ABSTRACT
Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.
Subject(s)
Antineoplastic Agents/therapeutic use , Benzamides/therapeutic use , Colorectal Neoplasms/genetics , Diphenylamine/analogs & derivatives , MAP Kinase Signaling System , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Cell Line, Tumor , Clonal Evolution , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Diphenylamine/pharmacology , Diphenylamine/therapeutic use , Drug Resistance, Neoplasm , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , MAP Kinase Signaling System/drug effects , Mice , Mutation , RetroviridaeABSTRACT
Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.
Subject(s)
Antibodies, Monoclonal , Receptor, Fibroblast Growth Factor, Type 1 , Signal Transduction , Animals , Humans , Mice , Neoplasms , Receptor, Fibroblast Growth Factor, Type 1/agonists , Receptor, Fibroblast Growth Factor, Type 1/antagonists & inhibitors , Antibodies, Monoclonal/pharmacologyABSTRACT
B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Immunoconjugates/therapeutic use , Oligopeptides/therapeutic use , Animals , Antineoplastic Agents/chemistry , Blotting, Western , Cell Line, Tumor , Female , Flow Cytometry , Humans , Immunoconjugates/chemistry , Immunohistochemistry , Mice , Mice, SCID , Oligopeptides/chemistry , Rats , Rats, Sprague-Dawley , Triple Negative Breast Neoplasms/drug therapy , Xenograft Model Antitumor AssaysABSTRACT
Herein we report the optimization efforts to ameliorate the potent CYP3A4 time-dependent inhibition (TDI) and low aqueous solubility exhibited by a previously identified lead compound from our NAMPT inhibitor program (1, GNE-617). Metabolite identification studies pinpointed the imidazopyridine moiety present in 1 as the likely source of the TDI signal, and replacement with other bicyclic systems was found to reduce or eliminate the TDI finding. A strategy of reducing the number of aromatic rings and/or lowering cLogD7.4 was then employed to significantly improve aqueous solubility. These efforts culminated in the discovery of 42, a compound with no evidence of TDI, improved aqueous solubility, and robust efficacy in tumor xenograft studies.
Subject(s)
Cytochrome P-450 CYP3A/chemistry , Enzyme Inhibitors/chemistry , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Animals , Binding Sites , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Cell Proliferation/drug effects , Crystallography, X-Ray , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 CYP3A Inhibitors/chemistry , Cytochrome P-450 CYP3A Inhibitors/pharmacokinetics , Cytochrome P-450 CYP3A Inhibitors/toxicity , Dogs , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Female , Half-Life , Humans , Kinetics , Madin Darby Canine Kidney Cells , Mice , Mice, Nude , Molecular Dynamics Simulation , Neoplasms/drug therapy , Neoplasms/pathology , Nicotinamide Phosphoribosyltransferase/metabolism , Protein Binding , Protein Structure, Tertiary , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Pyrimidines/toxicity , Solubility , Structure-Activity Relationship , Thermodynamics , Transplantation, Heterologous , Water/chemistryABSTRACT
Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a differentiated HER2 ADC, we chose an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. In our study, the PBD dimer is modified to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrates in vivo efficacy in models of HER2-positive and HER2-low cancers and is well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study (ClinicalTrials.gov: NCT03451162) in patients with HER2-positive metastatic breast cancer, with the primary objective of evaluating the safety and tolerability of DHES0815A and secondary objectives of characterizing the pharmacokinetics, objective response rate, duration of response, and formation of anti-DHES0815A antibodies, is reported herein. Despite early signs of anti-tumor activity, patients at higher doses develop persistent, non-resolvable dermal, ocular, and pulmonary toxicities, which led to early termination of the phase 1 trial.
