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SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

Protein tyrosine phosphatase SHP2 is an oncoprotein associated with cancer as well as a potential immune modulator because of its role in the programmed cell death PD-L1/PD-1 pathway. In the preceding manuscript, we described the optimization of a fused, bicyclic screening hit for potency, selectivity, and physicochemical properties in order to further expand the chemical diversity of allosteric SHP2 inhibitors. In this manuscript, we describe the further expansion of our approach, morphing the fused, bicyclic system into a novel monocyclic pyrimidinone scaffold through our understanding of SAR and use of structure-based design. These studies led to the identification of SHP394 (1), an orally efficacious inhibitor of SHP2, with high lipophilic efficiency, improved potency, and enhanced pharmacokinetic properties. We also report other pyrimidinone analogues with favorable pharmacokinetic and potency profiles. Overall, this work improves upon our previously described allosteric inhibitors and exemplifies and extends the range of permissible chemical templates that inhibit SHP2 via the allosteric mechanism.

In breast cancer, estrogen receptor alpha (ERα) positive cancer accounts for approximately 74% of all diagnoses, and in these settings, it is a primary driver of cell proliferation. Treatment of ERα positive breast cancer has long relied on endocrine therapies such as selective estrogen receptor modulators, aromatase inhibitors, and selective estrogen receptor degraders (SERDs). The steroid-based anti-estrogen fulvestrant (5), the only approved SERD, is effective in patients who have not previously been treated with endocrine therapy as well as in patients who have progressed after receiving other endocrine therapies. Its efficacy, however, may be limited due to its poor physicochemical properties. We describe the design and synthesis of a series of potent benzothiophene-containing compounds that exhibit oral bioavailability and preclinical activity as SERDs. This article culminates in the identification of LSZ102 (10), a compound in clinical development for the treatment of ERα positive breast cancer.

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

SHP099, a selective inhibitor of signalling meditator SHP2 with drug-like properties, has an allosteric mechanism of action whereby it stabilizes SHP2 in an auto-inhibited conformation, and suppresses RAS–ERK signalling and proliferation in receptor-tyrosine-kinase-driven cancer cell lines and mouse tumour xenograft models. The tyrosine phosphatase SHP2 is a key mediator of receptor tyrosine kinase (RTK) signalling, as well as being important in immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth, and SHP2 is a potential, but so far elusive, therapeutic target in cancer. Pascal Fortin and colleagues report the development of a selective SHP2 inhibitor with drug-like properties. The inhibitor, SHP099, has an allosteric mechanism of action whereby it stabilizes SHP2 in an auto-inhibited conformation. It also suppresses RAS–ERK signalling to inhibit RTK-driven proliferation in human cancer cell lines and mouse tumour xenograft models. The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1,2,3,4,5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2,3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6,7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8,9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

SHP2 is a nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript.

[Image: see text] Mutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma, and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1(R132H). This Letter describes SAR exploration focused on improving both the in vitro and in vivo metabolic stability of the compounds, leading to the identification of 19 as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, 19 efficiently inhibited the production of the biomarker 2-HG.

Autophagy is a dynamic process that regulates lysosomal-dependent degradation of cellular components. Until recently the study of autophagy has been hampered by the lack of reliable pharmacological tools, but selective inhibitors are now available to modulate the PI 3-kinase VPS34, which is required for autophagy. Here we describe the discovery of potent and selective VPS34 inhibitors, their pharmacokinetic (PK) properties, and ability to inhibit autophagy in cellular and mouse models.

Inhibition of mutant IDH1 is being evaluated clinically as a promising treatment option for various cancers with hotspot mutation at Arg132. Having identified an allosteric, induced pocket of IDH1R132H, we have explored 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors for in vivo modulation of 2-HG production and potential brain penetration. We report here optimization efforts toward the identification of clinical candidate IDH305 (13), a potent and selective mutant IDH1 inhibitor that has demonstrated brain exposure in rodents. Preclinical characterization of this compound exhibited in vivo correlation of 2-HG reduction and efficacy in a patient-derived IDH1 mutant xenograft tumor model. IDH305 (13) has progressed into human clinical trials for the treatment of cancers with IDH1 mutation.

