Cancerous cells can undergo Therapy-Induced Senescence (TIS) when challenged with low doses of genotoxic drugs such as doxorubicin (DOX). Senescence is characterized by cell cycle arrest, increased immunogenicity and production of inflammatory cytokines, growth factors and metalloproteinases, collectively referred to as Senescence-Associated Secretory Phenotype (SASP). In multiple myeloma (MM), a plasma cell malignancy that develops within the bone marrow, TIS is able to increase NK cells recognition and killing of tumor cells. Emerging evidence indicate that senescent cells accumulate cytosolic DNA, which in turn activates the cGAS-STING pathway, leading to production of IFN-I and other immune-stimulating cytokines. cGAS activation by cytosolic DNA triggers the production of 2'3'-cGAMP, a soluble small molecule that seems to be exported by tumor cells, activating STING in recipient cells. cGAMP and other STING agonists show impressive antitumoral activity in several in vivo models for their capacity to broadly stimulate the immune system. Our preliminary data suggest that DOX-treated senescent MM cells increase the production of cGAMP compared to untreated cells. Moreover, we demonstrated that DOX-conditioned medium can increase NK cells degranulation and induce senescence on bystander MM cells. However, whether cGAMP is responsible for these phenomena is still to be demonstrated. Interestingly, we have some evidence that low doses of cGAMP can induce senescence in MM cells, whereas higher doses seem to trigger apoptosis.
The objective of our proposal is therefore to correlate these phenomena and to investigate the effectiveness of cGAMP released by senescent cells in activating NK cells. This would be the first evidence of cGAMP being an immune-activating SASP component. Furthermore, we aim at enhancing the activity of cGAMP by modulating the function of known or suspected ectoenzymes responsible for cGAMP degradation in the extracellular environment.
Cellular senescence is a complex, diverse, and dynamic process triggered by a wide variety of stressors in many different cell types. The current view is that "acutely" induced senescence represents a protective mechanism, whereas the accumulation of senescent cells due to ineffective elimination by the immune system can worsen the pathology of cancer and age-related disorders.
Several connections between cellular senescence and cGAS-STING pathway have emerged and are continuing to emerge. What is becoming clear is the regulatory role of STING in the establishment of SASP. However, the role of cGAMP as a local immunotransmitter (the first discovered) has just started to be investigated, mainly because of the difficulty of measuring and studying the activity of such small molecule. Recently, a specific importer of cGAMP and other cyclic dinucleotides, named SLC19A1, has been discovered by genome-wide CRISPR screening (Ritchie C. et al. 2019), suggesting how a negatively charged, non-lipophilic molecule can be exchanged by neighbouring cells. We propose cGAMP as a novel SASP factor, released at higher amounts by senescent cells and directly able to evoke a protective anti-tumoral immune response, acting on both the innate immune system and the stromal compartment.
Regarding bystander senescence, this represents a notorious and widely investigated phenomenon. Nevertheless, considering the complexity and variability of senescent cell's secretome, it is not surprising that the causal agents underlying it are often incompletely understood. Even so, it is plausible that the combination of several factors is responsible for paracrine induction of senescence. The golden standard of SASP investigation is represented by mass spectrometry-based proteomics; indeed, most publications focused on unraveling the proteome of SASP, neglecting the contribution of small molecules that, in principle, may be equally important. Based on our preliminary data, we hypothesize that secreted cGAMP can contribute to bystander senescence in multiple myeloma.
The induction of senescence by cGAMP is particularly plausible in the model of multiple myeloma, since it has been demonstrated that excessive STING activation in B-cell malignancies can lead to mitochondria-mediated apoptosis. The evidence that signalling strength of STING pathway can trigger apoptosis, independently from type 1 IFNs, has been demonstrated in normal and malignant T cells as well (Gulen et al. 2017). We therefore propose that a weaker activation of STING pathway by secreted cGAMP might result in senescence rather than apoptosis.
Targeting enzymes that degrade cGAMP is currently a hot topic in immunology. Only recently, ENPP1 was identified as the first ectoenzyme able to hydrolyze and inactivate cGAMP revealing an important role in cancer immunotherapy for their ability to strengthen STING signalling; no other enzymes capable of doing so have been described so far.
We have preliminary evidence suggesting that CD38, a well-known ectoenzyme expressed by lymphocytes and multiple myeloma cells, can degrade cGAMP as well. Although such role of CD38 is, to our knowledge, unprecedented, it is fully in accordance with the notion of an immunosuppressive microenvironment created by multiple myeloma. Therefore, we suppose that multiple myeloma may be the perfect disease model for studying and targeting the regulation of cGAMP stability in tumor microenvironment. Interestingly, expression of CD38 correlates with negative prognosis in chronic linfocitic leukemia, another B-cell malignancy (Deaglio et al. 2008), but whether the correlation is based on its immunomodulatory function it is unknown.
In conclusion, we believe that, if these objectives will be reached, our work will not only set a rational base for possible therapeutic application in the treatment of multiple myeloma, but also shed light on general mechanisms that could be extended beyond our model.