pan-HER Inhibitor Research Articles

Targeting ErbB and tankyrase1/2 prevent the emergence of drug-tolerant persister cells in ALK-positive lung cancer

AbstractTargeting the drug tolerant persister (DTP) state in cancer cells should prevent further development of resistance mechanisms. This study explored combination therapies to inhibit alectinib-induced DTP cell formation from anaplastic lymphoma kinase–positive non-small cell lung cancer (ALK + NSCLC) patient–derived cells. After drug-screening 3114 compounds, pan-HER inhibitors (ErbB pathway) and tankyrase1/2 inhibitors (Wnt/β-catenin signaling) emerged as top candidates to inhibit alectinib-induced DTP cells growth. We confirmed knockdown of both TNKS1/2 in DTP cells recovered the sensitivity to alectinib. Further, our study suggested knockdown of TNKS1/2 increased stability of Axin1/2, which induced β-catenin degradation and decreased its nuclear translocation, thereby suppressing transcription of antiapoptotic and proliferation-related genes (survivin, c-MYC). Targeting both pathways with alectinib+pan-HER inhibitor and alectinib+TNKS1/2 inhibitor suppressed alectinib-induced DTP cells, and the triple combination almost completely prevented the appearance of DTP cells. In conclusion, combination with ALK-TKI, pan-HER and TNKS1/2 inhibitors has the potential to prevent the emergence of DTP in ALK + NSCLC.

Neratinib plus dasatinib is highly synergistic in HER2-positive breast cancer in vitro and in vivo

BackgroundHER2-targeted therapies have revolutionised the treatment of HER2-positive breast cancer. However, de novo resistance or the emergence of acquired resistance is a persistent clinical problem. Here we report that neratinib, an irreversible pan-HER inhibitor, in combination with the multi-kinase inhibitor dasatinib, currently used to treat certain leukemias, has strong anti-proliferative effects against models of HER2-positive breast cancer that are innately resistant to trastuzumab or have acquired resistance to neratinib. MethodsNeratinib plus dasatinib was examined in a panel of 20 breast cancer cell lines, including HER2-positive, estrogen-receptor-positive, triple negative, and acquired HER2-targeted therapy resistant models. Drug effects on migration and apoptosis induction was evaluated and signaling alterations were determined by reverse phase protein array (RPPA). In vivo efficacy was examined using orthotopically-implanted HCC1954 cells. ResultsSynergy was observed in cell lines innately resistant to trastuzumab, models with acquired resistance to neratinib, and in triple negative breast cancer cell lines. Further investigation showed that neratinib plus dasatinib induced apoptosis and inhibited cell migration to a greater degree than either drug alone. RPPA revealed that the combination caused suppression of key survival signaling through EGFR, Akt, and MAPK inhibition. In vivo, neratinib plus dasatinib was well tolerated and had a prolonged anti-tumor effect against HCC1954 xenografts. ConclusionsThis study provides a strong pre-clinical rationale for the clinical investigation neratinib and dasatinib in HER2+ breast cancer.

A retrospective study of first-line therapy and subsequent pyrotinib treatment in advanced lung adenocarcinoma with HER2 mutations.

HER2 is an infrequently mutated driver gene in non-small cell lung cancer (NSCLC). At present, there has been no comprehensive large-scale clinical study to establish the optimal first-line treatment strategy for advanced lung adenocarcinoma (LUAD) with HER2-Mutant. Besides that, the effectiveness and safety of pyrotinib, a pan-HER inhibitor, in the context of NSCLC are still undergoing investigation. In this study, we conducted a retrospective data collection of HER2-Mutated advanced LUAD who received first-line treatment and pyrotinib between May 2014 and June 2023. Patients treated with chemotherapy, chemotherapy + immune checkpoint inhibitors (ICIs), chemotherapy + bevacizumab and pyrotinib in first-line treatment. Furthermore, we collected data on the efficacy and safety of pyrotinib in these patients after disease progression. The main endpoint of the study was progression-free survival (PFS). In the final analysis, 89 patients were included in the first-line cohort and 30 patients were included in the pyrotinib cohort. In the first-line treatment cohort, chemotherapy + ICIs, chemotherapy + bevacizumab, and pyrotinib exhibited notable survival benefits compared to chemotherapy (median PFS: 9.87 vs. 7.77 vs. 7.10 vs. 5.40 months, p-value < 0.05). Furthermore, patients with a first-line treatment PFS of less than 6 months may potentially benefit from subsequent treatment with pyrotinib (median PFS: 7.467 vs. 3.000, p-value = 0.0490). In the first-line treatment of HER2-Mutant LUAD, regimens involving combinations like chemotherapy + ICIs, chemotherapy + bevacizumab, and pyrotinib may confer enhanced survival advantages compared to chemotherapy. Nevertheless, no significant distinctions were observed among these three treatment strategies, underscoring the imperative to identify biomarkers for the discerning selection of suitable therapeutic modalities. Moreover, patients with suboptimal response to first-line treatment may potentially derive more benefit from pyrotinib.

