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Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs.
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Acute myeloid leukemia (AML) has a poor prognosis and a heterogeneous mutation landscape. Although common mutations are well-studied, little research has characterized how the sequence of mutations relates to clinical features. Using published, single-cell DNA sequencing data from three institutions, we compared clonal evolution patterns in AML to patient characteristics, disease phenotype, and outcomes. Mutation trees, which represent the order of select mutations, were created for 207 patients from targeted panel sequencing data using 1 639 162 cells, 823 mutations, and 275 samples. In 224 distinct orderings of mutated genes, mutations related to DNA methylation typically preceded those related to cell signaling, but signaling-first cases did occur, and had higher peripheral cell counts, increased signaling mutation homozygosity, and younger patient age. Serial sample analysis suggested that NPM1 and DNA methylation mutations provide an advantage to signaling mutations in AML. Interestingly, WT1 mutation evolution shared features with signaling mutations, such as WT1-early being proliferative and occurring in younger individuals, trends that remained in multivariable regression. Some mutation orderings had a worse prognosis, but this was mediated by unfavorable mutations, not mutation order. These findings add a dimension to the mutation landscape of AML, identifying uncommon patterns of leukemogenesis and shedding light on heterogeneous phenotypes.
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Evolución Clonal , Metilación de ADN , Leucemia Mieloide Aguda , Mutación , Nucleofosmina , Fenotipo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Pronóstico , Evolución Clonal/genética , Masculino , Heterogeneidad Genética , Femenino , Persona de Mediana Edad , Adulto , AncianoRESUMEN
Acute myeloid leukemia (AML) has a poor prognosis and a heterogeneous mutation landscape. Although common mutations are well-studied, little research has characterized how the sequence of mutations relates to clinical features. Using published, single-cell DNA sequencing data from three institutions, we compared clonal evolution patterns in AML to patient characteristics, disease phenotype, and outcomes. Mutation trees, which represent the order of select mutations, were created for 207 patients from targeted panel sequencing data using 1 639 162 cells, 823 mutations, and 275 samples. In 224 distinct orderings of mutated genes, mutations related to DNA methylation typically preceded those related to cell signaling, but signaling-first cases did occur, and had higher peripheral cell counts, increased signaling mutation homozygosity, and younger patient age. Serial sample analysis suggested that NPM1 and DNA methylation mutations provide an advantage to signaling mutations in AML. Interestingly, WT1 mutation evolution shared features with signaling mutations, such as WT1-early being proliferative and occurring in younger individuals, trends that remained in multivariable regression. Some mutation orderings had a worse prognosis, but this was mediated by unfavorable mutations, not mutation order. These findings add a dimension to the mutation landscape of AML, identifying uncommon patterns of leukemogenesis and shedding light on heterogenous phenotypes.
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Measurable residual disease (MRD), defined as the population of cancer cells that persist following therapy, serves as the critical reservoir for disease relapse in acute myeloid leukemia and other malignancies. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of more effective curative treatments. Discriminating between residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools. Here, we developed a single-cell MRD (scMRD) assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated single-cell DNA sequencing and immunophenotyping. Our scMRD assay shows high sensitivity of approximately 0.01%, deconvolutes clonal architecture, and provides clone-specific immunophenotypic data. In summary, our scMRD assay enhances MRD detection and simultaneously illuminates the clonal architecture of clonal hematopoiesis/preleukemic and leukemic cells surviving acute myeloid leukemia therapy.
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Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Bioensayo , Citometría de Flujo , Genotipo , InmunofenotipificaciónRESUMEN
Chronic inflammation, although subtle, puts the body in a constant state of alertness and is associated with many diseases, including cancer and cardiovascular diseases. It leads hematopoietic cells to produce and release proinflammatory cytokines, which trigger specific signaling pathways in hematopoietic stem cells (HSCs) that cause changes in proliferation, differentiation, and migration. This response is essential when HSCs are needed to produce specific blood cells to eliminate an intruder, such as a pathogenic virus, but mutant HSCs can use these proinflammatory signals to their advantage and accelerate the development of hematologic disease or malignancy. Understanding this complex process is vital for monitoring and controlling disease progression in patients. In the 2023 International Society for Experimental Hematology winter webinar, Dr. Eric Pietras (University of Colorado Anschutz Medical Campus, United States) and Dr. Katherine Y. King (Baylor College of Medicine, United States) gave a presentation on this topic, which is summarized in this review article.
