1

Title

2

SHP2 inhibition enhances the effects of tyrosine kinase inhibitors in preclinical3

models of treatment-naïve ALK-, ROS1-, or EGFR-altered non-small-cell lung4

cancer5
6

Author’s name and affiliation

7

Hirohisa Kano1), Eiki Ichihara2), Hiromi Watanabe1), Kazuya Nishii1), Chihiro Ando1), Takamasa

8

Nakasuka1), Kiichiro Ninomiya1), Yuka Kato3), Toshio Kubo4), Kammei Rai2), Kadoaki Ohashi2),

9Katsuyuki Hotta3), Masahiro Tabata4), Yoshinobu Maeda1) and Katsuyuki Kiura2)

101) Department of Hematology, Oncology and Respiratory Medicine, Okayama University

11

Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Japan122) Department of Allergy and Respiratory Medicine, Okayama University Hospital, Japan133) Center for Innovative Clinical Medicine, Okayama University Hospital, Japan

144) Cancer Center, Okayama University Hospital, Japan

15
16

Running Title

17

Oncogene targeted therapy combined with SHP2 inhibitor in NSCLCs

18

1

19

Corresponding author:

20

Eiki Ichihara, MD, PhD

21

Okayama University Hospital, Okayama, Japan (Country code +81).

22

2-5-1 Shikata-cho Kita-ku Okayama City, Okayama 700-8558, Japan,

23

Tel.: +81-86-235-7227

E-mail: ichiha-e@md.okayama-u.ac.jp

24

2

25

Financial Information

26

This work was supported by grants from Project Mirai, the Ryobi Teien Memory Foundation,27

the Okayama Medical Foundation and the Japan Lung Cancer Society.

28

3

29

Abstract

30

After molecular-targeted therapy, some cancer cells may remain that are resistant to31

therapies targeting oncogene alterations, such as those in the genes encoding the32

epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) as

33

well as c-ros oncogene 1 (ROS1). The mechanisms underlying this type of resistance are34

unknown. Here we report the potential role of Src homology 2 domain-containing

35

phosphatase 2 (SHP2) in the residual cells of ALK/ROS1/EGFR-altered non-small-cell

36lung cancer (NSCLC). Molecular-targeted therapies failed to inhibit the ERK signaling

37

pathway in the residual cells whereas the SHP2 inhibitor SHP099 abolished their38

remaining ERK activity. SHP099 administered in combination with molecular-targeted

39

therapy resulted in marked growth inhibition of cancer cells both in vitro and in vivo.

40

Thus, treatment combining an SHP2 inhibitor and a tyrosine kinase inhibitor may be a

41

promising therapeutic strategy for oncogene-driven NSCLC.

42

4

43

Introduction

44

Agents targeting gene mutations/translocations in oncogenes, such as the genes

45

encoding epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase

46(ALK) as well as c-ros oncogene 1 (ROS1), remarkably inhibit the growth of47

non-small-cell lung cancer (NSCLC) (1-6). However, there is often residual tumor after48

these therapies, leading to acquired resistance and poor curative efficacy (7-11). Despite

49

efforts to develop post-resistance therapies to overcome acquired resistance (12-16),50

most therapies do not work well due to the diversity of resistance mechanisms,

51

difficulty of re-biopsy, and emergence of further resistance.

52

It is unclear why residual tumor remains after strong inhibition of oncogenic

53

signaling. A possible mechanism is that downstream signaling inhibited by the54

molecular agent is somehow compensated. This was demonstrated in an EGFR-mutant

55

NSCLC, in which treatment with the third-generation EGFR tyrosine kinase inhibitor

56(TKI) osimertinib induced reactivation of extracellular signal-regulated kinase (ERK)57

following transient ERK inhibition (17). Combination therapy consisting of a58

mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor and an EGFR59

TKI inhibited this reactivation and had a profound inhibitory effect on the cancer cells

60(18), suggesting its potential therapeutic efficacy in EGFR-mutant lung cancer (19).

561

Src homology 2 domain-containing phosphatase 2 (SHP2), a tyrosine

62

phosphatase encoded by protein-tyrosine phosphatase non-receptor type 11 (PTPN11),

63

mediates cell signaling from multiple receptor tyrosine kinases (RTKs) to their64

downstream pathways, including the MAPK pathway. Germline PTPN11 gene65

mutations are frequently found in Noonan syndrome (20), which is characterized by66

congenital malformations. Somatic PTPN11 gene mutations have also been implicated67

in cancers such as juvenile myelomonocytic leukemia (21), hepatocellular carcinoma

68(22), acute myeloid leukemia (23), colorectal cancer (24), breast cancer (25), melanoma

69(26) and lung cancer (27, 28). Recently developed allosteric SHP2 inhibitors such as

70

SHP099 and RMC-4550 have been shown to be highly effective, especially in

71

RAS/RAF/MAPK-pathway-driven cancers such as BRAF mutant melanoma (29-33). In

72

addition, in an RTK-driven NSCLC, an SHP2 inhibitor restored sensitivity to the ALK73

TKI ceritinib in ALK-rearranged NSCLC cells that had acquired resistance to the drug

74(34). However, only a few studies have examined the use of an SHP2 inhibitor

75

combined with an RTK inhibitor in a model of treatment-naïve RTK driven NSCLC

76(35-37). These include two reports of the efficacy of SHP2 inhibitor combined with an77

RTK inhibitor in EGFR-mutant NSCLC cell lines (36, 37), but there are no data78

regarding ALK/ROS1 rearranged NSCLC. Thus, in this study, we evaluated the efficacy

6

79

of the SHP2 inhibitor SHP099 against residual tumor cells following molecular-targeted

80

therapy in a model of treatment-naïve ALK/ROS1/EGFR-altered NSCLC.

