HER2-mutant Cancer Research Articles

Evaluation of the safety, pharmacokinetics, and efficacy of JSKN003 in patients with advanced solid tumors: A phase I/II clinical study.

3031 Background: JSKN003 is a bispecific HER2-directed antibody-drug conjugate (ADC) conjugated to a topoisomerase I inhibitor via a dibenzocyclooctyne tetrapeptide linker on the glycan of a humanized bispecific antibody. Pre-clinical studies showed that JSKN003 had a good serum stability, that may lead to a broader therapeutic window. Methods: JSKN003-102 (NCT05744427) is a phase I (dose escalation and dose expansion) and phase II (cohort expansion) study in Chinese patients (pts) with advanced solid tumors. Pts (ECOG PS 0-1) with HER2-expressing (IHC ≥ 1+) or HER2-mutant cancers who failed prior systemic therapies were recruited and received JSKN003 monotherapy intravenously Q3W. The objectives were safety, MTD or RP2D, pharmacokinetics, and preliminary antitumor activity. Here the results from phase I were reported. Results: As of 5th Jan 2024, 46 pts (25 breast cancers, 11 gastric cancers, 8 colorectal cancers, 1 lung cancer, and 1 ovarian cancer) were enrolled and received JSKN003 across 6 dose levels, including 2.1 (n=1), 4.2 (n=10), 5.2 (n=14), 6.3 (n=15), 7.3 (n=3), and 8.4 mg/kg (n=3), Q3W, in phase I period. Among the 46 pts, 18 were IHC 3+, 21 were IHC 2+, and 7 were IHC 1+. Most pts (73.9%) received ≥ 3 prior lines of therapy, including 60.9% and 45.6% of pts received prior anti-HER2 and anti-HER2 ADC therapy, respectively. The median duration of treatment was 13.1 (range, 2.1 - 42.4) weeks, and 37 pts (80.4%) remained on treatment. Treatment-related adverse events (TRAEs) occurred in 44 pts (95.7%), and the common, mostly grade 1 and 2, were diarrhea (37.0%) and nausea (32.6%). Only 6 pts (13.0%) experienced grade ≥3 TRAEs, and the common were lymphopenia (4.3%) and neutropenia(4.3%). 1 pt (2.2%) had treatment related SAE (nausea, grade 3). No pts experienced DLT or interstitial lung disease, and no TRAE led to death or discontinuation. Following a single dose, exposures (Cmax and AUC) of JSKN003 increased proportionally over a dose range of 4.2 mg/kg to 6.3mg/kg. T1/2 of JSKN003 is approximately 3-5 days. No significant accumulation was observed after 4 cycles treatment. The exposure of released payload was very low, demonstrating the stability of the JSKN003 in circulation. 37 pts had at least one post-baseline tumor assessment. The ORR and DCR was 51.4% (95%CI: 34.4, 68.1) and 91.9% (95%CI: 78.1, 98.3), respectively. The ORR in pts with HER2 IHC 1+, 2+ and 3+ was 20.0% (95% CI: 0.5, 71.6), 33.3% (95% CI: 11.8, 61.6), and 76.5% (95% CI: 50.1, 93.2), respectively. For pts who received prior anti-HER2 ADC, the ORR was 57.9% (95% CI: 33.5, 79.7). For HER2 positive breast cancer and gastric cancer, the ORR was 66.7% (95% CI: 38.4, 88.2) in 15 pts and 100% (95% CI: 39.8, 100) in 4 pts, respectively. Conclusions: MTD of JSKN003 was not reached yet. And safety of JSKN003 was extremely excellent with encouraging preliminary antitumor activity in heavily pretreated pts with advanced solid tumors. Clinical trial information: NCT05744427 .

Abstract CT179: Safety and efficacy of JSKN003 in patients with advanced/metastatic solid tumors: A first-in-human, dose-escalation, multicenter, open-label, phase I study

