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Background: Although immune checkpoint inhibitors have been a breakthrough in clinical oncology, these therapies fail to produce durable responses in a significant fraction of patients. This lack of long-term efficacy may be due to a poor pre-existing network linking innate and adaptive immunity. Here, we present an antisense oligonucleotide (ASO)-based strategy that dually targets toll-like receptor 9 (TLR9) and programmed cell death ligand 1 (PD-L1), aiming to overcome resistance to anti-PD-L1 monoclonal therapy. Methods: We designed a high-affinity immunomodulatory IM-TLR9:PD-L1-ASO antisense oligonucleotide (hereafter, IM-T9P1-ASO) targeting mouse PD-L1 messenger RNA and activating TLR9. Then, we performedin vitroandin vivostudies to validate the IM-T9P1-ASO activity, efficacy, and biological effects in tumors and draining lymph nodes. We also performed intravital imaging to study IM-T9P1-ASO pharmacokinetics in the tumor.ResultsIM-T9P1-ASO therapy, unlike PD-L1 antibody therapy, results in durable antitumor responses in multiple mouse cancer models. Mechanistically, IM-T9P1-ASO activates a state of tumor-associated dendritic cells (DCs), referred to here as DC3s, which have potent antitumor potential but express the PD-L1 checkpoint. IM-T9P1-ASO has two roles: it triggers the expansion of DC3s by engaging with TLR9 and downregulates PD-L1, thereby unleashing the antitumor functions of DC3s. This dual action leads to tumor rejection by T cells. The antitumor efficacy of IM-T9P1-ASO depends on the antitumor cytokine interleukin-12 (IL-12), produced by DC3s, andBatf3, a transcription factor required for DC development. Conclusions: By simultaneously targeting TLR9 and PD-L1, IM-T9P1-ASO amplifies antitumor responses via DC activation, leading to sustained therapeutic efficacy in mice. By highlighting differences and similarities between mouse and human DCs, this study could serve to develop similar therapeutic strategies for patients with cancer.

© 2020 American Association for the Advancement of Science. All rights reserved. Interpreting how multicellular interactions in the tumor affect resistance pathways to BRAF and MEK1/2 MAPK inhibitors (MAPKi) remains a challenge. To investigate this, we profiled global ligand-receptor interactions among tumor and stromal/immune cells from biopsies of MAPK-driven disease. MAPKi increased tumor-associated macrophages (TAMs) in some patients, which correlated with poor clinical response, and MAPKi coamplified bidirectional tumor-TAM signaling via receptor tyrosine kinases (RTKs) including AXL, MERTK, and their ligand GAS6. In xenograft tumors, intravital microscopy simultaneously monitored in situ single-cell activities of multiple kinases downstream of RTKs, revealing MAPKi increased TAMs and enhanced bypass signaling in TAM-proximal tumor cells. As a proof-of-principle strategy to block this signaling, we developed a multi-RTK kinase inhibitor nanoformulation that accumulated in TAMs and delayed disease progression. Thus, bypass signaling can reciprocally amplify across nearby cell types, offering new opportunities for therapeutic design.

© 2020 The Authors Targeted inhibition of oncogenic pathways can be highly effective in halting the rapid growth of tumors but often leads to the emergence of slowly dividing persister cells, which constitute a reservoir for the selection of drug-resistant clones. In BRAFV600E melanomas, RAF and MEK inhibitors efficiently block oncogenic signaling, but persister cells emerge. Here, we show that persister cells escape drug-induced cell-cycle arrest via brief, sporadic ERK pulses generated by transmembrane receptors and growth factors operating in an autocrine/paracrine manner. Quantitative proteomics and computational modeling show that ERK pulsing is enabled by rewiring of mitogen-activated protein kinase (MAPK) signaling: from an oncogenic BRAFV600E monomer-driven configuration that is drug sensitive to a receptor-driven configuration that involves Ras-GTP and RAF dimers and is highly resistant to RAF and MEK inhibitors. Altogether, this work shows that pulsatile MAPK activation by factors in the microenvironment generates a persistent population of melanoma cells that rewires MAPK signaling to sustain non-genetic drug resistance. Gerosa et al. show that pulsatile MAPK activation makes it possible for slow-growing drug-resistant persisters to emerge when BRAF-mutant melanoma cells are exposed to RAF and MEK inhibitors at clinically relevant doses. Computational modeling shows that MAPK signaling exists in two configurations, one activated by oncogenic BRAF that is drug sensitive and the other activated by autocrine/paracrine growth factors and transmembrane receptors that is drug resistant.

© 2020 American Association for the Advancement of Science. All rights reserved. Interpreting how multicellular interactions in the tumor affect resistance pathways to BRAF and MEK1/2 MAPK inhibitors (MAPKi) remains a challenge. To investigate this, we profiled global ligand-receptor interactions among tumor and stromal/immune cells from biopsies of MAPK-driven disease. MAPKi increased tumor-associated macrophages (TAMs) in some patients, which correlated with poor clinical response, and MAPKi coamplified bidirectional tumor-TAM signaling via receptor tyrosine kinases (RTKs) including AXL, MERTK, and their ligand GAS6. In xenograft tumors, intravital microscopy simultaneously monitored in situ single-cell activities of multiple kinases downstream of RTKs, revealing MAPKi increased TAMs and enhanced bypass signaling in TAM-proximal tumor cells. As a proof-of-principle strategy to block this signaling, we developed a multi-RTK kinase inhibitor nanoformulation that accumulated in TAMs and delayed disease progression. Thus, bypass signaling can reciprocally amplify across nearby cell types, offering new opportunities for therapeutic design.