Rebecca Hong

MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD-L1 Checkpoint Blockade

Targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) can induce regression of tumors bearing activating mutations in the Ras pathway but rarely leads to tumor eradication. Although combining MEK inhibition with T-cell-directed immunotherapy might lead to more durable efficacy, T cell responses are themselves at least partially dependent on MEK activity. We show here that MEK inhibition did profoundly block naive CD8(+) T cell priming in tumor-bearing mice, but actually increased the number of effector-phenotype antigen-specific CD8(+) T cells within the tumor. MEK inhibition protected tumor-infiltrating CD8(+) T cells from death driven by chronic TCR stimulation while sparing cytotoxic activity. Combining MEK inhibition with anti-programmed death-ligand 1 (PD-L1) resulted in synergistic and durable tumor regression even where either agent alone was only modestly effective. Thus, despite the central importance of the MAP kinase pathway in some aspects of T cell function, MEK-targeted agents can be compatible with T-cell-dependent immunotherapy.

Targeting Activated Akt with GDC-0068, a Novel Selective Akt Inhibitor That Is Efficacious in Multiple Tumor Models

Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. Experimental Design: The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. Results: GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents. Conclusions: GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling. Clin Cancer Res; 19(7); 1760–72. ©2012 AACR.

Metabolite profiling stratifies pancreatic ductal adenocarcinomas into subtypes with distinct sensitivities to metabolic inhibitors

Significance Targeting cancer metabolism requires personalized diagnostics for clinical success. Pancreatic ductal adenocarcinoma (PDAC) is characterized by metabolism addiction. To identify metabolic dependencies within PDAC, we conducted broad metabolite profiling and identified three subtypes that showed distinct metabolite profiles associated with glycolysis, lipogenesis, and redox pathways. Importantly, these profiles significantly correlated with enriched sensitivity to a variety of metabolic inhibitors including inhibitors targeting glycolysis, glutaminolysis, lipogenesis, and redox balance. In primary PDAC tumor samples, the lipid subtype was strongly associated with an epithelial phenotype, whereas the glycolytic subtype was strongly associated with a mesenchymal phenotype, suggesting functional relevance in disease progression. Our findings will provide valuable predictive utility for a number of metabolic inhibitors currently undergoing phase I testing. Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional ∼200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.

Bcl-2/Bcl-xL Inhibition Increases the Efficacy of MEK Inhibition Alone and in Combination with PI3 Kinase Inhibition in Lung and Pancreatic Tumor Models

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.

Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice

Expression of PD-L1, the ligand for T-cell inhibitory receptor PD-1, is one key immunosuppressive mechanism by which cancer avoids eradication by the immune system. Therapeutic use of blocking antibodies to PD-L1 or its receptor PD-1 has produced unparalleled, durable clinical responses, with highest likelihood of response seen in patients whose tumour or immune cells express PD-L1 before therapy. The significance of PD-L1 expression in each cell type has emerged as a central and controversial unknown in the clinical development of immunotherapeutics. Using genetic deletion in preclinical mouse models, here we show that PD-L1 from disparate cellular sources, including tumour cells, myeloid or other immune cells can similarly modulate the degree of cytotoxic T-cell function and activity in the tumour microenvironment. PD-L1 expression in both the host and tumour compartment contribute to immune suppression in a non-redundant fashion, suggesting that both sources could be predictive of sensitivity to therapeutic agents targeting the PD-L1/PD-1 axis.

Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Abstract S6-04: The PI3K inhibitor, taselisib, has enhanced potency in PIK3CA mutant models through a unique mechanism of action

