Detecting and exploiting amino acid vulnerabilities in cancer - PowerPoint PPT Presentation

Detecting and exploiting amino acid vulnerabilities in cancer Oncode Institute - NKI-AVL Reuven Agami Course Basic and Translational Oncology 2018

How to -specifically- kill cancer cells? Target DNA replication (chemotherapy) Suffocate them (inhibit signalling pathways) Re-activate the immune system (immunotherapy) ? Starve them to death

Amino acid usage Proteins poly-peptide cell Nucleotides proliferation Essential Lipids Non-essential Oxidative stress amino acids Epigenetics

Cancer Proteins poly-peptide cell Nucleotides proliferation Essential Lipids Oxidative stress Non-essential amino acids Epigenetics

Nature 1953

Nature 1961 L-Asparaginase Broome J.D. et al., Nature 1961

Since 1977 (introduction of high dose Asparaginase treatment) cure of childhood ALL was raised to >90% Resistance does occur: mostly related to re-expression of Asparagine synthetase (ASNS) in tumours

Diet/ Synthesis in liver ALL tumor cell Asparagine circulating in Asparagine blood Asparagine synthetase ASNS Asparaginase Glutamine Aspartic acid Aspartic acid

Asparagine Depletion affects BC metastasis 2018 Hannon and colleagues Nature 2018

Future » Sense bottlenecks in the demand of (solid) tumours » Exploit to identify cancer vulnerabilities

The problem amino acid vulnerability tumour vascularisation uptake use for mutant energy tRNA defect in use for How to sense amino acid shortage? production replication demand tRNA for protein synthetase synthesis

Ribosomes - the translation machinery protein subunits Ribosomal RNA David Goodsell, RCSB PDB ~ 4000 kDa

Ribosome profiling Ingolia NT et al Science 2009

DIfferential RIbosome COdon REading Cell state Cell state #1 #2 Potential amino acid shortage Loayza-Puch et al., Nature 2016

Sensing amino acid shortages in cancer State I (Normal tissue) State 2 (Tumor tissue)

Diricore analysis Position-based Codon-based analysis analysis Measure changes in codon usage Infer changes in amino acid related pathways Loayza. et al., Nature 2016

diricore treated/ctrl. 3-AT Evaluation 1 HIS Position-based b † analysis His 3-AT vs Control 10 abundance shift Sub-sequence Position 15 A P E A P E 5 0 -5 GCC GCG AGG CGC CGG GGC GGG CCG GCA GCT AGA CGA CGT GAC TGC CAG GAA GAG GGA GGT CTG AAG CCA CCC AGC AGT TCG ACA ACC ACG TGG GTG AAC TGT CAA CAC ATC CTC TTG AAA ATG TTC CCT TCA TCC ACT GTC AAT GAT CAT ATA CTA CTT TTT TCT TAC GTA GTT ATT TTA TAT A R N D C Q E G H I L K M F P S T WY V Codons Codons Codon-based d analysis abundance shift Sub-sequence CAC (His) CAT (His) 2 3-Amino-1,2,4-triazole 0 -2 TTC (Phe) TTT (Phe) 2 0 -2 -30 -15 0 15 30 -30 -15 0 15 30

d Evaluation 2 Sub-sequence abundance shift 4 -2 2 0 GCA GCC A A GCG GCT AGA Position-based AGG CGA R R CGC Harringtonine CGG analysis CGT AAC N N AAT GAC D C Q E D C Q E GAT TGC TGT CAA CAG GAA GAG GGA GGC G G GGG GGT CAC H I H I CAT ATA Codons Codons Codons ATC ATT CTA CTC CTG L L CTT TTA TTG AAA K K Met AAG M M ATG TTC F F TTT CCA † CCC P P CCG CCT AGC AGT Position 12 Control Harringtonine vs TCA S S TCC TCG TCT ACA ACC T WY T WY ACG ACT TGG TAC TAT GTA Cephalotaxus harringtonia GTC V V GTG GTT e Relative read density Harringtonine vs Control e -4 -4 0 4 0 4 -30 -15 Codon-based TTC (Phe) Position respect to codon ATG(Start) analysis 0 15 30 -30 -15 ATG (Other) TTT (Phe) 0 15 30

