University of Groningen Kinome directed target discovery and validation in unique ovarian clear cell carcinoma models Caumanns, Joost

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University of Groningen

Kinome directed target discovery and validation in unique ovarian clear cell carcinoma models

Caumanns, Joost

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Publication date: 2019

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Caumanns, J. (2019). Kinome directed target discovery and validation in unique ovarian clear cell carcinoma models. University of Groningen.

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526956-L-sub01-bw-Caumanns 526956-L-sub01-bw-Caumanns 526956-L-sub01-bw-Caumanns 526956-L-sub01-bw-Caumanns Processed on: 7-12-2018 Processed on: 7-12-2018 Processed on: 7-12-2018

Processed on: 7-12-2018 PDF page: 97PDF page: 97PDF page: 97PDF page: 97

CHAPTER 5

Low-dose triple drug combination

targeting the PI3K/AKT/mTOR pathway and

WKH0$3.SDWKZD\LVDKLJKO\HႇHFWLYH

DSSURDFKLQRYDULDQFOHDUFHOOFDUFLQRPD

Joseph J. Caumanns

1

_{, Anne van Wijngaarden}

1,2

_{, Arjan Kol}

1

_{, Gert J.}

Meersma

1

_{, Mathilde Jalving}

2

_{, René Bernards}

3

_{, Ate G.J. van der Zee}

1

_,

G. Bea A. Wisman

1*

_{and Steven de Jong}

2*

1_{Department of Gynecologic Oncology and}2_{Department of Medical Oncology,}

Cancer Research Centre Groningen, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, the Netherlands.

3_{Division of Molecular Carcinogenesis and Oncode Institute, the Netherlands}

Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. *The last two authors share senior authorship

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98

INTRODUCTION

Ovarian clear cell carcinoma (OCCC) is the second most common epithelial ovarian cancer subtype. Advanced stage OCCC is chemo-resistant and has a worse overall survival compared to the most common ovarian cancer subtype (high-grade serous) (1). There is an urgent need for targeted treatment strategies. Recent genomic analysis uncovered a heterogeneous mutation- and copy number alteration (CNA) spectrum in OCCC. DNA binding AT-rich interactive domain 1A gene (ARID1A), a key subunit of the SWI-SNF chromatin remodeling complex, is deleteriously mutated in 40-57% of OCCC tumors (2-4). This is the highest frequency

NOVELTY AND IMPACT

The unresponsiveness of advanced stage ovarian clear cell carcinoma (OCCC) to platinum-based chemotherapy urges development of alternative treatment strategies. The broad spectrum of JHQHWLF DOWHUDWLRQV LQ 3,.$.7P725 and MAPK pathways prompted the authors to evaluate a strategy combining 3,.P725&DQG0(.LQKLELWLRQ at sub-optimal doses. This combination HႇHFWLYHO\ LQKLELWHG SUROLIHUDWLRQ LQ OCCC cell lines and patient-derived xenografts, and prevented feedback DFWLYDWLRQ RI NH\ 3,.$.7P725 DQG MAPK kinases, demonstrating the potential of this approach for treatment of OCCC patients.

Low-dose triple drug combination targeting the PI3K/AKT/

mTOR pathway and the MAPK pathway is a highly effective

approach in ovarian clear cell carcinoma

Advanced stage ovarian clear cell carcinoma (OCCC) is poorly responsive to platinum-based chemotherapy and has an unfavorable prognosis. Previous studies revealed heterogeneous mutations in PI3K/AKT/mTOR and MAPK pathway nodules converging in mTORC1/2 activation. Here, we aimed to identify an effective low-dose combination of PI3K/AKT/mTOR and MAPK kinase inhibitors to simultaneously target key kinases in OCCC. Small molecule inhibitors of mTORC1/2 (AZD8055), PI3K (GDC0941) and MEK1/2 (selumetinib), were combined at monotherapy IC20 doses in a panel

of genetically diverse OCCC cell lines (n=7) to determine an optimal low-dose combination. IC20 combinations of AZD8055, GDC0941 and selumetinib

effectively inhibited proliferation in all seven cell lines. The triple combination reduced kinase activity in PI3K/AKT/mTOR and MAPK pathways, prevented single inhibitor induced feedback mechanisms and inhibited short- and long-term proliferation. Finally, this low-dose triple drug combination treatment VLJQLÀFDQWO\UHGXFHGWXPRUJURZWKLQWZRJHQHWLFDOO\FKDUDFWHUL]HG2&&& patient-derived xenograft (PDX) models without resulting in weight loss in these mice. In conclusion, PI3K, mTORC1/2 and MEK1/2 inhibitors combined at low-dose reduced proliferation of OCCC both in vitro and in vivo. The effectiveness and tolerability of this combined therapy in PDX models warrants clinical exploration of this treatment strategy for OCCC.

Joseph J. Caumanns, Anne van Wijngaarden, Arjan Kol, Gert J. Meersma, Mathilde Jalving, René Bernards, Ate G.J. van der Zee, G. Bea A. Wisman and Steven de Jong

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Processed on: 7-12-2018 PDF page: 99PDF page: 99PDF page: 99PDF page: 99 EFFECTIVE LOW-DOSE DRUG COMBINATIONS IN OCCC

5

99 JHQRPLFODQGVFDSHRI3,.$.7P725 and MAPK pathways in OCCC patients and cell lines and demonstrated high HႈFDF\ RI P725& LQKLELWLRQ LQ YLWUR and in vivo (4), we hypothesized that in 2&&& WKH DGGLWLRQ RI 3,. RU 0(. LQKLELWRUV WR P725& WDUJHWLQJ agents may further prevent feedback

mechanisms and cross-activation.

,QGHHG GXDO 3,.P725 LQKLELWRUV e.g. BEZ235 and XL765, exhibit high potency in in vitro models including ovarian cancer. Unfortunately, severe

3,.P725& LQKLELWRU UHODWHG

toxicity, especially hyperglycemia and gastrointestinal toxicity, was observed DW HႇHFWLYH GRVHV LQ FDQFHU SDWLHQWV (19-22). This may well be related to WKH LPSRUWDQW UROH RI 3,.$.7P725 and MAPK pathway signaling in normal tissue. These observations suggest a careful titration of combined single targeted inhibitors to search for optimal WXPRUHႈFDF\ZLWKDFFHSWDEOHV\VWHPLF toxicity.

)LUVWO\ ZH LQYHVWLJDWHG WKH HႈFDF\

RI 3,.$.7P725 SDWKZD\ DQG MAPK pathway inhibitors in a panel RI 2&&& FHOO OLQHV WKDW UHÀHFWV WKH genetic diversity of OCCC. We tested if low concentrations of the inhibitors demonstrated potentiating interactions in OCCC cell lines. Next, two established OCCC patient-derived xenograft (PDX) models were treated with low-doses RI WKH PRVW HႇHFWLYH LQ YLWUR WULSOH GUXJ combination, i.e. concentrations far below the maximum tolerated doses of single agents in mice, to demonstrate antitumor activity while minimizing systemic toxicity in these mice.

