ErbB Family Of Kinases Research Articles

A CRISPR/Cas9-based kinome screen identifies ErbB signaling as a new regulator of human naïve pluripotency and totipotency

Abstract Regulation of totipotency and naïve pluripotency is crucial for early human embryo development. However, the mechanisms of naïve pluripotency and totipotency regulation in humans, especially the signaling pathways involved in these processes, remain largely unknown. Here, using the conversion of human extended pluripotent stem cells (hEPSCs) to naïve pluripotent stem cells as a model, we performed a CRISPR/Cas9-based kinome knockout screen to analyze the effect of disrupting 763 kinases in regulating human naïve pluripotency. Further validation using small molecules revealed that the inhibition of ErbB family kinases promoted the transition of hEPSCs to human naïve pluripotent stem cells. More importantly, chemical inhibition of the ErbB family also promoted induction of totipotent signatures in human pluripotent cells under different culture conditions. Our findings provide new mechanistic insights into the regulation of naïve pluripotency and totipotency in humans.

Abstract 1102: Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC

Abstract ALK/ROS1 TKIs have significantly improved outcomes for patients with ALK/ROS1+ lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately 1/3 of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in most patients remains unknown/poorly characterized. To model and characterize acquired resistance to crizotinib, we used ROS1+ and ALK+ primary, patient tumor-derived non-small cell lung cancer (NSCLC) cell lines to generate acquired resistance models to the ALK/ROS1 inhibitor crizotinib. We used several techniques to probe the mechanisms driving resistance: DNA and RNA sequencing (seq), fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. The crizotinib-resistant line initially driven by a TPM3-ROS1 fusion (CUTO28-CR) displayed exquisite sensitivity to Src family kinase (SFK) TKIs in proliferation assays, which was modestly improved by addition of ERBB TKI afatinib. Bulk RNA seq revealed upregulation of SFKs Fyn and Src transcripts (141- and 2.31-fold, respectively). Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells vs parental (EGFR:CEP7 ratio 2.1 vs 1.0), and RNA seq revealed upregulation of EGFR and HER2 transcripts (1.79- and 2.45-fold, respectively). Western blot analysis confirmed upregulation of Fyn at the protein level. CUTO28-CR cells treated with a SFK TKI revealed dose-dependent deactivation of Akt signaling, and addition of afatinib caused inhibition of MAPK signaling. Stimulation with epidermal growth factor (EGF) enhanced MAPK signaling in CUTO28-CR cells more than in parental cells, consistent with EGFR/HER2 dependence. Because of a noted propensity for the EML4-ALK line (H3122) to upregulate ERBB kinase activity under sustained ALK TKI treatment, we maintained these cells in low dose (25nM) afatinib while driving crizotinib resistance to identify additional bypass signaling pathways. These H3122-CAR cells were partially resensitized to crizotinib with the addition of SFK TKIs, and the addition of afatinib with SFK TKIs further resensitized these resistant cells to crizotinib. Bulk RNA seq of H3122-CAR cells revealed upregulation of Fyn and Src (2.64- and 3.63-fold, respectively), and western blot analysis confirmed upregulation at the protein level. In conclusion, we demonstrated increased SFK (particularly Fyn and/or Src) expression and/or activation is a mechanism utilized to drive bypass signaling, in cooperation with ERBB family kinases, to circumvent treatment with ALK/ROS1 TKI crizotinib. To our knowledge, this is the first report of SFK-driven bypass signaling as a mechanism of acquired resistance to an ALK/ROS1 TKI in NSCLC. The upregulation of Src and/or Fyn in NSCLC biopsies may guide the exploration of combination SFK TKIs with ALK/ROS1 TKIs. Citation Format: Logan C. Tyler, Nan Chen, Anh T. Le, Hala Nijmeh, Liming Bao, Robert C. Doebele. Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC [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 1102.