Subject(s)
Antibodies, Monoclonal, Humanized , Antineoplastic Agents , Benzodiazepines , Breast Neoplasms , Immunoconjugates , Humans , Animals , Female , Breast Neoplasms/genetics , Macaca fascicularis/genetics , Receptor, ErbB-2/metabolism , Trastuzumab/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , DNAABSTRACT
Trastuzumab-DM1 (T-DM1) is a novel antibody-drug conjugate under investigation for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. One challenge in oncologic drug development is determining the optimal dose and treatment schedule. A novel dose regimen-finding strategy was developed for T-DM1 using experimental data and pharmacokinetic/pharmacodynamic modeling. To characterize the disposition of T-DM1, pharmacokinetic studies were conducted in athymic nude and beige nude mice. The pharmacokinetics of T-DM1 were described well by a two-compartment model. Tumor response data were obtained from single-dose, multiple-dose and time-dose-fractionation studies of T-DM1 in animal models of HER2-positive breast cancer, specifically engineered to be insensitive to trastuzumab. A sequential population-based pharmacokinetic/pharmacodynamic modeling approach was developed to describe the anti-tumor activity of T-DM1. A cell-cycle-phase nonspecific tumor cell kill model incorporating transit compartments captured well the features of tumor growth and the activity of T-DM1. Key findings of the model were that tumor cell growth rate played a significant role in the sensitivity of tumors to T-DM1; anti-tumor activity was schedule independent; and tumor response was linked to the ratio of exposure to a concentration required for tumor stasis.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antineoplastic Agents/administration & dosage , Immunotoxins/therapeutic use , Maytansine/analogs & derivatives , Models, Biological , Neoplasms/drug therapy , Ado-Trastuzumab Emtansine , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Humans , Maytansine/administration & dosage , Maytansine/pharmacokinetics , Mice , Mice, Nude , Time Factors , Trastuzumab , Xenograft Model Antitumor AssaysABSTRACT
Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents, Immunological/pharmacology , Endoplasmic Reticulum Stress/physiology , Endoribonucleases/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Triple Negative Breast Neoplasms/therapy , Animals , Antineoplastic Agents, Immunological/immunology , Cell Line, Tumor , Endoplasmic Reticulum Stress/drug effects , Endoribonucleases/genetics , Female , Gene Knockout Techniques , Humans , Mice , Mice, SCID , Neovascularization, Pathologic/therapy , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/genetics , Triple Negative Breast Neoplasms/blood supply , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Tumor Microenvironment , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/immunology , X-Box Binding Protein 1/antagonists & inhibitors , X-Box Binding Protein 1/genetics , Xenograft Model Antitumor AssaysABSTRACT
Squamous cell carcinomas (SCCs) account for the majority of cancer mortalities. Although TP63 is an established lineage-survival oncogene in SCCs, therapeutic strategies have not been developed to target TP63 or it's downstream effectors. In this study we demonstrate that TP63 directly regulates NRG1 expression in human SCC cell lines and that NRG1 is a critical component of the TP63 transcriptional program. Notably, we show that squamous tumors are dependent NRG1 signaling in vivo, in both genetically engineered mouse models and human xenograft models, and demonstrate that inhibition of NRG1 induces keratinization and terminal squamous differentiation of tumor cells, blocking proliferation and inhibiting tumor growth. Together, our findings identify a lineage-specific function of NRG1 in SCCs of diverse anatomic origin.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Differentiation , Neuregulin-1/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice, Nude , Receptor, ErbB-3/metabolismABSTRACT
The receptor tyrosine kinase HER2 is overexpressed in approximately 20% of breast cancer, and its amplification is associated with reduced survival. Trastuzumab emtansine (Kadcyla, T-DM1), an antibody-drug conjugate that is comprised of trastuzumab covalently linked to the antimitotic agent DM1 through a stable linker, was designed to selectively deliver DM1 to HER2-overexpressing tumor cells. T-DM1 is approved for the treatment of patients with HER2-positive metastatic breast cancer following progression on trastuzumab and a taxane. Despite the improvement in clinical outcome, many patients who initially respond to T-DM1 treatment eventually develop progressive disease. The mechanisms that contribute to T-DM1 resistance are not fully understood. To this end, we developed T-DM1-resistant in vitro models to examine the mechanisms of acquired T-DM1 resistance. We demonstrate that decreased HER2 and upregulation of MDR1 contribute to T-DM1 resistance in KPL-4 T-DM1-resistant cells. In contrast, both loss of SLC46A3 and PTEN deficiency play a role in conferring resistance in BT-474M1 T-DM1-resistant cells. Our data suggest that these two cell lines acquire resistance through distinct mechanisms. Furthermore, we show that the KPL-4 T-DM1 resistance can be overcome by treatment with an inhibitor of MDR1, whereas a PI3K inhibitor can rescue PTEN loss-induced resistance in T-DM1-resistant BT-474M1 cells. Our results provide a rationale for developing therapeutic strategies to enhance T-DM1 clinical efficacy by combining T-DM1 and other inhibitors that target signaling transduction or resistance pathways. Mol Cancer Ther; 17(7); 1441-53. ©2018 AACR.