RESULTS

AZD8055, GDC0941 and selumetinib susceptibility in OCCC cell lines

7R HYDOXDWH VHQVLWLYLW\ WR P725& 3,. DQG 0(. LQKLELWRUV D SDQHO RI seven OCCC cell lines was selected that among all cancer types and strategies

VSHFL¿FDOO\ WDUJHWLQJ ARID1A mutated OCCC are being explored extensively 7KH PRVW IUHTXHQW 3,.$.7 mTOR related alterations are found in the PI3K catalytic domain encoding subunit PIK3CA, which is mutated in 30-40% of OCCC, and the PI3K antagonist

PTEN, with loss of expression in 40%

of OCCC (3,4,7,8). Other alterations include deleterious mutations in the PI3K regulatory subunit PIK3R1 and DPSOL¿FDWLRQV DQG PXWDWLRQV LQ WKH 3,. GRZQVWUHDP HႇHFWRUV AKT1 and

AKT2 (4,9-11). MAPK related alterations

are primarily found in the oncogene

KRAS, which is mutated in 5-14% and

DPSOL¿HGLQRI2&&& Occasionally, NRAS and BRAF mutations have been found (4,7,9,10). Additionally, ERBB family of receptor tyrosine NLQDVHV DPSOL¿FDWLRQV EGFR, ERBB2 and ERBB3) and mutations (EGFR,

ERBB2, ERBB3 and ERBB4) have

been described in OCCC (4,9,10,13,14). Unfortunately, EGFR and EGFR-ERBB2 GXDO LQKLELWRUV GLG QRW VKRZ HႈFDF\ in pretreated non-subtype selected ovarian cancer patients (15-17). PI3K, LWV HႇHFWRU $.7 DQG WKH GRZQVWUHDP P725& DUH DWWUDFWLYH WDUJHWV IRU treatment given the high frequency of mutations present in regulatory proteins DQG NLQDVHV RI WKH 3,.$.7P725 pathway in OCCC. Similarly, inhibition of VLJQDOLQJ QRGHV VXFK DV 0(. FRXOG be of interest considering the substantial amount of MAPK pathway alterations in OCCC. Single targeted inhibition of 3,.P725&RU0(.FDQUHVXOW LQ UHDFWLYDWLRQ RI 3,.$.7P725 RU MAPK signaling via negative feedback

mechanisms. Moreover, cross-talk

EHWZHHQ WKH 3,.$.7P725 SDWKZD\ and the MAPK pathway induces cross-activation, complicating sustainable inhibition of both proliferation pathways (18).

Based on our recent report, in which we described the heterogeneous

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100

(selumetinib) using a short-term (96 h) viability assay. All cell lines revealed nanomolar range AZD8055 sensitivity (Fig. 1C). GDC0941 and selumetinib sensitivity varied more widely across the cell lines (Fig. 1D-E). Gene alteration and protein phosphorylation status (Fig. 1A-B) did not correlate with sensitivity for these inhibitors (Mann-Whitney U test, data not shown).

6\QHUJLVWLFDQWLSUROLIHUDWLYHH௺HFWVRI,&₂₀ AZD8055, GDC0941 and selumetinib combinations

Considering the high AZD8055

susceptibility in OCCC cell lines, we searched for additive or synergistic HႇHFWV RI$=' LQ FRPELQDWLRQ ZLWK GDC0941 and selumetinib. The inhibitors ZHUH FRPELQHG DW FHOO OLQH VSHFL¿F ,&₂₀ concentrations calculated from short-term viability assay results (Fig. 2A). largely resembles the genetic makeup

of OCCC. We recently characterized 3,.$.7P725 SDWKZD\ DQG 0$3. pathway alterations in these cell lines

(4). Alterations include activating

KRWVSRW PXWDWLRQV LQ WKH 3,.$.7 mTOR pathway signaling node PIK3CA,

deleterious mutations in PTEN,

DPSOL¿FDWLRQVLQAKT1 and AKT2 and a hotspot mutation in the MAPK pathway signaling node KRAS (Fig. 1A). Western blot analysis demonstrated universal H[SUHVVLRQ RI P725& GRZQVWUHDP target p-S6, indicating mTOR activity in all seven OCCC cell lines. Activity of PI3K (p-AKT308_{and p-AKT}473_{) and}

MAPK (p-ERK) pathways, upstream of P725&YDULHGDFURVVWKHFHOOOLQHV depending on their mutational status (Fig. 1B). We subsequently determined

VHQVLWLYLW\ WRZDUGV P725&

LQKLELWLRQ $=' 3,.Įį LQKLELWLRQ *'& DQG 0(. LQKLELWLRQ AZD8055 0 0.01 0.1 1 10 0 20 40 60 80 100 120 (μM) Su rv iv al (%) C GDC0941 0 0.1 1 10 0 20 40 60 80 100 120 (μM) Su rv iv al (%) D Selumetinib 0 0.1 1 10 0 20 40 60 80 100 120 ES2 HAC2 JHOC5 KOC7C RMG1 SMOV2 OVMANA (μM) Su rv iv al (%) E A PIK3CA ES2 HAC2 JHOC5 KOC7C RMG1 SMOV2 OVMANA Cell line Alteration

AKT1 AKT2 PIK3R1 PTEN NRAS BRAF

wt wt CNgain wt CNgain wt CNgain wt wt CNgain wt CNgain CNgain CNgain wt wt CNloss Q5R wt wt wt wt wt wt 233*, 323fs wt wt wt wt wt wt wt wt CNgain wt V600E CNgain wt wt wt wt wt wt H1047R wt K111N wt H1047L E545V B pAkt308 pErk pAkt473 pS6 p4ebp1 ȕActin ES2 HAC2 JHOC 5 KOC7 C RMG1 SMOV2 OVMA NA

Figure 1 | Determination of monotherapy sensitivity in seven OCCC cell lines. (A) Mutations and

&1$VLQ3,.$.7P725SDWKZD\DQG0$3.SDWKZD\VLJQDOLQJQRGHVDFURVVVHYHQ2&&&FHOOOLQHV (4). * indicates stop-gained alteration, fs indicates frameshift alteration. (B) Expression of p-AKT308_, p-AKT473_{S(5.S6DQGS(%3LQWKH2&&&FHOOOLQHSDQHOGHWHUPLQHGE\:HVWHUQEORWȕ$FWLQ} was used as loading control. Methyl thiazolyl tetrazolium (MTT) assays with AZD8055 (C), GDC0941

(D) and selumetinib (E) in the OCCC cell line panel. Error bars indicate SD and are derived from three

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101 2D). These results indicate that combined treatment is essential to prevent single treatment induced pathway cross-activation. Interestingly, a near-universal reduction of p-AKT308_{, p-AKT}473_{, and}

p-ERK was observed with AGS resulting in a downstream reduction of p-S6 and p-4EBP1 across all our OCCC cell lines (Fig. 2D). MLN0128 in combination with GDC0941 and selumetinib demonstrated similar results (Supplementary Fig. 1C).

In conclusion, synergistic proliferation inhibition was observed with AGS in ¿YH 2&&& FHOO OLQHV DQG DQ DGGLWLYH HႇHFWLQWZRRWKHU2&&&FHOOOLQHV7KLV observation was supported by long-term proliferation assays and extensive downregulation of phosphorylated target SURWHLQVRIWKH3,.$.7P725SDWKZD\ and MAPK pathway.

