Pharmacologically targeting the HER2 oncoprotein with therapeutics such as the mAb, trastuzumab, provides clinical benefit for patients with HER2-positive (HER2+) cancers. However, a significant number of patients eventually progress on these therapies. Efforts to overcome therapeutic resistance through combination therapy with small-molecule inhibitors of HER2 have been limited by toxicities associated with off-target activity and/or limited efficacy. In this preclinical study, we explore single-agent and combined activity of tucatinib, a novel HER2-selective small-molecule inhibitor. Tucatinib demonstrated potent, selective activity in a panel of 456 human cancer cell lines, with activity restricted to cell lines (breast and non-breast) with HER2-amplification, including models of acquired resistance to trastuzumab. Within the HER2+ population, tucatinib response tracked strongly with HER2-driven signaling. Single-agent tucatinib induced tumor regressions in xenograft models of HER2+ breast cancer and combination with trastuzumab induced a complete and sustained blockade of HER2/PI3K/AKT signaling. Efficacy of the tucatinib/trastuzumab combination matched that induced by current standard-of-care trastuzumab/pertuzumab/docetaxel combination, with the exception that the chemotherapy-sparing tucatinib/trastuzumab combination did not require a dosing holiday to achieve the same efficacy. In xenograft models of HER2+ breast cancer that also express estrogen receptor (ER; HER2+/ER+), tucatinib showed combined efficacy with inhibitors of CDK4/6 and ER, indicating potential novel therapeutic strategies for difficult-to-treat subtypes of HER2+ breast cancer. These data support expanded clinical investigations of tucatinib as a combination partner for other novel and approved targeted therapies for HER2-driven malignancies.
This article is featured in Highlights of This Issue, p. 701
Overexpression of the HER2 oncoprotein occurs in approximately 20% of all breast cancers and defines an aggressive subtype of this malignancy (1–3). Hyperactivation of HER2 signaling occurs mainly through amplification of the ERBB2 gene resulting in overexpression. Rare activating mutations may also occur in the absence of gene amplification (4). HER2 drives cell proliferation via homodimerization or heterodimerization with other type-1 receptor tyrosine kinases (RTK) such as EGFR, HER3 or HER4 (5). Because of the lack of a natural ligand, activation of HER2 must occur via a dimerization partner before downstream signaling through RAS/MAPK and PI3K/AKT/mTOR signaling pathways (5). Pharmacologically targeting HER2 provides significant clinical benefit for patients with HER2-amplified (HER2+) breast cancer. The benefits of targeting HER2 outside of breast cancer, where the gene alterations are also known to occur, are less clear (6, 7). Use of approved therapeutics such as the HER2-directed mAbs trastuzumab (Herceptin, Genentech) and pertuzumab (Perjeta, Genentech) in combination with chemotherapy has changed the natural course of HER2+ breast cancer (8–10). Binding of these mAbs to the extra cellular domain of HER2 leads to both recruitment of antibody-dependent cell-mediated cytotoxicity and downregulation of HER2 signaling likely due to increased endocytosis (11). Second-line therapies using trastuzumab-based antibody–drug conjugates (ADC) such as T-DM1 (Kadcyla, Genentech) or fam-trastuzumab deruxtecan (Enhertu, Daiichi-Sankyo) can provide improvements in both progression-free survival (PFS) and overall survival (OS) after disease progression on first-line therapies (12–15). Approved small-molecule inhibitors that target HER2, such as lapatinib (Tykerb, Novartis), afatinib (Gilotrif, Boehringer Ingelheim), and neratinib (Nerlynx, Puma Biotechnology), can also provide clinical benefit, but their use is limited by toxicities attributed to off target activity against other RTKs such as EGFR (HER1) and HER4 (12, 16).
Despite the improvements in PFS and OS associated with HER2-targeted therapies in breast cancer, overcoming therapeutic resistance remains an unmet clinical need, particularly in the metastatic setting (8, 17–20). Furthermore, currently appoved antibody-based therapuetics have limited CNS exposure and efficacy against CNS metastases (21–23). As such, the development of molecules that can target these metastases, overcome therapuetic resistance and be safely combined with existing agents is urgently required.
Tucatinib (Tukysa, Seattle Genetics) is an orally available, potent, selective, reversible, ATP-competitive small-molecule inhibitor of HER2 that has been shown to exhibit >50-fold selectivity for HER2 over EGFR and cross the blood–brain barrier (24–26). Data reported from the HER2CLIMB phase II clinical study showed that addition of tucatinib to trastuzumab/capecitabine induced a significant improvement in PFS and OS in patients with advanced metastatic HER2+ breast cancer who had progressed on previous HER2-targeted therapies. Moreover, significant improvement in PFS was also observed in a cohort of patients with CNS metastases (26). Tucatinib as a single agent and in combination with trastuzumab/capecitabine showed an acceptable safety profile in this patient cohort. On the basis of these data, the FDA approved tucatinib in April 2020, for use in combination with trastuzumab and capecitabine for patients with advanced metastatic HER2+ breast cancer.