Abstract Background: JSKN003 is a bispecific HER2-directed antibody-drug conjugate (ADC) conjugated to a topoisomerase I inhibitor (TOP1i) via a dibenzocyclooctyne tetrapeptide linker on the glycan of a humanized bispecific antibody. Pre-clinical studies showed that JSKN003 had a good serum stability and strong anti-tumor activity. Hence JSKN003 could be effective in HER2 expressing tumors with a potentially broader therapeutic window. Methods: JSKN003-101 is a first-in-human, dose-escalation and -expansion study in Australian patients (pts) with advanced/metastatic solid tumors. Pts (ECOG PS 0-1) with histologically documented HER2-expressing (IHC ≥ 1+) or pts with HER2-mutant cancers who failed prior systemic therapies were recruited and received JSKN003 monotherapy intravenously Q3W. The objectives were safety, tolerability, maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D), pharmacokinetics (PK), and preliminary antitumor activity. Here the results from dose-escalation part were reported. Results: As of 15th December 2023, 32 pts (15 breast cancers, 5 ovarian cancers, 3 bladder cancers, 2 esophageal cancers, 2 non-small cell lung cancers, 1 gastric cancer, 1 head and neck cancer, and 3 other cancers) were enrolled and received JSKN003 across 7 dose levels (1.0, 2.1, 4.2, 5.2, 6.3, 7.3, and 8.4 mg/kg, Q3W). Among the 32 pts, 9 were IHC 1+, 16 were IHC 2+, and 7 were IHC 3+. Most pts (22/32, 68.8%) received ≥ 3 prior lines of therapy. The median duration of treatment was 15.8 (range, 6-54) weeks, and 19 pts (59.4%) remain on treatment. Treatment-related adverse events (TRAEs) occurred in 27 pts (84.4%) and 2 pts (6.3%) in 4.2mg/kg and 7.3mg/kg experienced grade 3 TRAE (anemia and fatigue); the most common TRAEs were diarrhea (62.5%) and nausea (50.0%),which were all grade 1-2. Only 1 pt experienced interstitial lung disease (7.3mg/kg, Grade 2). To date, no DLT events were found yet, and no TRAE led to death or treatment discontinuation. MTD has not been reached in 8.4 mg/kg.Following a single dose, exposures (Cmax and AUC) of JSKN003 and released TOP1i increased proportionally over a dose range of 4.2 mg/kg to 8.4 mg/kg. T1/2 of JSKN003 is approximately 5-6 days for 6.3 mg/kg and higher doses and increases with dose escalation. Accumulation of JSKN003 was minimal within the range of 1.1-1.5. The exposure of released payload was significantly lower than JSKN003 ADC, demonstrating the stability of the JSKN003 in circulation. All pts had at least one post-baseline tumor assessment. 16 pts achieved partial response as per RECIST 1.1 by investigator. The objective response rate (ORR) and disease control rate (DCR) was 50.0% (95%CI: 31.9%, 68.1%) and 90.6% (95%CI: 75.0%, 98.0%), respectively. The ORR in pts with IHC 1+, 2+ and 3+ was 55.6% (5/9), 37.5% (6/16), and 71.4% (5/7), respectively. As for the efficacy of the HER2+ BC and HER2-low BC, the ORR was 80% (4/5) and 40.0% (4/10), respectively. Conclusions: JSKN003 was well tolerated with encouraging preliminary antitumor activity in heavily pretreated pts with advanced/metastatic solid tumors, especially in pts with HER2 amplified tumors. As of the cut-off date, no DLT was observed, MTD has not been reached yet. Clinical trial information: NCT05494918 Citation Format: Claire Beecroft, Bo Gao, John Park, Kate Wilkinson, Karl Zhang, Xiangyun Yan, Yuan Lv. Safety and efficacy of JSKN003 in patients with advanced/metastatic solid tumors: A first-in-human, dose-escalation, multicenter, open-label, phase I study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT179.

Targeting HER2-mutant metastatic cervical cancer with neratinib: Final results from the phase 2 SUMMIT basket trial

ObjectiveHER2 mutations are associated with poor prognosis and are detected in 3–6% of cervical cancers. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, had activity in several HER2-mutant cancer types in the phase 2 SUMMIT basket study. We present updated and final results from the cervical cancer cohort of SUMMIT. MethodsEligible patients had HER2-mutant, metastatic or recurrent cervical cancer progressing after platinum-based treatment for advanced/recurrent disease. Patients received neratinib 240 mg/day; loperamide was mandatory during cycle 1. Confirmed objective response rate (ORR) was the primary endpoint. Duration of response (DoR), clinical benefit rate (CBR), progression-free survival (PFS), and safety were secondary endpoints. ResultsTwenty-two patients were enrolled; 18 (81.8%) had endocervical adenocarcinoma; median two prior systemic chemotherapy regimens (range 1–4). The most common HER2 variant was S310F/Y mutation (n = 13; 59.1%). Four patients had confirmed partial responses (ORR 18.2%; 95% CI 5.2–40.3); 6 had stable disease ≥16 weeks (CBR 45.5%; 95% CI 24.4–67.8). Median DoR was 7.6 months (95% CI 5.6–12.3). Median PFS was 5.1 months (95% CI 1.7–7.2). All-grade diarrhea (90.9%), nausea (54.5%), and constipation (54.5%) were the most common adverse events. Five patients (22.7%) reported grade 3 diarrhea. There were no grade 4 adverse events, no diarrhea-related treatment discontinuations, and two grade 5 adverse events, unrelated to neratinib: dyspnea (n = 1) and embolism (n = 1). ConclusionsNeratinib resulted in durable responses and disease control in patients with HER2-mutant metastatic/recurrent cervical cancer in SUMMIT. These findings support next-generation sequencing and tailored therapy for select patients with advanced cervical cancer. All responses occurred in patients with endocervical adenocarcinoma. Further assessment of neratinib in this setting is warranted.Trial registration number.NCT01953926 (ClinicalTrials.gov), 2013-002872-42 (EudraCT).

