Tyrosine Kinase Inhibitor Neratinib Research Articles

Biomimetic Modification of siRNA/Chemo Drug Nanoassemblies for Targeted Combination Therapy in Breast Cancer.

The development and progression of tumors are characterized by intricate biological processes. Monotherapy not only struggles to achieve effective treatment but also tends to precipitate a series of issues, including multidrug resistance and limited antitumor effect. Consequently, it is imperative to adopt a synergistic multitherapy approach to enhance the efficacy of tumor treatment. The integration of chemotherapy drug with oligonucleotide drug for combinational treatment has shown significant promise in improving tumor therapeutic efficiency. However, the effective in vivo codelivery of oligonucleotide drugs and chemotherapy drugs faces substantial challenges such as poor stability of oligonucleotide drugs during the circulation time, limited tumor accumulation, and uncertain delivery ratios of different payloads. To overcome these obstacles, we have engineered cyclic Arg-Gly-Asp (cRGD)-modified red blood cell membrane (RBCm)-coated multidrug nanocomplexes, which were self-assembled from the Polo-like kinase 1 siRNA (siPlk1) and an irreversible tyrosine kinase inhibitor neratinib targeted to human epidermal growth factor receptor 2 (HER2) overexpressed in breast cancer. Through electrostatic and amphiphilic interactions between the positively charged neratinib and negatively charged siPlk1, we have successfully fabricated uniform multidrug nanoparticles. The cRGD-modified red blood cell membranes coated on the surface of the multidrug nanoparticles could enhance drug stability in circulation and tumor accumulation. This targeted combinational therapy significantly enhanced the antitumor efficiency in HER2-positive breast cancer in vitro and in vivo.

Neratinib Alone or in Combination with Immune Checkpoint Inhibitors with or without Mammalian Target of Rapamycin Inhibitors in Patients with Fibrolamellar Carcinoma

<b><i>Introduction:</i></b> Fibrolamellar carcinoma (FLC) displays upregulation of several oncogenes, including <i>HER2</i>, and multiple immune-suppressive mechanisms. We investigated the efficacy and safety of the pan-HER tyrosine kinase inhibitor neratinib as monotherapy (SUMMIT phase 2 basket study) or with immune checkpoint and/or mammalian target of rapamycin (mTOR) inhibitors (compassionate-use program) in patients with FLC. <b><i>Methods:</i></b> Patients received neratinib 240 mg/day orally in SUMMIT, or as doublet or triplet combinations with pembrolizumab 2 mg/kg intravenously every 3 weeks, nivolumab 240 mg intravenously every 2 weeks, everolimus 7.5 mg/day orally, or sunitinib 37.5 mg/day orally under compassionate use. The primary endpoint in SUMMIT was objective response rate; safety was a secondary endpoint. <b><i>Results:</i></b> Fifteen patients with FLC received neratinib monotherapy in SUMMIT. The objective response rate was 5% (95% confidence interval [CI]: 0–21.8) and the disease control rate was 13.3% (95% CI: 1.7–40.5). Upon progression, five had added immune checkpoint inhibitors with or without everolimus or sunitinib. Two additional patients received neratinib-based combinations outside of SUMMIT, for a total of 17 neratinib-treated patients. One patient who received neratinib plus pembrolizumab had a confirmed partial response, one treated with neratinib plus everolimus had stable disease lasting 6 months, and one who received neratinib plus pembrolizumab plus sunitinib had stable disease lasting 16 months. Grade 3/4 adverse events with neratinib monotherapy occurred in 10 (66.7%)/2 (13.3%) patients, respectively. Grade 3 adverse events with neratinib-based combinations were hyperglycemia (<i>n</i> = 1; neratinib plus pembrolizumab), hepatic failure, and anaphylaxis (<i>n</i> = 1 each, neratinib plus pembrolizumab plus everolimus). There were no grade 4 adverse events with combination therapy. <b><i>Conclusion:</i></b> In patients with FLC, single-agent neratinib had limited efficacy, but clinical benefit was observed with neratinib in combination with immunotherapy and/or mTOR-targeted agents.

Preclinical and clinical evaluation through serial colonoscopic evaluation of neratinib-induced diarrhea in HER2-positive breast cancer-A pilot study.

The irreversible pan-HER tyrosine kinase inhibitor neratinib is approved for patients with HER2-positive, early-stage and metastatic breast cancer (BC). Neratinib-associated diarrhea is the most common reason for early discontinuation. Preclinical studies identified mechanisms of neratinib-induced diarrhea and rationale for prophylactic and preventive measures. We studied effects of neratinib on rat intestines and conducted a phase 2 study of colon pathogenesis in patients with HER2-positive BC treated with neratinib (NCT04366713). Colon samples from female albino Wistar rats receiving neratinib or vehicle were examined for histopathological changes. Patients with HER2-positive BC received neratinib 240 mg once daily for up to 1 year. Colonoscopy biopsies were collected at baseline and at Day 28 to identify changes consistent with rat pathologies. Rat colons were markedly altered in appearance, with similar short circuit currents (Isc) and responses to carbachol and forskolin. Mucosal barrier loss and/or significant increase in secretory propensity in neratinib- versus control-treated animals were not seen. Two of four endpoint-evaluable patients presented with mild pathological changes, largely comparable with the rat model. Preclinical evidence supports an inflammatory component of neratinib-induced diarrhea without mucosal barrier function loss. Colonoscopy findings in patients with BC indicate mild or no pathological changes in the colon due to neratinib treatment.

