key: cord-0325980-4972o82u
authors: Alam, Sk. Kayum; Zhang, Yongchang; Wang, Li; Zhu, Zhu; Hernandez, Christina E.; Zhou, Yuling; Yang, Nong; Lei, Jian; Chen, Xiaoyan; Zeng, Liang; Klein, Mark A.; Hoeppner, Luke H.
title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma
date: 2021-02-13
journal: bioRxiv
DOI: 10.1101/2021.02.12.430856
sha: 561b19a319e3f1dea5487b40428f5d0ceaca64b4
doc_id: 325980
cord_uid: 4972o82u

While molecular targeted therapies have improved prognoses of advanced stage lung adenocarcinoma expressing oncogenic driver mutations, acquired therapeutic resistance continues to be a major problem. Epidermal growth factor receptor (EGFR) activating mutations are among the most common targetable genetic alterations in lung adenocarcinoma, and EGFR tyrosine kinase inhibitors (TKIs) are recommended first-line therapy for EGFR mutation positive cancer patients. Unfortunately, most patients develop resistance to EGFR TKIs and rapid disease progression occurs. A better mechanistic understanding of therapy refractory cancer progression is necessary to develop new therapeutic approaches to predict and prevent acquired resistance to EGFR TKIs. Here, we identify a new mechanism of ERBB3-mediated resistance to EGFR TKIs in human lung adenocarcinoma. Specifically, we show that dopamine and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32) physically recruits ERBB3 to EGFR to mediate a switch from EGFR homodimers to EGFR:ERBB3 heterodimers to bypass EGFR TKI-mediated inhibition to potentiate ERBB3-dependent activation of oncogenic AKT and ERK signaling that drives therapy refractory tumor cell survival. In a cohort of paired tumor specimens derived from 30 lung adenocarcinoma patients before and after the development of EGFR TKI refractory disease progression, we reveal that DARPP-32 as well as kinase-activated EGFR and ERBB3 proteins are overexpressed upon acquired EGFR TKI resistance. In vivo studies suggest that ablation of DARPP-32 protein activity sensitizes gefitinib-resistant lung tumor xenografts to EGFR TKI treatment, while DARPP-32 overexpression increases gefitinib-refractory lung cancer progression in gefitinib-sensitive lung tumors orthotopically xenografted into mice. Taken together, our findings introduce a DARPP-32-mediated, ERBB3-dependent mechanism used by lung tumor cells to evade EGFR TKI-induced cell death, potentially paving the way for the development of new therapies to prevent or overcome therapy-refractory lung adenocarcinoma progression.

Email: hoepp005@umn.edu 27 28 Sk. Kayum Alam, Ph.D. 29 The Hormel Institute, University of Minnesota 30 801 16th Avenue NE 31

Austin, MN 55912 32

Phone: +1 (507) 355-5223 33

Email: skalam@umn.edu 34 35 36 37 38 Introduction gastric cancer. The N-terminally truncated isoform of DARPP-32, termed t-DARPP, was found to utilize a 122 unique alternative first exon located within intron 1 of phosphoprotein phosphatase-1 regulatory subunit 1B 123 (PPP1R1B), the gene that transcribes DARPP-32 and t-DARPP proteins 35 . t-DARPP lacks the first 36 amino 124 acids of DARPP-32, including the T34 phosphorylation residue required for DARPP-32-mediated PP-1 125 inhibition 35 . Elevated expression of t-DARPP isoform in NSCLC is associated with poor overall survival and 126

increasing tumor (T) stage 30 . Our findings presented in this report suggest that overexpression of DARPP-32 127 isoforms in EGFR-mutated NSCLC promotes EGFR:ERBB3 "bypass signaling" that enables tumor cells to 128 evade EGFR TKI monotherapy-induced apoptosis by potentiating oncogenic AKT and ERK signaling. 129 cycles, and fed a standard diet. Eight-to twelve-week-old male and female mice were anesthetized with 260 pharmaceutical grade ketamine (90-120 mg/kg) and xylazine (5-10 mg/kg) via intraperitoneal injection under a 261 laminar flow hood in an SPF room within the animal facility. Each fully anesthetized mouse was placed in the 262 right lateral decubitus position and the left lateral chest was sterilized. One-million luciferase-labeled human 263 HCC827GR and HCC827P lung cancer cells suspended in 80 μl PBS and high concentration Matrigel 264 (Corning; Cat. no.: 354248) were orthotopically injected in the left thoracic cavity of each mouse. Based on the 265 captured luminescence images of mice using an In-Vivo Xtreme xenogen imaging system (Bruker) as 266 described 28 , mice were randomly divided into two groups with nearly same average luminescence intensity. 267