7

81

Materials and Methods

82

Cell lines and reagents83

PC-9 cells (EGFR Ex19 del E746_A750) were purchased from the European Collection

84

of Authenticated Cell Cultures (Salisbury, UK). HCC827 (EGFR Ex19 del E746_A750),

85

H3122 (EML4-ALK fusion), and HCC78 (SLC34A2-ROS1 fusion) cells were kindly

86

provided by Dr. William Pao (Vanderbilt University, Nashville, TN, USA). ABC-19

87(EML4-ALK fusion) and ABC-20 (CD74-ROS1 fusion) cells were obtained from88

NSCLC patients and established as cell lines in our laboratory (38). All cell lines were89

cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented

90with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin and were91

maintained at 37℃ in a 5% CO2 humified atmosphere. Alectinib, crizotinib, osimertinib,

92

SHP099, selumetinib, RMC-4550 and DS-6051b were purchased from Selleck

93

Chemicals (Houston, TX, USA). All compounds were dissolved in dimethyl sulfoxide

94

for in vitro studies.

95
96

Cell imaging and cell proliferation assay

97

The cells were seeded in a 10-cm dish (PC-9: 250,000 cells/dish; H3122 and HCC78:98

1,000,000 cells/dish) and treated the following day. After 48 h, cell mass and899morphology were observed under an inverted phase-contrast microscope, and images

100

were

101

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

102

Briefly, the cells were seeded in 96-well plates at a density of 1,500–10,000 cells/well103(PC-9: 1,500 cells/well; H3122, HCC78, HCC827: 3,000 cells/well; ABC-19: 4,000

104

cells/well; ABC-20: 10,000 cells/well) and continuously treated with each drug for 96 h.

obtained.

Growthinhibitionwas

measuredinamodified
(39).

105
106

Crystal violet assay

107

The cells were seeded in six-well plates at a density of 10,000–600,000 cells/well108(H3122: 600,000 cells/well; ABC19: 80,000 cells/well; HCC78: 20,000 cells/well;109

ABC-20: 200,000 cells/well; PC-9: 10,000 cells/well; HCC827: 20,000 cells/well) and110

treated the following day. Media and drugs were replaced 3 days after the first treatment.

111

After fixation in 10% formalin for 10 min, the cells were stained for 10 min with crystal

112

violet solution (Sigma-Aldrich) and then washed with H2O. The plates were dried

113

overnight and the stained cells were then quantified using ImageJ software (version

114

1.52a; National Institutes of Health, Bethesda, MD, USA).

115
116

Combination index (CI)

9

117

A constant ratio design for the combination assay was used because it enables efficient

118

data analysis (40). The multiple-drug-effect analysis, developed by Chou and Talaly, is119based on the median-effect principle and was used in this study to calculate the120

combined drug effect (41). Cells seeded in triplicate in 96-well plates were treated for

121

96 h with TKIs and/or SHP099 at the indicated concentrations. Cell viability was122assessed using the MTT assay. The CIs were calculated using CompuSyn software

123(CompuSyn, Paramus, NJ, USA), developed by Chou et al. in 2010 (42). In the plots

124

generated by CompuSyn, the y-axis represents the CI, with CI < 1, 1 or > 1 indicating a125

synergistic, additive or antagonistic effect respectively. The x-axis represents the126

fractional activity (Fa), which is the proportion of cells inhibited by the treatment

127

relative to that inhibited by the vehicle control.

128
129

Immunoblotting and RTK array

130

Immunoblotting was conducted using the following antibodies: phosphorylated

131(p)-ALK Tyr1282/1283 (#9687), ALK (#3633), p-ROS1 Tyr2274 (#3078), ROS1

132(#3287), p-EGFR Tyr1068 (#3777), EGFR (#2232), SHP2 (#3752), p-MEK1/2

133

Ser217/221 (#9154), MEK1/2 (#9126), p-ERK1/2 Thr202/Tyr204 (#9101), ERK1/2

134(#9102), p-AKT Ser473 (#9271), AKT (#9272), and GAPDH (#2118), obtained from

10

135

Cell Signaling Technology (Danvers, MA, USA), and anti-SHP2 (phosphor Y542)

136

antibody (ab62322), obtained from Abcam (Cambridge, MA, USA). Anti-rabbit IgG

137

horseradish peroxidase-linked whole antibody (from donkey; NA934) was purchased

138

from GE Healthcare Biosciences (Pittsburgh, PA, USA). For immunoblotting, the139

harvested

140

radioimmunoprecipitation assay buffer (1% IGEPAL CA-630 [MP Biochemicals, Irvine,

141

CA, USA], 0.1% sodium dodecyl sulphate, 1% okadaic acid, 50 mM Tris-HCl, pH 7.5,

142

75 mM NaCl, 40 mM NaF and 1 mM sodium orthovanadate) containing a protease143

inhibitor cocktail (Roche Applied Sciences, Penzberg, Germany). The proteins were144

separated by SDS-PAGE and then electrotransferred onto membranes, which were then145

incubated with the indicated primary and secondary antibodies. Chemiluminescence146

was performed using Enhanced Chemiluminescence Plus reagent (GE Healthcare

147

Biosciences, Chicago, IL, USA). Bands were detected using an ImageQuant LAS-4000

148

imager (GE Healthcare Biosciences) and quantified using ImageJ software (version

149

1.52a). A Phospho-RTK Array Kit was purchased from R&D Systems and used150

according to the manufacturer's recommendations.

cells

were

washedinphosphate-bufferedsaline

and

lysed

in

151
152

Xenograft mouse models

153

Female BALB/c nu/nu mice (6 weeks old) were purchased from Charles River
11

154

Laboratories, Yokohama, Japan). All mice were provided with sterilized food and water155

and housed in a barrier facility under a 12-h light/dark cycle. Cancer cells (H3122,

156

PC-9: 2×106 cells/tumor; ABC-20: 4×106 cells/tumor) were injected subcutaneously into157

the backs of the mice on the right and left sides. For combination treatment with

158

SHP099 and TKI, when the average tumor volume reached ~200 mm3, the mice were159

randomly allocated into four treatment groups (five mice/group): vehicle, SHP099 (50160

mg/kg/day), TKI (alectinib, 10 mg/kg/day; crizotinib, 50 mg/kg/day; osimertinib, 5161

mg/kg/day), and SHP099 plus TKI. Both SHP099 and the TKI were administered by

162

gavage once daily, five times weekly. Tumor volume (width2 × length/2) was measured

163

periodically. Statistical analyses were conducted based on the tumor volume measured

164

on day 28. For comparison of SHP099 and selumetinib in combination with osimertinib,

165

when the average tumor volume reached ~200 mm3, the mice were randomly allocated166

into three treatment groups (five mice/group): osimertinib (5 mg/kg/day), selumetinib

167(50 mg/kg/day) plus osimertinib, and SHP099 (50 mg/kg/day) plus osimertinib.