In this study, we evaluate the preclinical activity of tucatinib in a panel of over 450 molecularly characterized human cancer cell lines representing 16 different malignant histologies. Tucatinib activity in the panel was compared with that of other approved small-molecules inhibitors of HER2. Molecular predictors of response to tucatinib were identified through comprehensive biomarker analyses. Also, the potential for tucatinib to be further combined with standard of care (SOC) and novel therapuetics for breast cancer was investigated both in vitro and in vivo. These data provide insight on the optimal selection of patients for tucatinib-based therapies and identify combination strategies for the clinical development of this selective HER2-targeting small molecule.
Materials and Methods
Cell lines, cell culture, and reagents
Human cancer cell lines were maintained in appropriate culture media (e.g., RPMI-1640, DMEM, L-15) and supplemented with 10% to 15% heat-inactivated FBS, 2 mmol/L glutamine, and 1% penicillin G-streptomycin–fungizone solution (PSF, Irvine Scientific). Cells were routinely assessed for mycoplasma contamination using a multiplex PCR method (27) and STR profiling by the GenePrint 10 System (Promega) was used for cell line authentication. Tucatinib (ONT-380), ribocilcib (LEE011 succinate), abemaciclib (LY2835219 mesylate), palbociclib HCl (PD-0332991) were all purchased from BioChemPartner. Erlotinib HCl (OSI-744), neratinib (HKI-272), and lapatinib (GW-572016) were purchased from Selleck Chemicals. Trastuzumab (Herceptin, Genentech, Inc.), pertuzumab (Perjeta, Genentech, Inc.), and fulvestrant (Faslodex, AstraZeneca) were purchased from the UCLA pharmacy. Trastuzumab-resistant BT-474 (BT-474-TR) and SKBR3 (SK-BR3-TR) cell lines were established as previously described (28, 29). For molecular analysis, TR cells were removed from drug for 7 days before experiments.
Molecular characterization of the cancer cell line panel and biomarker analysis
Each of the cell lines in the panel were characterized at baseline for point mutations by whole-exome sequencing, DNA–copy-number alterations by comparative genomic hybridization microarray assays (a-CGH) and proteomic profiling by reverse phase protein array (RPPA) as previously described (30). RPPA data are presented as normalized, median-centered log (expression) values. For a-CGH, log2 ratios of 0.5–1.0 were considered gain, >1 (2-fold) were considered amplified and ratios of greater than 2 were considered highly amplified. Molecular markers commonly associated with HER2 signaling were examined for association with response to tucatinib using linear regression analysis in R.
In vitro proliferation assays
Response to tucatinib, lapatinib, and neratinib was measured in a custom 6-day proliferation assay. For treatment, drugs were prepared at a concentration of 10 mmol/L in DMSO (Sigma-Aldrich). Cells were seeded in 48-well plates at a density previously determined to maximize growth over a 6-day treatment window. After 24 hours, baseline cell counts were performed and the cells were treated with six 1:5 dilutions of each drug starting at 10 µmol/L. After six days of treatment cells were counted on a custom automation platform designed by Tecan. Using this robotic system, cells were trypsinized, centrifuged, and counted via brightfield image segmentation on a Synentec Cellavista imaging system. Data are presented as the percentage of inhibition of cell generation/doubling time; inhibition of >100% is considered to be induction of lethality. Assays were performed in duplicate. Drug combination studies were conducted as described above by treating with fixed molar ratios of each compound.
Western blotting and biochemical assays
To obtain the protein lysates, cells growing in log-phase in 10-cm culture dish were washed twice with ice-cold PBS and lysed for 15 minutes on ice with a RIPA lysis and extraction buffer (Thermo Fisher Scientific) containing working concentrations of cOmplete Protease Inhibitor Cocktail (Sigma-Aldrich) and PhosSTOP (Sigma-Aldrich) tablets. Supernatant was extracted after centrifugation at 10,000 × g for 10 minutes. Protein was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Snap-frozen tumor tissues, obtained from xenograft studies, were homogenized with a mortar and pestle followed by isolation of protein lysates using a (1% v/v Triton X-100, 50 mmol/L HEPES pH7.4, 150 mmol/L NaCl, 1.5 mol/L MgCl2, 1 mmol/L EGTA, 100 mmol/L NaF, 10 mmol/L Na pyrophosphate, 1 mmol/L Na3VO4, 10% glycerol) supplemented with working concentrations of cOmplete Protease Inhibitor Cocktail (Sigma-Aldrich) and PhosSTOP (Sigma-Aldrich) tablets. Samples were immunoblotted for HER2 (#2165), pHER2Y1248 (#2247), HER3 (#12708), pHER3Y1289 (#4791), EGFR (#2085), pEGFRY1068 (#2234), pEGFRY1173 (#2244), AKT (#4685), pAKTS473 (#4060), pAKTT308 (#4056), S6 (#2217), pS6 (#5364), ERK (#4695), pERK (#4370), PARP (#9532), Caspase-9 (#9502), Caspase-8 (#4790), retinoblastoma (RB) (#9309), and pRbS807/811 (#8516) using specific primary antibodies form Cell Signaling Technology (CST), followed by probing with a horseradish peroxidase (HRP)–linked anti-rabbit IgG antibody (#7074, CST). Each blot was incubated with 1 µL/mL of primary antibody in 5% BSA and 200 nL/mL of secondary antibody in BSA. Each experiment was repeated in duplicate.
