SCIENCE ADVANCES | RESEARCH ARTICLE CANCER Copyright © 2022 The Authors, some Targeting HER2-AXL heterodimerization to overcome rights reserved; exclusive licensee resistance to HER2 blockade in breast cancer American Association for the Advancement of Science. No claim to Anna Adam-Artigues1, Enrique J. Arenas2,3, Alex Martínez-Sabadell2, Fara Brasó-Maristany4,5, original U.S. Government Raimundo Cervera1, Eduardo Tormo1,3, Cristina Hernando1,6, María Teresa Martínez1,6, Works. Distributed Juan Carbonell-Asins1, Soraya Simón6, Jesús Poveda6, Santiago Moragón6, Sandra Zazo7, under a Creative Débora Martínez4,5, Ana Rovira3,8,9, Octavio Burgués1,3,10, Federico Rojo3,7, Joan Albanell3,8,9,11, Commons Attribution Begoña Bermejo1,3,6, Ana Lluch1,3,6,12, Aleix Prat4,5,13, Joaquín Arribas2,3,9,14,15, NonCommercial License 4.0 (CC BY-NC). Pilar Eroles1,3,16*, Juan Miguel Cejalvo1,3,6*† Anti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mecha- nism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer. INTRODUCTION Metastasis is a complex multistep process that involves intra- HER2 is a member of the human epidermal growth factor trans- tumoral cell invasion followed by circulatory spread and is completed membrane receptor family and is overexpressed in about 20% of breast once cancer cells invade and survive in distant tissues to grow as cancer (BC) (1). The HER2-positive (HER2+) BC subtype correlates macrometastatic lesions in permissive niches (5). Epithelial-to-­ with an aggressive phenotype and poor prognosis (2). Several anti-­ mesenchymal transition (EMT) plays an important role in this malig- HER2 drugs have been developed and are currently being used fol- nant progression, allowing cells to metastasize and promoting acquired lowing positive clinical outcomes in both localized and metastatic resistance to several treatment strategies (6, 7). HER2+ BC (3). HER2 is thus a well-established therapeutic target, AXL is a member of the TAM (TYRO3, AXL, MER) receptor and anti-HER2 treatment has become the standard of care for patients tyrosine kinase family that has been linked to EMT. AXL signaling with HER2-amplified BC. Despite a large fraction of HER2+ BC promotes cell survival, resistance to anoikis, drug resistance, inva- patients benefiting from anti-HER2 therapy, a subset of these patients sion, and metastasis across different cancers (8). AXL is activated develop resistance and ultimately experience disease progression through multiple mechanisms, including binding to its ligand growth (4). Hence, furthering our understanding and defining the molecular arrest–specific protein 6 (GAS6) precursor and extracellular domain– mechanisms involved in treatment response and the metastatic mediated dimerization or cross-talk with other transmembrane re- process may reveal novel personalized and more efficient thera- ceptor tyrosine kinase (9). Furthermore, AXL up-regulation causes peutic strategies. both targeted agents and chemotherapy resistance in several types of cancer (10–19). In BC, AXL expression correlates with metastasis and worse survival (20–22). In addition, AXL inhibition has been 1 INCLIVA Biomedical Research Institute, Valencia 46010, Spain. 2Preclinical Research proposed as a therapeutic strategy in triple-negative BC (23–25) Program, Vall d’Hebron Institute of Oncology (VHIO), Barcelona 08035, Spain. 3Center and other cancers (26). However, there is still a lack of clinical infor- for Biomedical Network Research on Cancer (CIBERONC), Madrid 28019, Spain. mation on the role of AXL in HER2+ BC. 4 August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona 08036, Spain. 5Department of Medical Oncology, Hospital Clinic de Barcelona, Barcelona In this study, we demonstrate that AXL high expression inde- 08036, Spain. 6Department of Medical Oncology, Hospital Clínico Universitario de pendently predicts poor prognosis in HER2+ BC patients. We present València, Valencia 46010, Spain. 7Department of Pathology, IIS Fundación Jiménez our findings, which delineate in a systematic fashion how AXL acti- Díaz, Madrid 28040, Spain. 8Department of Medical Oncology, Hospital del Mar, Barcelona 08003, Spain. 9Cancer Research Program, IMIM (Hospital del Mar Medical vation modulates trastuzumab response in in vitro and in vivo Research Institute), Barcelona 08003, Spain. 10Department of Pathology, Hospital patient-derived xenograft (PDX) models. Concordant with this Clínico Universitario de València, Valencia 46010, Spain. 11Pompeu Fabra University observation, neoadjuvant treatment with lapatinib and trastuzumab (UPF), Barcelona 08002, Spain. 12Department of Medicine, Universidad de Valencia, Valencia 46010, Spain. 13SOLTI Breast Cancer Research Group, Barcelona 08008, led to an increase in AXL mRNA levels at week 2 in HER2+ BC Spain. 14Department of Biochemistry and Molecular Biology, Universitat Autónoma patients from the PAMELA trial. The rapid increase in AXL expres- de Barcelona, Barcelona 08193, Spain. 15Institució Catalana de Recerca i Estudis Avançats sion suggests its potential role in acquired resistance to anti-HER2 (ICREA), Barcelona 08010, Spain. 16Department of Physiology, Universidad de Valencia, treatment in BC patients. Valencia 46010, Spain. *Corresponding author. Email: pilar.eroles@uv.es (P.E.); jmcejalvo@incliva.es (J.M.C.) We propose AXL overexpression as a new mechanism of resistance †Lead author. to anti-HER2 therapy and as a new potential druggable prognostic Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 1 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE biomarker. AXL inhibition plus anti-HER2 could be a new therapeutic the combination of trastuzumab with TP-0903 in acquired resistant approach in HER2+ BC. cell lines reached growth inhibition levels observed in parental cell lines with trastuzumab (fig. S2C). Subsequently, TP-0903 combined with increasing trastuzumab concentrations led to trastuzumab RESULTS IC50 values in acquired resistant cell lines similar to those in sensi- Characterization of trastuzumab-resistant HER2+ tive cells (Fig. 2D and table S1). However, growth inhibition and BC cell lines IC50 values for trastuzumab were not modified in parental cell lines To identify novel mechanisms of trastuzumab resistance, we used or HCC1954 upon addition of AXL inhibitor (fig. S2, C and D). three in vitro BC cell lines with acquired resistance to trastuzumab: Conversely, AXL gain of function in parental cell lines signifi- AU565R, BT474R, and SKBR3R. In resistant models, trastuzumab cantly reduced growth rate inhibition after trastuzumab treatment, treatment decreased the growth rate by less than 20%, in contrast with thus leading to trastuzumab resistance (Fig. 2E and fig. S2E). More- the approximately 50% reduction observed in parental cell lines over, treatment with a range of trastuzumab concentrations showed (Fig. 1A). Given that standard of care for HER2+ BC consists of dual an increased IC50 value in AXL-overexpressed cell lines compared HER2 blockade with trastuzumab plus pertuzumab, we checked re- to controls (AU565, P = 0.0012; BT474, P < 0.0001; SKBR3, P = 0.0001) sponse to pertuzumab and combination of both agents in trastuzumab-­ (Fig. 2F and table S1). On the other hand, AXL overexpression had resistant cell lines to give clinical value to our study. Results showed no effect on trastuzumab response in resistant cell lines (fig. S2, a significantly lower response of AU565R, BT474R, and SKBR3R to E and F). Proliferation was not affected by AXL levels (fig. S3). these treatments in comparison to parental cells (fig. S1A). In addition, Collectively, these results suggest that AXL promotes acquired re- the three acquired trastuzumab-resistant cell lines were significantly sistance to trastuzumab, and AXL inhibition restores sensitivity in more proliferative at basal level than parental cell lines, as demonstrated in vitro acquired trastuzumab-resistant models. by Ki67+ staining and cell proliferation (Fig. 1, B and C). Immunohistochemistry (IHC) analysis revealed no substantial AXL-mediated trastuzumab resistance is associated changes in pathological biomarkers between resistant and parental with EMT-like phenotype cell lines. BT474 and BT474R cell lines express HER2, estrogen re- Acquisition of mesenchymal characteristics by epithelial cells, in ceptor (ER), and progesterone receptor (PR). In contrast, AU565, particular, the ability to migrate and invade the extracellular matrix, AU565R, SKBR3, SKBR3R, and an innate trastuzumab-resistant cell is a well-defined mechanism of drug resistance (28). Given that the line (HCC1954) were HER2+, while ER and PR were not expressed mesenchymal phenotype is related to worse response to trastuzum- (Fig. 1D). All cell lines were HER2 amplified, yet trastuzumab-resistant ab and that AXL has been described as a component of the acquired cell lines expressed lower HER2 total expression and cell membrane mesenchymal phenotype (29), we next analyzed the association be- expression (Fig. 1, E and F). Notably, ERBB2 mRNA remains lower tween AXL expression and EMT markers. in our acquired resistant cell lines compared to the parental sensi- First, we observed that acquired trastuzumab-resistant cell lines, tive cell lines (Fig. 1G). The above data suggest that our resistant models which overexpressed AXL, also showed an EMT-like phenotype by have an increased proliferation rate and lower ERBB2 expression despite up-regulation of vimentin (VIM), fibronectin (FN1), -catenin maintaining HER2 amplification and the same molecular subtype. (CTNNB1), and N-cadherin (CDH2) and a decrease in E-cadherin (CDH1) at mRNA and protein levels compared with their respec- AXL promotes trastuzumab resistance in HER2+ BC cell lines tive parental cell lines (Fig. 3, A and B). As expected, the SKBR3R Given that AXL has been previously associated with resistance to cell line presented an enhanced cancer cell invasion and migration therapies (27), we compared AXL expression between