Protein tyrosine phosphatase SHP2 is an oncoprotein associated with cancer as well as a potential immune modulator due to its role in the programmed cell death PD-L1/PD-1 pathway. Small molecule inhibition of SHP2 has been widely investigated including our previous reports describing SHP099, which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies led to the identification of multiple, potent inhibitor chemotypes, an additional and distinct allosteric binding site, and the identification of SHP394, an orally efficacious inhibitor of SHP2 with improved potency and enhanced pharmacokinetic properties. Overall, this work improves upon our previously described allosteric inhibitors, and exemplifies and extends the range of permissible chemical templates that inhibit SHP2 via the allosteric mechanism. Citation Format: Matthew J. LaMarche, Jeff Bagdanoff, Mike Acker, Ying-Nan Chen, Homan Chan, Michael Dore, Brant Firestone, Michelle Fodor, Jorge Garcia-Fortanet, Murphy Hentemann, Mitsunori Kato, Robert Koenig, Laura La Bonte, Shumei Liu, Movarid Mohseni, Rukundo Ntaganda, Patrick Sarver, Troy Smith, Martin Sendzik, Travis Stams, Stan Spence, Chris Towler, Hongyun Wang, Ping Wang, Sarah Williams, Zhouliang Chen, Huaixiang Hao, Gang Liu, Chen Liu, Eric McNeill, Bing Yu. Allosteric SHP2 phosphatase inhibition: Multiple mechanisms and chemotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-005.

High throughput screening and subsequent hit validation identified 4-isopropyl-3-(2-((1-phenylethyl)amino)pyrimidin-4-yl)oxazolidin-2-one as a potent inhibitor of IDH1R132H. Synthesis of the four separate stereoisomers identified the (S,S)-diastereomer (IDH125, 1f) as the most potent isomer. This also showed reasonable cellular activity and excellent selectivity vs IDH1wt. Initial structure–activity relationship exploration identified the key tolerances and potential for optimization. X-ray crystallography identified a functionally relevant allosteric binding site amenable to inhibitors, which can penetrate the blood–brain barrier, and aided rational optimization. Potency improvement and modulation of the physicochemical properties identified (S,S)-oxazolidinone IDH889 (5x) with good exposure and 2-HG inhibitory activity in a mutant IDH1 xenograft mouse model.

Over 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publically available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment called the Public Repository of Xenografts (PRoXe; www.proxe.org). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional and proteomic biomarkers in both treatment-naïve and relapsed/refractory disease.

Histone deacetylase (HDAC) inhibitors have shown promise in treating various forms of cancer. However, many HDAC inhibitors from diverse structural classes have been associated with QT prolongation in humans. Inhibition of the human ether a-go-go related gene (hERG) channel has been associated with QT prolongation and fatal arrhythmias. To determine if the observed cardiac effects of HDAC inhibitors in humans is due to hERG blockade, a highly potent HDAC inhibitor devoid of hERG activity was required. Starting with dacinostat (LAQ824), a highly potent HDAC inhibitor, we explored the SAR to determine the pharmacophores required for HDAC and hERG inhibition. We disclose here the results of these efforts where a high degree of pharmacophore homology between these two targets was discovered. This similarity prevented traditional strategies for mitigating hERG binding/modulation from being successful and novel approaches for reducing hERG inhibition were required. Using a hERG homology model, two compounds, 11r and 25i, were discovered to be highly efficacious with weak affinity for the hERG and other ion channels.

The predictive value of non-clinical cancer pharmacology models has been poor with many drugs failing in the clinic that previously demonstrated anti-tumor responses in animal models. In the age of targeted drug discovery, the cancer pharmacologist is challenged with improving the translation of these models to a clinical setting. Various model systems currently utilized in preclinical cancer drug discovery are discussed and emphasis is placed on selecting models tailored to interrogate hypothesis-driven scientific questions.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