HER2-Targeted Therapy Shows Promise across Tumor Types.

The antibody-drug conjugate trastuzumab deruxtecan shows promising activity across multiple types of HER2-expressing advanced solid tumors, including traditionally hard-to-treat malignancies, according to data from the DESTINY-PanTumor02 trial. The ongoing study may pave the way for approval of a tumor-agnostic therapy for HER2-expressing and HER2-mutated cancers.

Efficacy of anti-HER2 drugs in the treatment of patients with HER2-mutated cancers: a systematic review and meta-analysis.

Anti-human epidermal growth factor receptor-2 (anti-HER2) therapy has shown excellent efficacy in patients with HER2 overexpression and amplification. Although HER2 mutations are rarely expressed in several cancers, when they occur, they can activate the HER2 signaling pathway. In recent years, studies have shown that anti-HER2 drugs have promising efficacy in patients with HER2 mutations. Based on keywords, we searched databases, such as PubMed, Embase, and Cochrane Library, and the main conference abstracts. We extracted data on objective response rate (ORR), clinical benefit rate (CBR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS) from studies on the efficacy of anti-HER2 therapies in patients with HER2-mutated cancers, and analyzed grade 3 or higher adverse events (AEs). We included 19 single-arm clinical studies and 3 randomized controlled trials (RCTs), containing a total of 1017 patients with HER2 mutations, involving seven drugs and nine cancers, and 18 studies enrolled a high proportion of heavily pretreated patients who had received multiple lines of therapy. Our results showed pooled ORR and CBR of 25.0% (range, 3.8-72.7%; 95% CI, 18-32%) and 36.0% (range, 8.3-63.0%; 95% CI, 31-42%) for anti-HER2 therapy in HER2-mutated cancers. The pooled median PFS, OS, DOR were 4.89 (95% CI, 4.16-5.62), 12.78 (95% CI, 10.24-15.32), and 8.12 (95% CI, 6.48-9.75) months, respectively. In a subgroup analysis, we analyzed the ORR for different cancers, showing 27.0, 25.0, 23.0, and 16.0% for breast, lung, cervical, and biliary tract cancers, respectively. ORR analyses were performed for different drugs as monotherapy or in combination, showing 60.0% for trastuzumab deruxtecan (T-DXd), 31.0% for pyrotinib, 26.0% for neratinib combined with trastuzumab, 25.0% for neratinib combined with fulvestrant, 19.0% for trastuzumab combined with pertuzumab, and 16.0% for neratinib. In addition, we found that diarrhoea, neutropenia, and thrombocytopenia were the most common grade ≥ 3 AEs associated with anti-HER2 therapeutic agents. In this meta-analysis of heavily pretreated patients with HER2 mutations, anti-HER2 therapies, DS-8201 and trastuzumab emtansine, showed promising efficacy and activity. Anti-HER2 therapies showed different efficacies in different or the same cancer settings and all had a tolerable safety profile.

559P Neratinib in HER2-mutant, recurrent/metastatic cervical cancer (R/M CC): Updated findings from the phase 2 SUMMIT basket trial

Somatic HER2 mutations are oncogenic drivers in 3–6% of CC. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has demonstrated single-agent activity in multiple HER2-mutant cancers [Hyman et al. Nature 2018], including HER2-mutant R/M CC [Oaknin et al. doi.org/10.1016/j.ygyno.2020.07.025]. We describe updated findings from the HER2-mutant R/M CC cohort from SUMMIT (cut-off date: Mar 18, 2022). Patients (pts) with R/M CC and HER2 mutations documented by local testing received oral neratinib 240 mg once daily with up to 2 cycles of mandatory loperamide prophylaxis. Tumor response was assessed by investigators using RECIST 1.1 and/or PET Response Criteria. Endpoints: confirmed objective response rate (ORR); duration of response; clinical benefit rate (CBR); progression-free survival (PFS); safety. Clinicaltrials.gov: NCT01953926. As of Mar 18, 2022, 22 pts with HER2-mutant R/M CC were enrolled (adenocarcinoma, 82%; squamous cell carcinoma, 18%). Single HER2 mutations: S310F/Y (n=10); R678Q (n=2); D769H/N (n=2); other (n=4). Four pts had 2 HER2 mutations, 3 of which included S310F/Y. Prior treatments included platinum-based chemotherapy (100%), bevacizumab (73%), and pembrolizumab (18%). Median duration of neratinib therapy was 3.7 months (range 0.5–64.7 months). Efficacy results (efficacy evaluable population) are shown in the Table. ORR was 18% (95% CI 5–40%) and CBR was 46% (95% CI 24–68%). Median PFS was 5.1 months (95% CI 1.7–7.2 months). Prior treatment with bevacizumab did not appear to influence outcomes. Diarrhea was the most commonly reported adverse event (all grade, 90.9%; grade 1, 31.8%; grade 2, 36.4%, grade 3, 23%; grade 4, 0%). No patients discontinued treatment due to diarrhea.Table: 559PEndpointPrior bevacizumabTotal (N=22)Yes (n=16)No (n=6)Confirmed objective response, n (%)3 (19)1 (17)4 (18)CR1 (6)01 (5)PR2 (13)1 (17)3 (14)ORR, % (95% CI)19 (4–46)17 (0–64)18 (5–40)Median DoR, months (95% CI)5.9 (5.6–9.3)12.3 (NE)7.6 (5.6–12.3)Clinical benefit, n (%)7 (44)3 (50)10 (46)CR1 (6)01 (5)PR2 (13)1 (17)3 (14)SD (≥16 weeks)4 (25)2 (33)6 (27)Clinical benefit rate, % (95% CI)44 (20–70)50 (12–88)46 (24–68)Median PFS, months (95% CI)4.4 (1.7–7.2)5.5 (1.7–20.1)5.1 (1.7–7.2)CBR, CR + PR + SD ≥16 weeks; CR, complete response; DoR, duration of response; NE, not estimable; ORR, objective response rate; PFS, progression-free survival; PR, partial response; SD, stable disease Open table in a new tab CBR, CR + PR + SD ≥16 weeks; CR, complete response; DoR, duration of response; NE, not estimable; ORR, objective response rate; PFS, progression-free survival; PR, partial response; SD, stable disease These encouraging results support the clinical benefit of neratinib in this patient population and warrant further investigation following platinum failure.