Binding constants (Kd) for tucatinib, lapatinib, and neratinib on the EGFR, HER2, HER3, and HER4 kinases were determined using KINOMEscan technology at LeadHunter Discovery Services. The relative selectivity of tucatinib at 200 and 2,000 nmol/L was measured using standard protocols for the scanELECT platform for 51 different kinases at LeadHunter Discovery Services.
Flow cytometry assays
Cells (0.25–1 × 106 per 6-cm dish) were incubated with increasing concentrations of tucatinib for 24 or 48 hours. For the apoptosis assays, culture media were collected in addition to the trypsinized cells. Cells were subsequently washed and stained with Annexin V-FITC and propidium iodide (PI; Thermo Fisher Scientific) at room temperature for 5 minutes in the dark before measurement. For the cell-cycle assays, cells were incubated in PI solution at room temperature for 10 minutes before measurement. Samples were measured with the Attune NxT Flow Cytometer from the UCLA Flow Cytometry Core Facility. Assays were repeated in duplicate.
Cell line xenograft studies
HER2/+ER− and HER2+/ER+ cell line xenograft models were established in 6-week-old CD-1 athymic nude mice (Charles River Laboratories) by subcutaneous injection of each cell line at 1.0 × 107 cells per mouse in a 50% Matrigel/media mix. For ER-positive models, 17-ß-estradiol 60-day release pellets (Innovative Research of America) were implanted subcutaneously into the left flank 7 days before inoculation with cells. When tumors reached an average size of 150–300 mm3, mice were randomized into treatment groups. Tumor xenografts were measured with calipers 3 times/wk, and tumor volume in mm3 was determined by multiplying height, width, and length. Drugs were dosed as follows: Tucatinib at 50 mg/kg (0.5% carboxymethylcellulose, pH 3.0) PO BID, trastuzumab (Herceptin, Genentech Inc.) by intraperitoneal injection at 10 mg/kg once per week (QW), pertuzumab (Perjeta, Genentech, Inc.) by intraperitoneal injection at 10 mg/kg once per week (QW), docetaxel (Taxotere, Hospira) at clinically achievable doses of 10 mg/kg by intravenous injection once weekly, palbociclib at 50 mg/kg (50 m/mol/L sodium lactate, pH 4.0), ribociclib at 50 mg/kg (0.5% w/v methylcellulose/water) PO daily, abemaciclib at 50 mg/kg (1% HEC in 25 mmol/L phosphate buffer (pH = 2), and fulvestrant (Faslodex) at 5 mg/wk (castor oil/ethanol) by subcutaneous injection once per week. Xenograft data were analyzed using StudyLog Software. All animal work was conducted in accordance with and with the approval of UCLA's Institutional Animal Care and Use Committee (IACUC). Statistical differences between treatment arms at specific time points were performed using a one-tailed unpaired Student t test (Supplementary Tables S1–S4). Differences between groups were considered statistically significant at P < 0.05. All statistics were calculated in R.
IHC and immunofluorescent analyses
Paraffin-embedded sections were cut at 4-μm thickness and paraffin removed with xylene and rehydrated through graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 minutes. Heat-induced antigen retrieval (HIER) was carried out for all sections in 0.001 mol/L EDTA, pH = 8 using a Biocare Decloaking Chamber at 95°C for 25 minutes. The slides were then incubated with HER2/ErB2 antibody (CST #4290) at 1/100 dilution overnight at 4°C. The signal was detected using EnVision kit HRP-conjugated polymer anti rabbit (Agilent, K4003) and visualized with the diaminobenzidine reaction. Slides were then counterstained with hematoxylin, dehydrated and coversliped.
IHC slides were imaged using Aperio ScanScope AT (Leica Biosystems) and were then quantified using Definiens Tissue Studio software (Definiens) that measures the number of positive cells and intensity of staining. Counts were analyzed using Microsoft Excel.
For immunofluorescent analysis, 10,000 EFM192A cells were seeded in a µ-Slide 4-well (Ibidi) and labeled with Transfect CellLight Lysosomes-GFP and BacMam 2.0 (Invitrogen) and DNA dye Hoechst 33342 (Abcam). The following day, cells were treated with either DMSO vehicle control or 200 nmol/L of tucatinib for either 2 or 24 hours. Cells were incubated with Alexa Fluor 647 (Invitrogen) labeled trastuzumab at 21 µg/mL for 30 minutes. HER2 receptor internalization was visualized using the Keyence microscope starting at 2 hours after dosing whilst cells were incubated at 37°C and 5% CO2.
