Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC

Abstract

Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common. By using genome-wide CRISPR/Cas9 library screening, we identify phosphoglycerate dehydrogenase (PHGDH), the first committed enzyme in the serine synthesis pathway (SSP), as a critical driver for Sorafenib resistance. Sorafenib treatment activates SSP by inducing PHGDH expression. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that inactivation of PHGDH paralyzes the SSP and reduce the production of αKG, serine, and NADPH. Concomitantly, inactivation of PHGDH elevates ROS level and induces HCC apoptosis upon Sorafenib treatment. More strikingly, treatment of PHGDH inhibitor NCT-503 works synergistically with Sorafenib to abolish HCC growth in vivo. Similar findings are also obtained in other FDA-approved tyrosine kinase inhibitors (TKIs), including Regorafenib or Lenvatinib. In summary, our results demonstrate that targeting PHGDH is an effective approach to overcome TKI drug resistance in HCC.

Fig. 1

CRISPR library screening identified PHGDH as a driver for Sorafenib resistance. a Schematic diagram illustrates the workflow of genome-wide CRISPR/Cas9 knockout library screening (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats). Human genome-wide CRISPR/Cas9 knockout library (GeCKO v2A) containing 65,386 sgRNAs was packed into lentiviral particle and transduced into Cas9-overexpressing MHCC97L cells (MHCC97L-Cas9) at low multiplicity of infection (MOI). The sgRNA transduced cells were selected by puromycin to generate a mutant cell pool. Mutant cells were cultured in vehicle and Sorafenib for 7 days for genetic screening. Genomic DNA was extracted from the treated cells and the sgRNA fragment was amplified by PCR. Copy number of sgRNAs was determined by high-throughput sequencing and analyzed by MAGeCK v0.5.7 algorithm. b PHGDH (phosphoglycerate dehydrogenase) was identified as the most significant gene in the library screening as indicated by the red dot. The sgRNAs targeting PHGDH were consistently depleted in Sorafenib-treated cells. c Volcano plots revealed that PHGDH targeting sgRNAs were negatively selected during Sorafenib treatment, suggesting that PHGDH is an essential gene for HCC cells to survive from Sorafenib treatment. Source data are provided as a Source Data file

Fig. 2

Activation of serine synthesis pathway was involved in Sorafenib resistance. a MHCC97L cells were treated with Sorafenib for 112 days. RNA-Seq was performed in Sorafenib treated MHCC97L cells at the indicated time points. b DAVID gene ontology and c Gene Set Enrichment Analysis (GSEA) showed that genes involved in serine biosynthesis were significantly deregulated in Sorafenib-resistant cells (Day 112) when compare to the parental cells (Day 0). d RNA-Seq analysis revealed that the key enzymes in serine synthesis pathway, PHGDH, PSAT1 (phosphoserine aminotransferase 1), and PSPH (phosphoserine phosphatase), were all up-regulated in Sorafenib-resistant cells. The upregulation of PHGDH was also confirmed in protein level by western blot. The error bar represents the mean with upper and lower limit. Source data are provided as a Source Data file

Fig. 3

Depletion of PHGDH-sensitized HCC cells to Sorafenib treatment. a Knockout of PHGDH in MHCC97L cells by CRISPR/Cas9 gene editing system. b Knockout of PHGDH showed mild effect on cell proliferation in in vitro cell culture. However, knockout of PHGDH significantly suppressed MHCC97L cell proliferation in the presence of Sorafenib (black connected dots: non-target control; red connected dots: sgPHGDH#2; deep blue connected dots: sgPHGDH#3; green connected dots: sgPHGDH#4; purple connected dots: sgPHGDH#12; light blue connected dots: sgPHGDH#32). c Knockout of PHGDH induced apoptosis significantly upon Sorafenib treatment (gray bar: vehicle-treated group; red bar: Sorafenib-treated group). d Knockdown of PHGDH by lentiviral-based shRNA approach. e Knockdown of PHGDH suppressed HCC cell proliferation under Sorafenib treatment (black connected dots: non-target control; red connected dots: shPHGDH#20; deep blue connected dots: shPHGDH#32). f Knockdown of PHGDH augmented Sorafenib induced apoptosis in HCC cells (gray bar: vehicle-treated group; red bar: Sorafenib-treated group). g Knockdown of PHGDH sensitized HCC cell to Sorafenib in nude mice (gray connected dots: Vehicle-NTC, non-target control treated with vehicle; blue connected dots: Sora-NTC, non-target control treated with Sorafenib; red connected dots: Vehicle-shPHGDH, PHGDH knockdown clones treated with vehicle; purple connected dots: Sora-shPHGDH, PHGDH knockdown clones treated with Sorafenib). The error bar in panels b, c, d, f represents the standard error of mean (SEM), n = 3 biologically independent samples. The error bar in panel g represents the standard deviation (SD), n = 6 mice. Source data are provided as a Source Data file. (Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

