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One of the most recent advances in the treatment of HER2+ breast cancer is the development of the antibody–drug conjugate, T-DM1. T-DM1 has proven clinical benefits for patients with advanced and/or metastatic breast cancer who have progressed on prior HER2-targeted therapies. However, T-DM1 resistance ultimately occurs and represents a major obstacle in the effective treatment of this disease. Because anti-apoptotic BCL-2 family proteins can affect the threshold for induction of apoptosis and thus limit the effectiveness of the chemotherapeutic payload, we examined whether inhibition of BCL-2/XL would enhance the efficacy of T-DM1 in five HER2-expressing patient-derived breast cancer xenograft models. Inhibition of BCL-2/XL via navitoclax/ABT-263 significantly enhanced the cytotoxicity of T-DM1 in two of three models derived from advanced and treatment-exposed metastatic breast tumors. No additive effects of combined treatment were observed in the third metastatic tumor model, which was highly sensitive to T-DM1, as well as a primary treatment-exposed tumor, which was refractory to T-DM1. A fifth model, derived from a treatment naïve primary breast tumor, was sensitive to T-DM1 but markedly benefited from combination treatment. Notably, both PDXs that were highly responsive to the combination therapy expressed low HER2 protein levels and lacked ERBB2 amplification, suggesting that BCL-2/XL inhibition can enhance sensitivity of tumors with low HER2 expression. Toxicities associated with combined treatments were significantly ameliorated with intermittent ABT-263 dosing. Taken together, these studies provide evidence that T-DM1 cytotoxicity could be significantly enhanced via BCL-2/XL blockade and support clinical investigation of this combination beyond ERBB2-amplified and/or HER2-overexpressed tumors.

Most patients with late-stage high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy but inevitably relapse and develop resistance, highlighting the need for novel therapies to improve patient outcomes. The MEK/ERK pathway is activated in a large subset of HGSOC, making it an attractive therapeutic target. Here, we systematically evaluated the extent of MEK/ERK pathway activation and efficacy of pathway inhibition in a large panel of well-annotated HGSOC patient–derived xenograft models. The vast majority of models were nonresponsive to the MEK inhibitor cobimetinib (GDC-0973) despite effective pathway inhibition. Proteomic analyses of adaptive responses to GDC-0973 revealed that GDC-0973 upregulated the proapoptotic protein BIM, thus priming the cells for apoptosis regulated by BCL2-family proteins. Indeed, combination of both MEK inhibitor and dual BCL-2/XL inhibitor (ABT-263) significantly reduced cell number, increased cell death, and displayed synergy in vitro in most models. In vivo, GDC-0973 and ABT-263 combination was well tolerated and resulted in greater tumor growth inhibition than single agents. Detailed proteomic and correlation analyses identified two subsets of responsive models—those with high BIM at baseline that was increased with MEK inhibition and those with low basal BIM and high pERK levels. Models with low BIM and low pERK were nonresponsive. Our findings demonstrate that combined MEK and BCL-2/XL inhibition has therapeutic activity in HGSOC models and provide a mechanistic rationale for the clinical evaluation of this drug combination as well as the assessment of the extent to which BIM and/or pERK levels predict drug combination effectiveness in chemoresistant HGSOC.

One of the most recent advances in the treatment of HER2+ breast cancer is the development of the antibody-drug conjugate, T-DM1. T-DM1 has proven clinical benefits for patients with advanced and/or metastatic breast cancer who have progressed on prior HER2-targeted therapies. However, T-DM1 resistance ultimately occurs and represents a major obstacle in the effective treatment of this disease. Since anti-apoptotic BCL-2 family proteins can affect the threshold for induction of apoptosis and thus limit the effectiveness of the chemotherapeutic payload, we examined whether inhibition of BCL-2/X(L) would enhance the efficacy of T-DM1 in five HER2-expressing patient-derived breast cancer xenograft models. Inhibition of BCL-2/X(L) via navitoclax/ABT-263 significantly enhanced the cytotoxicity of T-DM1 in two of three models derived from advanced and treatment-exposed metastatic breast tumors. No additive effects of combined treatment were observed in the third metastatic tumor model which was highly sensitive to T-DM1, as well as a primary treatment-exposed tumor, which was refractory to T-DM1. A fifth model, derived from a treatment naïve primary breast tumor, was sensitive to T-DM1 but markedly benefited from combination treatment. Notably, both PDXs that were highly responsive to the combination therapy expressed low HER2 protein levels and lacked ERBB2 amplification, suggesting that BCL-2/X(L) inhibition can enhance sensitivity of tumors with low HER2 expression. Toxicities associated with combined treatments were significantly ameliorated with intermittent ABT-263 dosing. Taken together, these studies provide evidence that T-DM1 cytotoxicity could be significantly enhanced via BCL-2/X(L) blockade and support clinical investigation of this combination beyond ERBB2-amplified and/or HER2-overexpressed tumors.

Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line–Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody–drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti–GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo. The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line–derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker–payload based ADCs. Overall, these data suggest that anti–GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638–49. ©2017 AACR.

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene, loss or mutation of phosphatase and tensin homolog (PTEN), and deregulation of mammalian target of rapamycin (mTOR) complexes. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K and mTOR are promising therapeutic targets for cancer. We discovered GDC-0980, a selective, potent, orally bioavailable inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2) with excellent pharmacokinetic and pharmaceutical properties. GDC-0980 potently inhibits signal transduction downstream of both PI3K and mTOR, as measured by pharmacodynamic (PD) biomarkers, thereby acting upon two key pathway nodes to produce the strongest attainable inhibition of signaling in the pathway. Correspondingly, GDC-0980 was potent across a broad panel of cancer cell lines, with the greatest potency in breast, prostate, and lung cancers and less activity in melanoma and pancreatic cancers, consistent with KRAS and BRAF acting as resistance markers. Treatment of cancer cell lines with GDC-0980 resulted in G1 cell-cycle arrest, and in contrast to mTOR inhibitors, GDC-0980 induced apoptosis in certain cancer cell lines, including those with direct pathway activation via PI3K and PTEN. Low doses of GDC-0980 potently inhibited tumor growth in xenograft models including those with activated PI3K, loss of LKB1 or PTEN, and elicited an exposure-related decrease in PD biomarkers. These preclinical data show that GDC-0980 is a potent and effective dual PI3K/mTOR inhibitor with promise for the clinic. Mol Cancer Ther; 10(12); 2426–36. ©2011 AACR.

20-25% of acute myeloid leukemia (AML) patients harbor FLT3 internal tandem duplication (ITD) mutations, which confer poorer prognosis. FLT3-ITD is a constitutively activated kinase with differential signaling compared to wild type (WT) FLT3. Notably, FLT3-ITD activates STAT5, which can regulate the prosurvival proteins BCL-XL and MCL-1. Venetoclax, a potent, selective inhibitor of the prosurvival protein BCL-2, demonstrated monotherapy activity in relapsed/refractory AML (ORR 19%); however, no activity was seen in FLT3 mutant cases. BCL-XL and MCL-1 are known venetoclax resistance factors and targeting their regulation in combination with venetoclax may enhance cell death. Based on this hypothesis, we interrogated the combination of venetoclax and quizartinib, a potent FLT3 inhibitor, in FLT3-ITD+ AML models. In vitro, quizartinib reduced expression of BCL-XL and MCL-1, but not BCL-2, in FLT3-ITD+ cells (Molm13 and MV4;11). However, quizartinib had no effect on BCL-XL or MCL-1 expression in FLT3 WT cells (HL60 and OCI-AML3). Combination treatment with venetoclax led to significant reduction in growth and increased apoptosis in FLT3-ITD+ cells compared to single agents while FLT3 WT cells were not sensitive to quizartinib alone or in combination with venetoclax. Consistent with FLT3-ITD regulating BCL-XL and MCL-1, cotreatment of quizartinib with either BCL-XL or MCL-1 specific inhibitors had no combination effect in FLT3-ITD+ cells. Additionally, quizartinib and venetoclax were assessed in vivo utilizing MV4;11 and Molm13 orthotopic xenograft models. Mice were treated with venetoclax (100 mg/kg), quizartinib (5 mg/kg), or the combination of both drugs for 21 continuous days. Quizartinib treatment significantly increased survival compared to the vehicle group in both xenograft models (56 days vs. 26 days and 32 days vs. 14 days for MV4;11 and Molm13 xenografts, respectively). However, venetoclax only marginally improved survival (34 days vs. 26 days) in the MV4;11 model and was not efficacious in the Molm13 model. Despite lack of single-agent activity, addition of venetoclax to quizartinib led to a further improvement in survival when compared to quizartinib (91 days vs. 56 days and 40 days vs. 32 days in MV4;11 and Molm13 xenografts, respectively). These data underscore the dependency of these models on FLT3-ITD for survival and its regulation of survival pathways that confer resistance to venetoclax, suggesting FLT3-ITD could be a predictive biomarker of venetoclax resistance. To determine mechanisms of venetoclax resistance mediated by FLT3-ITD, mutant cells were treated with PI3K, MEK or JAK2 kinase selective inhibitors alone or in combination with venetoclax. Each pathway inhibitor combined with venetoclax to reduce cell growth compared to single agents. This observation suggests that multiple pathways may contribute to venetoclax resistance rather than a single pathway dependency to confer the prosurvival effects of FLT3-ITD. Additional mechanistic studies are under way to define the role of each pathway in regulating survival. Together, our data demonstrate that FLT3-ITD inhibition combines with venetoclax in vitro and in vivo in FLT3-ITD+ AML models. These results provide a strong mechanistic rationale to support clinical investigation of FLT3 inhibitors in combination with venetoclax to treat FLT3-ITD+ AML. Citation Format: Raghuveer Mali, Elisabeth A. Lasater, Kelly Doyle, Ritu Malla, Erwin Boghaert, Andrew Souers, Joel Leverson, Deepak Sampath. FLT3-ITD activation mediates resistance to the BCL-2 selective antagonist, venetoclax, in FLT3-ITD mutant AML models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B052.

