Autophagy induction by thiostrepton for the improvement of anticancer therapy

ABSTRACT

Macroautophagy/autophagy induction by caloric restriction mimetics (CRMs) is a strategy to stimulate anticancer immune responses of immunogenic cell death (ICD)-inducing chemotherapeutics. We designed a phenotypic screening campaign in which we identified pharmacological agents that have CRM properties (i.e., non-cytotoxic induction of autophagic flux that reduces cytoplasmic protein acetylation) and simultaneously act as ICD amplifiers (i.e. with the capacity to enhance the release of adenosine triphosphate, ATP, from stressed and dying cancer cells). This approach led to the identification of thiostrepton, a natural cyclic oligopeptide antibiotic, as an agent that enhances chemotherapy-induced anticancer immune responses in vivo, in immunocompetent mice bearing syngeneic tumors. Interestingly, both the pro-autophagic and the anticancer effects of thiostrepton rely on the activation of TFEB (transcription factor EB) and TFE3 (transcription factor E3). In summary, thiostrepton represents a novel CRM and ICD amplifier that may be useful for cancer therapy.

In contrast to prevailing belief, anticancer chemotherapies do not achieve remission from malignancy and (occasionally) definitive cure because they simply kill neoplastic cells. Rather, their beneficial effects beyond treatment discontinuation rely on the induction of a durable antitumor immune response. Often, chemotherapeutics stimulate such an anticancer immune response by triggering “immunogenic cell death” (ICD), a process whereby malignant cells are stressed and killed in a fashion such that the danger-associated molecular patterns (DAMPs) emanating from the cancer cells can activate innate and later cognate immune effectors. One such DAMP is extracellular adenosine triphosphate (ATP), which acts on purinergic receptor expressed by myeloid cells to attract them into the tumor bed, hence starting the first step for igniting an anticancer immune response.

In the context of chemotherapy, the release of ATP from dying cells is favored by premortem autophagy. Indeed, cancer cells in which the autophagic machinery has been inactivated fail to elicit tumor antigen-specific immune responses in mouse models. Moreover, so-called “caloric restriction mimetics” (CRMs), which induce autophagy through a reduction of cytoplasmic protein acetylation, improve the efficacy of immunogenic chemotherapies because they stimulate ATP release from stressed and dying cancer cells, thereby boosting the local immune response. Such CRMs include a panel of chemically unrelated natural compounds including spermidine (a polyamine that inhibits the acetyl transferase EP300), hydroxycitrate (a modified metabolite that inhibits the ATP citrate lyase, ACLY, the enzyme that generates acetyl coenzyme A), resveratrol (a polyphenol that augments the deacetylase activity of SIRT1), as well as 3,4-dimethoxychalcone (a chalcone that stimulates the nuclear translocation, and transactivation, of gene expression by, TFEB (transcription factor EB), and TFE3 (transcription factor E3). Of note, more specific synthetic inhibitors of EP300 (such as C646) and ACLY (such as SB-204990) also are able to improve the immunological and therapeutic outcome of chemotherapy with anthracyclines and oxaliplatin in vitro.

Stimulated by these premises, we initiated campaigns to identify novel CRMs using a phenotypic screening approach in which cell lines expressing autophagic biosensors (such as green fluorescent protein, GFP, fused to MAP1LC3B/LC3 (microtubule-associated protein 1 light chain 3 beta), as well as the monomeric red-fluorescent protein (mRFP)-GFP-LC3 tandem reporter) are exposed to compound libraries [1]. Agents that induce GFP-LC3 puncta without cell loss (a sign of cytotoxicity) are then tested for their capacity to induce autophagic flux as well as to reduce cytoplasmic protein acetylation, which can be easily quantified by immunofluorescence. We applied this method to a total of 1560 compounds (the Prestwick library complemented by home-made libraries of flavonoids and fatty acids), screening them on two different cell lines, H4 neuroblastoma cells equipped with the GFP-LC3 biosensor and U2OS osteosarcoma cells expressing the mRFP-GFP-LC3 tandem reporter. Thirty-four hits resulting from this screen were then tested for their capacity to increase ATP release by cultured cancer cells exposed to an anthracycline, identifying thiostrepton as an interesting candidate.

Thiostrepton is a cyclic oligopeptide antibiotic that is naturally produced by several Streptomyces species and has been used, mostly in veterinary medicine, in ointments to treat dermatitis and mastitis. Thus, thiostrepton potentially represents a novel class of CRMs. Validation experiments confirmed that thiostrepton induces autophagic flux, causing, for instance, the clearance of mCherry-GFP-SQSTM1/p62 (sequestosome 1) and that of Q74-HTT (huntingtin)-GFP reporters. Thiostrepton reduces cytoplasmic protein acetylation, as well as the translocation of TFEB and TFE3 from the cytoplasm to the nucleus. In TFEB TFE3 double-knockout cells, thiostrepton loses the capacity to induce GFP-LC3 puncta, indicating that these transcription factors are indeed important for autophagy induction. Moreover, the capacity of thiostrepton to increase ATP release by cancer cells responding to anthracycline-based chemotherapy is lost in such TFEB TFE3 double-knockout cells, as well as in cells lacking the essential autophagy protein ATG5. TFEB and TFE3 are also required for thiostrepton to stimulate the release of another DAMP, HMGB1 (high mobility group box 1), from cancer cells treated with anthracyclines or oxaliplatin in vitro (Figure 1).

Figure 1.

Thiostrepton enhances immunogenic chemotherapy. Unbiased screening led to the discovery of thiostrepton as an inducer of TFEB- and TFE3-dependent autophagy that is able to enhance immunogenic cell death (ICD) by boosting the chemotherapy-induced emission of damage-associated molecular patterns (DAMPs). Specifically, the release of ATP and the exodus of HMGB1 are enhanced by the combination treatment. Altogether, the combination of ICD-inducers with thiostrepton has the ability to overcome immunosuppression and to reinstate anticancer immunity.

In vivo experiments confirmed that thiostrepton induces autophagy and that it enhances tumor growth control by oxaliplatin in a T cell-dependent fashion, accompanied by an increase in the ratio of cytotoxic T lymphocytes over regulatory T cells in the tumor bed. Improved tumor growth control by thiostrepton is not observed when cancer cells are engineered to lack TFEB and TFE3, confirming the importance of these two related pro-autophagic transcription factor for the immune response-stimulatory action of thiostrepton.

It may be important to note that thiostrepton acts as an antibiotic on 23S rRNA, inhibiting elongation factor-dependent reactions in both archaea and bacteria. We found that thiostrepton partially inhibits mRNA and protein synthesis in human cancer cells, coupled to the phosphorylation of EIF2A/eIF2 (eukaryotic translation initiation factor 2A) as a sign of endoplasmic reticulum stress. Partial inhibition of mRNA and protein synthesis as well as EIF2A phosphorylation are common hallmarks of ICD, hence confirming that thiostrepton acts not only as a CRM, but also as a bona fide ICD inducer.

Disclosure statement

GK and OK are cofounders of Samsara Therapeutics. GK is a cofounder of Therafast Bio.