trastuzumab-­ capability through AXL signaling (Fig. 3C and fig. S4A). sensitive and trastuzumab-resistant cell lines, finding that both AXL Second, we determined whether AXL expression was sufficient mRNA and protein expression were up-regulated in the three resis- to modulate a mesenchymal-like phenotype. The results showed that tant cell lines compared to their respective parental cell lines. In ad- AXL inhibition by two different siRNAs or by TP-0903 was suffi- dition, AXL phosphorylation was also higher in resistant cell lines cient to reverse the mesenchymal-like phenotype (Fig. 3, E and F, (Fig. 1, H and I). These data suggest that AXL expression could and fig. S4B). AXL down-regulation by siRNA and inhibition by mediate trastuzumab resistance in our models. To address this TP-0903 reduced cell migration and invasion in the SKBR3R cell question, AXL was inhibited using both a genetic and a pharma- line (Fig. 3, D and G, and fig. S4, C and D). cological approach. First, we depleted AXL expression using two Consistent with the above results, AXL overexpression increased independent small interfering RNAs (siRNAs): In both cases, AXL both EMT markers expression (Fig. 3H) and migration and invasion down-regulation restored the sensitivity of resistant cells to trastu- capability in the SKBR3 cell line (Fig. 3I and fig. S4E). Moreover, zumab, and we observed over 20% growth inhibition in AXL-depleted modulation of migration and invasion ability by AXL was also vali- acquired resistant cell lines after trastuzumab treatment (Fig. 2A and dated in BT474 and BT474R cells (fig. S5). These results suggest that fig. S2A). Trastuzumab IC50 (half-maximal inhibitory concentration) AXL orchestrates a mesenchymal-like phenotype in acquired resist­ values in the three acquired resistant cell lines with AXL knockdown ance to trastuzumab in HER2+ BC cell lines. were similar to IC50 of their parental cell lines (Fig. 2B and table S1). However, AXL down-regulation in sensitive cell lines and in the innate Acquired trastuzumab resistance is mediated by resistant cell line HCC1954 did not enhance trastuzumab growth ligand-independent heterodimerization of AXL and HER2 rate inhibition, underscoring its role as mechanism of acquired re- Several studies have demonstrated that both ligand-dependent and sistance (fig. S2, A and B). ligand-independent AXL activation initiates downstream signaling These results were confirmed using a selective AXL tyrosine ki- in several cancer types (30, 31). We undertook to determine the nase inhibitor (TP-0903) (Fig. 2C). Reduced proliferation caused by biological mechanism underlying the resistance to trastuzumab Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 2 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 1. Trastuzumab-resistant HER2+ BC cell line characterization. (A) Cell proliferation analysis by WST-1 in trastuzumab-sensitive and trastuzumab-resistant cell lines with and without trastuzumab treatment at 15 g/ml for 7 days. (B) IHC staining of Ki67 in the indicated cell lines. (C) Comparison of basal cell proliferation by WST-1 in trastuzumab-sensitive and trastuzumab-resistant cell lines over 144 hours. (D) IHC staining ER, PR, and HER2 in the indicated cell lines. MDA-MB-231 was used as a nega- tive control for HER2, ER, and PR staining. Flow cytometry analysis of HER2 expression in cell membrane (E), Western blot analysis for total HER2 expression (F) and RT-qPCR analysis of ERBB2 mRNA expression (G) in trastuzumab-sensitive and trastuzumab-resistant cell lines. Western blot analysis of AXL and phospho-AXL (H) and RT-qPCR analysis of AXL mRNA expression (I) and in trastuzumab-sensitive and trastuzumab-resistant cell lines. Scale bars, 100 m; magnifications, ×20 (B) and ×10 (D). GAPDH was used as an endogenous control in (F) to (I). Flow cytometry negative control was stained with isotype primary antibody in (E). *P < 0.05, **P < 0.01, ***P < 0.001 by two- tailed Student’s t test in (A), (C), (E), (G), and (I). Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 3 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 2. AXL has a role in trastuzumab response in HER2+ BC cell lines. (A) Western blot analysis of AXL and phospho-AXL protein expression after 72 hours of siRNA-­ mediated AXL knockdown. (B) Cell proliferation analysis by WST-1 in sensitive cell lines and siRNA-mediated AXL knockdown acquired resistant cell lines treated with trastuzumab for 7 days. (C) Western blot analysis of AXL and phospho-AXL protein expression after 72 hours of TP-0903 treatment. (D) Cell proliferation analysis by WST- 1 in sensitive cell lines and TP-0903–treated resistant cell lines with trastuzumab for 7 days. (E) Western blot analysis of AXL and phospho-AXL protein expression after 72 hours of AXL overexpression. (F) Cell proliferation analysis by WST-1 in resistant cell lines and AXL-overexpressed sensitive cell lines with trastuzumab treatment for 7 days. GAPDH was used as an endogenous control in (A), (C), and (E). Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 4 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 3. AXL expression is associated with EMT-like phenotype. RT-qPCR analysis of mRNA expression (A) and Western blot analysis of protein expression (B) of EMT markers: VIM, FN1, CTNNB1, CDH2, and CDH1 in sensitive versus trastuzumab-resistant cell lines. Transwell migration and invasion assay in SKBR3 versus SKBR3R (C), siRNA-­ mediated AXL knockdown (D), and pharmacological AXL inhibition by TP-0903 (G) in the SKBR3R cell line. Western blot analysis of EMT markers in siRNA-mediated AXL knockdown (E) and pharmacological AXL inhibition by TP-0903 (F) in SKBR3R and in AXL-overexpressed SKBR3 (H). (I) Transwell migration and invasion assay in AXL-overexpressed SKBR3. GAPDH was used as an endogenous control in (A), (B), (E), (F), and (H). Images show representative migrating/invading cells, and bars show percentage of the mean (±SD, n = 8) of migrating/invading cells. Scale bar, 100 m; magnification, ×10 (C, D, G, and I). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test in (A), (C), (D), (G), and (I). Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 5 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE mediated by AXL overexpression. To do so, we evaluated the role of of PI3K/AKT and MAPK/ERK pathways. Furthermore, growth in- its single ligand GAS6 without detecting consistent overexpression hibition caused by trastuzumab in PDX118 was independent of AXL of GAS6 mRNA or protein in acquired resistant versus parental cell blockade (Fig. 5, D and F, and table S1). These results were next lines (fig. S6, A and B). Neither GAS6 protein expression nor the validated in three-dimensional (3D) in vitro models from PDX118, percentage of GAS6-positive cells changed after treatment with protein TR1, and TR2 cell lines. In resistant organoids, trastuzumab or TP- transport inhibitor, thus confirming that GAS6 ligand production 0903 alone did not induce significant growth inhibition, while the did not vary between resistant and sensitive cell lines (fig. S6, B to E). combination achieved a decrease to 27% in both models compared On the basis of these data, we hypothesized that AXL activation to control. In PDX118 organoids, trastuzumab significantly decreased is ligand independent and proposed the heterodimerization between cell numbers with no greater effect of combination with TP-0903 AXL and HER2 as the trigger in our acquired trastuzumab-resistant (Fig. 5, E and G). Besides, apoptosis assay revealed a significant in- models. To test this hypothesis, we co-immunoprecipitated AXL and crease in the percentage of apoptotic cells achieved by the combina- HER2 demonstrating a direct interaction between these two proteins tion of trastuzumab plus TP-0903 compared to any single agent in (Fig. 4A). In agreement with this result, AXL-HER2 interaction was both parental and resistant organoids (Fig. 5H). also detectable by proximity ligation assay (PLA). Flow cytometry Second, an in vivo PDX-resistant model (PDX118-TR4) was es- and immunofluorescence confirmed the presence in the plasma tablished by chronic treatment with trastuzumab for 4 months and membrane of AXL-HER2 heterodimer in our acquired trastuzumab-­ two serial passages in mice (Figs. 5A and 6A). The level of HER2 resistant models (Fig. 4, B and C, and fig. S7A). These results sug- amplification was similar in parental PDX118 and PDX118-TR4, but gest a ligand-independent interaction between AXL and HER2. ERBB2 expression was down-regulated (32, 33). Consistent with the phenotype observed in vitro, these resistant PDX tumors up-regulated AXL-HER2 dimerization induces phosphorylation of AXL and VIM (Fig. 6B) and presented AXL-HER2 heterodimers PI3K/AKT and MAPK/ERK pathways (fig. S8B). Notably, GAS6 was undetectable in our PDX models, We next studied signaling on HER2-AXL downstream pathways. which supports the ligand-independent mechanism. In 3D organoid AXL-HER2 heterodimer downstream adapter proteins, such as AKT, models from PDX118-TR4, trastuzumab or TP-0903 alone did not RPS6, extracellular signal–regulated kinase (ERK), and mitogen-­ significantly reduced cell numbers, whereas in combination they de- activated protein kinase (MAPK), were found to be hyperphos- creased cell numbers to 38.2% compared to control (Fig. 6, C and D). phorylated in trastuzumab-resistant models compared to sensitive In a PDX118-TR4 in vivo model experiment, mice were treated parental cell lines (Fig. 4D). Furthermore, genetic knockdown or with vehicle, trastuzumab, TP-0903, or a combination of these two pharmacological inhibition of AXL plus trastuzumab achieved the drugs. The results showed that trastuzumab and the AXL inhibitor greatest inactivation of HER2 downstream pathways (Fig. 4, E and F, alone had no significant effect on tumor growth; however, the two and fig. S7, B and C). Although AXL overexpression alone was not drugs in combination abrogated tumor growth and