The non-receptor protein tyrosine phosphatase (PTP) SHP2 is an important component of RTK signaling in response to growth factor stimulus and sits just upstream of the RAS-MAPK signaling cascade. The first oncogenic phosphatase to be identified, SHP2 is dysregulated in multiple human diseases including the developmental disorders Noonan and Leopard syndromes, as well as leukemia, lung cancer and neuroblastoma where aberrant activity of SHP2 leads to uncontrolled MAPK signaling. Cancer-associated activating mutations in SHP2 impart an “auto-on” state of the enzyme, boosting basal activity by shifting the equilibrium away from the auto-inhibited state. Reduction of SHP2 activity through genetic knockdown suppresses tumor growth, validating SHP2 as a target for cancer therapy. SHP099, a recently reported potent and selective allosteric inhibitor of SHP2, stabilizes the auto-inhibited form of SHP2 through interactions with the N-terminal SH2 and C-terminal PTP domains of the protein. SHP099 suppresses MAPK signaling in RTK amplified cancers resulting in suppressed proliferation in vitro and inhibition of tumor growth in mouse tumor xenograft models. Together, these data demonstrate the therapeutic potential of SHP2 inhibition in the treatment of cancer and other RAS/MAPK-linked diseases. Citation Format: Michael G. Acker, Ying-Nan P. Chen, Matthew J. LaMarche, Ho Man Chan, Peter Fekkes, Jorge Garcia-Fortanet, Jonathan R. LaRochelle, Brandon Antonakos, Christine Hiu-Tung Chen, Zhuoliang Chen, Vesselina G. Cooke, Jason R. Dobson, Zhan Deng, Fei Feng, Brant Firestone, Michelle Fodor, Cary Fridrich, Hui Gao, Huai-Xiang Hao, Jaison Jacob, Samuel Ho, Kathy Hsiao, Zhao B. Kang, Rajesh Karki, Mitsunori Kato, Jay Larrow, Laura R. La Bonte, Gang Liu, Shumei Liu, Dyuti Majumdar, Matthew J. Meyer, Mark Palermo, Minying Pu, Edmund Price, Subarna Shakya, Michael D. Shultz, Kavitha Venkatesan, Ping Wang, Markus Warmuth, Sarah Williams, Guizhi Yang, Jing Yuan, Ji-Hu Zhang, Ping Zhu, Stephen C. Blacklow, Timothy Ramsey, Nicholas J. Keen, William R. Sellers, Travis Stams, Pascal D. Fortin. Conformational activation and allosteric inhibition of SHP2 in RTK-driven cancers [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 2084. doi:10.1158/1538-7445.AM2017-2084

Tankyrase 1 and 2 have been shown to be redundant, druggable nodes in the Wnt pathway. As such, there has been intense interest in developing agents suitable for modulating the Wnt pathway in vivo by targeting this enzyme pair. By utilizing a combination of structure-based design and LipE-based structure efficiency relationships, the core of XAV939 was optimized into a more stable, more efficient, but less potent dihydropyran motif 7. This core was combined with elements of screening hits 2, 19, and 33 and resulted in highly potent, selective tankyrase inhibitors that are novel three pocket binders. NVP-TNKS656 (43) was identified as an orally active antagonist of Wnt pathway activity in the MMTV-Wnt1 mouse xenograft model. With an enthalpy-driven thermodynamic signature of binding, highly favorable physicochemical properties, and high lipophilic efficiency, NVP-TNKS656 is a novel tankyrase inhibitor that is well suited for further in vivo validation studies.

In an attempt to combat the resistance of tumors to chemotherapy, we have already described at this year's AACR, the systematic evaluation of over 11,000 different compound combinations using the human cancer cell line encyclopedia. Correlations of synergy with genetic features were identified and some novel synergies discovered. Here, we describe the efficacy, tolerability and PK-PD of 23 different in vivo combinations selected from the screens to determine the predictability of in vitro screening, including two examples from the combination of a MEK1/2-inhibitor (MEK162) with the taxane, docetaxel. In vitro screens were conducted as previously described using 3-day viability assays, and inhibition of proliferation determined relative to untreated samples, and the degree of synergy scored using different types of analyses: Gaddum, Bliss, Loewe. Only those combinations showing synergy over a wide range of concentrations were chosen for in vivo study. For in vivo studies, cells were injected s.c. in the flank of athymic nude mice, and once tumors reached a mean size of at least 100 mm3 were treated for 2-4 weeks with the appropriate dose and schedule of the compounds either as monotherapy, or in combination. Efficacy and tolerability were determined at the endpoint using the T/CTVol and T/CBW respectively to derive a combination-index as previously described by Clarke (1997), where a negative-value (-CCI) indicated synergy. In most cases, PK-PD was also measured in plasma and tumour either at steady-state and/or the endpoint to study the mechanism of the interaction and to check for drug-drug interactions and their eventual impact on PD and efficacy. Thus far, we have studied in vivo 13 different molecular targets across 6 different histotypes to give 23 different combinations. No antagonism was seen in vivo (+CCI), and 19/23 were deemed synergistic (CCI ≤-0.1), of which 8 showed regression which was not seen with the individual monotherapies. Of the 4 combinations showing no interaction, 2 were predicted by the in vitro score and the other 2 showed negative drug-drug interactions. There were no significant correlations between the CCI and the different types of in vitro score (p>0.35), but perhaps more importantly, cut-offs could be identified suggesting synergy could be predicted (p≤0.02) although not the extent of the interaction. Several novel combinations were identified for clinical investigation, including MEK162 combined with docetaxel in KRAS-mutant NSCLC, which in two different models in vivo had a CCI≤ -0.1, with PD-analyses showing that cytotoxic doses of the taxane activated the MAPK-pathway which was blocked by the combination. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A140. Citation Format: Paul Martin J. Mcsheehy, Alex Cao, Giordi Caponigro, David Duhl, Brant Firestone, Tom Gesner, Daniel Guthy, Jocelyn Holash, Fred King, Joseph Lehar, Christopher Leroy, Manway Liu, Lilli Petruzzelli, Dale Porter, Daniel Menezes, Anupama Reddy, Johannes Roesel, Christian Schnell, Timothy Smith, Mark Stump, Markus Wartmann, Marion Wiesmann. Evaluation of prediction of in vivo activity from in vitro combinations: Examples using a MEK1/2 inhibitor combined with docetaxel in NSCLC models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A140.