168

Osimertinib, selumetinib and SHP099 were administered by gavage once daily, five169

times weekly. Tumor volume (width2 × length/2) was measured periodically. Statistical170

analyses were conducted based on the tumor volume measured on day 14. All

171

experiments involving animals were performed under the auspices of the Institutional

12

172

Animal Care and Research Advisory Committee at the Department of Animal Resources,

173

Okayama University Advanced Science Research Center (Okayama, Japan). The

174

experiments were performed under the Policy on the Care and Use of Laboratory

175

Animals, Okayama University, and Fundamental Guidelines for Proper Conduct of176

Animal Experiment and Related Activities in Academic Research Institutions, Ministry177

of Education, Culture, Sports, Science and Technology of Japan. The experimental

178

protocol was approved by the Animal Care and Use Committee of Okayama University

179(OKU-2019521).

180
181

Statistical analysis

182

The statistical analyses were performed using STATA software (version 15; StataCorp,

183

College Station, TX, USA). Differences between two groups were compared using the184

two-tailed paired Student’s t-test, and differences among three groups were compared in

185

a one-way analysis of variance (ANOVA) followed by the Bonferroni method. A P

186value < 0.05 was considered to indicate statistical significance.

187

13

188

Results

189

Characterization of residual ALK/ROS1/EGFR-altered NSCLC cells after

190

molecular-targeted therapy

191

To investigate whether TKI tolerance in the clinical setting could be simulated in a

192

preclinical model, ALK/ROS1/EGFR-altered NSCLC cells were treated with the193

following drugs: alectinib for ALK-rearranged cells, crizotinib for ROS1-rearranged

194

cells and osimertinib for EGFR-mutant cells. Each agent effectively inhibited the195

majority of the cancer cells, but in each case some cells remained even after 48 h of196

drug exposure (Fig. 1A). The cell signaling status in the residual cells was determined197

by treating each cell line as described followed by immune blotting at different time

198

points thereafter. After 4 h of treatment, phosphorylation of the following driver199

oncogenes was effectively inhibited (Fig. 1B). In ALK-rearranged NSCLC cells (H3122

200

and ABC-19), ALK-TKI alectinib completely inhibited ALK phosphorylation over the201

studied period, whereas ERK activity was partially sustained or reactivated despite

202

treatment with the inhibitor for 24 and 72 h (Fig. 1B, left). ERK phosphorylation was

203

investigated in ROS1-rearranged or EGFR-mutant NSCLC cells. In the former (HCC78

204

and ABC-20), treatment with the ROS1-TKI crizotinib completely inhibited ROS1

205

phosphorylation, whereas ERK phosphorylation was partially reactivated without

206

upstream ROS1 activation (Fig. 1B, center). ERK phosphorylation was also partially
14

207

sustained or reactivated over time in EGFR-mutant cells (PC-9 and HCC827) treated

208with the EGFR-TKI osimertinib, whereas EGFR phosphorylation was completely

209

inhibited (Fig. 1B, right). These results demonstrate the persistent activity of the ERK

210

pathway after TKI exposure in all of the examined cell lines and imply that a sustained211

ERK pathway plays a role in the resistance of NSCLC treated with TKIs. In the212

experiment focusing on the AKT pathway, while in some cells phosphorylated AKT was

213

completely inhibited, in others it was not (Fig. 1B).

214
215

An SHP2 inhibitor suppresses sustained ERK and AKT phosphorylation in the216

presence of TKI

217

We hypothesized a role for another RTK in sustaining or reactivating the ERK pathway

218

in the residual tumor cells after TKI exposure. However, a search using a219

phosphorylated RTK array showed simultaneous and slight activation of various bypass

220

RTKs following TKI treatment, with none of them targeted by a single agent

221(Supplementary Fig. 1). Thus, to control this broadly activated signaling in the residual222

cells, we focused on SHP2 as a common targetable node of multiple RTK signaling,223

given that this phosphatase serves as an important hub that mediates RTK signaling to224

the downstream ERK pathway.

15

225

First, the efficacy of the SHP2 inhibitor SHP099 on oncogene activation in residual226

cells under TKI therapy was tested. ALK- or ROS1-rearranged cells as well as

227

EGFR-mutant cells were treated for 24 h with the corresponding TKI with or without

228

SHP099, after which signal activation was evaluated (Fig. 2). Alectinib with or without

229

SHP099 completely inhibited p-ALK in ALK-rearranged NSCLC cells (H3122 and230

ABC19). Although alectinib or SHP099 alone did not completely suppress ERK and its

231

upstream activation of MEK, both proteins were strongly suppressed by a combination

232of the two drugs (Fig. 2, left). In ROS1-rearranged cells (HCC78 and ABC-20), p-ROS1

233

levels were strongly suppressed by crizotinib, but whereas crizotinib or SHP099

234

treatment alone had a partial effect on p-MEK and p-ERK, their combined use resulted

235

in strong suppression of the proteins (Fig. 2, center). Because crizotinib is a236

multi-targeting agent that inhibits ROS1, ALK and MET, the efficacy of another ROS1

237

TKI,

238

ROS1-rearranged HCC78 and ABC-20 cells (Supplementary Fig. 2A). Consistent with239

the effect of crizotinib, greater inhibition of p-ERK was obtained with DS-6051b in