Abstract A026: An alternative to HER2 IHC 0/1+/2+ status to predict which clinically HER2-negative patients will respond to anti-HER2 therapies: A rationale for the likely superiority of quantitative HER2 pathway RPPA measurements

Abstract Immunohistochemistry (IHC) may not accurately measure HER2 levels and does not reflect HER2 activity/phosphorylation in HER2-negative patients in the HER2 LOW (IHC 1+/2+) and ULTRA-LOW (IHC 0) range [PMID: 35595825). Yet, accurate quantification of HER2 may be key for predicting response to antibody drug conjugates (ADC) such as fam-trastuzumab-deruxtecan-nxki (T-DxD). We showed previously that response to the receptor tyrosine kinase inhibitor (RTKI) neratinib and the ADC ado-trastuzumab emtansine (TDM-1) can be predicted by phospho(p) HER2 Y1248 and pEGFR Y1173, but not total HER2, in TN and HER2+ pts, respectively (PMID: 34741023; 32914002). We now utilize I-SPY2 TRIAL data to explore further relationships between HER2 IHC, Reverse Phase Protein Array (RPPA)-based quantitative HER2 measurement and a HER2 Activation Response Predictive Signature (HARPS) as candidate response predictors for HER2low-targeted agents. Tumors from 395 HER2-negative patients (HR+=200, TN=195) from 8 arms of the I-SPY2 TRIAL had HER2 IHC/FISH data, along with RPPA-based HER2, pHER2 Y1248 and pEGFR Y1173 data from Laser Capture Microdissection (LCM)-enriched tumor epithelium. Of these tumors, 37% (145/395) were HER2 IHC 0, 42% (168/395) were IHC 1+ and 21% (82/395) were IHC 2+. RPPA-based HARPS was categorized as HARPS+ (pHER2-high/pEGFR-high) or HARPS- (pHER2-low and/or pEGFR-low) using previously established combined cut points. Association between quantitative protein values and HER2 IHC was assessed using ANOVA/Tukey tests. Association between endpoint quartiles and IHC was assessed using chi-squared/posthoc tests. RPPA quantitative HER2 total protein levels were associated with HER2 IHC (Tukey p<1E-09), however, the median and ranges of HER2 expression highly overlapped between HER2 IHC 0, 1+ and 2+ (e.g. HER2=0.44 [0.27 - 0.6] in IHC 0 vs 0.69 [0.42-1.0] in IHC 2+). A significant number (34%) of HER2 IHC 0 tumors expressed HER2 above the median level in the HER2 LOW population. While pHER2 and HARPS status were not associated with HER2 IHC class, HARPS associated with total HER2 overall (chi-squared p=8E-05) and in TNBC (chi-squared p=2.4E-06). We found that 42% (81/195) of TNBC were HARPS+, as were 44% (40/89) of HER2 ULTRA-LOW and 40% (41/104) of HER2 LOW tumors. Our results reveal general disconcordance between IHC and RPPA quantitative HER2, with a third of HER2 IHC 0 tumors expressing HER2 and thus, potentially responsive to ADCs such as T-DxD or TDM1. The high proportion of HER2 ULTRA-LOW TNBC tumors classified HARPS+ by our CLIA-based assay identifies tumors predicted to respond to HER2-directed TKI therapies, representing a cohort with actionable HER2 biology missed by IHC. As HER2Low targeted therapies enter ISPY 2.2, we will test the hypothesis that quantitative HER2 is most predictive for HER2 ADCs, and HARPS is most predictive for RTKIs. Citation Format: Julia Wulfkuhle, Denise M. Wolf, Angela DeMichele, Christina Yau, Laura van ‘t Veer, Hope S. Rugo, Lajos Pusztai, Lamorna Brown-Swigart, Gillian L. Hirst, Rosa I. Gallagher, Amy L. Delson, Alexander D. Borowsky, Laura J. Esserman, Paula R. Pohlmann, Emanuel F. Petricoin. An alternative to HER2 IHC 0/1+/2+ status to predict which clinically HER2-negative patients will respond to anti-HER2 therapies: A rationale for the likely superiority of quantitative HER2 pathway RPPA measurements [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A026.

Activation of the integrated stress response by inhibitors of its kinases

Phosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases.

Abstract 3962: Development of selection-free HER4-mutated CRISPR/Cas9 knock-ins in cutaneous melanoma cell lines

Abstract Background: Cutaneous melanoma showed the highest HER4 genomic alteration frequency (17.34%) across 33 different cancer types (cBioPortal). HER4 is a tyrosine kinase receptor belonging to the human epidermal growth factor receptor (HER/ErbB) family. The role of HER4 in cancer has not been fully studied, potentially due to its complex biology (four isoforms, different stability/tissue-specific expression patterns, and oncogenic/tumor suppressor roles). Using computational analysis, we identified two potentially oncogenic HER4 mutations in melanoma (HER4 R106C and R711C). We then developed CRISPR/Cas9-modified selection-free melanoma cell lines for interrogating the role of these mutations as biomarkers of response to the oral, irreversible pan-HER (EGFR, HER2, HER4) tyrosine kinase inhibitor (TKI) neratinib (NER). Methods: Two melanoma cell lines (WM115, SKMEL24) were genome-edited by using a selection-free CRISPR/Cas9 approach. Single guide RNAs (sgRNAs) were designed by using the CRISPRon (v1.0) web tool, and Homology Directed Repair (HDR) single-stranded OligoDeoxyriboNucleotides (ssODNs), containing the HER4 mutations, were designed with the SnapGene Software (v6.1.1). The RNP complex (Cas9 and sgRNAs) and the HDR_ssODNs, for each HER4 mutation, were delivered into the melanoma cells through electroporation. Upon validation of the CRISPR/Cas9-efficiency via PCR and Sanger Sequencing, the CRISPR/Cas9-modified cells were single-cell sorted via FACS, by using two singlets gating (FSC-A/FSC-H and SSC-A/SSC-H) and a live/dead (propidium iodide) gating, to perform single-cell cloning. Colonies were then amplified and screened via PCR and Sanger sequencing to isolate pure selection-free HER4 CRISPR/Cas9 knock-ins clones of melanoma cells. Results: The WM115 and SKMEL24 melanoma cell lines were found to be wild-type for the HER4 receptor in the Depmap Portal (mutation 22Q2, 2022 data release 2). R106C and R711C CRISPR/Cas9 knock-ins were created in the melanoma cell lines three days after the electroporation of the CRISPR/Cas9 complex. The efficiency of the HER4 R106C CRISPR/Cas9 knock-in was 22% and 7% in the SKMEL24 and WM115 melanoma cells, respectively, with a respective total indel percentage of 76% (SKMEL24) and 55% (WM115). The HER4 R711C CRISPR/Cas9 knock-in efficiency was 15% in the SKMEL24 (with 26% total indels) and 1% in the WM115 (with 33% total indels). Single-cell colonies of the HER4 CRISPR/Cas9-mediated knock-ins (HER4 R106C and R711C), were formed at four days (SKMEL24) and at one week (WM115) after single-cell sorting. Conclusions: CRISPR/Cas9 technology can be utilised to introduce single-point mutations in HER4 to melanoma cell lines. In vitro functional characterisation of the isolated HER4 CRISPR/Cas9-edited melanoma cell lines will be performed in order to understand the activating potential of HER4 R106C and R711C and their sensitivity to NER. Citation Format: Marta Valenti, Yonglun Luo, Neil T. Conlon, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Development of selection-free HER4-mutated CRISPR/Cas9 knock-ins in cutaneous melanoma cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3962.