After establishment of the lung tumor, mice were administered either vehicle or gefitinib (25mg/Kg) every other 268 day for 2 weeks. Upon completion of the study, mice were euthanized using asphyxiation by CO2 inhalation to 269 effect with a flow rate displacing less than 30% of the chamber volume per minute in accordance with IACUC 

Eight-to twelve-week-old male and female mice were subcutaneously injected with 2×10 6 luciferase-labeled 279 human PC9P lung cancer cells suspended in 80 μl PBS and high concentration Matrigel. To determine tumor 280 growth, the tumor volume was measured every week using the formula: (length x width 2 )/2. After establishment 281 of palpable tumor (≥150mm 3 ), mice were randomly divided into two groups and administered either vehicle or 282 gefitinib (25mg/Kg). At the endpoint, mice were euthanized by CO2 asphyxiation. Extirpated tumors were 283 photographed, weighed, and preserved in formalin for immunohistochemistry analysis. This study was 284 performed in accordance with approved University of Minnesota IACUC protocols. 285

Patients who met the following criteria were enrolled in this study: (1) pathologically confirmed advanced lung 288 adenocarcinoma; (2) NGS identified EGFR exon 21 L858R mutation; (3) treatment with gefinitib or erlotinib in 289 the first-line setting; and (4) accessed with disease progression and available tumor sample at baseline and 290 progression. Patients were examined every two weeks after EGFR TKI administration and 20% incensement of 291 tumor burden is considered as disease progression according to RECIST 

To compare differences between two groups, two-way unpaired t-test was performed and values of P ≤0.05 308 were considered significant. One-way analysis of variance (ANOVA) followed by Dunnett's test was used to 309 determine statistically significant differences between multiple groups (greater than two). Data expressed as 310 mean ±SEM are representative of at least three independent experiments. 311

The authors declare that the data supporting the findings of this study are available within the article and its 313 supplementary information. 314

Given the ability of DARPP-32 to modulate oncogenic signaling 50, 51 , we hypothesized that DARPP-32 318 contributes to acquired EGFR TKI resistance in NSCLC. To test this hypothesis, we utilized two well-319 characterized NSCLC models of EGFR TKI resistance. Gefitinib-resistant HCC827 (EGFR ΔE746-A750 ) human 320 NSCLC cells were previously generated through six months of exposure to increasing concentrations of 321 gefitinib and shown to have acquired gefitinib resistance through a c-MET amplification 43 . Secondly, we relied 322 on gefitinib-sensitive PC9 (EGFR L858R ) human NSCLC cells and their corresponding PC9 gefitinib-resistant 323 (PC9GR2 and PC9GR3) counterparts, which acquired gefitinib resistance through a secondary EGFR T790M 324 mutation following prolonged parental cell exposure to this first-generation EGFR TKI 44 . We observed reduced 325 DARPP-32 protein levels in gefitinib-sensitive, HCC827 parental (HCC827P) and PC9 parental (PC9P) cells 326 upon treatment with EGFR inhibitor, gefitinib ( Fig. 1a-b; Supplementary Fig. 1a-b) . Based on this result, we 327 sought to examine DARPP-32 protein levels in gefitinib-resistant EGFR-mutated NSCLC cells. By 328 immunoblotting, we observed elevated DARPP-32 protein expression in gefitinib-resistant cells relative to 329 parental counterparts (Supplementary Fig. 2a-b) . 330

Given that DARPP-32 is upregulated in gefitinib-resistant NSCLC cells, we designed experiments to assess 332 the functional effects of DARPP-32 overexpression in the presence of EGFR TKI. We stably silenced DARPP-333 32 protein expression in HCC827GR cells via lentiviral-mediated transduction of two previously validated 334 Collectively, our findings suggest DARPP-32 reduces gefitinib-induced apoptosis of EGFR-mutated NSCLC 356

We next sought to determine the molecular basis of DARPP-32-mediated cell survival in the presence of 359 EGFR inhibition. DARPP-32 has been shown to promote resistance of gastric cancer cells to EGFR inhibitors 360 by promoting an interaction between EGFR and ERBB3, which drives PI3K-AKT signaling 53 to "bypass" EGFR 361 TKI resistance. Importantly, we observe concomitant decreases in DARPP-32 protein expression and 362 phosphorylation of EGFR, ERBB2, and ERBB3 over time when gefitinib-sensitive HCC827P and PC9P cells 363