240

combination with SHP099 than with DS-6051b alone. Similarly, in EGFR-mutant

241

NSCLC cells (PC-9 and HCC827), combination of the EGFR TKI osimertinib and

242

SHP099 strongly suppressed p-MEK and p-ERK, even after 24 h (Fig. 2, right). These

DS-6051b

(16),

was

investigated16

incombination

with

SHP099in243

data imply that TKIs in combination with the SHP2 inhibitor SHP099 strongly

244

suppressed the ERK pathway. RAS activity in ALK/ROS1/EGFR-altered cells was also

245

determined, using a RAS-GTP assay (Supplementary Fig. 2B). Consistent with MEK

246

and ERK, RAS activity was not completely inhibited by an oncogene-targeted TKI

247

when used alone. A similar investigation of the AKT pathway showed that SHP099

248

reduced the level of p-AKT to varying extents in most of the investigated cell lines

249

except ABC-19. However, SHP099 in combination with a TKI showed stronger

250

inhibition of p-AKT (Fig. 2).

251
252

The SHP2 inhibitor SHP099 reduces residual cells associated with TKIs

253

The ability of the SHP2 inhibitor SHP099 to prevent residual cells was also assessed.

254

Oncogene-driven cancer cells were treated with the corresponding TKI with or without

255

SHP099 for 6 days, followed by crystal violet staining to determine the extent of cell

256

proliferation (Fig. 3A). In ALK-rearranged H3122 and ABC-19 cells, SHP099

257

monotherapy resulted in little growth inhibition, whereas alectinib monotherapy

258

significantly inhibited growth but with residual cells after treatment. The combination of

259

SHP099 and alectinib effectively inhibited cell growth, with fewer residual cells

260

remaining than obtained with either monotherapy (Fig. 3A, left). Similarly, SHP099

17

261

combined with crizotinib effectively inhibited ROS1-rearranged cancer cells (Fig. 3A,

262

center), as also observed with SHP099 combined with osimertinib in EGFR-mutant

263

cancer cells (Fig. 3A, right). Based on the proportion of stained cells, addition of

264

SHP099 to the TKI treatment significantly inhibited residual proliferating cells of265

ALK/ROS1/EGFR-altered NSCLC (Fig. 3B).

266

To determine whether the activity of SHP099 was synergistic or additive, a cell

267

proliferation assay was performed to obtain the CI (Fig. 4). Each TKI, i.e., alectinib for268

ALK (Fig. 4A, left), crizotinib or DS-6051b for ROS1 (Fig. 4A, center and

269

Supplementary Fig. 2C) or osimertinib for EGFR (Fig. 4A, right), effectively inhibited

270

cell proliferation, whereas SHP099 alone had little effect, with a 50% inhibitory271

concentration (IC50) > 10 µM in all of the examined cell lines. However, SHP099

272

shifted the viability curve of cells treated with alectinib, crizotinib/DS-6051b or

273

osimertinib to the left, indicative of a decreased IC50 for these drugs in

274

ALK/ROS1/EGFR-altered cells. In addition, combined use of SHP099 (1 or 5 µM) with

275

alectinib, crizotinib or osimertinib suppressed cell proliferation to a significantly greater276

extent than any of the agents alone (Supplementary Fig. 3). The CI was < 1 in the277

fraction affected (Fa), with a range ≥ 0.5 in all cell lines tested (Fig. 4B), suggesting that278

the TKIs and SHP099 have synergistic effects in these cell lines.

18

279

To confirm that SHP099 acted by inhibiting SHP2 and not through an off-target effect,280

SHP2 was genetically disrupted using siPTPN11. Because siPTPN11 did not281

sufficiently inhibit SHP2 expression in the investigated cells (Supplementary Fig. 4), we

282

used another SHP2 inhibitor RMC4550 (33) (Supplementary Fig. 5). Consistent with

283

SHP099, RMC-4550 enhanced the effects of the TKIs in the investigated cell lines;284

specifically, combined use of RMC-4550 and a TKI yielded greater inhibition of p-ERK

285

and p-AKT than achieved with the TKI alone (Supplementary Fig. 5A) and significantly

286

suppressed cell proliferation (Supplementary Fig. 5B). This result supports the finding

287

that the effects of SHP099 were mediated by SHP2 inhibition, not an off-target activity.

288
289

SHP099 enhances the effects of TKIs on xenograft tumors

290

To examine the combined effects of SHP099 and TKIs in vivo, xenograft models were291

prepared using BALB/c-nu/nu mice. Xenografted tumors derived from ALK-rearranged

292

H3122 were treated with vehicle, SHP099, alectinib or a combination of SHP099 and293

alectinib (Fig. 5). Consistent with the in vitro results (Figs. 3, 4A), SHP099 alone had

294

little effect on tumor growth, SHP099 plus alectinib resulted in significantly greater

295

inhibition of the tumors than SHP099 or alectinib alone, but only the two agents used in296

combination decreased the size of the tumors (Fig. 5). The experiment was then

19

297

repeated using xenografted tumors derived from ROS1-rearranged ABC-20 cells treated298with vehicle, SHP099, crizotinib or their combination. Tumor volume was significantly

299

smaller after combination therapy than after SHP099 or crizotinib monotherapy (Fig. 5).

300

Finally, xenograft tumors derived from EGFR-mutant PC-9 cells were treated with

301

vehicle, SHP099, osimertinib or their combination. Tumor growth was more strongly

302

inhibited by the combination therapy than with either monotherapy (Fig. 5). Increased

303

toxicity was not observed with the combination therapy in any of the xenograft models

304(Supplementary Fig. 6).