Subject(s)
Breast Neoplasms/drug therapy , Receptor, ErbB-2/genetics , Trastuzumab/pharmacology , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Bridged-Ring Compounds/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunoconjugates/pharmacology , Mice , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Signal Transduction/drug effects , Taxoids/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Breast Neoplasms/drug therapy , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Immunoconjugates/pharmacology , Xenograft Model Antitumor Assays , Animals , Antibodies/chemistry , Antibodies/immunology , Antibodies/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/immunology , HEK293 Cells , Humans , Immunoconjugates/immunology , Immunoconjugates/pharmacokinetics , MCF-7 Cells , Macaca fascicularis , Mice, Nude , Mice, SCID , Rats, Sprague-Dawley , Receptors, Steroid/metabolism , Tumor Burden/drug effects , Tumor Burden/geneticsABSTRACT
Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity.Experimental Design: A panel of cell lines representative of TNBC was tested for in vitro and in vivo sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging.Results: The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both in vitro (P < 0.001 LAR vs. basal-like) and in vivo in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in PIK3CA-mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes.Conclusions: Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC. Clin Cancer Res; 23(18); 5561-72. ©2017 AACR.
Subject(s)
Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Triple Negative Breast Neoplasms/metabolism , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Female , Humans , Mice , Mitosis/drug effects , Molecular Imaging , Phenotype , Phosphatidylinositol 3-Kinases/metabolism , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Single-Cell Analysis , Time-Lapse Imaging , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathologyABSTRACT
PURPOSE: Chemotherapies are limited by a narrow therapeutic index resulting in suboptimal exposure of the tumor to the drug and acquired tumor resistance. One approach to overcome this is through antibody-drug conjugates (ADC) that facilitate greater potency via target-specific delivery of highly potent cytotoxic agents. EXPERIMENTAL DESIGN: In this study, we used a bioinformatics approach to identify the lymphocyte antigen 6 complex locus E (LY6E), an IFN-inducible glycosylphosphatidylinositol (GPI)-linked cell membrane protein as a promising ADC target. We developed a monoclonal anti-LY6E antibody and characterized in situ LY6E expression in over 750 cancer specimens and normal tissues. Target-dependent anti-LY6E ADC killing was investigated both in vitro and in vivo using patient-derived xenograft models. RESULTS: Using in silico approaches, we found that LY6E was significantly overexpressed and amplified in a wide array of different human solid tumors. IHC analysis revealed high LY6E protein expression in a number of tumor types, such as breast, lung, gastric, ovarian, pancreatic, kidney and head/neck carcinomas. Characterization of the endocytic pathways for LY6E revealed that the LY6E-specific antibody is internalized into cells leading to lysosomal accumulation. Consistent with this, a LY6E-specific ADC inhibited in vitro cell proliferation and produced durable tumor regression in vivo in clinically relevant LY6E-expressing xenograft models. CONCLUSIONS: Our results identify LY6E as a highly promising molecular ADC target for a variety of solid tumor types with current unmet medical need.
Subject(s)
Antigens, Neoplasm/pharmacology , Antigens, Surface/immunology , Antineoplastic Agents/pharmacology , Immunoconjugates/pharmacology , Neoplasms/immunology , Animals , Antibodies, Monoclonal/pharmacology , Antigens, Neoplasm/immunology , Cell Line, Tumor , Flow Cytometry , GPI-Linked Proteins/immunology , Humans , Immunoblotting , Immunohistochemistry , Mice , Mice, SCID , Polymerase Chain Reaction , RNA, Small Interfering , Transfection , Xenograft Model Antitumor AssaysABSTRACT
Phosphoinositide-3 kinase (PI3K) signaling pathway alterations occur broadly in cancer and PI3K is a promising therapeutic target. Here, we investigated acquired resistance to GDC-0941, a PI3K inhibitor in clinical trials. Colorectal cancer (CRC) cells made to be resistant to GDC-0941 were discovered to secrete amphiregulin, which resulted in increased EGFR/MAPK signaling. Moreover, prolonged PI3K pathway inhibition in cultured cells over a period of months led to a secondary loss of PTEN in 40% of the CRC lines with acquired resistance to PI3K inhibition. In the absence of PI3K inhibitor, these PTEN-null PI3K inhibitor-resistant clones had elevated PI3K pathway signaling and decreased sensitivity to MAPK pathway inhibitors. Importantly, PTEN loss was not able to induce resistance to PI3K inhibitors in the absence of amphiregulin, indicating a multimodal mechanism of acquired resistance. The combination of PI3K and MAPK pathway inhibitors overcame acquired resistance in vitro and in vivo.
