Week 4 progress update

This week, we have continued analyzing our RNA-seq data, we have completed and sequenced our CRISPR screens, and we have cloned and are performing initial RdRP experiments. Stay tuned for updates on the CRISPR screen in the next few days.

Regarding RNA-seq, we are working on how best to develop coherent GSEA analysis (Fig. 1a). While we move forward in this area, our most tangible progress has been in evaluating the RNA-seq finding that HCQ impairs cholesterol uptake in our cell lines. We have performed fluorescent LDL uptake assays, showing significantly reduced LDL uptake upon HCQ dosing by flow cytometry (Fig. 1b) and an LDL endosome retention phenotype by fluorescent microscopy (Fig. 1c) that support the mechanistic hypothesis that HCQ impairment of endosome to lysosome processing underlies impairs LDL uptake. 

Because statins are widely prescribed drugs that impair cholesterol metabolism, we tested whether there is synthetic lethality between HCQ and Simvastatin in our cell lines as a result of excessive cholesterol starvation. We find reproducible synthetic lethality between HCQ and Simvastatin in several cell lines (Fig. 1d). We are currently testing whether this lethality can be alleviated by providing mevalonate pathway supplements. Certain mevalonate supplements such as Coenzyme Q10 that have been shown to alleviate statin-induced toxicity in cells and have shown mixed effects in reducing statin side effects in clinical studies. 

It is important to note that our findings at this point are on cancer cell lines, and even robust evidence in primary cells of synthetic lethality between HCQ and statins that can be alleviated be a supplement known to be safe for human use is insufficient to suggest that patients taking statins and HCQ should take this supplement. Instead, such data should be combined with the CRISPR screening and human genetic association analyses we will perform. If gene-drug or drug-drug interactions are highlighted by these multiple orthogonal approaches, it raises confidence that they are robust and worth relaying to the medical community to assess their clinical value.