305
306

SHP099 is comparable to or more effective than MEK inhibitor in enhancing the307

efficacy of TKIs

308

In EGFR-mutant NSCLC, inhibitors of MEK, a protein downstream of SHP2, enhance309

the effects of EGFR TKIs by increasing the inhibition of ERK activity (18). To compare

310

the inhibitory effect of SHP2 and MEK inhibitors on residual cells, EGFR-mutant

311

NSCLC cells were treated with SHP099 or the MEK inhibitor selumetinib. Either agent,

312

when combined with osimertinib, resulted in more potent inhibition of ERK activity

313

than that achieved with osimertinib alone (Fig. 6A). However, while AKT activity was314strongly suppressed by the combination of SHP099 and osimertinib, the combination of

20

315

selumetinib and osimertinib was not better than osimertinib alone (Fig. 6A). To

316

determine the effect in vivo, xenograft tumors derived from PC-9 cells (EGFR ex19317

deletion) were treated with osimertinib alone, osimertinib plus selumetinib, or

318

osimertinib plus SHP099. The strongest inhibition of tumor growth was obtained with319

the combination of osimertinib and SHP099 (Fig. 6B). A signaling analysis of the320

xenograft tumors (Supplementary Fig. 7A) showed that, unlike the in vitro results,

321

combination therapy did not result in the further inhibition of ERK in the xenograft322

tumor. A possible reason for this difference is that residual cells with persisting ERK

323

cell signaling are more tightly concentrated in the xenograft tumors and thus less324

responsive to combination therapy with SHP2 or selumetinib. A comparison of the325

effects of SHP099 and selumetinib in ALK (H3122) xenograft tumors showed that, as in326

the EGFR mutant tumors, SHP099 was more potent than selumetinib in enhancing the327

effects of TKI therapy, even after 6 weeks of treatment (Fig. 6C). Similar results were328

obtained in ROS1 rearranged ABC-20 xenograft tumors, in which the combination of

329

SHP099 and crizotinib showed the most effective tumor inhibition, although the330

difference was not significant compared to the combination of selumetinib and331

crizotinib (Fig. 6D). There was no difference in the body weights of the xenografted

332

mice (Supplementary Fig. 7B-C).

21

333

Discussion

334This study demonstrated the ability of the allosteric SHP2 inhibitor to enhance335

the effects of TKIs in a model of treatment-naïve ALK/ROS1/EGFR-rearranged NSCLC.

336

SHP2 inhibitors have been evaluated mainly for their ability to overcome

337

acquired resistance in oncogene-driven NSCLC, such as ALK-rearranged or

338

EGFR-mutant NSCLC (34, 35). Standard oncogene-driven NSCLC treatment involves

339

administration of a molecular-targeting agent to overcome acquired resistance. However,

340

that strategy has not been completely successful, in part because tumor heterogeneity341

leads to the development of multiple resistance mechanisms within a single tumor. Thus,

342

SHP2 is an effective target for overcoming acquired resistance because it mediates the343transduction of multiple RTKs to their downstream pathways, such that its inhibition

344

acts on many resistance mechanisms simultaneously. However, some acquired

345

resistance mechanisms, such as the small cell transition or the epithelial-mesenchymal

346

transition, may be difficult to overcome even with an SHP2 inhibitor.

347

Residual cells that persist following molecular-targeted therapies have recently

348

garnered attention as a potential cause of acquired resistance. Although the precise349

mechanisms underlying the survival of residual cells under molecular-targeted therapy

350

remain to be elucidated, reactivated ERK after TKI therapy is thought to play a role (17,351

18). Our study showed that in ALK/ROS1/EGFR-altered cancer cells, ERK remained
22

352

activated or was reactivated even after 48 h of cell exposure to TKIs (Fig. 1B). SHP2

353

mediates RTK signaling, forms a complex with GRB2 and SOS, converts RAS-GDP to

354

RAS-GTP and activates downstream ERK signaling (43, 44). Based on these key

355

activities, treating ALK/ROS1/EGFR-altered cancer cells with the SHP2 inhibitor

356

SHP099 was expected to inhibit remaining ERK activity in the residual cells under TKI

357

therapy. While neither the SHP2 inhibitor nor TKI alone completely suppressed ERK

358

activity in ALK/ROS1/EGFR-altered NSCLC cells, this was achieved by their359

combination. Moreover, combination treatment was effective in inhibiting residual

360

cancer cells both in vitro (Figs. 3, 4) and in vivo (Fig. 5). Supporting our observations,361

combination therapy with erlotinib, an older generation EGFR-TKI, and a SHP2

362

inhibitor led to synergistic inhibition of EGFR-mutant cell proliferation in vitro (37).

363Collectively, our findings indicate that SHP2 inhibition combined with molecular364

targeting agents represent a promising treatment strategy for ALK/ROS1/EGFR-altered

365

NSCLC.

366

SHP099 acts not only on the ERK pathway but also on the PI3K/AKT pathway

367(45, 46) and in this study it suppressed AKT phosphorylation in most of the investigated368

cell lines, especially in ROS1-rearranged HCC78 and ABC-20 cells (Fig. 2 and369

Supplementary Fig. 4). Other studies have reported that SHP099 did not inhibit pAKT

23

370

in EGFR or ALK-altered models (34, 47), which suggests that the impact of an SHP2

371

inhibitor on AKT differs in different cell lines.

372

Combination therapy of osimertinib and the MEK inhibitor selumetinib is373

currently being evaluated in patients with EGFR-mutant NSCLC (19). A comparison of374

the efficacy of SHP099 vs. selumetinib when used in combination with osimertinib in375

the treatment of EGFR-mutant NSCLC showed the superiority of SHP099 (Fig. 6A). Of

376

note, this more potent effect was not limited to EGFR-mutant NSCLC but was also

377

observed in ALK/ROS1-altered NSCLC (Fig. 6C, D). These results imply that combined378

use of a TKI and an SHP2 inhibitor, rather than an MEK inhibitor, is the better379

therapeutic strategy.

380

In conclusion, the results of this study demonstrate that SHP2 plays an

381

important role in maintaining ALK- or ROS1-altered or EGFR-mutant NSCLC cells that382

remain under TKI treatment, and that targeting SHP2 enhances the actions of TKIs.

