Abstract
Neratinib is an irreversible, pan-HER tyrosine kinase inhibitor that is FDA approved for HER2-overexpressing/amplified (HER2+) breast cancer. In this preclinical study, we explored the efficacy of neratinib in combination with inhibitors of downstream signaling in HER2+ cancers in vitro and in vivo.
Cell viability, colony formation assays, and Western blotting were used to determine the effect of neratinib in vitro. In vivo efficacy was assessed with patient-derived xenografts (PDX): two breast, two colorectal, and one esophageal cancer (with HER2 mutations). Four PDXs were derived from patients who received previous HER2-targeted therapy. Proteomics were assessed through reverse phase protein arrays and network-level adaptive responses were assessed through Target Score algorithm.
In HER2+ breast cancer cells, neratinib was synergistic with multiple agents, including mTOR inhibitors everolimus and sapanisertib, MEK inhibitor trametinib, CDK4/6 inhibitor palbociclib, and PI3Kα inhibitor alpelisib. We tested efficacy of neratinib with everolimus, trametinib, or palbociclib in five HER2+ PDXs. Neratinib combined with everolimus or trametinib led to a 100% increase in median event-free survival (EFS; tumor doubling time) in 25% (1/4) and 60% (3/5) of models, respectively, while neratinib with palbociclib increased EFS in all five models. Network analysis of adaptive responses demonstrated upregulation of EGFR and HER2 signaling in response to CDK4/6, mTOR, and MEK inhibition, possibly providing an explanation for the observed synergies with neratinib.
Taken together, our results provide strong preclinical evidence for combining neratinib with CDK4/6, mTOR, and MEK inhibitors for the treatment of HER2+ cancer.
Neratinib is an irreversible, pan-HER tyrosine kinase inhibitor that is FDA approved for HER2-overexpressing/amplified breast cancer in the adjuvant and metastatic setting. This preclinical study explored the combinatorial therapies of neratinib. Results from our in vitro HER2+ cell lines showed that neratinib was synergistic with the mTOR inhibitors everolimus and sapanisertib, CDK4/6 inhibitor palbociclib, MEK inhibitor trametinib, and PI3Kα inhibitor alpelisib. In vivo tumor growth study of HER2+ tumor patient-derived xenografts further confirmed the enhanced therapeutic efficacy when neratinib was combined with everolimus, trametinib, or palbociclib. Reverse phase protein array assay and network-level adaptive response analysis revealed potential molecular mechanisms for the observed synergies with neratinib. Taken together, the promising outcomes of this preclinical study provide strong rationale for combining neratinib with a number of pathway inhibitors.
Introduction
HER2/neu, also known as ERBB2, is a member of EGFR/ERBB family of four structurally related transmembrane HER receptors—HER1 (EGFR), HER2, HER3, and HER4 (1). By autophosphorylation, receptor dimerization causes activation of their intrinsic intracellular receptor tyrosine kinase activity, leading to ultimate activation of downstream signaling cascades, principally through the MAPK and the PI3K/Akt pathways (2, 3), resulting in the activation of gene expression, proliferation, cell migration, differentiation, and regulation of apoptosis. Amplification or overexpression of HER2 is found in about 15%–25% of breast cancers, which is associated with aggressive biology and worse overall survival in the absence of HER2-targeted therapy (4–6).
Neratinib (HKI-272, Puma Biotechnology) is an oral, small-molecule, irreversible pan-inhibitor of the EGFR, HER2, and HER4 members of the ERBB tyrosine kinase family (7–9). Neratinib covalently binds to cysteine residues (Cys773 and Cys805) which are conserved in these receptors, preventing receptors from phosphorylation, thus blocking activation of tyrosine kinase activity, and subsequent downstream signaling cascades (8, 10). In addition to the MAPK and PI3K/Akt pathways, neratinib also induces cell-cycle arrest through cyclin D and Rb (11). Neratinib is FDA approved for extended adjuvant treatment of high-risk HER2-overexpressing or -amplified (HER2+) breast cancer in monotherapy, and in combination therapy with capecitabine for advanced or metastatic breast cancer patients who have received two or more anti-HER2 agents. Combination therapies of neratinib with trastuzumab or other chemotherapeutic agents such as paclitaxel have demonstrated clinical benefit (12–15). Furthermore, neratinib has been shown to have antitumor efficacy alone and in combination with endocrine therapy for selected tumors with activating HER2 mutations (16, 17).
Although many patients benefit from neratinib, as with other targeted therapies, patients often ultimately progress. Findings from a recent study indicated that an acquired ERBB2 T7981 mutation may contribute to the acquired resistance to neratinib (18). Therefore, pursuing new therapeutic strategies that can enhance initial efficacy of neratinib therapy is necessary.
Several cell signaling inhibitors targeting HER2 downstream signaling have already been approved by the FDA for indications other than HER2+ cancers. MEK inhibitor trametinib in combination with dabrafenib is approved for melanoma as well as lung cancer with BRAF_V600E mutations (19). PI3K inhibitor alpelisib in combination with fulvestrant is approved for hormone receptor–positive breast cancers bearing PIK3CA mutations (20). Everolimus is approved for multiple types of solid tumors including hormone receptor–positive breast cancer (in combination with exemestane; ref. 21), neuroendocrine tumors, renal cell carcinoma, and subependymal giant cell astrocytoma associated with tuberous sclerosis. Of three FDA-approved cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, palbociclib is approved in combination with aromatase inhibitors as initial endocrine-based therapy for metastatic hormone receptor–positive cancer, and in combination with fulvestrant in patients with disease progression on endocrine therapy (22).