The influence of verapamil on the pharmacokinetics of the pan-HER tyrosine kinase inhibitor neratinib in rats: the role of P-glycoprotein-mediated efflux.

Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been approved for the treatment of HER2-positive (HER2+) early-stage and brain metastatic breast cancer. Thus far, the pharmacology effects and pharmacodynamics of neratinib have been well studied. However, the disposition of neratinib and its influencing factors in vivo remain unclear. P-glycoprotein (P-gp), one of the most extensively studied transporters, substantially restricts penetration of drugs into the body or deeper compartments (i.e., blood-brain barrier, BBB), regarding drug resistance and drug-drug interactions. Thereby, the aim of this study was to investigate the influence of verapamil (a P-gp inhibitor) on the pharmacokinetics of neratinib in rats. Here, we have established a high specific, selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify plasma concentrations of neratinib in rats. Pharmacokinetic results showed that verapamil significantly increased the system exposure of neratinib, as Cmax increased by 2.09-fold and AUC0-t increased by 1.64-fold, respectively. Additionally, the in vitro transport of neratinib was evaluated using Madin-Darby canine kidney II (MDCK II) andhumanMDR1 gene overexpressed MDCK (MDCK-MDR1) cell line models. As a result, the net flux ratio was over than 2 and decreased over 50% by verapamil, suggesting that neratinib was a substrate of P-gp. Hence, our findings have highlighted the important role of P-gp in the system exposure of neratinib in vivo, and drug-drug interaction should be considered when coadministration of P-gp inhibitors with neratinib. These findings may support the further clinical development and application of neratinib.

Poziotinib Inhibits HER2-Mutant-Driven Therapeutic Resistance and Multiorgan Metastasis in Breast Cancer.

Evaluation of the functional impact of HER2 mutations on therapy-induced resistance and metastasis identifies robust antitumor activity of poziotinib and supports the clinical evaluation of poziotinib in ER+ HER2 mutant breast cancer.

Abstract 3988: Identification of HER4 mutations with potential as biomarkers of response to neratinib

Abstract Background: HER4 is a member of the human epidermal growth factor receptor (HER/ErbB) family of receptor tyrosine kinases. HER4 is activated through ligand binding, transmitting intracellular signals by homodimerizing, or by heterodimerizing with other family members (EGFR, HER2, HER3). The role of HER4 in cancer or normal tissue has not been studied as comprehensively as EGFR and HER2, potentially due to a more complex biology that includes four different isoforms and different stability/tissue-specific expression patterns, and oncogenic or oncosuppressor roles. The oral, irreversible pan-HER (EGFR, HER2, HER4) tyrosine kinase inhibitor (TKI) neratinib (NER) is clinically active against HER2-mutant and HER2-amplified cancers. Using publicly available datasets including cBioPortal and COSMIC and predicted structural and functional impacts, this study aimed to identify HER4 mutations with the greatest oncogenic potential that may have value as potential biomarkers of response to NER. Methods: The frequency of HER4 mutations across 33 cancer types was determined by using the clinical database cBioPortal and TCGA PanCancer Atlas datasets (n=10967). A shortlist of potentially oncogenic HER4 mutations was created based on the occurrence in cBioPortal and COSMIC databases, annotation as putative driver and cancer hotspot, structural and functional impacts according to OncoKB, Mutation Assessor, SIFT and Polyphen-2 software, and HER4 protein folding studies. Analysis was carried out using the R-studio statistical environment. Results: HER4 amplification is rare in cancer, with the majority of genetic changes reported as point mutations; particularly missense (31.93%), synonymous (6.43%) and nonsense (3%) substitutions. Melanoma and esophagogastric (EG) cancers showed the highest HER4 alteration frequency, 17.34% and 13.42%, respectively, across different cancer types in the cBioPortal database. Melanoma and EG cancers showed the second and third highest absolute count of HER4 genomic alterations (n=77 and n=69, respectively). Based on the criteria outlined in methods, a priority list of HER4 mutations with greatest oncogenic potential was curated for melanoma (HER4 R106C, R306C, R711C, G936E, F1003I), stomach (HER4 C217R, S774N, K1002R, Y1150C) and esophageal (HER4 R488W, S449Y) adenocarcinomas. Mutations at these sites, located across ligand-binding, dimerization and intracellular signaling domains, have the potential to impact HER4 activity and thus possibly act as biomarkers of response to an irreversible pan-HER TKI like NER. Conclusions: These data suggest that melanoma and EG cancers are the cancer types with the highest rate of HER4 mutations. In vitro functional characterization of the identified HER4 mutations is now required to further explore their value as biomarkers of response to NER. Citation Format: Marta Valenti, Neil T. Conlon, Lisa D. Eli, Irmina Diala, John Crown, Denis M. Collins. Identification of HER4 mutations with potential as biomarkers of response to neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3988.

