RESUMEN
Resistance to targeted therapy remains a major clinical challenge in melanoma. To uncover resistance mechanisms, we perform single-cell RNA sequencing on fine-needle aspirates from resistant and responding tumors of patients undergoing BRAFi/MEKi treatment. Among the genes most prominently expressed in resistant tumors is POSTN, predicted to signal to a macrophage population associated with targeted therapy resistance (TTR). Accordingly, tumors from patients with fast disease progression after therapy exhibit high POSTN expression levels and high numbers of TTR macrophages. POSTN polarizes human macrophages toward a TTR phenotype and promotes resistance to targeted therapy in a melanoma mouse model, which is associated with a phenotype change in intratumoral macrophages. Finally, polarized TTR macrophages directly protect human melanoma cells from MEKi-induced killing via CD44 receptor expression on melanoma cells. Thus, interfering with the protective activity of TTR macrophages may offer a strategy to overcome resistance to targeted therapy in melanoma.
Asunto(s)
Resistencia a Antineoplásicos , Macrófagos , Melanoma , Melanoma/tratamiento farmacológico , Melanoma/patología , Melanoma/genética , Melanoma/metabolismo , Humanos , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Animales , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Línea Celular Tumoral , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Hialuranos/metabolismo , Receptores de Hialuranos/genéticaRESUMEN
Transposable element (TE) invasions have shaped vertebrate genomes over the course of evolution. They have contributed an extra layer of species-specific gene regulation by providing novel transcription factor binding sites. In humans, SINE-VNTR-Alu (SVA) elements are one of three still active TE families; approximately 2800 SVA insertions exist in the human genome, half of which are human-specific. TEs are often silenced by KRAB zinc finger (KZNF) proteins recruiting corepressor proteins that establish a repressive chromatin state. A number of KZNFs have been reported to bind SVAs, but their individual contribution to repressing SVAs and their roles in suppressing SVA-mediated gene-regulatory effects remains elusive. We analyzed the genome-wide binding profile for ZNF91 in human cells and found that ZNF91 interacts with the VNTR region of SVAs. Through CRISPR-Cas9-mediated deletion of ZNF91 in human embryonic stem cells, we established that loss of ZNF91 results in increased transcriptional activity of SVAs. In contrast, SVA activation was not observed upon genetic deletion of the ZNF611 gene encoding another strong SVA interactor. Epigenetic profiling confirmed the loss of SVA repression in the absence of ZNF91 and revealed that mainly evolutionary young SVAs gain gene activation-associated epigenetic modifications. Genes close to activated SVAs showed a mild up-regulation, indicating SVAs adopt properties of cis-regulatory elements in the absence of repression. Notably, genome-wide derepression of SVAs elicited the communal up-regulation of KZNFs that reside in KZNF clusters. This phenomenon may provide new insights into the potential mechanisms used by the host genome to sense and counteract TE invasions.
Asunto(s)
Células Madre Embrionarias Humanas , Factores de Transcripción de Tipo Kruppel/deficiencia , Familia de Multigenes/genética , Proteínas Represoras/genética , Retroelementos/genética , Activación Transcripcional , Regulación hacia Arriba , Genoma Humano , Humanos , Dedos de Zinc/genéticaRESUMEN
PURPOSE: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. EXPERIMENTAL DESIGN: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. RESULTS: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. CONCLUSIONS: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.