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Enfermedades Hematológicas , Células Madre Hematopoyéticas , Humanos , Células Madre Hematopoyéticas/metabolismo , Diferenciación Celular , Transducción de Señal , Enfermedades Hematológicas/metabolismo , Inflamación/patologíaRESUMEN
PURPOSE: The Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) polycythemia vera, essential thrombocythemia, and primary myelofibrosis are characterized by JAK/STAT pathway activation. JAK inhibitors are approved for MPN treatment, but persistence has been observed, due to JAK/STAT reactivation. EXPERIMENTAL DESIGN: Using MPN patient samples, JAK2-mutated cell lines, and MPN mouse models, we examined both the efficacy and mechanism by which crizotinib, the ALK/MET/RON/ROS1 inhibitor approved for the treatment of non-small cell lung cancer, alters MPN cell proliferation and JAK/STAT activation. RESULTS: We found that crizotinib suppresses proliferation and activation of JAK/STAT signaling, and decreases the disease burden in the JAK2V617F mouse model of MPN. Furthermore, we found that crizotinib could overcome JAK inhibitor persistence to ruxolitinib. Interestingly, phosphorylation of the crizotinib target RON kinase was enhanced in ruxolitinib-persistent cells. We show that phospho-JAK2 and phospho-RON can physically interact to sustain JAK/STAT signaling, and that the combination of crizotinib and ruxolitinib disrupts this interaction. Furthermore, RON knockdown suppresses proliferation and activation of JAK/STAT signaling in JAK2-mutated cells, and RON deletion in a JAK2V617F mouse MPN model decreases the disease burden. We also observed RON hyperactivation in MPN patient cells, suggesting that RON may be an important target of crizotinib in MPN. CONCLUSIONS: In summary, we demonstrate that crizotinib has preclinical efficacy in MPN patient cells, JAK2-mutated cell lines, and a JAK2-mutated mouse model, and that the combination of crizotinib with JAK inhibitors suppresses JAK inhibitor persistence. Our work suggests that crizotinib should be investigated for the treatment of patients with MPN.
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Carcinoma de Pulmón de Células no Pequeñas , Inhibidores de las Cinasas Janus , Neoplasias Pulmonares , Trastornos Mieloproliferativos , Animales , Ratones , Inhibidores de las Cinasas Janus/uso terapéutico , Crizotinib/farmacología , Crizotinib/uso terapéutico , Quinasas Janus/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Transducción de Señal , Factores de Transcripción STAT/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Janus Quinasa 2/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , MutaciónRESUMEN
Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD- remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD- remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD- remission. Patients with fewer individual clones were more likely to achieve MRD- remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD- after induction or later MRD- after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered.
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Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Humanos , Pronóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Trasplante de Células Madre , Inducción de Remisión , Trasplante Homólogo , Neoplasia Residual/genéticaRESUMEN
In adult acute myeloid leukemia (AML), the acquisition of driver somatic mutations may be preceded by a benign state termed clonal hematopoiesis (CH). To develop therapeutic strategies to prevent leukemia development from CH, it is important to understand the mechanisms by which CH-driving and AML-driving mutations cooperate. Here, we use mice with inducible mutant alleles common in human CH (DNMT3AR882; mouse Dnmt3aR878H) and AML (NPM1c; mouse Npm1cA). We find that Dnmt3aR878H/+ hematopoietic stem cells (HSCs), but not multipotent progenitor cell (MPP) subsets, have reduced cytokine expression and proinflammatory transcriptional signatures and a functional competitive advantage over their wild-type counterparts. Dnmt3aR878H/+ HSCs are the most potent cell type transformed by Npm1cA, generating myeloid malignancies in which few additional cooperating somatic mutation events were detected. At a molecular level, Npm1cA, in cooperation with Dnmt3aR878H, acutely increased the accessibility of a distinct set of promoters in HSCs compared with MPP cells. These promoters were enriched for cell cycling, PI3K/AKT/mTOR signaling, stem cell signatures, and targets of transcription factors, including NFAT and the chromatin binding factor HMGB1, which have been implicated in human AML. These results demonstrate cooperativity between preexisting Dnmt3aR878H and Npm1cA at the chromatin level, where specific loci altered in accessibility by Npm1cA are dependent on cell context as well as Dnmt3a mutation status. These findings have implications for biological understanding and therapeutic intervention in the transformation from CH to AML.