ABT-737 sensitizes OCCC cells to apoptosis induction by AGS

Although AGS strongly reduced

SUROLIHUDWLRQ QR VLJQL¿FDQW DSRSWRVLV induction was observed, as determined

by DiIC₁SURSLGLXP LRGLGH ÀRZ

cytometry analysis (Fig. 3C). Next, we investigated whether AGS sensitized OCCC cells to cisplatin, since platinum-based chemotherapy is still the standard of care for OCCC patients, despite its OLPLWHGHႈFDF\$*6GLGQRWHQKDQFH proliferation inhibition and apoptosis induction by cisplatin, suggesting that the mechanisms through which AGS and FLVSODWLQDFWGRQRWEHQH¿FLDOO\LQWHUDFWLQ OCCC (Supplementary Fig. 2A-B).

Finally, we investigated whether

apoptosis inducing agents could

sensitize OCCC cells towards AGS, taking into account that 89% of OCCC tumors are TP53 wild-type (4,23,24). 7RWKLVHQGWKHHႈFDF\RI$%7DQ inhibitor of the anti-apoptotic proteins

Bcl-2, Bcl-X_L and Bcl-w (25), was

evaluated in our cell line panel. ABT-737 treatment inhibited proliferation and WKLV HႇHFW ZDV DGGLWLYH WR WKH HႇHFW RI The combination of AZD8055 and

*'& LQGXFHG V\QHUJLVWLF HႇHFWV (CI < 0.75) in two cell lines and AZD8055 in combination with selumetinib in three cell lines (Fig. 2B, Supplementary Fig. 1A). Interestingly, the IC₂₀ combination of AZD8055, GDC0941 and selumetinib (from here on referred to as AGS),

demonstrated strongly synergistic

proliferation inhibition (CI < 0.25) of JHOC5, KOC7C and RMG1 cells and synergistic proliferation inhibition (CI RI (6 DQG 6029 FHOOV 1R V\QHUJLVWLFHႇHFWZDVREVHUYHGLQ+$& DQG 290$1$ FHOOV ZKLFK PLJKW EH related to their initial high sensitivity to all three inhibitors.

Single inhibitor IC₂₀ treatment in long-term proliferation reduced growth in ES2 (GDC0941), HAC2 (AZD8055,

GDC0941), JHOC5 (AZD8055,

GDC0941), KOC7C (GDC0941) and 290$1$ VHOXPHWLQLE )LJ & 7KH combination of AZD8055 with either

GDC0941 or selumetinib caused

DGGLWLRQDO JURZWK LQKLELWLRQ LQ 6029 Importantly, AGS induced a persistent growth reduction in all cell lines, FRQ¿UPLQJWKHUHVXOWVLQVKRUWWHUPDVVD\V (Fig. 2C). MLN0128 (sapanisertib), DQRWKHU P725& LQKLELWRU FXUUHQWO\ in clinical development, reduced growth in combination with GDC0941 and selumetinib at IC₂₀concentrations as well (Supplementary Fig. 1B). The inhibitory HႇHFW RI $*6 ZDV OHVV SURIRXQG LQ immortalized human retinal epithelial RPE1 cells, when treated with AGS at intermediate and high concentrations (Fig. 2C). At the molecular level, AZD8055 treatment led to upregulation of either p-ERK, p-AKT308_{or p-AKT}473

or combinations of these responses in HAC2, JHOC5, KOC7C, RMG1 and 6029 FHOOV 0RUHRYHU *'&

treatment induced p-ERK or p-AKT473

in three cell lines (JHOC5, KOC7C and RMG1) while selumetinib induced

p-AKT473_{in four cell lines (JHOC5,}

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102 No synergy CI < 0.75 CI < 0.25 AZD8055 GDC0941 Selumetinib KOC7C RMG1 JHOC5 HAC2 SMOV2 ES2 OVMANA X X -X -X X X X B C AZD8055 GDC0941 Selumetinib X -KOC7C RMG1 JHOC5 HAC2 SMOV2 ES2 OVMANA -X -X -X -X X X -X X X RPE1 (25%) OVMANA X X X X X X pAkt308 pErk pAkt473 pS6 p4ebp1 ȕActin AZD8055 GDC0941 Selumetinib D ES2 HAC2 X X X X X X X X X X X X pAkt308 pErk pAkt473 pS6 p4ebp1 ȕ$FWLQ AZD8055 GDC0941 Selumetinib JHOC5 KOC7C X X X X X X X X X X X X pAkt308 pErk pAkt473 pS6 p4ebp1 ȕActin AZD8055 GDC0941 Selumetinib RMG1 SMOV2 X X X X X X X X X X X X pAkt308 pErk pAkt473 pS6 p4ebp1 ȕActin AZD8055 GDC0941 Selumetinib IC20 <100 nM IC20 100-1000 nM IC20 >1000 nM KOC7C RMG1 JHOC5 HAC2 SMOV2 ES2 OVMANA AZD8055 mTORC1/2 5 7.5 20 2.5 7.5 5 5 Selumetinib MEK1/2 N.A. 25 490 15 40 50 115 GDC0941 2180 60 675 20 225 2160 50 3,.Įį A RPE1 (75%)

Figure 2 | Synergystic IC₂₀ FRPELQDWLRQ HႇHFWV RI$=' *'& DQG VHOXPHWLQLE $ IC₂₀

concentrations as determined from MTT assay data. KOC7C IC₂₀ for selumetinib could not be determined ZLWKLQWKHXVHGFRQFHQWUDWLRQUDQJH$FFRUGLQJO\.2&&ZDVWUHDWHGZLWKȝ0VHOXPHWLQLELQVXEVH-quent experiments. (B) Synergy determination of AZD8055, GDC0941 and selumetinib IC₂₀ combinations in the OCCC cell line panel with the Talalay–Chou method (24). Combinations were regarded synergistic when Combination Index (CI) < 0.75 or < 0.25 was observed in at least two out of three MTT assay ex-periments. (C)$*6SUROLIHUDWLRQLQKLELWRU\HႇHFWVZHUHPHDVXUHGLQDORQJWHUPFRORQ\IRUPDWLRQDVVD\ RPE1 cells were treated with 25% (5 nM AZD8055, 540 nM GDC0941 and 119 nM selumetinib) and 75% Q0$='Q0*'&DQGQ0VHOXPHWLQLERIWKHGLႇHUHQFHEHWZHHQWKHORZHVWDQG highest IC₂₀ from our OCCC cell line panel. Results shown are representative of two experiments (D) Ex-pression of p-AKT308_{, p-AKT}473_{, p-ERK, p-S6 and p-4EBP1 after 48 h of treatment with IC}

20 concentration RI$='*'&VHOXPHWLQLERU$*6DVGHWHUPLQHGE\:HVWHUQEORWȕ$FWLQZDVXVHGDVORDGLQJ control. Results shown are representative of three experiments.

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103 other cell lines (HAC2 and KOC7C) (Fig. 3C and Supplementary Fig. 2D). This observation was supported by an increase in the apoptotic markers Cleaved PARP and Cleaved Caspase-3 (Supplementary Fig. 2E).