Abstract 153: An unbiased in vitro screen for activating ERBB4 mutations

Abstract Introduction: Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites across different samples. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hotspots within their respective genes. However, a vast majority of mutations in cancer tissues are rare and their functional significance remains unknown. Several lines of in vitro and clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations which could serve as predictive markers in oncology. Here, we performed an unbiased functional screen to identify potential activating mutations of ERBB4, a frequently mutated member of the epidermal growth factor receptor family. Method: To identify functional driver mutations of ERBB4, the previously published pipeline, in vitro screen for activating mutations (iSCREAM) [1], was utilized. iSCREAM is a functional genetics screen based on the expression of random cDNA variants and the ability of driver mutations to promote cellular growth in vitro. The expression library encoding randomly mutated ERBB4 variants was retrovirally introduced into murine lymphoid Ba/F3 cells, that normally require interleukin-3 (IL-3) for survival but can exploit ectopic expression of activated variants of oncogenic kinases to compensate for the deficiency of exogenous IL-3. When expressed under an appropriate promoter, the wild-type ERBB4 receptor failed to promote IL-3-independent survival in the presence of the neuregulin-1 ligand, while the cells expressing activating mutations readily proliferated. The identity and frequency of the activating ERBB4 mutations were subsequently determined from the proliferating cell pool using targeted next-generation sequencing. Results: Ten candidate activating mutations were identified out of the over 7000 random ERBB4 missense or nonsense mutations present in the original library. The candidate activating mutations were individually characterized using functional assays both in vitro and in vivo, as well as by structural analyses. The activating mutation were sensitive to clinically used pan-ERBB tyrosine kinase inhibitors afatinib, dacomitinib and neratinib. Conclusions: A subset of ERBB4 missense mutations are activating and sensitive to tyrosine kinase inhibitor drugs. Citation Format: Deepankar Chakroborty, Veera K. Ojala, Anna M. Knittle, Kari J. Kurppa, Klaus Elenius. An unbiased in vitro screen for activating ERBB4 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 153.

Abstract 3248: Acquired resistance to targeted inhibitors in EGFR-driven glioblastoma: Identification of dual kinase targets

Abstract Glioblastoma (GBM) is a devastating primary brain tumor with <5% 5-year survival. CDKN2A deletion (~60%) and EGFR amplification (~55%) mutations frequently co-occur in these tumors. EGFR is an attractive therapeutic target due to its mutational frequency and availability of brain-penetrant tyrosine kinase inhibitors (TKI). Several EGFR TKI have failed clinically, due in part to acquired resistance. To mechanistically examine this type of resistance, we used a panel of ten genetically engineered mouse astrocyte lines harboring Cdkn2a deletion and EGFRvIII, a common (~30%) activating mutation. Resistant cells were generated via long-term exposure to gefitinib or erlotinib, either in vitro or in vivo. Both transcriptomic (RNAseq) and proteomic (multiplexed inhibitor beads with mass spectrometry, MIB-MS) experiments showed that cell lines clustered primarily by resistance phenotype and secondarily by method of resistance development when analyzed using principal component analysis and unsupervised hierarchical clustering. Kinases involved in proliferation and differentiation signaling pathways (ex: Pdgfrb, Pdk2, Tnik, Mapk3, Fgfr2) were upregulated in both RNAseq and MIB-MS datasets and thus represent putative druggable targets for dual kinase inhibition. Analysis of commonly upregulated kinases and their commercially available inhibitors revealed dovitinib and dasatinib, two brain-penetrant drugs approved for other cancer indications, as candidates for dual inhibition with an EGFR TKI. Resistant cell lines were all more sensitive to dovitinib than their drug-naïve parents; however, sensitivity to dasatinib varied. BLISS analysis of dual treatment with EGFR TKI neratinib and dasatinib or dovitinib revealed synergistic drug interactions in most lines. Additionally, drug-naïve cells displayed a robust, acute proteomic response to EGFR TKI afatinib over 48h, while the response of resistant lines was significantly blunted. This model system can also be used to examine acute vs. long-term kinome response to EGFR TKI. Acute response was examined by treating drug-naïve cells with afatinib over 48h, and long-term kinome rewiring was observed by comparing untreated cells to gefitinib- and erlotinib-resistant cell lines. Combing both RNAseq datasets for kinases upregulated in both drug-naïve cells over a 48h EGFR TKI treatment course and in resistant cell lines compared to their sensitive parents reveals 21 and 13 common kinases, respectively, at p<0.001. Eight of these kinases (Cdk19, Ddr1, Kalrn, Khk, Mapk3, Pink1, Tnik, Ulk2) appear in both the in vitro and in vivo datasets, indicating a conserved kinome response regardless of method of resistance generation. Overall, integrated kinome profiling in GBM models with defined mutational profiles provides a powerful framework to identify novel therapeutic targets that could significantly alter current treatment paradigms. Citation Format: Abigail K. Shelton, Erin Smithberger, Madison Butler, Allie Stamper, Ryan E. Bash, Steve P. Angus, Michael P. East, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, Ryan Miller. Acquired resistance to targeted inhibitors in EGFR-driven glioblastoma: Identification of dual kinase targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3248.

Pyrotinib monotherapy or pyrotinib in combination with capecitabine could significantly prolong progression-free survival and overall survival in patients with HER2-positive metastatic breast cancer.

1034 Background: In the NALA phase III trial, irreversible pan-ErbB tyrosine kinase inhibitor (TKI) neratinib plus capecitabine demonstrated significant improvement in the progression-free survival (PFS), but no significant benefit in overall survival (OS) compared to lapatinib plus capecitabine. However, another TKI pyrotinib plus capecitabine showed significant benefits in PFS and a trend of OS benefits in the PHOEBE phase III study. But in general, current data on survival of irreversible TKIs in patients with HER2-positive metastatic breast cancer (MBC) was limited. Thus, we performed a pooled analysis of individual patient data from the phase I to III trials in HER2-positive MBC patients receiving pyrotinib or pyrotinib combined with capecitabine, to provide a cumulative assessment on long-term outcomes of irreversible TKI. Methods: Individual patient data was collected and analyzed from the phase I trial for pyrotinib and pyrotinib plus capecitabine, the Pivotal phase II trial and the PHOEBE phase III trials that enrolled patients in China National Cancer Center. Next-generation sequencing was performed on circulating tumor DNA for predictive biomarkers in the phase I trial. Results: Between August 2013 and October 2018, a total of 120 patients were assigned to received pyrotinib (n = 38), pyrotinib plus capecitabine (n = 53) and lapatinib plus capecitabine (n = 29) across the above four trials. The median follow-up duration for OS was 73.6 months (95% CI:69.9-77.3 months). The estimated median PFS was 8.2 months (95% CI:5.6-10.9 months) in the pyrotinib monotherapy group and 22.0 months (95% CI:13.2-30.8 months) in the pyrotinib plus capecitabine group ( P= 0.002), while the median OS was 27.1 months (95% CI: 21.6-32.5 months) in the pyrotinib monotherapy group and 44.5 months (95% CI: 30.8-58.1 months) in the pyrotinib plus capecitabine group ( P= 0.065). In this pooled analysis, pyrotinib 400mg in combination with capecitabine, recommended for phase II and III trials, had significantly longer PFS (22.0 vs 6.9 months, P＜0.001) and OS (59.9 vs 31.2 months, P= 0.035) than lapatinib plus capecitabine. Analysis of all genetic alterations in baseline blood samples suggested that the patients harbored concomitant mutations in HER2-related signaling network (including HER2 bypass signaling pathway, PI3K/Akt/mTOR pathway and TP53) were observed with significantly poorer PFS and OS compared to none or one genetic alteration (median PFS, 7.3 vs. 26.1 months, P= 0.003; median OS, 25.1 vs. 48.0 months, P= 0.013). Conclusions: This pooled analysis based on phase I to III trials revealed promising PFS and OS was achieved in pyrotinib and pyrotinib plus capecitabine. Concomitant mutations in HER2-related signaling network may be a potential efficacy and prognosis biomarker for pyrotinib in HER2-positive MBC. Clinical trial information: NCT01937689,NCT02361112,NCT02422199,NCT03080805.