Hitting the target in HER2 mutant cancers.

Hitting the target in HER2 mutant cancers.

Computational and Functional Analyses of HER2 Mutations Reveal Allosteric Activation Mechanisms and Altered Pharmacologic Effects.

This study identified new oncogenic HER2 allosteric mutations, including ECD mutations that share covalent dimerization as a mechanism of oncogenicity, suggesting the need for novel inhibitors to treat HER2-mutant cancers.

CHMFL-26 is a highly potent irreversible HER2 inhibitor for use in the treatment of HER2-positive and HER2-mutant cancers.

Oncogene HER2 is amplified in 20%-25% of human breast cancers and 6.1%-23.0% of gastric cancers, and HER2-directed therapy significantly improves the outcome for patients with HER2-positive cancers. However, drug resistance is still a clinical challenge due to primary or acquired mutations and drug-induced negative regulatory feedback. In this study, we discovered a potent irreversible HER2 kinase inhibitor, CHMFL-26, which covalently targeted cysteine 805 of HER2 and effectively overcame the drug resistance caused by HER2 V777L, HER2 L755S, HER2 exon 20 insertions, and p95-HER2 truncation mutations. CHMFL-26 displayed potent antiproliferation efficacy against HER2-amplified and mutant cells through constant HER2-mediated signaling pathway inhibition and apoptosis induction. In addition, CHMFL-26 suppressed tumor growth in a dose-dependent manner in xenograft mouse models. Together, these results suggest that CHMFL-26 may be a potential novel anti-HER2 agent for overcoming drug resistance in HER2-positive cancer therapy.

Clinical Activity of Anti-HER2-Antibody Drug Conjugates in HER2-Mutated Metastatic Cancer Patients

Background/Objective: Antibody drug conjugates (ADCs) deliver potent cytotoxic therapy in a highly targeted manner by binding cancer-specific cell surface antigens. HER2 is a receptor tyrosine kinase that is commonly amplified in breast and gastric cancers, but more recently has been shown to harbor gain-of-function activating mutations in several cancer types. Recent observations have suggested that HER2-ADCs may be viable therapies for patients that harbor these activating mutations. This research retrospectively explores the efficacy of HER2-ADCs in metastatic cancer patients in a real-world setting.
 Methods: Patient information was gathered from the Precision Genomics program at IU Health, narrowed for specific mutation criteria (HER2 pathogenic mutation), cross referenced with OncoKB to legitimize oncogenicity, and selected for patients prescribed HER2-ADCs. For these patients, pre-treatment and mid-treatment CT-scans were analyzed following RECIST protocols. The primary outcomes were overall response rate (ORR), clinical benefit rate (CBR), and progression free survival (PFS). Secondary outcomes were to tabulate the frequency and clinical characteristics of HER2-mutated tumors.
 Results: Out of 517 patients with somatic HER2 mutations, 60 patients had a pathogenic HER2 mutation. Of these 60 patients, the most common tumor types were 26.67% Breast and 11.67% Bladder/Urothelial. 11 of 60 patients were prescribed a HER2-ADC (Trastuzumab Emtansine = 10, Trastuzumab Deruxtecan = 1). 8 of 11 patients were evaluable for response with RECIST criteria with 1 patient having a partial response, 4 patients having stable disease and 3 patients having progressive disease. ORR=13%, CBR=63%, Median PFS = 2.77 months (95% CI: 2.15-3.39 months).
 Conclusion/Implications: To our knowledge, this is the first report of HER2-ADCs demonstrating clinical activity in HER2-mutated cancers across tumor types. Further clinical trials are ongoing that will validate these initial findings.