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Leucemia Mieloide Aguda , Trastornos Mieloproliferativos , Animales , Cromatina , Hematopoyesis Clonal , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Trastornos Mieloproliferativos/patología , Nucleofosmina , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/uso terapéuticoRESUMEN
Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.
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Proteínas de Unión al GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Infección por el Virus Zika/enzimología , Virus Zika/fisiología , Proteína de Unión al GTP rhoB/metabolismo , Células A549 , Sistemas CRISPR-Cas , Proteínas de Unión al GTP/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Internalización del Virus , Replicación Viral , Virus Zika/genética , Infección por el Virus Zika/genética , Infección por el Virus Zika/virología , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rhoB/genéticaRESUMEN
Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.
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Picornaviridae/fisiología , Receptores de Péptidos/química , Receptores de Péptidos/metabolismo , Acoplamiento Viral , Internalización del Virus , Glicosilación , Humanos , Picornaviridae/genética , Receptores de Superficie Celular/metabolismo , Receptores de Péptidos/genéticaRESUMEN
Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.
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Infecciones por Flavivirus/genética , Flavivirus/fisiología , Proteínas de la Membrana/metabolismo , Animales , Pueblo Asiatico/genética , Autofagia , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Sistemas CRISPR-Cas , Línea Celular , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/metabolismo , Infecciones por Flavivirus/virología , Técnicas de Inactivación de Genes , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , SARS-CoV-2/fisiología , Replicación Viral , Virus de la Fiebre Amarilla/fisiología , Virus Zika/fisiologíaRESUMEN
Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1-3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.
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Células Clonales/patología , Análisis Mutacional de ADN , Mutación , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Análisis de la Célula Individual , Separación Celular , Células Clonales/metabolismo , Humanos , InmunofenotipificaciónRESUMEN
Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTS: TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection.TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes.TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection.TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.
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Clonal hematopoiesis (CH) is a common aging-associated condition with increased risk of hematologic malignancy. Knowledge of the mechanisms driving evolution from CH to overt malignancy has been hampered by a lack of in vivo models that orthogonally activate mutant alleles. Here, we develop independently regulatable mutations in DNA methyltransferase 3A (Dnmt3a) and nucleophosmin 1 (Npm1), observed in human CH and AML, respectively. We find Dnmt3a mutation expands hematopoietic stem and multipotent progenitor cells (HSC/MPPs), modeling CH. Induction of mutant Npm1 after development of Dnmt3a-mutant CH causes progression to myeloproliferative disorder (MPD), and more aggressive MPD is observed with longer latency between mutations. MPDs uniformly progress to acute myeloid leukemia (AML) following transplant, accompanied by a decrease in HSC/MPPs and an increase in myeloid-restricted progenitors, the latter of which propagate AML in tertiary recipient mice. At a molecular level, progression of CH to MPD is accompanied by selection for mutations activating Ras/Raf/MAPK signaling. Progression to AML is characterized by additional oncogenic signaling mutations (Ptpn11, Pik3r1, Flt3) and/or mutations in epigenetic regulators (Hdac1, Idh1, Arid1a). Together, our study demonstrates that Npm1 mutation drives evolution of Dnmt3a-mutant CH to AML and rate of disease progression is accelerated with longer latency of CH.