Phase 1b trial of anti-HER2 antibody inetetamab and pan-HER inhibitor pyrotinib in HER2-positive advanced lung cancer.

There remains an unmet need for targeted therapies against advanced non-small-cell lung cancer (NSCLC) with HER2 mutations. To improve the antitumor activity of single anti-HER2 agent, this prospective, single-arm clinical trial (NCT05016544) examined the safety profile and efficacy of anti-HER2 antibody inetetamab and pan-HER TKI pyrotinib in HER2-posivite advanced NSCLC patients. Enrolled patients received inetetamab every 3weeks and pyrotinib once per day (pyrotinib, dose-escalation part, 240mg, 320mg; dose-expansion part, 320mg). Primary endpoints were dose-limiting toxicity (DLT) dosage and safety. Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR). A total of 48 patients were enrolled. During the dose-escalation period, no DLT occurred. Diarrhea was the most commonly reported treatment-related adverse event (TRAE). Grade 3 TRAEs occurred in seven patients. The median PFS (mPFS) was 5.5months [95% confidence interval (CI): 4.4-8.6months]. The confirmed ORR and DCR reached 25% (11/44) and 84.1% (37/44), respectively. Responses were shown in patients with distinct HER2 aberrations. In summary, inetetamab in combination with pyrotinib demonstrated acceptable safety and antitumor activity among patients with advanced HER2-mutant NSCLC.

Neratinib could be effective as monotherapy or in combination with trastuzumab in HER2-low breast cancer cells and organoid models

BackgroundPrevious studies have suggested that patients with HER2-low breast cancers do not benefit from trastuzumab treatment although the reasons remain unclear.MethodsWe investigated the effect of trastuzumab monotherapy and its combination with different HER2 targeting treatments in a panel of breast cancer cell lines and patient-derived organoids (PDOs) using biochemical methods and cell viability assays.ResultsCompared to sensitive HER2 over-expressing (IHC3 + ) breast cancer cells, increasing doses of trastuzumab could not achieve IC50 in MDA-MB-361 (IHC 2 + FISH + ) and MDA-MB-453 (IHC 2 + FISH-) cells which showed an intermediate response to trastuzumab. Trastuzumab treatment induced upregulation of HER ligand release, resulting in the activation of HER receptors in these cells, which could account for their trastuzumab insensitivity. Adding a dual ADAM10/17 inhibitor to inhibit the shedding of HER ligands in combination with trastuzumab only showed a modest decrease in the cell viability of HER2-low breast cancer cells and PDOs. However, the panHER inhibitor neratinib was an effective monotherapy in HER2-low breast cancer cells and PDOs, and showed additive effects when combined with trastuzumab.ConclusionThis study demonstrates that neratinib in combination with trastuzumab may be effective in a subset of HER2-low breast cancers although further validation is required in a larger panel of PDOs and in future clinical studies.

TH-4000, a hypoxia-activated pan-HER inhibitor, shows excellent preclinical efficacy for the treatment of HER2+ breast cancer.

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is correlated with poor prognosis, the current treatment of which is still based on surgery and adjuvant targeted therapy with monoclonal antibody. Problems of drug resistance hinder the use of monoclonal antibodies. Subsequently, tyrosine kinase inhibitors (TKIs) have been noticed, TKIs have the advantages of multi-targets and reduced drug resistance. However, TKIs that target HER family proteins often cause adverse effects such as liver damage and diarrhea. Thus, TKIs with high selectivity are being developed. TH-4000, a prodrug that generated an active form TH-4000Effector (TH-4000E) under hypoxic condition, was evaluated in this research. We found that TH-4000E ([(E)-4-[[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino]-4-oxobut-2-enyl]-dimethyl-[(3-methyl-5-nitroimidazol-4-yl)methyl]azanium) (1-1000nM) had potent and highly selective toxic effects on HER2+ breast cancer cells and inhibited the phosphorylation of HER family kinases at lower doses than that of Lapatinib and Tucatinib. TH-4000E activated Caspase-3 and induced apoptosis through a reactive oxygen species (ROS)-dependent pathway. The prodrug TH-4000 ([(E)-4-[[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino]-4-oxobut-2-enyl]-dimethyl-[(3-methyl-5-nitroimidazol-4-yl)methyl]azanium;bromide) (50mg/kg) effectively suppressed the tumor growth with less liver damage in mouse tumor models. This hypoxia-targeted strategy has possessed advantage in avoiding drug-induced liver damage, TH-4000 could be a promising drug candidate for the treatment of HER2+ breast cancer.