AGS treatment on proliferation of all cell lines, with ES2 being the exception (Fig. 3A and Supplementary Fig. 2C). AGS in combination with ABT-737 strongly HQKDQFHG DSRSWRVLV LQ 6029 FHOOV compared to AGS or ABT-737 single treatment (Fig. 3B-C). The combination

VLJQL¿FDQWO\ HQKDQFHG DSRSWRVLV

compared to single treatments in two B Propidium iodide 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Control AGS DilC 1 (5) 91.3% 0.7% 2.7% 5.4% 68.3% 1.8% 3.4% 26.4% 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 ABT-737 Combination 62.4% 1.7% 9.1% 26.8% 13.7% 7.3% 3.3% 75.7% A 0 0.1 1 10 (μM) 0 20 40 60 80 100 120 Su rv iv al (%) ABT-737 ES2 HAC2 JHOC5 KOC7C RMG1 SMOV2 OVMANA AGS + ABT-737 0 0.1 1 10 ABT737 (μM) 0 20 40 60 80 100 N orm al iz ed s urv iv al ( % ) 120 140

C HAC2 SMOV2 OVMANA

ES2 JHOC5 KOC7C RMG1

0 20 40 60 80 100 D IL C 5-/P I+ (% ) Control AGS ABT-737 Combination 0 20 40 60 80 100 D IL C 5-/P I+ (% ) 0 20 40 60 80 100 D IL C 5-/P I+ (% ) 0 20 40 60 80 100 D IL C 5-/P I+ (% ) 0 20 40 60 80 100 D IL C 5-/P I+ (% ) ** ** ** * ** ** * 0 20 40 60 80 100 D IL C 5-/P I+ (% ) **** **** **** **** 0 20 40 60 80 100 D IL C 5-/P I+ (% ) * * * * * * ** * ** * * ES2 HAC2 JHOC5 KOC7C RMG1 SMOV2 OVMANA

Figure 3 | ABT-737 added to IC₂₀ AZD8055, GDC0941 and selumetinib combination. (A) Sensitivity

of ABT-737 and ABT-737 with AGS (normalized to AGS) as determined by MTT assay. Error bars indicate SD and are derived from two experiments. Flow cytometry plots from DiIC₁3,ÀRZF\WRPHWU\DQDO\VLV RI6029(B)DIWHUKWUHDWPHQWZLWK$*6ȝ0$%7RUWKHFRPELQDWLRQ(C)4XDQWL¿FDWLRQRIWKH bottom two quadrants (DiIC₁3,DQG'L,&₁3,IURP'L,&₁3,ÀRZF\WRPHWU\DQDO\VLVDIWHU KWUHDWPHQWZLWK$*6$%7RUWKHFRPELQDWLRQLQWKHFHOOOLQHSDQHO+$&6029DQG290$1$ ZHUHWUHDWHGZLWKȝ0$%7(6-+2&.2&&DQG50*ZHUHWUHDWHGZLWKȝ0$%7 Error bars indicate SD and are derived from three experiments. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001 and **** indicates p<0.0001.

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104

Low-dose AZD8055, GDC0941 and VHOXPHWLQLE FRPELQDWLRQ LV H௺HFWLYH LQ OCCC PDX models

1H[WZHH[SORUHGWKHHႇHFWRIFRPELQLQJ AZD8055, GDC0941 and selumetinib at low-dose in vivo in an unbiased manner by using two unique OCCC PDX models. Both PDX.180 and PDX.247 tumors KDYH 3,.$.7P725 SDWKZD\ UHODWHG alterations (Fig. 4A). To mimic the IC₂₀ concentrations used in vitro, dosing for each drug was set at 20% of the maximum

dose daily used for monotherapy in mice, as described previously (26-28). Growth inhibition was observed in AZD8055, GDC0941 and selumetinib single treatment arms as compared to vehicle. 7KH HႇHFW RI PRQRWKHUDS\ WUHDWPHQW EHFDPHVLJQL¿FDQWDWGD\LQERWK3'; models (Supplementary Fig. 3A). AGS low-dose combination treatment caused VLJQL¿FDQWWXPRUUHJUHVVLRQLQ3'; DQG VLJQL¿FDQWO\ UHGXFHG WXPRU JURZWK in PDX.247 bearing mice (Fig. 4B-C). Importantly, weight of the mice remained

A PDX.180 PIK3CA AKT2 Alteration PDX.247 wt CNgain K111E CNgain PIK3R2 CNgain wt 0 5 10 15 20 25 30 35 We ig h t ( g ) PDX.180 mouse weight 0 2 4 6 8 10 12 14 16 18 20 22 PDX.247 0 100 200 300 400 500 600 0 2 4 6 8 10 12 14 16 18 20 22 Time (days) AZD8055 4 mg/kg (n=4) Vehicle (n=5) GDC0941 30 mg/kg (n=4) Selumetinib 20 mg/kg (n=3) Combination (n=5) *** *** *** * T um or vo lu m e o ve r baseline vo lu m e ( % ) 0 100 200 300 400 500 0 2 4 6 8 10 12 14 16 18 20 22 Time (days) *** *** *** ** *** Vehicle (n=7) AZD8055 4 mg/kg (n=4) GDC0941 30 mg/kg (n=4) Selumetinib 20 mg/kg (n=4) Combination (n=5) PDX.180 T um or vo lu m e o ve r baseline vo lu m e ( % ) B C D E Vehicle (n=7) AZD8055 4 mg/kg (n=4) GDC0941 30 mg/kg (n=4) Selumetinib 20 mg/kg (n=4) Combination (n=5) 0 5 10 15 20 25 30 35 We ig h t ( g ) PDX.247 mouse weight 0 2 4 6 8 10 12 14 16 18 20 22 Time (days) Vehicle (n=5) AZD8055 4 mg/kg (n=4) GDC0941 30 mg/kg (n=4) Selumetinib 20 mg/kg (n=3) Combination (n=5) Time (days) )LJXUH_/RZGRVH$='*'&DQGVHOXPHWLQLEFRPELQDWLRQHႈFDF\LQ2&&&3';PRG-els. (A)0XWDWLRQVDQG&1$VLQ3,.$.7P725VLJQDOLQJQRGHVLQ3';DQG3';3'; (B) and PDX.247 (C) NSG mice treated with vehicle, AZD8055, GDC0941, selumetinib or the combination

in F5 generation. Tumor volume is represented as percentage of initial tumor volume at start of treatment. In the PDX.180 vehicle arm three mice received oral gavage vehicle treatment. Four mice received in-traperitoneal vehicle treatment and are previously described by Caumanns et al. (4). Error bars indicate SEM, * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001 of combination treatment arm relative to vehicle treatment arm. PDX.180 (D) and PDX.247 (E) mouse weight represented for the afore-mentioned treatment groups. Error bars indicate SD.

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105 data. A similar growth reduction was observed with the combination treatment DQG PJNJ $=' LQ 3'; mice (Supplementary Fig. 3A-B).