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Transformación Celular Neoplásica/patología , Evolución Clonal , ADN (Citosina-5-)-Metiltransferasas/genética , Modelos Animales de Enfermedad , Leucemia Mieloide Aguda/etiología , Mutación , Trastornos Mieloproliferativos/patología , Proteínas Nucleares/genética , Animales , Biomarcadores de Tumor/genética , Transformación Celular Neoplásica/genética , ADN (Citosina-5-)-Metiltransferasas/fisiología , ADN Metiltransferasa 3A , Progresión de la Enfermedad , Femenino , Hematopoyesis , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Progenitoras Mieloides/patología , Células Progenitoras Mieloides/trasplante , Trastornos Mieloproliferativos/genética , Proteínas Nucleares/fisiología , NucleofosminaRESUMEN
Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. It has shown promise as a cancer therapeutic in preclinical studies and early-phase clinical trials. Here, we have identified anthrax toxin receptor 1 (ANTXR1) as the receptor for SVV using genome-wide loss-of-function screens. ANTXR1 is necessary for permissivity in vitro and in vivo. However, robust SVV replication requires an additional innate immune defect. We found that SVV interacts directly and specifically with ANTXR1, that this interaction is required for SVV binding to permissive cells, and that ANTXR1 expression is necessary and sufficient for infection in cell lines with decreased expression of antiviral IFN genes at baseline. Finally, we identified the region of the SVV capsid that is responsible for receptor recognition using cryoelectron microscopy of the SVV-ANTXR1-Fc complex. These studies identify ANTXR1, a class of receptor that is shared by a mammalian virus and a bacterial toxin, as the cellular receptor for SVV.
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Proteínas de la Cápside/química , Proteínas de Neoplasias/química , Picornaviridae , Receptores de Superficie Celular/química , Receptores Virales/química , Animales , Línea Celular Tumoral , Supervivencia Celular , Microscopía por Crioelectrón , Femenino , Perfilación de la Expresión Génica , Genoma , Proteínas Fluorescentes Verdes/química , Humanos , Ratones , Ratones Desnudos , Proteínas de Microfilamentos , Viroterapia Oncolítica , Virus OncolíticosRESUMEN
Small cell lung cancer is initially highly responsive to cisplatin and etoposide but in almost every case becomes rapidly chemoresistant, leading to death within 1 year. We modeled acquired chemoresistance in vivo using a series of patient-derived xenografts to generate paired chemosensitive and chemoresistant cancers. Multiple chemoresistant models demonstrated suppression of SLFN11, a factor implicated in DNA-damage repair deficiency. In vivo silencing of SLFN11 was associated with marked deposition of H3K27me3, a histone modification placed by EZH2, within the gene body of SLFN11, inducing local chromatin condensation and gene silencing. Inclusion of an EZH2 inhibitor with standard cytotoxic therapies prevented emergence of acquired resistance and augmented chemotherapeutic efficacy in both chemosensitive and chemoresistant models of small cell lung cancer.
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Proteína Potenciadora del Homólogo Zeste 2/fisiología , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Nucleares/fisiología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Animales , Resistencia a Antineoplásicos , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Humanos , Ratones , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Proteína 1 Relacionada con Twist/fisiologíaRESUMEN
The recently described clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has proven to be an exquisitely powerful and invaluable method of genetic manipulation and/or modification. As such, many researchers have realized the potential of using the CRISPR/Cas9 system as a novel screening method for the identification of important proteins in biological processes and have designed short guide RNA libraries for an in vitro screening. The seminal papers describing these libraries offer valuable information regarding methods for generating the short guide RNA libraries, creating cell lines containing these libraries, and specific details regarding the screening workflow. However, certain considerations are often overlooked that may be important when planning and performing a screen, including which CRISPR library to use and how to best analyze the resulting screen data. In this review, we offer suggestions to answer some of these questions that are not covered as deeply in the papers describing the available CRISPR libraries for an in vitro screening.
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Sistemas CRISPR-Cas , Animales , Línea Celular , Edición Génica , Técnicas de Inactivación de Genes , Biblioteca de Genes , Pruebas Genéticas , Humanos , Interferencia de ARN , ARN Guía de Kinetoplastida/genética , Activación TranscripcionalRESUMEN
Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.