achieved a com- sufficient for promoting the phosphorylation of phosphoinositide plete regression after 21 days of treatment (Fig. 6, E to H, and fig. S8, 3-kinase (PI3K)/AKT and MAPK/ERK, pathway inhibition by tras- C to E). Notably, animals from the combined treatment group re- tuzumab was considerably less effective in AXL-overexpressed cell mained alive with no evidence of tumor growth after therapy during lines than in control cell lines (Fig. 4G). Together, our data indicate a total of 321 days of follow-up (Fig. 6I); likewise, treatment did not that AXL-HER2 dimerization activates PI3K/AKT and MAPK/ERK affect animal weight, showing that AXL inhibition did not lead to a signaling pathways, thus leading to a reduced trastuzumab treat- significant toxic effect in preclinical models (Fig. 6J). ment efficacy. With these results, we conclude that the combination of TP-0903 with trastuzumab completely abrogates tumor growth in acquired AXL inhibition overcomes trastuzumab resistance trastuzumab-resistant PDX models and achieves complete tumor in PDX models regression safely. Therefore, this is a promising therapeutic strategy On the basis of the previous results demonstrating that AXL leads to treat HER2+ BC resistant to standard of care. to trastuzumab resistance, we tested whether the combination of trastuzumab and AXL inhibition could be used to blunt tumor growth AXL expression in HER2+ BC patients correlates with a strong in different in vitro and in vivo acquired trastuzumab-resistant negative prognostic factor PDX models from a HER2+ BC patient to obtain more translational On the basis of these preclinical data, we hypothesized that up-­ results. First, two in vitro PDX-derived resistant models were established regulation of AXL might promote acquired resistance to trastuzumab by chronic treatment for 6 months with trastuzumab (Fig. 5, A and B, treatment in HER2+ BC patients. To test this hypothesis and assess and table S1). The two obtained PDX-derived trastuzumab-resistant the clinical relevance of our preclinical findings, we analyzed AXL cell lines (PDX118-TR1 and PDX118-TR2) also presented decreased expression in a retrospective cohort of HER2+ BC patients (n = 50) response to pertuzumab and dual HER2 blockade (fig. S1, B and C) (table S2). The median age at BC diagnosis was 51 years (range, 33 and maintained HER2 overexpression but exhibited up-regulation to 73); median follow-up was 7 years. Median disease-free survival of AXL and VIM in comparison to the parental cells (PDX118) (DFS) was 65.5 months (range, 5 to 177), and median overall sur- (Fig. 5C). Co-immunoprecipitation assay confirmed AXL-HER2 vival (OS) was 80.5 months (range, 7 to 177). dimerization in these PDX-derived trastuzumab-resistant models AXL expression was significantly up-regulated at the time of di- (fig. S8A). These results reproduced those previously obtained in agnosis in primary tumor tissue from patients who later experi- our panel of acquired trastuzumab-resistant cell lines. In addition, enced disease relapse (P = 0.003) (Fig. 7A). Receiver operating curve the in vitro combination of TP-0903 plus trastuzumab resensitized (ROC) analysis also revealed that AXL expression may discriminate resistant cells by significantly decreasing trastuzumab IC50 (PDX118-­ between HER2 + BC patients who will relapse from those who TR1 and PDX118-TR2 P < 0.0001) and inhibiting phosphorylation will be free of disease with area under the curve (AUC) of 96.96% Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 6 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 4. AXL-HER2 heterodimer regulates PI3K/AKT and MAPK/ERK pathways. (A) Co-immunoprecipitation (IP) of AXL, HER2, and IgG as a negative control (NC) fol- lowed by immunoblotting of AXL and HER2 in acquired trastuzumab-resistant cell lines. (B) PLA of AXL and HER2 (red) in acquired resistant cell lines by flow cytometry. Negative control: primary antibodies were omitted (gray). (C) PLA of AXL and HER2 in trastuzumab-sensitive and acquired resistant cell lines by confocal microscopy. Red spots indicate AXL-HER2 interaction. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (D) Western blot analysis of AXL-HER2 downstream proteins in trastuzumab-sensitive versus trastuzumab-resistant cell lines at basal level. Western blot analysis of AXL-HER2 downstream proteins with and without trastu- zumab for 72 hours in siRNA-mediated AXL knockdown (E) after pharmacological AXL inhibition by TP-0903 (F) in acquired trastuzumab-resistant cell lines and in AXL-overexpressed sensitive cell lines (G). GAPDH was used as an endogenous control in (D) to (G). Scale bar, 25 m; magnification, ×40 (C). Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 7 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 5. AXL inhibition overcomes trastuzumab resistance in PDX-derived HER2+ BC cell lines. (A) Schematic representation of in vitro trastuzumab–resistant PDX cell lines (PDX118-TR1 and TR2) and in vivo trastuzumab–resistant PDX model (PDX118-TR4) generation. (B) Cell proliferation assay of PDX-derived cell lines treated with trastuzumab for 6 days. Cell numbers were estimated with the crystal violet staining assay. (C) RT-qPCR analysis of ERBB2, AXL, and VIM mRNA expression of PDX-derived cell lines. (D) Western blot analysis of HER2 downstream pathways with and without trastuzumab and TP-0903 treatment for 72 hours in acquired trastuzumab-resistant PDX-derived cell lines. (E) Representative images of organoids treated with trastuzumab, TP-0903, and combination. (F) Cell proliferation analysis of PDX-derived cell lines treated with trastuzumab combined with pharmacological AXL inhibition by TP-0903. Cell number (G) and apoptosis assay (H) of PDX118, TR1, and TR2 organoids treated with trastuzumab (100 g/ml), TP-0903 (1 M in PDX118 and TR1, 0.15 M in TR2), or combination. Viable and apoptotic cells were quantified by flow cytometry using propidium iodide and annexin V staining. GAPDH was used as an endogenous control in (C) and (D). Scale bar, 100 m; magnification, ×4 (E). *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student’s t test in (B), (C), (G), and (H). Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 8 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 6. AXL inhibition overcomes trastuzumab resistance in HER2+ BC PDX models. (A) Generation of an in vivo trastuzumab–resistant model (PDX118-TR4). Parental PDX118 was implanted in an immunocompromised mouse, and when tumors reached ~200 mm3, animals were treated intraperitoneally with trastuzumab (left). Next, resistant tumors were reimplanted in immunocompromised mice and treated with trastuzumab (right). Red arrows represent days of trastuzumab treatment. (B) RT-qPCR analysis of ERBB2, AXL, and VIM mRNA expression of in vivo PDX–resistant model PDX118-TR4. GAPDH was used as an endogenous control. (C) Cell number analysis of trastuzumab-resistant patient-derived organoids treated with trastuzumab at 100 g/ml, TP-0903 at 150 nM, and a combination of these two drugs. Viable cells were quantified by flow cytometry using EpCAM as a marker. (D) Representative images of organoids treated with trastuzumab, TP-0903, and combination of these two drugs. Tumor growth of PDX118-TR4 in vivo model injected in NOD-SCID mice treated with vehicle, trastuzumab alone (10 mg/kg), TP-0903 alone (50 mg/kg), or a combination of these two drugs represented in time-course line (E), endpoint dot plot (G), and percentage change from baseline of each individual (H). (F) Tumor growth of PDX118- TR4 in vivo model in NOD-SCID mice treated with trastuzumab and TP-0903 combination. Black line represents treatment time period. (I) OS Kaplan-Meier curve of PDX mice divided by treatment. (J) Animals’ weight in grams during treatment and follow-up period. Treatment was performed between days 89 and 110 after injection. N = 30 mammary fat pad tumors; data represent means ± SEM in (E) to (G). Scale bar, 100 m; magnification, ×4 (D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed Student’s t test in (B) and (C). ****P < 0.0001 by two-way ANOVA with Bonferroni correction posttest in (E). **P < 0.01 and ***P < 0.001 by Mann-Whitney test in (G). Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 9 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 7. AXL expression correlates with a strong negative prognostic factor in HER2+ BC patients. (A) RT-qPCR analysis of AXL mRNA expression in HER2+ BC patients’ FFPE samples. (B) ROC curve analysis for relapse prediction based on AXL mRNA expression in HER2+ BC patients. DFS (C) and OS (D) Kaplan-Meier curves of HER2+ BC patients stratified by median AXL expression levels. (E) RT-qPCR analysis of VIM mRNA expression in HER2+ BC patients’ FFPE samples. DFS (F) and OS (G) Kaplan-Meier curves of HER2+ BC patients stratified by median VIM expression levels. (H) Correlation matrix of AXL, VIM, ERRB2, and GAS6 mRNA expression in HER2+ BC patients. N = 50. Red lines represent median and interquartile range. GAPDH was used as an endogenous control in (A) and (E). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test with Welch’s correction in (A), (E), and (H). Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 10 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE [95% confidence interval (CI), 0.9292 to 1.00; P < 0.0001]. Applying the dose to surgery, during which the tumor samples were obtained, best cutoff value, biomarker performance parameters showed 100% was 29.5 days (range, 7 to 76 days). AXL was found to be differen- sensitivity, 84.62% specificity, and 92% accuracy (Fig. 7B). We found tially expressed in residual tumors at surgery compared to day 14 that patients with high levels of AXL had significantly shorter DFS (P = 6.04 × 10−14) (Fig. 8D). In agreement with previous gene ex- and OS (for DFS: log-rank P < 0.001; hazard ratio, 12.56; 95% CI, 3.70 pression analysis in the PAMELA trial (36), we found a rebound to 42.66; for OS: log-rank P = 0.006; hazard ratio, 6.91; 95% CI, 1.50 effect between day 14 and surgery samples (P = 6.04 × 10−14) poten- to 31.78). Median DFS and OS were 30 and 136 months, respectively, tially due to that discontinuation of dual HER2 inhibition, which in patients with high AXL expression, whereas in patients with low reverses its biological effects. According to our previous preclinical AXL expression these were not reached (Fig. 7, C and D). data, VIM expression presented similar biological changes than AXL, The prognostic value of AXL was also confirmed in silico in an whereas ERBB2 expression decreased after dual HER2 blockade independent public early HER2+ BC patients’ cohort (34). Patients (fig. S10, A and B). with high AXL levels had significantly shorter DFS (log-rank P = 0.0018; Other interesting findings were that AXL and VIM expression hazard ratio, 2.03; 95% CI, 1.29 to 3.19) and distant metastasis–free were strongly correlated (cor = 0.67, P = 1.15 × 10−55), and ERBB2 survival (log-rank P = 0.0043; hazard ratio, 2.62; 95% CI, 1.32 to 5.19) had a negative correlation with both AXL and VIM (cor = −0.362, (fig. S9, A and B). P = 2.23 × 10−14; cor = −0.48, P = 2.31 × 10−25, respectively) (Fig. 8E). As AXL expression correlated with VIM in our resistant models, These observations were particularly significant as the biological changes we sought to validate this correlation in our patients’ cohort. We observed in our preclinical models recapitulated the biological changes found a strong positive correlation between VIM and AXL expres- observed in patients from a phase 2 clinical trial. sion [correlation (cor) = 0.91, P < 0.001] (Fig. 7H), and VIM expres- sion was associated with worse prognosis. The results revealed that higher VIM expression in primary tumor was related to relapse DISCUSSION (P = 0.0033), worse DFS (log-rank P < 0.001; hazard ratio, 50.63; Trastuzumab is an essential and effective targeted anticancer agent 95% CI, 6.74 to 380.5), and OS (log-rank P < 0.001; hazard ratio, for HER2+ BC patients. Unfortunately, despite the efficacy of this 16.48; 95% CI, 2.11 to 128.80) compared to low VIM HER2+ BC anti-HER2 treatment, a significant number of patients develop pro- patients (Fig. 7, E to G). gressive disease (2), requiring additional therapeutic strategies. Several Next, we analyzed GAS6 mRNA expression in our patients’ co- molecular mechanisms such as alternative signaling pathways, acti- hort. No association between GAS6 and AXL expression was found vation, and/or down-regulation of ERBB2 could contribute to de- (cor = −0.069, P = 0.647) (Fig. 7H), in accordance with our preclinical velopment of resistance to anti-HER2 agents (37, 38). This scenario data. Furthermore, GAS6 was not a negative prognostic factor; there highlights an urgent need to develop new therapeutic approaches to was no significant relationship between GAS6 levels and DFS or OS effectively treat these patients. The current study was conducted to (fig. S9, C to F). A similar result was observed with ERBB2, whose investigate the potential role of AXL activation in acquired resistance expression was not strongly correlated with either AXL (cor = 0.43, to trastuzumab with the aim of exploring AXL inhibition as a new P = 0.003) or VIM (cor = 0.32, P = 0.031) (Fig. 7H). potential therapeutic strategy for HER2+ BC patients. Several tyro- In the multivariable model, AXL was selected the best model for sine kinase inhibitors, monoclonal antibodies, and antibody-drug both DFS (hazard ratio, 22.95; 95% CI, 3.07 to 171.87; P = 0.002) conjugates against AXL are currently under development in differ- and OS (hazard ratio, 15.92; 95% CI, 0.849 to 298.32; P = 0.064) ac- ent clinical trials as effective targeted therapy in solid tumors (39). cording to Akaike’s information criterion (AIC). Collectively, these We demonstrated that acquired trastuzumab-resistant HER2+ findings reveal a ligand-independent association between high AXL BC cells have significantly higher AXL expression than sensitive expression and worse prognosis in patients with HER2+ BC treated cells. AXL up-regulation, together with a mesenchymal phenotype, with trastuzumab and taxanes plus anthracycline-based chemotherapy. has been associated with acquired resistance to several drugs in can- cer, including triple-negative BC (17, 19, 40). However, the role of Early in vivo biological changes during dual HER2 blockade AXL in the EMT process remains unclear (14). We found that AXL in BC patients up-regulation occurred in the context of an EMT-associated tran- To identify early molecular changes induced by dual HER2 blockade scriptional program. The data obtained are consistent with previous in patients with HER2+ BC, gene expression profiling was performed studies in which VIM up-regulation was associated with AXL over- in 139 tumor samples obtained before treatment and at day 14 with expression in BC cells (41). Trastuzumab-resistant cell lines showed lapatinib and trastuzumab [and endocrine therapy in hormonal re- increased migration and invasion capability and an EMT-like phe- ceptor–positive (HR+) tumors] in the PAMELA phase 2 clinical trial notype, which are related to metastatic behavior. AXL gain and loss (35). AXL gene expression was explored at both time points. Of par- of function experiments across our in vitro models corroborated ticular interest was that AXL significantly increased at day 14 com- that AXL activity is associated with trastuzumab response and pared to baseline (P = 5.8 × 10−20) (Fig. 8A). Overall, this effect was induces a mesenchymal-like phenotype. AXL genetic or therapeutic observed in HER2+/HR+ (P = 5.63 × 10−7), in HER2+/HR− (P = 1.446 inhibition is sufficient to reduce migration and invasion capacity, × 10−6), and across all PAM50 subtypes (luminal A, P = 0.0192; EMT marker expression, and acquired trastuzumab resistance in luminal B, P = 0.00247; HER2-enriched, P = 6.73 × 10−8; basal-like, our models. Together, these results highlight the role of AXL in P = 0.042) (Fig. 8, B and C). This rapid response suggests that AXL metastatic cascade in HER2+ BC, in agreement with previous find- is involved in determining resistance to HER2 inhibition. ings by Goyette et al. (20). We completed the analysis of AXL in residual tumors from the Previous studies show evidence that AXL could be activated via surgery in those patients failing to achieve pathological complete both ligand-dependent and ligand-independent mechanisms (42). response (n = 97). Of note, the median time from the last treatment Recent data suggest that AXL heterodimerization with other Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 11 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 8. AXL mRNA biological changes after dual HER2 blockade. AXL mRNA expression changes between baseline and day 14 of treatment in all 139 tumor samples from PAMELA clinical trial (A) and across histological subtypes (B) and PAM50 subtypes (C). (D) AXL mRNA expression changes between baseline, day 14, and surgery in 97 residual tumors at surgery. (E) Correlation between AXL, VIM, and ERBB2 in 96 tumor samples from the PAMELA clinical trial. Each line represents a tumor sample. Increases are represented in orange, and decreases in blue. P values were determined by two-tailed paired t tests. Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 12 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE transmembrane receptors occurs in several solid tumors (42–44). trastuzumab with AXL inhibitor overcomes resistance to trastuzumab Our results provide evidence that AXL activation arises through both in vitro and in vivo. This dual inhibition induced complete AXL-HER2 heterodimerization followed by the trigger of PI3K and tumor regression in in vivo PDX models without tumor regrowth MAPK cascades. This is consistent with previous work showing after 321 days of total follow-up. The strong independent correla- that AXL can drive oncogenic signaling of these pathways in other tion of AXL expression with reduced DFS and OS in HER2+ BC cancer types (45). In addition, we demonstrated in our models that patients suggests that patients with high AXL expression could AXL activation occurs in a ligand-independent manner, and no benefit from AXL-targeted therapy. Moreover, we proved that high correlation was observed between GAS6 and AXL expression in AXL expression is not genetically driven (neither amplified nor either in vitro or in vivo models and patients’ samples. activating mutations) (50); thus, this key drug target will be missed We further elucidated the contribution of AXL in acquired resistance when using DNA-based tests. to trastuzumab in a PDX model derived from a HER2+ BC patient, Given the retrospective nature of this study, and to determine and interestingly, AXL overexpression was confirmed in acquired the potential heterogeneity of treatment effects associated with AXL trastuzumab-resistant PDX in vitro and in vivo models. Moreover, functions, our results warrant further validation in a prospective we reported that simultaneous therapeutical ablation of AXL and clinical trial. Despite this limitation, our findings provide a strong HER2 activity results in complete regression of trastuzumab-resistant rationale for developing and testing AXL inhibitors for clinical use HER2+ tumors. The potential effect of TP-0903 has been studied in AXL up-regulated HER2+ BC patients to either prevent or over- in vivo in other cancers such as leukemia (46, 47) and lung cancer come resistance to trastuzumab. (48). To our knowledge, this is the first report to demonstrate the in vivo efficacy and tolerability of TP-0903 combined with trastu- zumab in the treatment of HER2+ BC. All PDX animals treated with MATERIALS AND METHODS this combination remained tumor free for over 211 days after therapy Cell lines and reagents discontinuation. This observation also suggests that the combina- The BC cell lines BT474, SKBR3, AU565, HCC1954, and MDA- tion of HER2 and