In this study, we sought to explore effective combination therapies with neratinib for HER2+ cancer. We examined potential synergistic combinations of neratinib with targeted inhibitors of multiple downstream adaptive cell survival pathways, including the MAPK and PI3K/Akt pathways by testing in vitro cell line models and in vivo patient-derived xenografts (PDX) of multiple histologies.
Materials and Methods
Cell lines, drugs, and other reagents
Breast cancer cell lines, including BT-474, SK-BR-3, HCC-1954, MDA-MB-361, MDA-MB-453, and CAMA-1 were obtained from the ATCC. All the cell lines tested Mycoplasma negative. Cells were cultured in DMEM/F-12 supplemented with 10% FBS at 37°C and humidified 5% CO2. Neratinib was obtained from Puma Biotechnology Inc. Everolimus (NSC733504) and trametinib (NSC758246) were obtained from NCI Developmental Therapeutics Program. Palbociclib was obtained as a gift from Pfizer as well as from the NCI Developmental Therapeutic Program (https://dtp.cancer.gov). Alpelisib was obtained through the AACR PI3K SU2C Dream Team. Sapanisertib was purchased from Selleck Chemicals. The following antibodies for Western blotting were purchased from Cell Signaling Technology, including anti-HER2 (#2242), anti-phospho-Akt/T308 (#4056), anti-Akt (#9272), anti-phospho-S6K1/T389 (#9234), anti-S6K1 (#9202), anti-phospho-S6/S235/236 (#4858), anti-S6 (#2217), anti-phospho-4E-BP1/S65 (#9456), anti-4E-BP1 (#9452), anti-phospho-ERK1/2/T202/Y204 (#4370), anti-ERK1/2 (#9102), anti-phospho-MEK1/2/S217/221 (#9154), anti-MEK1/2 (#9126), and anti-phospho-Rb/S780 (#9307). Anti-β-actin antibody (#A5441) was purchased from Sigma. Second antibodies Goat-anti-Rabbit-Alexa Fluor-680 (#A21076) and Goat-anti-Mouse-Dylight-800 (#610145-121) were purchased from Life Technologies and Rockland Immunochemicals, respectively.
Cell viability assay
Cells were seeded in 96-well plates at densities of 0.5–1.0 × 104 cells/100 μL per well in triplicates for each treatment dose. After adhering overnight, for single-drug treatment, 100 μL of drug at serially diluted concentrations were added to the wells and incubated at 37°C for 72 hours. Cells were then fixed with 50% trichloroacetic acid followed by staining with 0.4% sulforhodamine B (SRB) solution. Optical density (OD) values were read at 490 nm by plate reader Synergy 4 (BioTek). The half maximal inhibitory concentration (IC50) was determined on the basis of the sigmoid drug-inhibition curve using GraphPad Prism v6.05 software. For combinatorial drug treatment, the cells were treated with neratinib together with individual drugs at individual combinatorial dose ratios based on single-drug sigmoid dose–response curves. Each combination treatment group had six doses with a fixed combination ratio and a fixed serial dilution. For example, in combination of neratinib + alpelisib, we chose 10,000 and 90,000 nmol/L as the highest doses for neratinib and alpelisib, respectively (combination ratio: 0.11). Then we had five serial dilution at 10-fold from the highest combination doses. In combination of neratinib with everolimus, 10,000 and 20 nmol/L were chosen as the highest doses for neratinib and everolimus, respectively (combination ratio: 500; Supplementary Table S3). To evaluate combination efficacy, combination index (CI) was determined using CalcuSyn program based on Chou-Talalay IC50 model. CI < 1.0 (curve left shift): synergistic; CI = 1.0, additive; CI > 1.0 (curve right shift): antagonistic; ref. 23).
Colony formation assay
Cells were seeded in 6-well plates at a density of 1,000 cells per well in triplicates for each treatment group. Next day, cells were treated with the individual drugs, or in combination at the different concentrations. Culture medium was changed with fresh drugs twice a week. Cells were cultured for 3 weeks. Cell colonies were then fixed in 10% formalin and stained with 0.05% crystal violet in 25% methanol. The stained colonies were scanned and total colony area was quantitated using NIH ImageJ v.1.48 software. Effect-based CI was calculated by inhibition percentage of single-drug and combination treatments using Bliss combination model. CI = ((EA + EB) – (EA*EB))/EAB, where EA, EB, and EAB are effects of drug A and B and combination AB inhibition percentage, respectively. Here the effect is inhibition percentage of colony formation compared to vehicle controls. (CI < 1.0: synergistic; CI = 1.0: additive; CI > 1.0: antagonistic; ref. 24).
Western blot analysis
In immunoblotting assay to investigate the effect of drug combination on cell signaling, we first tested single agents at different doses and selected a proper dose for each agent for combination. After treated with single drug or combination for 24 hours, cells were washed with cold PBS and lysed in 2× Laemmli buffer. The protein concentrations in the cell lysates were measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The same amount of protein for each group was loaded to the gel (20–50 μg/lane). After SDS-PAGE, the protein was transferred to a 0.2 μmol/L nitrocellulose membrane (Bio-Rad Laboratories). Membranes were blocked with 0.1% casein blocking buffer at room temperature for 1 hour, followed by immunoblotting with the primary antibodies at room temperature overnight. After washing, the immunoblotting membrane was probed with the secondary antibodies with fluorescence conjugation. The immunoblots were visualized and the immunoblotting signal intensity quantitated using the Odyssey IR imaging system (Li-Cor Biosciences; refs. 25, 26). Several drug treatment experiments were performed and Fig. 2A is one of these immunoblotting.
