ABSTRACT
Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.
Subject(s)
Cyclin E , Membrane Proteins , Ovarian Neoplasms , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , CDC2 Protein Kinase , Cyclin E/genetics , Female , Gene Amplification , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/genetics , Neoplasms/genetics , Ovarian Neoplasms/pathology , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Synthetic Lethal MutationsABSTRACT
Triple-negative breast cancer (TNBC) is typically aggressive, difficult to treat, and commonly metastasizes to the visceral organs and soft tissues, including the lungs and the brain. Taxanes represent the most effective and widely used therapeutic class in metastatic TNBC but possess limiting adverse effects that often result in a delay, reduction, or cessation of their use. DZ-2384 is a candidate microtubule-targeting agent with a distinct mechanism of action and strong activity in several preclinical cancer models, with reduced toxicities. DZ-2384 is highly effective in patient-derived taxane-sensitive and taxane-resistant xenograft models of TNBC at lower doses and over a wider range relative to paclitaxel. When comparing compound exposure at minimum effective doses relative to safe exposure levels, the therapeutic window for DZ-2384 is 14-32 compared with 2.0 and less than 2.8 for paclitaxel and docetaxel, respectively. DZ-2384 is effective at reducing brain metastatic lesions when used at maximum tolerated doses and is equivalent to paclitaxel. Drug distribution experiments indicate that DZ-2384 is taken up more efficiently by tumor tissue but at equivalent levels in the brain compared with paclitaxel. Selective DZ-2384 uptake by tumor tissue may in part account for its wider therapeutic window compared with taxanes. In view of the current clinical efforts to combine chemotherapy with immune checkpoint inhibitors, we demonstrate that DZ-2384 acts synergistically with anti-CTLA-4 immunotherapy in a syngeneic murine model. These results demonstrate that DZ-2384 has a superior pharmacologic profile over currently used taxanes and is a promising therapeutic agent for the treatment of metastatic TNBC.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , CTLA-4 Antigen/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , Oxazoles/pharmacology , Triple Negative Breast Neoplasms/drug therapy , Animals , Antineoplastic Agents, Immunological/administration & dosage , Brain/metabolism , CTLA-4 Antigen/immunology , Cell Line, Tumor , Drug Synergism , Female , Humans , Lactams, Macrocyclic/administration & dosage , Lactams, Macrocyclic/pharmacokinetics , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, SCID , Neoplasm Metastasis , Oxazoles/administration & dosage , Oxazoles/pharmacokinetics , Random Allocation , Triple Negative Breast Neoplasms/immunology , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
We describe the asymmetric synthesis of a highly substituted ω-octynoic acid derivative and demonstrate its utility for generating complex macrocycles from unprotected peptides. The molecule harbors an isolated quaternary center that displays four uniquely functionalized arms, each of which can be reacted orthogonally in sequence as the molecule is integrated into peptide structure. These processing sequences entail (1) scaffold ligation, (2) macrocyclization via internal aromatic alkylations or catalyzed etherifications, (3) acyliminium ion mediated embedding of condensed heterocycles, and (4) terminal alkyne derivatization or dimerization reactions. Numerous polycycles are prepared and fully characterized in this study. Factors that influence reaction efficiencies and selectivity are also probed. We construct a novel mimic of the second mitochondria derived activator of caspase using these techniques, wherein subtle variations in macrocycle connectivity have a marked impact on performance. In general, the chemistry is an important step toward facile, systematic access to complex peptidomimetics synthesized by directly altering the structure and properties of machine-made oligomers.