Although mitogen-activated protein (MAP)–extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the efficacy of a MEK inhibitor, we evaluated a panel of 53 non–small cell lung cancer and pancreatic cancer cell lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-xL (BCL2/BCL2L1) antagonist, and a novel MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment combination increases cell killing compared with double- or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with a Bcl-2 family inhibitor. Mol Cancer Ther; 12(6); 853–64. ©2013 AACR.

The ability of a cancer cell to avoid apoptosis is crucial to tumorigenesis and can also contribute to chemoresistance. The Bcl-2 family of prosurvival proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1) plays a key role in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small-molecule inhibitor of Bcl-2, Bcl-XL, and Bcl-w. While navitoclax exhibits single-agent activity in tumors dependent on Bcl-2 or Bcl-XL for survival, the expression of Mcl-1 has been shown to confer resistance to navitoclax, most notably in solid tumors. Thus, therapeutic agents that can downregulate or neutralize Mcl-1 are predicted to synergize potently with navitoclax. Here, we report the activity of navitoclax in combination with 19 clinically relevant agents across a panel of 46 human solid tumor cell lines. Navitoclax broadly enhanced the activity of multiple therapeutic agents in vitro and enhanced efficacy of both docetaxel and erlotinib in xenograft models. The ability of navitoclax to synergize with docetaxel or erlotinib corresponded to an altered sensitivity of the mitochondria toward navitoclax, which was associated with the downmodulation of Mcl-1 and/or upregulation of Bim. These data provide a rationale to interrogate these combinations clinically. Mol Cancer Ther; 10(12); 2340–9. ©2011 AACR.