Simultaneously targeting ErbB family kinases and PI3K in HPV-positive head and neck squamous cell carcinoma

ObjectivesTo identify the most effective PI3K and EGFR inhibitors in HPV-positive head and neck squamous cell carcinoma (HNSCC) and investigate the efficacy of a combination of an ErbB family kinase inhibitor and a PI3K inhibitor to inhibit cell proliferation of HPV-positive HNSCC. Materials and methodHPV-positive HNSCC cell lines were treated with the FDA approved ErbB kinase inhibitor, Afatinib or FDA-approved PI3K inhibitor, Copanlisib, alone or in combination, and phosphorylation and total protein levels of cells were assessed by Western blot analysis.Cell proliferation and apoptosis were examined by MTS assay, flow cytometry, and Western blots, respectively. ResultsCopanlisib more effectively inhibited cell proliferation in comparison to other PI3K inhibitors tested. HPV-positive HNSCC cells differentially responded to cisplatin, Afatinib, or Copanlisib. The combination of Afatinib and Copanlisib more effectively suppressed cell proliferation and induced apoptosis compared to either treatment alone. Mechanistically, the combination of Afatinib and Copanlisib completely blocked phosphorylation of EGFR, HER2, HER3, and Akt as well as significantly decreased the HPV E7 expression compared to either treatment alone. ConclusionAfatinib and Copanlisib more effectively suppress cell proliferation and survival of HPV-positive HNSCC in comparison to either treatment alone.

Systematic analysis and molecular profiling of EGFR allosteric inhibitor cross-reactivity across the proto-oncogenic ErbB family kinases by integrating dynamics simulation, energetics calculation and biochemical assay.

Human ErbB family of proteins contains four receptor tyrosine kinases (EGFR, Her2, Her3 and Her4) and has been established as a group of attractive druggable targets against diverse cancers. Over the past decades, a variety of ATP-competitive inhibitors have been discovered to target the orthosteric site of EGFR, which, however, would eventually develop acquired drug resistance due to the missense mutations T790M/C797S occurring in orthosteric site. In recent years, a number of forth-generation inhibitors have been successfully designed to overcome the T790M/C797S-induced drug resistance by targeting EGFR allosteric site instead of orthosteric site. Considering that the four proto-oncogenic ErbB kinases share a high conservation in sequence, structure and function, we herein attempted to investigate the binding potency and cross-reactivity of cognate EGFR allosteric inhibitors over noncognate Her2, Her3 and Her4 kinases--they are closely related to gynecological tumors such as ovarian cancer but no allosteric inhibitors have been reported specifically for them to date. A systematic affinity profile of 12 allosteric inhibitors and 4 orthosteric inhibitors to the 4 ErbB kinases was created by integrating dynamics simulations, energetics calculations and biochemical assays, which was then used to derive a systematic inhibitor selectivity profile for EGFR over other three kinases. It is found that allosteric and orthosteric inhibitors exhibit moderate and modest cross-reactivity across the ErbB family, respectively, but the former generally has a higher binding potency than the latter due to the additional energy cost used for inducing kinase conformational change. Moreover, most allosteric inhibitors can be sensitized by Her2 T798M gatekeeper mutation, a counterpart of EGFR T790M gatekeeper mutation that has been previously reported to cause generic drug resistance for orthosteric inhibitors.

Noncognate HER2 sensitivity to cognate EGFR allosteric inhibitors at molecular level: New uses for old drugs in gynecological tumors