Evaluation 3 f Sub-sequence PC3 Cells abundance shift -0.5 0.5 0 1 GCA GCC A GCG GCT AGA AGG CGA R CGC CGG CGT Asn AAC N AAT GAC D C Q E GAT † TGC TGT CAA CAG GAA GAG GGA GGC G GGG GGT CAC H I CAT ATA Codons ATC ATT CTA CTC CTG L CTT TTA TTG AAA K AAG M ATG TTC F Position 15 ASNase vs Control TTT CCA CCC P CCG CCT AGC AGT TCA S TCC TCG h TCT ACA ACC Relative tRNA T WY ACG ACT TGG uncharging levels TAC TAT GTA GTC V 0 2 4 GTG GTT Asn-GTT Asn-ATT Leu-CAG Val-TAC ** un-charged tRNA levels Position respect to codon Relative read density ASNase vs Control ** -0.5 0.5 -0.5 0.5 0 0 -30 Position respect to codon -15 TTC (Phe) AAC (Asn) 0 15 ASNase Control 30 -30 -15 TTT (Phe) AAT (Asn) 0 15 30 g

logFC=−1.4, FDR=2.6e−08 Kidney Cancer tumor/normal Differential ribosome codon reading (Diricore) logFC=0.1, FDR=8.1e−01 Met T1 vs N2 1 M Pro Position 12 P 0.5 P P P A Q G R D D 0 R H R S R G G A Q T A H A K T W R G T E S R E S V C L L S S I N L L V I S T V V C N Y K Y L L F F I -0.5 GGG GCA GCC GCG AGG CGC CGG GAG GGA GGC CCG AGC ACG TGG GCT AGA CGA CGT AAC GAC TGC CAG GAA GGT CAC CTG AAG CCA CCC CCT AGT TCG ACA ACC ACT GTC GTG 1 GAT TGT CAA ATC CTC CTT TTG AAA ATG TTC TCA TCC TCT TAC GTA GTT AAT CAT ATA ATT CTA TTA TTT TAT A R N D C Q E G H I L K M F P S T WY V Codons Loayza. et al., Nature 2016 β-a

High production = Shortage High production Shortage Prolin production pathway

Poor survival of PYCR1 high cancers Overall survival: Kidney tumors PYCR1 low PYCR1 high

