25 Jan 2023
by Richa

Summary- paper 12: A CRISPRi/a screening platform to study cellular nutrient transport in diverse microenvironments

Christopher Chidley, Alicia M. Darnell, Benjamin L. Gaudio, Evan C. Lien, Anna M. Barbeau, Matthew G. Vander Heiden, Peter K. Sorger

Biorxiv, 2023

Questions/gaps addressed:

  • Aggressive growth of cancerous cells means dependence on teh environment for supplies of non-essential amino acids. This includes an increase in the rate of nutrient uptake, by upregulation of SLC transporters and a rewiring of intracellular metabolism.

  • Which SLC transporters are more important for tumor growth, and can they used as therapeutic targets.

Major hypotheses:

  • Key SLC and ABC transporters play an essential role in proliferation of cancerous cells.

Key methods:

  • CRISPR interference (CRISPRi) and activation (CRISPRa) growth-based pooled screens (10 sgRNAs/gene and 730 non-targeting controls) to systematically interrogate the individual contributions to nutrient transport and growth by 489 annotated members of the SLC and ABC transporters in human cells.

  • Identified all amino acids in RPMI-1640 medium whose absence limited K562 cell proliferation by performing proliferation assays with a series of single amino acid dropout RPMI media to which amino acids were added back to between 0.1% and 100% of their normal levels.

  • Performed pooled CRISPRi/a screens at amino acid concentrations that reduced K562 cell proliferation by 50% for all 13 growth-limiting amino acids to identify transporter perturbations that caused either a decrease or increase in proliferation. Three rounds of low amino acid exposure lasting for four days with daily media changes followed by one recovery day in complete medium, enrichment analysis between starved and complete RPMI conditions.

  • Validated phenotypes in PAA-RPMI medium formulated to contain all amino acids as well as five other known SLC7 family substrates (citrulline, ornithine, creatine, creatinine, and carnitine) at levels found in human plasma.

  • Mass spectrometry-based transport assays to follow amino acid transport at the plasma membrane. Quantified amino acid import by incubating K562 cells in medium containing amino acids labeled with heavy isotopes and measuring intracellular isotope accumulation by gas chromatography-mass spectrometry (GC-MS)

  • Tested the effect of two ferroptosis inducers: erastin, which inhibits SLC7A11, and RSL3, which inhibits glutathione peroxidase 4 (GPX4).

  • Compared transporter essentiality in: (1) RPMI at high confluence, (2) RPMI with regular fetal bovine serum (FBS) replaced with FBS that was dialyzed to remove small molecules (dFBS), (3) Dulbecco’s Modified Eagle Medium (DMEM), an alternative synthetic medium, and (4) in different cell lines- K562 vs A375 (BRAFV600E melanoma)

Major takeaways:

  • Removal of Asn, Asp, Glu, Gly, and Pro from the RPMI-1640 medium has no effect on the proliferation of K562 cells. Only these five amino acids are also net secreted by K562s. Cystine (oxidized cysteine more abundant in culture medium)deprivation induces significant cell death (ferroptosis?). A reduction in media levels of 13 of the 19 amino acids found in RPMI decreased proliferation.

  • Strong CRISPRi hits were also hits in the corresponding CRISPRa screens. CRISPRi/a combination screens also allow identification of genes expressed in a particular cell line.

  • RPMI which contains many amino acids at 0.4× to 10× the levels found in human plasma but only trace amounts of alanine, cysteine.

  • Serotonin (5-HT) and transporter SLC6A4 act as an endogenous antioxidant to protect cells from ferroptosis.

  • Specific transporters differentially affected depending on the influence of the environment on metabolism, and differences in metabolism between cell lines.