were treated with various doses of gefitinib ( Fig. 1a-b; Supplementary Fig. 1a-b) . We next replicated these 364 PC9GR3 all express high levels of DARPP-32, p-EGFR, and p-ERBB3 upon treatment with 100 nM gefitinib 368 ( Fig. 1c-d) . Total ERBB3 protein levels were markedly increased upon EGFR TKI treatment in parental cells 369 and these high ERBB3 levels were maintained in gefitinib-treated HCC827GR, PC9GR2, and PC9GR3 cells ( Fig. 1c-d) . Changes observed in p-ERBB2 and total ERBB2 protein expression were less uniformly consistent 371 across the two resistance models, HCC827 and PC9 (Fig. 1c-d) , and ERBB4 protein was undetectable in 372 these cells (data not shown). Taken together, our observations suggest that upregulation of p-ERBB3, total 373 ERBB3 and total DARPP-32 protein levels positively correlate with an EGFR TKI resistance phenotype in 374 EGFR-mutated NSCLC cells. 375

Given that DARPP-32 is overexpressed and ERBB3 is activated during gefitinib treatment in EGFR-mutated 377 NSCLC cells, we propose a mechanism of acquired resistance to EGFR TKIs in NSCLC, in which DARPP-32 378 mediates a switch from EGFR TKI-sensitive EGFR homodimers to TKI-resistant EGFR:ERBB3 heterodimers. 379

This hypothesis is supported by findings showing that the physical association of EGFR and ERBB3 promotes 380 resistance to gefitinib in NSCLC 54 . To test this hypothesis, we assessed the phosphorylation status of EGFR 381

and ERBB3 by immunofluorescence studies in EGFR TKI-sensitive PC9P human NSCLC cells overexpressing 382 DARPP-32 or t-DARPP upon gefitinib treatment. We observed a substantial reduction of p-EGFR intensity in 383 gefitinib-treated LacZ-overexpressed control PC9P cells, whereas p-EGFR intensity in EGFR-mutated cells 384

overexpressing DARPP-32 isoforms remained unchanged upon gefitinib treatment ( Fig. 3a-b) . Overexpression 385 of DARPP-32 and t-DARPP promotes increased p-ERBB3 upon EGFR TKI treatment (Fig. 3a,c) , suggesting 386 DARPP-32 upregulation may be associated with increased activation of ERBB3 in the presence of EGFR TKIs. 387

We next asked whether stable shRNA-mediated depletion of DARPP-32 in gefitinib-resistant PC9GR3 cells 388 affects phosphorylation of ERBB3 and EGFR upon gefitinib treatment. We observed a decrease in p-ERBB3 389 expression in gefitinib-treated DARPP-32-ablated PC9GR3 cells, whereas changes of p-ERBB3 levels were 390 not detectable in corresponding LacZ shRNA control PC9GR3 cells upon treatment with gefitinib 391 ( Supplementary Fig. 7a,c) . Others have reported that p-EGFR is not responsive to gefitinib in PC9GR3 cells 44 . 392

Correspondingly, knockdown of DARPP-32 in gefitinib-treated PC9GR3 cells did not affect p-EGFR levels 393

(Supplementary Fig. 7a-b) . Collectively, our results demonstrating changes in activation of ERBB3 upon 394 DARPP-32 modulation in the presence of EGFR TKI support a model in which DARPP-32 contributes to 395 ERBB3-driven "bypass signaling" to promote EGFR-mutated NSCLC cell survival. 396

To better understand the mechanism of resistance to gefitinib in EGFR-mutated NSCLC cells, we aimed to 399 determine how DARPP-32 activates ERBB3 signaling to suppress gefitinib-mediated EGFR inhibition. To 400 address our hypothesis that DARPP-32 drives EGFR:ERBB3 heterodimerization to evade EGFR TKI-mediated 401 cell death, we performed immunoprecipitation studies to assess potential EGFR and ERBB3 interactions in 402 DARPP-32-modulated EGFR-mutated NSCLC cells. Immunoprecipitation using anti-ERBB3 antibody 403 demonstrates that EGFR and ERBB3 physically interact and that the EGFR:ERBB3 association increases 404 upon overexpression of DARPP-32 isoforms in HCC827P and PC9P cells ( Fig. 4a- 