Subject(s)
Macrocyclic Compounds/chemistry , Peptides/chemistry , CyclizationABSTRACT
Enhanced protein synthesis capacity is associated with increased tumor cell survival, proliferation, and resistance to chemotherapy. Cancers like multiple myeloma (MM), which display elevated activity in key translation regulatory nodes, such as the PI3K/mammalian target of rapamycin and MYC-eukaryotic initiation factor (eIF) 4E pathways, are predicted to be particularly sensitive to therapeutic strategies that target this process. To identify novel vulnerabilities in MM, we undertook a focused RNAi screen in which components of the translation apparatus were targeted. Our screen was designed to identify synthetic lethal relationships between translation factors or regulators and dexamethasone (DEX), a corticosteroid used as frontline therapy in this disease. We find that suppression of all three subunits of the eIF4F cap-binding complex synergizes with DEX in MM to induce cell death. Using a suite of small molecules that target various activities of eIF4F, we observed that cell survival and DEX resistance are attenuated upon eIF4F inhibition in MM cell lines and primary human samples. Levels of MYC and myeloid cell leukemia 1, two known eIF4F-responsive transcripts and key survival factors in MM, were reduced upon eIF4F inhibition, and their independent suppression also synergized with DEX. Inhibition of eIF4F in MM exerts pleotropic effects unraveling a unique therapeutic opportunity.
Subject(s)
Dexamethasone/therapeutic use , Eukaryotic Initiation Factor-4F/metabolism , Multiple Myeloma/drug therapy , Cell Death/drug effects , Cell Line, Tumor , Dexamethasone/pharmacology , Genes, Modifier , Humans , Molecular Targeted Therapy , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference/drug effects , Suppression, Genetic/drug effects , Triterpenes/pharmacologyABSTRACT
BACKGROUND: Obatoclax is a clinical stage drug candidate that has been proposed to target and inhibit prosurvival members of the Bcl-2 family, and thereby contribute to cancer cell lethality. The insolubility of this compound, however, has precluded the use of many classical drug-target interaction assays for its study. Thus, a direct demonstration of the proposed mechanism of action, and preferences for individual Bcl-2 family members, remain to be established. METHODS: Employing modified proteins and lipids, we recapitulated the constitutive association and topology of mitochondrial outer membrane Mcl-1 and Bak in synthetic large unilamellar liposomes, and measured bakdependent bilayer permeability. Additionally, cellular and tumor models, dependent on Mcl-1 for survival, were employed. RESULTS: We show that regulation of bilayer permeabilization by the tBid - Mcl-1 - Bak axis closely resemblesthe tBid - Bcl-XL - Bax model. Obatoclax rapidly and completely partitioned into liposomal lipid but also rapidly exchanged between liposome particles. In this system, obatoclax was found to be a direct and potent antagonist of liposome-bound Mcl-1 but not of liposome-bound Bcl-XL, and did not directly influence Bak. A 2.5 molar excess of obatoclax relative to Mcl-1 overcame Mcl-1-mediated inhibition of tBid-Bak activation. Similar results were found for induction of Bak oligomers by Bim. Obatoclax exhibited potent lethality in a cellmodel dependent on Mcl-1 for viability but not in cells dependent on Bcl-XL. Molecular modeling predicts that the 3-methoxy moiety of obatoclax penetrates into the P2 pocket of the BH3 binding site of Mcl-1. A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1. Systemic treatment of mice bearing Tsc2(+) (/) (-) Em-myc lymphomas (whose cells depend on Mcl-1 for survival) with obatoclax conferred a survival advantage compared to vehicle alone (median 31 days vs 22 days, respectively; p=0.003). In an Akt-lymphoma mouse model, the anti-tumor effects of obatoclax synergized with doxorubicin. Finally, treatment of the multiple myeloma KMS11 cell model (dependent on Mcl-1 for survival) with dexamethasone induced Bim and Bim-dependent lethality. As predicted for an Mcl-1 antagonist, obatoclax and dexamethasone were synergistic in this model. CONCLUSIONS: Taken together, these findings indicate that obatoclax is a potent antagonist of membranerestricted Mcl-1. Obatoclax represents an attractive chemical series to generate second generation Mcl-1 inhibitors.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Lymphoma/drug therapy , Membrane Proteins/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Pyrroles/administration & dosage , Animals , Bcl-2-Like Protein 11 , Cell Line, Tumor , Disease Models, Animal , Doxorubicin/administration & dosage , Drug Synergism , Humans , Indoles , Lymphoma/metabolism , Mice , Myeloid Cell Leukemia Sequence 1 Protein/chemistry , Pyrroles/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
The Bcl2 pro-survival protein family has long been recognized for its important contributions to cancer. At elevated levels relative to pro-apoptotic effector members, the survival proteins prevent cancer cells from initiating apoptosis in the face of many intrinsic tumour-suppressing pathways and extrinsic therapeutic treatments aimed at controlling tumorigenesis. Recent studies, including genome-wide analyses, have begun to focus attention on a particularly enigmatic member of the family-myeloid cell leukaemia 1 (Mcl1). For reasons that are not clear, Mcl1 in cancer cells is turned over rapidly, eliminated primarily through the ubiquitin-proteasome pathway. Moreover, the mechanistic aspects of this constitutive membrane-associated protein have not been fully elucidated. As the pro-cancer activity of Mcl1 requires elevated expression levels of the protein, the cancer genome adapts to ensure either high levels of synthesis or evasion of degradation, or both. Here, we focus on the complex strategies at play and their therapeutic implications.