Isocitrate dehydrogenase 1 and 2 (IDH1/2) oxidize isocitrateto α-ketoglutarate (α-KG), a cofactor needed for the function of over 100 enzymes1. Heterozygous mutations of IDH1/2 occur in a variety of tumor types including glioma, acute myeloid leukemia (AML), cholangiosarcoma, chondrosarcoma and melanoma2. Mutant IDH is crippled for wild-type function but gains the ability to convert α-KG to 2-hydroxyglutarate (2-HG), a believed “oncometabolite” that may alter cell biology through, in part, changes in global histone and DNA methylation 1,3,4. However, the dearth of in vivo models dependent upon mutant IDH has made understanding the biological relevance of IDH mutations and 2-HG production in cancer challenging. To better understand the role that IDH mutations play in oncogenesis, and the potential effects of inhibition of these mutations, we introduced a heterozygous point mutation of IDH1 (R132H) into the endogenous locus of the HCT116 colon carcinoma cell line. When compared with the parental IDH1 wild-type HCT116 model, HCT116 IDH1R132H/+ xenograft tumors produce significantly higher levels of 2-HG. In addition, we have identified a patient-derived melanoma model, HMEX2838, which harbors an endogenous IDH1R132C/+ mutation and expresses high levels of 2-HG. To better explore the biology of mutant IDH inhibition, we developed a mutant-selective IDH inhibitor with favorable in vivo properties. Using this compound we demonstrate that 2-HG production is strongly inhibited in both the engineered HCT116 IDH1R132H/+ xenograft model and the endogenously mutant HMEX2838 patient-derived xenograft model. The data we have generated suggests that this inhibition of 2-HG directly correlates with the area under the curve (AUC) of free-drug above the HCT116 IDH1R132H/+ cellular IC50, as determined in vitro. Using these models and our IDH inhibitors we hope to gain a better understanding of how IDH mutations contribute to cancer and how inhibition of the mutant enzyme could benefit patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A144. Citation Format: Kelly L. Slocum, Julia Downall, Ty Gould, Mohammad Zafari, Stephanie Dodd, Brant Firestone, Ray Pagliarini, Julian Levell. Inhibition of 2-HG production in IDH mutant xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A144.

