Lighting the way to tumor destruction

Abstract

Achieving tumor-specific protein loss remains a challenge in the delivery of PROteolysis-TArgeting Chimeras (PROTACs) as cancer therapeutics. As a solution, Wang et al. developed nanoformulated PROTACs, a novel photoactivatable degradation approach. This innovative class of compounds harnesses near-infrared light for precise PROTAC release and protein degradation in mouse tumors.

PROteolysis-TArgeting Chimeras (PROTACs) are a powerful therapeutic approach to directly control protein levels and are currently in clinical trials for several cancers and autoimmune diseases [1]. These heterobifunctional small molecule degraders function by recruiting an E3 ligase to a target protein to induce ubiquitination and subsequent degradation by the proteasome [1]. PROTACs are a breakthrough approach and have advantages over inhibitors due to the ability to completely abolish all functions of a protein [2]. Nevertheless, one clinical drawback of PROTACs is that on-target protein loss can occur across normal tissues and organs, leading to toxicity [3]. Innovative approaches are needed to specifically degrade target proteins in the diseased tissue of interest and emerging technologies aim to overcome this limitation.

Strategies to activate small molecules with light such as photodynamic therapy (PDT) are used as a therapeutic strategy in diseases including dermatological and ocular cancers [4]. The application of light-based therapies in medicine has inspired several groups to develop photoactivatable PROTACs incorporating either reversible photoswitches [5, 6] or irreversible photocleavage sites [7]. These compounds respond to light irradiation to achieve spatiotemporal control of protein degradation and have remarkable potential to mitigate toxicity concerns of PROTACs by achieving tissue-specific protein degradation [4]. However, for translation into the clinic, these approaches are not compatible with deeper tissue penetration, and their efficacy in preclinical mouse models has not been demonstrated. To achieve deeper tissue penetration, approaches that respond to red-shifted light are necessary. One application has been development of semiconducting polymer nano-PROTACs. This approach combines a PROTAC with a cancer-biomarker-cleavable peptide for tumor release, and a semiconducting polymer that generates singlet oxygen (¹O₂) to initiate an immune response upon near-infrared (NIR) irradiation [8]. Recently, in the Journal of the American Chemical Society, Wang et al. harness PDT to advance photoactivable degradation approaches by creating novel nanoformulated PROTACs (NAPs). This new class of compounds are precisely activated by NIR irradiation to induce the destruction of target proteins in tumor-bearing mice [9].

To develop their NAP, Wang et al. focused on the bromodomain and extraterminal (BET) protein family (BRD2/3/4/T), which are prototypical substrates in the field of targeted protein degradation. These essential transcriptional co-activators are involved in several malignancies including cancer and autoimmune diseases [10]. While targeted degradation of BET proteins with PROTACs is advantageous over inhibition [10], on-target toxicity that results from BET protein depletion limits their potential clinical advancement [3]. Interestingly, the BET proteins mediate resistance to PDT, leading to the hypothesis that BET proteins degradation in the tumor may also have synergistic effects with PDT. The use of NIR light is particularly powerful as it allows for deeper tissue penetration, which other wavelengths of light used in existing approaches are unable to achieve [4].

NAPs are composed of a PROTAC conjugated to a ¹O₂-responsive cleavable linker and a pheophorbide a (PA) moiety, which is used in PDT as a photosensitizer. The hydrophobic and rigid structure of PA mediates self-assembly in aqueous solution and in response to NIR-irradiation generates ¹O₂ that induces linker cleavage and release of the PROTAC. ARV-771, which is a hydrophilic BRD2/3/4 PROTAC that recruits the E3 ubiquitin ligase von Hippel-Lindau (VHL), was selected as the first candidate for development of NAPs (Figure 1A). Administration of ARV-771 has been reported to have toxicity in tumor-bearing mouse models manifesting as deterioration of skin health, hunching of the spine, lethargy, and decreased mobility [3]. Therefore, precise delivery and release of ARV-771 in the tumor may reduce these observed toxicities in healthy tissues, while also synergizing with PDT.

Figure 1. Overview of the self-assembly and release of a nanoformulated PROTAC (NAP) for targeted protein degradation in vivo.