In vivo studies
PDX TMR-248 was developed by implantation of postneoadjuvant therapy surgical samples through a collaboration with Champions Oncology. The other four PDXs (MDA-PDX.003.025, B8086, MDA-PDX.003.087, MDA-PDX.003.164) were generated from core biopsy samples obtained from patients with metastatic cancer as described previously (27, 28). Alterations shown in Table 1 are selected actionable genes. Genomic alterations for TMR-248, B8086, and PDX.003.164 were identified in lab-based whole-exome sequencing of developed PDXs. Genomic alterations for PDX.003.025 and PDX.003.087 were identified in patient samples prior to PDX development. After implantation in NSG mice, with subsequent passaging, models underwent short tandem repeat testing to confirm genomic match to the parental tumor, with subsequent passaging in nu/nu mice. Fragments (3 × 3 × 3 mm3) of the PDXs were surgically implanted on the flank of nude mice as described previously (27, 28). Once tumors grew to approximately 200 mm3, they were randomized for treatment. Neratinib, palbociclib, trametinib, and everolimus were suspended in water containing 0.5% methyl cellulose and were administered via oral gavage (100 μL) daily for the length of experiment. Tumor sizes were assessed twice a week and body weight was measured once a week. Tumor volumes were calculated using the formula (length × width2)/2. All experiments were approved by Institutional Animal Care and Use Committee.
Model . | Tumor type . | ER/PR . | Selected genomic coalterations . | Prior to collection . |
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MDA-PDX.003.025 | Breast, invasive lobular carcinoma | ER(−), PR(−) | PIK3CA H1047R, RPTOR amplification |
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TMR-248 | Breast, invasive ductal carcinoma | ER(+), PR(−) | ERBB2 V777L, PIK3CA H1047Q, AKT1 deletion, BRAF E26D |
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MDA-PDX.003.087 | Colorectal cancer | N/A | ERBB2 R678Q, IDH1 R132C, PIK3CA H1047R, TSC2 T1462I, R73Q, MET amplification |
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B8086 | Colorectal adenocarcinoma | N/A | KRAS G12V, PTEN 79+1G>A, MAP2K4 Q163* |
|
MDA-PDX.003.164 | GEJ adenocarcinoma | N/A | CDKN2A A97V, TP53 Y236C |
|
Model . | Tumor type . | ER/PR . | Selected genomic coalterations . | Prior to collection . |
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MDA-PDX.003.025 | Breast, invasive lobular carcinoma | ER(−), PR(−) | PIK3CA H1047R, RPTOR amplification |
|
TMR-248 | Breast, invasive ductal carcinoma | ER(+), PR(−) | ERBB2 V777L, PIK3CA H1047Q, AKT1 deletion, BRAF E26D |
|
MDA-PDX.003.087 | Colorectal cancer | N/A | ERBB2 R678Q, IDH1 R132C, PIK3CA H1047R, TSC2 T1462I, R73Q, MET amplification |
|
B8086 | Colorectal adenocarcinoma | N/A | KRAS G12V, PTEN 79+1G>A, MAP2K4 Q163* |
|
MDA-PDX.003.164 | GEJ adenocarcinoma | N/A | CDKN2A A97V, TP53 Y236C |
|
Abbreviations: 5-FU, 5-fluorouracil; AC, anthracycline/cyclophosphamide; Folfiri, folinic acid (leukovorin), 5-FU, irinotecan; Folfox, folinic acid (leukovorin), 5-FU, oxaliplatin; GEJ, gastroesophageal junction.
RPPA analysis
Tumor tissues of PDXs [TMR-248 (n = 4–5), PDX-003.025 (n = 5), B8086 (n = 3–4), PDX-003.087 (n = 5–7)] were harvested from mice treated with neratinib and its combinations. The extracted tumor proteins were detected by reverse phase protein array (RPPA), which was conducted by the MD Anderson Functional Proteomics core facility as described previously (29). The protein levels were normalized for protein loading and then log transformed. The two group comparisons were performed by fitting linear mixed effect model using “Group” as the fixed effect and “Model” as the random effect. The differentially expressed proteins were identified with specified FDR.
The network-level adaptive responses were computed using the Target Score algorithm as described in Yan and colleagues (30). The method is developed on the rationale that collective molecular responses of functionally associated molecule have a higher likelihood of therapeutic relevance than changes in abundances of individual molecules. On the basis of the collective upregulation of network modules in response to targeted therapies, the algorithm nominates combination therapies, involving the agent that induces the adaptive response and a second agent that targets vulnerabilities induced by the adaptive response. An adaptive response score, termed target score, is quantified for each molecular entity as a sum of the self-change and cumulative change in its network neighborhood. The highest target scores correspond to potential adaptive responses (drug-induced activation of oncogenic processes or deactivation of tumor suppressors). In summary, the method uses molecular profiles of response to a perturbation as input. In parallel, a reference network which captures potential relations between all measured molecular entities of interest is constructed. Here, the interactions between the measured entities are extracted from the SignedPC module of the Pathway Commons database (31). The collective adaptive responses to a perturbation are quantified as a sample/context-specific adaptation score (target score) that links protein interactions to drug response on the reference network using proteomic drug response data. Next, we identified the network modules that have a significantly high target score (i.e., collectively participate in adaptive responses) in each sample. The actionable targets that participate in sample-specific adaptive responses and drug combinations are selected.
Statistical analysis
For in vitro studies, Student t test was performed to compare groups. Pearson test was used for correlation between groups. For in vivo studies, one-way ANOVA tests followed by Tukey was used by Dunnett multiple comparison. Data were presented as means ± SEM. Log-rank test was used for comparison of Kaplan–Meier survival curves.