Multicenter phase I dose escalation and expansion study of pyrotinib in combination with camrelizumab and chemotherapy as first-line treatment for HER2-positive advanced gastric and gastroesophageal junction adenocarcinoma

Pembrolizumab plus trastuzumab and chemotherapy showed remarkable efficacy as first-line therapy for advanced HER2-positive gastric cancer. Pyrotinib is an irreversible pan-HER inhibitor. This single-arm, open-label phase 1 dose-escalation (1a) and expansion (1b) study investigated camrelizumab, an anti-PD-1 antibody, plus pyrotinib and chemotherapy as first-line treatment for advanced HER2-positive gastric and gastroesophageal junction (G/GEJ) adenocarcinoma. Between June 2020 and June 2022, 41 patients with previously untreated HER2-positive locally advanced unresectable or metastatic G/GEJ adenocarcinoma were enrolled. In phase 1a, patients underwent a 3+3 escalating dose design, receiving oral pyrotinib (240mg, 320mg, or 400mg daily), intravenous camrelizumab (200mg), and CapeOX (oxaliplatin 130mg/m2 on day 1 and capecitabine 1000mg/m2 twice daily for two weeks) every 3 weeks until progression, intolerable toxicity or consent withdrawal. The recommended phase 2 dose (RP2D) of pyrotinib was determined and used in the phase 1b. The primary endpoints were the safety, maximum tolerated dose (MTD), RP2D, and confirmed objective response rate (ORR). This trial was registered with chictr.org, number ChiCTR2000029717. Among 41 patients, 10 were in phase 1a (3at 240mg, 3at 400mg, and 4at 320mg due to one patient withdrawing consent), and 31 were in phase 1b. In phase 1a, the MTD of pyrotinib was 320mg daily due to dose-limiting toxicities (diarrhea [n=3] and vomiting [n=1]) observed at 400mg. Based on all available data, the RP2D of pyrotinib was set at 320mg. Among 41 patients, 20 patients (48.8%) developed grade ≥3 treatment-emergent adverse events (TEAEs), and four patients (9.8%) had any grade serious adverse events. No deaths occurred due to TEAEs. Among 27 patients who received the RP2D of pyrotinib and had a post-baseline tumor assessment, two patients (7.4%) achieved a confirmed complete response, and 19 patients (70.4%) achieved a confirmed partial response, resulting in a confirmed ORR of 77.8% (95% CI: 57.7-91.4). Pyrotinib plus camrelizumab and chemotherapy showed promising efficacy in the first-line treatment of advanced HER2-positive G/GEJ cancer. The safety profile was consistent with known toxicities of the agents, and no new or unexpected safety signals were identified. This study was funded by the Beijing Xisike Clinical Oncology Research Foundation (Y-HR2019-0377).

Pyrotinib versus placebo in combination with trastuzumab and docetaxel as first line treatment in patients with HER2 positive metastatic breast cancer (PHILA): randomised, double blind, multicentre, phase 3 trial

ObjectiveTo assess the efficacy and safety of pyrotinib (an irreversible pan-HER (human epidermal growth factor receptor) inhibitor), trastuzumab, and docetaxel compared with placebo, trastuzumab, and docetaxel for untreated HER2 positive...

Afatinib in patients with recurrent/metastatic (RM) squamous cell carcinoma of the head and neck (SCCHN) harboring alterations in the HER pathway: Results of the B1 cohort of the EORTC-HNCG-1559 trial (UPSTREAM).