383

Thus, combination therapy consisting of a TKI and an allosteric SHP2 inhibitor may be384

a novel treatment strategy for TKI-naïve ALK/ROS1/EGFR-altered NSCLC.

385
386

Conflicts of Interest

387

EI received honoraria from Chugai Pharmaceutical. EI received additional research funding

24

388

from Eli Lilly Japan. KO received a research grant from Boehringer-Ingelheim, Japan. KH

389

received honoraria and research funding from Chugai Pharmaceutical. YM received honoraria

390

from Chugai Pharmaceutical. KK received honoraria from Chugai Pharmaceuticals. All other391

authors declare no conflicts of interest regarding this study.

392
393

Acknowledgements

394

We are grateful to Hiromi Nakashima and Kyoko Maeda for their technical support.

395

25

396

References

397

1.

398

in East Asian patients with ROS1-positive advanced non-small-cell lung cancer. J Clin Oncol.

399

2018;36(14):1405-11.

400

2.

401

ROS1-rearranged non-small-cell lung cancer. N Engl J Med. 2014;371(21):1963-71.

402

3.

403

crizotinib in untreated ALK-positive non-small-cell lung cancer. N Engl J Med.

404

2017;377(9):829-38.

405

4.

406

crizotinib

407

2014;371(23):2167-77.

408

5.

409

et al. Osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer. N Engl

410

J Med. 2018;378(2):113-25.

411

6.

412

as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell413

lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study.

414

Lancet Oncol. 2011;12(8):735-42.

415

7.

416

resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second

417

mutation in the EGFR kinase domain. PLoS Med. 2005;2(3):e73.

418

8.

419

amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.

420

Science. 2007;316(5827):1039-43.

421

9.

422

with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but

423lack mutations in KRAS, NRAS, or MEK1. Proc Natl Acad Sci U S A. 2012;109(31):E2127-33.

424

10.

425

Molecular mechanisms of resistance to first- and second-generation ALK inhibitors in

426

ALK-rearranged lung cancer. Cancer Discov. 2016;6(10):1118-33.

427

11.

428

cell lung cancer cells acquire resistance to the ALK inhibitor alectinib by activating429

alternative receptor tyrosine kinases. Cancer Res. 2016;76(6):1506-16.

430

12.

Wu YL, Yang JC, Kim DW, Lu S, Zhou J, Seto T, et al. Phase II study of crizotinib

Shaw AT, Ou SH, Bang YJ, Camidge DR, Solomon BJ, Salgia R, et al. Crizotinib inPeters S, Camidge DR, Shaw AT, Gadgeel S, Ahn JS, Kim DW, et al. Alectinib versus

Solomon BJ, Mok T, Kim DW, Wu YL, Nakagawa K, Mekhail T, et al. First-line
versuschemotherapyinALK-positivelungcancer.

N

Engl

J

Med.

Soria JC, Ohe Y, Vansteenkiste J, Reungwetwattana T, Chewaskulyong B, Lee KH,

Zhou C, Wu YL, Chen G, Feng J, Liu XQ, Wang C, et al. Erlotinib versus chemotherapy

Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al. Acquired

Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al. MET

Ohashi K, Sequist LV, Arcila ME, Moran T, Chmielecki J, Lin YL, et al. Lung cancers

Gainor JF, Dardaei L, Yoda S, Friboulet L, Leshchiner I, Katayama R, et al.

Isozaki H, Ichihara E, Takigawa N, Ohashi K, Ochi N, Yasugi M, et al. Non-small

Mok TS, Wu YL, Ahn MJ, Garassino MC, Kim HR, Ramalingam SS, et al. Osimertinib

26

431

or

432

2017;376(7):629-40.

433

13.

434

mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med.

435

2005;352(8):786-92.

436

14.

437

crizotinib-refractory ALK-rearranged non-small-cell lung cancer: A phase II global study.

438

J Clin Oncol. 2016;34(7):661-8.

439

15.

440

of ALK mutations mediating ALK-TKIs resistance and drug re-purposing to overcome the441resistance. EBioMedicine. 2019;41:105-19.

442

16.

443

new-generation selective ROS1/NTRK inhibitor DS-6051b overcomes crizotinib resistant

444

ROS1-G2032R mutation in preclinical models. Nat Commun. 2019;10(1):3604.

445

17.

446

attenuates osimertinib efficacy in both drug-sensitive and drug-resistant models of447

EGFR-mutant lung cancer. Cancer Res. 2017;77(11):2990-3000.

448

18.

449

inhibition prevents the emergence of resistance in EGFR-mutant lung cancer. Cancer Discov.

450

2015;5(9):960-71.

451

19.

452

phase Ib trial of osimertinib combined with selumetinib, savolitinib, or durvalumab in

453

EGFR-mutant lung cancer. Ann Oncol. 2020;31(4):507-16.

454

20.

455

Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan

456

syndrome. Nat Genet. 2001;29(4):465-8.

457

21.

458

Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood.

459

2004;103(6):2325-31.

460

22.

461

enhances liver cancer progression and predicts poor prognosis of patients. J Hepatol.

462

2015;63(3):651-60.

463

23.

464

mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and465

acute myeloid leukemia. Nat Genet. 2003;34(2):148-50.

466

24.

platinum-pemetrexed

in

EGFR

T790M-positivelungcancer.

N

Engl

J

Med.

Kobayashi S, Boggon TJ, Dayaram T, Jänne PA, Kocher O, Meyerson M, et al. EGFR

Ou SH, Ahn JS, De Petris L, Govindan R, Yang JC, Hughes B, et al. Alectinib in

Okada K, Araki M, Sakashita T, Ma B, Kanada R, Yanagitani N, et al. Prediction

Katayama R, Gong B, Togashi N, Miyamoto M, Kiga M, Iwasaki S, et al. The

Ichihara E, Westover D, Meador CB, Yan Y, Bauer JA, Lu P, et al. SFK/FAK Signaling

Tricker EM, Xu C, Uddin S, Capelletti M, Ercan D, Ogino A, et al. Combined EGFR/MEK

Oxnard GR, Yang JC, Yu H, Kim SW, Saka H, Horn L, et al. TATTON: a multi-arm,

Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, Kremer H, et al.