The taxanes, paclitaxel (PTX) and docetaxel (DTX), belong to a novel class of anticancer drugs that stabilize microtubules and lead to tumor cell death. While both agents are widely used for the treatment of lung, breast, and ovarian cancer, many tumor types are refractory or develop resistance to these drugs. We describe here a novel analogue of DTX, designated MAC-321 [Microtubule/Apoptosis/Cytotoxic: 5beta, 20-epoxy-1, 2alpha-, 4-, 7beta-, 10beta-, 13alpha-hexahydroxytax-11-en-9-one 4 acetate 2 benzoate 7-propionate 13-ester with (2R,3S)-N-tertbutoxycarbonyl-3-(2-furyl)isoserine], that overcomes P-glycoprotein-mediated resistance to PTX and DTX in preclinical model systems. Similar to PTX or DTX, MAC-321 enhanced the rate of tubulin polymerization in vitro and caused the bundling of microtubules in cells. MAC-321 inhibited proliferation of a panel of 14 tumor cell lines with minimal variation in potency (IC(50) = 2.2 +/- 1.4 nM; range = 0.6-5.3 nM). Unlike PTX or DTX, the IC(50) of MAC-321 did not vary in cells that expressed low to moderate levels of P-glycoprotein. Even under extraordinary conditions in KB-V1 cells, which highly overexpress P-glycoprotein, resistance to MAC-321 was 80-fold compared with that of PTX (1400-fold) and DTX (670-fold). In addition, equivalent or less resistance to MAC-321 compared with PTX or DTX was observed in four cell lines that contain distinct point mutations within the taxane-binding site of beta-tubulin. Most importantly, MAC-321 displayed superior in vivo efficacy because: (a) MAC-321 either partially or completely inhibited tumor growth in three tumor models that overexpressed P-glycoprotein and were resistant to PTX; and (b) unlike PTX or DTX, MAC-321 was highly effective when given orally. MAC-321 was also highly effective when given as single i.v. dose. Our findings suggest that MAC-321, which is currently under clinical evaluation, may have broad therapeutic value.

Nicotinamide adenine dinucleotide (NAD) is a metabolite essential for cell survival and is generated de novo from tryptophan or recycled from nicotinamide (NAM) through the nicotinamide phosphoribosyl transferase (NAMPT)-dependent salvage pathway. Alternatively, tumors can convert nicotinic acid (NA) [also referred in the literature as vitamin B3 or niacin] to NAD through the nicotinic acid phosphoribosyltransferase 1 (NAPRT1)-dependent pathway. Tumor cells do not efficiently utilize the de novo synthesis pathway, rendering them more reliant on NAMPT and, therefore, making this enzyme an attractive therapeutic target. Moreover, it has been proposed that the therapeutic index of NAMPT inhibitors may be increased in tumors that lack NAPRT1 upon co-administration of NA as normal tissues can generate NAD through NAPRT1. Using novel and potent orally bioavailable NAMPT small molecule inhibitors, GNE-617 and GNE-618, we evaluated the feasibility of the latter approach in vivo using xenograft models (n=5) derived from tumor cells lines (n=3) and primary patient samples (n=2) that lacked NAPRT1. All xenograft models tested were sensitive to GNE-617 or GNE-618 demonstrated by rapid induction of tumor regressions upon treatment with NAMPT inhibitors. While NA did not protect NAPRT1-deficient tumor models in vitro or ex vivo from GNE-617 and GNE-618, it rescued the anti-tumor effects of these inhibitors in the same tumor models grown as xenografts in vivo. Co-administration of NA with GMX-1778, a previously reported NAMPT small molecule inhibitor, also resulted in complete loss of efficacy suggesting that rescuability with NA in vivo is not an inhibitor-specific phenomenon. Importantly, tumor xenografts remained NAPRT1-deficient upon NA co-treatment, indicating that tumors were not reactivating the NAPRT1-dependent salvage pathway to compensate for NAMPT inhibition. However, NA co-treatment increased NAD and NAM levels in NAPRT1-deficient tumor xenografts to levels that were sufficient to support robust tumor growth even in the presence of GNE-617. Furthermore, NAM co-administration with GNE-617 led to increased tumor NAD levels in vivo and afforded the same degree of protection as NA. Given that NAMPT inhibitors compete biochemically with NAM, a modest increase in tumor NAM levels may be sufficient to reduce NAMPT inhibition to adequately sustain NAD production. Thus, it appears that the tumor models tested in this study maybe utilizing exogenously supplied metabolites in vivo to circumvent the effects of NAMPT inhibition. The unexpected protection of NAPRT1-deficient tumors in vivo may be due to enhanced circulating levels of metabolites generated by normal tissue, such as liver, in response to NA. Our results have important implications for the development of NAMPT inhibitors when considering NA co-treatment as an antidote and strategy for increasing the therapeutic index. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B154. Citation Format: Deepak Sampath, Jason Oeh, Yang Xiao, Xiaorong Liang, Alexander Vanderbilt, Ann Qin, LuLu Yang, Leslie Lee, Justin Ly, Ely Cosino, Jennifer LaCap, Annie Ogasawara, Simon Williams, Michelle Nannini, Bianca Liederer, Peter Jackson, Peter Dragovich, Thomas O'Brien. Co-administration of nicotinic acid with NAMPT small molecule inhibitors results in loss of in vivo efficacy in NAPRT1-deficient tumor 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 B154.