Given that ERBB3 has limited kinase activity and relies on heterodimerization with EGFR for activation 55 , we 417 postulate that DARPP-32 promotes ERBB3 phosphorylation by increasing physical association between p-418 EGFR and p-ERBB3. To address our theory, we performed proximity ligation assay (PLA) using anti-p-EGFR 419 and anti-p-ERBB3 antibodies in PC9GR3 cells. PLA is a powerful tool for identifying protein-protein interaction 420 in situ with high specificity and sensitivity. Our PLA findings suggest that gefitinib treatment induces p-EGFR/p-421 ERBB3 heterodimer complex formation in PC9GR3 cells ( Fig. 5a-b) . However, ablation of DARPP-32 in 422 PC9GR3 cells abolishes gefitinib-induced p-EGFR/p-ERBB3 dimerization, suggesting DARPP-32 plays a 423 significant role in the formation of these active heterodimers ( Fig. 5a-b) . We next sought to determine how DARPP-32 regulates MEK/ERK and PI3K/AKT signaling pathways in the presence of gefitinib. It has been 425 reported that ligand-independent EGFR activation initiates intracellular signaling via Ras/Raf/MEK/ERK and 426 PI3K/AKT signaling pathways 13 . We show by immunoblotting that overexpression of DARPP-32 isoforms 427 increases p-AKT and p-ERK expression in gefitinib-treated sensitive cells (Fig. 6a) . Knockdown of DARPP-32 428 reduces p-AKT and p-ERK expression in gefitinib-treated resistant cells (Fig. 6b) . EGFR-dependent PI3K 429 activation requires dimerization with the ERBB3 receptor because docking sites of PI3K (i.e. p85 subunit) are 430 abundant on ERBB3 and absent within EGFR 13 . To test our hypothesis that DARPP-32 activates the PI3K 431 signaling pathway in EGFR-mutated NSCLC cells, we used a bioinformatics approach to assess DARPP-32 432 transcript expression in specimens derived from 80 EGFR-mutated NSCLC patients cataloged in The Cancer 433

Genome Atlas (TCGA). We first subdivided patient-derived specimens into two groups based on high versus 434 low DARPP-32 mRNA expression ( Supplementary Fig. 8a ). Interestingly, we found that expression of 435 RPS6KB2 transcripts, but not RPS6KB1 transcripts, increases in lung tumor specimens with high expression of 436 DARPP-32 ( Supplementary Fig. 8b-c) 

Based on our findings suggesting that DARPP-32 increases ERBB3 phosphorylation to bypass gefitinib-448

induced EGFR inhibition, we next sought to understand whether DARPP-32 drives NSCLC resistance to EGFR 449

TKIs in vivo. To this end, we tested whether DARPP-32 ablation increases EGFR TKI sensitivity in a gefitinib-450 resistant orthotopic xenograft mouse model. Briefly, we injected luciferase-labeled human gefitinib-resistant 451 (HCC827GR) NSCLC cells into the left thorax of anesthetized SCID mice, confirmed establishment of lung tumors via luciferase imaging, administered gefitinib over the course of two weeks, and measured tumors 453 through non-invasive luciferase imaging (Fig. 7a ). Mice challenged with HCC827GR cells with DARPP-32 454 stably silenced by shRNA show decreased tumor growth when treated every other day with gefitinib relative to 455 vehicle controls ( Fig. 7b; Supplementary Fig. 9a ). DARPP-32 knockdown sensitizes gefitinib-resistant NSCLC 456 tumors to EGFR inhibition in vivo, whereas no such effect was observed in mice challenged with control LacZ 457 shRNA transduced HCC827GR cells ( Fig. 7b; Supplementary Fig. 9a ). Histological sections from these mice 458

were immunostained for Ki-67. We observed decreased tumor cell proliferation in the lungs of gefitinib-treated 459 mice challenged with DARPP-32-silenced HCC287GR cells (Fig. 7c) , confirming that DARPP-32 knockdown 460 enhances EGFR TKI-induced anti-cancer effects in gefitinib-resistant tumors in vivo. 461

We next sought to determine whether overexpression of DARPP-32 isoforms promotes resistance to gefitinib 462 in vivo. Gefitinib-sensitive HCC827 parental (HCC827P) tumors overexpressing DARPP-32 or t-DARPP that 463

were implanted orthotopically into the lungs of mice exhibit gefitinib resistance relative to controls ( 