Abstract P2-13-36: Comparative time course analysis of the effects of neratinib, lapatinib and tucatinib in an in vitro HER2+ breast cancer model

Abstract Introduction: Human epidermal growth factor 2 (HER2/ErbB2) is overexpressed in approximately 20% of breast cancers. The HER2-targeted tyrosine kinase inhibitors (TKIs) neratinib, lapatinib and tucatinib are all FDA-approved for the treatment of HER2-positive breast cancer. All three inhibit the HER2 kinase domain but differ in binding characteristics (the irreversible neratinib vs reversible lapatinib and tucatinib) and their affinity for other HER family members. The aim of this study was to carry out kinetic analysis of the effect of the three TKIs on HER family signalling, cellular proliferation, and apoptosis induction in a cell model of breast cancer. Methods: In this study, the anti-proliferative effects of the three TKIs were assessed in the HER2-amplified breast cancer cell line SKBR3 by acid phosphatase assay (5-day endpoint assay) and with the IncuCyte→ live-cell imaging system over 120 hours. TKI IC50 values were calculated using CalcuSyn software. Apoptotic cells were detected every 3 hours for 5 days using the Incucyte→ Caspase 3/7 dye. Western blotting was used to investigate the time course effects (15 min, 6 h, 24 h, 72 h) of the TKIs on the expression and phosphorylation of EGFR, HER2 and on their downstream effectors ERK1/2 and Akt. Results: All three TKIs displayed nanomolar activity against SKBR3 cells. Neratinib showed the greatest anti-proliferative effect (IC50 = 3.4 ± 1.1 nM), followed by tucatinib (IC50 = 37.5 ± 18.4 nM) and lapatinib (IC50 = 51.0 ± 23.0 nM). 50 nM neratinib induced higher activation of caspase 3/7 than 500 nM lapatinib or 500 nM tucatinib. Total levels of HER2 and EGFR were increased in the presence of lapatinib and tucatinib, but decreased in the presence of neratinib. The three drugs (500 nM) significantly inhibited the activation of EGFR and HER2 at 15 min. From 6 h to 72 h, treatment with 5 nM and 50 nM neratinib resulted in greater inhibition of phosphorylated HER2 and EGFR, compared to the equivalent concentration of lapatinib or tucatinib. All three TKIs (500 nM) inhibited the activity of phospho-Akt at the earliest timepoint (15 min), but a strong impact on downstream ERK1/2 phosphorylation was not observed until the 6h timepoint. 50 and 500 nM neratinib was more effective at preventing reactivation of ERK1/2 and Akt phosphorylation than either 500 nM lapatinib or tucatinib at 72 h. Conclusion: These data highlight the differences in in vitro efficacy of the three HER2-targeting TKIs currently approved for the treatment of HER2+ breast cancer in the clinic. Neratinib inhibits HER2 and EGFR at lower concentrations and for longer compared to lapatinib and tucatinib, resulting in increased apoptosis induction. This may be due to the irreversible nature of neratinib binding, as opposed to the reversible nature of lapatinib and tucatinib binding. Further comparative studies between neratinib, lapatinib and tucatinib are warranted to explore the impact of varied pre-clinical characteristics on the emergence of therapeutic resistance. Citation Format: Myra E Castel, Neil T Conlon, Naomi Walsh, Irmina Diala, Lisa Eli, John Crown, Denis M Collins. Comparative time course analysis of the effects of neratinib, lapatinib and tucatinib in an in vitro HER2+ breast cancer model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-36.

Abstract PD8-06: Acquired resistance to tucatinib is associated with EGFR amplification in HER2+ breast cancer (BC) models and can be overcome by a more complete blockade of HER receptor layer