AXL inhibition might avoid treatment escape by MB-231 were purchased from the American Type Culture Collection tumor cells with acquired trastuzumab resistance, leading to a po- (Manassas, VA, USA). All cell lines were maintained as recommended tential long-term benefit for HER2+ BC patients. by the suppliers. TP-0903 was purchased from Selleckchem (#S7846, In agreement with previous research, our data further current Houston, TX, USA). Trastuzumab was obtained from Roche Pharma understanding of the role of AXL as a prognostic biomarker in (Basel, Switzerland). Trastuzumab-resistant cell lines (AU565R, human cancer (21). Analysis of primary tumor HER2+ BC samples BT474R, and SKBR3R) were obtained from F.R.’s group at the showed that high AXL expression at the moment of diagnosis was Fundación Jiménez Díaz Hospital. Cells were established by treat- strongly related with poorer prognosis in terms of DFS and OS. This ing with trastuzumab for 6 months, starting at 10 g/ml trastuzum- observation demonstrates a direct association between AXL activa- ab for the first 30 days and increasing the dose at each passage until tion and poor response to anti-HER2 therapies, leading to metastatic resistant clones emerged. The final trastuzumab concentration was disease development. In our patients’ cohort, AXL was also found to 15 g/ml (51). In vitro trastuzumab treatment was maintained at be strongly linked to VIM overexpression, which has been described 15 g/ml. TP-0903 was given at IC50 for each cell line for 72 hours. as a poor prognosis biomarker in triple-negative BC (49). However, little is known about its relationship with prognosis in HER2+ In vitro cell proliferation assays BC. Here, we demonstrated that VIM is also related to poor prog- Cell proliferation was measured using WST-1 Assay Reagent Cell nosis in HER2+ BC patients. Proliferation (#155902, Abcam, Cambridge, UK). A total of 5000 cells Another marked observation across tumor sample studies deriving per well were seeded in a 96-well plate with at least four technical from the PAMELA clinical trial is the increase of AXL expression in replicates for each condition. After the treatment, WST-1 was added residual disease during dual anti-HER2 therapy. This biological ac- to each well under sterile conditions (to a final concentration of 7% quisition was relatively rapid in the first 2 weeks of treatment, sug- of the total volume) and the plates were incubated for 4 hours at gesting the importance of AXL overexpression in development of 37°C. Absorbance was measured at 450 nm in a microplate reader anti-HER2 resistance and the consequent high risk of metastases. with background correction at 650 nm. This underlines that the selective pressures of therapy could shape cancer evolution, leading to selection of tumor subclones enriched Immunohistochemistry with aberrations causing drug resistance. IHC of cell lines was performed in a -slide VI plate (#80826, ibidi The increase in AXL mRNA expression upon trastuzumab resistance GmbH, Martinsried, Germany) for HR and HER2 staining and in observed in our in vitro and in vivo models and also in patients’ Cytospin cytocentrifuge for Ki67 staining. Cells were fixed with samples from the PAMELA trial indicates enhanced transcription methanol/acetone before antibody staining. For expression of HR activity. On that basis, further investigation exploring potential tran- and HER2, Benchmark XT instrument in a fully automated system scription factors regulating AXL in the context of trastuzumab re- (Ventana System, Roche) was used with the UltraView Universal sistance would be of special interest. DAB Detection Kit (#0526980600, Roche) and corresponding anti- In conclusion, this study demonstrates the substantial biological bodies (see table S3). For IHC analysis of tumor samples from significance of AXL activation in acquired trastuzumab resistance in vivo experiment, tissue specimens were embedded in paraffin in HER2+ BC. AXL plays a critical role in EMT and in development and sectioned at 4-m thickness. Ki67 staining was performed by of metastatic disease. Our findings demonstrate cross-talk between automated Ventana System, and for phospho-Axl and cleaved AXL and HER2 receptors. This heterodimerization induces resistance caspase 3 staining, tissue specimens were pretreated in the DAKO through PI3K and MAPK pathways activation. The combination of PT Link Pre-treatment module (#PT100/PT101, Dako, Glostrup, Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 13 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Denmark) and epitope retrieval was performed with Dako Low Re- Flow cytometry trieval Solution (citrate buffer, pH 6) for 30 min. After quenching For intracellular staining, fixation and permeabilization were per- endogenous peroxidase with EnVision FLEX Peroxidase-Blocking formed with the Cytofix/Cytoperm Plus Fixation/Permeabilization Reagent (#SM801, Dako), slides were incubated for 1 hour with Solution Kit (#555028, Becton Dickinson, Franklin Lake, NJ, USA) primary antibody (table S4) diluted in EnVision FLEX antibody before staining. For GAS6 staining, cells were previously treated with diluent (#K8006, Dako). Slides were incubated with Real EnVision GolgiStop (#554724, Becton Dickinson). For antibody staining, HRP Rabbit (#K5007, Dako) for 30 min following the manu- cells were incubated for 30 min in fluorescence-activated cell sorting facturer’s protocol. Detection of peroxidase was revealed with (FACS) buffer at the preferred antibody dilution (table S3). Samples 3,3′-­diaminobenzidine EnVision FLEX DAB+ Chromogen (#DM827, were analyzed using a BD LSRFortessa flow cytometer (Becton Dako), and samples were counterstained with Harris hematoxylin Dickinson), and data were analyzed by FlowJo V10 (FlowJo LLC). and mounted for visualization in Dako Coverstainer. Annexin V assay RNA isolation and RT-qPCR To assay the number of apoptotic cells, cells were stained with allo- mRNA expression was measured by reverse transcription quantita- phycocyanin–annexin V (#550475, BD Pharmingen) for 15 min and tive polymerase chain reaction (RT-qPCR) on RNA isolated from then washed and resuspended in 1:500 propidium iodide (#81845, BC cells and patients’ samples. RNA from eight to ten 10-m for- Sigma-Aldrich) viability marker and acquired on LSRFortessa. Data malin-fixed paraffin-embedded (FFPE) slides was extracted using were analyzed with FlowJo software. Annexin V+ cells were considered the High Pure FFPET RNA Isolation Kit (#06650775001, Roche) as apoptotic cells. following the manufacturer’s protocol. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract RNA from cell lines accord- siRNA and plasmid transfection ing to the manufacturer’s protocol. RNA was quantified with a Cells were transfected with Lipofectamine 2000 (#11668019 Invitrogen) NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, according to the manufacturer’s instructions. We used pcDNA AXL MA, USA). RNA (1000 ng) was retro-transcribed to complementary construct for overexpression donated by R. M. Melillo [#105932, DNA (cDNA) using a High-Capacity cDNA Reverse Transcription Addgene (52)] and AXL siRNA (#AM16708 Id1313 and Id1218, kit (#34368814, Applied Biosystems, Waltham, MA, USA). Gene expres- Invitrogen) or scramble siRNA (#AM4635, Invitrogen). Knockdown sion was quantified with TaqMan Universal Master Mix (#0123456, Applied and overexpression efficiency was assessed at 72 hours after trans- Biosystems) using TaqMan probes (see table S3). Glyceraldehyde-3-­ fection by RT-qPCR or Western blotting as previously described. phosphate dehydrogenase (GAPDH) expression was used as an in- ternal reference to normalize input cDNA. The threshold cycle Wound-healing assay value (CT) was determined for each measurement, and mRNA Cells were seeded at 100% confluence on a 24-well plate. For AXL expression was calculated relative to the control using the compar- gain or loss of function, cells were seeded 72 hours after transfec- ative critical threshold (2−CT) method, where CT = CTmRNA – tion, and for pharmacological inhibition of AXL, the drug was added CThousekeeping control. Triplicates were performed for each sample. to media during the assay. The wound was induced by scratching the monolayer with a micropipette tip, and cells were allowed to move Protein extraction, Western immunoblotting, and co- into the gap. Migration rate was calculated as the proportion of ini- immunoprecipitation tial scratch distance and the mean distance between the two borders Protein expression was measured through Western blot. Cells were remaining cell free after migration. Triplicates were performed for lysed with radioimmunoprecipitation assay buffer (#89900, Thermo each condition. Fisher Scientific) and sonicated for five cycles of 10 s at 50% pulse and 50 s of pause. Protein quantity was measured with the Pierce In vitro cell migration and invasion assay BCA Protein Assay Kit (#23227, Thermo Fisher Scientific). For im- Invasion and migration assay was performed using an 8-m-pore munoprecipitation, whole-cell lysates were precleared with the ap- Boyden chamber (24-well, #353097, Corning, NY, USA). For inva- propriate control immunoglobulin G (IgG) together with agarose sion assay, the upper chamber was coated with Matrigel (#356235, conjugate (Protein G PLUS-Agarose; sc-2002, Santa Cruz Biotech- Corning). For pharmacological inhibition of AXL, cells were pre- nology, Dallas, TX, USA) at 4°C for 30 min. After bead pelleting, treated with dimethyl sulfoxide (DMSO) or TP-0903 for 24 hours 500 g of total cellular protein was incubated rotating head over tail before seeding. For AXL down-regulation or overexpression, cells for 2 hours at 4°C with anti-HER2 or anti-AXL antibodies and then were seeded 72 hours after transfection. A total of 50,000 cells were reacted overnight with Protein G PLUS-Agarose beads (#2002, seeded in serum-free medium in the top chamber and allowed to Santa Cruz Biotechnology). These beads