DISCUSSION

New therapeutic strategies for the treatment of OCCC are urgently QHHGHG EHFDXVH RI WKH ORZ HႈFDF\ RI standard platinum-based chemotherapy in advanced stage OCCC. Here, ZH GHPRQVWUDWH V\QHUJLVWLF HႇHFWV

RI P725& $=' 3,.

*'& DQG 0(. VHOXPHWLQLE inhibitors combined at low-dose in a stable during the course of treatment and

none of the mice had to be prematurely VDFUL¿FHG VXJJHVWLQJ QR FRPELQDWLRQ treatment related systemic toxicity (Fig. 4D-E). Reduced expression of the proliferation marker Ki67 and an increase in Cleaved Caspase-3 accompanied tumor regression in PDX.180 mice in the AGS low-dose combination treatment arm (Fig. 5A). Reduced Ki67 expression was also observed in the AGS low-dose combination treatment arm of PDX.247 mice (Fig. 5B). The inhibitory HႇHFWRIWKHFRPELQDWLRQWUHDWPHQWZDV VWURQJHUWKDQWKDWRIPJNJ$=' treatment in PDX.180 mice in historical

A

100 μm 100 μm 100 μm 100 μm

Vehicle AZD8055 GDC0941 Selumetinib Combination

Ki67 positive cells (% ) **** ** *** 80 60 40 20 0 * 100 μm 100 μm 100 μm 100 μm

Cleaved Caspase-3 positive cells

(% ) 30 20 10 0 **_* ** * PDX.180 100 μm 100 μm Vehicle GDC0941 Selumetinib Combination AZD8055 Ki67 Cleaved Caspase-3 B PDX.247 100 μm 100 μm 100 μm 100 μm Ki67 positive cells (% ) 80 60 40 20 0 * * Vehicle GDC0941 Selumetinib Combination AZD8055 4x 100 μm 4x 100 μm 4x 100 μm 100 μm

Cleaved Caspase-3 positive cells

(% ) 30 20 10 0

Vehicle AZD8055 GDC0941 Selumetinib Combination

100 μm

Ki67

Cleaved Caspase-3

Figure 5 | Ki67 and Cleaved Caspase-3 expression in OCCC PDX models.

Repre-sentative expression DQG TXDQWL¿FDWLRQ RI Ki67 and Cleaved Caspase-3 in the vehicle, AZD8055, GDC0941, selume-tinib or combination treatment groups in PDX.180 (A) and PDX.247 (B) as de-termined by IHC. Er-ror bars indicate SD. * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001.

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pathway nodes in OCCC. Moreover, the in vivo well tolerated low-dose AGS FRPELQDWLRQ HႈFLHQWO\ UHGXFHG WXPRU growth in two OCCC PDX models. We used PDX models instead of cell line based xenografts, since these models more accurate represent patient tumors (33). Fortunately, we could establish a few of these unique OCCC PDX models and demonstrate their feasibility in GUXJ WHVWLQJ 3,.P725& GXDO LQKLELWRU DQG 0(. LQKLELWRU combinations have been assessed in ovarian cancer models, including ES2 xenografts where synergistic inhibition of tumor growth was found after PI3K-P725& LQKLELWLRQ E\ 3) DW KLJK GRVH DQG 0(. LQKLELWLRQ E\ PD-0325901 at low-dose (35,36). Taken together, these results suggest that 2&&&SDWLHQWVFRXOGSRWHQWLDOO\EHQH¿W IURPDFRPELQDWLRQRI3,.P725& DQG0(.LQKLELWRUVXVLQJORZGRVHV

3,.P725& GXDO LQKLELWRU DQG

0(. LQKLELWRU FRPELQDWLRQV KDYH been explored clinically. The PI3K-P725& GXDO LQKLELWRU JHGDWROLVLE VKRZHG SUHOLPLQDU\ DQWLWXPRU HႈFDF\ in a subset of KRAS mutant ovarian cancer patients when combined with PD-0325901. Unfortunately, adverse event related treatment interruptions (mainly gastrointestinal complications) and dose-limiting toxicity occurred in over half of the patients. Notably, the treatment arm combining the other PI3K-P725& LQKLELWRU 3) ZLWK PD-0325901 was prematurely closed due to low tolerability (37). Dose-limiting toxicity or treatment discontinuation occurred in other combination trials with 3,.P725& GXDO LQKLELWRUV DQG MEK inhibitors as well, which included ovarian cancer (NCT01936363 and NCT01248858) (38-40). Importantly, stable disease or partial responses were observed in some patients, indicating that combined inhibition of PI3K, P725& DQG 0(. PD\ KDYH WKHUDSHXWLFEHQH¿W7KHVHFOLQLFDOUHVXOWV genetically diverse panel of OCCC cell

lines and OCCC PDX models. Our study VLJQL¿HV WKDW FRPELQLQJ P725& 3,.DQG0(.LQKLELWRUVDWORZGRVH LV DQ HႇHFWLYH WUHDWPHQW VWUDWHJ\ WKDW warrants further exploration in OCCC.

In our recent report, we demonstrated KLJKVXVFHSWLELOLW\RI2&&&WRP725& inhibition, however we also observed 3,.$.7P725 SDWKZD\ UHDFWLYDWLRQ (4). Therefore, we evaluated whether 3,. DQG 0(. LQKLELWRUV FDQ DGG WR WKH SRWHQF\ RI P725& LQKLELWRUV WR prevent pathway re-activation and cross-activation at suboptimal concentrations.

Indeed, IC₂₀ combinations of AZD8055,

GDC0941 and selumetinib (AGS) decreased single inhibitor related re-DFWLYDWLRQ RI 3,.$.7P725 DQG MAPK pathways at the molecular level. We observed that exposure of OCCC cell lines to the apoptosis inducing agent ABT-737 increased responsiveness towards AGS. These results are in line with previous work in serous ovarian cancer cell lines and PDX models

FRPELQLQJ GXDO 3,.P725&

inhibitors with ABT-737 or navitoclax (ABT-263) (29,30). Interestingly, the low-dose AGS combination induced apoptosis in our most sensitive OCCC PDX model, suggesting that apoptosis enhancers may not always be necessary for apoptosis induction. Recently, we found that BET bromodomain inhibitors KDYH V\QWKHWLF OHWKDO HႇHFWV LQ ARID1A mutant OCCC (6). BET bromodomain inhibitors have been shown to reduce

kinase inhibition related pathway

rewiring and re-activation in multiple cancer models, including ARID1A mutant ovarian cancer cells (31,32). Therefore, the combination of AGS with BET bromodomain inhibitors may be especially of interest in the context of

ARID1A mutant OCCC.

Our results of the AGS combination LQYLWURLQGLFDWHWKDWWKHHႈFDF\RI$*6 covers the mutational spectra found in 3,.$.7P725 SDWKZD\ DQG 0$3.

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Inhibitors

AZD8055, GDC0941, selumetinib and MLN0128 were obtained from Axon Medchem (the Netherlands). ABT-737 was obtained from MedchemExpress (United Kingdom). All inhibitors were dissolved in DMSO and stored at -80°C.