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.

Co-Occurring Gain-of-Function Mutations in <i>HER2</i> and <i>HER3</i> Modulate HER2/HER3 Activation, Breast Cancer Progression, 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. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrated computational structural modeling with biochemical and cell biological analyses. Computational modeling predicted that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3, stabilizes the HER2 active conformation, and reduces binding of HER2 to its inhibitor neratinib, potentially explaining the lower clinical response of HER2/HER3 co-mutant tumors. Co-expression of mutant HER2 and HER3 enhanced 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 was reversed by treatment with PI3Kα inhibitors.SignificanceThe HER2 TKI neratinib has shown clinical activity against cancers harboring HER2 activating mutations. However, not all HER2-mutant tumors respond, suggesting that co-occurring mutations may limit sensitivity. We found that co-occurring HER3 mutations resulted in reduced sensitivity to HER2 TKIs and HER2 antibodies by disrupting neratinib binding and preventing the dissociation of HER2/HER3, respectively. Cells expressing HER2/HER3 mutations were sensitive to the combination of a HER2 TKI and a PI3Kα inhibitor, suggesting a potential therapeutic strategy against these refractory tumors. 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 lesser response to neratinib in HER2-mutant cancers.

Neratinib in patients with HER2-mutant, metastatic cervical cancer: Findings from the phase 2 SUMMIT basket trial

ObjectiveSomatic HER2 mutations occur in ~5% of cervical cancers and are considered oncogenic and associated with poor prognosis. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, is active in multiple HER2-mutant cancers. SUMMIT is a phase II basket trial investigating the efficacy and safety of neratinib in solid tumors. MethodsPatients with HER2-mutant, persistent, metastatic/recurrent cervical cancer with disease progression after platinum-based treatment for advanced/recurrent disease received oral neratinib 240 mg/day with mandatory loperamide prophylaxis during cycle 1. The primary endpoint was confirmed objective response rate (ORR). Secondary endpoints included: response duration (DOR); clinical benefit rate (CBR); progression-free survival (PFS); overall survival (OS); safety. ResultsSixteen eligible patients were enrolled; 10 (62.5%) had endocervical adenocarcinoma. The most common HER2 mutation was S310F (63% of patients). Three of 12 RECIST-measurable patients had confirmed partial responses (ORR 25%; 95%CI 5.5–57.2%); 3 had stable disease ≥16 weeks (CBR 50%; 95%CI 21.1–78.9%). DOR for responders were 5.6, 5.9, and 12.3 months. Median PFS was 7.0 months (95%CI 0.7–18.3 months); median OS was 16.8 months (95%CI 4.1–NE months). Diarrhea (75%), nausea (44%), and decreased appetite (38%) were the most common adverse events. One patient (6%) reported grade 3 diarrhea. There were no grade 4 events, and no diarrhea-related treatment discontinuations. ConclusionsNeratinib monotherapy showed evidence of activity in heavily pretreated patients with HER2-mutant cervical cancer, with no new safety signals. Given the few effective options for cervical cancer after platinum-based therapy failure, neratinib warrants further investigation in this molecularly defined patient population.Trial registration number.NCT01953926 (ClinicalTrials.gov), 2013–002872-42 (EudraCT).

Hyperactivation of TORC1 Drives Resistance to the Pan-HER Tyrosine Kinase Inhibitor Neratinib in HER2-Mutant Cancers

We developed neratinib-resistant HER2-mutant cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Primary and acquired neratinib resistance in HER2-mutant breast cancer patient-derived xenografts (PDXs) was also associated with TORC1 hyperactivity. Genetic suppression of RAPTOR or RHEB ablated P-S6 and restored sensitivity to the tyrosine kinase inhibitor. The combination of the TORC1 inhibitor everolimus and neratinib potently arrested the growth of neratinib-resistant xenografts and organoids established from neratinib-resistant PDXs. RNA and whole-exome sequencing revealed RAS-mediated TORC1 activation in a subset of neratinib-resistant models. DNA sequencing of HER2-mutant tumors clinically refractory to neratinib, as well as circulating tumor DNA profiling of patients who progressed on neratinib, showed enrichment of genomic alterations that converge to activate the mTOR pathway.