were then washed three migrate for 48 hours toward the bottom chamber containing times with lysis buffer, and immunoblotting was performed with 10% fetal bovine serum medium. Cells were fixed and permeabilized anti-HER2 and anti-AXL antibodies. Cell lysates or immunoprecip- with cold 70% ethanol and methanol and stained with 0.4% crystal itates were resolved by SDS–polyacrylamide gel electrophoresis gel, violet. The average number of migrating cells was evaluated from at transferred to nitrocellulose membrane, and blocked with 5% least four independent microscope fields. bovine serum albumin on tris-buffered saline–Tween. Membranes were incubated with the primary antibody overnight at 4°C and Proximity ligation assay then washed and incubated with secondary antibody. Blots were de- Cells were fixed and permeabilized with the Cytofix/Cytoperm Plus veloped with a chemiluminescence system. GAPDH expression was Fixation/Permeabilization Solution Kit. Primary antibodies (see used as an internal endogenous control. The antibodies used are table S3) were incubated overnight at 4°C. Hybridization with listed in table S3. the PLA probes and negative control conditions, ligation, and Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 14 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE amplification were performed with Duolink reagents according to with either trastuzumab (10 mg/kg, intraperitoneally) biweekly, the manufacturer’s recommendations (#DUO92007, Sigma-Aldrich, TP-0903 orally (50 mg/kg) daily 5/7, or a combination of both St. Louis, MO, USA). Technical negative controls were included by across 21 days (from days 89 to 110). A follow-up of 321 days was omission of primary antibodies. PLA results were obtained with a recorded. Animal experiments received ethical approval from Leica TCS SP8 confocal microscope and by flow cytometry with BD Generalitat de Catalunya/Comissió d’Experimentació Animal Òrgan FACSAria III instrument (Becton Dickinson) and analyzed by Habilitat (Authorised Establishment B9900062, ethical protocol FlowJo V10. CEA-OH/10303). In vitro PDX–resistant model Clinical samples PDX118 (ER+/PR−/HER23+) was derived from skin metastasis col- Samples were obtained from patients with early-stage HER2+ BC lected by core needle biopsy. PDXs were established at Vall treated at the Hospital Clínico de Valencia Department of Medical d’Hebron Institute of Oncology (VHIO) following institutional Oncology from 2004 to 2016 by standard guidelines. Patients re- guidelines. The Institutional Review Board at Vall d’Hebron Hospital ceived (neo)adjuvant chemotherapy and anti-HER2 therapy provided approval for this study in accordance with the Declaration (trastuzumab ± pertuzumab) for 6 months, followed by surgery. of Helsinki. Written informed consent was obtained from all pa- Adjuvant treatment was completed with trastuzumab for up to 1 year, tients who provided tissue samples. Trastuzumab in vitro PDX– and a minimum of 5 years of hormonal therapy for patients with resistant models (PDX118-TR1 and PDX118-TR2) were established HR+ tumors. Radiation therapy was administered according to local by culturing cells in the presence of increasing concentration of guidelines. Patients provided signed informed consent for experi- trastuzumab (starting from 5 to 1000 ng/ml). In both models, resistance mental analysis of samples. The study is compliant with all relevant was obtained after 4 months of treatment. For proliferation experi- ethical regulations regarding research involving human participants ments, PDX118-TR1 and PDX118-TR2 cell lines were treated with and received ethical approval from the Hospital Clínico Research trastuzumab for 7 days combined with TP-0903 at a concentration Ethics Committee. All samples were FFPE and had been analyzed of 150 nM during the last 72 hours of treatment. by an expert pathologist to ensure >30% tumor infiltration. HR sta- tus was evaluated by IHC (ER+ and PR+ were defined as ≥1% posi- Patient-derived organoid generation and treatment tively stained nuclei), and HER2 was assessed by IHC and/or Tumor cells were seeded in 48-well plates (5 × 104 cells per well) in fluorescence in situ hybridization (FISH) (IHC: complete 3+ mem- a drop of 20 l of Matrigel. Each drop was dispensed in the center of brane staining ≥10% invasive cells; FISH:HER2:CEP17 ratio ≥ 2.0). the well and incubated for 15 min at room temperature. After the Clinicopathological data of this cohort are detailed in table S2. Matrigel solidified, 250 l of 3D breast tumor organoid medium was added to each well (53). Medium was replaced twice weekly, and 3D Analysis of public human data formation was assessed after 15 days. Organoids per well were counted HER2+ BC cohort from Kaplan-Meier plotter software was ana- and each one assumed to contain approximately 50 cells. Organoid lyzed. The prognostic value of AXL and GAS6 mRNA expression medium was removed, and 3D structures were cultured with trastu- was analyzed in 252 HER2+ by IHC BC patients with available data. zumab and/or TP-0903. At the end point, organoids were disaggre- The median value was used to identify the high and low groups. gated by adding 500 l of trypsin for 30 min at 37°C, after which The hazard ratio with 95% CI and P value were calculated, and cells were collected and incubated for 30 min on ice to liquify the Kaplan-Meier curves were plotted (34). Matrigel. Fully disaggregated organoids were washed and stained as previously described in full (53). The number of live cells was quan- PAMELA study tified with FlowJo software by means of EpCAM counts. Viability The main results of the PAMELA neoadjuvant phase 2 study have marker Zombie Aqua was used at 1:1000 (#423101, BioLegend, San been previously reported (35). This completed study is registered Diego, CA, USA). with ClinicalTrials.gov (NCT01973660). The study protocol was approved by independent ethics committees at each center. In this In vivo PDX–resistant model trial, 151 patients with early HER2+ BC were treated with neoadjuvant To generate the PDX118 model resistant to trastuzumab, nonobese lapatinib (1000 mg daily) and trastuzumab (8 mg kg−1 intravenous diabetic (NOD)–severe combined immunodeficient (SCID) mice loading dose followed by 6 mg kg−1) for 18 weeks. Patients with were transplanted with fragments of PDX118 tumors. Once tumor HR+ BC received letrozole or tamoxifen according to menopausal size reached 300 mm3, animals were intraperitoneally injected with status. Tumor samples were collected at baseline, day 14, and sur- trastuzumab (10 mg/kg) twice per week. After two passages, resistant gery and then FFPE. HER2+ was defined as 3+ or 2+ overexpression tumors (termed PDX118-TR4) were obtained. Samples for flow cy- by IHC and positive for chromogenic in situ hybridization. RNA tometry and reinjection were digested in collagenase IA (300 U/ml; samples of the PAMELA trial at baseline, at day 14 of treatment, #C2674, Sigma-Aldrich) and hyaluronidase IS (100 U/ml; #H3506, and in residual tumors were previously analyzed using a panel of Sigma-Aldrich) in Dulbecco's modified Eagle’s medium/F-12. After 560 genes (including AXL, VIM, and ERBB2) at the nCounter plat- 1 hour of incubation at 37°C with shaking at 80 rpm, the mixture form (NanoString Technologies, Seattle, WA, USA). was filtered through 100-m strainers. Red blood cells were lysed with 1× RBC lysis buffer for 5 min at room temperature. After a Statistical analysis wash with 1× phosphate-buffered saline, samples were counted and Statistical analysis was performed using GraphPad Prism software either reinjected or stained for HER2 and EpCAM as previously de- (version 6.01; GraphPad Software Inc., La Jolla, CA, USA) and R scribed and acquired on LSRFortessa. Data were analyzed with (version 3.6.2). Results were expressed as means ± SD. Normality was FlowJo software. For the in vivo experiment, mice were injected checked using Shapiro-Wilks’s test. Mean comparison was carried Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 15 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE out using two-tailed Student’s t test. All experiments were repro- mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors duced at least three times. and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clin. Cancer Res. 19, 279–290 (2013). GAS6, ERBB2, AXL, and VIM gene expression in the retrospec- 14. H. Taniguchi, T. Yamada, R. Wang, K. Tanimura, Y. Adachi, A. Nishiyama, A. Tanimoto, tive cohort was dichotomized by the median. Prognostic potential S. Takeuchi, L. H. Araujo, M. Boroni, A. Yoshimura, S. Shiotsu, I. Matsumoto, S. Watanabe, was evaluated by ROC analysis, and optimal cutoff was calculated T. Kikuchi, S. Miura, H. Tanaka, T. Kitazaki, H. Yamaguchi, H. Mukae, J. Uchino, H. Uehara, with Youden index (54). DFS and OS were described graphically K. Takayama, S. Yano, AXL confers intrinsic resistance to osimertinib and advances using Kaplan-Meier curves, and differences were evaluated using the emergence of tolerant cells. Nat. Commun. 10, 1–14 (2019). 