ABSTRACT
Inhibiting NAD biosynthesis by blocking the function of nicotinamide phosphoribosyl transferase (NAMPT) is an attractive therapeutic strategy for targeting tumor metabolism. However, the development of drug resistance commonly limits the efficacy of cancer therapeutics. This study identifies mutations in NAMPT that confer resistance to a novel NAMPT inhibitor, GNE-618, in cell culture and in vivo, thus demonstrating that the cytotoxicity of GNE-618 is on target. We determine the crystal structures of six NAMPT mutants in the apo form and in complex with various inhibitors and use cellular, biochemical and structural data to elucidate two resistance mechanisms. One is the surprising finding of allosteric modulation by mutation of residue Ser165, resulting in unwinding of an α-helix that binds the NAMPT substrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The other mechanism is orthosteric blocking of inhibitor binding by mutations of Gly217. Furthermore, by evaluating a panel of diverse small molecule inhibitors, we unravel inhibitor structure activity relationships on the mutant enzymes. These results provide valuable insights into the design of next generation NAMPT inhibitors that offer improved therapeutic potential by evading certain mechanisms of resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Cytokines/antagonists & inhibitors , Cytokines/chemistry , Drug Resistance, Neoplasm , Enzyme Inhibitors/pharmacology , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Nicotinamide Phosphoribosyltransferase/chemistry , Catalytic Domain , Cell Line, Tumor , Cytokines/genetics , Humans , Models, Molecular , Mutation , Nicotinamide Phosphoribosyltransferase/geneticsABSTRACT
PURPOSE: Targeting HER2 with multiple HER2-directed therapies represents a promising area of treatment for HER2-positive cancers. We investigated combining the HER2-directed antibody-drug conjugate trastuzumab emtansine (T-DM1) with the HER2 dimerization inhibitor pertuzumab (Perjeta). EXPERIMENTAL DESIGN: Drug combination studies with T-DM1 and pertuzumab were performed on cultured tumor cells and in mouse xenograft models of HER2-amplified cancer. In patients with HER2-positive locally advanced or metastatic breast cancer (mBC), T-DM1 was dose-escalated with a fixed standard pertuzumab dose in a 3+3 phase Ib/II study design. RESULTS: Treatment of HER2-overexpressing tumor cells in vitro with T-DM1 plus pertuzumab resulted in synergistic inhibition of cell proliferation and induction of apoptotic cell death. The presence of the HER3 ligand, heregulin (NRG-1ß), reduced the cytotoxic activity of T-DM1 in a subset of breast cancer lines; this effect was reversed by the addition of pertuzumab. Results from mouse xenograft models showed enhanced antitumor efficacy with T-DM1 and pertuzumab resulting from the unique antitumor activities of each agent. In patients with mBC previously treated with trastuzumab, lapatinib, and chemotherapy, T-DM1 could be dosed at the maximum tolerated dose (MTD; 3.6 mg/kg every 3 weeks) with standard dose pertuzumab. Adverse events were mostly grade 1 and 2, with indications of clinical activity. CONCLUSIONS: Dual targeting of HER2 with the combination of T-DM1 and pertuzumab in cell culture and mouse xenograft models resulted in enhanced antitumor activity. In patients, this combination showed an encouraging safety and tolerability profile with preliminary evidence of efficacy.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Neuregulins/antagonists & inhibitors , Receptor, ErbB-2/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Autocrine Communication/drug effects , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Therapy, Combination , Female , Humans , Mice , Neuregulin-1/antagonists & inhibitors , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/metabolism , Signal Transduction , Trastuzumab , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Potent, trans-2-(pyridin-3-yl)cyclopropanecarboxamide-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using fragment-based screening and structure-based design techniques. Multiple crystal structures were obtained of initial fragment leads, and this structural information was utilized to improve the biochemical and cell-based potency of the associated molecules. Many of the optimized compounds exhibited nanomolar antiproliferative activities against human tumor lines in in vitro cell culture experiments. In a key example, a fragment lead (13, KD = 51 µM) was elaborated into a potent NAMPT inhibitor (39, NAMPT IC50 = 0.0051 µM, A2780 cell culture IC50 = 0.000 49 µM) which demonstrated encouraging in vivo efficacy in an HT-1080 mouse xenograft tumor model.