AbstractHuman epidermal growth factor receptor (EGFR) of the ErbB family kinases is a well‐established therapeutic target for malignant lung cancer, which, however, has been observed to cause the dramatic drug resistance to almost all first‐ to third‐generation ATP‐competitive inhibitors with the T790M/C797S double‐mutation at the peripheral region of its kinase active site. In recent years, allosteric inhibition of the EGFR kinase domain has evolved rapidly to overcome these clinically significant drug‐resistant mutations, leading to the discovery of a number of EGFR allosteric inhibitors. In the present study, we attempted to systematically evaluate the molecular sensitivity of existing cognate EGFR allosteric inhibitors to their noncognate HER2 target –– another druggable ErbB member that is a potential therapeutic target of diverse gynecological tumors such as breast, cervical and ovarian cancers, and shares high conservation with EGFR but still has no cognate allosteric inhibitors specifically developed for it. An integrative strategy that combined molecular modeling and biochemical inhibition assay was described to investigate the structural basis, energetics property, and dynamics behavior involved in the intermolecular interaction between the HER2 kinase domain and nine reported EGFR allosteric inhibitors at a molecular level. The inhibitor response to HER2 T798M/C805S double‐mutation was also examined and compared with their response to counterpart EGFR T790M/C797S double‐mutation. It is revealed that the EGFR allosteric inhibitors can also effectively target HER2 kinase with a similar or moderately decreased potency; they exhibit a consistent response profile to EGFR and HER2 counterpart mutations. In addition, the HER2 T798M mutation was found to considerably sensitize allosteric inhibitors EAI045 and JBJ‐04‐125‐02 by creating additional noncovalent interactions between them, while the HER2 C805S mutation was observed to have no essential effect on most allosteric inhibitors due to its spatial separation from the kinase allosteric site.

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

70. A PHASE 1–2 CLINICAL TRIAL OF EO1001, A NOVEL IRREVERSIBLE pan-ErbB INHIBITOR WITH PROMISING BRAIN PENETRATION

CNS metastasis has become a prominent driver of morbidity and mortality in recent years as new targeted therapies have improved systemic outcomes. Mutations in the ErbB family of kinases are known oncodrivers in many of these cancers. ErbB family member “crosstalk” is associated with rapid development acquired resistance to ErbB TKIs. The development of agents targeting multiple ErbB receptors has shown promise but has been limited by toxicity and poor brain penetration. EO1001 is a first-in-class, oral, brain penetrating, irreversible pan-ErbB inhibitor with superior CNS penetration targeting ErbB1, ErbB2 and ErbB4. Preclinical data suggests a favorable pharmacokinetic and safety profile and promising activity against ErbB-driven cancers in patient-derived xenograft models. Male or female adult participants with confirmed ErbB-positive cancer, including patients with CNS involvement, who have progressed after standard of care therapy, with adequate bone marrow, renal and liver function are eligible. An accelerated dose-escalation design is employed. One subject per dose cohort will be recruited until drug related toxicity (≥G2) is observed in the first dosing cycle, after which dose escalation will revert to a 3 + 3 design to determine the maximum tolerated dose (MTD). Dose Escalation: Cycle 1: Patients will receive a single dose EO1001 on day 1 and single dose pharmacokinetics will be performed. Beginning on day 8, EO1001 will be administered once daily for 21 days; multi-dose pharmacokinetics will be performed. Cycles 2–6: Oral EO1001 will be administered once daily in continuous 28-day cycles for up to 20 weeks. MTD Expansion: Oral EO1001 will be administered once daily at the MTD in continuous 28-day cycles for up to 6 cycles (24 weeks). Toxicity is assessed based on NCI CCTCAEv5 and tumor response will be assessed by RECIST 1.1 or RANO for CNS disease. CNS exposure is evaluated in patients via CSF collection with confirmed CNS disease involvement.

Systematic molecular profiling of inhibitor response to the clinical missense mutations of ErbB family kinases in human gastric cancer