15. S. Terry, A. Abdou, A. S. T. Engelsen, S. Buart, P. Dessen, S. Corgnac, D. Collares, the log-rank test. Cox regression was conducted to calculate hazard G. Meurice, G. Gausdal, V. Baud, P. Saintigny, J. B. Lorens, J. P. Thiery, F. Mami-Chouaib, ratio and 95% CI. Cox multivariable model included dichotomized S. Chouaib, AXL targeting overcomes human lung cancer cell resistance to NK- And CTL- GAS6, ERBB2, AXL, VIM, tumor size, and affected nodes. Selection mediated cytotoxicity. Cancer Immunol. Res. 7, 1789–1802 (2019). of the best model was done according to AIC using a stepwise 16. J. Boshuizen, L. A. Koopman, O. Krijgsman, A. Shahrabi, E. G. Van Den Heuvel, M. A. Ligtenberg, D. W. Vredevoogd, K. Kemper, T. Kuilman, J. Y. Song, N. Pencheva, forward-backward approach. J. T. Mortensen, M. G. Foppen, E. A. Rozeman, C. U. Blank, M. L. Janmaat, D. Satijn, To identify AXL gene expression changes in clinical samples of E. C. W. Breij, D. S. Peeper, P. W. H. I. Parren, Cooperative targeting of melanoma the PAMELA trial across time points (baseline versus day 14 or day heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors. Nat. Med. 14 versus surgery), we used two-tailed paired t tests. Two-way anal- 24, 203–212 (2018). ysis of variance (ANOVA) with Bonferroni correction post hoc 17. Z. Zhang, J. C. Lee, L. Lin, V. Olivas, V. Au, T. Laframboise, M. Abdel-Rahman, X. Wang, A. D. Levine, J. K. Rho, Y. J. Choi, C. M. Choi, S. W. Kim, S. J. Jang, Y. S. Park, W. S. Kim, was used for in vivo statistical analysis. The cutoff for statistical sig- D. H. Lee, J. S. Lee, V. A. Miller, M. Arcila, M. Ladanyi, P. Moonsamy, C. Sawyers, nificance in all tests was 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, T. J. Boggon, P. C. Ma, C. Costa, M. Taron, R. Rosell, B. Halmos, T. G. Bivona, Activation ****P < 0.0001). of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nat. Genet. 44, 852–860 (2012). 18. N. Okura, N. Nishioka, T. Yamada, H. Taniguchi, K. Tanimura, Y. Katayama, A. Yoshimura, SUPPLEMENTARY MATERIALS S. Watanabe, T. Kikuchi, S. Shiotsu, T. Kitazaki, A. Nishiyama, M. Iwasaku, Y. Kaneko, Supplementary material for this article is available at https://science.org/doi/10.1126/ J. Uchino, H. Uehara, M. Horinaka, T. Sakai, K. Tanaka, R. Kozaki, S. Yano, K. Takayama, sciadv.abk2746 ONO-7475, a Novel AXLInhibitor, suppresses the adaptive resistance to initial EGFR-TKI View/request a protocol for this paper from Bio-protocol. treatment in EGFR-mutated non-small cell lung cancer. Clin. Cancer Res. 26, 2244–2256 (2020). 19. M. Elkabets, E. Pazarentzos, D. Juric, Q. Sheng, R. A. Pelossof, S. Brook, A. O. Benzaken, REFERENCES AND NOTES J. Rodon, N. Morse, J. J. Yan, M. Liu, R. Das, Y. Chen, A. Tam, H. Wang, J. Liang, J. M. Gurski, 1. M. M. Moasser, The oncogene HER2: Its signaling and transforming functions and its role D. A. Kerr, R. Rosell, C. Teixidó, A. Huang, R. A. Ghossein, N. Rosen, T. G. Bivona, in human cancer pathogenesis. Oncogene 26, 6469–6487 (2007). M. Scaltriti, J. Baselga, AXL mediates resistance to PI3K inhibition by activating 2. E. A. Perez, E. H. Romond, V. J. Suman, J. H. Jeong, N. E. Davidson, C. E. Geyer, S. Martino, the EGFR/PKC/mTOR axis in head and neck and esophageal squamous cell carcinomas. E. P. Mamounas, P. A. Kaufman, N. Wolmark, Four-year follow-up of trastuzumab plus Cancer Cell 27, 533–546 (2015). adjuvant chemotherapy for operable human epidermal growth factor receptor 2-positive 20. M. A. Goyette, S. Duhamel, L. Aubert, A. Pelletier, P. Savage, M. P. Thibault, R. M. Johnson, breast cancer: Joint analysis of data from NCCTG N9831 and NSABP B-31. J. Clin. Oncol. P. Carmeliet, M. Basik, L. Gaboury, W. J. Muller, M. Park, P. P. Roux, J. P. Gratton, J. F. Côté, 29, 3366–3373 (2011). The receptor tyrosine kinase AXL is required at multiple steps of the metastatic cascade 3. J. Wang, B. Xu, Targeted therapeutic options and future perspectives for her2-positive during HER2-positive breast cancer progression. Cell Rep. 23, 1476–1490 (2018). breast cancer. Signal Transduct. Target. Ther. 4, 34 (2019). 21. C. Gjerdrum, C. Tiron, T. Høiby, I. Stefansson, H. Haugen, T. Sandal, K. Collett, S. Li, 4. T. Mukohara, Mechanisms of resistance to anti-human epidermal growth factor receptor E. McCormack, B. T. Gjertsen, D. R. Micklem, L. A. Akslen, C. Glackin, J. B. Lorens, Axl is 2 agents in breast cancer. Cancer Sci. 102, 1–8 (2011). an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer 5. R. N. Kaplan, B. Psaila, D. Lyden, Bone marrow cells in the “pre-metastatic niche”: Within metastasis and patient survival. Proc. Natl. Acad. Sci. U.S.A. 107, 1124–1129 (2010). bone and beyond. Cancer Metastasis Rev. 25, 521–529 (2006). 22. V. Davra, S. Kumar, K. Geng, D. Calianese, D. Mehta, V. Gadiyar, C. Kasikara, K. C. Lahey, 6. A. Dongre, R. A. Weinberg, New insights into the mechanisms of epithelial–mesenchymal Y. J. Chang, M. Wichroski, C. Gao, M. S. de Lorenzo, S. V. Kotenko, T. Bergsbaken, transition and implications for cancer. Nat. Rev. Mol. Cell Biol. 20, 69–84 (2019). P. K. Mishra, W. C. Gause, M. Quigley, T. E. Spires, R. B. Birge, Axl and mertk receptors 7. M. A. Nieto, Epithelial-mesenchymal transitions in development and disease: Old views cooperate to promote breast cancer progression by combined oncogenic signaling and new perspectives. Int. J. Dev. Biol. 53, 1541–1547 (2009). and evasion of host antitumor immunity. Cancer Res. 81, 698–712 (2021). 8. J. Antony, R. Y. J. Huang, AXL-driven EMT state as a targetable conduit in cancer. 23. W. Leconet, M. Chentouf, S. Du Manoir, C. Chevalier, A. Sirvent, I. Aït-Arsa, M. Busson, M. Jarlier, Cancer Res. 77, 3725–3732 (2017). N. Radosevic-Robin, C. Theillet, D. Chalbos, J. M. Pasquet, A. Pèlegrin, C. Larbouret, B. Robert, 9. M. Asiedu, F. Beauchamp-Perez, J. Ingle, D. Radisky, K. Knutson, AXL induces epithelial-to- Therapeutic activity of anti-AXL antibody against triple-negative breast cancer patient- mesenchymal transition and regulates the function of breast cancer stem cells. Oncogene derived xenografts and metastasis. Clin. Cancer Res. 23, 2806–2816 (2017). 33, 1316–1324 (2014). 24. M. A. Goyette, R. Cusseddu, I. Elkholi, A. Abu-Thuraia, N. El-Hachem, B. Haibe-Kains, 10. L. Liu, J. Greger, H. Shi, Y. Liu, J. Greshock, R. Annan, W. Halsey, G. M. Sathe, A. M. Martin, J. P. Gratton, J. F. Côté, AXL knockdown gene signature reveals a drug repurposing T. M. Gilmer, Novel mechanism of lapatinib resistance in HER2-positive breast tumor cells: opportunity for a class of antipsychotics to reduce growth and metastasis of triple- Activation of AXL. Cancer Res. 69, 6871–6878 (2009). negative breast cancer. Oncotarget 10, 2055–2067 (2019). 11. M. A. Miller, M. J. Oudin, R. J. Sullivan, S. J. Wang, A. S. Meyer, H. Im, D. T. Frederick, 25. J. Wei, H. Sun, A. Zhang, X. Wu, Y. Li, J. Liu, Y. Duan, F. Xiao, H. Wang, M. Lv, L. Wang, J. Tadros, L. G. Griffith, H. Lee, R. Weissleder, K. T. Flaherty, F. B. Gertler, C. Wu, A novel AXL chimeric antigen receptor endows T cells with anti-tumor effects D. A. Lauffenburger, Reduced proteolytic shedding of receptor tyrosine kinases is a post-translational mechanism of kinase inhibitor resistance. Cancer Discov. 6, 383–399 against triple negative breast cancers. Cell. Immunol. 331, 49–58 (2018). (2016). 26. M. Schoumacher, M. Burbridge, Key roles of AXL and MER receptor tyrosine kinases 12. J. Müller, O. Krijgsman, J. Tsoi, L. Robert, W. Hugo, C. Song, X. Kong, P. A. Possik, in resistance to multiple anticancer therapies. Curr. Oncol. Rep. 19, 19 (2017). P. D. M. Cornelissen-Steijger, M. H. G. Foppen, K. Kemper, C. R. Goding, U. McDermott, 27. D. Kim, D. H. Bach, Y. H. Fan, T. T. T. Luu, J. Y. Hong, H. J. Park, S. K. Lee, AXL degradation C. Blank, J. Haanen, T. G. Graeber, A. Ribas, R. S. Lo, D. S. Peeper, Low MITF/AXL ratio in combination with EGFR-TKI can delay and overcome acquired resistance in human predicts early resistance to multiple targeted drugs in melanoma. Nat. Commun. 5, 1–15 non-small cell lung cancer cells. Cell Death Dis. 10, 1–12 (2019). (2014). 28. T. Brabletz, R. Kalluri, M. A. Nieto, R. A. Weinberg, EMT in cancer. Nat. Rev. Cancer 18, 13. L. A. Byers, L. Diao, J. Wang, P. Saintigny, L. Girard, M. Peyton, L. Shen, Y. Fan, U. Giri, 128–134 (2018). P. K. Tumula, M. B. Nilsson, J. Gudikote, H. Tran, R. J. G. Cardnell, D. J. Bearss, S. L. Warner, 29. Y. del Pozo Martin, D. Park, A. Ramachandran, L. Ombrato, F. Calvo, P. Chakravarty, J. M. Foulks, S. B. Kanner, V. Gandhi, N. Krett, S. T. Rosen, E. S. Kim, R. S. Herbst, B. Spencer-Dene, S. Derzsi, C. S. Hill, E. Sahai, I. Malanchi, Mesenchymal cancer cell-stroma G. R. Blumenschein, J. J. Lee, S. M. Lippman, K. K. Ang, G. B. Mills, W. K. Hong, crosstalk promotes niche activation, epithelial reversion, and metastatic colonization. J. N. Weinstein, I. I. Wistuba, K. R. Coombes, J. D. Minna, J. V. Heymach, An epithelial- Cell Rep. 13, 2456–2469 (2015). Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 16 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE 30. J. Antony, T. Z. Tan, Z. Kelly, J. Low, M. Choolani, C. Recchi, H. Gabra, J. P. Thiery, of leukemic cells to FLT3-targeted therapy in acute myeloid leukemia. Leukemia 29, R. Y. J. Huang, The GAS6-AXL signaling network is a mesenchymal (Mes) molecular 2382–2389 (2015). subtype-specific therapeutic target for ovarian cancer. Sci. Signal. 9, ra97 (2016). 47. J. Y. Jeon, D. R. Buelow, D. A. Garrison, M. Niu, E. D. Eisenmann, K. M. Huang, 31. M. Scaltriti, M. Elkabets, J. Baselga, Molecular pathways: AXL, a membrane receptor M. E. Zavorka Thomas, R. H. Weber, C. J. Whatcott, S. L. Warner, S. J. Orwick, B. Carmichael, mediator of resistance to therapy. Clin. Cancer Res. 22, 1313–1317 (2016). E. Stahl, L. T. Brinton, R. Lapalombella, J. S. Blachly, E. Hertlein, J. C. Byrd, B. Bhatnagar, 32. R. Vicario, V. Peg, B. Morancho, M. Zacarias-Fluck, J. Zhang, Á. Martínez-Barriocanal, S. D. Baker, TP-0903 is active in models of drug-resistant acute myeloid leukemia. A. Navarro Jiménez, C. Aura, O. Burgues, A. Lluch, J. Cortés, P. Nuciforo, I. T. Rubio, JCI Insight 5, e140169 (2020). E. Marangoni, J. Deeds, M. Boehm, R. Schlegel, J. Tabernero, R. Mosher, J. Arribas, Patterns 48. T. Sen, P. Tong, L. Diao, L. Li, Y. Fan, J. Hoff, J. V. Heymach, J. Wang, L. A. Byers, Targeting of HER2 gene amplification and response to anti-HER2 therapies. PLOS ONE 10, e0129876 AXL and mTOR pathway overcomes primary and acquired resistance to WEE1 inhibition (2015). in small-cell lung cancer. Clin. Cancer Res. 23, 6239–6253 (2017). 33. S. A. Kang, J. S. Guan, H. J. Tan, T. Chu, A. A. Thike, C. Bernado, J. Arribas, C. Y. Wong, 49. N. Yamashita, E. Tokunaga, H. Kitao, Y. Hisamatsu, K. Taketani, S. Akiyoshi, S. Okada, P. H. Tan, M. Gudi, T. C. Putti, J. Sohn, S. H. Lim, S. C. Lee, Y. P. Lim, Elevated WBP2 S. Aishima, M. Morita, Y. Maehara, Vimentin as a poor prognostic factor for triple-negative expression in HER2-positive breast cancers correlates with sensitivity to trastuzumab- breast cancer. J. Cancer Res. Clin. Oncol. 