RESUMEN
AML is characterized by mutations in genes associated with growth regulation such as internal tandem duplications (ITD) in the receptor kinase FLT3. Inhibitors targeting FLT3 (FLT3i) are being used to treat patients with FLT3-ITD+ but most relapse and become resistant. To elucidate the resistance mechanism, we compared the gene regulatory networks (GRNs) of leukemic cells from patients before and after relapse, which revealed that the GRNs of drug-responsive patients were altered by rewiring their AP-1-RUNX1 axis. Moreover, FLT3i induces the upregulation of signaling genes, and we show that multiple cytokines, including interleukin-3 (IL-3), can overcome FLT3 inhibition and send cells back into cycle. FLT3i leads to loss of AP-1 and RUNX1 chromatin binding, which is counteracted by IL-3. However, cytokine-mediated drug resistance can be overcome by a pan-RAS inhibitor. We show that cytokines instruct AML growth via the transcriptional regulators AP-1 and RUNX1 and that pan-RAS drugs bypass this barrier.
RESUMEN
Targeting hard-to-drug proteins, such as proteins functioning by protein-protein interactions (PPIs) with small molecules, is difficult because of the lack of well-defined pockets. Fragment or computational-based methods are usually employed for the discovery of such compounds, but no generic method is available to quickly identify small molecules interfering with PPIs. Here, we provide a protocol describing a generic method to discover small molecules inhibiting the interaction between an intracellular antibody and its target, in particular for proteins that are hard to make in recombinant form. This protocol reports a versatile and generic method that can be applied to any target/intracellular antibody. Because it is a cell-based assay, it identifies chemical matters that are already displaying advantageous cell permeability properties. Graphic abstract: Cell-based intracellular antibody-guided small molecule screening.
RESUMEN
Refractory T cell acute leukaemias that no longer respond to treatment would benefit from new modalities that target T cell-specific surface proteins. T cell associated surface proteins (the surfaceome) offer possible therapy targets to reduce tumour burden but also target the leukaemia-initiating cells from which tumours recur. Recent studies of the T cell leukaemia surfaceome confirmed that CD7 is highly expressed in overt disease. We have used an anti-CD7 antibody drug conjugate (ADC) to show that the binding of antibody to surface CD7 protein results in rapid internalization of the antigen together with the ADC. As a consequence, cell killing was observed via induction of apoptosis and was dependent on cell surface CD7. The in vitro cytotoxic activity (EC50) of the anti-CD7 ADC on T cell acute leukaemia (T-ALL) cells Jurkat and KOPT-K1 was found to be in the range of 5-8 ng/mL. In a pre-clinical xenograft model of human tumour growth expressing CD7 antigen, growth was curtailed by a single dose of ADC. The data indicate that CD7 targeting ADCs may be developed into an important second stage therapy for T cell acute leukaemia, for refractory CD7-positive leukaemias and for subsets of acute myeloid leukaemia (AML) expressing CD7.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos CD7/inmunología , Apoptosis , Liberación de Fármacos , Inmunoconjugados/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Antígenos CD7/metabolismo , Proliferación Celular , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The use of intracellular antibodies as templates to derive surrogate compounds is an important objective because intracellular antibodies can be employed initially for target validation in pre-clinical assays and subsequently employed in compound library screens. LMO2 is a T cell oncogenic protein activated in the majority of T cell acute leukaemias. We have used an inhibitory intracellular antibody fragment as a competitor in a small molecule library screen using competitive surface plasmon resonance (cSPR) to identify compounds that bind to LMO2. We selected four compounds that bind to LMO2 but not when the anti-LMO2 intracellular antibody fragment is bound to it. These findings further illustrate the value of intracellular antibodies in the initial stages of drug discovery campaigns and more generally antibodies, or antibody fragments, can be the starting point for chemical compound development as surrogates of the antibody combining site.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos de Neoplasias/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Proteínas con Dominio LIM/metabolismo , Leucemia de Células T/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/metabolismo , Anticuerpos/metabolismo , Unión Competitiva , Células Cultivadas , Descubrimiento de Drogas , Humanos , Fragmentos de Inmunoglobulinas/genética , Espacio Intracelular , Conformación Proteica , Bibliotecas de Moléculas Pequeñas , Resonancia por Plasmón de Superficie , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismo , Linfocitos T/inmunologíaRESUMEN
Intracellular antibodies are valuable tools for target validation studies for clinical situations such as cancer. Recently we have shown that antibodies can be used for drug discovery in screening for chemical compounds surrogates by showing that compounds could be developed to the so-called undruggable RAS protein family. This method, called Antibody-derived compound (Abd) technology, employed intracellular antibodies binding to RAS in a competitive surface plasmon resonance chemical library screen. Success with this method requires a high affinity interaction between the antibody and the target. We now show that reduction in the affinity (dematuration) of the anti-active RAS antibody facilitates the screening of a chemical library using an in vitro AlphaScreen method. This identified active RAS specific-binding Abd compounds that inhibit the RAS-antibody interaction. One compound is shown to be a pan-RAS binder to KRAS, HRAS and NRAS-GTP proteins with a Kd of average 37 mM, offering the possibility of a new chemical series that interacts with RAS in the switch region where the intracellular antibody binds. This simple approach shows the druggability of RAS and is generally applicable to antibody-derived chemical library screening by affording flexibility through simple antibody affinity variation. This approach can be applied to find Abd compounds as surrogates of antibody-combining sites for novel drug development in a range of human diseases.