Subject(s)
Gene Expression Regulation, Neoplastic , Neoplasms/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Apoptosis , Gene Expression , Humans , Molecular Targeted Therapy , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasms/drug therapy , Neoplasms/pathology , Protein Processing, Post-Translational , Protein Structure, Tertiary , Proteolysis , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/geneticsABSTRACT
PURPOSE: The importance of the DNA damage response in mediating effects of radiotherapy (RT) has galvanized efforts to target this pathway with radiosensitizers. Yet early clinical trials of this approach have failed to yield a benefit in unselected populations. We hypothesized that ataxia-telangiectasia mutated (Atm)-null tumors would demonstrate genotype-specific synergy between RT and an inhibitor of the DNA damage response protein ataxia-telangiectasia and Rad3-related (ATR) kinase. EXPERIMENTAL DESIGN: We investigated the synergistic potential of the ATR inhibitor (ATRi) RP-3500 and RT in two Atm-null and isogenic murine models, both in vitro and in vivo. Staining of γ-H2AX foci, characterization of the immune response via flow cytometry, and tumor rechallenge experiments were performed to elucidate the mechanism of interaction. To examine genotype specificity, we tested the interaction of ATRi and RT in a Brca1-null model. Finally, patients with advanced cancer with ATM alterations were enrolled in a phase I/II clinical trial to validate preclinical findings. RESULTS: Synergy between RP-3500 and RT was confirmed in Atm-null lines in vitro, characterized by an accumulation of DNA double-strand breaks. In vivo, Atm-null tumor models had higher rates of durable control with RT and ATRi than controls. In contrast, there was no synergy in tumors lacking Brca1. Analysis of the immunologic response indicated that efficacy is largely mediated by cell-intrinsic mechanisms. Lastly, early results from our clinical trial showed complete responses in patients. CONCLUSIONS: Genotype-directed radiosensitization with ATRi and RT can unleash significant therapeutic benefit and could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments.
ABSTRACT
ATR is a key kinase in the DNA-damage response (DDR) that is synthetic lethal with several other DDR proteins, making it an attractive target for the treatment of genetically selected solid tumors. Herein we describe the discovery of a novel ATR inhibitor guided by a pharmacophore model to position a key hydrogen bond. Optimization was driven by potency and selectivity over the related kinase mTOR, resulting in the identification of camonsertib (RP-3500) with high potency and excellent ADME properties. Preclinical evaluation focused on the impact of camonsertib on myelosuppression, and an exploration of intermittent dosing schedules to allow recovery of the erythroid compartment and mitigate anemia. Camonsertib is currently undergoing clinical evaluation both as a single agent and in combination with talazoparib, olaparib, niraparib, lunresertib, or gemcitabine (NCT04497116, NCT04972110, NCT04855656). A preliminary recommended phase 2 dose for monotherapy was identified as 160 mg QD given 3 days/week.