Tucatinib has selective activity in HER2-driven cancers
The antiproliferative activity of tucatinib was compared with that of two approved HER2-targeting agents, lapatinib and neratinib, in a panel of 456 human cancer cell lines. Although a broad range of IC50 values, covering 4 logs of separation, was determined for tucatinib, only a small percentage of cell lines (28/456; 6%) were considered sensitive to tucatinib using a cutoff value of <1 µmol/L (Fig. 1A), which is equivalent to clinically achievable plasma concentrations (31). Using the same cutoff value, far broader response profiles were observed for lapatinib (16%) and neratinib (92%; Fig. 1B and C). A blinded screen of 51 receptors and non-RTKs confirmed HER2 and EGFR as the only studied kinases inhibited by tucatinib in competitive binding assays (Supplementary Table S5). Kinase assays showed tucatinib has Kds for HER2 over 290-fold lower than for EGFR, and >1,000-fold lower than HER3 (which has weak kinase activity) and HER4 (Fig. 1D and Supplementary Table S6). In contrast, lapatinib demonstrated equal potency on HER2 and EGFR and neratinib showed equal potency on all four HER family receptors (Fig. 1D). Despite these significant specificity differences, presence of the HER2-amplicon is the dominant correlate of response to each molecule (Fig. 1E and F; Supplementary Fig. S1). Strong positive correlations were observed between each molecule in the breast cancer cell lines, due to the high frequency of HER2 amplifications (Fig. 1G and H). Within the HER2+ breast cell line subgroup, response to tucatinib showed strong positive correlations with high levels of total HER2, phosphorylated HER2Y1248, and phosphorylated EGFRY1068 (Supplementary Table S7 and Supplementary Fig. S2), indicating a significant association between HER2-driven signaling and tucatinib response.
To investigate the utility of tucatinib outside of breast cancer, we analyzed tucatinib response in upper gastrointestinal cancer cell lines with and without HER2 amplification, and observed a similar robust association between activated HER2 signaling and tucatinib response (Fig. 1I and Supplementary Table S8). Of the 21 gastric cancer cell lines tested, the five most sensitive carry the HER2-amplicon. High levels of total HER2 and phosphorylated HER2Y1248, total EGFR and phosphorylated EGFRY1068/Y1173 and phosphorylated AKTS473/T308 also correlated with response to tucatinib in HER2+ gastric cancer cell lines (Supplementary Table S8 and Supplementary Fig. S3).
To explore additional drivers of tucatinib response outside of breast cancer, we investigated the role of HER2 receptor mutations. We found that response to tucatinib was not associated with the presence of mutations in the HER2 receptor across a diverse range of cell lines. The lack of response to tucatinib in the HER2-mutated cell lines could be attributed to the relatively low levels of phosphorylated HER2, EGFR, and HER3 at baseline compared with tucatinib sensitive, HER2+ breast cancer cell lines (Supplementary Table S9).
Tucatinib selectively blocks HER2 signaling to induce cellular apoptosis in HER2+ cell lines
Treatment with tucatinib induced a complete block of HER2-autophosphorylation within 30 minutes of dosing HER2+ breast cancer cells. Blockage of HER2-phosphorylation was accompanied by loss of EGFRY1068/Y1173, HER3Y1289, AKTS473/T308, ERK1/2T202/Y204, and reduction in S6S225/236 phosphorylation (Fig. 2A). Treatment of the triple-negative breast cancer (TNBC) cell line (MDA-MB-468) with tucatinib blocked only phosphorylation of the HER2Y1221/1222 protein, and not downstream AKT, ERK or S6 phosphorylation (Fig. 2B). In contrast, lapatinib, and to a lesser degree erlotinib, a selective EGFR inhibitor, effectively blocked EGFR and downstream ERK signaling in this EGFR-amplified model (Fig. 2B). These data, coupled with the kinome screening data (Supplementary Table S5) provide further proof that tucatinib selectively targets HER2 and blocks autophosphorylation of EGFR indirectly. In HER2+ cells, tucatinib treatment induced only marginal changes in the percentage of cells in G0–G1 of the cell cycle but effectively blocked the cells from entry into S-phase (Fig. 2C). Tucatinib treatment also induced a significant increase in annexin V/PI-positive staining, indicating induction of apoptosis (Fig. 2D).
Tucatinib selectively inhibits cell proliferation and HER2-driven signaling in trastuzumab-resistant cell lines
BT-474 and SKBR3, HER2+ breast cancer cell lines, were conditioned through long-term drug exposure to acquire resistance to trastuzumab (BT-474-TR & SKBR3-TR; ref. 29). Tucatinib showed activity in these cells similar to that observed in the parental, non-resistant clones (Fig. 3A and B). Tucatinib treatment induced complete loss of HER2-phosphorylation in the resistant cells, which was accompanied by loss of EGFR, HER3, AKT, ERK1/2, and S6 phosphorylation (Fig. 3C). Blockade of progression through cell cycle by tucatinib was confirmed by a loss of phosphorylated RB (pRb) in cells treated for 24 hours (Fig. 3D). Furthermore, treatment of the BT-474-TR cells with tucatinib induced time-dependent cleavage of both caspase-9 and PARP (Fig. 3D), indicating effective induction of apoptosis in trastuzumab-resistant HER2+ breast cancer cell lines.