Abstract A046: Pre-clinical assessment of neratinib sensitivity and biomarkers of response

Abstract Introduction: Neratinib is an irreversible pan-HER inhibitor that has shown clinical activity against HER2-amplified (HER2+) and HER2-mutated cancers. Alteration in HER2 is currently the only biomarker of sensitivity to neratinib. To examine other potential biomarkers of sensitivity, we evaluated neratinib as a single agent in a panel of 108 cancer cell lines from 25 different primary tumour sites. In order to determine possible biomarkers of response, correlation analysis was performed to assess the relationship between genomic alterations or gene expression levels of oncogenes and neratinib response. Methods: Sensitivity to neratinib was assessed in 108 cancer cell lines (Oncolines™) by ATPlite assay after 72-hour treatment. Pearson correlation analysis was carried out between the gene expression profile of 361 relevant cancer genes for 94 of the 108 cell lines (CCLE) and neratinib sensitivity (10logIC50 values). The neratinib gene expression profile was then compared to > 160 reference compound profiles to exclude false positives. The mutation and copy number status of established oncogenes and tumour suppressor genes was sourced from COSMIC cell lines project and CCLE and compared to neratinib IC50 values by ANOVA and t-test. Results: Neratinib had a potent anti-proliferative effect across numerous cancer types, achieving an IC50 value of < 500 nM in 18 of the 108 cell lines; including breast, lung, pancreatic, colorectal, bladder, head and neck, and cervical cancers. Overall, no significant difference was observed between solid and haematological cancers. As expected, ERBB2-amplification, over-expression, and mutation correlated with neratinib response. However, sensitivity was also significantly correlated with EGFR mutations/ amplification/over-expression, amplification of MRPS28 (p=5.2 x 10-5), mutations in BRCA2 (p=0.02) and FBXW7 (p=0.008), and with both CNVs and mutations in SMAD4 (p=0.007) and PTEN (p=0.02). Gene expression of ERBB2 and RAC1, which can be activated downstream of HER2, positively correlated to neratinib response compared to reference compounds. Conclusions: Neratinib displayed broad-spectrum pre-clinical efficacy across numerous cancer types outside of HER2+ breast cancer. This study identified several novel markers of response to neratinib, which warrant further validation. Citation Format: Neil T Conlon, Jeffrey J Kooijman, Guido JR Zaman, Irmina Diala, Alshad Lalani, John Crown, Denis M Collins. Pre-clinical assessment of neratinib sensitivity and biomarkers of response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A046. doi:10.1158/1535-7163.TARG-19-A046

P2.14-10 Identification of Frequent, Activating HER2 Mutations and HER2 Inhibitor Response and Resistance in Canine Lung Cancer

Naturally occurring primary canine lung cancers share clinical and pathologic features with human never-smoker lung cancer, but their genetic and biologic underpinnings have been unknown. We mapped the genomic landscape of canine lung cancer, discovering somatic HER2 (ERBB2) mutations in ∼38% of canine pulmonary adenocarcinomas (cPACs). We describe our genomic findings, effects of HER2 mutations on cell signaling and HER2 inhibitor response/resistance, and results of pharmacokinetic (PK) studies of the HER2 inhibitor neratinib in healthy dogs. We performed multi-platform genomic analysis of 92 primary canine lung tumors or cell lines. We evaluated HER2 signaling by Western blot. Neratinib and lapatinib responses were assessed in HER2-mutant and wild-type cPAC cell lines in vitro. HER2-mutant cell lines were continuously treated with IC50 neratinib doses to select for drug resistance, then evaluated for resistance mutations. Neratinib tolerability and PK were evaluated in healthy, mixed-breed, middle-aged dogs given a single 6 mg/kg oral neratinib dose with or without maropitant citrate or loperamide. Plasma samples for PK were collected pre-dose and timepoints from 0.5-24h after dosing. Canine lung cancers exhibited low tumor mutation burden. HER2 was the most commonly mutated gene in cPACs, occurring in ∼38%, but was absent from canine adenosquamous or squamous cell carcinomas. HER2 hotspot V659E transmembrane domain (TMD) mutations were most common and comparable to activating TMD mutations in human cancer. HER2 V659E correlated with constitutive phosphorylation of Akt in cPAC cell lines. HER2 V659E lines displayed hypersensitivity to neratinib and lapatinib relative to wild-type lines. Continuous treatment of HER2-mutant cell lines led to rapid development of resistance. Characterization of resistance-driving alterations is ongoing, as are efficacy studies in mouse xenografts. A dose of 6 mg/kg neratinib in healthy dogs showed that neratinib was tolerable and achieved therapeutic levels from IC50 studies. Evaluation of neratinib tolerability and efficacy in dogs bearing HER2-mutant lung cancer is ongoing. HER2 TMD mutations are common in otherwise low-mutation-burden primary canine lung cancers. They confer constitutive HER2 signaling and HER2 inhibitor response in cell lines. Dosing and tolerability studies in healthy dogs have shown that therapeutically relevant doses are achievable. Study of HER2 inhibitor response/resistance in cell lines and mouse xenografts is ongoing as is clinical evaluation of neratinib activity in dogs with spontaneous lung cancer. These data offer immediate diagnostic and therapeutic opportunities for pet dogs and bear implications for comparative understanding of human never-smoker lung cancers and other HER2-mutant cancers.

