was combined with selective PI3Kδ inhibition, selective JAK1 or JAK1/2 inhibition, or cytarabine. Based on these data, pan-PIM kinase inhibitors, including INCB053914, may have therapeutic utility in hematologic malignancies when combined with other inhibitors of oncogenic kinases or standard chemotherapeutics.

Abstract 5414: Activity of the pan-PIM kinase inhibitor INCB053914 in models of multiple myeloma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnThe PIM family of serine-threonine protein kinases (PIM1, PIM2 and PIM3) mediates responses to cytokines and growth factors and drives cell proliferation and survival in a number of hematologic malignancies. Overexpression of PIM kinases in these malignancies, including multiple myeloma (MM), has been associated with poor overall survival. Given the overlapping functions of these kinases, the ability of one family member to compensate for the loss of another as well as the relatively benign phenotype of mice deficient in all three PIM isoforms, discovery of pan-PIM kinase inhibitors is warranted. The in vitro and in vivo activity of the pan-PIM kinase inhibitor, INCB53914, was determined in MM cell lines. The antiproliferative potencies for INCB053914 were <200 nM in the majority of MM cell lines tested. INCB053914 potently suppressed the phosphorylation of multiple PIM substrates in MM cell lines, however in contrast, a PIM2-sparing compound, INCB050646, was unable to impact signaling in the KMS12 MM cell line, suggesting the importance of the PIM2 isoform in myeloma growth and survival. An assay was established to measure the inhibition of the phosphorylation of the PIM substrate, BAD, in KMS12 cells when spiked into whole blood (WB) to assess the shift in compound potency due to protein binding. The IC50 for INCB053914 in this assay was similar to its potency in suppressing BAD phosphorylation in KMS12 tumors in vivo. Dose dependent tumor growth inhibition (TGI) was seen in mice bearing KMS12 tumors, with maximal TGI achieved with 24 hours of KMS12 WB IC50 coverage. Similar data were obtained in a second MM model, OPM2.nnTo understand the impact of inhibiting the PIM pathway in combination with other pathways dysregulated in hematological malignancies, an unbiased in vitro screen was performed and the potential synergy of INCB053914 in combination with 65 cytotoxic or targeted agents was determined. This screen identified several agents active against the PI3K pathway or which impacted cell cycle progression. In addition, the combinatorial activity of selected targeted agents hypothesized to exhibit significant interactions with the PIM pathway was assessed in vivo. Since PIM family members are STAT regulated genes, enhanced activity may be expected upon combined PIM and JAK inhibition. In fact, synergistic activity was seen with this combination in the INA6 multiple myeloma model, and pharmacodynamic analyses revealed enhanced suppression of both pBAD and c-myc levels in tumors from treated mice. Additionally, c-myc levels are regulated both by PIM and the BET family member, BRD4. The expected synergistic efficacy of PIM and BET inhibitors was also observed in the KMS12 model, again with enhanced reduction in pBAD and c-myc levels in the tumors of treated mice. Taken together, these data support the utility of PIM inhibition in MM patients, both as monotherapy and in combination with other targeted agents.nnCitation Format: Holly Koblish, Niu Shin, Leslie Hall, Xiaoming Wen, Sybil OConnor, Valerie Dostalik, Qian Wang, Kathy Wang, Maryanne Covington, Cindy Marando, Kevin Bowman, Jason Boer, Krista Burke, Ke Zhang, Hao Feng, Chu-Biao Xue, Yun-Long Li, Wenqing Yao, Reid Huber, Kris Vaddi, Peggy Scherle. Activity of the pan-PIM kinase inhibitor INCB053914 in models of multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5414. doi:10.1158/1538-7445.AM2015-5414

An Inhibitor of ADAMs (a Distintegrin and Metalloproteinase) Overcomes HER3-Mediated Resistance to Trastuzumab and Lapatinib.

Background: Inhibitors of HER-2/neu and EGF receptors such as trastuzumab, lapatinib, and erlotinib have demonstrated clinical efficacy but not all HER-2/neu or EGFR positive tumors respond and many that initially respond develop resistance. Ligand mediated HER-3 signaling results in tumor growth and survival and is a proposed mechanism of resistance to trastuzumab and lapatinib. Proteolytic cleavage of both ErbB ligands and receptors has been shown to be a critical event that results in ErbB pathway activation. Cleavage is necessary for the generation of soluble, functionally active forms of ErbB ligands and in the case of HER-2/neu, cleavage results in a shed extracellular domain (ECD) and a membrane bound fragment (p95) that contains a kinase domain with significant constitutive activity. In addition, it has been shown that the preferential association between HER3 and p95 can further activate the pathway. Both ErbB ligand and HER-2/neu cleavage are mediated by the ADAM family of proteases. Further, we have previously shown that the ADAM protease inhibitor, INCB7839, provides synergistic inhibition of HER2 + breast cancer cell line growth when combined with either trastuzumab or lapatinib. Materials and Methods: The HER-2 overexpressing BT-474 human breast cancer cell line was treated with either lapatinib or trastuzumab in the presence or absence of the HER-3 ligand, heregulin, and the effects on cell growth measured. The effects of the ADAM protease inhibitor INCB7839 in this system were also examined. Results: The addition of heregulin overcame the anti-proliferative effects of both lapatinib and trastuzumab on the growth of the BT-474 cell line in vitro. Addition of INCB7839 synergized with lapatinib or trastuzumab and importantly, restored the anti-proliferative effects of these agents in the presence of heregulin. Further, pretreatment of BT-474 cells with INCB7839 for 6 days, which we have previously shown completely eliminates the presence of the p95 form of HER2, amplified these effects, presumably by eliminating p95 before stimulating the cells with heregulin. Discussion: Together, these results confirm that heregulin can overcome the anti-proliferative effects of both trastuzumab and lapatinib as previously reported. Prevention of p95 formation by ADAM protease inhibitors appears to restore the anti-proliferative effects of both trastuzumab and lapatinib when heregulin/HER3 signaling occurs, negating a proposed mechanism of resistance to these agents in the clinic. These results suggest that combining an ADAM inhibitor with targeted inhibitors of the ErbB family can overcome HER-3-mediated resistance and enhance the clinical efficacy of approved HER2-targeted agents in the clinical setting. INCB7839 is currently under clinical investigation in combination with trastuzumab for women with metastatic breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3138.