The combination of tucatinib and trastuzumab induces sustained blockade of HER2/AKT/ERK1/2 signaling
To assess the potential for tucatinib to be used in combination, we treated a panel of HER2+ cell lines with a range of tucatinib concentrations and a fixed concentration of trastuzumab for 5 days (Fig. 4A). In BT-474 cells that are highly sensitive to trastuzumab, the combination of both molecules provided only marginal increase in growth inhibition over single-agent tucatinib. A similar level of increase in growth inhibtion was induced by the combination in trastuzumab insensitive BT-474-TR cells. However, the benefit of the combination was more obvious in three HER2+ cell lines (EFM192A, MDA-MB-361, and MDA-MB-453) with moderate responses to trastuzumab, particularly at lower concentrations of tucatinib (Fig. 4A). Treatment with single-agent tucatinib inhibited the phosphorylation of HER2, HER3, and EGFR as well as downstream AKT, ERK1/2, and S6, with low level re-activation of signaling observed at 48–72 hours after dosing. Partial block of each of these signaling pathways was observed between 24 and 72 hours after treatment with trastuzumab alone (Fig. 4B). In contrast, combination treatment with tucatinib and trastuzumab resulted in complete block, without feedback activation, of phosphorylation of HER2, HER3, EGFR, AKT, S6, and ERK1/2 out to 72 hours. Selective block of signaling in response to treatment with the combination was shown to be sustained long-term in drug washout studies. Whereas HER2/AKT/S6/ERK1/2 signaling returned to baseline between 48 and 72 hours after drug washout of either tucatinib or trastuzumab, sustained inhibition of these proteins was observed at 72 hours after washout of the tucatinib/trastuzumab combination. Combination treatment also induced a sustained loss of pRb and FOXM1 levels, indicating continued inhibition of cell-cycle progression (Fig. 4C). These data were reproduced in a second HER2+ cell line model (Supplementary Fig. S4). Interestingly, it appears that sustained block of signaling by the combination is not due to effects on recycling or endcytosis of the HER2-protein at the cell surface. Treatment for either 2 or 24 hours with tucatinib, followed by trastuzumab, had no discernable effect on the internalization of cell surface HER2 as measured by quantification of fluorescently labeled trastuzumab (Fig. 4D).
Combined efficacy of tucatinib with currently approved therapies for HER2-amplified breast cancer
The activity of tucatinib in combination with other approved therapies for HER2 was evaluated in SUM-190PT (HER2+) breast cell line xenografts. Tucatinib and trastuzumab each induced significant tumor growth inhibition (TGI) relative to non-targeting antibody control treated mice. However, the combination of tucatinib with trastuzumab induced an increase in TGI over either single agent that was similar to that induced by the combination of the two mAbs, trastuzumab, and pertuzumab (Fig. 5A). The addition of tucatinib to trastuzumab and pertuzumab, induced significant improvement in antitumor responses over the trastuzumab/pertuzumab doublet (Fig. 5A). In a second HER2+ breast cancer cell line xenograft model, single-agent tucatinib induced significant antitumor responses, with sustained tumor regressions observed after 4 weeks of treatment (Fig. 5B). Responses to single-agent tucatinib were equal to that achieved by trastuzumab monotherapy or trastuzumab in combination with pertuzumab and docetaxel, which is considered SOC of HER2+ breast cancer (Fig. 5B). The addition of tucatinib to trastuzumab/pertuzumab/docetaxel triplet did not result in further efficacy. However, it should be noted that the chemotherapy-sparing dosing arms induced significantly less body weight loss in the mice compared with the docetaxel containing arms (Supplementary Tables S1–S4).
Inhbition of HER2 and HER3 phosphorylation, and reduction in phosphorylation of AKT, ERK1/2, and RB were observed in EFM192A xenograft tissue samples collected from mice treated continuously for 4 days with single-agent tucatinib (Fig. 5C). Only marginal impact on HER2 signaling was observed in samples collected from mice treated with single-agent trastuzumab treatment for the same period. However, a more potent block of HER2/HER3/AKT/ERK/Rb signaling was observed in xenograft tissue samples collected from mice treated with the tucatinib/trastuzumab combination. Loss of total HER2 and HER3 protein expression was also unique to the combination at the day 4 timepoint (Fig. 5C). Western blot analysis of a second set of samples, collected 5 days after the final of 7 days of continuous treatment, confirmed that signaling returned to baseline in samples treated with single-agent tucatinib, whereas sustained block of HER2 signaling was observed in response to treatment with trastuzumab or the tucatinib/trastuzumab combination. Inhibition of HER2 signaling in these samples was marked by downstream loss of phosphorylated AKT, ERK1/2 and ultimately loss of RB phosphorylation (Fig. 5D). IHC analyses of xenograft tissues from the same experiment identified a loss of total HER2 protein that is unique to tucatinib/trastuzumab combination at the day 4 timepoint. Five days after treatment washout, HER2 protein is lost in both the trastuzumab single-agent and combination arms (Fig. 5E). However, Western analysis of samples collected at this later timepoint also show a loss of CK-19 protein, indicating a loss of human epithelial cell burden in the xenograft tissues that could account for the loss of HER2 and other signaling proteins (Fig. 5D).