6034 Background: Afatinib (A) is an irreversible pan-HER inhibitor that improved PFS in second line RM SCCHN after platinum (LUX-H&amp;N1 trial). In a retrospective biomarker analysis, subgroups of pts with p16 negative, PTEN high or EGFR amplification SCCHN seem to benefit the most. Other studies suggest that A has activity in tumors with HER2 gene activation. Methods: The UPSTREAM trial is a biomarker-driven umbrella trial of targeted therapies and immunotherapy for RM SCCHN (post-platinum, ECOG 0-1, measurable disease). The B1 cohort was a phase II, randomized, open-label substudy evaluating the efficacy of afatinib (orally, 40mg/day) vs physician’s choice (ctrl) (2:1 ratio) in pts with p16 negative RM SCCHN harboring one of the following biomarkers (fresh biopsy): EGFR mutation(mut)/amplification(amp) or HER2 mut/amp or PTEN high by immunohistochemistry (IHC, H-score &gt; 150). Only KRAS/ HRAS/ NRAS wild type tumors were included. The primary endpoint was progression-free survival rate (PFSR) at 16 weeks after randomization. A 2-stage Simon optimal design was applied to arm A (H1 40%, H0 20%, 1-sided α 10%, power 90%) with 17 and 20 pts enrolled in the first and second stage respectively. Secondary endpoints included ORR, toxicity, PFS, and OS. Results: 59 RM SCCHN pts were included in the B1 cohort (A: n = 40, ctrl: n = 19). Out of the 59 patients, EGFR amp, HER2 amp and HER2 mut were found in 6 pts (10%), 1 pt (2%) and 1 pt (2%), respectively. PTEN (IHC) was high in 97%. 55 pts were evaluable (A: n = 38, ctrl: n = 17) (median age: 62, oral cavity: 29%, oropharynx: 36%, hypopharynx: 24%, larynx: 11%). 69% of pts had received ≥ 2 lines of systemic treatment for RM disease. 80% were pretreated with cetuximab. In arm A, 13/38 patients were alive and free of objective progression at 16 weeks (34.2%, 1-sided 90%CI, 23.9±inf), meeting the predefined criteria of success. The PFSR at 16W in ctrl arm was 29.4% (5/17). 4 objective responses were observed in arm A (3 PR and 1 CR) (ORR 11%, 95%CI 2.9-24.8%) and 1 PR in ctrl arm (6%). The median duration of response under A was 5.7 mo (range: 3.7-25.9 mo). All responding patients had previously been treated with cetuximab, were PTEN high but none had EGFR or HER2 alterations. The median PFS and OS were 2.2/2.4 mo and 7.2/5.0 mo in the A/ctrl arms, respectively. Grade ≥3 drug-related AEs were reported in 31% in arm A and 12% in ctrl arm. The most frequent drug-related AE with A were diarrhea (67%, G3:8%), rash (41%, G3: 3%), fatigue (31%, no G3) and mucositis (23%, G3: 3%). Conclusions: The B1 substudy met its primary endpoint of PFSR at 16 weeks. Responses were seen in cetuximab pre-treated pts. However, the clinical activity of A in this biomarker-selected population remains limited. Translational research is ongoing to better define potential biomarkers. Clinical trial information: NCT03088059 .

HER2 inhibition increases non-muscle myosin IIA to promote tumorigenesis in HER2+ breast cancers.

HER2 is over-expressed in around 15% to 20% of breast cancers. HER3 plays a critical role in HER2 mediated tumorigenesis. Increased HER3 transcription and protein levels occur upon inhibition of HER2. We aimed to identify proteins that bound to HER3 upon inhibition of the HER family with the pan-HER inhibitor neratinib in HER2+ breast cancer cells. Immunoprecipitation of HER3 followed by mass spectrometry experiments found non-muscle myosin IIA (NMIIA) increased upon neratinib treatment relative to vehicle DMSO treatment. MYH9 is the gene that encodes for the heavy chain of NMIIA. Breast cancer patients with high MYH9 were significantly associated with a shorter disease specific survival compared to patients with low MYH9 expression from the METABRIC cohort of patients. In addition, high MYH9 expression was associated with HER2+ tumors from this cohort. Immunoblots of whole cell lysates of BT474 and MDA-MB-453 HER2+ breast cancer cells demonstrated elevated HER3 and NMIIA protein levels upon neratinib treatment for 24 hours. To examine the role of NMIIA in HER2+ breast cancer, we modulated NMIIA levels in BT474 and MDA-MB-453 cells using doxycycline inducible shRNA targeting MYH9. MYH9 knockdown reduces HER3 protein levels and concomitant reduction in downstream P-Akt. In addition, loss of MYH9 suppresses cell growth, proliferation, migration, and invasion. Our data reveals that NMIIA regulates HER3 and loss of NMIIA reduces HER2+ breast cancer growth.

Abstract 6132: Impact of treatment with agents targeting different members of HER family, CDKs and downstream cell signaling molecules on growth and migration of stomach cancer cells

Abstract Stomach cancer ranks fifth and fourth for the incidence and mortality rate for cancer worldwide, respectively. In recent years, several inhibitors with specificity to one or more members of human epidermal growth factor receptor (HER) family have been approved for the treatment of patients with different cancer types. Of these, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients and with tumor heterogeneity being one contributing factor. In this study, we investigated the effect of various types of targeted agents on the growth in vitro of a panel of human stomach cancer cells (HSCCLs) and the impact of stomach cancer proliferation rate on the anti-tumor activities of these agents. Moreover, we investigated the cell surface expression of the HER family members, other biomarkers such as C-Met, Alk-7 and cancer stem cell makers CD44 and CD133 by flow cytometry and the effect various targeted agents on tumor migration using Incucyte. Of the 18 agents examined, the CDK 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at IC50 values between 1nM to 9nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual EGFR/HER2 TKI lapatinib and the EGFR specific TKI erlotinib in inhibiting the growth of HSCCLs. Interestingly, the HER-2 overexpressing cells lines NCI-N87 (Mean Fluorescence Intensity =596) was most sensitive to the HER inhibitors. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs and with the IC50 values ranging from 2nM to 7uM. Interestingly, many of these agents were more effective in inhibiting the growth of HSCCLs when proliferating at slower rate. Of the agent examined, neratinib, afatinib, dinacicilib, dasatinib, stattic and miransertib also inhibited the migration of stomach cancer cells. Finally, treatment with a combination of afatinib with dinaciclib, capmatinib, dasatinib, stattic, ponatinib or miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. As stomach cancer cells are heterogenous in nature, our results support further research on the therapeutic potential of the CDK dinaciclib in combination with a pan-HER inhibitor in stomach cancer. Citation Format: Tina Al-Janaby, Narmin Nahi, Said A. Khelwatty, Alan Seddon, Izhar Bagwan, Helmout Modjtahedi. Impact of treatment with agents targeting different members of HER family, CDKs and downstream cell signaling molecules on growth and migration of stomach cancer cells. [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 6132.