Abstract 329: Hyperactivation of mTORC1 drives acquired resistance to the pan-HER tyrosine kinase inhibitor neratinib in HER2-mutant cancers

Abstract Background: The HER2 tyrosine kinase inhibitor (TKI) neratinib has exhibited clinical activity in patients with metastatic HER2-mutant cancers. However, responses are heterogeneous across tumor types and not generally prolonged, suggesting mechanisms of de novo and acquired drug resistance. Methods: Neratinib-resistant 5637 (HER2S310F) bladder cancer and OVCAR8 (HER2G776V) ovarian cancer cells were developed after gradual dose escalation. Candidate pathways associated with drug resistance identified by RNA sequencing were validated in a panel of HER2-mutant cell lines and in the SUMMIT basket trial in patients with HER2-mutant cancers. Results: Neratinib-resistant 5637 and OVCAR8 cells were cross-resistant to the HER2 TKIs afatinib and lapatinib. Immunoblot analysis showed that neratinib was still able to suppress HER2, EGFR and HER3 phosphorylation. Gene Set Enrichment and Connectivity Map analyses of RNA-seq data suggested mTORC1 signaling as a druggable pathway driving neratinib resistance. Immunoblot analysis of drug-resistant cells revealed a striking increase in S6K and S6 phosphorylation compared to parental cells. P-S6 levels and viability of drug resistant cells/tumors were ablated upon combining neratinib with the TORC1i everolimus both in vitro and in vivo. Similar results were obtained in cells transfected with Raptor or Rheb siRNAs. Further, neratinib resistance was induced by TSC2 knockdown and resultant TORC1 hyperactivation in parental 5637, OVCAR8, and MCF7 cells expressing L755S or V777L HER2 mutations. RNA-seq also revealed significant enrichment of RAS pathway in neratinib resistant cells which was confirmed by RAS-GTP pulldown. Pharmacological inhibition of RAS signaling using the PI3Ki buparlisib and the MEKi trametinib, or genetic suppression using H-, K-, and N-RAS isoform-specific siRNAs, ablated P-S6 and viability of neratinib resistant cells, suggesting RAS is causally associated with TORC1 hyperactivity and drug resistance. Further, intrinsically neratinib-resistant HER2-mutant cell lines with KRAS or PIK3CA co-mutations [DV90 (ERBB2V842I, KRASG13D), SNUC2A (ERBB2R678Q, KRASG12D, TSC2P1521T), MCF7 (HER2L755S/V777L, PIK3CAH1047R)] were sensitized to neratinib upon the addition of everolimus. Finally, DNA sequencing of tumors (MSK-IMPACT panel; 410 genes) from 141 patients enrolled in the SUMMIT trial showed enrichment of somatic alterations associated with aberrant activation of TORC1 pathway (KRAS, NRAS, NF1, PIK3CA, PIK3R1, AKT1/2, PTEN) in patients exhibiting primary resistance to neratinib. Conclusions: These data suggest that hyperactivation of TORC1 pathway promotes de novo and acquired resistance to neratinib across histologically distinct HER2-mutant cancers. Thus, we propose the combination of neratinib with TORC1 inhibitors is worthy of investigation in patients with HER2-mutant cancers. Citation Format: Dhivya R. Sudhan, Angel Guerrero-Zotano, Helen Won, Paula Gonzales Ericsson, Qi Liu, Teresa Dugger, James Koch, Alison Schram, Alberto Servetto, Richard Cutler, Alshad Lalani, Richard Bryce, Alan Auerbach, Ariella Hanker, Carlos L. Arteaga. Hyperactivation of mTORC1 drives acquired resistance to the pan-HER tyrosine kinase inhibitor neratinib in HER2-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 329.

Abstract 347: Identification of poziotinib alone or in combination with TDM1 as a pan-HER2 inhibitor