Tucatinib has combined activity in vivo with inhibitors of CDK4/6 in HER2+/ER+ breast cancer cell line xenografts
Combination of tucatinib with the CDK4/6 inhibitor, palbociclib, induced an increase in in vitro cell growth inhibition over single-agent treatment in three tucatinib-sensitive HER2+ breast cancer cell lines (EFM192A, MDA-MB-361, and BT-474-TR). Combined inhibition of cell growth was also observed in the moderately sensitive MDA-MB-453 cell line. In contrast, the combination of tucatinib and palbociclib had only minimal impact on the proliferation of the tucatinib and palbociclib-insensitive TNBC cell line, MDA-MB-468 (Fig. 6A). Similar results were observed when tucatinib was combined with other CDK4/6 inhibitors, specifically abemaciclib and ribociclib. Cell-cycle analysis also confirmed a lack of antagonism between tucatinib and the CDK4/6 inhibitors (Supplementary Fig. S5).
In xenograft models of HER2+/ER+ breast cancer, single-agent tucatinib or palbociclib (p) induced significant inhibition of tumor growth relative to vehicle control–treated mice (Fig. 6B and C). In both models, the combination of tucatinib (t) and palbociclib (p) induced significant improvement in TGI over either single agent with significant tumor regressions observed in the MDA-MB-361 study (Fig. 6C). The addition of trastuzumab or trastuzumab (tz) plus fulvestrant (f; ER-antagonist) to the tucatinib/palbociclib doublet further increased efficacy in both models. Responses to the non-chemotherapy containing doublet (T+Tz), triplet (T+Tz+P), and quadruplet (T+Tz+P+F) combinations were comparable with that observed with the clinically used regimen of trastuzumab/pertuzumab/docetaxel. Once again, there was markedly less mouse body weight loss observed in the absence of docetaxel (Supplementary Tables S1–S4). Responses to each of these combinations were maintained during a 14-day follow up monitoring after dosing (Fig. 6C). Western blot analysis of xenograft tissues collected after 4 days of treatment confirmed that the combination of tucatinib plus palbociclib leads to improved inhibition of HER2/AKT/ERK and downstream RB signaling (Fig. 6D). Similar findings were observed in separate studies when palbociclib was substituted with either abemaciclib or ribociclib (Supplementary Fig. S6), indicating that responses can be achieved with each of the currently approved CDK4/6 inhibitors.
The development of the HER2-specific mAb, trastuzumab, has “revolutionized” therapy for patients with HER2-amplified breast cancers (32). The addition of trastuzumab to chemotherapy has demonstrated significant improvements in OS in both early-stage and metastatic breast cancer (9, 10). The subsequent inclusion of pertuzumab therapy induced further improvements in clinical outcomes in the metastatic setting with statistically significant but clinically marginal improvements in early HER2+ breast cancer (8). The use of HER2-directed ADCs has provided additional benefit as second-line therapeutics (15, 33). Despite the impact that these molecules have had on this aggressive subtype of breast cancer, high rates of therapeutic resistance, particularly in the metastatic setting, indicate the need for additional treatment options. Efforts to overcome resistance by combining HER2-directed mAbs with small-molecule inhibitors of HER2 have thus far been limited by toxicities associated with off-target inhibition of other RTKs such as EGFR and HER4 (12, 16). The advent of tucatinib, a highly selective small-molecule inhibitor of HER2, provides a unique opportunity to investigate the potential of this drug to provide clinical benefit as a single-agent or as a combination partner with the other current approved anti-HER2 therapies.
In the present study, we used a panel of 456 molecularly characterized cell lines representing 16 different malignant histologies to identify those cancers most likely to benefit from tucatinib-based therapies. In contrast with other less selective HER2 small-molecule inhibitors such as lapatinib and neratinib, we found that tucatinib activity is restricted almost entirely to the HER2-amplified cell lines. The differences in response profiles seen with each HER2 therapeutic molecule can be explained by differences in their respective selectivity for HER-family RKTs. Direct comparison of Kds for each of these three molecules confirmed that tucatinib is the most selective for HER2 over EGFR, HER3 and HER4 (24). In addition, a screen of 51 RTKs confirmed that HER2 is the protein most competitively inhibited by tucatinib. By contrast, lapatinib has greater potency for EGFR than HER2 whereas neratinib inhibits each of the 4 receptors at relatively equal potency. As such, lapatinib has considerable activity outside of HER2+ cell lines whereas neratinib has the profile of a multi-kinase inhibitor with 92% of the cell lines presenting with IC50 values of <1 µmol/L. The selectivity of tucatinib for a known oncogene, coupled with the specific response profile in the cell line panel indicates a potentialy “wide” therapeutic window for this molecule. Moreover, the selective nature of tucatinib likely limits its growth inhibitory activity to patients with HER2+ cancers. Biomarker analysis within the HER2+ cell population showed that those lines with the highest levels of phosphorylated HER2 and EGFR at baseline were most sensitive to tucatinib. These data suggest that a dependence on activated HER2 signaling, in addition to gene amplification, is required for optimal response to tucatinib. We found that this was also true in non-breast HER2+ cancers, where the cell lines most sensitive to tucatinib carry the HER2-amplificon and have high levels of phosphorylated HER2 and EGFR as well as downstream AKT activation at baseline. Interestingly, each of the nine HER2-mutated cell lines in the panel had relatively low levels of these markers at baseline and were not responsive to tucatinib treatment. These data indicate that it may be possible to further enrich for response to tucatinib-based therapies (beyond standard HER2 testing) by selecting for cancers with a HER2-driven signature. It should be possible to test this hypothesis in clinical material collected from patients receiving tucatinib therapy.
