Targeting of Nucleotide-Binding Proteins by HAMLET - a Conserved Tumor Cell Death Mechanism

SUPPLEMENTAL INFORMATION

Targeting of nucleotide-binding proteins by HAMLET - a conserved tumor cell death mechanism

James Ho Chin Shing1,2, Aftab Nadeem1,2, Anna Rydström1, Manoj Puthia1, Catharina Svanborg1,*

1 Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden

*To whom correspondence should be addressed:

Prof. Dr. Catharina Svanborg, Phone: +46-709426549. Fax: +46-46137468.

E-mail:

2These authors contributed equally to this work.

Running Title: Kinome inhibition by HAMLET

SupplementaryTable S1. Kinases targeted by HAMLET, defined by an M1 > 10 (fold change with mean negative value control).

Gene Name / Gene Symbol / Fold change / Z-Score
discoidin domain receptor family, member 2 (DDR2), transcript variant 1 / DDR2 / 63.40 / 7.28
bone morphogenetic protein receptor, type IA (BMPR1A) / BMPR1A / 63.39 / 7.31
megakaryocyte-associated tyrosine kinase (MATK), transcript variant 2 / MATK / 61.97 / 7.01
spleen tyrosine kinase (SYK) / SYK / 61.76 / 6.34
v-raf murine sarcoma viral oncogene homolog B1 (BRAF) / BRAF / 61.02 / 6.31
epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) (EGFR), transcript variant 1; mutant protein:EGFR L861Q / EGFR L861Q / 56.86 / 6.42
dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 4 (DYRK4) / DYRK4 / 55.48 / 5.30
sterile alpha motif and leucine zipper containing kinase AZK (ZAK), transcript variant 1 / ZAK / 53.62 / 5.68
epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) (EGFR), transcript variant 1 / EGFR / 47.64 / 4.52
calcium/calmodulin-dependent protein kinase kinase 2, beta (CAMKK2), transcript variant 1 / CAMKK2 / 46.85 / 5.02
polo-like kinase 1 (Drosophila) (PLK1) / PLK1 / 44.95 / 4.06
v-raf-1 murine leukemia viral oncogene homolog 1 (RAF1) / RAF1 / 43.25 / 4.25
myosin light chain kinase 2, skeletal muscle (MYLK2) / MYLK2 / 38.26 / 4.18
WEE1 homolog (S. pombe) (WEE1) / WEE1 / 36.41 / 3.17
dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), transcript variant 1 / DYRK1A / 36.05 / 5.36
zeta-chain (TCR) associated protein kinase 70kDa (ZAP70) / ZAP70 / 35.11 / 3.26
spleen tyrosine kinase (SYK) / SYK / 34.06 / 2.95
NIMA (never in mitosis gene a)-related kinase 4 (NEK4) / NEK4 / 33.95 / 4.02
protein kinase C, iota (PRKCI) / PRKCI / 33.87 / 3.16
polo-like kinase 2 (Drosophila) (PLK2) / PLK2 / 33.75 / 2.92
MAP/microtubule affinity-regulating kinase 4 (MARK4) / MARK4 / 33.55 / 3.46
aurora kinase C (AURKC), transcript variant 1 / AURKC / 32.42 / 2.72
protein kinase N2 (PKN2) / PKN2 / 31.98 / 2.96
aurora kinase A (AURKA), transcript variant 2 / AURKA / 31.75 / 2.58
serum/glucocorticoid regulated kinase 2 (SGK2) / SGK2 / 31.43 / 2.92
protein kinase N1 (PKN1), transcript variant 2 / PKN1 / 31.21 / 3.25
feline sarcoma oncogene (FES) / FES / 30.72 / 2.72
protein kinase D3 (PRKD3) / PRKD3 / 30.40 / 2.56
mitogen-activated protein kinase kinase kinase 10 (MAP3K10) / MAP3K10 / 30.01 / 2.88
src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (SRMS) / SRMS / 29.99 / 2.51
v-akt murine thymoma viral oncogene homolog 1 (AKT1) / AKT1 / 29.87 / 2.64
testis-specific serine kinase 2 (TSSK2) / TSSK2 / 29.80 / 2.91
NIMA (never in mitosis gene a)-related kinase 3 (NEK3), transcript variant 1 / NEK3 / 29.67 / 3.65
muscle, skeletal, receptor tyrosine kinase (MUSK) / MUSK / 29.27 / 3.04
casein kinase 1, alpha 1 (CSNK1A1), transcript variant 1 / CSNK1A1 / 28.83 / 2.74
TTK protein kinase (TTK) / TTK / 28.59 / 2.88
serine/threonine kinase 25 (STE20 homolog, yeast) (STK25) / STK25 / 28.08 / 2.56
AXL receptor tyrosine kinase (AXL), transcript variant 2 / AXL / 27.44 / 2.57