Loh ML, Vattikuti S, Schubbert S, Reynolds MG, Carlson E, Lieuw KH, et al.

Han T, Xiang DM, Sun W, Liu N, Sun HL, Wen W, et al. PTPN11/Shp2 overexpression

Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, Jung A, et al. Somatic

Cai P, Guo W, Yuan H, Li Q, Wang W, Sun Y, et al. Expression and clinical

27

467

significance of tyrosine phosphatase SHP-2 in colon cancer. Biomed Pharmacother.

468

2014;68(3):285-90.

469

25.

470

et al. Tyrosine phosphatase SHP2 promotes breast cancer progression and maintains

471

tumor-initiating cells via activation of key transcription factors and a positive feedback472

signaling loop. Nat Med. 2012;18(4):529-37.

473

26.

474

novel therapeutic target for melanoma treatment. Oncotarget. 2016;7(45):73817-29.

475

27.

476

markers in non-small cell lung cancer: a tissue microarray study of 80 cases. Appl

477

Immunohistochem Mol Morphol. 2013;21(5):386-94.

478

28.

479

et al. SHP2 is required for growth of KRAS-mutant non-small-cell lung cancer in vivo. Nat

480

Med. 2018;24(7):961-7.

481

29.

482

SHP2 drives adaptive resistance to ERK signaling inhibition in molecularly defined subsets

483

of ERK-dependent tumors. Cell Rep. 2019;26(1):65-78.e5.

484

30.

485

inhibition of SHP2 and MEK is effective in models of NF1-deficient malignant peripheral

486

nerve sheath tumors. Cancer Res. 2020;80(23):5367-79.

487

31.

488

al. NRAS status determines sensitivity to SHP2 inhibitor combination therapies targeting489

the RAS-MAPK pathway in neuroblastoma. Cancer Res. 2020;80(16):3413-23.

490

32.

491

overcomes RTK-mediated pathway reactivation in KRAS-mutant tumors treated with MEK492

inhibitors. Mol Cancer Ther. 2019;18(7):1323-34.

493

33.

494

nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and

495

RAS-driven cancers. Nat Cell Biol. 2018;20(9):1064-73.

496

34.

497

restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK

498

inhibitors. Nat Med. 2018;24(4):512-7.

499

35.

500

inhibitor,

501

mechanisms toward osimertinib. Cancer Res. 2020;80(21):4840-53.

502

36.

Aceto N, Sausgruber N, Brinkhaus H, Gaidatzis D, Martiny-Baron G, Mazzarol G,

Zhang RY, Yu ZH, Zeng L, Zhang S, Bai Y, Miao J, et al. SHP2 phosphatase as aTang C, Luo D, Yang H, Wang Q, Zhang R, Liu G, et al. Expression of SHP2 and related

Mainardi S, Mulero-Sánchez A, Prahallad A, Germano G, Bosma A, Krimpenfort P,

Ahmed TA, Adamopoulos C, Karoulia Z, Wu X, Sachidanandam R, Aaronson SA, et al.

Wang J, Pollard K, Allen AN, Tomar T, Pijnenburg D, Yao Z, et al. Combined

Valencia-Sama I, Ladumor Y, Kee L, Adderley T, Christopher G, Robinson CM, et

Lu H, Liu C, Velazquez R, Wang H, Dunkl LM, Kazic-Legueux M, et al. SHP2 inhibition

Nichols RJ, Haderk F, Stahlhut C, Schulze CJ, Hemmati G, Wildes D, et al. RAS

Dardaei L, Wang HQ, Singh M, Fordjour P, Shaw KX, Yoda S, et al. SHP2 inhibition

Sun Y, Meyers BA, Czako B, Leonard P, Mseeh F, Harris AL, et al. Allosteric SHP2
IACS-13909,

overcomes EGFR-dependent and EGFR-independent

resistance

Liu C, Lu H, Wang H, Loo A, Zhang X, Yang G, et al. Combinations with allosteric

28

503

SHP2 inhibitor TNO155 to block receptor tyrosine kinase signaling. Clin Cancer Res.

504

2021;27(1):342-54.

505

37.

506

D,

507

mutation-positive508

2020;1(4):100084.

509

38.

510

effect of cabozantinib and gefitinib in crizotinib-resistant lung tumors harboring ROS1

511

fusions. Cancer Sci. 2018;109(10):3149-58.

512

39.

513

to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63.

514

40.

515

combinations: a review. Acta Paediatr Taiwan. 2000;41(6):294-302.

516

41.

517

inhibitions of Michaelis-Menten kinetic systems. J Biol Chem. 1977;252(18):6438-42.

518

42.

519

Chou-Talalay method. Cancer Res. 2010;70(2):440-6.

520

43.

521

tyrosine phosphatase function in promoting growth factor stimulation of Erk activity. Mol

522

Cell Biol. 2000;20(5):1526-36.

523

44.

524

Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway. Cell Signal.

525

2008;20(3):453-9.

526

45.

527

phosphatidylinositide 3'-kinase/Akt pathway and suppresses caspase 3-mediated apoptosis.

528

J Cell Physiol. 2004;199(2):227-36.

529

46.

530

2 mediates cisplatin-related drug resistance by inhibiting apoptosis and activating the531

Ras/PI3K/Akt1/survivin pathway in lung cancer cells. Oncol Rep. 2018;39(2):611-8.

532

47.

533

Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine

534

kinases. Nature. 2016;535(7610):148-52.

Ito M, Codony-Servat J, Giménez-Capitán A, Serra-Mitjans M, Pérez-Ochoa F, Lligeetal.

Src-homology
lung2

domain-containing

adenocarcinoma.

JTO

phosphatase
Clinical

2

and

in

resected

Research

EGFR

Reports.