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutational activation of the PIK3CA gene. The dysregulation of this pathway has been implicated in tumor cell growth and survival, thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha sparing inhibition of p110beta, is investigated in these preclinical studies. Taselisib demonstrates greater potency in cancer cell lines harboring activating mutations in PIK3CA vs. wild-type lines, and induces regressions at tolerated doses in both PIK3CA mutant xenograft and patient-derived xenograft (PDX) models. When comparing taselisib to other clinical-stage PI3K inhibitors at Maximum Tolerated Dose (MTD) in vivo, taselisib confers greater activity in PIK3CA mutant models, which may indicate a larger therapeutic index. Unlike other PI3K inhibitors, taselisib has a gain of potency in PI3K alpha mutant SW48 isogenic cells compared to wildtype SW48 parental cells. Pathway inhibition and increased apoptosis are associated with the enhanced activity observed in PI3K alpha mutant cells. Other clinical PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not have improved potency in PI3K alpha mutant cells due to their inability to maintain pathway suppression after alleviation of negative feedback. The unique mechanism of action of taselisib is most notable when comparing signaling suppression at 24 hours vs. 1 hour of drug exposure. In mutant cells, Taselisib displays greater pathway suppression at 24 hours and is more effective at maintaining pathway suppression upon re-activation of growth factor RTK signaling. This mechanism is specific to PIK3CA mutant cells and not observed in wildtype cells. Ongoing studies to further elucidate this mechanism of action will be presented. Citation Format: Kyle A. Edgar, Kyung Song, Stephen Schmidt, Don Kirkpatrick, Lilian Phu, Michelle Nannini, Rebecca Hong, Eric Cheng, Amy Young, Deepak Sampath, Lori S. Friedman. The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 370.

Abstract P4-15-02: The PI3K inhibitor GDC-0032 enhances the efficacy of standard of care therapeutics in PI3K alpha mutant breast cancer models

The phosphoinositide 3-kinases (PI3Ks) are lipid kinases that activate the PI3K signaling pathway and play an essential role in regulating breast tumor cell growth, migration, and survival. Activating and transforming mutations in the PIK3CA gene (PI3K alpha) are commonly found in HER2+ breast cancer and ER+ breast cancer. GDC-0032 is a selective, orally bioavailable inhibitor with a Ki of 0.29 nM for PI3K alpha with 30 fold less inhibition of PI3K beta. In addition, GDC-0032 has increased single agent activity against PI3K alpha mutant and HER2 amplified cancer models in vitro and in vivo. The aim of our study was to evaluate the efficacy of GDC-0032 in breast cancer models in combination with standard of care therapeutics including taxanes, endocrine therapies, and HER2 targeted therapies. We evaluated cell viability for a range of dose concentrations of GDC-0032, as single agent and in combinations, in eleven breast cancer cell lines that harbor mutations in PI3K alpha (K111N, E545K, H1047R) and/or over-expressed HER2. GDC-0032 was combined with taxanes (paclitaxel and docetaxel), endocrine therapies (fulvestrant and tamoxifen) or anti-HER2 agents (trastuzumab, pertuzumab or ado-trastuzumab-emtansine) and synergy quantified using the Chou and Talalay method of Combination Index (C.I.). The combination of GDC-0032 with taxanes, endocrine therapies or anti-HER2 therapies were synergistic in the breast cancer cell lines tested based on C.I. values of less than 1.0. The combination of GDC-0032 with docetaxel in vitro resulted in decreased viability as a result of increased cell death. The in vitro results translated in vivo as GDC-0032 enhanced the anti-tumor activity of docetaxel and paclitaxel in MCF7 (E545K) xenografts that resulted in increased tumor regressions when GDC-0032 was dosed daily or intermittently on the same schedule as the taxane. In addition, the combination of GDC-0032 and fulvestrant or tamoxifen resulted in greater efficacy in the MCF7 xenograft model when compared to either agent alone. Enhanced efficacy was also observed when GDC-0032 was combined with trastuzumab or ado-trastuzumab-emtansine in the HER2+ BT474M1 (K111N) xenograft model. Moreover, the triple combinations of GDC-0032/trastuzumab/pertuzumab or GDC-0032/trastuzumab/docetaxel resulted in durable tumor regressions that were sustained in the HER2+ KPL-4 (H1047R) and BT474M1 xenograft models, respectively. All drug combinations with GDC-0032 were well tolerated in vivo based on minimal changes in body weight. Collectively, our preclinical data demonstrate that GDC-0032 enhances the efficacy of standard of care therapeutics in PI3K alpha mutant breast cancer models and provides a strong rationale for further evaluation in patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-02.