Results
Neratinib combination magnified inhibition of cell growth
First, we tested the efficacy of neratinib in a panel of breast cancer cell lines that are known to express HER2 protein at various levels, along with varying genomic alterations in the PI3K/Akt and MAPK pathways (Supplementary Table S1). Immunoblotting analysis showed that BT-474, SK-BR-3, and HCC-1954 cells that are known to be HER2+ cell lines (32, 33), strongly expressed HER2 protein, while MDA-MB-361 and MDA-MB-453 cells displayed lower HER2 expression levels (Supplementary Fig. S1A and S1B). Cell viability assay showed that IC50s of neratinib in these cell lines ranged from 4.5 to 2,024 nmol/L (Supplementary Fig. S1C; Supplementary Table S2). Among these, HER2+ BT-474, SK-BR-3, and HCC-1954 cells were the most sensitive cell lines to neratinib with low nanomolar IC50s. Pearson correlation analysis showed that that cell sensitivity to neratinib is significantly and positively correlated with HER2 levels (P = 0.042; Supplementary Fig. S1D). We have also examined in parallel the sensitivity of these breast cancer cells to multiple kinase inhibitors targeting the MAPK and PI3K/Akt pathways. The results showed varying sensitivities in these cell lines to different inhibitors (Supplementary Table S2).
The activity of neratinib in combination therapy was then evaluated in the three HER2+ breast cancer cell lines. Cell viability assay showed that in HCC-1954 and BT-474 cell lines, combinatorial treatments of neratinib with multiple targeting agents, including the MEK inhibitor trametinib, the PI3Kα inhibitor alpelisib, the mTOR inhibitors everolimus (allosteric mTOR inhibitor) and sapanisertib (catalytic mTOR inhibitor), and the CDK4/6 inhibitor palbociclib, produced antiproliferative activity. CI analysis using a Chou-Talalay model revealed that adding these drugs markedly synergized with neratinib-induced growth inhibition with low CI values ranging from 0.003 to 0.228 (Fig. 1A and B). Dose–response curves in HCC-1954 cells showed that neratinib combinations caused a left shift with substantially reduced IC50s, compared with single-drug treatment (Supplementary Fig. S2A–S2E). On the other hand, the combinatorial efficacy in SK-BR-3 cells was relatively less potent in general than that observed in other two cell lines. We did not observe a synergy between neratinib and trametinib in this cell line (Fig. 1C).
Next, we examined the combination effects on cell capacity of colony formation using HCC-1954 cells. First, we determined dose response of single-drug treatment for all the six agents. After 3-week exposure to individual drugs at different doses, colony quantitation showed that neratinib and other targeting agents dose dependently inhibited colony formation at appropriate dose ranges (Supplementary Fig. S3A–S3F). In the subsequent combination assay, combination dose for each drug was selected on the basis of the response of single-agent treatment. Cell colony staining showed that the cell ability to form colony was further reduced by combination of neratinib (5 nmol/L) with trametinib (2 nmol/L), alpelisib (500 nmol/L), everolimus (0.1 nmol/L), and sapanisertib (10 nmol/L), compared with the single-agent treatment (Fig. 1D and E). These results were confirmed by colony area quantitation (Supplementary Fig. S4A–S4D). Combination efficacy was analyzed using a Bliss combination model. CI values (CIBliss) proved that neratinib was capable of synergizing with trametinib, alpelisib, everolimus, and sapanisertib at their relatively lower doses with CIBliss ranging from 0.46 to 0.83 (Fig. 1G; Supplementary Fig. S4A–S4D). For combination of neratinib plus palbociclib, although the colony staining revealed a further reduction in colony formation with this combination compared with single-agent treatment (Fig. 1F), Bliss analysis showed a CIBliss value of 1.04, indicating an additive combinatorial effect between these two agents (Fig. 1G; Supplementary Fig. S4E).
Neratinib combination intensified inhibition of downstream signaling
We next evaluated the impact of neratinib and its combination on intracellular signaling. We treated three HER2+ cell lines, HCC-1954, BT-474, and SK-BR-3, with single agent or combinations for 24 hours, followed by Western blot analysis. Neratinib itself reduced phosphorylation levels of ERK1/2, Akt, and S6K in all three cell lines, compared with vehicle controls (Fig. 2A; Supplementary Fig. S5A, S5C and S5E), indicating that the HER2-targeting drug neratinib inhibited HER2 signaling through both MAPK and PI3K/Akt pathways. Neratinib also mildly inhibited MEK1/2 phosphorylation in HCC-1954 cells, while increased it in BT-474 cells (Fig. 2A; Supplementary Fig. S5F). All three cell lines displayed a substantial reduction of phospho-ERK1/2, a downstream substrate of MEK kinase, when they were subjected to MEK inhibitor trametinib, in comparison with vehicle controls (Fig. 2A; Supplementary Fig. S5F). However, trametinib was shown to increase phosphorylated MEK1/2 levels in all three cell lines. In parallel, single-drug treatment with mTOR inhibitors everolimus or sapanisertib attenuated phospho-4E-BP1 expression in HCC-1954 and BT-474 cells (Fig. 2A; Supplementary Fig. S5B). Everolimus alone also almost completely abolished phosphorylation of S6K protein in all three cell lines, with consequently reduced phospho-S6 protein levels (Fig. 2A; Supplementary Fig. S5B and S5D). However, the PI3K inhibitor alpelisib did not show signaling inhibition activity at the dose tested in these cells (Fig. 2A; Supplementary Fig. S5).