To investigate the clinical relevance of DARPP-32 given its role in promoting resistance to EGFR first-476 generation TKIs in mouse models of human NSCLC, we assessed DARPP-32, p-EGFR, total EGFR, p-477 ERBB3, and total ERBB3 protein expression by immunostaining in paired EGFR TKI-naïve and -resistant 478 specimens from 30 lung adenocarcinoma patients (Supplementary Table 1 ). Briefly, lung tumor specimens were biopsied from lung adenocarcinoma patients before EGFR TKI treatment (i.e. baseline) and following the 480 development of progressive disease after first-line gefitinib or erlotinib therapy. For immunostaining of each 481 protein, three pathologists independently scored the percentage of tumor cells staining positive and 482 corresponding staining intensity (i.e., 0 = none, 1 = weak, 2 = moderate, 3 = strong expression). We calculated 483 an immune reactive (IR) score for each specimen based on the percentage of tumor cells staining positive and 484 the staining intensity in those cells (IR score = percentage of tumor cells x staining intensity). We found that 485 DARPP-32, kinase-activated EGFR, total EGFR, kinase-activated ERBB3, and total ERBB3 proteins are 486 upregulated in 1st generation EGFR TKI-resistant NSCLC patient-derived specimens relative to individual 487 patient-matched (i.e. paired) baseline samples biopsied prior to frontline gefitinib or erlotinib treatment (Fig 9a-488 b). Collectively, our results suggest that DARPP-32 overexpression and increased EGFR and ERBB3 489 activation is associated with EGFR TKI resistance in NSCLC patients. Lung cancer is the deadliest and most frequently diagnosed type of tumor worldwide, with 1.6 million deaths 494 reported annually 57 . The molecular targeting of specific oncogenic drivers has emerged as a major 495 advancement in the treatment of NSCLC. Patients diagnosed with advanced non-squamous cell NSCLC are 496 tested for oncogenic alternations and treated accordingly 20, 58 . Single oncogenic driver mutations in EGFR that 497 confer sensitivity to TKIs are the most common targetable molecular alteration in lung adenocarcinoma. 498

Although EGFR mutation positive patients initially respond well to EGFR TKI therapy, most patients inevitably 499 develop resistance and experience rapid advanced disease progression. Developing acquired resistance to 500 lung cancer therapy is a major problem. The development of effective strategies to circumvent the emergence 501 of this resistance is needed to improve survival rates and the quality of life of NSCLC patients. 502

The spectrum of identified EGFR resistance mechanisms includes on-target EGFR gatekeeper mutations (i.e. . ERBB3, specifically, has been implicated in the initiation of EGFR TKI resistance. Unlike its fellow family 514 members, ERBB3 was initially believed to be an inactive kinase because its kinase domain lacks certain 515 residues known to be essential for catalytic activity 67 . However, ERBB3 forms heterodimers with other ERBB 516 family members to become transphosphorylated and transactivated to sustain transduction of downstream 517 oncogenic signaling that would otherwise be inhibited by EGFR TKIs acting upon EGFR homodimers [68] [69] [70] . 518

Several known mechanisms of ERBB3-induced TKI resistance exist by which ERBB3 compensates for TKI-inhibited EGFR to trigger and sustain PI3K/Akt signal transduction. First, MET amplification has been shown to 520 result in constitutive activation of ERBB3 signaling to promote gefitinib resistance in lung cancer cell lines 43 . 521

Second, ERBB3 heterodimerization with ERBB2 has been demonstrated to drive oncogenic signaling in breast 522 cancer 71 as the effects of ERBB2 inhibition could be reversed by increasing ERBB3 phosphorylation and 523 activity to drive a TKI resistance phenotype 70 . Third, ligand-mediated activation of ERBB3 has been shown to 524 result in PI3K/Akt-mediated resistance to TKIs in a variety of cancers, including ERBB2-amplified breast 525 cancer cells stimulated with ERBB3 ligands, NRG1 72 or HRG 73 . We identify a new mechanism of ERBB3-526 mediated TKI resistance in which DARPP-32 physically stimulates this process of EGFR:ERBB3 heterodimer 527 formation to promote PI3K/Akt and MAPK signaling to overcome the inhibitory effects of EGFR TKIs. 528

We were the first to report that DARPP-32 overexpression in lung cancer contributes to oncogenic growth 30 . 529

While DARPP-32 is virtually undetectable in normal human lung 28 , DARPP-32 is overexpressed in human 530 EGFR-mutated NSCLC. Specifically, we previously demonstrated that DARPP-32 proteins promote NSCLC 531 cell survival through increased Akt and Erk1/2 signaling 30 . Given that these PI3K and MAPK signaling 532 pathways are upregulated during resistance, we hypothesized that overexpression of DARPP-32 proteins in 533 EGFR-mutated NSCLC may promote EGFR:ERBB3 "bypass signaling" that enables tumor cells to evade 534 EGFR TKI monotherapy. In this report, we provide the first evidence that DARPP-32 overexpression in EGFR-535 mutated lung adenocarcinoma promotes ERBB3-mediated oncogenic signaling to drive EGFR TKI therapy 536 refractory cancer progression. In vivo studies reveal that ablation of DARPP-32 protein activity sensitizes 537 gefitinib-resistant lung tumor xenografts to EGFR TKI treatment, while DARPP-32 overexpression increases 538 gefitinib-refractory lung cancer progression in gefitinib-sensitive lung tumors orthotopically xenografted into 539 mice. Findings from proximity ligation assays, immunoprecipitation studies, and immunofluorescence 540 experiments presented here support a model in which DARPP-32 mediates a switch from EGFR TKI-sensitive 541 EGFR homodimers to TKI-resistant EGFR:ERBB3 heterodimers to potentiate oncogenic AKT and ERK 542 signaling that drives therapy refractory tumor cell survival. To our knowledge, no proteins have been identified 543 that are capable of mediating such a "dimerization switch" in EGFR-mutated NSCLC. Here, we take 544 advantage of a unique cohort of paired tumor specimens derived from 30 lung adenocarcinoma patients before 545 and after the development of EGFR TKI refractory disease progression to reveal that DARPP-32 as well as 546 kinase-activated EGFR and ERBB3 proteins are overexpressed upon acquired EGFR TKI resistance. This 547 observation coincides with our published report that increased t-DARPP immunostaining positively correlates 548 with increasing T stage among unknown EGFR mutation status NSCLC patients 30 . There is no precedent of 549 DARPP-32 isoform immunostaining in molecular targeted therapy naïve vs. resistant patients in other tumor 550