Ephrin receptor A3 (EPHA3), transcript variant 1 / EPHA3 / 26.90 / 2.42
mitogen-activated protein kinase 11 (MAPK11), transcript variant 2 / MAPK11 / 26.65 / 2.51
NIMA (never in mitosis gene a)-related kinase 1 (NEK1) / NEK1 / 25.04 / 1.96
mitogen-activated protein kinase kinase kinase 4 (MAP3K4), transcript variant 1 / MAP3K4 / 24.69 / 2.24
serine/threonine kinase 3 (STE20 homolog, yeast) (STK3) / STK3 / 24.29 / 2.42
fms-related tyrosine kinase 4 (FLT4), transcript variant 2 / FLT4 / 24.27 / 2.00
PAS domain containing serine/threonine kinase (PASK) / PASK / 23.85 / 2.03
TANK-binding kinase 1 (TBK1) / TBK1 / 23.31 / 2.91
protein kinase, X-linked (PRKX) / PRKX / 23.27 / 2.09
serum/glucocorticoid regulated kinase 2 (SGK2), transcript variant 1 / SGK2 / 23.26 / 2.09
mitogen-activated protein kinase kinase kinase 9 (MAP3K9) / MAP3K9 / 23.05 / 2.07
v-akt murine thymoma viral oncogene homolog 1 (AKT1), transcript variant 1 / AKT1 / 22.71 / 2.26
death-associated protein kinase 2 (DAPK2) / DAPK2 / 22.32 / 2.02
phosphorylase kinase, gamma 1 (muscle) (PHKG1) / PHKG1 / 22.13 / 1.93
calcium/calmodulin-dependent protein kinase (CaM kinase) II delta (CAMK2D), transcript variant 3 / CAMK2D / 22.09 / 1.68
mitogen-activated protein kinase kinase kinase 3 (MAP3K3), transcript variant 2 / MAP3K3 / 21.95 / 1.61
fyn-related kinase (FRK) / FRK / 21.50 / 1.77
serine/threonine kinase 17b (STK17B) / STK17B / 21.48 / 1.90
activin A receptor, type IB (ACVR1B), transcript variant 1 / ACVR1B / 20.94 / 1.90
STE20-like kinase (yeast) (SLK) / SLK / 20.82 / 1.79
inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) / IKBKB / 20.80 / 2.15
mitogen-activated protein kinase kinase 3 (MAP2K3), transcript variant A / MAP2K3 / 20.73 / 1.99
anaplastic lymphoma kinase (Ki-1) (ALK) / ALK / 20.67 / 2.69
epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) (EGFR), transcript variant 1; mutant protein:EGFR L858R / EGFR L858R / 20.12 / 1.88
conserved helix-loop-helix ubiquitous kinase (CHUK) / CHUK / 20.05 / 1.56
protein kinase D1 (PRKD1) / PRKD1 / 19.54 / 1.60
NUAK family, SNF1-like kinase, 1 (NUAK1) / NUAK1 / 19.46 / 1.92
protein kinase C, epsilon (PRKCE) / PRKCE / 19.15 / 2.14
neurotrophic tyrosine kinase, receptor, type 3 (NTRK3), transcript variant 3 / NTRK3 / 18.76 / 1.48
v-abl Abelson murine leukemia viral oncogene homolog 1 (ABL1), transcript variant a; mutant protein:ABL1 G250E / ABL1 G250E / 18.64 / 1.41
mitogen-activated protein kinase kinase kinase 11 (MAP3K11) / MAP3K11 / 18.47 / 1.53
SCY1-like 1 (S. cerevisiae) (SCYL1) / SCYL1 / 18.31 / 1.29
insulin receptor-related receptor (INSRR) / INSRR / 18.10 / 1.40
serine/threonine kinase 31 (STK31), transcript variant 1 / STK31 / 17.60 / 1.50
v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), transcript variant 2 / ERBB2 / 17.20 / 1.42
cyclin-dependent kinase 2 (CDK2) / CDK2 / 17.17 / 1.26
ARP1 actin-related protein 1 homolog B, centractin beta (yeast) (ACTR1B) / ACTR1B / 16.91 / 1.28
serine/threonine kinase 17b (STK17B) / STK17B / 16.68 / 1.50
v-abl Abelson murine leukemia viral oncogene homolog 1 (ABL1), transcript variant a; mutant protein:ABL1 Y253F / ABL1 Y253F / 16.48 / 1.29
casein kinase 1, epsilon (CSNK1E), transcript variant 2 / CSNK1E / 16.23 / 1.16
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), transcript variant 1; mutant protein: KIT V654A / KIT V654A / 16.10 / 1.51
fer (fps/fes related) tyrosine kinase (phosphoprotein NCP94) (FER) / FER / 15.80 / 1.09
ribosomal protein S6 kinase, 70kDa, polypeptide 2 (RPS6KB2), transcript variant 2 / RPS6KB2 / 15.65 / 1.16
MAP kinase interacting serine/threonine kinase 1 (MKNK1) / MKNK1 / 15.57 / 1.22