The oncogenic receptor tyrosine kinase family ErbB consists of four members (ErbB1, ErbB2, ErbB3 and ErbB4); they are involved in the tumorgenesis of diverse cancers. A variety of missence mutations have been clinically observed in ErbB kinases, which would shift drug sensitivity to these kinases and cause drug resistance in targeted cancer therapy. In this study, systematic inhibitor response to ErbB missense mutations in gastric cancer (GC) is investigated by combining computational analysis and experimental assay. The response profile is created for 6 ATP-competitive, reversible inhibitors against 9, 17, 5 and 17 GC-associated missense mutations of ErbB1, ErbB2, ErbB3 and ErbB4 kinase domains, respectively. From the profile a number of potential resistant and sensitive responses are identified theoretically. It is suggested that most ErbB mutations have only a modest effect on inhibitor binding, but few that are located around the kinase active site can influence the binding significantly. Structural examination reveals that steric hindrance and allosteric effect are primarily responsible for inhibitor resistance and sensitivity, respectively. Two ErbB2 mutations, namely V777L and T862A, are predicted to cause effective resistance on inhibitors TAK285 and Lapatinib, respectively. Kinase assays consistently observe that the mutations can reduce inhibitor activity by 4.9-fold and 2.4-fold, with IC50 changing from 29 to 16 nM (wild type) to 83 and 39 nM (mutant) for TAK285 and Lapatinib, respectively.

Abstract 384: Identification of a lung adenocarcinoma cell line addicted to ERBB4, an actionable target for cancer therapy

Abstract Cancer cells often survive by getting addicted to specific genes. In most cases, such genes function as an enzyme, especially kinases that are specifically activated in cancers by genetic aberrations; e.g., BCR-ABL fusion kinase found in chronic myelogenous leukemias and epidermal growth factor receptor (EGFR) with activating mutations found in non-small cell lung cancers (NSCLCs). These activated kinases would be an ideal target for cancer therapy and indeed imatinib and gefitinib displayed good clinical benefits to the patients in these diseases. However, only a limited population of cancer cells harbor such well-characterized driver genes which could be attacked by targeted drugs developed so far and there remain unmet medical needs to identify novel therapeutic targets and drugs for the rest of cancers. To this end, we exploited our original database of drug efficacy of >100 kinase inhibitors across a panel of 39 human cancer cell lines (JFCR39) to characterize each of the cell line in terms of susceptibility to these kinase inhibitors. Expectedly, cell lines harboring PIK3CA hotspot mutation and PTEN mutation were susceptible to PI3K and mTOR inhibitors, whereas those harboring KRAS or BRAF mutation to MEK inhibitors, respectively. Of note, we found four of the cells responding to a series of EGFR family tyrosine kinase inhibitors (TKIs) including gefitinib and lapatinib. Gefitinib exert selective effect on NSCLCs addicted to activated EGFR mutant; however, none of the four gefitinib-sensitive cell lines possessed such EGFR mutation. Of these, an NSCLC cell line NCI-H522 hardly expressed EGFR but overexpressed ERBB4 instead, while an ovarian cancer cell line SK-OV3 overexpressed ERBB2 as reported previously. Actually, treatment with a certain TKI in this class efficiently dephosphorylated ERBB4 in NCI-H522 along with inhibition of cell growth and suppression of downstream factor ribosomal S6 protein. Moreover, knockdown of ERBB family kinases revealed that cell growth was dependent on overexpressed ERBB4 in NCI-H522 cells. These results indicate that NCI-H522 cells exhibited susceptibility to EGFR family TKIs via inhibition of ERBB4. Of note, NCI-H522 cells exhibited hypersensitivity to three of these TKIs, sapitinib, PF-7775 and GW583340, as compared to gefitinib. Especially, GW583340 suppressed growth of NCI-H522 cells at the same or lower concentration than in HCC827 cells harboring an EGFR active mutation. Consistent with this observation, in vitro kinase assay revealed that sapitinib and PF-7775 exhibited ~10-fold higher ERBB4-inhibitory effect than gefitinib did while GW583340 displayed selective inhibition on ERBB4, with ~10-fold higher activity than that on activated EGFR. In conclusion, we identified NCI-H522 as an ERBB4-dependent cell line, and found ERBB4 as a novel therapeutic cancer target that could be attacked by ERBB4-directing TKIs. Citation Format: Noritaka Tanaka, Kanami Yamazaki, Yuko Uno, Yoshimi Ohashi, Yumiko Nishimura, Masaaki Sawa, Shingo Dan. Identification of a lung adenocarcinoma cell line addicted to ERBB4, an actionable target for cancer therapy [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 384.