Abstract 873: Combined targeting of Akt and MEK with the Akt inhibitor GDC-0068 and MEK inhibitor GDC-0973 demonstrates synergistic anti-tumor effects

Background: Akt, a serine/threonine protein kinase, and MEK, a dual specificity kinase, are key signaling nodes in the PI3K/Akt/mTOR and the Ras/Raf/MAPK pathway, respectively. Both Akt and MEK play key roles in regulating numerous cellular processes such as cell proliferation, growth, survival, protein synthesis and oncogenic transformation. Many cancer types have alterations in both of these pathways and inhibiting only one of these pathways can result in up-regulation of the other pathway. We previously reported on a potent highly selective ATP-competitive pan-Akt inhibitor, GDC-0068 as well as a potent selective inhibitor of MEK, GDC-0973 that are currently in Phase 1 clinical trials. We hypothesized that dual inhibition of both MEK and Akt pathways with the combination of these two agents would induce synergistic antitumor activity. Methods: In these studies, we evaluated the efficacy of GDC-0068 and GDC-0973 both individually and in combination on a panel of cancer cell lines. Cells were treated with either GDC-0068 or GDC-0973 or in combination at increasing concentrations and assayed after 4 days for viability. For in vivo studies, tumor cells were subcutaneously implanted in the flank of female NCR.nude mice. Once tumors reached sufficient size, mice were dosed orally for 21 days with GDC-0068, GDC-0973 or the combination of both compounds. Results: In vitro, the combination of GDC-0068 and GDC-0973 results in enhanced inhibition of cell viability compared to either single agent alone. Synergistic effects are observed in multiple cell lines; especially in cell lines that have activation of the Ras/Raf/MAPK pathway or both pathways (e.g. via the combination of PTEN loss or PI3K mutations and Ras or BRaf mutations). Combined knockdown of downstream targets of both Akt and MEK is observed at concentrations where a synergistic effect is observed, with enhanced knockdown of several converged targets. Increased cell death is also observed. These results were recapitulated in vivo in xenograft models, where the combination of GDC-0068 and GDC-0973 resulted in increased tumor growth inhibition or regression compared to either single agent alone. All combinations tested were well tolerated as assessed by animal body weights and mortalities. Conclusions: Our studies demonstrate significant combination benefit between GDC-0068 and GDC-0973 on cell viability in vitro and tumor growth in vivo. These data support the clinical development of the combination of these two compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 873. doi:1538-7445.AM2012-873

Abstract 156: Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Dysregulation of the PI3K/Akt/mTOR signaling pathway through multiple mechanisms has been described in solid tumor malignancies, including activating and transforming “hotspot” mutations as well as amplification of PIK3CA that encodes the p110 alpha subunit of PI3K. Hotspot mutations of PIK3CA mutation are frequently observed in breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent inhibitor of PI3K alpha (IC50 = 0.038 + 0.003 nM) and exerts its activity by binding to the ATP binding site of PI3K, thereby inhibiting the phosphorylation of PIP2 to PIP3. Biochemically, GDC-0077 is more than 300-fold selective over other Class I PI3K isoforms such as beta, delta, and gamma and more than 2000 fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell based assays. Compared to the PI3K inhibitor, taselisib, the improved biochemical selectivity of GDC-0077 against PI3K delta is demonstrated in human CD69+ B-cells, which are primarily dependent on PI3K delta for proliferation and survival and were more sensitive to taselisib than GDC-0077. Mechanism of action studies indicate that GDC-0077 induces depletion of mutant PI3K alpha protein resulting in reduction of PI3K pathway biomarkers such as pAkt and pPRAS40, inhibition of cell proliferation and increased apoptosis in human PIK3CA mutant breast cancer cell lines to a greater extent when compared to PIK3CA wild-type cells. In vivo, daily oral treatment with GDC-0077 in cell-culture-derived and patient derived PIK3CA mutant breast cancer xenograft models, resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo efficacy in a PIK3CA-mutant human breast cancer xenograft model was also improved when GDC-0077 was combined with standard-of-care therapies for hormone-receptor positive (HR+) breast cancer such as anti-estrogens (fulvestrant) or CDK4/6 inhibitor (palbociclib). Collectively, the preclinical data provide rationale for evaluating GDC-0077, a PI3K alpha mutant selective inhibitor, as a single agent and in combination with standard-of-care endocrine and targeted therapies that may provide additional benefit to patients that harbor PIK3CA mutations. Citation Format: Kyle Edgar, Emily Hanan, Steven Staben, Stephen Schmidt, Rebecca Hong, Kyung Song, Amy Young, Patricia Hamilton, Alfonso Arrazate, Cecile de la Cruz, Marcia Belvin, Michelle Nannini, Lori S. Friedman, Deepak Sampath. Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development [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 156. doi:10.1158/1538-7445.AM2017-156