139, 739–746 (2013). based neoadjuvant therapy: A retrospective and multicentric study. Clin. Cancer Res. 25, 50. S. Garcia-Recio, A. Thennavan, M. P. East, J. S. Parker, J. M. Cejalvo, J. P. Garay, 2588–2600 (2019). D. P. Hollern, X. He, K. R. Mott, P. Galván, C. Fan, S. R. Selitsky, A. R. Coffey, D. Marron, 34. B. Györffy, A. Lanczky, A. C. Eklund, C. Denkert, J. Budczies, Q. Li, Z. Szallasi, An online F. Brasó-Maristany, O. Burgués, J. Albanell, F. Rojo, A. Lluch, E. M. De Dueñas, J. M. Rosen, survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer G. L. Johnson, L. A. Carey, A. Prat, C. M. Perou, FGFR4 regulates tumor subtype prognosis using microarray data of 1,809 patients. Breast Cancer Res. Treat. 123, 725–731 differentiation in luminal breast cancer and metastatic disease. J. Clin. Invest. 130, (2010). 4871–4887 (2020). 35. A. Llombart-Cussac, J. Cortés, L. Paré, P. Galván, B. Bermejo, N. Martínez, M. Vidal, 51. S. Zazo, P. González-Alonso, E. Martín-Aparicio, C. Chamizo, I. Cristóbal, O. Arpí, A. Rovira, S. Pernas, R. López, M. Muñoz, P. Nuciforo, S. Morales, M. Oliveira, L. de la Peña, A. Peláez, J. Albanell, P. Eroles, A. Lluch, J. Madoz-Gúrpide, F. Rojo, Generation, characterization, A. Prat, HER2-enriched subtype as a predictor of pathological complete response and maintenance of trastuzumab-resistant HER2+ breast cancer cell lines. Am. J. Cancer following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive Res. 6, 2661–2678 (2016). breast cancer (PAMELA): An open-label, single-group, multicentre, phase 2 trial. 52. G. P. Krishnamoorthy, T. Guida, L. Alfano, E. Avilla, M. Santoro, F. Carlomagno, Lancet Oncol. 18, 545–554 (2017). R. M. Melillo, Molecular mechanism of 17-allylamino-17-demethoxygeldanamycin 36. F. Brasó-Maristany, G. Griguolo, T. Pascual, L. Paré, P. Nuciforo, A. Llombart-Cussac, (17-AAG)-induced AXL receptor tyrosine kinase degradation. J. Biol. Chem. 288, B. Bermejo, M. Oliveira, S. Morales, N. Martínez, M. Vidal, B. Adamo, O. Martínez, S. Pernas, 17481–17494 (2013). R. López, M. Muñoz, N. Chic, P. Galván, I. Garau, L. Manso, J. Alarcón, E. Martínez, 53. E. J. Arenas, A. Martínez-Sabadell, I. Rius Ruiz, M. Román Alonso, M. Escorihuela, S. Gregorio, R. R. Gomis, P. Villagrasa, J. Cortés, E. Ciruelos, A. Prat, Phenotypic changes A. Luque, C. A. Fajardo, A. Gros, C. Klein, J. Arribas, Acquired cancer cell resistance to of HER2-positive breast cancer during and after dual HER2 blockade. Nat. Commun. 11, T cell bispecific antibodies and CAR T targeting HER2 through JAK2 down-modulation. 1–11 (2020). Nat. Commun. 12, 1237 (2021). 37. G. Griguolo, F. Brasó-Maristany, B. González-Farré, T. Pascual, N. Chic, T. Saurí, R. Kates, 54. W. J. Youden, Index for rating diagnostic tests. Cancer 3, 32–35 (1950). O. Gluz, D. Martínez, L. Paré, V. Tsvetkova, D. Pesantez, M. Vidal, B. Adamo, M. Muñoz, P. Galván, L. Barberá, M. Cuatrecasas, M. Christgen, H. Kreipe, I. Monge-Escartín, P. Villagrasa, D. Soy, T. Giarratano, M. V. Dieci, P. Conte, N. Harbeck, V. Guarneri, A. Prat, Acknowledgments: We thank the INCLIVA Biobank (PT17/0015/0049; B.000768 ISCIII) and the ERBB2 mRNA expression and response to ado-trastuzumab emtansine (T-DM1) Valencian Biobanking Network integrated into the Spanish National Biobanks Network for its in HER2-positive breast cancer. Cancers 12, 1902 (2020). collaboration. We are also grateful to SOLTI. We are especially grateful to the patients who 38. A. Prat, T. A. Pascual, C. De Angelis, C. Gutierrez, A. Llombart-Cussac, T. Wang, J. Cort, agreed to participate in this research and to Associations Amunt Contra el Cáncer, Asociación B. Rexer, L. Par, A. Forero, A. C. Wolff, S. In Morales, B. Adamo, F. B. O-Maristany, M. Vidal, Cultural Falla Palleter, and Asociación en tu Seno. Funding: A.A.-A., E.J.A., and F.B.-M. were J. Veeraraghavan, I. Krop, P. Galv, A. C. Pavlick, N. Bermejo, M. Izquierdo, V. Rodrik-Outmezguine, supported by Asociación Española contra el Cáncer AECC (PRDVA18013LLUC to A.A.-A., J. S. Reis-Filho, S. G. Hilsenbeck, M. Oliveira, M. V. Dieci, G. Griguolo, R. Fasani, P. Nuciforo, POSTD211413AREN to E.J.A., and AECC_Postdoctoral17-1062 to F.B.-M.). A.M.-S. was funded J. S. Parker, P. Conte, R. Schiff, V. Guarneri, C. K. Osborne, M. F. Rimawi, HER2-enriched by the Spanish Government (PFIS FI20/00188). J.Ar. is supported by Breast Cancer Research Foundation (BCRF-20-08), Instituto de Salud Carlos III Project reference number AC15/00062, subtype and ERBB2 expression in HER2-positive breast cancer treated with dual HER2 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative cofinanced by FEDER, blockade. J. Natl. Cancer Inst. 112, 46–54 (2020). Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra 39. J. Boshuizen, N. Pencheva, O. Krijgsman, D. D. Altimari, P. G. Castro, B. de Bruijn, el Cáncer (AECC). A.P. was supported by Instituto de Salud Carlos III—PI19/01846, Breast M. A. Ligtenberg, E. Gresnigt-Van den Heuvel, D. W. Vredevoogd, J.-Y. Song, N. Visser, Cancer Now—2018NOVPCC1294. P.E. and A.L. were funded by Instituto de Salud Carlos III and G. Apriamashvili, M. L. Janmaat, T. S. Plantinga, P. Franken, M. Houtkamp, A. Lingnau, cofinanced by FEDER (PI18/01219 to P.E. and CB16/12/00481 to A.L.). J.M.C. was funded by M. Jure-Kunkel, D. S. Peeper, Cooperative targeting of immunotherapy-resistant Sociedad Española de Oncología Médica (Rio Hortega-SEOM) and Compromiso ADAMED. melanoma and lung cancer by an AXL-targeting antibody–drug conjugate and immune Author contributions: A.A.-A., P.E., and J.M.C. conceptualized the study. A.A.-A., E.J.A., F.B.-M., checkpoint blockade. Cancer Res. 81, 1775–1787 (2021). A.M.-S., J.Ar., A.P., and J.M.C. designed the study methodology. A.A.-A., E.J.A., F.B.-M., A.M.-S., 40. C. Wang, H. Jin, N. Wang, S. Fan, Y. Wang, Y. Zhang, L. Wei, X. Tao, D. Gu, F. Zhao, J. Fang, J.C.-A., O.B., and J.M.C. interpreted the data and/or performed statistical analysis. A.A.-A., E.J.A., M. Yao, W. Qin, Gas6/Axl axis contributes to chemoresistance and metastasis in breast R.C., F.B.-M., A.M.-S., D.M., and E.T. conducted experiments. A.A.-A., E.J.A., F.B.-M., A.M.-S., C.H., cancer through Akt/GSK-3/- catenin signaling. Theranostics 6, 1205–1219 (2016). M.T.M., S.S., J.P., S.M., S.Z., A.R., O.B., F.R., J.Al., B.B., A.L., A.P., J.Ar., P.E., and J.M.C. interpreted 41. K. Vuoriluoto, H. Haugen, S. Kiviluoto, J. P. Mpindi, J. Nevo, C. Gjerdrum, C. Tiron, data and/or wrote the manuscript. A.A.-A., E.J.A., and J.M.C. were responsible for data curation J. B. Lorens, J. Ivaska, Vimentin regulates EMT induction by Slug and oncogenic H-Ras and supervised the project. A.L., B.B., J.Ar., A.P., and P.E. provided resources. J.M.C. supervised and migration by governing Axl expression in breast cancer. Oncogene 30, 1436–1448 the study. Competing interests: A.P. reports advisory and consulting fees from Roche, Pfizer, (2011). Novartis, Amgen, BMS, Puma, Oncolytics Biotech, MSD, Guardan Health, Peptomyc, and 42. A. S. Meyer, M. A. Miller, F. B. Gertler, D. A. Lauffenburger, The receptor AXL diversifies Lilly; lecture fees from Roche, Pfizer, Novartis, Amgen, BMS, NanoString Technologies, and EGFR signaling and limits the response to EGFR-targeted inhibitors in triple-negative Daiichi Sankyo; institutional financial interests from Boehringer, Novartis, Roche, NanoString, breast cancer cells. Sci. Signal. 6, ra66 (2013). Sysmex Europa GmbH, Medica Scientia inno. Research, SL, Celgene, Astellas, and Pfizer; and a 43. G.-X. Ruan, A. Kazlauskas, Axl is essential for VEGF-A-dependent activation of PI3K/Akt. leadership role in Reveal Genomics, SL; A.P. is an inventor on a patent related to this work filed EMBO J. 31, 1692–1703 (2012). by SOLTI Breast Cancer Research Group, Hospital Clinic de Barcelona, Vall d’Hebron Institute of 44. S. Salian-Mehta, M. Xu, M. E. Wierman, AXL and MET crosstalk to promote gonadotropin Oncology (VHIO) University and FISABIO (no. PCT/EP2016/080056, filed on 7 December 2016, releasing hormone (GnRH) neuronal cell migration and survival. Mol. Cell. Endocrinol. 374, published on 14 June 2018). J.Al. has received consulting or advisory role fees from Roche, 92–100 (2013). Pfizer, Amgen, MSD, Lilly, AstraZeneca, and Daiichi-Sankyo; research funding or grant support 45. A. K. Keating, G. K. Kim, A. E. Jones, A. M. Donson, K. Ware, J. M. Mulcahy, D. B. Salzberg, trials by Roche, Pfizer, Amgen, MSD, Lilly, and Daiichi-Sankyo; and travel and accommodation N. K. Foreman, X. Liang, A. Thorburn, D. K. Graham, Inhibition of Mer and Axl receptor support from Roche, Pfizer, Amgen, MSD, Lilly, and Daiichi-Sankyo. A.L. reports advisory and tyrosine kinases in astrocytoma cells leads to increased apoptosis and improved consulting fees from Novartis, Pfizer, Roche/Genentech, Eisai, and Celgene. J.M.C. has received chemosensitivity. Mol. Cancer Ther. 9, 1298–1307 (2010). honoraria as Speaker from Pfizer and Novartis, and travel expenses from Roche. The other 46. I. K. Park, B. Mundy-Bosse, S. P. Whitman, X. Zhang, S. L. Warner, D. J. Bearss, W. Blum, authors declare that they have no competing interests. Data and materials availability: All G. Marcucci, M. A. Caligiuri, Receptor tyrosine kinase Axl is required for resistance data needed to evaluate the conclusions in the paper are present in the paper and/or the Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 17 of 18 SCIENCE ADVANCES | RESEARCH ARTICLE Supplementary Materials. The patient-derived xenografts (PDXs) can be provided by J.Ar. Submitted 2 July 2021 (VHIO) pending scientific review and a completed material transfer agreement. Requests for Accepted 5 April 2022 the PDX models should be submitted to the corresponding author (jmcejalvo@incliva.es) and Published 20 May 2022 to J.Ar. (jarribas@vhio.net). 10.1126/sciadv.abk2746 Adam-Artigues et al., Sci. Adv. 8, eabk2746 (2022) 20 May 2022 18 of 18