The importance of PYCR1 for tumor growth e Control KO1 KO3 T P PYCR1 9 5 1 - M f g β-a ctin U S g a c d f † CCA (Pro) CCC (Pro) 5 Control Pro Control Control Tumor 1 vs Cultured 1 1 y d 0.5 t abundance shift Position 15 PYCR1 expression i e PYCR1 KO1 Sub-sequence PYCR1 KO1 s PYCR1 KO1 4 r n 0 0.5 u e t PYCR1 KO3 PYCR1 KO3 d l 0.5 PYCR1 KO3 u ) 3 C d 3 0 400 m TGC (Cys) TGT (Cys) a s Cell proliferation e m Cell proliferation v 0.5 r 10000 300 2 10000 -0.5 ( 1 e 0 e v r 1000 m i u GCC GCG AGG CGC CGG GGA GGC GGG CCG AGC ACG TGG 1000 GCA GCT AGA CGA CGT AAC GAC TGC CAG GAA GAG GGT CAC CTC CTG AAG CCA CCC CCT AGT TCG ACA ACC ACT GTC GTG GAT TGT CAA ATC CTA CTT TTG AAA ATG TTC TCA TCC TCT TAC GTA GTT AAT CAT ATA ATT TTA TTT t 200 TAT 0.5 a 1 o u l m e 100 -30 -15 0 15 30 -30 -15 0 15 30 l A R N D C Q E G H I L K M F P S T WY V o 100 R u 100 V Position respect to codon Codons T 0 10 r *** 10 u ‡ o 0 1 Breast IDBC Tumor 2 vs Cultured 2 Pro m 1 CCA (Pro) CCC (Pro) 2 u y b Position 15 abundance shift 0.1 d 0.5 T Sub-sequence t 0.1 i 0 4 6 8 e 5 10 15 20 25 30 35 s 0.5 0 4 6 8 Days r n 0 eGFP RPL10a Days u e Days t d 0.5 l u h 0 d C h Gln (4mM) Gln (1mM) TGC (Cys) TGT (Cys) a s e v 0.5 -0.5 r * e 2 0 v * CGG GGG GCA GCC GCG AGG CGA CGC GAC CAG GAG GGA GGC GGT CCC CCG AGC ACC ACG TGG GCT AGA CGT AAC TGC TGT CAA GAA CAC CTC CTG AAA AAG ATG CCA CCT AGT TCC TCG ACA ACT GTC GTG AAT GAT CAT ATC CTA CTT TTG TTC TCA TCT TAC GTA GTT r ATA ATT TTA TTT TAT i u t 0.5 a o NS * * A R N D C Q E G H I L K M F P S T WY V l m e -30 -15 0 15 30 -30 -15 0 15 30 1.2 R u l Codons T Position respect to codon l e c h 0.8 e t Cultured 1 vs Cultured 2 2 CCA (Pro) w CCC (Pro) v y i o d t abundance shift Position 15 0.5 t a r Sub-sequence e i 0.4 NS g s l Pro r e 0.5 n u 0 R e t l u d 0.5 Cell Lysate Tumor Lysate 0 C 0 d Pro (10mM): - - + - - + - - + TGC (Cys) TGT (Cys) a s e v 0.5 r -0.5 Control PYCR1 KO1 PYCR1 KO3 1 RNAse I e GFP IP Ribo-Seq 0 d v GCG AGG CGG GGC GGG GCA GCC GCT AGA CGA CGC CGT GAC TGC CAG GAG GGA GGT CTG AAG CCA CCC CCG AGC AGT TCG ACA ACC ACG TGG GTG AAC GAT TGT CAA GAA CAC ATC CTC TTG AAA ATG TTC CCT TCA TCC ACT GTC GTT AAT CAT ATA ATT CTA CTT TTA TTT TCT TAC GTA digestion TAT i e i t 0.5 a r u Gln (4mM) Gln (1mM) l Gln (1mM) + Pro (4mM) A R N D C Q E G H I L K M F P S T WY V e -30 -15 0 15 30 -30 -15 0 15 30 t A l A R A u A A Diricore A Codons Position respect to codon A GFP- C A A * * A * * A A A A Trap A A A A 4 A A s A A A A Tumor 1 vs Tumor 2 l A A e A CCA (Pro) CCC (Pro) A A A A 2 v y 3 abundance shift Position 15 N e Sub-sequence 0.5 t NS r e i l l R u Pro o s 0.5 g r o n 0 1 t t 3 n 2 n O m e O e o i K d 0.5 g C v K u 0 i r T d t a a 1 T GTA (Val) a GTC (Val) h s l P e PYCR1 e c v -0.5 0.5 R r 9 - 1 n 0 e 5 u 0 1 v GGG r GCA GCC GCG AGG CGC CGG GAG GGA GGC CCG AGC ACG TGG GCT AGA CGA CGT AAC GAC TGC CAA CAG GAA GGT CAC CTC CTG AAG CCA CCC CCT AGT TCC TCG ACA ACC ACT GTC GTG GAT TGT ATC CTA CTT TTG AAA ATG TTC TCA TCT TAC GTA GTT AAT CAT ATA ATT TTA TTT TAT u i - t Control PYCR1 KO3 Control PYCR1 KO3 0.5 M o a �-actin L A R N D C Q E G H I K M F P S T WY V m l U e -30 -15 0 15 30 -30 -15 0 15 30 R u S Codons tRNA Pro (AGG) tRNA Pro (TGG) T Position respect to codon β-a