Abstract 3999: Combination of T-DXd with the irreversible pan-HER TKI afatinib drives combination benefit in HER2-low gastric and lung tumors

Abstract Background: Trastuzumab deruxtecan (T-DXd) has been approved for advanced or metastatic breast, gastric and lung cancers. It has been postulated that irreversible tyrosine kinase inhibitors (TKIs) increase HER2 internalization, thus enhancing T-DXd efficacy. Here we report studies evaluating the combination of TKIs with T-DXd in preclinical models of HER2-low disease. Methods: Surface HER2 was measured with flow cytometry quantifying fluorescently labelled trastuzumab on the surface of HER2-low cell lines HCC38 and HCC1171 treated with afatinib, neratinib, lapatinib or tucatinib for 12 h. HER2 receptor dynamics on cell surface was also measured in NCI-N87 (HER2 IHC 3+) and HCC38 at 24, 48, 72 and 96 h post-treatment with afatinib, and recovery was measured up to 72 h after drug washout. In vivo efficacy was established with T-DXd alone and in combination with afatinib or tucatinib in two gastric patient-derived xenograft (PDX) models, HD-2 (HER2 IHC 1+) and FU (HER2 IHC 2+), and one lung PDX model, MEDI-NSCLC-04 (HER2 IHC 1+). Results: In both HER2-low cell lines, treatment with irreversible inhibitors neratinib or afatinib led to enhanced internalization with a greater than 50% surface HER2 reduction, while treatment with the reversible inhibitors lapatinib or tucatinib led to 13-35% higher surface HER2 levels. Washout experiments indicated that receptor recovery at the plasma membrane begins between 4-12h post-washout of afatinib, with complete recovery at about 72h. Tumor shrinkage was significant in HER2-low gastric PDXs when T-DXd was combined with afatinib, but not with tucatinib. In HD-2, T-DXd 10mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 74% and -3% (with respect to T-DXd) tumor growth inhibition (TGI), respectively. In FU, T-DXd 3mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 80.3% and 24.7% TGI, respectively. Similarly, in MEDI-NSCLC-04 PDX model, benefit was observed in 5mg/kg T-DXd combination with 20mg/kg QD afatinib (95.3% TGI with respect to monotherapy T-DXd), but not with tucatinib (0.37% TGI). Intermittent schedules (QW, MWF) were also tested for afatinib in combination with T-DXd, with the MWF showing 49.2% TGI and QW showing 33.0 %TGI. In tissue samples from MEDI-NSCLC-04 efficacy study, γH2AX was detected by IHC in T-DXd and T-DXd+afatinib combination. In western blots, a decrease in HER2 as well as pHER2 was observed in response to T-DXd and 20mg/kg afatinib treatment, but not with tucatinib. Conclusions: In HER2 low gastric and lung preclinical models, T-DXd combined with irreversible pan-HER inhibitor induced superior antitumor effects compared to single agents or the combination of T-DXd with reversible inhibitors. These data support the rationale to test combination of T-DXd and pan-HER irreversible inhibitors in the clinical setting. Citation Format: Deepa Bhavsar, Laura Kazlauskas, Flavia Michelini, Matt Griffin, Matt Wilson, Fabiola Cecchi, Liz Croydon, Kim Maratea, Elisa de Stanchina, Yelena Y. Janjigian, Maurizio Scaltriti, Theresa Proia, Jerome Mettetal. Combination of T-DXd with the irreversible pan-HER TKI afatinib drives combination benefit in HER2-low gastric and lung tumors. [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 3999.