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), transcript variant 1; mutant protein: KIT T670I / KIT T670I / 15.28 / 1.03
kinase insert domain receptor (a type III receptor tyrosine kinase) (KDR) / KDR / 14.93 / 1.18
v-abl Abelson murine leukemia viral oncogene homolog 1 (ABL1), transcript variant a / ABL1 / 14.88 / 1.11
tec protein tyrosine kinase (TEC) / TEC / 14.87 / 1.09
mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) / MAP4K2 / 14.70 / 0.89
platelet-derived growth factor receptor, alpha polypeptide (PDGFRA) / PDGFRA / 14.60 / 0.88
mitogen-activated protein kinase kinase kinase 8 (MAP3K8) / MAP3K8 / 14.59 / 1.08
platelet-derived growth factor receptor, alpha polypeptide (PDGFRA); mutant protein:PDGFRA T674I / PDGFRA T674I / 14.30 / 0.99
RIO kinase 3 (yeast) (RIOK3) / RIOK3 / 14.20 / 1.13
casein kinase 1, gamma 2 (CSNK1G2) / CSNK1G2 / 14.08 / 1.18
p21(CDKN1A)-activated kinase 4 (PAK4), transcript variant 1 / PAK4 / 13.85 / 0.92
serine/threonine kinase 24 (STE20 homolog, yeast) (STK24), transcript variant 2 / STK24 / 13.84 / 1.02
casein kinase 1, delta (CSNK1D), transcript variant 1 / CSNK1D / 13.79 / 0.91
interleukin-1 receptor-associated kinase 4 (IRAK4) / IRAK4 / 13.78 / 1.21
v-abl Abelson murine leukemia viral oncogene homolog 1 (ABL1), transcript variant a; mutant protein: ABL1 E255K / ABL1 E255K / 13.76 / 0.87
calcium/calmodulin-dependent protein kinase I (CAMK1) / CAMK1 / 13.69 / 0.90
glycogen synthase kinase 3 beta (GSK3B) / GSK3B / 13.62 / 0.72
cyclin-dependent kinase 3 (CDK3) / CDK3 / 13.57 / 1.03
protein kinase C, zeta (PRKCZ), transcript variant 1 / PRKCZ / 13.56 / 0.99
mitogen-activated protein kinase kinase 2 (MAP2K2) / MAP2K2 / 13.34 / 0.84
PTK2 protein tyrosine kinase 2 (PTK2), transcript variant 2 / PTK2 / 13.24 / 0.76
polo-like kinase 3 (Drosophila) (PLK3) / PLK3 / 13.17 / 0.89
tribbles homolog 1 (Drosophila) (TRIB1) / TRIB1 / 13.11 / 0.75
Ephrin receptor A2 (EPHA2) / EPHA2 / 12.79 / 0.78
serine/threonine kinase 40 (STK40) / STK40 / 12.68 / 0.76
NUAK family, SNF1-like kinase, 1 (NUAK1) / NUAK1 / 12.67 / 1.06
v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) (ERBB4), transcript variant JM-a/CVT-2 / ERBB4 / 12.33 / 0.89
misshapen-like kinase 1 (zebrafish) (MINK1), transcript variant 4 / MINK1 / 12.32 / 0.75
v-yes-1 Yamaguchi sarcoma viral related oncogene homolog (LYN) / LYN / 12.32 / 0.79
p21 (CDKN1A)-activated kinase 2 (PAK2) / PAK2 / 12.28 / 0.76
polo-like kinase 1 (Drosophila) (PLK1) / PLK1 / 12.08 / 0.72
zeta-chain (TCR) associated protein kinase 70kDa (ZAP70) / ZAP70 / 12.08 / 0.84
protein kinase C, theta (PRKCQ) / PRKCQ / 12.02 / 0.73
LIM domain kinase 1 (LIMK1), transcript variant dLIMK / LIMK1 / 11.90 / 0.78
v-ros UR2 sarcoma virus oncogene homolog 1 (avian) (ROS1) / ROS1 / 11.69 / 0.65
dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3 (DYRK3), transcript variant 2 / DYRK3 / 11.47 / 2.20
p21 (CDKN1A)-activated kinase 3 (PAK3) / PAK3 / 11.47 / 0.67
death-associated protein kinase 1 (DAPK1) / DAPK1 / 11.46 / 0.75
receptor tyrosine kinase-like orphan receptor 1 (ROR1), transcript variant 1 / ROR1 / 11.33 / 0.59
BMX non-receptor tyrosine kinase (BMX), transcript variant 2 / BMX / 11.16 / 0.72
ret proto-oncogene (RET), transcript variant 4 / RET / 10.94 / 0.63
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), transcript variant 1 / KIT / 10.86 / 0.71
protein kinase C, delta (PRKCD), transcript variant 1 / PRKCD / 10.60 / 0.55
aurora kinase C (AURKC), transcript variant 3 / AURKC / 10.57 / 0.59
neurotrophic tyrosine kinase, receptor, type 2 (NTRK2), transcript variant b / NTRK2 / 10.04 / 0.45