Data reported from the HER2CLIMB phase II clinical study showed that the addition of tucatinib to trastuzumab and capecitabine leads to significant improvements in OS and PFS in patients that have previously progressed on other currently approved HER2-targeted therapies (26). In this study, we used cell lines and xenograft models of acquired resistance to trastuzumab to further investigate the potential of tucatinib to overcome resistance. Tucatinib monotherapy inhibited the proliferation of two HER2-amplified breast cancer cell lines, conditioned to acquire resistance to trastuzumab (BT-474-TR and SK-BR3-TR), at concentrations similar to that required in the parental cell lines (BT-474 and SK-BR3). We have previously shown that resistance to trastuzumab in these models is acquired through upregulation of PI3K/AKT signaling (29). Here, we show that treatment with tucatinib induced a block of HER2, HER3, and EGFR phosphorylation and downstream PI3K/AKT signaling in the trastuzumab-resistant BT474-TR cells. Inhibition of HER2-driven signaling by tucatinib was followed by induction of apoptosis through cleavage of caspase 9 and PARP1/2 as previously shown (24). Low level feedback activation of PI3K/AKT signaling was observed within 72 hours of dosing with tucatinib. Treatment of the same cell line with tucatinib plus trastuzumab induced a stronger block of HER2/PI3K/AKT signaling without any feedback activation at 72 hours after dosing. Drug washout experiments indicate that between 24 and 48 hours after treatment, PI3K/AKT/mTOR/ERK signaling returns to baseline in the cells treated with either tucatinib or trastuzumab as single agents. However, complete block of PI3K/AKT/mTOR/ERK signaling is maintained after washout of the combination treatment. Sustained loss of pRb and FOXM1 after washout indicates that cells are also maintained in state of growth arrest following treatment with the trastuzumab/tucatinib combination. We made similar observations in xenograft tissues collected from mice treated with the tucatinib/trastuzumab combination. After 4 days of continuous treatment with the combination, sustained block of HER2/PI3K/AKT signaling and loss of expression of total HER2 protein was observed by both Western blot and IHC. The combination also appears to bring about a more rapid loss of HER2+ cells than that observed for trastuzumab monotherapy. The prolonged block of HER2 signaling in these models is likely due to the complementary mechanisms of action of the antibody and small-molecule TKI at the cell surface HER2 protein. However, our data indicate that tucatinib does not appear to inhibit recycling of HER2 protein to the cell surface.
Clinical data show that tucatinib can be safely combined with trastuzumab-based therapies (26, 34). However, studies are still required to identify the optimal treatment regimen for tucatinib as a combination partner in HER2+ breast cancer. To address this, we compared the in vivo single-agent and combined efficacy of tucatinib with each of the components of a currently used SOC regimen for early-stage HER2+ breast cancer, that is, trastuzumab/pertuzumab/docetaxel. Tucatinib monotherapy induced sustained tumor regressions from baseline that were slightly less than responses achieved with the SOC triple combination. Combinations of tucatinib with trastuzumab, pertuzumab or docetaxel also induced efficacy similar to the SOC control arm. However, major differences were detected in terms of tolerability of the various combination regimens. Each combination schedule that contained docetaxel required a dosing holiday, whereas the chemo-sparing combinations were well tolerated and allowed for continuous dosing. These data are consistent with clinical studies that show the use of cytotoxic chemotherapy in combination with HER2-targeted therapies induces significant toxicity (8, 35). The data presented here support the use of tucatinib in chemotherapy-sparing regimens for HER2+ cancers and that these regimens maintain efficacy while significantly reducing toxicity. Furthermore, our data highlight the potential for tucatinib to replace costly therapeutics such as pertuzumab, which despite approval, provides only marginal clinical benefit over trastuzumab alone in early HER2+ breast cancer and adds nothing to treatment of HER2+ gastric cancer (36, 37).