Abstract Clinical studies of HER2 targeting agents have shown mutational variant & cancer-specific differences in patient outcomes. Furthermore, the drug sensitivity profiles of HER2 mutational variants have not been fully characterized. To this end, we expressed the 16 most frequently detected HER2 mutations across exons 19-21 into Ba/F3 cells & determined activating potential & drug sensitivity to 13 HER1/2 targeting agents including afatinib, neratinib, dacomitinib, tarloxotinib-TKI, poziotinib, pyrotinib, TDM-1, & trastuzumab. Third generation EGFR TKIs, osimertinib, ibrutinib, & nazartinib were not effective at inhibiting cell viability in cells expressing exon 20 mutations; however, 3rd generation TKIs demonstrated activity against cells expressing D769 exon 19 variants & exon 21 variants. By comparison, covalent, quinazoline-based TKIs, afatinib, neratinib, dacomitinib, tarloxotinib-TKI, & poziotinib, differentially inhibited HER2 mutants across all three exons. Across all HER2 mutation variants & TKIs tested, poziotinib had the lowest average IC50 (1.73nM) & was significantly more effective in reducing cell viability than neratinib & tarloxotinib-TKI (p<0.001 & p=0.018). In addition, while poziotinib was more efficacious than either neratinib or tarloxotinib-TKI against HER2 exon 19 & 20 mutations, there was no significant difference in average IC50 for exon 21 mutants, suggesting that mutation location impacts drug binding. Furthermore, within exon 19, L755S & L755P variants had significant differences in drug sensitivity across all TKIs tested, & L755P & exon 20 insertion mutations were refractory to most inhibitors, but remained sensitive to poziotinib. We next tested the anti-tumor activity of HER2 targeting agents in human breast (MCF10A), colorectal (CW-2), & kidney (TUHR14TKB) cell lines with HER2 mutations in exons 19-20 & observed that poziotinib was the most potent inhibitor of the 13 TKIs tested. Furthermore, in a CW-2 xenograft model, poziotinib treatment resulted in a 58% reduction in tumor volume compared to the control group (p=0.011). In comparison, neratinib & afatinib treatment did not reduce tumor growth compared to control animals. Lastly, in a HER2 mutant NSCLC PDX model (HER2 Y772dupYVMA), the combination of low dose poziotinib (2.5mpk) & a single dose of TDM1 (10mpk) resulted in complete tumor regression in 9/9 mice, compared to 2/9 mice receiving TDM1 alone or 0/9 mice receiving low dose poziotinib (p<0.0001). In conclusion, HER2 exon 20 insertions & L755P mutations are resistant to the majority of HER2 TKIs, but remain sensitive to poziotinib. Combination of low dose poziotinib & TDM1 caused complete tumor regression in a HER2 exon 20 mutant PDX model. These data suggest that poziotinib alone & in combination with TDM1 has activity against the most common HER2 variants across diverse malignancies & that clinical studies testing these agents in HER2 mutant cancers are warranted. Citation Format: Jacqulyne P. Robichaux, Monique B. Nilsson, Fahao Zhang, Limei Hu, Junqin He, Marlese Pisegna, Alissa Poteete, John V. Heymach. Identification of poziotinib alone or in combination with TDM1 as a pan-HER2 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 347.

Hyperactivation of Torc1 Drives Resistance to the Pan-Her Tyrosine Kinase Inhibitor Neratinib in Her2-Mutant Cancers

The HER2 tyrosine kinase inhibitor (TKI) neratinib is clinically active in patients with HER2-mutant cancers. However, responses are heterogeneous across tumor types and generally short lived, suggesting mechanisms of de novo and acquired resistance. We developed neratinib-resistant HER2-mutant 5637 bladder and OVCAR8 ovarian cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Treatment with the TORC1 inhibitor everolimus or genetic inactivation of RAPTOR or RHEB ablated p-S6 levels and restored sensitivity to the TKI. The combination of everolimus and neratinib potently arrested growth of neratinib-resistant xenografts. RNA-seq and RAS activity assays revealed significant enrichment of the RAS pathway in neratinib-resistant cells. siRNA mediated knockdown of RAS isoforms also abrogated p-S6 and neratinib resistance. Finally, DNA sequencing of HER2-mutant tumors from patients clinically refractory to neratinib showed enrichment of somatic alterations associated with aberrant activation of the mTOR pathway.

Mutations in the Kinase Domain of the HER2/ERBB2 Gene Identified in a Wide Variety of Human Cancers

The HER2 (official name ERBB2) gene encodes a membrane receptor in the epidermal growth factor receptor family amplified and overexpressed in adenocarcinoma. Activating mutations also occur in several cancers. We report mutation analyses of the HER2 kinase domain in 7497 histologically diverse cancers. Forty-five genes, including the kinase domain of HER2 with HER2 IHC and dual in situ hybridization, were analyzed in tumors from 7497 patients with cancer, including 850 breast, 770 colorectal, 910 non-small cell lung, 823 uterine or cervical, 1372 ovarian, and 297 pancreatic cancers, as well as 323 melanomas and 2152 other solid tumors. Sixty-nine HER2 kinase domain mutations were identified in tumors from 68 patients (approximately 1% of all cases, ranging from absent in sarcomas to 4% in urothelial cancers), which included previously published activating mutations and 13 novel mutations. Fourteen cases with coexisting HER2 mutation and amplification and/or overexpression were identified. Fifty-two of 68 patients had additional mutations in other analyzed genes, whereas 16 patients (23%) had HER2 mutations identified as the sole driver mutation. HER2 mutations coexisted with HER2 gene amplification and overexpression and with mutations in other functionally important genes. HER2 mutations were identified as the only driver mutation in a significant proportion of solid cancers. Evaluation of anti-HER2 therapies in nonamplified, HER2-mutated cancers is warranted.