SupplementaryTable S2. Functional categories of nucleotide-binding proteins targeted by HAMLET in the protein array,defined by an M1 > 10 (fold change with mean negative value control)

ATP Binding Proteins / GTP Binding Proteins
Receptors / MAP Kinase Pathway / Mitotic proteins / Non-kinases / Ras superfamily
DDR2 / 74.20 / BRAF / 71.41 / EGFR / 55.76 / MCM7 / 56.95 / RASL11B / 147.44
EGFR / 55.76 / RAF1 / 50.61 / WEE1 / 42.61 / KIFC3 / 45.76 / RAB3C / 89.07
MUSK / 34.25 / ZAK / 41.08 / NEK4 / 39.74 / DDX17 / 40.47 / RAP1B / 71.67
AXL / 32.11 / MAP3K10 / 35.12 / PLK2 / 39.49 / UBE2O / 36.1 / RHEBL1 / 60.06
EPHA3 / 31.48 / AKT1 / 34.96 / MARK4 / 39.27 / TBK1 / 27.28 / RASL12 / 53.44
ALK / 24.19 / STK25 / 32.86 / MAPRE2 / 39.21 / WARS / 24.5 / ARL5A / 49.92
NTRK3 / 21.95 / MAPK11 / 31.19 / AURKA / 37.15 / HSPD1 / 23.37 / RAB43 / 47.45
INSRR / 21.18 / MAP3K4 / 28.89 / AKT1 / 34.96 / INSRR / 21.18 / RAB28 / 34.96
KDR / 17.47 / STK3 / 28.42 / NEK3 / 34.72 / KIF2C / 20.16 / RASL10B / 34.96
PDGFRA / 17.08 / PRKX / 27.23 / TTK / 33.45 / ACTR1B / 19.78 / RAB20 / 33.75
PTK2 / 15.49 / MAP3K9 / 26.97 / NEK1 / 29.3 / TOP1 / 17.14 / RAB39 / 28.84
EPHA2 / 14.96 / MAP3K3 / 25.69 / CAMK2D / 25.85 / UBE2M / 16.35 / RAB2B / 28.63
ROS1 / 13.68 / IKBKB / 24.34 / ACVR1B / 24.5 / KIF26A / 16.05 / ARL1 / 26.99
ROR1 / 13.25 / MAP2K3 / 24.26 / MAP3K11 / 21.62 / TRIB1 / 15.34 / RRAGA / 24.98
NTRK2 / 11.75 / MAP3K11 / 21.62 / KIF2C / 20.16 / ERCC6L / 14.92 / RAB7L1 / 24.72
EPHA4 / 10.89 / STK31 / 20.59 / CDK2 / 20.1 / ITM2C / 14.6 / RAB40B / 21.85
STK17B / 19.52 / ABL1 / 17.41 / G3BP1 / 12.55 / RND2 / 20.7
MAP4K2 / 17.2 / CDK3 / 15.88 / TAF9 / 12.35 / RAB14 / 19.59
MAP3K8 / 17.07 / PLK1 / 14.14 / CLP1 / 11.02 / RAB1B / 19.1
STK24 / 16.2 / PSMC4 / 11.88 / RRAGB / 18.66
MAP2K2 / 15.61 / AURKB / 10.05 / RAB33A / 17.88
STK40 / 14.84 / RAB10 / 16.07
HRAS / 15.67
RABL2B / 12.19
RRAGC / 11.44
RAB2A / 11.4
GIMAP6 / 10.47

SupplementaryTable S3. Properties of Ras-related proteins: Z-scores (ProtoArray), amino acid sequences (lipidation site) and subcellular localization.