In recent years great advances have been made in developing targeted cancer therapeutics that produce dramatic responses in a subset of rationally selected patients. The initial breakthrough of the targeted design concept was established by the treatment of chronic myelogenous leukemia with Abl inhibitors and has been expanded to other cancer indications. As a consequence of this early success in CML, the identification and targeting of genetic lesions that confer cancer dependence has become an established strategy for drug discovery efforts. However, this approach has met with mixed degrees of success as confounding factors, such as tumor heterogeneity, have often resulted in partial responses and/or the emergence of resistance when targeted therapies were employed as single agents. To improve the therapeutic benefit in cancer, rationally-devised novel combinations of two or more agents are being explored clinically. To discover combinations that may be more effective therapies, an unbiased, systematic approach was used to identify drug combinations in vitro, using a panel of genetically diverse, and well characterized cell lines from the cancer cell line encyclopedia (CCLE: Barretina et al. Nature 2012). For three cancer indications, all pairwise combinations of 18 selected drugs (both novel inhibitors and standards of care) were tested as dose matrices in a proliferation assay. Synergistic interactions were scored using isobologram/Loewe's excess inhibition and synergistic concentration ranges for each agent were identified. However, the clinical translation of positive combinations from in vitro matrix-based screens into clinically-relevant doses and schedules are challenging, due to host biology, tumor-stroma interactions, and the pharmacokinetic and pharmacodynamics of drug delivery. To explore this higher complexity, we evaluated the in vitro to in vivo translation of drug synergies in immune-compromised mouse tumor xenograft models. To recapitulate the pharmacological combination effects in vivo, mouse pharmacokinetic data and simulation was used to determine single agent doses that would result in the desired compound plasma concentration range and duration of action. Pharmakokinetics, pharmacodynamics, antitumor activity and tolerability of the combinations were then tested in tumor-bearing mice. Observed combination effects in vivo could in some cases be explained by either the expected biological pathway interactions or partially by physiological effects relating to drug-drug interactions. Citation Format: Marion Wiesmann, Mark Stump, Giordano Caponigro, David Duhl, Brant Firestone, Tom Gesner, Bjoern Gruenenfelder, Daniel Alexander Guthy, Jocelyn Holash, Fred King, Joseph Lehar, Christophe Leroy, Manway Liu, Lilli Petruzelli, Dale Porter, Paul McSheehy, Daniel Menezes, Anupama Reddy, Johannes Roesel, Christian Schnell, Timothy R. Smith, Markus Wartmann. Systematic evaluation of drug combinations in vitro and in vivo. [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 2058. doi:10.1158/1538-7445.AM2013-2058

The inhibitor of apoptosis proteins (IAPs) have recently been shown to modulate nuclear factor κB (NF-κB) signaling downstream of tumor necrosis factor (TNF) family receptors, positioning them as essential survival factors in several cancer cell lines, as indicated by the cytotoxic activity of several novel small molecule IAP antagonists. In addition to roles in cancer, increasing evidence suggests that IAPs have an important function in immunity; however, the impact of IAP antagonists on antitumor immune responses is unknown. In this study, we examine the consequences of IAP antagonism on T cell function in vitro and in the context of a tumor vaccine in vivo. We find that IAP antagonists can augment human and mouse T cell responses to physiologically relevant stimuli. The activity of IAP antagonists depends on the activation of NF-κB2 signaling, a mechanism paralleling that responsible for the cytotoxic activity in cancer cells. We further show that IAP antagonists can augment both prophylactic and therapeutic antitumor vaccines in vivo. These findings indicate an important role for the IAPs in regulating T cell–dependent responses and suggest that targeting IAPs using small molecule antagonists may be a strategy for developing novel immunomodulating therapies against cancer.