Subject(s)
Neoplasms , Humans , Ataxia Telangiectasia Mutated Proteins , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , GemcitabineABSTRACT
Combinations of ataxia telangiectasia- and Rad3-related kinase inhibitors (ATRis) and poly(ADP-ribose) polymerase inhibitors (PARPis) synergistically kill tumor cells through modulation of complementary DNA repair pathways, but their tolerability is limited by hematological toxicities. To address this, we performed a genome-wide CRISPR-Cas9 screen to identify genetic alterations that hypersensitize cells to a combination of the ATRi RP-3500 with PARPi, including deficiency in RNase H2, RAD51 paralog mutations, or the "alternative lengthening of telomeres" telomere maintenance mechanism. We show that RP-3500 and PARPi combinations kill cells carrying these genetic alterations at doses sub-therapeutic as single agents. We also demonstrate the mechanism of combination hypersensitivity in RNase H2-deficient cells, where we observe an irreversible replication catastrophe, allowing us to design a highly efficacious and tolerable in vivo dosing schedule. We present a comprehensive dataset to inform development of ATRi and PARPi combinations and an experimental framework applicable to other drug combination strategies.
Subject(s)
Antineoplastic Agents , Poly(ADP-ribose) Polymerase Inhibitors , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacology , RibonucleasesABSTRACT
Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC50 values of 1.0 and 0.33 nmol/L in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mammalian target of rapamycin (mTOR) and more than 2,000-fold selectivity over ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and phosphatidylinositol 3-kinase alpha (PI3Kα) kinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5 to 7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor inhibition of phosphorylated checkpoint kinase 1 (pCHK1) (IC80 = 18.6 nmol/L) and induction of phosphorylated H2A.X variant histone (γH2AX), phosphorylated DNA-PK catalytic subunit (pDNA-PKcs), and phosphorylated KRAB-associated protein 1 (pKAP1). RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC80 for 10 to 12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.
Subject(s)
Ataxia Telangiectasia , Poly(ADP-ribose) Polymerase Inhibitors , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , DNA-Activated Protein Kinase/metabolism , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic useABSTRACT
PKMYT1 is a regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1. To address this need compound 1 was identified as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol increased potency on PKMYT1. These dimethylphenol analogs were found to exist as atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design enabled optimization of cell-based potency. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1. RP-6306 inhibits CCNE1-amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials for treatment of various solid tumors.
Subject(s)
Neoplasms , Protein-Tyrosine Kinases , Cell Line, Tumor , Cell Proliferation , Humans , Membrane Proteins , Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine KinasesABSTRACT
DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of RP-6685: a potent, selective, and orally bioavailable Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2-/- mouse tumor xenograft model.
Subject(s)
DNA-Directed DNA Polymerase , Ovarian Neoplasms , Animals , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Drug Design , Drug Discovery , Female , Humans , MiceABSTRACT
Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Drug Resistance, Neoplasm/drug effects , Neoplasm Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Pyrroles/pharmacology , Animals , Boronic Acids/pharmacology , Bortezomib , Cell Line, Tumor , Cysteine Proteinase Inhibitors/pharmacology , Humans , Indoles , Melanoma/metabolism , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Proteins/metabolism , Proteasome Inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyrazines/pharmacology , bcl-2 Homologous Antagonist-Killer Protein/antagonists & inhibitors , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
GMX1778 was recently shown to function as a potent inhibitor of nicotinamide phosphoribosyl transferase. To translate the discovery of GMX1778 mechanism of action into optimal clinical use of its intravenously administered prodrug, GMX1777, the efficacy of GMX1777 was evaluated in xenograft models and the pharmacokinetic profile of GMX1778 and its effect on nicotinamide adenine dinucleotide cellular levels was measured by liquid chromatography/mass spectrometry. Consistent with the requirement for a prolonged exposure for cytotoxicity in vitro, a dose of 75 mg/kg of GMX1777 administered as two bolus intravenous injections in 1 day were not effective at reducing the growth of multiple myeloma (IM-9) tumors, whereas the same dose of GMX1777 administered over a 24 h intravenous infusion caused tumor regression in the IM-9 model, a small-cell lung cancer (SHP-77) model, and a colon carcinoma (HCT-116) model. A 72 h continuous intravenous infusion of GMX1777 was also effective in the IM-9 model, but was associated with a smaller therapeutic index. GMX1777 at a dose of 75 mg/kg administered over a 24 h intravenous infusion produced GMX1778 steady-state plasma levels of approximately 1 microg/ml and caused nicotinamide adenine dinucleotide levels to decrease significantly in tumors. Consistent with the GMX1778 mechanism of action, nicotinic acid protected mice treated with a lethal dose of GMX1777. These data support the design of an open-label, dose-escalation trial, in which patients with refractory solid tumors and lymphomas receive 24 h infusions of GMX1777 as a single agent in 3-week cycles. Furthermore, these results indicate that nicotinic acid is a potent antidote to treat GMX1777 overdose.