In combination treatment, neratinib plus trametinib showed a further reduction in phosphorylation of ERK1/2 in all three cell lines, compared with single-drug treatment (Fig. 2A; Supplementary Fig. S5E). The data also revealed that when neratinib was combined with mTOR inhibitors everolimus or sapanisertib, they produced an enhanced effect in inhibiting phosphorylation of 4E-BP1 and S6 over the single-drug treatment in BT-474 and HCC-1954 cells (Fig. 2A; Supplementary Fig. S5B and S5D). Combinations of neratinib with mTOR inhibitors also further reduced phospho-ERK1/2. Finally, neratinib combination with PI3K inhibitor alpelisib exhibited an enhanced inhibition of phosphorylation of 4E-BP1 in HCC-1954 and BT-474 and ERK1/2 in all three cell lines (Fig. 2A; Supplementary Fig. S5B and S5E). In separate immunoblotting assays, we examined combination efficacy of neratinib with palbociclib. We found that when neratinib was combined with palbociclib, it substantially enhanced palbociclib-induced inhibition of Rb phosphorylation in SK-BR-3 and BT-474 cells (Fig. 2B; Supplementary Fig. S5G). Neratinib/palbociclib combination also revealed greater inhibition of AKT phosphorylation in all three cell lines (Fig. 2B; Supplementary Fig. S5H). However, this combination showed enhanced reduction of phospho-EKR1/2 levels only in BT-474 cells (Fig. 2B; Supplementary Fig. S5I).
Neratinib combination enhanced antitumor efficacy on tumor growth of HER2+ PDXs
In vivo studies were designed to explore optimal therapeutic regimens of neratinib combination on tumor growth. The efficacy of the combinations was tested in five HER2+ PDXs that have been recently established (Table 1) from patients with HER2+ as defined by IHC score of 3+ and/or gene amplification by FISH. This PDX panel consisted of two breast cancer PDXs (TMR-248 and MDA-PDX.003.025), two colorectal cancer PDXs (B8086 and MDA-PDX.003.087), and one gastroesophageal junction (GEJ) cancer PDX (MDA-PDX.003.164). Both breast cancer models and one colorectal cancer model (MDA-PDX.003.087) had PIK3CA mutations, reported to confer relative resistance to trastuzumab-based therapy in several preclinical as well as clinical studies (34, 35). In addition, two of the models had HER2 missense mutations (ERBB2_V777L in TMR-248 and ERBB2_R678Q in MDA-PDX.003.087) which are both known to be activating mutations [36, 37]. It is also notable that four PDXs was generated from tumors of HER2+ patients who previously were treated with standard-of-care HER2-targeted therapy. TMR-248 was generated from a patient who did not achieve pathologic complete response with neoadjuvant pertuzumab/trastuzumab and paclitaxel therapy. PDX.003.025 was derived from a locally recurrent breast cancer in a patient previously treated with trastuzumab/pertuzumab, docetaxel, and carboplatin as well as T-DM1. PDX-003-087 had already been treated with standard-of-care colorectal cancer treatments as well as lapatinib/trastuzumab, a HER2-directed therapy that has been shown to have efficacy in HER2+, KRAS wild-type colorectal cancer (38) and that has recently been incorporated into National Cancer Center Network guidelines (39). PDX-003-164 was derived from an esophageal cancer from a patient previously treated with not only standard therapies including trastuzumab, but also two investigational HER2-targeted therapies. While B8086 had not been previously treated with prior HER2-targeted therapy, concomitant KRAS mutations have been shown to limit the efficacy of HER2-targeted therapy (40).
Nude mice bearing these PDXs were treated with orally administered single-agent neratinib (10 mg/kg) or combinations with trametinib (0.3 mg/kg), everolimus (5 mg/kg), or palbociclib (50 mg/kg) daily. The combinations were well tolerated as evidenced by monitoring of body weight and lack of treatment-related deaths. The therapeutic benefit of these combinations was evidenced by lower tumor volumes in these PDXs, compared with the single agent alone. The combination of neratinib with palbociclib significantly reduced tumor volume with T/C ratios from 0.03 to 0.16 across all five PDXs (Fig. 3A–E; Supplementary Fig. S6A–S6E). The neratinib combination with trametinib showed enhanced efficacy to reduce tumor volume in PDX.003.164 (Fig. 3E; Supplementary Fig. S6E), while neratinib plus everolimus further reduced tumor volume in both PDX.003.025 and PDX.003.164 models (Fig. 3B and E; Supplementary Fig. S6B and S6E).
We also compared event-free survival (EFS) defined as the tumor doubling using Kaplan–Meier survival curves between treatment groups (41). The survival curves showed that combination treatment of neratinib with palbociclib significantly extended EFS in all five PDX models, compared with the single-agent treatment and control groups (Fig. 3F–J). The combinatorial regimen of neratinib with everolimus significantly increased the EFS in PDX.003.025 when compared with the everolimus single-agent group but not the neratinib group. In contrast, the combination of neratinib with everolimus showed a significant increase in EFS in PDX.003.164 when compared with the neratinib single-agent group but not the everolimus group (Supplementary Fig. S7B and S7D). The neratinib plus trametinib combination significantly increased the PFS in PDX.003.164 (Supplementary Fig. S7I). For TMR-248, the combination of neratinib and tramentinib also increased the EFS in this group when compared with the trametinib treatment group but not to the neratinib group. (Supplementary Fig. S7E). In summary: (i) combination of neratinib with palbociclib demonstrated substantially enhanced antitumor efficacy in all five HER2+ PDX models, (ii) combinations of neratinib with everolimus or with trametinib provided varying therapeutic benefit, (iii) the GEJ cancer PDX (PDX.003.164) had the best responses to all three combinatorial treatments.