Our data comprehensively suggests that DARPP-32 overexpression promotes EGFR TKI resistance by 552 stimulating formation of EGFR:ERBB3 heterodimers, which are less sensitive to EGFR inhibition and drive 553 oncogenic signaling. Therefore, dual inhibition of EGFR and ERBB3 may better prevent treatment-refractory 554 cancer progression as opposed to solely targeting EGFR, especially in tumors overexpressing DARPP-32. A 555 precision oncology approach could be used to identify EGFR-mutated lung adenocarcinomas with high 556 DARPP-32 and phosphorylated ERBB3 expression with the highest likelihood to benefit from dual EGFR and 557 ERBB3 inhibition. For example, duligotuzumab is a human IgG1 monoclonal "two-in-one" antibody with high 558 affinity for EGFR (KD ~ 1.9 nM) and ERBB3 (KD ~ 0.4 nM) developed to improve treatment response of solid 559 tumors exhibiting ERBB3-mediated resistance to EGFR-targeted treatment 74 . Partial responses to 560 duligotuzumab were achieved in patients with squamous cell carcinoma of the head and neck that had become 561 resistant to cetuximab, an antibody therapy that inhibits EGFR 75 . Efficacy was also observed in tumors 562 refractory to both radiation and long-term EGFR-targeted treatment 76, 77 . Duligotuzumab monotherapy has 563 been shown to be well-tolerated in patients with locally advanced or metastatic solid tumors of epithelial 564 origin 75 . However, a recent randomized phase II study of duligotuzumab vs. cetuximab in squamous cell 565 carcinoma of the head and neck (i.e. MEHGAN; NCT01577173) found duligotuzumab did not improve disease 566 free survival compared to cetuximab 78 and antibody-reactive protein bands were detected using anti-p-EGFR, EGFR, p-ERBB2, ERBB2, p-ERBB3, 608

ERBB3, DARPP-32, and α-tubulin antibodies. Immunoblotting experiments were repeated independently at 609 least three times, and a representative experimental result is shown. 610 , and ERBB3. Immunoprecipitated protein complexes and total cell lysates (input) were immunoblotted using anti-EGFR, ERBB3, FLAG, and α-tubulin antibodies. c-d Human lung adenocarcinoma HCC827P, HCC827GR (c), PC9P, PC9GR2, and PC9GR3 (d) cells were lysed and immunoprecipitated with anti-DARPP-32 (recognizes endogenous DARPP-32 and t-DARPP) and anti-ERBB3 antibodies. Immunoprecipitated lysates along with total cell lysates were separated on SDS-PAGE followed by immunoblot analysis using antibodies against EGFR, ERBB3, DARPP-32, and α-tubulin. Immunoprecipitation experiments were repeated at least three times. : Expression of DARPP-32, p-EGFR, and p-ERBB3 proteins is elevated in EGFR TKI resistant lung adenocarcinoma. a Tumor tissue was biopsied before EGFR TKI treatment (i.e. baseline) and following EGFR TKI resistance (i.e. progressive disease after first-line gefitinib or erlotinib therapy) from lung adenocarcinoma patients with EGFR activating mutations (n=30 patients in each group). Paired baseline (top) and EGFR TKI resistance (bottom) lung tumor specimens were immunostained for DARPP-32, phosphorylated EGFR (p-EGFR), total EGFR, p-ERBB3, and total ERBB3. b IHC score was calculated by multiplying the staining intensity score were immunoblotted with anti-DARPP-32 and -α-tubulin (loading control) antibodies. b HCC827GR cells were transduced with control (LacZ) or DARPP-32 shRNAs and seeded into 96-well cell culture plates. Cells were treated with increasing concentration of gefitinib and colorimeter-based cell survival assay was conducted using MTS1 reagents. c The half maximal inhibitory concentration (IC50) of gefitinib was determined from MTS1 survival assays and plotted. d HCC827P cells were transduced with retrovirus encoding control (LacZ), DARPP-32 or t-DARPP overexpressing clones. Cells were lysed and immunoblotting was performed to detect α-tubulin and DARPP-32 isoforms. e Cell survival assays were performed using HCC827P cells stably overexpressing LacZ, DARPP-32 or t-DARPP proteins exposed to increasing concentrations of gefitinib. f Gefitinib-treated HCC827P cells overexpressing LacZ or DARPP-32 isoforms were subjected to MTS1-based cell survival assays and IC50 of gefitinib was calculated. Each open circle on a graph represents an independent experiment. All bar graphs represent mean ± SEM (n=3). *P<0.05, **P<0.01, and ***P<0.001, one-way ANOVA followed by Dunnett's test for multiple comparison. a DARPP-32-depleted luciferase-labeled human HCC827GR cells were orthotopically injected into the left thoracic cavity of SCID mice. Mice administered either vehicle or gefitinib (25mg/Kg) were imaged for luminescence before and after treatment. b Retrovirus encoding control (LacZ), DARPP-32 or t-DARPP cDNAs were transduced in luciferase-labeled human HCC827P cells. After establishment of the tumor, mice were treated with vehicle or gefitinib (25mg/Kg) three times in a week. Luminescence images of mice were taken pre-and post-treatment. The colored bar represents the numerical value of luminescence.