Pseudo-temporal dynamics of chemoresistant triple negative breast cancer cells reveal EGFR/HER2 inhibition as synthetic lethal during mid-neoadjuvant chemotherapy

In the absence of targetable hormonal axes, chemoresistance for triple-negative breast cancer (TNBC) often compromises patient outcomes. To investigate the underlying tumor dynamics, we performed trajectory analysis on the single-nuclei RNA-seq (snRNA-seq) of chemoresistant tumor clones during neoadjuvant chemotherapy (NAC). It revealed a common tumor trajectory across multiple patients with HER2-like expansions during NAC. Genome-wide CRISPR-Cas9 knock-out on mammary epithelial cells revealed chemosensitivity-promoting knock-outs were up-regulated along the tumor trajectory. Furthermore, we derived a consensus gene signature of TNBC chemoresistance by comparing the trajectory transcriptome with chemoresistant transcriptomes from TNBC cell lines and poor prognosis patient samples to predict FDA-approved drugs, including afatinib (pan-HER inhibitor), targeting the consensus signature. We validated the synergistic efficacy of afatinib and paclitaxel in chemoresistant TNBC cells and confirmed pharmacological suppression of the consensus signature. The study provides a dynamic model of chemoresistant tumor transcriptome, and computational framework for pharmacological intervention.

A small-molecule pan-HER inhibitor alone or in combination with cisplatin exerts efficacy against nasopharyngeal carcinoma.

The abnormal activation of HER family kinase activity is closely related to the development of human malignancies. In this study, we used HER kinases as targets for the treatment of nasopharyngeal carcinoma (NPC) and explored the anti-tumor effects of the novel pan-HER inhibitor HM781-36B, alone or in combination with cisplatin. We found that HER family proteins were positively expressed in tumor tissues of some NPC patients, and the high levels of those proteins were significantly related to poor prognosis. HM781-36B inhibited NPC in vitro and in vivo. HM781-36B exerted synergistic effects with cisplatin on inhibiting proliferation and promoting apoptosis of NPC cells. In NPC xenograft models in nude mice, HM781-36B and cisplatin synergistically inhibited tumor growth. Downregulating the activity of HER family proteins and their downstream signaling pathways and regulating tumor microenvironment may explain the synergistic anti-tumor effects of HM781-36B and cisplatin. In conclusion, our study provides evidence for HER family proteins as prognostic biomarkers and potential therapeutic targets for NPC. The pan-HER inhibitor HM781-36B alone or in combination with cisplatin represents promising therapeutic effects for the treatment of NPC patients, which provides a new idea for the comprehensive treatment of NPC.

180 (PB060) - Neratinib plus dasatinib has pre-clinical efficacy against HER2-positive breast cancer

Background: HER2-targeted therapies have revolutionised the treatment of HER2-positive breast cancer. However, de novo resistance or the emergence of acquired resistance is a persistent clinical problem. The aim of this study was to investigate neratinib, an irreversible pan-HER inhibitor, in combination with the multi-kinase inhibitor dasatinib, currently used to treat certain leukemias, as a new treatment option for patients with HER2-targetted therapy resistant HER2-positive breast cancer.

Pan-HER inhibitors overcome lorlatinib resistance caused by NRG1/HER3 activation in ALK-rearranged lung cancer.

Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) with a broad coverage against ALK mutations, has demonstrated dramatic effects in patients with ALK-rearranged lung cancer. The mechanisms of acquired resistance to lorlatinib by secondary ALK compound mutations have recently been reported; however, resistance mechanisms other than secondary mutations remain unclear. Here, we investigated the molecular mechanisms of the acquired resistance in ALK-rearranged lung cancer cells in vitro. We established two different lorlatinib-resistant ALK-rearranged lung cancer cell lines (H3122LR and A925LLR) via long-term administration of lorlatinib. These resistant cells did not harbor the secondary ALK mutations and showed cross-resistance to the other kinds of ALK-TKIs (crizotinib or alectinib) compared with the parental cells; however, these resistant cells overexpressed the phosphorylated human epidermal growth factor receptor 3 (HER3) protein and the ligand of HER3 (neuregulin 1; NRG1). Pharmacological inhibition of HER3 with pan-HER inhibitors or genetic knockdown of HER3 with siRNA resensitized H3122LR and A925LLR cells to lorlatinib in vitro, indicating that H3122LR and A925LLR acquired resistance by NRG1/HER3 activation. These findings demonstrated that targeting NRG1/HER3 is a potential novel therapeutic option for lorlatinib-resistant ALK-rearranged lung cancer.

An ultra-performance LC-MS/MS method for determination of JRF103 in human plasma: application in first in-patient study.

Background: JRF103, a novel pan-HER inhibitor, has shown potent activity against HER1, HER2, HER4 and EGFR in vitro. To support its first in-patient trial, a sensitive and rapid method was developed and validated using ultra-performance LC-MS/MS. Materials & methods: JRF103 was extracted from plasma using protein precipitation. Extracts were subjected to ultra-performance LC-MS/MS with electrospray ionization. Results: Separation of analyte was achieved using a 1.7-μm C18 column (2.1×50-mm internal diameter) with a gradient elution. The developed method was fully validated following the international guides. Conclusion: The developed method was sensitive, specific and suitable for measuring JRF103 concentration in patients with advanced solid tumors in the first in-patient study of JRF103.