The tumor vasculature is a key component in maintaining tumor growth and regulating the tumor microenvironment through the supply of nutrients and oxygen. ASA404 (vadimezan, 5,6-dimethylxanthenone-4-acetic acid) is a flavonoid, non-tubulin-binding Tumor-Vascular Disrupting Agent (Tumor-VDA) that induces breakdown of the established tumor vasculature through the induction of tumor endothelial cell apoptosis, resulting in the inhibition of tumor blood supply, leading to tumor ischemia and extensive necrosis of the tumor core. In contrast to antiangiogenics, ASA404 has limited effects on angiogenesis at the tumor periphery and so the remaining peripheral tumor rim represents a source of viable cells for regrowth of the tumor upon cessation of ASA404 treatment. Preclinical and clinical studies have highlighted the effectiveness of combining chemotherapies, especially taxanes, with ASA404 for marked tumor inhibition. Currently, the benefits of adding ASA404 to the standard of care in first- and second-line NSCLC indications are being evaluated in the ATTRACT-1 and ATTRACT-2 Phase III trials respectively. Despite the prior clinical efficacy observed with ASA404, the detailed molecular mechanism by which ASA404 selectively targets the tumor vasculature still remains to be defined. To investigate this, we initiated studies to explore the function of ASA404 both in vitro using human endothelial cells (HUVEC) and in ASA404-treated MDA-MB-231 breast cancer xenografts examined ex vivo. Consistent with previous reports, we observed inhibition of HUVEC cell proliferation along with rapid cell death in vitro as assessed using FACS analysis and newly demonstrated the rapid induction of caspase 3 assessed by immunocytochemistry. Treatment of the MDA-MB-231 breast xenografts in vivo with ASA404 resulted in significant tumor growth inhibition, tumor necrosis and elevation of the hypoxia marker carbonic anhydrase 9 (CAIX). The nature of ASA404-induced endothelial cell death is likely to be due to apoptosis since rapid caspase 3 cleavage was observed in the endothelial cells both in vitro and in vivo. In addition, the mitochondrial potential of cultured HUVEC cells in vitro was found to be seriously compromised after 1-2 hours treatment with ASA404 as monitored by Mito-tracker staining. This was accompanied by cytochrome C release into the cytosol as evidenced by immunohistochemical co-localization of cytochrome C and mitochondria markers. We also detected a moderate, but concentration-dependent increase in ceramide levels in HUVEC cells following 2 hours of ASA404 treatment, which may serve as a link to the observed mitochondrial depolarization phenotype. Lastly, a pooled-based shRNA screen was also conducted to search for genes that could modulate ASA404 activity in HUVEC cells. Some of the candidates identified may provide new insights into the pathways that are critical in the ASA404 mechanism of action. 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 4443.

Inhibitor of Apoptosis Proteins (IAP) proteins negatively regulate cell death through a variety of mechanisms. The prototypical IAP family member XIAP binds and inhibits the catalytic activity of caspases 3/7 and caspase 9 via the BIR2-linker region and BIR3 domains, respectively. CIAP1 and CIAP2 do not directly inhibit caspases but negatively regulate death receptor mediated apoptosis via intrinsic E3 ligase activity towards RIPK and NIK among other client proteins. IAP inhibitors (IAPi) are low molecular weight compounds that mimic Smac and bind to the IAP binding motif in the BIR3 domain of XIAP, CIAP1 and CIAP2. Smac mimetics induce apoptosis as a single agent in a subset of tumor cell lines in vitro. Cell death is preceded by the rapid proteosome-mediated degradation of CIAP1 followed by activation of both canonical and non-canonical NFKB pathway activation, TNF production and robust activation of caspase 3/7 activity. Multiple nodes in the NFKB signaling pathway were interrogated following IAPi treatment in sensitive and resistant cancer cells to delineate the basis for differential responses. Although canonical and non-canonical NFKB signaling was activated in both sensitive and resistant cells, TNF was induced only in the former. LCL161 is a second generation orally bioavailable IAPi with nM affinity for XIAP, CIAP1, CIAP2. Consistent with above, tumor cell lines with high baseline TNF levels are predisposed to IAPi sensitivity. Curiously, addition of exogenous TNF can sensitize many otherwise resistant tumor cell lines, but not normal cells, to LCL161. We undertook an unbiased study of the entire TNF super family to determine what other TNF-like cytokines could sensitize tumor cells to LCL161- induced cell death. In addition to TNF, several cytokines synergized with LCL161 and in each case RIPK appeared to play a central role. LCL161 showed potent single agent activity in the MDA-MB-231 tumor xenograft model. In vivo efficacy was accompanied by a series of tumor pharmacodynamic readouts including CIAP1 elimination, activation of an NFKB transcriptional program and caspase activation. In primary patient derived human tumor xenograft models of triple negative breast cancer and NSCLC, LCL161 had a range of responses from no effect to tumor stasis. Consistent with in vitro mechanistic studies, tumor models which were sensitive had high basal TNF levels. LCL161 lacked single agent activity in the A2058 melanoma model but significantly enhanced the anti-tumor activity of paclitaxel. The LCL161-Taxol combination triggered synergistic activation of caspases and near complete regressions in xenograft tumors. Clinical trials in man with LCL161 are ongoing in patients with solid tumors. A range of pharmacodynamic readouts have been observed which are consistent with preclinical observations. These findings show promise for IAP inhibitor therapy in humans. 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 138.