Supplementary Figure 10 : Overexpression of DARPP-32 suppresses gefitinib efficacy in vivo. a-c SCID mice were subcutaneously injected with PC9P cells stably overexpressing control (LacZ) or DARPP-32 cDNAs and treated with vehicle or Gefitinib (25mg/Kg). At endpoint of the experiments, mice were sacrificed and xenografted tumors were extirpated. Photographs of extirpated tumors were taken to visualize gross morphology (a). Tumor volume was calculated from calipers measurement after extirpation (b). Weight of extirpated tumors from sacrificed mice was measured using a digital balance (c). Each open circle on bar graphs represents an individual mouse. Bar diagrams show Mean±SEM. **P<0.01, 2-way unpaired t-test. L858R  60  0  0  0  0  180  80  10  30  0  gefitinib  SD  -10%  25  12 IV  66  2 ADC  L858R  0  210  0  0  0  30  150  20  10  30  gefitinib  PR  -60%  8  13 IV  55  2 ADC  L858R  240  100  0  10  5  240  270  0  60  10  erlotinib  PR  -30%  8  14 IV  66  2 ADC  L858R  90  120  0  0  0  150  270  20  0  0  erlotinib  PR  -80%  11  15 IV  47  2 ADC  L858R  0  0  0  0  0  240  100  20  0  0  gefitinib  SD  -15%  3  16 IV  66  2 ADC  L858R  180  0  0  0  0  150  180  30  15  0  erlotinib  PR  -30%  21  17 IV  63  1 ADC  L858R  120  120  40  0  0  180  180  10  10  0  gefitinib  PR  -35%  9  18 IV  62  1 ADC  L858R  180  30  0  0  0  150  210  10  100  10  erlotinib  PR  -35%  24  19 IV  70  1 ADC  L858R  100  30  20  0  0  90  150  70  50  0  gefitinib  PR  -30%  22  20 IV  62  1 ADC  L858R  210  30  0  0  0  30  30  50  10  0  gefitinib  PR  -30%  4  21 IV  81  1 ADC  L858R  240  80  0  0  0  60  120  10  0  30  erlotinib  PR  -50%  12  22 IV  61  1 ADC  L858R  100  180  0  10  15  60  270  50  10  10  erlotinib  SD  -15%  26  23 IV  54  2 ADC  L858R  240  80  0  0  0  180  100  0  60  0  gefitinib  PR  -50%  15  24 IV  48  2 ADC  L858R  20  160  0  0  0  60  210  60  60  5  erlotinib  SD  -20%  3  25 IV  66  1 ADC  L858R  20  270  30  0  0  90  150  0  30  0  gefitinib  PR  -70%  18  26 IV  55  2 ADC  L858R  240  60  0  5  5  210  210  0  15  25  gefitinib  PR  -35%  13  27 IV  66  1 ADC  L858R  240  50  0  5  5  270  100  10  10  0  erlotinib  PR  -30%  12  28 IV  59  2 ADC  L858R  40  300  0  0  0  45  210  25  40  40  gefitinib  PR  -35%  21  29 IV  62  1 ADC  L858R  40  80  0  10  0  150  120  0  0  20  erlotinib  PR  -42%  7.8  30 IV  70  1 ADC  L858R  60  40  0  0  0  270  160  0  0  0  erlotinib  SD  -20%  19   Supplementary Table 1 