MTT assays

Cells were seeded in 96-wells plates and cultured overnight (16-18 h) before 96 h treatment with increasing inhibitor concentrations. Plating concentrations were: ES2, JHOC5 and KOC7C 2000 FHOOVZHOO 50* DQG 6029 FHOOVZHOO +$& DQG 290$1$ FHOOVZHOO$IWHUK077ZDVDGGHGWR D ¿QDO FRQFHQWUDWLRQ RI PJPO DQG cells were incubated for four additional hours. Cells were then dissolved in DMSO, and the produced formazan was measured at 520 nm with a Bio-Rad iMark spectrometer. IC₂₀ inhibitory concentrations were determined in Graphpad Prism® 6.01.

Long-term proliferation assays

Cells were seeded in 12-well plates and cultured overnight (16-18 h) before treatment was added. Plating FRQFHQWUDWLRQV ZHUH (6 FHOOV ZHOO+$&FHOOVZHOO-+2& FHOOVZHOO.2&&FHOOVZHOO50* FHOOVZHOO6029FHOOVZHOO 290$1$FHOOVZHOODQG53( FHOOVZHOO53(FHOOVZHUHWUHDWHGZLWK AZD8055, GDC0941 and selumetinib concentrations at 25% and 75% of the GLႇHUHQFH EHWZHHQ WKH ORZHVW DQG highest IC₂₀ from our OCCC cell line SDQHO $IWHU GD\V FHOOV ZHUH ¿[HG with 4% formaldehyde and stained with 0.1% crystal violet and subsequently scanned.

Western blotting

For Western blot primary antibodies

against p-AKT308_{(9275), p-AKT}473

S(5. S6 p-4EBP1 (9455), PARP (9532) and advocate the use of separate inhibitors

RI3,.DQGP725&WRSUHYHQWWDUJHW related toxicity as observed with PI3K-P725& GXDO LQKLELWRUV 0RUHRYHU WRVDIHO\DGGD0(.LQKLELWRUWR3,. DQG P725& LQKLELWRU FRPELQDWLRQV careful titration of such a putative low-dose triple combination will be crucial. Titrated low-dose combinations of targeted drugs have already been used LQ FDQFHU SDWLHQWV DQG HႈFDF\ ZDV observed with minimal dose-limiting toxicity (41-43). For this low-dose strategy multiple inhibitors of PI3K, P725&RU0(.FXUUHQWO\LQSKDVH II trials (NCT01737450, NCT02101788, NCT02465060, NCT02725268, NCT03128619 and NCT03264066), are available. &ROOHFWLYHO\WKHHႇHFWLYHQHVVRIORZ GRVH 3,. P725& DQG 0(. inhibitor combinations in OCCC cell lines DQG 3'; PRGHOV VLJQL¿HV H[SORUDWLRQ of such single targeted inhibitor combinations at low-doses in future clinical trials in OCCC.

MATERIALS AND METHODS

Cell lines

We obtained seven human OCCC cell OLQHV 50* 290$1$ DQG +$& (JCRB Cell Bank, Japan); JHOC5 5,.(1&HOO%DQN-DSDQ6029DQG KOC7C (Dr. Hiroaki Itamochi, Tottori University School of Medicine, Tottori, Japan) and ES2 (Dr. E. Berns, Erasmus MC, Rotterdam, the Netherlands). All OCCC cells were maintained in RPMI supplemented with 10% fetal calf serum. The non-transformed human retinal pigment epithelial cell line (RPE1) was maintained in DMEM-low medium supplemented with 10% fetal calf serum. All the cell lines were tested by STR SUR¿OLQJDQGWHVWHGP\FRSODVPDIUHH$OO cells were kept in culture for a maximum of 50 passages.

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Cleaved Caspase-3 (9661) were

obtained from Cell Signaling Technology 86$ ȕ$FWLQ & ZDV REWDLQHG IURP MPbiomedicals (USA).

$SRSWRVLVÀRZF\WRPHWU\

Cells were seeded in 6-wells plates

and incubated overnight (16-18

h) before treatment was added. Cells were harvested in their own media using trypsine, stained with ƍƍƍKH[DPHWK\OLQGRGLFDUER

cyanine iodide (DiIC₁(5)) dye (Thermo

Fischer, USA) for 30 minutes at 37°C and subsequently stained with propidium Iodide (Thermo Fischer, USA). At least 10,000 events were measured on a FACS Calibur (Becton Dickinson, USA).

DiIC₁(5), a dye that accumulates in

mitochondria, is lost when cells undergo apoptosis via the intrinsic apoptosis pathway. By combining the quartiles of cells that are propidium iodide positive

or have lost DiIC₁(5) dye, both apoptotic

DQG QHFURWLF FHOOV FRXOG EH TXDQWL¿HG FlowJo software was used for data DQDO\VLV 6WDWLVWLFDO VLJQL¿FDQFH ZDV determined using repeated measures RQHZD\ $129$ ZLWK SRVWKRF 7XNH\¶V multiple comparison test.

Combination treatment in vivo

All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Groningen (Groningen, the Netherlands) and carried out in accordance with the approved guideline “code of practice: animal experiments in cancer research” (Netherlands Inspectorate for Health 3URWHFWLRQ&RPPRGLWLHVDQG9HWHULQDU\ Public Health, 1999). Before surgery, all patients from whom tumor samples were obtained for PDX modeling gave written informed consent. PDX.180 was derived at interval debulking surgery from a FIGO stage IIIC OCCC patient treated with QHRDGMXYDQW FDUERSODWLQSDFOLWD[HO &3 chemotherapy and 10 months disease-VSHFL¿F VXUYLYDO DIWHU LQLWLDO GLDJQRVLV

PDX.247 was obtained at primary surgery from a FIGO stage IIB OCCC patient, who had a complete debulking followed by CP chemotherapy and no evidence of disease after 24 months of follow-up. Surgical tissues were implanted and propagated successfully to the F3 or F4 generation according to previously described methods (44). The PDX models were sequenced for 40 genes, including genes with a high mutation frequency in OCCC, ARID1A and other cancer-related genes using Haloplex

custom kit (Agilent technologies®,

USA) (4). Additional SNP genotyping of the PDX models was performed

with

HumanOmniExpressExome-8BeadChip (Illumina, USA) (4). F3 or F4

tumor pieces were cut into 3x3x3 mm3 sections and subcutaneously implanted LQ WKH ÀDQN RI WR ZHHN ROG 12' &%3UNGFVFLG1&U+VG 16* PLFH (internally bred, Central Animal Facility, University Medical Centre Groningen). Weight, fur quality and overall activity of the mice were monitored at least once per three days. Tumor growth was TXDQWL¿HG DW OHDVW RQFH SHU WKUHH GD\V by caliper measurements according WR WKH IRUPXOD ZLGWK [ OHQJWK When tumors demonstrated sustained growth (on average after 27 days), mice were randomized into vehicle FRQWURORUWUHDWPHQWJURXSVQ PLFH JURXS $=' PJNJ *'& PJNJ VHOXPHWLQLE PJNJ or the combination were diluted in 0.2% Tween-80 0.5% hydroxyl-propyl methylcellulose (Sigma-Aldrich, USA) and administered by oral gavage daily. Treatment with AZD8055, GDC0941, selumetinib, the combination or vehicle was continued for 21 days, after which DOO PLFH ZHUH VDFUL¿FHG 2QH PRXVH LQ the selumetinib treated group died on WUHDWPHQW GD\ IRXU GXH WR XQGH¿QHG reasons. In historical data, AZD8055 (10 PJNJLQ'0623RO\HWK\OHQH

glycol 300) was administered

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5

109 Aperia ImageScope software (Leica). 6WDWLVWLFDO VLJQL¿FDQFH ZDV GHWHUPLQHG XVLQJ RQHZD\ $129$ ZLWK SRVWKRF Tukey’s multiple comparison test.