LBA19 Pyrotinib or placebo in combination with trastuzumab and docetaxel for HER2-positive metastatic breast cancer (PHILA): A randomized phase III trial

Pyrotinib (pyro, an irreversible pan-HER inhibitor) plus capecitabine has demonstrated antitumor activity as 2L treatment in HER2-positive metastatic breast cancer (mBC) (Lancet Oncol 2021). Pertuzumab combined with trastuzumab (H) and docetaxel (T) is the standard 1L dual anti-HER2 therapy for HER2-positive mBC. Here, we report the efficacy and safety of dual anti-HER2 regimen of Pyro+H+T (PyroHT) compared with placebo+H+T (HT) in untreated HER2-positive mBC. Eligible pts were randomized (1:1) to receive oral Pyro (400 mg QD) or placebo, both combined with intravenous H (8 mg/kg in cycle 1 and 6 mg/kg in subsequent cycles) and T (75 mg/m2) on day 1 of each 21-day cycle. The primary endpoint was PFS per investigator (INV). Data cutoff for this prespecified interim analysis was May 25, 2022. Between May 2019 and Jan 2022, 590 eligible pts from 40 centers were randomized and treated (297 with PyroHT and 293 with HT). Median follow-up was 15.8 mo and 14.9 mo, respectively. PFS per INV of PyroHT was significantly longer than HT (median, 24.3 mo [95% CI 19.1-33.0] vs 10.4 mo [95% CI 9.3-12.3]; HR, 0.41 [95% CI 0.32-0.53]; P < 0.0001; subgroup results are in the Table), which was consistent with PFS per IRC (median, 33.0 vs 10.4 mo; HR, 0.35 [95% CI 0.27-0.46]). The most common grade ≥3 TRAEs were decreased neutrophil count (62.6% vs 65.2%), decreased WBC count (53.2% vs 50.9%), and diarrhea (46.5% vs 3.1%). Grade 3 diarrhea occurred mainly during cycle 1 and substantially decreased in cycle 2 and thereafter. No grade 4 or 5 diarrheas occurred. Serious TRAEs occurred in 24.9% vs 6.1% of pts and treatment-related deaths occurred in 0 vs 0.3% of pts, respectively.Table: LBA19Subgroup analyses of PFS per INVPyroHTHTHR (95% CI)n/NPFS (median, mo)n/NPFS (median, mo)ECOG performance status055/16427.596/15510.40.42 (0.30-0.59)144/13322.282/13810.30.39 (0.27-0.57)Prior neoadjuvant or adjuvant therapyYes49/14827.891/15310.60.43 (0.30-0.62)No50/14919.587/14010.30.38 (0.26-0.54)Prior neoadjuvant or adjuvant trastuzumabYes11/46NA32/429.30.23 (0.12-0.46)No88/25121.9146/25110.40.45 (0.34-0.59)Treatment-free interval with prior adjuvant therapy≥12 - <24 mo8/30NA25/388.30.22 (0.10-0.50)≥24 mo34/9922.258/10013.00.57 (0.37-0.87)n/N=events/patients. HRs are from unstratified analyses. Open table in a new tab n/N=events/patients. HRs are from unstratified analyses. PyroHT significantly prolonged PFS over HT in pts with HER2-positive mBC. The safety is manageable. To our knowledge, this is the second phase 3 trial to demonstrate PFS benefit from dual HER2 inhibition in 1L treatment of HER2-positive mBC.

Abstract 5349: Single-cell analysis sheds light on the resistance mechanisms of a novel pan-HER inhibitor, pyrotinib, in non-small cell lung cancer