Abstract Background: With recent approval of the irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (N) and the HER2-specific TKI tucatinib (T) in the advanced setting and their edging towards the early setting in HER2+ BC, resistance will likely emerge as a challenge, as illustrated by the HER2CLIMB study, where only one patient with brain metastasis remained progression free after 2 years on T. We set out to define the mechanisms of resistance to T and treatment strategies to overcome it. Materials and Methods: Our previously characterized HER2+ BT474 models with acquired resistance to lapatinib (L; LapR) or N (NrbR) (SABCS20-PD3-09), and our recently developed models of BT474 and SKBR3 with acquired resistance to T (TucaR) developed through long-term exposure to increasing doses of T (up to 200nM) and their naïve parental (P) were used. Genomic (DNA-seq), transcriptomic (RNA-seq), and proteomic (RPPA, western blot) characterization were performed. Drug efficacy studies involved cell growth assays by the imaging-based IncuCyte system. Results: We recently reported that while LapR is associated with acquisition of HER2 L755S mutation, which partially reactivates the HER pathway, NrbR is associated with the additional co-acquisition of a pathogenic PIK3CA mutation. Preliminary analysis of 2 BT474 TucaR models (ATCC and AZ) showed highly elevated levels of phosphorylated (p) and total (t) EGFR, suggesting EGFR signaling activation. Levels of pHER2, pHER3, and downstream pAKT and pS6 were also markedly higher in the TucaR models compared to P or short-term T. The TucaR but not LapR or NrbR models exhibited EGFR amplification, explaining the higher EGFR levels and signaling. Further, the elevated pEGFR, pHER2, pHER3, pAKT, and pS6 levels in TucaR models were substantially suppressed by the EGFR-specific TKI gefitinib (G) (50, 500nM) or even further when combined with T (500 nM G+200nM T). These results suggest that the higher pHER2 levels in TucaR models is probably due to heterodimerization of the amplified EGFR with HER2 and subsequent HER2 phosphorylation. In contrast to the P cells where the apoptotic marker cleaved (c)-PARP was not induced with G alone (50, 500nM), but with T (200nM) or 500nM G+T, in the TucaR model, 500nM G alone was enough to induce c-PARP, which was further enhanced when combined with T, implying the survival dependence of TucaR cells on EGFR signaling. The TucaR models were hypersensitive to G compared to P cells, and this growth inhibition was further enhanced with G+T. Whilst we previously reported that the LapR and NrbR cells were cross-resistant to T, the TucaR cells remained highly sensitive to the pan-HER TKIs N, poziotinib, and pyrotinib. Finally, in a second HER2+ model SKBR3, at 200nM TucaR, we observed elevated pEGFR, pHER2, pHER3, and pAKT levels, the underlying mechanism of which is under investigation by genomic and molecular analysis. In-depth characterization of our TucaR models to determine the differential gene expression and signatures is ongoing to gain additional mechanistic insights. Conclusions: Our findings suggest that whilst complete blockade of the HER layer using N is evaded by acquisition of HER and PIK3CA mutations, resistance to the HER2 TKI T is associated with EGFR amplification, a finding that underscores the HER signaling pathway redundancy and cross-talk between HER receptors to compensate for partial blockade of the pathway. Further, while resistance to L and N confers cross-resistance to T, resistance to T may be overcome using pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Together, our findings hold crucial implications in light of the current treatment landscape of HER2+ BC, with a particular emphasis on the considerations to strategize the treatment sequence of currently available TKIs. Citation Format: Jamunarani Veeraraghavan, Sreyashree Bose, Ragini Mistry, Pier Selenica, Sarmistha Nanda, Lanfang Qin, Andrea Gazzo, Yingjie Zhu, Michael A Mancini, Fabio Stossi, Britta Weigelt, Jorge S Reis-Filho, C. Kent Osborne, Mothaffar F Rimawi, Rachel Schiff. Acquired resistance to tucatinib is associated with EGFR amplification in HER2+ breast cancer (BC) models and can be overcome by a more complete blockade of HER receptor layer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD8-06.

Abstract P2-13-28: Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines

Abstract Introduction: Treatment strategies for HER2+ breast cancer (BC) involves first line treatment with the monoclonal antibody trastuzumab, however resistance to treatment can occur. The pan-HER tyrosine kinase inhibitor neratinib is an approved adjuvant treatment for early stage HER2+ BC after one year of treatment with trastuzumab. Recent studies have highlighted the potential benefits of using retinoic acid as an adjuvant treatment for BC. The retinoic acid receptor (RAR) family of nuclear transcription factors consists of RARα, RARβ and RARγ. Interestingly ~30% of HER2+ BC have a co-amplification of the RARA gene, which codes for RARα. We examined the effect of RARα agonists (Fenretinide & AM580) and antagonist (AGN194310) in combination with neratinib in two HER2+ cell lines: SKBR3, has co-amplification of ERBB2 and RARA, and HCC1569 has amplified ERBB2 but are not RARA-amplified. Methods: SKBR3 and HCC1569 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. 10mM stock concentrations of fenretinide (H7779-Sigma), AM580 (A8843-Sigma), AGN194310 (SML2665-Sigma) and neratinib (Puma Biotechnology, Inc) were prepared in DMSO. For proliferation assays, cells were seeded in 96 well plates at a density of 3 X 104 cells per well for 24h. Cells were treated with a drug alone (2X concentration) or combination of drugs (4X concentration) in 100μL of medium. Proliferation was measured using an acid phosphatase-based assay after 5 days as percentage growth versus DMSO control. The half maximal inhibitory concentration (IC50) was calculated for each drug, using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50), using CalcuSyn. Values < 1 represent a synergistic effect, a value of 1 is additive and values > 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research has found that the synthetic retinoic acid fenretinide, a pan-RAR (α, β and γ) activator, was more potent in the HCC1569 cells compared to the SKBR3 cell line with an IC50 of 0.21 ± 0.03μM compared to 4.55 ± 0.87 μM, respectively. However, when combined with neratinib there was a strong antagonistic effect observed in the HCC1569 cell line (CI value: 15.63 ± 9.5). Conversely, fenretinide enhanced the effect of neratinib in the SKBR3 cell line (CI value: 0.83 ± 0.4). The RARα-specific activator AM580 had an IC50 >10μM for both cell lines. When combined with neratinib there was an additive effect observed in the RARα negative HCC1569 cells (CI value: 0.97 ± 0.4), and a synergistic effect observed in the RARα upregulated SKBR3 cell line (CI value: 0.78 ± 0.08). Next we wanted to determine if inhibiting RAR activity would have any effect on the proliferation of these cell lines. The IC50 for the pan-RAR (α, β and γ) antagonist AGN194310 was >10μM for each cell line. However, when combined with neratinib treatment there was a strong synergistic effect observed in the HCC1569 cell line (CI value: 0.52 ± 0.17) and the SKBR3 cell line (CI value: 0.66 ± 0.19). Conclusions: This study suggests a differential effect of RAR agonists when combined with neratinib in RARα amplified versus non-amplified HER2+ BC. Targeting RAR signalling, particularly with a RAR antagonist, in combination with the pan-HER inhibitor warrants further investigation in HER2+ BC. Citation Format: Debbie O'Reilly, Nicola Gaynor, Neil Conlon, Irmina Diala, Lisa D Eli, John Crown, Denis M Collins. Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-28.