Subject(s)
Amides/chemical synthesis , Antineoplastic Agents/chemical synthesis , Cyclopropanes/chemical synthesis , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Pyridines/chemical synthesis , Sulfones/chemical synthesis , Amides/chemistry , Amides/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Cyclopropanes/chemistry , Cyclopropanes/pharmacology , Drug Screening Assays, Antitumor , Heterografts , Humans , Mice , Mice, Nude , Models, Molecular , Neoplasm Transplantation , Protein Conformation , Pyridines/chemistry , Pyridines/pharmacology , Stereoisomerism , Structure-Activity Relationship , Sulfones/chemistry , Sulfones/pharmacologyABSTRACT
Although standard chemotherapies are commonly used to treat most types of solid tumors, such treatment often results in inadequate response to, or relapse after, therapy. This is particularly relevant for lung cancer because most patients are diagnosed with advanced-stage disease and are treated with frontline chemotherapy. By studying the residual tumor cells that remain after chemotherapy in several in vivo non-small cell lung cancer models, we found that these cells have increased levels of human epidermal growth factor receptor (HER) signaling due, in part, to the enrichment of a preexisting NRG1(HI) subpopulation. Neuregulin 1 (NRG1) signaling in these models can be mediated by either the HER3 or HER4 receptor, resulting in the differential activation of downstream effectors. Inhibition of NRG1 signaling inhibits primary tumor growth and enhances the magnitude and duration of the response to chemotherapy. Moreover, we show that inhibition of ligand-mediated Her4 signaling impedes disease relapse in cases where NRG1 inhibition is insufficient. These findings demonstrate that ligand-dependent Her4 signaling plays an important role in disease relapse.
Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/metabolism , Lung Neoplasms/drug therapy , Neuregulin-1/antagonists & inhibitors , Signal Transduction , Animals , Antibodies, Blocking/pharmacology , Antibodies, Blocking/therapeutic use , Autocrine Communication/drug effects , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , ErbB Receptors/antagonists & inhibitors , Humans , Ligands , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Nude , Neoplasm, Residual/drug therapy , Neoplasm, Residual/metabolism , Neoplasm, Residual/pathology , Neuregulin-1/metabolism , Receptor, ErbB-4 , Signal Transduction/drug effects , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: MEHD7945A is a novel dual-action monoclonal antibody in which each of the two antigen-binding fragments is capable of binding to EGFR and HER3 with high affinity. It is being evaluated as a potential therapy for human cancer. The purpose of these studies was to characterize the pharmacokinetics (PK) of MEHD7945A in mouse and monkey and predict its human PK and efficacious dose. METHODS: PK of MEHD7945A was determined in SCID beige mice and cynomolgus monkeys after administration of single intravenous doses. Human PK profiles were projected from monkey PK profiles using a species-invariant time method, and human population PK parameters were estimated using a nonlinear, two-compartment model comprising specific (target-mediated) and nonspecific clearance pathways. The antitumor efficacy in mice bearing human tumor xenografts was used in conjunction with human PK projections to estimate human efficacious doses. RESULTS: The total clearance of MEHD7945A decreased with increase in dose in both mouse and monkey. The nonspecific clearance in monkey was estimated to be 14 mL/day/kg. The predicted nonspecific clearance range in humans was 6-10 mL/day/kg. Doses of 8-12 mg/kg administered every 2 weeks in humans were predicted to achieve exposure of 300 day µg/mL per week to match the efficacious exposure observed in xenograft models. CONCLUSIONS: The PK of MEHD7945A was nonlinear in mouse and monkey in the dose range tested. The nonspecific clearance in monkey was approximately twofold higher than typical humanized IgG1 antibodies. The projected human efficacious dose and dose regimen appear to be achievable in patients.
Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Antineoplastic Agents/pharmacokinetics , ErbB Receptors/immunology , Immunoglobulin G/metabolism , Receptor, ErbB-3/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/immunology , Dose-Response Relationship, Drug , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Female , Humans , Immunoglobulin G/administration & dosage , Immunoglobulin G/immunology , Macaca fascicularis , Mice , Mice, SCID , Receptor, ErbB-3/metabolismABSTRACT
Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.