Rational creation and systematic analysis of cervical cancer kinase-inhibitor binding profile.

The kinase-regulatory cell signaling networks play a central role in the pathogenesis of human cervical cancer (hCC). However, only few kinase inhibitors have been successfully developed for treatment of this cancer to date. Considering that the active sites of protein kinases are highly conserved and small-molecule inhibitors should generally exhibit high promiscuity and broad specificity across the hCC-related kinase array, it is supposed that the established kinase targets of hCC can be targeted unexpectedly by certain noncognate kinase inhibitors. This provides a novel idea to practice the new uses for old drugs in anti-cancer chemotherapy. Here, we create a systematic kinase-inhibitor binding profile in a high-throughput manner by molecular docking and consensus scoring, where the kinases have been collected as therapeutic targets of hCC and the inhibitors are reversible, ATP-competitive and readily available. The docking/scoring scheme is tested rigorously with structure-solved and affinity-known kinase-inhibitor complex samples, which is later demonstrated to be effective in inferring unexpected inhibitor response to hCC-related kinases. Few promising kinase-inhibitor pairs are identified from the profile and tested experimentally at cellular and molecular levels. It is found that the kinase-inhibitor promiscuity is a common phenomenon but only few can interaction effectively and inhibit potently. In addition, the high-scoring inhibitors generally exhibit good suppressing potency on hCC cell viability as compared to those low-scoring ones, imparting that the created profile can well reflect the tumor cytotoxicity of noncognate kinase inhibitors. A further kinase assay suggests that the ErbB family kinases are the potential targets of these high-scoring inhibitors, with noncognate inhibitory activity up to nanomolar level. Structure analysis reveals that the nonbonded interactions of potent noncogante kinase-inhibitor binding can divided into a polar tail and a nonpolar lobe, which confer specificity and stability to the binding, respectively.

P2.13-18 A Multicenter Prospective Biomarker Study to Explore Mechanisms of Afatinib Resistance Based on Digita PCR and Next-Generation Sequencing

Afatinib is an oral irreversible blocker of ErbB-family kinases and shows a pronounced anti-tumor efficacy for advanced non–small cell lung cancer (NSCLC) positive for activating mutations of EGFR. We applied digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) to explore mechanisms of afatinib resistance. Eligible patients had advanced lung adenocarcinoma with EGFR activating mutations. Tumor and plasma samples were collected before afatinib treatment and after treatment failure with disease progression (systemic progressive disease, SPD). DNA from the samples was analyzed by dPCR and NGS. Thirty-five patients were enrolled, with a median follow-up time of 15.8 months. Among 25 patients with SPD, tumor, plasma, or both samples were available for 18, 23, and 16 individuals, respectively. dPCR and NGS detected EGFR T790M mutation in 13 (56.5%) and 11 (47.8%) of 23 plasma samples at SPD, with sensitivity and specificity compared with tumor samples being 83.3% and 70.0% (dPCR) and 50.0% and 70.0% (NGS), respectively. Applying the ratio of the number of T790M alleles to that of activating mutations (T/A) for determination of the T790M positivity improved the sensitivity and specificity of plasma analysis compared with tumor analysis to 83.3% and 100% (dPCR) and 57.1% and 100% (NGS), respectively. Among 25 patients with SPD, the T790M mutation of EGFR alone (n = 11), copy number gain (CNG) of NRAS (n = 1), CNG of MET (n = 1), CNG of EGFR plus T790M (n = 1), and CNG and E545K of PIK3CA plus T790M of EGFR (n = 1) were identified by NGS as putative resistance mechanisms against afatinib. No tumor showed transformation to small cell carcinoma. Median progression-free survival was longer in patients with than in those without T790M at SPD (15.1 versus 10.9 months, P =0.25). Median time to SPD was much longer in patients with than in those without T790M at SPD (17.9 versus 10.9 months, P =0.18). Assessment of T/A ratio with dPCR or NGS improved specificity of plasma analysis for determination of T790M positivity compared with tumor analysis. dPCR and NGS analysis in tumor and plasma samples shed light on exploring mechanisms of afatinib resistance.