Nicotinamide adenine dinucleotide (NAD) is a critical cellular metabolite and the key enzyme in the NAD salvage pathway is nicotinamide phosphoribosyl transferase (NAMPT). Here we describe GNE-617, a novel orally bioavailable NAMPT inhibitor, and show that this compound promotes rapid depletion of NAD both in cells and in xenograft tumor models. Moreover, GNE-617 rapidly induced tumor regressions in colorectal (HCT-116), fibrosarcoma (HT-1080), prostate (PC3) and pancreatic (MiaPaca2) xenograft models. Despite increasing interest in exploiting cancer cell metabolism as a therapeutic strategy, there is still a poor understanding of cell death mechanisms following disruption of key metabolic processes. Here we show that depletion of NAD in cells induces a series of cellular events that leads to oncosis-Blister Cell Death (oncosis-BCD). In all cell lines examined, NAD was depleted >95% within 25-39 hours, which was rapidly followed by a loss of ATP (>95% depleted by 41-76 hours). Depletion of NAD correlates with loss of cell motility and a reduction in mitotic index, which may be explained by reduced activity of NAD-dependent protein deacetylases. In support of this, we find that α-tubulin-K40 is hyper-acetylation, a modification that is known to reduce tubulin dynamics and cell motility. Additionally, there is no evidence of histone H3-K9 deacetylation, which normally occurs as cells enter mitosis. Using a combination of approaches including live cell imaging and electron microscopy we found that cell lines with a more rapid depletion of ATP display signs of necrosis and oncosis-BCD, while cell lines with a slower depletion of ATP show signs of autophagy, apoptosis, necrosis and oncosis-BCD. In all cases however, oncosis-BCD is the predominant form of cell death and is characterized by plasma membrane swelling to form large organelle-free blisters. Finally, we conclude that cell death is due to loss of plasma membrane ion-homeostasis associated with ATP depletion. In conclusion, our data shows that there is an ordered progression of events that occurs in cancer cells following NAD depletion and reveals that oncosis-BCD is the predominant form of cell death in response to NAD depletion. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C150. Citation Format: Thomas O'Brien, Christopher Del Nagro, Yang Xiao, Jason Oeh, Linda Rangell, Mike Reichelt, Xiaorong Liang, Bianca M. Liederer, Peter S. Dragovich, Deepak Sampath. Depletion of cellular NAD using the NAMPT inhibitor GNE-617 leads to oncosis blister cell death. [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 C150.

Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody induces a high level of direct cell death. As a result of glycoengineering, GA101 has increased affinity for FcγRIIIa on effector cells resulting in enhanced direct cell death and ADCC induction. GA101 is currently in pivotal clinical trials in chronic lymphocytic leukemia (CLL), indolent non-Hodgkins lymphoma (iNHL) and diffuse large B-cell lymphoma (DLBCL). ABT-199 (GDC-0199) is a novel, orally bioavailable, selective Bcl-2 inhibitor that induces robust apoptosis in preclinical models of hematological malignancies and is currently in clinical trials for CLL, NHL and multiple myeloma. Based on their complementary mechanisms of action involving increased apoptosis (GDC-0199) or direct cell death (GA101) the combination of anti-CD20 therapy with a Bcl 2 inhibitor has the potential for greater efficacy in treating B lymphoid malignancies. The combination of GA101 or rituximab with GDC-0199 was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (AxV/Pi positivity) on WSU-DLCL2 NHL, SU-DHL4 DLBCL and Z138 mantle cell lymphoma cells by FACS and the impact of Bcl-2 inhibition on ADCC induction. In vivo efficacy of the combination of GA101 or rituximab and GDC-0199 was evaluated in SU-DHL4 and Z138 xenograft models. GA101 and rituximab enhanced cell death induction when combined with GDC-0199 in SU-DHL4, WSU-DLCL2 and Z138 cell lines. When combined at optimal doses of GA101 and GDC-0199 an additive effect of the two drugs was observed. GDC-0199 did not negatively impact the capability of GA101 or rituximab to induce NK-cell mediated ADCC. Combination of GDC 0199 with GA101 or rituximab in vivo induced greater than additive anti-tumor effects in the SU DHL4 and Z138 xenograft models resulting in tumor regressions and delay in tumor regrowth when compared to monotherapy. Importantly, continued single-agent treatment with GDC-0199 after combination with GA101 resulted in sustained in vivo efficacy in the SU-DHL4 xenograft model. Our data demonstrate that the combination of GA101 with GDC-0199 results in enhanced cell death and robust anti-tumor efficacy in xenograft models representing NHL sub-types that is comparable to combination therapy with rituximab. In addition, single-agent treatment with GDC-0199 following combination with GA101 sustains efficacy in vivo suggesting a potential benefit in continued maintenance therapy with GDC-0199. Collectively, our preclinical data strongly supports clinical investigation of GA101 and GDC-0199 combination therapy, which is currently being evaluated in a phase Ib clinical trial (clinical trial.gov identifier [NCT01685892][1]). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A245. Citation Format: Deepak Sampath, Sylvia Herter, Ellen Ingalla, Frank Herting, Marina Bacac, Michelle Nannini, Wayne J. Fairbrother, Christian Klein. Combination of the glycoengineered Type II CD20 antibody obinutuzumab (GA101), and the novel Bcl-2 selective inhibitor, ABT-199 (GDC-0199), results in superior in vitro and in vivo anti-tumor activity in models of B-cell malignancies. [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 A245. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01685892&atom=%2Fmolcanther%2F12%2F11_Supplement%2FA245.atom