(A) Structures of NAP and NCP are indicated. NAP and NCP are composed of ARV-771, which includes the BET bromodomain protein binder (green) conjugated with a linker (black) to the VHL binder (blue). NAP is also conjugated with a linker that is ¹O₂-cleavable (red) to a NIR photosensitizer (orange), while NCP is conjugated with a linker that is not ¹O₂-cleavable (purple) to a NIR photosensitizer (orange). (B) Schematic illustrating that in aqueous solution NAP self-assembles. Similar self-assembly occurs with NCP. (C) Schematic illustrating the methodology of NAP in its inactive (non-irradiated) and activated (NIR-irradiated) states. Upon NIR irradiation of NAP, ARV-771 is released in the tumor to induce BRD4 ubiquitination and degradation. Degradation of BRD4 synergizes with PDT to lead to a decrease in tumor burden. Figure created with BioRender.com.

Wang et al. used a wide range of in vitro and in vivo assays to test the photophysical properties, dispersion, and anti-cancer activity of their BET protein-targeting NAP. An important feature of NAPs is the ability to develop negative control compounds (NCPs). NCP is almost identical to NAP except that it contains a noncleavable alkyl linker to connect ARV-771 and PA, and was used as a comparison in all experiments (Figure 1A). It should be noted that although NCP with NIR irradiation did not induce degradation of BRD4, NCP with NIR irradiation decreased cell viability and tumor burden. This may be due to the ability of NCP to still inhibit the BET proteins, which was not evaluated by the authors. First, they showed that NAP and NCP self-assemble in aqueous solutions and release ¹O₂ upon NIR irradiation (Figure 1B). Upon a 5-minute NIR irradiation, NAP disassembled and released ARV-771 with 85% efficiency after a 12-hour incubation. Subsequently, NAP and NCP activity was assessed in MCF-7 cells, a human breast cancer cell line. While NAP and NCP were similarly able to penetrate MCF-7 cells and release ¹O₂ upon NIR irradiation, near complete degradation of BRD4 was observed at doses ranging from 1 to 5 μM NAP, with evidence of degradation 30 to 60 minutes after NIR irradiation. The proteasome and VHL were required for the observed BRD4 degradation. Due to the pan-BET protein binder used to develop ARV-771, potent degradation of BRD2 and BRD3 was also observed. Antiproliferation and apoptosis assays revealed that NAP or NCP alone had minimal activity, which was significantly enhanced upon NIR irradiation. While ARV-771 or NCP with NIR irradiation decreased proliferation and increased apoptosis, NAP with NIR irradiation was superior to both compounds.

These promising results inspired evaluation of NAP distribution and anti-tumor activity in mouse models following subcutaneous implantation of MCF-7 cells. NAP and NCP were equivalently detected in the tumor and organs including liver and lung but were not observed in the kidney, lung, spleen, or heart. They also similarly induced ¹O₂ release upon NIR irradiation in the tumor. In an efficacy study, partial reductions in tumor weight and volume were observed upon administration of ARV-771 alone or NCP with NIR irradiation. These responses were significantly enhanced when NAP was administered with NIR irradiation (Figure 1C). Pronounced reductions in BRD4 levels were observed in the tumor without significant body weight loss or impact to the integrity of the heart, liver, spleen, lung, or kidney. While it was not tested whether BET protein levels were impacted in these organs upon NAP with NIR irradiation, these results highlight the promise of this approach to reduce tumor burden without toxicity.

In this work, Wang et al. made important progress in the development of a novel photoactivatable degradation strategy. For broad applicability of NAPs in precision medicine, it will be critical to expand this approach beyond the BET proteins and to demonstrate applicability to PROTACs that recruit other E3 ligases including cereblon, which is commonly co-opted in PROTACs in clinical evaluation [1]. It will also be important to understand the mechanisms and properties of NAP release and endosome escape after cellular internalization. Finally, studies are necessary to demonstrate activity using orthotopic implantation or genetically engineered mouse models rather than subcutaneous implantation mouse models of cancers against which PDT is currently deployed. This future work will identify limitations in the delivery of NAPs with NIR irradiation for degrading target proteins in the tumor relative to healthy organs. Overall, this initial characterization of NAPs highlights the potential of photoactivatable degraders to reduce drug toxicities in vivo. This promising advance contributes to the broader goal of improving the quality of life for patients with cancer.