Knockdown of PHGDH impaired serine biosynthesis and induced oxidative stress. a Knockdown of PHGDH impaired serine synthesis pathway (SSP), leading to accumulation of 3-phosphoglyceric acid (3-PG) and reduced production of nicotinamide adenine dinucleotide+hydrogen (NADH), α-ketoglutarate (α-KG), and serine. b Glycolytic metabolites cannot be branched into SSP upon knockdown of PHGDH, causing the accumulation of most glycolytic metabolites glucose 1-phosphate (G1P), glucose-6-phosphate (G6P), fructose 6-phosphate (F6P), fructose 1,6-bisphosphate (F1,6P), glyceraldehyde 3-phosphate (G3P), phosphoenolpyruvate (PEP), and pyruvate (gray bar: non-target control; red bar: PHGDH knockdown clones). c Knockdown of PHGDH resulted in reduced nicotinamide adenine dinucleotide phosphate (NADPH) production. d Sorafenib treatment augmented reactive oxygen species (ROS) level. Knockdown of PHGDH intensified the Sorafenib-induced oxidative stress in HCC cells (black line: non-target control treated with vehicle; light red line: non-target control treated with Sorafenib; blue line: PHGDH knockdown clones treated with vehicle; deep red line: PHGDH knockdown clones treated with Sorafenib). e H₂O₂ and tert-butyl hydroperoxide (tBHP) treatment at 20 and 200 μM for 24 h respectively induced PHGDH expression in HCC cells in both mRNA and protein level. f Knockdown of NRF2 (Nuclear factor erythroid 2-related factor 2) alleviated Sorafenib-induced PHGDH up-regulation in HCC cells in both mRNA and protein level. g Treatment of α-KG at 4 mM and NAC at 5 mM for 48 h substantially inhibited Sorafenib (5 μM) and NCT-503(40 μM)-induced apoptosis in HCC cells. The error bar represents SEM. n = 3 biologically independent samples in panels a–f and 5 in panel g. Source data are provided as a Source Data file (Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

PHGDH inhibitor sensitized HCC cells to Sorafenib treatment. a NCT-503 inhibited HCC cell proliferation in a dose-dependent manner with 50% growth inhibiting (GI50) at 50 µM. b Treatment of NCT-503 at 40 μM for 48 h significantly reduced the relative ratio of NAPDH/NAD+in HCC cells. c Annexin V-PI staining showed that NCT-503 worked synergistically with Sorafenib to induce HCC cell apoptosis. d Both NCT-503 and Sorafenib inhibited HCC tumorigenicity. NCT-503 and Sorafenib combination therapy effectively abolished HCC growth in the nude mice model (blue connected dots: vehicle-treated group; red connected dots: Sorafenib-treated group; green connected dots: NCT-503-treated group; purple connected dots: Sorafenib and NCT-503-treated group). The error bar in panels b and c represents the SEM, n = 3 biological independent samples. The error bar in panel d represents the SD, n = 6 mice. Source data are provided as a Source Data file (Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 6

PHGDH contributed to Regorafenib and Lenvatinib resistances in HCC. a Treatment of Regorafenib at 10 μM and Lenvatinib at 40 μM for 48 h induced PHGDH, PSAT1, and PSPH expression in HCC cells. b Co-treatment of NCT-503 intensified Regorafenib and Lenvatinib-induced apoptosis in HCC cells, which can be rescued by addition of 5 mM NAC of 48 h treatment. c In total, 2514 compounds are ranked by their ability of inducing SSP in nine cell lines. The Sorafenib-like small molecules (n = 52) were significantly enriched in the highly ranked SSP-inducible compounds as suggested by GSEA pre-ranked enrichment analysis. d Most of the Sorafenib-like small molecules profoundly induced the mRNA expression of PHGDH, PSAT1, and PSPH in all cell lines. The error bar represents the SEM, n = 3 biological independent samples. Source data are provided as a Source Data file. (Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001)