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Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers

AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer

Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer

Resistance mechanisms to osimertinib in EGFR-mutated non-small cell lung cancer

Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M

Fighting cancer drug resistance: Opportunities and challenges for mutation-specific EGFR inhibitors

ASCL1-regulated DARPP-32 and t-DARPP stimulate small cell lung cancer growth and neuroendocrine tumour cell proliferation

Dopamine D2 receptor agonists inhibit lung cancer progression by reducing angiogenesis and tumor infiltrating myeloid derived suppressor cells

DARPP-32 and t-DARPP promote non-small cell lung cancer growth through regulation of IKKalpha-dependent cell migration

Overexpression of the 32-kilodalton dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein in common adenocarcinomas

Darpp-32: a novel antiapoptotic gene in upper gastrointestinal carcinomas

Gastric tumour-derived ANGPT2 regulation by DARPP-32 promotes angiogenesis

t-Darpp overexpression in HER2-positive breast cancer confers a survival advantage in lapatinib

Gastric cancers overexpress DARPP-32 and a novel isoform, t-DARPP

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis

t-DARPP regulates phosphatidylinositol-3-kinase-dependent cell growth in breast cancer

Overexpression of DARPP-32 in colorectal adenocarcinoma

Regional distribution of calcium-and cyclic adenosine 3':5'-monophosphate-regulated protein phosphorylation systems in mammalian brain. I. Particulate systems

DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1

Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons

The neurobiology of slow synaptic transmission

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

Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) peceptor-1 down-modulates VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase-dependent pathways

ASCL1-regulated DARPP-32 and t-DARPP stimulate small cell lung cancer growth and neuroendocrine tumor cell survival

The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data

Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal

Oncogenic Signaling Pathways in The Cancer Genome Atlas

Darpp-32 and t-Darpp protein products of PPP1R1B: Old dogs with new tricks

DARPP-32: from neurotransmission to cancer

Caspases: opening the boxes and interpreting the arrows

DARPP-32 increases interactions between epidermal growth factor receptor and ERBB3 to promote tumor resistance to gefitinib

Heterodimerization to the EGFR Mutation-Induced Gefitinib-and Erlotinib-Resistance in Non-Small-Cell Lung Carcinoma Treatments

The EGFR family: not so prototypical receptor tyrosine kinases. Cold Spring Harbor perspectives in biology

Molecular mechanisms of mTOR-mediated translational control

Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012

Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Antitumour immunity regulated by aberrant ERBB family signalling

The next tier of EGFR resistance mutations in lung cancer

Targeting the EGFR signaling pathway in cancer therapy

Analysis of Somatic Mutations in Cancer: Molecular Mechanisms of Activation in the ErbB Family of Receptor Tyrosine Kinases

The interplay of HER2/HER3/PI3K and EGFR/HER2/PLC-gamma1 signalling in breast cancer cell migration and dissemination

Activated human hydroxy-carboxylic acid receptor-3 signals to MAP kinase cascades via the PLC-dependent PKC and MMP-mediated EGFR pathways

Cross talk of signals between EGFR and IL-6R through JAK2/STAT3 mediate epithelial-mesenchymal transition in ovarian carcinomas

EGFR phosphorylates tumor-derived EGFRvIII driving STAT3/5 and progression in glioblastoma

Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3

Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer

EGFR-TKIs resistance via EGFR-independent signaling pathways

Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3

The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation

Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

An heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in HER2+ breast cancer models

A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies

Safety and Pharmacokinetics/Pharmacodynamics of the First-in-Class Dual Action HER3/EGFR Antibody MEHD7945A in Locally Advanced or Metastatic Epithelial Tumors

Dual targeting of EGFR and HER3 with MEHD7945A overcomes acquired resistance to EGFR inhibitors and radiation

Antitumor Effects of MEHD7945A, a Dual-Specific Antibody against EGFR and HER3, in Combination with Radiation in Lung and Head and Neck Cancers

Randomized Phase II Study of Duligotuzumab (MEHD7945A) vs. Cetuximab in Squamous Cell Carcinoma of the Head and Neck (MEHGAN Study)

Commentary: Randomized Phase II Study of Duligotuzumab (MEHD7945A) vs. Cetuximab in Squamous Cell Carcinoma of the Head and Neck (MEHGAN Study)

Imaging EGFR and HER3 through (89)Zr-labeled MEHD7945A (Duligotuzumab)