Abstract P2-13-24: Distinct HER2 allele specific therapeutic response and preclinical efficacy of poziotinib in metastatic ER+ HER2 mutant breast cancer

Abstract Background.Targeting HER2 gene amplification is one of the great achievements in oncology resulting in the use of a wide array of anti-HER2 agents in the clinic. Unfortunately, 20% of patients still relapse with secondary organ metastasis and are currently incurable. While only 1.6% of primary non-HER2-amplified ER+ breast cancers harbor HER2 mutations, 6-10% of all metastatic breast cancers harbor HER2 mutations, suggesting their causal role in contributing to metastasis. The clinical value of HER2 activating mutations is being tested with the pan-HER tyrosine kinase inhibitor neratinib in phase II clinical trials (NCT01670877, NCT01953926, and NCT02465060). To date, neratinib has elicited only modest responses in ER+ breast cancer, often rapidly followed by progression. This study characterizes HER2-mutant induced resistance to endocrine therapy or neratinib induced resistance and resulting metastasis, in order to determine a more effective rational therapeutic approach for treating ER+ HER2-mutant breast cancer. Methods.HER2 mutation frequency and its impact on patient outcome was determined using METABRIC primary breast cancer (BC) and MSK IMPACT metastatic BC ER+ sequencing studies. Effects of estrogen (E2), fulvestrant, or neratinib on cell growth and HER2 signaling were examined on ER+/ILC cells (MM134) stably expressing HER2/WT, HER2/S310F, and HER2/L755S. Cell growth was measured using CellTiter-Glo and HER2 signaling was analyzed by western blot analysis. Additionally, the effect of these ER+/ILC and IDC HER2 mutations on tumor growth and endocrine or neratinib treatment resistance was determined using fat pad injections and MIND xenografts in NOD-scid gamma mice. Results.We searched ER+ sequencing datasets and identified HER2 mutations that are highly enriched in ER+ ILC as compared to ER+ IDC. These activating HER2 mutations in ER+ ILC are associated with early relapse and poor overall survival. Moreover, we are finding that ILC patients harboring the recurrent HER2/L755S mutation have worse overall survival compared to non-mutant HER2 ILC. MM134 cells expressing HER2/S310F and HER2/L755S show increased cell growth, strongly activated autophosphorylation of HER2, and increased downstream signaling (pMAPK and pAKT) as compared to cells expressing HER2/WT upon treatment with fulvestrant (1μM). Three clinically relevant in vivo models including ILC HER2/L755S Mammary INtraDuctal (MIND) xenografts, IDC HER2/L755S fat pad xenografts, and IDC HCI-003 (an ER+ patient-derived xenograft (PDX) naturally harboring the exon 20 activating HER2G778_P780 dup) exhibit fulvestrant and neratinib resistance and lung and ovary metastases. In addition, we find that HER2 mutations induced mTOR signaling. In contrast, however, the pan-HER drug poziotinib does potently inhibit tumor growth and organ-specific metastasis and perturbs mTOR activation in these models. Conclusion.We demonstrate that clinically associated HER2 mutations drive endocrine therapy or neratinib resistance and poor patient outcome in ER+ patients. Our data propose the use of the irreversible pan-HER TKI poziotinib for treating endocrine therapy or neratinib refractory ER+ HER2-mutant metastatic breast cancer. Citation Format: Shyam. M Kavuri, Rashi Kalra, Ching Hui Chen, Junkai Wang, Ahmad Bin Salam, Lacey Dobrolecki, Alaina Lewis, Christina Sallas, Clayton Yates, Carolina Gutierrez, Balasubramanyam Karanam, Meenakshi Anurag, Bora Lim, Matthew Ellis. Distinct HER2 allele specific therapeutic response and preclinical efficacy of poziotinib in metastatic ER+ HER2 mutant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-24.

Abstract P4-01-02: A spectrum of secondary mutations in HER2 augment breast cancer cell growth and reduce neratinib sensitivity in HER2-mutant breast cancer

Abstract Activating mutations in HER2 (ERBB2) drive tumor growth and antiestrogen resistance in ~5% of metastatic ER+ breast cancers. Treatment with the irreversible HER2 tyrosine kinase inhibitor neratinib, in combination with fulvestrant, leads to objective responses in patients with HER2-mutant ER+ breast cancer. However, acquired resistance is common. The majority of HER2-mutant tumors progressing on neratinib-based therapy acquire secondary mutations in HER2. Except for the HER2T798I gatekeeper mutation, whether these secondary HER2 mutations are causal to neratinib resistance is not known. We hypothesized that secondary HER2 mutations augment HER2 pathway activation and promote neratinib resistance. We first studied acquired secondary HER2 mutations found in circulating tumor DNA (ctDNA) of 2 patients with breast cancer harboring the primary HER2L869R hotspot mutation. The following mutations were absent at baseline but detected in ctDNA at the time of progression on neratinib: S310Y, L755S, D769Y, and T798I (patient 1, partial response to neratinib/fulvestrant >16 months); and T862A, T798I, S310F, and I767M (patient 2, stable disease >7 months). We stably expressed each mutation alone or in combination with HER2L869R in MCF10A breast epithelial cells. All single mutations, with the exception of T862A and T798I, promoted growth factor-independent proliferation and growth in 3D Matrigel. Enhanced growth was blocked by treatment with 10 nM neratinib. Cells expressing HER2L869R together with S310F, I767M, and D769Y remained highly sensitive to neratinib (IC50 <6 nM). In contrast, HER2L869R/L755S and HER2L869R/T862A (double-mutant) cells formed large acini in 3D Matrigel in the presence of neratinib, and exhibited a 3- to 5-fold higher IC50 than HER2L869R in 2D cell viability assays. Western blot analysis revealed that HER2L869R/L755S and HER2L869R/T862A cells required a higher concentration of neratinib (50-100 nM; 4 h) to block phosphorylation of HER2 and downstream targets AKT, ERK, and S6 relative to cells transduced with HER2L869R alone (10 nM neratinib). While HER2T862A alone did not increase HER2 pathway activation or growth factor-independent growth compared to wild type (WT) HER2, phosphorylation of HER2, AKT, ERK, and S6 was elevated in cells expressing HER2L869R/T862A relative to each single-mutant. Likewise, cells expressing HER2L869R/T862A formed larger and more invasive acini in 3D Matrigel. Computational modeling predicted that the active state of HER2 was stabilized in the HER2L869R/T862A double-mutant relative to that of either single mutant. The overall energetic barrier for the active to inactive state transition also increased for the double mutant compared to that of each single mutant. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) simulations showed that neratinib binding affinity is higher for HER2L869R/T862Arelative to HER2L869R or HER2WT. Two secondary HER2 juxtamembrane domain mutations, E698del and P699L, were also acquired in HER2V777L-driven breast cancers that had progressed on neratinib. However, MCF10A cells co-transduced with V777L/E698del and with V777L/9669L remained highly sensitive to neratinib. Conclusions: In tumors with an oncogenic primary HER2 mutation, acquired secondary mutations in HER2L755S and HER2T862A reduced neratinib sensitivity. The HER2L869R/T862A double-mutant stabilizes the HER2 activation state and enhances HER2 signaling output which, in turn, was incompletely blocked by neratinib. Some but not all acquired secondary HER2 mutations enhance breast tumor growth and/or are causal to neratinib resistance. Citation Format: Arnaldo Andres Marin, Abdullah Al Mamun, Hiroaki Akamatsu, Dan Ye, Dhivya Sudhan, Benjamin Brown, Monica Red Brewer, Lisa Eli, Jens Meiler, Carlos L Arteaga, Ariella B Hanker. A spectrum of secondary mutations in HER2 augment breast cancer cell growth and reduce neratinib sensitivity in HER2-mutant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-02.

Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, oncogenesis, and HER2 inhibitor sensitivity.

Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against HER2-mutant tumors. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrate computational structural modeling with biochemical and cell biological analyses. Computational modeling predicts that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3 and reduces binding of HER2 to its inhibitor neratinib. Co-expression of mutant HER2/HER3 enhances HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which can be reversed by combined treatment with PI3Kα inhibitors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a poor response to neratinib in HER2-mutant cancers.

Abstract 1077: Acquired neratinib resistance is associated with acquisition of HER2 and PIK3CA mutations and can be overcome using potent drug combinations in HER2-positive breast cancer models

Abstract The role of HER2 and PIK3CA mutations in anti-HER2 resistance is gaining more importance in HER2-positive (+) breast cancer. We recently reported that acquired resistance to lapatinib (Lap)-containing regimens is mediated by HER2 L755S, which could be overcome using the irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (Nrb). However, less is known about the role of L755S in resistance to next-generation TKIs, particularly when co-occurring with PIK3CA mutations. HER2+ BT474 cell models with primary or sequential acquired resistance (R) to Lap (LapR) or Nrb (NrbR) and their parental (P) counterparts were profiled for alterations in signaling and gene expression by RPPA, western blot, and RNA-seq. For drug efficacy studies, change in cell growth was assessed using imaging-based high-throughput system. Proteomic profiling revealed partial restoration of HER2 phosphorylation and downstream signaling in the LapR and NrbR derivatives. RNA-seq analysis showed that the LapR and NrbR models, but not P cells, harbor HER2 L755S mutation. Importantly, the NrbR but not LapR cells also co-acquire a PIK3CA pathogenic mutation. GSEA analysis of RNA-seq data showed significant downregulation of G2/M checkpoint in the R derivatives compared to P cells, suggesting genetic instability. In line with the presence of HER2 and PIK3CA activating mutations and HER pathway reactivation in the R models, GSEA revealed an enrichment of mTORC1 and KRAS signaling in the R cells. Furthermore, enrichment of epithelial mesenchymal transition signature and downregulation of apical surface genes was observed in the R models compared to P cells, suggestive of their aggressive phenotype. Interestingly, the LapR cells remained sensitive to Nrb, though a higher dose (IC50: ~50nM) was required compared to P cells (IC50: ~2nM). The LapR and NrbR cells were cross-resistant to the HER2-selective TKI tucatinib, and trastuzumab. We recently showed that the NrbR cells were either partially or completely sensitive to poziotinib or TDM1, respectively, suggesting their therapeutic promise against HER2- and PIK3CA-mutant tumors. Of note, our studies using small molecule agents targeting HER and its downstream pathway to facilitate treatment of CNS lesions suggest that AKT or mutant PIK3CA inhibitors are effective only when combined with either neratinib or poziotinib, but not tucatinib, findings which we are currently expanding to xenograft-derived organoids. Overall, our findings suggest a complex disease evolution upon resistance to neratinib but indicate their potentially continued efficacy in overcoming resistance through drug combinations. Ongoing integrative omics analysis to determine the genomic and mutational complexity and landscape will uncover additional mechanistic insights and guide the discovery of other actionable targets. Citation Format: Jamunarani Veeraraghavan, Ragini Mistry, Sarmistha Nanda, Sreyashree Bose, Chia Chia Liu, Vidyalakshmi Sethunath, Martin J. Shea, Tamika Mitchell, Meenakshi Anurag, Michael A. Mancini, Irmina Diala, Alshad S. Lalani, Fabio Stossi, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. Acquired neratinib resistance is associated with acquisition of HER2 and PIK3CA mutations and can be overcome using potent drug combinations in HER2-positive breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1077.

How to Optimise Extended Adjuvant Treatment with Neratinib for Patients with Early HER2+ Breast Cancer.

Over the last 20 years, treatment of patients with human epidermal growth factor receptor 2-positive (HER2+) early breast cancer has considerably improved. The development and addition of (neo)adjuvant trastuzumab to chemotherapy in patients with early HER2+ breast cancer (EHBC) has been shown to provide improvements in both disease-free survival (DFS) and overall survival, with some patients having a good prognosis being candidates for chemotherapy de-escalation strategies. However, despite such promising clinical outcomes, a significant proportion of patients still recur calling for the development of new preventive approaches. To this aim, the use of (neo)adjuvant trastuzumab for longer than one year or followed by lapatinib were tested without additional clinical improvement. Based on more recent advances, therapeutic strategies for patients with HER2+ tumours are now incorporating the use of newer (neo)adjuvant treatments, such as pertuzumab and trastuzumab emtansine, which have shown to further improve the invasive DFS (iDFS) benefit gained with trastuzumab. In this context, the tyrosine kinase inhibitor neratinib is approved in Europe for the extended adjuvant treatment of adult patients with early-stage hormone receptor-positive HER2+ breast cancer who completed adjuvant trastuzumab-based therapy less than one year ago. Clinical data have demonstrated that neratinib significantly improves iDFS when used for the total recommended duration of 12 months. This review paper provides an overview of the treatment of patients with EHBC, with a focus on the post-trastuzumab use of neratinib.