Subject(s)
Antineoplastic Agents/therapeutic use , Cytokines/antagonists & inhibitors , Guanidines/therapeutic use , Neoplasm Proteins/antagonists & inhibitors , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Prodrugs/therapeutic use , Adenocarcinoma/drug therapy , Adenocarcinoma/pathology , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Carcinoma, Small Cell/drug therapy , Carcinoma, Small Cell/pathology , Cell Line, Tumor/transplantation , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Cyanides/administration & dosage , Cyanides/pharmacology , Cyanides/therapeutic use , Drug Screening Assays, Antitumor , Female , Guanidines/administration & dosage , Guanidines/pharmacokinetics , Guanidines/pharmacology , Humans , Infusions, Intravenous , Injections, Intravenous , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Mice, SCID , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , NAD/metabolism , Niacin/metabolism , Niacinamide/metabolism , Xenograft Model Antitumor AssaysABSTRACT
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
ABSTRACT
Tumor cells are particularly dependent on NAD+ due to higher rates of metabolism, DNA synthesis and repair. Nicotinamide phosphoribosyltransferase inhibitors (NAMPTis) inhibit NAD+ biosynthesis and represent promising new anti-cancer agents. However, clinical efficacy has been limited by toxicities demonstrating the need for drug combinations to broaden the therapeutic index. One potential combination involves niacin/NAMPTi co-administration. Niacin can rescue NAD+ biosynthesis through a parallel pathway that depends on nicotinic acid phosphoribosyltransferase (NAPRT) expression. Most normal tissues express NAPRT while a significant proportion of malignant cells do not, providing a possible selection marker for patients to achieve NAMPTi efficacy while minimizing toxicities. Here we identify and validate a novel highly NAPRT-specific monoclonal antibody (3C6D2) that detects functional NAPRT in paraffin embedded tissue sections by immunohistochemistry (IHC). NAPRT detection by 3C6D2 coincides with the ability of niacin to rescue cells from NAMPTi induced cytotoxicity in cell lines and animal xenograft models. 3C6D2 binds to an epitope that is unique to NAPRT among phosphoribosyltransferases. In a series of primary tumor samples from lung and brain cancer patients, we demonstrate that >70 % of human small cell lung carcinomas, glioblastomas and oligodendrogliomas lack NAPRT identifying them as potentially suitable indications for the NAMPT/niacin combination.
ABSTRACT
TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) has long been considered a tantalizing target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors DR4 or DR5. Despite initial promise, both recombinant human TRAIL (native TRAIL) and dimeric DR4/DR5 agonist monoclonal antibodies (mAbs) failed in multiple human clinical trials. Here we show that in-frame fusion of human C-propeptide of α1(I) collagen (Trimer-Tag) to the C-terminus of mature human TRAIL leads to a disulfide bond-linked homotrimer which can be expressed at high levels as a secreted protein from CHO cells. The resulting TRAIL-Trimer not only retains similar bioactivity and receptor binding kinetics as native TRAIL in vitro which are 4-5 orders of magnitude superior to that of dimeric TRAIL-Fc, but also manifests more favorable pharmacokinetic and antitumor pharmacodynamic profiles in vivo than that of native TRAIL. Taken together, this work provides direct evidence for the in vivo antitumor efficacy of TRAIL being proportional to systemic drug exposure and suggests that the previous clinical failures may have been due to rapid systemic clearance of native TRAIL and poor apoptosis-inducing potency of dimeric agonist mAbs despite their long serum half-lives.