Asunto(s)
Bibliotecas de Moléculas Pequeñas/metabolismo , Proteínas ras/metabolismo , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos/metabolismo , Afinidad de Anticuerpos , Regiones Determinantes de Complementariedad/química , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/química , Resonancia por Plasmón de Superficie , Proteínas ras/química , Proteínas ras/inmunologíaRESUMEN
Tumour-associated KRAS mutations are the most prevalent in the three RAS-family isoforms and involve many different amino-acids. Therefore, molecules able to interfere with mutant KRAS protein are potentially important for wide-ranging tumour therapy. We describe the engineering of two RAS degraders based on protein macromolecules (macrodrugs) fused to specific E3 ligases. A KRAS-specific DARPin fused to the VHL E3 ligase is compared to a pan-RAS intracellular single domain antibody (iDAb) fused to the UBOX domain of the CHIP E3 ligase. We demonstrate that while the KRAS-specific DARPin degrader induces specific proteolysis of both mutant and wild type KRAS, it only inhibits proliferation of cancer cells expressing mutant KRAS in vitro and in vivo. Pan-RAS protein degradation, however, affects proliferation irrespective of the RAS mutation. These data show that specific KRAS degradation is an important therapeutic strategy to affect tumours expressing any of the range of KRAS mutations.
Asunto(s)
Sustancias Macromoleculares/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Neoplasias/metabolismo , Proteolisis , Proteínas ras/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Humanos , Ratones Desnudos , Dominios Proteicos , Ingeniería de Proteínas , Transducción de SeñalRESUMEN
Rearrangements involving the mixed lineage leukemia gene (MLL) are found in the majority of leukemias that develop within the first year of age, known as infant leukemias, and likely originate during prenatal life. MLL rearrangements are also present in about 10% of other pediatric and adult acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). These translocations and others occurring in early life are associated with a dismal prognosis compared with adult leukemias carrying the same translocations. This observation suggests that infant and adult leukemias are biologically distinct but the underlying molecular mechanisms for these differences are not understood. In this work, we induced the same MLL chromosomal translocation in the embryo at the time of fetal liver hematopoiesis and in the adult hematopoietic tissues to develop disease models in mice that recapitulate human infant and adult leukemias, respectively. We successfully obtained myeloid leukemia in adult mice after MLL-ENL recombination induction using the interferon inducible Mx1-Cre line. Using this same Cre line, we generated embryonic MLL-ENL leukemias, which were more aggressive than the corresponding adult leukemias. In conclusion, we have developed a novel MLL-ENL embryonic leukemia model in mice that can be used to study some aspects of infant leukemia ontogeny.
Asunto(s)
Proteínas de Unión al ADN , Embrión de Mamíferos , N-Metiltransferasa de Histona-Lisina , Leucemia Mieloide Aguda , Proteína de la Leucemia Mieloide-Linfoide , Neoplasias Experimentales , Proteínas de Fusión Oncogénica , Leucemia-Linfoma Linfoblástico de Células Precursoras , Factores de Transcripción , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/embriología , Embrión de Mamíferos/patología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Leucemia Mieloide Aguda/embriología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Ratones Transgénicos , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Neoplasias Experimentales/embriología , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/embriología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The success of therapeutic antibodies is largely attributed for their exquisite specificity, homogeneity, and functionality. There is, however, a need to engineer antibodies to extend and enhance their potency. One parameter is functional affinity augmentation, since antibodies matured in vivo have a natural affinity threshold. Generation of multivalent antibodies is one option capable of surpassing this affinity threshold through increased avidity. In this study, we present a novel platform consisting of an array of multivalent antibody formats, termed Quads, generated using the self-assembling tetramerization domain from p53. We demonstrate the versatility of this tetramerization domain by engineering anti-tumor necrosis factor (TNF) Quads that exhibit major increases in binding potency and in neutralizing TNF-mediated cytotoxicity compared to parental anti-TNF molecules. Further, Quads are amenable to fusion with different binding domains, allowing generation of novel multivalent monospecific and bispecific formats. Quads are thus a novel group of molecules that can be engineered to yield potential therapeutics with novel modalities and potencies.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Ingeniería de Proteínas/métodos , Multimerización de Proteína/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Antígenos CD20/genética , Antígenos CD20/inmunología , Antígenos CD20/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células HEK293 , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/metabolismo , Ratones , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Macromolecules such as antibodies and antibody fragments have been reported to interfere with intracellular targets, but their use is limited to delivery systems where expression is achieved from vectors such as plasmids or viruses. We have developed PEGylated nanoparticles of poly-lactic acid (PLA), including the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), which are functionalized with monoclonal anti-CD7, anti-CD53, or anti-GPR56 antibodies for receptor-mediated endocytic delivery into T-cell leukemia cell lines. Incorporation of DOTAP as the lipid component of the PLA nanoparticles enhanced the release of the immuno-nanoparticles from the endosomes into the cytosol compared to nanoparticles made from PLA alone. Systemic delivery of these anti-CD7 immuno-nanoparticles into humanized CD7 transgenic mice resulted in localization in the spleen, enhanced uptake into CD7-expressing splenocytes, and release of low amounts of reporter mRNA for translation. These functionalized polymer lipid nanoparticles are the basis for elaboration and optimization for realizing their potential in therapeutic applications to carry specific macromolecules such as mRNAs for translation into therapeutic proteins that can target intracellular proteins which mediate disease.