Highlights of This Issue

Ibrutinib is an irreversible tyrosine kinase inhibitor that targets Bruton's tyrosine kinase (BTK) as well as ErbB family kinases. Chen and colleagues found that ibrutinib potently inhibited the activity of ErbB kinases and the growth of HER2 overexpressing cells similarly as ErbB-targetted inhibitors in clinic. Compare to ErB inhibitors, the dual activity toward TEC and ErbB family kinases was unique to ibrutinib. In addition, it was shown that the anti-tumor effects of ibrutinib in breast cancer models are at clinically achievable levels. Since TEC kinases modulate several types of tumor accessory cells, the utility of ibrutinib's unique inhibitory spectrum is worthy of further exploration.Advanced/metastatic gastrointestinal stromal tumors (GIST), driven mainly by KIT or PDGFRA mutations, are treated with tyrosine kinase inhibitors (TKI) such as imatinib. However, the development of resistance remains to be a major clinical challenge. Gebreyohannes, Schöffski and colleagues showed the in vivo activity of cabozantinib, a novel small molecule multi-targeted TKI, in three GIST patient-derived xenograft models, characterized by different KIT mutations and sensitivity to standard TKI. Cabozantinib decreased or stabilized tumor burden, mainly through the inhibition of cell proliferation and tumor vascularization. Moreover, the effect observed was significantly better than the one obtained with imatinib.T cell immunoglobulin mucin-1 (TIM-1) is highly up-regulated in renal cell carcinoma and ovarian clear cell carcinoma. To exploit the expression of TIM-1 on these malignancies, the Celldex Therapeutics team developed CDX-014, which is an antibody drug conjugate comprised of a fully-human monoclonal antibody directed against an epitope of TIM-1, conjugated to the cytotoxic agent MMAE. Preclinical studies with this ADC demonstrated in vitro and in vivo efficacy against a variety of TIM-1 expressing cell lines and tumor models. With the successful completion of IND-enabling studies, activities are on-going to advance CDX-014 in clinical studies in renal cell carcinomas and other TIM-1 expressing tumors.122 anti-cancer agents were profiled in proliferation assays with cancer cell lines from diverse tumor tissues. For 38 compounds this is the first comparative profiling study. Good correlations were observed with other cell panels. Curve fitting led to a large source of variation. Hierarchical clustering revealed common mechanisms of action. Nine clusters, including EZH2, BET and TTK inhibitors, have not been reported before. The lack of clustering of several Aurora and PI3K inhibitors could be explained by differences in biochemical selectivity. The study shows that cell line profiling is an excellent tool for the unbiased classification of anti-proliferative compounds.

Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth.

Ibrutinib is a potent, small-molecule Bruton tyrosine kinase (BTK) inhibitor developed for the treatment of B-cell malignancies. Ibrutinib covalently binds to Cys481 in the ATP-binding domain of BTK. This cysteine residue is conserved among 9 other tyrosine kinases, including HER2 and EGFR, which can be targeted. Screening large panels of cell lines demonstrated that ibrutinib was growth inhibitory against some solid tumor cells, including those inhibited by other HER2/EGFR inhibitors. Among sensitive cell lines, breast cancer lines with HER2 overexpression were most potently inhibited by ibrutinib (<100 nmol/L); in addition, the IC50s were lower than that of lapatinib and dacomitinib. Inhibition of cell growth by ibrutinib coincided with downregulation of phosphorylation on HER2 and EGFR and their downstream targets, AKT and ERK. Irreversible inhibition of HER2 and EGFR in breast cancer cells was established after 30-minute incubation above 100 nmol/L or following 2-hour incubation at lower concentrations. Furthermore, ibrutinib inhibited recombinant HER2 and EGFR activity that was resistant to dialysis and rapid dilution, suggesting an irreversible interaction. The dual activity toward TEC family (BTK and ITK) and ERBB family kinases was unique to ibrutinib, as ERBB inhibitors do not inhibit or covalently bind BTK or ITK. Xenograft studies with HER2+ MDA-MB-453 and BT-474 cells in mice in conjunction with determination of pharmacokinetics demonstrated significant exposure-dependent inhibition of growth and key signaling molecules at levels that are clinically achievable. Ibrutinib's unique dual spectrum of activity against both TEC family and ERBB kinases suggests broader applications of ibrutinib in oncology. Mol Cancer Ther; 15(12); 2835-44. ©2016 AACR.