Subject(s)
Antineoplastic Agents/administration & dosage , Colonic Neoplasms/drug therapy , Phosphopeptides/chemistry , Procollagen/chemistry , Recombinant Fusion Proteins/administration & dosage , TNF-Related Apoptosis-Inducing Ligand/metabolism , Animals , Antineoplastic Agents/pharmacokinetics , CHO Cells , Cell Line, Tumor , Colonic Neoplasms/metabolism , Cricetulus , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Recombinant Fusion Proteins/pharmacokinetics , TNF-Related Apoptosis-Inducing Ligand/chemistry , Xenograft Model Antitumor AssaysABSTRACT
Nicotinamide phosphoribosyltransferase (NAMPT) is crucial for nicotinamide adenine dinucleotide (NAD(+)) biosynthesis in mammalian cells. NAMPT inhibitors represent multifunctional anticancer agents that act on NAD(+) metabolism to shut down glycolysis, nucleotide biosynthesis, and ATP generation and act indirectly as PARP and sirtuin inhibitors. The selectivity of NAMPT inhibitors preys on the increased metabolic requirements to replenish NAD(+) in cancer cells. Although initial clinical studies with NAMPT inhibitors did not achieve single-agent therapeutic levels before dose-limiting toxicities were reached, a new understanding of alternative rescue pathways and a biomarker that can be used to select patients provides new opportunities to widen the therapeutic window and achieve efficacious doses in the clinic. Recent work has also illustrated the potential for drug combination strategies to further enhance the therapeutic opportunities. This review summarizes recent discoveries in NAD(+)/NAMPT inhibitor biology in the context of exploiting this new knowledge to optimize the clinical outcomes for this promising new class of agents.
ABSTRACT
Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.
Subject(s)
Antineoplastic Agents/pharmacology , Lactams, Macrocyclic/pharmacology , Microtubules/drug effects , Neurons/drug effects , Oxazoles/pharmacology , Vinca Alkaloids/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Crystallography, X-Ray , Dimerization , Genomics , Humans , Lactams, Macrocyclic/chemistry , Mice , Microscopy, Electron , Mitosis , Neoplasm Transplantation , Oxazoles/chemistry , Tubulin/chemistry , Vinblastine/analogs & derivatives , Vinblastine/chemistry , Vinblastine/pharmacology , Vinca Alkaloids/chemistry , VinorelbineABSTRACT
GMX1778 and its prodrug GMX1777 represent a new class of cancer drugs that targets nicotinamide phosphoribosyltransferase (NAMPT) as a new strategy to interfere with biosynthesis of the key enzymatic cofactor NAD, which is critical for a number of cell functions, including DNA repair. Using a genome-wide synthetic lethal siRNA screen, we identified the folate pathway-related genes, deoxyuridine triphosphatase and dihydrofolate reductase, the silencing of which sensitized non-small cell lung carcinoma (NSCLC) cells to the cytotoxic effects of GMX. Pemetrexed is an inhibitor of dihydrofolate reductase currently used to treat patients with nonsquamous NSCLC. We found that combining pemetrexed with GMX1777 produced a synergistic therapeutic benefit in A549 and H1299 NSCLC cells in vitro and in a mouse A549 xenograft model of lung cancer. Pemetrexed is known to activate PARPs, thereby accelerating NAD consumption. Genetic or pharmacologic blockade of PARP activity inhibited this effect, impairing cell death by pemetrexed either alone or in combination with GMX1777. Conversely, inhibiting the base excision repair pathway accentuated NAD decline in response to GMX and the cytotoxicity of both agents either alone or in combination. These findings provide a mechanistic rationale for combining GMX1777 with pemetrexed as an effective new therapeutic strategy to treat nonsquamous NSCLC.