Background: Akt, a serine/threonine protein kinase, is a key signaling node in the PI3K/Akt/mTOR pathway and plays an essential role in regulating tumor cell proliferation, migration and survival. PI3K/Akt signaling is up-regulated in gastric cancer, mainly due to loss of PTEN, and is associated with poor prognosis and chemoresistance. Chemotherapeutics such as doxorubicin and 5-FU/cisplatin have been shown to upregulate PI3K/Akt signaling in several solid cancers in response to DNA damage. We have previously reported on a potent highly selective ATP-competitive pan-Akt inhibitor, GDC-0068, which inhibits cell cycle progression and viability of cancer cell lines and xenografts driven by Akt signaling, including those with defects in the tumor suppressor PTEN, oncogenic mutations in PIK3CA, and amplification of HER2. Thus, we hypothesized that the combination of FOLFOX and GDC-0068 would induce anti-tumor activity in primary gastric cancer models. Methods: Gastric cancer cell lines (n=11) were treated with increasing concentrations of GDC-0068 and FOLFOX and evaluated for combinational efficacy over single agent via cell viability assay. For in vivo studies, primary gastric cancer xenograft models were implanted in immuno-compromised mice and treated with GDC-0068 and FOLFOX as single agents or in combination. Results: In vitro, the combination of GDC-0068 and FOLFOX results in enhanced inhibition of tumor cell viability in the cell lines with activation of the PI3K pathway compared to either single agent alone. These results were recapitulated in vivo, in the primary xenograft models STO#240 and STO#182, where the combination of GDC-0068 and FOLFOX resulted in increased tumor growth inhibition or regressions compared to single agents alone. All combinations tested were well tolerated in vivo based on minimal changes in body weights. Conclusions: Our preclinical studies demonstrate improved anti-tumor efficacy when GDC-0068 is combined with FOLFOX in gastric cancer models in vitro and in vivo. The data supports the clinical development of GDC-0068 in combination with FOLFOX for the treatment of gastric cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B190. Citation Format: Ellen Ingalla, Rebecca Hong, Heidi Savage, Elizabeth Punnoose, Sandra Rost, Hartmut Koeppen, Deepak Sampath, Michelle A. Nannini. GDC-0068 is a novel and selective Akt inhibitor that enhances the efficacy of FOLFOX in primary gastric cancer 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 B190.

