Functional Phosphoproteomics in Cancer Chemoresistance Using CRISPR‐Mediated Base Editors

Abstract

Selective inhibition of targeted protein kinases is an effective therapeutic approach for treatment of human malignancies, which interferes phosphorylation of cellular substrates. However, a drug-imposed selection creates pressures for tumor cells to acquire chemoresistance-conferring mutations or activating alternative pathways, which can bypass the inhibitory effects of kinase inhibitors. Thus, identifying downstream phospho-substrates conferring drug resistance is of great importance for developing poly-pharmacological and targeted therapies. To identify functional phosphorylation sites involved in 5-fluorouracil (5-FU) resistance during its treatment of colorectal cancer cells, CRISPR-mediated cytosine base editor (CBE) and adenine base editor (ABE) are utilized for functional screens by mutating phosphorylated amino acids with two libraries specifically targeting 7779 and 10 149 phosphorylation sites. Among the top enriched gRNAs-induced gain-of-function mutants, the target genes are involved in cell cycle and post-translational covalent modifications. Moreover, several substrates of RSK2 and PAK4 kinases are discovered as main effectors in responding to 5-FU chemotherapy, and combinational treatment of colorectal cancer cells with 5-FU and RSK2 inhibitor or PAK4 inhibitor can largely inhibit cell growth and enhance cell apoptosis through a RSK2/TP53BP1/𝜸-H2AX phosphorylation signaling axis. It is proposed that this screen approach can be used for functional phosphoproteomics in chemotherapy of various human diseases.

Proteome-wide base editor screens to assess phosphorylation site functionality in high-throughput

“…We next examined whether mutation of phosphosites important for cell division would affect cell viability or proliferation in our pooled format without specific stimuli or selective pressure 37 . Examination of phosphorylation sites on CDK1, a kinase whose activity is necessary for proper cell division, showed that Y160C;T161A and Y15C reduced cell viability post-ABE8e electroporation similar in magnitude and direction to levels previously seen using homology-directed recombination 38 ( Figure 3C ).…”

“…Our study focused on phosphosites empirically identified in a parallel experiment rather than from PTM repositories or databases, mitigating the introduction of indiscriminate coding mutations that can have PTM-independent effects 20,21,37 . Moreover, as phosphoproteomic analyses of novel, unstudied systems continue to grow (i.e.…”

“…Proteome-wide, PTM-centric base editing coupled to phenotypic screens provides a powerful experimental framework to untangle the vast network of biochemical signaling reactions and how they lead to the control of specific cellular functions. This approach directly assesses the impact of a phosphosite mutation on a given phenotype rather than relying purely on evolutionary or structural conservation, and provides interpretable results with high confidence for further mechanistic studies 37,77–80 . With currently available base editors it should be possible to functionally screen other PTMs including acetylation/ubiquitination/methylation (lysine) 81 , O-GlcNAcylation (Ser/Thr) 82 , cysteine 83 , or even specific proteolysis events (caspases) 84 , utilizing established infrastructure common to many research institutions.…”

Preclinical Anticipation of On- and Off-Target Resistance Mechanisms to Anti-Cancer Drugs: A Systematic Review

“…This allowed the discovery of essential mediators of sensitivity or resistance to IFN-γ, and, importantly, a strong overlap with available patient data was observed [112]. In another study, CRISPR-mediated screens with CBEs and ABEs targeting altogether nearly 18,000 phosphorylation sites were performed in the CRC HCT116 cells to understand resistance mechanisms to the anti-metabolite 5-fluorouracil, thus leading to the identification of RSK2 and PAK4 kinase substrates [113].…”

Cas9‐orthologue‐mediated cytosine and adenine base editors recognizing NNAAAA PAM sequences

“…[ 7,10 ] To expand their targeting scope, elevate their targeting efficiencies, and minimize their off‐targeting activities, various base editor derivates have been developed by replacing or engineering novel deaminases. [ 11‐14 ] Since then, base editors have been widely used for annotating the functional significance of SNVs or modifications in a low‐ or high‐throughput manner, [ 15‐19 ] as we have reported previously. Considering the restriction of protospacer adjacent motif (PAM) sequences and protein sizes for distinct Cas9 proteins, Cas9 is engineered or replaced by other orthologs or other Cas proteins to expand the versatility and efficiency of in vitro and in vivo genome editing.…”