How can Death-seq help to improve senolytic therapies?

New research identifies a screening method that helps in the systematic study of cell death and that will enhance the treatment of senescence.

Cellular senescence is a cell state characterized by extended and irreversible cell-cycle arrest and is considered to be a hallmark of aging. Senescence is most often triggered by different types of stresses or stimuli such as telomere shortening, mitochondrial dysfunction, epigenetic changes, chromatic disorganization and others [1]. Elimination of CDKN2A-positive senescent cells in mice has been found to restore tissue homeostasis in aging as well as treat several age-related diseases. Senolytics are a class of drugs that play an important role in the elimination of such senescent cells.

Senolytic drugs have been observed to accumulate at sites of chronic disorders as well as with age. Several studies have indicated senolytics to be effective in the treatment of serious conditions such as cancer, liver damage, cardiovascular disorders, skin conditions, kidney problems, lung disorders and others. The first senolytic drugs to be discovered included quercetin, navitoclax, fisetin and dasatinib [2].

Longevity.Technology: Little potency and systemic toxicity of senolytics limit their use for local administration and reduce their clinical application. Recent research has highlighted pooled genetic screens to be useful in the identification of senolytic targets. A recent RNAi approach study was used to identify new senolytic targets but it could only identify one target. Another Annexin V-based enrichment screen was also found to have several limitations. It was time to seek a new approach.

Seek and ye shall find – a new study published in Cell Metabolism developed a method termed ‘Death-seq’ that could positively select cells that die due to pharmacologic or genetic interventions. It then combined this approach with a genome-wide CRISPR screen for the identification of targets that would improve senolytic therapy [3].

Death-seq involved the collection of dying cells and allowed amplification of signal and reduction of noise. This helped to screen live and dying cells in more physiologically relevant models. This study used Death-seq to carry out a genome-wide CRISPR knockout (KO) screen in normal human lung fibroblast cells. The cells were induced to senescence by doxorubicin (Doxo-SEN) to identify whether KOs showed increased or decreased sensitivity to senolysis by ABT263, which was an inhibitor of several anti-apoptotic proteins. Thereafter, the distribution of sgRNA was determined in both live and dying cells using DNA-seq followed by the effect of each gene knockout.

The results of the study identified top hits that inhibited death of the cells which included APAF1, HCCS, CYCS, PPTC7, MCL1 and VHL. Genetic interaction networks and pathways analysis indicated the enrichment of pathways that were associated with mTOR signaling, intrinsic apoptosis pathway and SMAC-IAP-caspase interactions. However, Death-seq was observed to identify more hits as compared to traditional methods. Along with ABT-263, several parallel screens were developed that could also identify hits that inhibited cell deaths.

A combination of ABT-263 and a second mitochondria-derived activator of caspases (SMAC) mimetic, birinapant was observed to increase overall senolytic effects. However, a combination of another screen ABT-199, and birinapant was found to be non-toxic towards human and mouse platelets but could reduce senescent cell markers and improve the severity of conditions such as pulmonary fibrosis and liver damage. Death-seq was also observed to identify genetic modifiers of senescent cell death in the absence of any pharmacological initiation. Death-seq was reported to not only identify modifiers of the apoptosis pathway but also identify regulators of nonapoptotic cell death [3].

Therefore, this study shows that Death-seq allows the development of effective and efficient genome-wide screens which throws light into molecular mechanisms of regulated cell death procedure and can also help to identify drug targets for the treatment of senescence, fibrosis and cancer.

The paper authors note “the world’s dramatically increasing population of persons over the age of 65” and the subsequent need to develop strategies to extend and restore healthspan.

“The COVID-19 pandemic has demonstrated the enhanced vulnerability of persons over
65 and those with age-related disease to hospitalization, death, and other adverse events as a result of infections, in this case with severe acute respiratory syndrome coronavirus 2 (SARSCoV-2),” they explain. “Senescent cells have been highlighted as a potential reason for this enhanced vulnerability of persons over 65, with a recent study demonstrating that senescent cells have an amplified inflammatory response to SARS-CoV-2 and that in old mice infected with SARS-CoV-2 related virus, senolytics reduce mortality and increase antiviral antibodies [3].”

The authors propose that a “systemic senolytic treatment with minimal side effects” could be used to protect the aging population from future pandemics, and will be critical for clinical trials diseases such as in other diseases that cannot be treated with local administration IPF, COPD, atherosclerosis and neurodegeneration [3].

The authors also explain that because Death-seq compares guide composition in dying cells directly with live cells, the platform allows more efficient and effective genome-wide screens, unlocking “the potential for new findings in the senescence field and in a wide variety of fields outside of senescence.”

“As understanding of which damaged cell types, including senescent cell types, play pathogenic versus beneficial or negligible roles in age-related disease and aging continues to improve, we expect that Death-seq will be utilized in additional cell types based on their disease-specific pathogenic relevance,” they explain. “Just like the Cancer Dependency Project has profiled hundreds of cancer cell models to identify genetic vulnerabilities for therapeutic development, we anticipate that Death-seq will enable a similar project in the field of ‘‘senescent cells’’ across different models and inducers of senescence [3].”