Protein / Z-Score / Amino acid sequence / Cellular localization
RasL11B / 13.32 / MRLIQNMCTI……FKQALSAKVRTVTSV / Membranea
Rab3C / 7.73 / MRHEAPMQMA……RLKETPPPPQPNCAC / Membrane (plasmab, synapticc,), cytosolc,
Rap1B / 7.19 / MREYKLVVLG……PVPGKARKKSSCQLL / Membranea,e, cytosolb, lipid particleb
RhebL1 / 5.67 / MPLVRYRKVV……RVENSYGQERRCHLM / Membranea, b, cytoplasb
Rasgrp3 / 4.39 / MGSSGLGKAA……DQDEGEETRQDGEDG / Membrane (plasma)b, cytosola
RasL12 / 4.38 / MSSVFGKPRA……RKAPTLTLLKGFKIF / Membranea
Rab43 / 4.17 / MAGPGPGPGD……QLNSKDIGEGWGCGC / Membrane (Golgi, phagocytic vesicle)b
Arfgap3 / 4.32 / MGDPSKQDIL……FANGVVTSIQDRYGS / Membrane (Golgib), cytosolb
Arl5A / 4.31 / MGILFTRIWR……LCQGLEWMMSRLKIR / Intracellulara
RRAGA / 2.08 / MPNTAMKKKV……KLERVDGPKHSLLMR / Nucleus, cytoplasma, Golgid
Rab28 / 2.88 / MSDSEEESQD……SRTVNPPRSSMCAVQ / Membraneb
Rab20 / 2.54 / MRKPDSKIVL……SSHKPPKRTRSGCCA / Membrane (Golgi, phagocytic vesicle)b
Rab39 / 2.15 / METIWIYQFR……HSSEEAVKPRKECFC / Membrane (plasma, phagocytic vesicle)b
RasL10B / 2.95 / MVSTYRVAVL……LRFQGALRRNRCAIM / Membranea, b
Rab2B / 2.95 / MTYAYLFKYI……SQRNSRDIGSNSGCC / Membrane (plasma, Golgi, ER)b
Arl1 / 2.09 / MGGFFSSIFS……DEAMEWLVETLKSRQ / Membrane (Golgi, trans-Golgi network)b
aInterPro
bUniProtKB
cCompara
dHPA
eReactome

Supplementary Figure S1. Correlation of Z-score (M3) to fold change (M2).

SupplementaryFigure S2. Pilot study of HAMLET’s effect on kinase activity. (A) Dose-dependent inhibition of kinases by HAMLET (EPHA3, EGFR, ALK3/BMPR1A and Braf). (B) Inhibition by Staurosporine of EPHA3, EGFR, ALK3/BMPR1A and NEK3. (C) Lack of correlation between the effects of HAMLET and Staurosporine. (D) Lack of synergy between HAMLET and Staurosporin. Cell death was measured as cellular ATP levels and Prestoblue fluorescence.All data are represented as Mean±S.D. of three independent experiments.

SupplementaryFigure S3. Effects of HAMLET on in vivo phosphorylation. (A) Effects of HAMLET on kinase phosphorylation activity. About 17 % of the substrates show a decrease in phosphorylation (n = 57) and 22 % show an increase (n = 76). For the remaining substrates (61 %), the phosphorylation level is unchanged. (B) Proteins with changed phosphorylation levels in response to HAMLET were evaluated using kinase-to-phosphorylation-site relationships from a recently constructed activity-based phosphorylation network in human cells. Limited numbers of connections (blue lines) were found among the proteins, indicating that the proteins were involved in distinct signaling cascades (average number of neighbors = 0.6). Red dots, kinases; green dots, kinase substrates.

SupplementaryFigure S4. HAMLET perturbs cancer-related signaling cascades. By pathway analysis, HAMLET showed effects on multiple targets in the ‘molecular mechanism of cancer’ network. Inhibited kinases were predicted to affect the PI3K/Ras/Raf/MEK/ERK-, PI3K/AKT-, p38 MAPK- and JNK- pathways. Pathways involving CTNN b, SMAD and Rb were activated.

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