Effects of neratinib combination on functional proteomics of HER2+ PDXs
We also performed a proteomic study to explore the potential mechanisms underlying the enhanced combinatorial efficacy of neratinib therapeutics. RPPA was applied on 136 tumor tissue samples harvested from the treated PDX models as described above. When normalized protein expression was analyzed by unsupervised hierarchical clustering, tumors were clustered by PDX model, rather than treatment, demonstrating that the treatment-related differences were less robust than the intertumoral proteomic heterogeneity (Supplementary Fig. S8). With FDR 0.05, we assessed the differentially expressed proteins (DEP) with single-agent treatment compared with controls as well as single drug to combination treatment (Fig. 4). We found that neither neratinib nor trametinib induced significant DEPs. Everolimus treatment was associated with four differentially expressed proteins compared with untreated control PDXs (Fig. 4A). When everolimus was added to neratinib, 13 proteins were differentially expressed compared with neratinib alone (Fig. 4B). Everolimus treatment alone and in combination with neratinib led to upregulation of IR-b and phospho-NDRG1_T346, but downregulation of phospho-S6, a well-established pharmacodynamic marker of mTOR signaling.
Palbociclib treatment was associated with 26 DEPs compared with untreated controls (Fig. 4C), There were 44 DEPs identified in the group of neratinib/palbociclib combination, compared with neratinib alone (Fig. 4D). Both palbociclib single and combination treatments downregulated expression of Wee1 and phospho-Wee1_S642. Interestingly, some DEPs were presented in both everolimus and palbociclib combination groups, such as increased Bcl-XL, Glutamate-D1-2, IRS2, and IR-b, but decreased phospho-ATR_S428 and MelanA, suggesting an involvement of these DEPs in mechanisms or action of neratinib combination therapy.
We further analyzed adaptive responses with the Target Score algorithm, which quantifies network modules of functionally related molecular entities involved in adaptive responses to targeted perturbations (30). Using the Target Score algorithm, we analyzed the network-level adaptive responses to neratinib, palbociclib, everolimus, and trametinib (Fig. 5; Supplementary Fig. S9). The adaptation scores were significantly higher for EGFR and HER2 signaling in response to CDK4, MEK, and mTOR inhibitors possibly providing an explanation for the observed synergies with neratinib. Both palbociclib and everolimus led to statistically significant (Q < 0.05) increases in adaptive responses in EGFR, AKT, SHP2, SRC, RAF, STAT1/3, JNK, PRAS, FAK, and others. Trametinib treatment resulted in a unique adaptive response compared with palbociclib and everolimus with low scores for MAPK and AKT pathways and yet high scores associated with EphA2, PRAS40, and PEA15 phosphorylation as well as total levels of the kinases, EGFR, FAK, and DAPK2. In neratinib treatment, we did not observe a significant change in any actionable target involved in the adaptive responses. Albeit statistically insignificant, heregulin was increased in response to neratinib possibly suggesting a negative feedback loop activating other HER or other RTK family members. Overall, the adaptive responses to trametinib, palbociclib, and everolimus provided the potential predictive markers of synergistic interactions with neratinib particularly evidenced by increases in receptor tyrosine kinase levels and phosphorylation while the downstream events differed remarkably in trametinib compared with the other agents.
Discussion
Overexpression/amplification of the HER2 oncogene in approximately 25% of human breast cancers predicts response to therapies targeting HER2. HER2 signaling is primarily mediated through downstream PI3K/Akt and MAPK axis which govern cell proliferation and apoptosis. The importance of the PI3K/Akt and MAPK pathways in oncogenic signaling is becoming increasingly apparent, especially in the case of HER2+ breast cancer (42, 43). Meanwhile, the contribution of aberrations in these pathways to resistance of HER2-targeted therapies has been evidenced. For example, PIK3CA hotspot mutations are found in approximately 25% of breast cancers and can overlap with HER2 amplification (44, 45). The presence of these mutations confers relative resistance to trastuzumab or lapatinib (46–48). Hence, the PI3K/Akt and MAPK signaling pathways are potential targets for HER2+ breast cancer with drug resistance due to activating mutations in these pathways. A number of clinical trials have suggested that HER2-directed therapies in combination with agents targeting PI3K or MAPK signaling, or cell cycle, have clinical efficacy in patients, particularly those with hyperactivity in these pathways. For example, combinations of trastuzumab or lapatinib with everolimus have demonstrated encouraging antitumor activity in HER2-overexpressing tumors (49–52). A recent report demonstrated that trastuzumab in combination with palbociclib exhibits promising survival outcomes in trastuzumab pretreated estrogen receptor–positive (ER+)/HER2+ advanced breast cancer (53). As a novel HER2 inhibitor, neratinib has demonstrated powerful therapeutic efficacy. In view of this and the mechanisms potentially involved in its resistance as described above, this study is designed to examine the synergism between neratinib and inhibitors targeting the PI3K/Akt and MAPK pathways, establishing potential optimal combination regimens for HER2+ breast cancer.