Kato Y, Ninomiya K, Ohashi K, Tomida S, Makimoto G, Watanabe H, et al. Combined

Mosmann T. Rapid colorimetric assay for cellular growth and survival: application

Chang TT, Chou TC. Rational approach to the clinical protocol design for drug

Chou TC, Talaly P. A simple generalized equation for the analysis of multiple

Chou TC. Drug combination studies and their synergy quantification using theShi ZQ, Yu DH, Park M, Marshall M, Feng GS. Molecular mechanism for the Shp-2

Dance M, Montagner A, Salles JP, Yart A, Raynal P. The molecular functions ofIvins Zito C, Kontaridis MI, Fornaro M, Feng GS, Bennett AM. SHP-2 regulates theTang C, Luo H, Luo D, Yang H, Zhou X. Src homology phosphotyrosyl phosphatase

Chen YN, LaMarche MJ, Chan HM, Fekkes P, Garcia-Fortanet J, Acker MG, et al.

535
536

29

537

Figure Legends

538

Figure 1. Residual cells of ALK/ROS1/EGFR-altered NSCLC following TKI

539

therapy.

540(A) The cells were treated for 48 h with DMSO or a TKI. H3122 cells were treated with541

alectinib (Alec, 1 µM), HCC78 cells with crizotinib (Cri, 5 µM) and PC-9 cells with542

osimertinib (Osi, 1 µM). (B) Immunoblots of the indicated proteins in H3122 and543

ABC-19 cells treated with DMSO for 4 h or with alectinib (100 nM) for 4, 24 or 72 h;

544

HCC78 and ABC-20 cells treated with DMSO for 4 h or crizotinib (1 µM) for 4, 24 or

545

72 h and PC-9 and HCC827 cells treated with DMSO for 4 h or osimertinib (100 nM)

546

for 4, 24 or 72 h.

547
548

Figure 2. Inhibition of ERK and AKT activities by SHP099 combined with a TKI.

549

Immunoblots of the indicated proteins in H3122 and ABC-19 cells treated with alectinib

550(Alec, 100 nM), HCC78 and ABC-20 cells treated with crizotinib (Cri, 1 µM) and PC-9

551

and HCC827 cells treated with osimertinib (Osi, 100 nM) for 24 h. All cell lines were552

also treated with SHP099 (SHP, 5 µM) for 24 h. Comb, combination.

553
554

Figure 3. Inhibition of cancer cell proliferation by SHP099 combined with a TKI.

30

555

(A) Crystal-violet-stained cells after drug loading for 6 days. Cancer cells were treated556

with 1 or 5 µM SHP099 combined with 100 nM alectinib (H3122 and ABC-19), 1 µM

557

crizotinib (HCC78 and ABC-20) or 100 nM osimertinib (PC-9 and HCC827) for 6 days.

558

Alec, alectinib; Cri, crizotinib; Osi, osimertinib. (B) The crystal-violet-stained cells

559

were quantified (n=3). Error bars represent the standard error. *P < 0.01 (one-way

560

ANOVA and Bonferroni method). S, SHP099; A, alectinib; C, crizotinib; O,

561

osimertinib.

562
563

Figure 4. Enhanced effect of SHP099 on cell viability inhibition by TKIs in vitro.

564(A) Cell viability curves for cell lines treated with the molecular-targeted drug alone

565(circles), SHP099 alone (triangles) or the molecular-targeted drug and SHP099 (5 µM)

566(squares). All drugs were loaded for 96 h. Error bars represent the standard error. SHP,

567

SHP099; Alec, alectinib; Cri, crizotinib; Osi, osimertinib. (B) In the ALK-rearranged568cell lines (H3122 and ABC-19), the ratio of the alectinib and SHP099 concentrations569

was 1:50. In the ROS1-rearranged cell lines (HCC78 and ABC-20), the ratio of the570

crizotinib and SHP099 concentrations was 1:5. In the EGFR-mutant cell lines (PC-9 and571

HCC827 cells), the ratio of the osimertinib and SHP099 concentrations was 1:50. CI,

572

combination index; Fa, fraction affected.

31

573
574

Figure 5. Combination of a TKI and an SHP2 inhibitor in a xenograft mouse575

model. Mice transplanted with H3122, ABC-20 or PC-9 cells were orally administered

576

TKIs and SHP099 (50 mg/kg/day) five times weekly. Mice bearing H3122 tumors were577

treated with vehicle, alectinib (10 mg/kg/day), SHP099 or alectinib plus SHP099. Mice

578

bearing ABC-20 tumors were treated with vehicle, crizotinib (50 mg/kg/day), SHP099

579

or crizotinib plus SHP099. Mice bearing PC-9 tumors were treated with vehicle,580

osimertinib (5 mg/kg/day), SHP099 or osimertinib plus SHP099. Error bars represent581

the standard error. *P < 0.01 (Student’s t-test).

582
583

Figure 6. Comparison of the effects of SHP099 vs. selumetinib. (A) Immunoblots of584

the indicated proteins in PC-9 cells treated for 24 h with osimertinib (Osi, 100 nM),

585

selumetinib (Selu, 500 nM) or SHP099 (SHP, 5 µM). p-AKT was quantified using

586

ImageJ software. (B) Mice bearing PC-9 tumors were treated with osimertinib (5587

mg/kg/day), osimertinib plus selumetinib (50 mg/kg/day) or osimertinib plus SHP099

588(50 mg/kg/day). (C) Mice bearing H3122 tumors were treated with alectinib (10589

mg/kg/day), alectinib plus selumetinib (50 mg/kg/day) or alectinib plus SHP099 (50590

mg/kg/day). (D) Mice bearing ABC-20 tumors were treated with crizotinib (50

32

591

mg/kg/day), crizotinib plus selumetinib (50 mg/kg/day) or crizotinib plus SHP099 (50592

mg/kg/day). All drugs were orally administered five times weekly. Error bars represent593

the standard error. *P < 0.05 (Student’s t-test).

594

33