We further explored the potential of tucatinib to provide benefit in a subtype of HER2+ cancers that are also positive for ER (HER2+/ER+). This subtype has been shown to be less responsive than HER2+/ER− cancer to the current SOC HER2 therapies (35, 38, 39). Given the presence of high levels of ER in these tumors, there is sound rationale for the inclusion of hormonal blockade plus inhibition of CDK4/6. The combination of ER-signaling blockade and CDK4/6 inhibition is now the SOC for treatment of ER-positive HER2 normal (ER+/HER2−) breast cancers based on clinical data that demonstrate clear improvements PFS and OS in response to combination treatment (40, 41). In addition, the combination of fulvestrant, abemaciclib, and trastuzumab in patients with HER2+/ER+ advanced breast cancer improves PFS over trastuzumab plus chemotherapy (42). Activated ER signaling drives cell proliferation by increasing transcription of Cyclin D1 that binds to and activates the cyclin dependent kinases 4 and 6 (CDK4/6). Activated cyclin D1:CDK4/6 complexes phosphorylate retinoblastoma protein leading to transcription of E2F-regulated genes that promote progression through the G1–S checkpoint of the cell cycle (43). Signaling through HER2 also promotes transcription of cyclin D1 and drives proliferation through activation of CDK4/6 (44). Given these data, targeting CDK4/6 in combination with HER2-targeted therapy may add additional efficacy benefit in HER2+ cancers especially in this HER2+/ER+ subtype. Previous studies from our laboratory have shown that CDK4/6 inhibitors have single-agent activity in cell line models of HER2+/ER+ cancer and combined targeting of CDK4/6, HER2, and ER induced increased efficacy in xenograft models of HER2+/ER+ breast cancer (30). In the current study, we demonstrate that tucatinib has combined activity with each of the approved CDK4/6 inhibitors, palbociclib (Ibrance, Pfizer), ribociclib (Kisqali, Novartis), and abemaciclib (Verzenio, Eli-Lilly), in HER2+ breast cancer cell lines. In xenograft models of HER2+/ER+ breast cancer, treatment with tucatinib and palbociclib combination demonstrated significant improvement in efficacy over either single-agent therapy. Further addition of trastuzumab and fulvestrant to the tucatinib/palbociclib combination led to increases in antitumor responses that match the efficacy achieved by trastuzumab/pertuzumab/docetaxel without the trade off in tolerability. We have reproduced these findings in xenograft studies using either ribociclib or abemaciclib as the CDK4/6-inhibitor. We believe that these data support the hypothesis that inclusion of a tucatinib/trastuzumab backbone in combination therapy targeting HER2, ER, and CDK4/6 without the use of cytotoxic chemotherapy, may provide improved benefit to patients in terms of efficacy and more favorable safety. This hypothesis will be tested in an investigator-sponsored randomized, open label, phase II clinical trial designed to compare the efficacy and safety of tucatinib in combination with trastuzumab and palbociclib plus fulvestrant versus current SOC (trastuzumab/pertuzumab/docetaxel or carboplatin) in patients with early-stage HER2+/ER+ breast cancer. In addition, this study will explore the potential of tucatinib/trastuzumab in combination with CDK4/6 inhibition in the HER2+/ER− subtype by comparing a combination of tucatinib/trastuzumab/palbociclib with the same SOC regimen used in the HER2+/ER+ group. The inclusion of a tucatinib/trastuzumab/docetaxel arm in this study will allow for direct comparison of the efficacy/safety benefits of a relatively pure HER2 kinase inhibitor (tucatinib) in combination with trastuzumab versus a therapeutic that also inhibits EGFR (pertuzumab) with the attendant cutaneous and gastrointestinal toxicities. The arms with the best efficacy/safety profile will be included in a larger clinical study.
In summary, we present a comprehensive profile of tucatinib in vitro and in vivo activity as a single-agent and combination partner in a series of preclinical models of HER2+ human cancer. Tucatinib activity is restricted to breast and non-breast cancer cell lines carrying the HER2 gene amplification, and sensitivity is further enriched in HER2+ cell lines that demonstrate HER2-driven downstream signaling. The high degree of selectivity of tucatinib makes it an ideal combination partner for approved and novel therapeutics. Combination with trastuzumab leads to sustained blockade of downstream HER2 signaling and increased efficacy in xenograft models. Finally, we demonstrate that tucatinib combines effectively with inhibitors of CDK4/6 in cell line and xenograft models of HER2+/ER+ breast cancer. We believe these data support expanded clinical development of tucatinib in HER2+ cancers to include investigation of tucatinib as a combination partner with trastuzumab and inhibitors of CDK4/6 in HER2+/ER+ breast cancer.
N.A. O'Brien reports other support from 1200 Pharma and TORL Biotherapeutics outside the submitted work. M.S.J. McDermott reports other support from 1200 Pharma LLC and TORL Biotherapeutics LLC outside the submitted work. A.M. Madrid reports other support from 1200 Pharma and TORL Biotherapeutics outside the submitted work. T. Luo reports other support from 1200 Pharma and TORL Biotherapeutics outside the submitted work. R. Ayala reports other support from 1200 pharma and TORL BIOtherapeutics outside the submitted work. K. Gong reports other support from TORL Biotherapeutics outside the submitted work. J. Zhang reports other support from TORL Biotherapeutics outside the submitted work. D.J. Slamon reports other support from BioMarin, Pfizer, Novartis, Eli Lilly, Amgen, and Seattle Genetics during the conduct of the study. No disclosures were reported by the other authors.
N.A. O'Brien: Conceptualization, data curation, investigation, writing–original draft. H.K.T. Huang: Data curation, investigation, methodology, writing–review and editing. M.S.J. McDermott: Data curation, investigation, writing–review and editing. A.M. Madrid: Data curation, investigation, methodology, writing–review and editing. T. Luo: Data curation, investigation, methodology. R. Ayala: Investigation, methodology. S. Issakhanian: Investigation, methodology. K.W. Gong: Investigation, methodology. M. Lu: Investigation, methodology. J. Zhang: Investigation, methodology. D.J. Slamon: Conceptualization, writing–review and editing.
This work was supported by the Stacy and Donald Kivowitz Fund.
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