LCL161, a small molecule antagonist of inhibitor of apoptosis proteins (IAPs), induces TNF -mediated apoptosis in a subset of tumor cell lines including the MDA-MB-231 breast cancer line. To investigate the in vivo activity of LCL161, MDA-MB-231 tumor bearing mice were treated with a once weekly oral dose. We observed anti-tumor activity that was associated with pharmacodynamic responses including degradation of CIAP1 and induction of cleaved caspase 3. The loss of CIAP1, specifically the E3 ligase activity of this protein, has been shown to directly impact the stability of a number of client proteins including NIK (NF-κB inducing kinase), Mad1 (MAX-binding protein), and others. Gene expression analysis on LCL161 treated MDA-MB-231 tumors revealed a striking upregulation of NF-κB regulated target genes. These data were further supported by the observation that TNFα, a direct target of NF-κB, was induced in LCL161 treated MDAMB-231 tumor lysates. To explore whether TNFα expression levels could be used to predict single agent efficacy in a more clinically relevant context, a series of human primary tumor xenografts were profiled for baseline TNFα mRNA levels and evaluated for response to LCL161 in vivo. We observed that tumors with the highest TNFα expression levels showed the greatest sensitivity to LCL161. These studies demonstrate an essential role of the NF-κB pathway in IAP antagonist anti-tumor activity. Ongoing efforts focus on delineating the contribution of the canonical and non-canonical NF-κB pathways to efficacy and on leveraging this mechanism for LCL161 patient selection. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B27.

Panobinostat (LBH589) is a highly potent oral pan-deacetylase (DAC) inhibitor currently undergoing clinical development in hematologic and solid malignancies. Here we report the effects of panobinostat on multiple myeloma (MM) cells in vitro and in a murine xenograft model in vivo. Panobinostat exhibited potent cytotoxic activity (IC50

VX-944 is a small molecule, orally bioavailable, specific inhibitor of inosine monophosphate dehydrogenase (IMPDH), an essential rate-limiting enzyme in de novo guanine nucleotide synthesis. We have previously shown that VX-944 has broad anti-cancer properties in vitro and that its potency is not affected by MDR pumps (Jain et al, Blood 2002, 100:572a; ibid 2003, 102: 622a). Here, we describe studies that demonstrate its oncolytic activity in blast cells derived from patients with acute myeloid leukemia (AML). We also report the efficacy of VX-944 in an aggressive murine model of myeloproliferative disease. The anti-leukemic activity of VX-944 was established both in colony formation assays and in viability assays using primary cells from AML patients. VX-944 inhibited the clonogenic proliferation of acute myeloid progenitor cells in a dose-dependent manner. The mean and median IC50 values were 218±92 and 199 nM respectively (n=8), indicating that no samples were resistant to VX-944. In another study, VX-944 reduced the viability of AML blasts from 4 additional patient samples, with IC50 values ranging from 20–200 nM. VX-944 was observed to be 3–40-fold more potent than mycophenolic acid (MPA, MMF), another IMPDH inhibitor. Importantly, genotyping of these samples revealed that VX-944 is active against cancer cells with wild type and point- or ITD-mutations in Flt3 that are implicated in approx 30% of AML patients. The anti-proliferative activity of VX-944 was at least additive, and in some patient samples, synergistic, when tested in combination with Daunorubicin, a standard chemotherapy drug for AML. To determine the therapeutic potential of VX-944 in vivo, VX-944 was tested in a leukemia model using Ba/F3 cells transduced with an activating human Flt-3 mutation injected into Balb/c mice. The anti-proliferative activity of VX-944 was first established in vitro in the cell lines used in the model. VX-944 inhibited the proliferation of the human MV-4-11 and murine Ba/F3-Flt3-ITD-dependent cell lines with IC50 values of 26 and 30 nM, respectively. In PK studies, a dose-dependent increase in Cmax and AUC values were observed in Balb/c mice. In the leukemia model, VX-944 was administered orally at 75 or 150 mg/kg BID. Doxorubicin (3 mg/kg, weekly), a standard AML therapy, and MLN518 (60 mg/kg BID), a selective Flt3 inhibitor, were administered as reference compounds. All treatments began 5 days following cell implantation. Both dose groups of VX-944 provided a significant increase in median survival time as compared with the vehicle treated group (p In conclusion, we have demonstrated both potency and efficacy of VX-944 in in vitro and in vivo leukemia models. These preclinical results support further clinical development of VX-944 for the treatment of patients with leukemias and other rapidly proliferating hematological malignancies. VX-944 may provide significant therapeutic benefit when used alone, or in combination with approved chemotherapy agents such as Daunorubicin.