Introduction: The phosphoinositide-3 kinase (PI3K)/Akt signaling pathway is deregulated in a wide variety of cancers. Somatic activating mutations and amplifications in PI3K are common in multiple cancers including breast, colon, and lung cancer. PTEN, a phosphatase that converts PIP3 to PIP2 and thus has an opposing function to PI3K, is a commonly mutated tumor suppressor in multiple cancers including prostate and glioblastoma. Alterations of this pathway have been implicated in tumor initiation, progression, survival, angiogenesis, and metastasis. PI3K/Akt pathway activation has also been implicated in therapeutic resistance. Thus PI3K is considered to be a promising therapeutic target for cancer. Summary of Results: Medicinal chemistry efforts resulted in the discovery of GDC-0980, a selective, orally bioavailable inhibitor of PI3 Kinase and mTOR kinase with promising pharmacokinetic and pharmaceutical properties. Here we report the first pre-clinical profile of GDC-0980. This compound is a potent ATP-competitive Class I PI3 kinase inhibitor with an IC50 of 4.8 nM against the PI3K p110apha subunit, 26.8 nM p110beta, 6.7 nM p110delta, 13.8 nM p110gamma, and an mTOR Kiapp of 17.3 nM. GDC-0980 demonstrates selectivity against a large panel of protein kinases as well as selectivity over PIK family kinases including Class II, Class III, and DNA-PK. GDC-0980 inhibits the PI3K signaling pathway in vitro causing a reversible G1 cell cycle arrest, and apoptosis induction in a subset of tumor cell lines. Levels of signaling pathway markers such as phosphorylated AKT (pAKT), PRAS40 (pPRAS40), and S6 (pS6) are rapidly and dramatically reduced following exposure of cells to GDC-0980. Oral dosing of GDC-0980 potently inhibits tumor growth in xenograft models including those mutated in PI3K and PTEN, and elicits an exposure-related concomitant decrease in PD biomarkers. Conclusion: These preclinical data provide compelling evidence in support of GDC-0980 as a clinical candidate, and Phase I studies are ongoing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C201.

Background: The Janus-associated kinase (JAK) proteins are a family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that play an important role in cellular survival, proliferation, and differentiation. A gain of function mutation (V617F) in the pseudo-kinase domain of JAK2 is detected at high frequency in Philadelphia-negative myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. To further elucidate the role of JAK2V617F on cell growth in vivo we established and validated a novel xenograft model using the acute myeloid leukemia cell line, SET2, which is heterozygous for the mutation. Materials and Methods: SET2 cells containing stable doxycycline-inducible JAK2V617F shRNA clones were generated by lentiviral transduction. Cell proliferation and viability were assessed by incorporation of 3H-Thymidine or by ATP quantitation. Cell-cycle and apoptosis markers were evaluated by Western blotting using antibodies against cyclin D1, PIM1, cleaved caspase 3 and 7 and PARP. Phospho-STAT5 and total STAT5 were measured using both western blotting and ELISA. A SET2 shRNA clone was selected for in vivo growth in SCID beige female mice and treated with doxycycline or a JAK2 inhibitor (JAK2i). Plasma and tumor drug levels were measured by LC/MS. Results: Genetic knockdown using a doxycycline-inducible JAK2V617F shRNA confirmed SET2 cells are dependent on this gain of function mutation for growth based on inhibition of cell proliferation in the presence of doxycycline. The latter was corroborated with a selective JAK2i (Ki= 4nM) that potently inhibited SET2 cell viability (EC50 = 355nM). The JAK2i rapidly suppressed phosphorylation of STAT5 (EC50 = 313nM) and induced cycle arrest at the G0–G1 phase based on downregulation of cyclin D1 expression. The JAK2i also effectively inhibited expression of PIM-1 kinase, a STAT5 target gene, and activated cleavage of procaspase 3 and 7 and DNA-repair enzyme PARP to induce apoptosis in a time- and dose-dependent manner. A SET2 xenograft model was established by in vivo selection of a variant of the doxycycline- JAK2V617F shRNA parental cell line. Its dependence on JAK2V617F was confirmed by treatment with doxcycline and oral administration of a JAK2i, which resulted in significant tumor growth inhibition in vivo. Investigation of the PK/PD relationship underlying the activity of JAK2i provided direct evidence of phospho-STAT5 suppression and induction of apoptosis for its antitumor effect in vivo. Plasma drug levels correlated with the duration and magnitude of suppression. Conclusion: A JAK2V617F dependent SET2 xenograft model has been established and validated by shRNA gene knockdown and pharmacologically with a JAK2i in vivo. JAK2V617F inhibition results in suppression of STAT5 phosphorylation, cell-cycle arrest and induction of apoptosis in vitro and in vivo. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A20.