Abstract Recently, a novel oral, irreversible pan-HER tyrosine kinase inhibitor, pyrotinib, has shown anticancer effects on HER2-mutant non-small cell lung cancer (NSCLC) patients, but the explicit responses of lung cancer cells towards pyrotinib treatment are barely investigated. In our current study, we employed single-cell RNA sequencing to profile the longitudinal transcriptomic dynamics of lung cancer cells during the pyrotinib treatment, unraveling the potential resistance mechanisms. We treated a lung adenocarcinoma cell line, H358, with pyrotinib at doses of 100nM, 200nM, 500nM, and 1000nM every four days, respectively. Then, the untreated cells (P0) as well as longitudinal samples after each administration (P4, P8, P12, P16) were harvested for further analysis. After quality control, a total of 41,804 cells were sequenced and included for further analysis. The differentially expressed genes were determined using MAST and the differentially enriched cancer hallmark signatures based on single-sample Gene Set Variation Analysis (ssGSVA) were identified. The single-cell data revealed that the untreated cells (Sample P0) and persistent cells (Sample P16) had stable and distinct gene expression patterns, while cells during the course of treatment shared similar expression features at a transitional state. Several cancer driver genes were found to be aberrantly regulated during the administration, like EPHA2, CDKN2A, CDK4, PTEN, and MYC. Also, some pathways related to immune response were suppressed after pyrotinib treatment, including interferon alpha response, interferon gamma response, TNFA signaling via NF-κB, and inflammatory response. Intriguingly, CD274 (the gene encoding PD-L1) and LGALS9 were found to significantly decrease after pyrotinib treatment, suggesting that pyrotinib may be associated with outcomes of immunotherapy. With the large cell numbers analyzed, we distinguished an isolated, small subpopulation of cells characterized by the enriched expression of MGP, COL1A1, COL1A2, SPARC, and COL3A1 along with the elevated EMT and angiogenesis scores, suggesting its critical role in pyrotinib resistance. Collectively, our study for the first time reveals the transcriptomic evolution of NSCLC cells in response to pyrotinib administration at the single-cell resolution, providing new insights into potential mechanisms of resistance to this novel regimen in NSCLC. Citation Format: Xinfeng Wang, Yuan Li, Runsen Jin, Nan Sun, Jie He. Single-cell analysis sheds light on the resistance mechanisms of a novel pan-HER inhibitor, pyrotinib, in non-small cell lung cancer [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 5349.

Abstract 1182: Molecular landscape and ErbB family signaling in NRG fusion NSCLC: Therapeutic implications for pan-ErbB family inhibitors

Abstract NRG1 gene fusions are rare clinically actionable somatic alterations identified in 0.1% - 0.2% of all tumors. Currently, there are no approved targeted therapies for patients with NRG1 fusion-positive cancer. Previous studies have demonstrated that NRG1 fusions signal through ErbB/HER family members and that HER2 inhibition has anti-tumor activity in NRG1 fusion driven cancers. However, NRG1 binds HER4 in addition to HER3, and the role of individual HER proteins and NRG1 fusion partners have not been fully elucidated. We hypothesized that HER2 and HER4 signaling may have important roles in NRG1 fusion driven cancers, and pan-HER targeting therapies may be more effective for NRG1 fusion positive cancers than HER2-specific inhibitors alone. To test this hypothesis, we investigated the frequency of the NRG1 fusions across cBioPortal and AACR project GENIE datasets and MD Anderson’s GEMINI databases and found that the incidence of NRG1 fusions in all tumor types was 0.1%. NRG1 fusions were observed in 3.3% of patients with cervical cancers and in 1.8% of breast cancers and 1.3% of non-small cell lung cancers. The most common fusion partner was CD74 (9%) and the second most common fusion partner was SLC3A2 (4%). To determine the role of ErbB signaling in NRG1-fusion positive cancers, we engineered Ba/F3 cells to express various ErbB/HER family members and the most common NRG1 fusion partners. Then, we evaluated different strategies of targeting NRG1-fusion positive cell lines using drugs that predominantly target EGFR, HER2, EGFR/HER2, dimerization, or drugs that are pan-HER inhibitors. In Ba/F3 cells, poziotinib, a pan-HER TKI, showed potent activity against cells harboring NRG1 fusions, with IC50 values of 0.19 - 0.36 nM and relative IC50 values (IC50 value of NRG1 fusion/IC50 value of WT EGFR) of 0.04 – 0.07. TKIs targeting predominately EGFR/HER2 (afatinib, dacomitinib and neratinib) we observed modest activity with IC50 values of 6.8 – 42 nM and relative IC50 values of 0.11 – 0.68. The IC50 values of HER2-specific TKIs (58 – 122 nM) were higher than EGFR/HER2 targeting TKIs, but the relative IC50 values (0.005 – 0.015) were lower than poziotinib. Among antibodies, targeting HER2 dimerization with pertuzumab had better single agent activity (IC50 values of 95 – 156 μg/ml) than trastuzumab which had no in vitro activity. Lastly, an additive effect was seen when trastuzumab was used in combination with pertuzumab, with IC50 values of 0.79 – 1.0 μg/ml, and poziotinib with trastuzumab and pertuzumab further potentiated activity. Taken together, these data suggest that pan-HER inhibition with agents such as poziotinib alone and in combination with inhibition of dimerization appears more effective than selective EGFR/HER2 inhibition and highlights the potential of combining agents with multiple mechanisms of HER family targeting. This pan-HER strategy merits further investigation in the clinic. Citation Format: Hibiki Udagawa, Jacqulyne P. Robichaux, Yasir Y. Elamin, Junqin He, Monique B. Nilsson, John V. Heymach. Molecular landscape and ErbB family signaling in NRG fusion NSCLC: Therapeutic implications for pan-ErbB family inhibitors [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 1182.