Synergy determination

Drug synergy was assessed with the Chou-Talalay method in CompuSyn 9HUVLRQ&RPERV\QLQFRUSRUDWHGWR GHWHUPLQHWKHGRVHHႇHFWRIFRPELQDWLRQ therapy (45). For this analysis, cells were treated with single inhibitors in increasing concentrations to determine a standard curve, and in combination

using the IC₂₀ of each compound.

Strong synergism, synergism, additive HႇHFWVDQGDQWDJRQLVPZHUHGH¿QHGE\ CI < 0.25, CI < 0.75, CI = 1 or CI > 1, respectively.

ACKNOWLEDGEMENTS

We acknowledge Dr. Hiroaki Itamochi and Dr. Els Berns for their kind contribution of OCCC cell lines. The authors thank *HUGDGH9ULHVDQG)HUQDQGD;5RVDV Plaza for help with the PDX models. G. Bea A. Wisman and Steven de Jong are members of the EurOPDX Consortium. This research was supported by grants from the Dutch Cancer Society (KWF, RUG 2012-5477) and the Cock-Hadders foundation.

AUTHOR CONTRIBUTIONS

A.G.J.Z., G.B.A.W. and S.J. conceived and supervised the project. J.J.C. and A.W. performed the majority of experiments and data analysis with the assistance of G.J.M. and A.K. J.J.C., A.G.J.Z., G.B.A.W. and S.J. wrote the manuscript with the assistance of M.J. and R.B. All authors discussed the results and commented on the manuscript. JURZWKZDVGHWHUPLQHGE\WXPRUYROXPH

WXPRU YROXPH DW VWDUW RI ¿UVW WUHDWPHQW (day 0). For further analysis the tumors were resected, split into two parts and VQDSIUR]HQDW&DVZHOODVSDUDႈQ HPEHGGHG 6WDWLVWLFDO VLJQL¿FDQFH IRU tumor growth was determined using two-ZD\$129$ZLWK%RQIHURQLSRVWKRFWHVW correction in Graphpad Prism® 6.01.

Immunohistochemical analysis

)URP SDUDႈQHPEHGGHG 3';

WXPRUV VOLFHV ȝP WKLFN ZHUH FXW using a microtome and placed on 3-aminopropyltriethoxysilane-coated glass slides. Heat-induced antigen retrieval was performed in 10 mM FLWUDWH EXႇHU XVLQJ D : URWDU\ microwave. Endogenous peroxidase was blocked by 30 minutes incubation

with 0.3% H₂O₂ in PBS. Endogenous

DYLGLQELRWLQ DFWLYLW\ ZDV EORFNHG XVLQJ a commercially available blocking kit 9HFWRU /DERUDWRULHV 86$ 6OLGHV were incubated with primary antibodies detecting human Ki67 (DAKO M7240, USA) and Cleaved Caspase-3 (Cell Signaling #9661). Staining was visualized after incubation with biotinylated or peroxidase-bound secondary antibodies 'DNR 86$ XVLQJ VWUHSWDYLGLQELRWLQ

horseradish peroxidase complex

(Dako, USA) and 3,3’-diaminobenzidine

(Sigma-Aldrich, USA). Hematoxylin

counterstaining was applied routinely, and hematoxylin & eosin (H&E) staining was used to analyze tissue viability and morphology. Photographs were acquired by digitalized scanning of slides using the NanoZoomer 2.0-HT multi-slide scanner (Hamamatsu, Japan). Ki67 VWDLQLQJV ZHUH TXDQWL¿HG E\ VFRULQJ .L SRVLWLYH QXFOHL LQ ¿YH UDQGRPO\ DVVLJQHG[PDJQL¿HGDUHDVIURPHDFK slide. For Cleaved Caspase-3 stainings, the positive cytoplasm was scored with

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40. Grilley-Olson JE, Bedard PL, Fasolo A, Cornfeld M, Cartee L, Razak AR, et al. A phase Ib dose-esca-ODWLRQVWXG\RIWKH0(.LQKLELWRUWUDPHWLQLELQFRPELQDWLRQZLWKWKH3,.P725LQKLELWRU*6. in patients with advanced solid tumors. Invest New Drugs. 2016 Dec;34(6):740-9.

41. Hong DS, Sebti SM, Newman RA, Blaskovich MA, Ye L, Gagel RF, et al. Phase I trial of a combina-tion of the multikinase inhibitor sorafenib and the farnesyltransferase inhibitor tipifarnib in advanced malignancies. Clin Cancer Res. 2009 Nov 15;15(22):7061-8.

'XPPHU5%H\HU0+\PHV.(SSLQJ07%HUQDUGV56WHLQKRႇ0HWDO9RULQRVWDWFRPELQHGZLWK bexarotene for treatment of cutaneous T-cell lymphoma: in vitro and phase I clinical evidence sup-SRUWLQJDXJPHQWDWLRQRIUHWLQRLFDFLGUHFHSWRUUHWLQRLG;UHFHSWRUDFWLYDWLRQE\KLVWRQHGHDFHW\ODVH inhibition. Leuk Lymphoma. 2012 Aug;53(8):1501-8.

*RPH]$OPDJXHU ' 6DOGDQD9D]TXH] 5 7DULQ$U]DJD / +HUUHUD5RMDV 0$ 9D]TXH]0HOODGR de Larracoechea A, Cantu-Rodriguez OG, et al. Combination of low-dose imatinib plus nilotinib for the treatment of chronic-phase chronic myeloid leukaemia after imatinib failure. Hematology. 2016 Aug;21(7):411-4.

44. Alkema NG, Tomar T, Duiker EW, Jan Meersma G, Klip H, van der Zee AG, et al. Biobanking of patient DQGSDWLHQWGHULYHG[HQRJUDIWRYDULDQWXPRXUWLVVXHHႈFLHQWSUHVHUYDWLRQZLWKORZDQGKLJKIHWDOFDOI serum based methods. Sci Rep. 2015 Oct 6;5:14495.

&KRX7&'UXJFRPELQDWLRQVWXGLHVDQGWKHLUV\QHUJ\TXDQWL¿FDWLRQXVLQJWKH&KRX7DODOD\PHWKRG Cancer Res. 2010 Jan 15;70(2):440-6.