In cell viability assay, we found that neratinib displayed powerful synergistic efficacy when combined with all these targeted inhibitors in HCC-1954 and BT-474 cell lines, compared to SK-BR-3 cell line which showed less synergistic response to these combinations (Fig. 1). The discrepancy of combination efficiency could be presumably resulted from the varying genomic mutations in the PI3K/Akt and MAPK pathways. As shown in Supplementary Table S1, HCC-1954 and BT-474 cells have multiple mutations in MAPK signaling molecules. This could reasonably account for the less synergistic actions in SK-BR-3 cells than the other two cell lines. These phenotypic differences of cell line viabilities in response to the treatment are correlated with cell signaling activity. For example, in HCC-1954 and BT-474 cells when neratinib was combined with these MEK and mTOR inhibitors, they demonstrated enhanced signaling inhibition in both MAPK and PI3K/Akt pathways in all these combination groups. On the other hand, SK-BR-3 cells had less response to most of these combinations in both pathways, except for the neratinib/trametinib combination which only affected phospho-ERK. Compared with the other two cell lines, SK-BR-3 cells have much fewer mutations in both pathways which may lead to less responses in cell survival and signaling. We also realized that variations of CI values in cell survival assay are bigger in SK-BR-3 cells than those in the other two cell lines. Whether this is a biological or technical variation needs further study.
We noticed that, in contrast to trametinib, everolimus, or sapanisertib, all of which diminished phosphorylation of their own downstream kinase target proteins, the PI3Kα inhibitor alpelisib lacked activity on signaling inhibition across the cell lines. This could possibly be attributed to the occurrence of activating PIK3CA mutations in these HER2+ cell lines, except SK-BR-3 cells. Intriguingly, in BT-474 cells, combinations of neratinib with mTOR inhibitors synergized not only on the mTOR targets 4E-BP1 and S6, but also on the MEK target ERK1/2, demonstrating signaling cross-talk between the PI3K/Akt and MAPK pathways. Signaling cross-talk has been previously described to contribute to resistance in HER2-targeted therapies (54, 55). We also noticed that palbociclib was able to reduce Rb phosphorylation in HCC-1954 and SK-BR-3 cell lines, but not in BT-474 cells. Both HER2 and ER are found to ultimately impinge activity of CDK4/6. While all three cell lines are HER2+, only BT-474 cells are ER+. Whether different ER status plays a role in Rb phosphorylation by CDK4/6 needs to be further clarified. DNA damage repair pathway is also known to be involved in regulation of Rb activity. Unlike the other two cell lines, BT-474 has a deleterious BRCA2 mutation. Further study is needed to determine whether this differential BRCA2 mutational status may play a role in the differential response of cell lines to the CDK4/6 inhibitor on Rb phosphorylation.
From multiple drug combinations that were demonstrated synergistic in vitro on cell lines, we tested three combination regimens in vivo on PDX models. The tumor growth results showed enhanced antitumor efficacy by these combinatorial therapies which are generally consistent with the in vitro results. However, we observed diverse responses to combination therapies across the five PDX models. As shown in Table 1, in addition to HER2 amplification status, these PDXs have a variety of genomic alterations in multiple pathways, many of which are known to be cancer drivers. Some of these alterations overlapped between the PDXs, such as both breast models and one colorectal cancer PDX have PIK3CA mutations. Some PDXs have MAPK pathway mutations including BRAF and KRAS while some have activating ERBB2 mutations. These diverse genomic backgrounds may contribute to the observed inconsistency in therapeutic outcomes in these in vivo models. Therefore, the association between genomics and therapeutic efficacy needs to be further established.
To elucidate the potential mechanisms responsible for enhanced combinatorial efficacy, in addition to the cell signaling study on cell lines as described above, we have also performed RPPA assay on in vivo tumor samples. Previous studies have demonstrated that neratinib and other targeting agents such as palbociclib and everolimus are capable of inducing cell-cycle arrest and apoptosis (56–58). The evidence from our RPPA data suggests that the enhanced antitumor efficacy of combinatorial neratinib therapeutics could be attributed at least to boosted cell apoptosis. For example, two combination treatments (neratinib + everolimus and neratinib + palbociclib) share the same apoptosis-related DEPs, including Bcl-XL, Glutamate-D1-2, IRS2, IR-b, phospho-ATR, and MelanA. Bcl-xL and Glutamate-D1-2 are known to be required for apoptosis (59–62). Increased IR-b expression was also found to induce apoptosis (63). It is interesting that while palbociclib alone was able to decrease Wee1 and Wee1_pS642 levels, combination of palbociclib and neratinib downregulated expression of multiple molecules in the DNA repair pathway, including Wee1, Wee1_pS642, ATR_pS428, CHK2_pT68, Cdc25C, and CDK1_pT114. The DNA repair pathway is well known to play an important role in regulating cell apoptosis (64, 65). Thus, the enhanced cell apoptosis induced by these shared DEPs could be a potential mechanism responsible for the synergistic efficacy of combinatorial neratinib therapeutics. We noticed that single neratinib or trametinib did not induce DEPs, suggesting that the two drugs do not have powerful impact on DEPs as when they act alone at the doses and at the time points we tested at the FDR cutoff of 0.05.
We also performed network-level adaptive response analysis using RPPA data. The results demonstrated feedback loops between drug inhibitions and HER2/EGFR signaling as well as its downstream pathways, providing the rationale for drug combinations. The results also verified the involvement of an apoptosis mechanism in the combinatorial therapy which we identified in RPPA assay. Data showed that combination treatments, such as neratinib with palbociclib or neratinib with everolimus, triggered survival stress response, as evidenced by enhanced Bcl-2 and Bcl-xL. Moreover, findings of DNA damage checkpoint molecule SLFN11 with a high Target Score, indicates that modulation of DNA repair pathways may contribute to functional mechanisms for the combination therapy. Overall, the results indicate that multiple signaling pathways are associated with the enhanced antitumor efficacy of neratinib combination therapeutics. The functional involvement of these pathways and their sophisticated network in neratinib therapy warrant further investigation. Future study is also needed to determine whether individual adaptive responses can be used to help personalize optimal combination therapy.