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113 SUPPLEMENTARY DATA ES2 _HAC2 JHOC5KOC 7C RMG 1 SMO V2 OVM ANA Sy ne rg y ( C I) 24 22 20 18 16 14 12 10 8 6 4 2 0 AG ES2 _HAC2 JHOC5KOC 7C RMG 1 SMO V2 OVM ANA Sy ne rg y ( C I) 24 22 20 18 16 14 12 10 8 6 4 2 0 AS ES2 _HAC2 JHOC5KOC 7C RMG 1 SMO V2 OVM ANA Sy ne rg y ( C I) 24 22 20 18 16 14 12 10 8 6 4 2 0 AGS A B C KOC7C X X X X X X pAkt308 pErk pAkt473 pS6 ȕActin MLN0128 GDC0941 Selumetinib RMG1 X X X X X X SMOV2 X X X X X X MLN0128 GDC0941 Selumetinib X X X X X X X X X X KOC7C RMG1 SMOV2

Figure S1 | (A) Combination index (CI) data from Figure 1A represented in barplots. Error bars indicate

SEM and are derived from three experiments. (B) 3UROLIHUDWLRQ LQKLELWRU\ HႇHFW RI 0/1 DW Q0 .2&&RUQ050*DQG6029ZLWK*'&DQGVHOXPHWLQLE,&₂₀ concentration in a long-term colony formation assay. Results shown are representative of two experiments. (C) Expression of p-AKT308_{, p-AKT}473_{, p-ERK and p-S6 after 48h of treatment with MLN0128 at 5 nM (KOC7C) or 7.5 nM} 50*DQG6029ZLWK*'&DQGVHOXPHWLQLE,&₂₀ concentration as determined by Western blot. ȕ$FWLQZDVXVHGDVORDGLQJFRQWURO5HVXOWVVKRZQDUHUHSUHVHQWDWLYHRIWZRH[SHULPHQWV

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114 A E _KOC7C _SMOV2 X X X X ȕActin AGS ABT Cleaved &DVS X X X X X X X X OVMANA Cleaved PARP B Cisplatin AGS + + + + + + + + + KOC7C RMG1 SMOV2 0 20 40 60 80 100 DI LC 5 /P I+ (% ) 0 20 40 60 80 100 120 N orm al iz ed s urv iv al Cisplatin AGS + + + + + + + + + KOC7C RMG1 SMOV2 D ES2 0 20 40 60 80 100 DI LC 5 /P I+ (% ) RMG1 0 20 40 60 80 100 DI LC 5 /P I+ (% ) KOC7C 0 20 40 60 80 100 DI LC 5 /P I+ (% ) JHOC5 0 20 40 60 80 100 DI LC 5 /P I+ (% ) Control AGS ABT Combination * ES2 HAC2 JHOC5 KOC7C RMG1 SMOV2 OVMANA 0 0.1 1 10 ABT77 (μM) 0 20 40 60 80 100 Su rv iv al (%) AGS + ABT-737 120 C Figure S2 | (A)6HQVLWLYLW\RIFLVSODWLQȝ0RUȝ0DGGHGWR$*6GHWHUPLQHGZLWK077DVVD\(UURU

bars indicate SD and are derived from two experiments. (B)4XDQWL¿FDWLRQRIWKHERWWRPWZRTXDGUDQWV (DiIC₁3,DQG'L,&₁3,IURP'L,&₁3,ÀRZF\WRPHWU\DQDO\VLVDIWHUKWUHDWPHQWZLWKFLVSODWLQ ȝ0RUȝ0$*6RUWKHFRPELQDWLRQ(UURUEDUVLQGLFDWH6'DQGDUHGHULYHGIURPWKUHHH[SHULPHQWV

(C) Sensitivity of ABT-737 with AGS as determined by MTT assay. Error bars indicate SD and are derived

from two experiments. (D)4XDQWL¿FDWLRQRIWKHERWWRPWZRTXDGUDQWV'L,&₁3,DQG'L,&₁3, IURP'L,&3,ÀRZF\WRPHWU\DQDO\VLVDIWHUKWUHDWPHQWZLWK$*6ȝ0$%7RUWKHFRPELQD-tion in ES2, JHOC5, KOC7C and RMG1. Error bars indicate SD and are derived from two experiments performed in duplicate. (E) Expression of Cleaved PARP and Cleaved Caspase-3 after 48h of treatment ZLWK$*6ȝ0$%7RUȝ0$%7DGGHGWR$*6DVGHWHUPLQHGE\:HVWHUQEORW)ORDWLQJFHOOV ZHUHLQFOXGHGLQO\VDWHȕ$FWLQZDVXVHGDVORDGLQJFRQWURO5HVXOWVVKRZQDUHUHSUHVHQWDWLYHRIWZR experiments.

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5

115 B Vehicle (n=7) AZD8055 10 mg/kg (n=5) *** *** * 0 100 200 300 400 500 T um or vo lu m e o ve r baseline vo lu m e ( % ) 0 2 4 6 8 10 12 14 16 18 20 22 Time (days) PDX.180 PDX.247 0 100 200 300 400 500 600 0 2 4 6 8 10 12 14 16 18 20 22 Time (days) AZD8055 4 mg/kg (n=4) Vehicle (n=5) GDC0941 30 mg/kg (n=4) Selumetinib 20 mg/kg (n=3) Combination (n=5) T um or vo lu m e o ve r baseline vo lu m e ( % ) A 0 100 200 300 400 500 0 2 4 6 8 10 12 14 16 18 20 22 Time (days) Vehicle (n=7) AZD8055 4 mg/kg (n=4) GDC0941 30 mg/kg (n=4) Selumetinib 20 mg/kg (n=4) Combination (n=5) PDX.180 T um or vo lu m e o ve r basline vo lu m e ( % ) ** **_*** *** *** *** *** *** Vehicle (n=5) AZD8055 10 mgkg/day (n=4) * *** *** *** *** 0 2 4 6 8 10 12 14 16 18 20 22 Time (days) 0 100 200 300 400 500 T um or vo lu m e o ve r baseline vo lu m e ( % ) *** PDX.247

Figure S3 | (A) 7XPRUJURZWKJUDSKVIURP)LJXUH%DQG&ZLWKLQGLFDWHGVLJQL¿FDQWGLႇHUHQFHVEHWZHHQ

all treatment groups at day 21. Error bars indicate SEM, * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001. (B)3';16*PLFHWUHDWHGZLWKYHKLFOHIURP)LJXUH%RUPJNJ$=' treatment in F5 generation, derived from Caumanns et al. (1). PDX.247 NSG mice treated with vehicle DQG$='PJNJLQWUDSHULWRQHDOLQ)JHQHUDWLRQGHULYHGIURP&DXPDQQVet al. (1). Tumor vol-ume is represented as percentage of initial tumor volvol-ume at start of treatment. Error bars indicate SEM, * indicates p<0.05, *** indicates pRIFRPELQDWLRQWUHDWPHQWDUPUHODWLYHWR$='PJNJ treatment arm.

SUPPLEMENTARY REFERENCES

1. Caumanns JJ, Berns K, Wisman GBA, Fehrmann RSN, Tomar T, Klip H, et al. Integrative kinome SUR¿OLQJLGHQWL¿HVP725&LQKLELWLRQDVWUHDWPHQWVWUDWHJ\LQRYDULDQFOHDUFHOOFDUFLQRPD&OLQ Cancer Res. 2018 Apr 23.

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