In summary, while the in vitro cell line and in vivo PDX approaches consistently demonstrated therapeutic benefits from these neratinib combinations in this study, the in silico assays identified potential molecular mechanisms and predictive biomarkers. In particular, from these in vivo results, we have several take-home messages: (i) combination of neratinib with palbociclib demonstrated substantially enhanced antitumor efficacy in all these HER2+ PDX models, (ii) combinations of neratinib with everolimus or with trametinib provided varying therapeutic benefits, and (iii) esophageal adenocarcinoma (PDX.003.164) had the best responses to all three combinatorial treatments. Although the combination of HER2-targeted therapy with ER-targeted therapy or a triplet could make sense for ER+/HER2+ breast cancer, we believe that simply based on HER2+ status the combination of HER2 inhibitor and palbociclib may be worth exploring across tumor types. The promising outcomes of this preclinical study provide guidance of clinical application of combinatorial therapies of neratinib for HER2+ cancer. A phase I trial evaluating the safety and clinical activity of neratinib in combination with everolimus, trametinib, or palbociclib in patients with solid tumors is ongoing (NCT03065387).
Authors' Disclosures
S. Kopetz reports personal fees from Roche, Genentech, Merck, Karyopharm Therapeutics, Amal Therapeutics, Novire Pharma, Symphogen, Holy Stone, Biocartis, Amgen, Novartis, Lilly, Boehringer Ingelheim, Boston Biomedical, AstraZeneca/MedImmune, Bayer Health, Pierre Fabre, EMD Serono, Redx Pharma, Jacobio, Natera, Repare Therapeutics, Daiichi Sankyo, Lutris, Pfizer, Ipsen, and HalioDx outside the submitted work. A.S. Lalani reports other from Puma Biotechnology Inc. during the conduct of the study, as well as other from Puma Biotechnology Inc. outside the submitted work. S. Piha-Paul reports other from AbbVie, Inc., ABM Therapeutics, Inc., Acepodia, Inc., Alkermes, Aminex Therapeutics, Inc., Amphivena Therapeutics, Inc., BioMarin Pharmaceutical, Inc., Boehringer Ingelheim, Bristol Myers Squibb, Cerulean Pharma, Inc., Chugai Pharmaceutical Co., Inc., Curis, Inc., Daiichi Sankyo, Inc., Eli Lilly, ENB Therapeutics, Five Prime Therapeutics, Gene Quantum, Genmab A/S, GlaxoSmithKline, Helix BioPharma Corp., Incyte Corp., Jacobio Pharmaceuticals Co., Ltd., Medimmune, LLC, Medivation, Inc., Merck Sharp & Dohme Corp., Novartis Pharmaceuticals, Pieris Pharmaceuticals, Inc., Pfizer, Principia Biopharma, Inc., Puma Biotechnology, Inc., Rapt Therapeutics, Inc., Seattle Genetics, Silverback Therapeutics, Taiho Oncology, Tesaro, Inc., and TransThera Bio, as well as grants from NCI/NIH P30CA016672—Core Grant (CCSG Shared Resources) outside the submitted work. F. Meric-Bernstam reports personal fees from Arduro BioTech, Alkermes, F. Hoffman-La Roche Ltd., IBM Watson, Jackson Laboratory, Kolon Life Science, OrigiMed, PACT Pharma, Parexel International, Pfizer Inc., Samsung Bioepis, Seattle Genetics Inc., Tyra Biosciences, Xencor, Zymeworks, Immunomedics, Inflection Biosciences, Mersana Therapeutics, Puma Biotechnology Inc., Seattle Genetics, Silverback Therapeutics, Zentalis, Chugai Biopharmaceuticals, Mayo Clinic, and Rutgers Cancer Institute of New Jersey; grants and personal fees from AstraZeneca, DebioPharm, eFFECTOR Therapeutics, and Genentech Inc.; grants from Aileron Therapeutics, Bayer Healthcare Pharmaceutical, Calithera Biosciences, Curis Inc., CytomX Therapeutics Inc., Daiichi Sankyo Co. Ltd., Guardant Health Inc., Millennium Pharmaceuticals, Novartis, Puma Biotechnology Inc., and Taiho Pharmaceutical Co.; and other from Beth Israel Deaconess Medical Center outside the submitted work. No disclosures were reported by the other authors.
Authors' Contributions
M. Zhao: Conceptualization, data curation, formal analysis, investigation, writing-original draft, writing-review and editing. S. Scott: Investigation, writing-review and editing. K.W. Evans: Data curation, investigation, writing-review and editing. E. Yuca: Investigation. T. Saridogan: Investigation. X. Zheng: Formal analysis. H. Wang: Formal analysis. A. Korkut: Formal analysis. C.X. Cruz Pico: Writing-review and editing. M. Demirhan: Data curation, project administration. B. Kirby: Data curation, project administration. S. Kopetz: Writing-review and editing. I. Diala: Writing-review and editing. A.S. Lalani: Conceptualization, writing-review and editing. S. Piha-Paul: Writing-review and editing. F. Meric-Bernstam: Conceptualization, data curation, supervision, writing-review and editing.
Acknowledgments
This work was funded by PUMA Biotechnology Inc., U54-CA224065, Cancer Prevention and Research Institute Precision Oncology Decision Support Core (RP150535), Nellie B. Connally Breast Cancer Endowment, CTSA (1UL1TR003167), and the NIH through MD Anderson's Cancer Center Support Grant (CA016672). We appreciate the financial support from these funders. We also thank Susanna Brisendine for coordination in submission of this manuscript.
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