Abstract LB-77: The Met receptor tyrosine kinase drives signaling through EGFR and ErbB3 in Met-amplified non-small-cell lung cancer

Abstract Receptor tyrosine kinases are canonically activated by dimerization induced by the binding of a cognate ligand. This leads to autophosphorylation of the receptor molecules by their intrinsic kinase domains and the recruitment of intracellular protein complexes to initiate signaling. In many human cancers, receptor tyrosine kinases are aberrantly activated to promote proliferative signaling, thus driving tumor growth. The MET proto-oncogene is amplified in approximately 5% of non-small-cell lung cancers (NSCLC), a disease in which aberrant signaling through the epidermal growth factor receptor (EGFR) or the anaplastic lymphoma kinase is known to be oncogenic. The Met receptor tyrosine kinase is overexpressed and constitutively active in cell lines derived from MET-amplified tumors, and Met activity is required for proliferation. Furthermore, Met activity is sufficient to promote the phosphorylation of EGFR and its homologs ErbB2 and ErbB3 in the presence of inhibitors targeting EGFR, ErbB2, and Src family kinases. We show that Met utilizes the EGFR, ErbB2 and ErbB3 proteins as scaffolds to broaden downstream oncogenic signaling and promote cancer cell proliferation. The contribution of EGFR and its homologs to Met signaling in NSCLC will be presented with a focus on ErbB family kinases required for proliferation in MET-amplified NSCLC. Understanding the mechanisms by which Met drives proliferative signaling in NSCLC will become crucial as Met-targeted therapies become available in the clinic. Citation Format: Yaakov E. Stern, Andrea Z. Lai, Morag Park. The Met receptor tyrosine kinase drives signaling through EGFR and ErbB3 in Met-amplified non-small-cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-77. doi:10.1158/1538-7445.AM2014-LB-77

Abstract C258: BTK inhibitor ibrutinib inhibits breast cancer growth by inhibiting ErbB2 kinase.

Abstract Ibrutinib is a BTK (Bruton tyrosine kinase) targeting inhibitor that has been developed for the treatment of B cell malignancies. Ibrutinib's specificity is enhanced by its ability to bind covalently to a cysteine residue in the BTK active site, which is conserved among 9 other kinases. Although BTK is the most sensitive of these to ibrutinib inhibition, with IC50 = 0.4 nM, some of the other 9 kinases may also be inhibited by ibrutinib at clinically achievable plasma levels. IC50s of ibrutinib for ErbB1, ErbB2 and ErbB4 in particular are 18, 22, and 0.6 nM, respectively. Among 13 breast cancer lines screened for sensitivity, significant inhibition of in vitro growth at ibrutinib concentrations &amp;lt;100 nM was noted of the ErbB2 over-expressing and gene amplified lines MDA-MB-453, BT-474, UACC-893 and SK-BR-3. In vivo growth of MB-453 in mice was inhibited &amp;gt;60% by 5 mg/kg po QD of ibrutinib, and nearly completely by 50 mg/kg/day. Ibrutinib inhibited in vitro phosphorylation of ErbB2 and ErbB4, accompanied by downstream phosphorylation of AKT, MEK and ERK, by Western blot. Neither CC-292, another BTK inhibitor which lacks significant ErbB1 or 2 inhibitory activity, nor the ErbB1 inhibitor gefitinib, were inhibitory at concentrations comparable to ibrutinib. Although ErbB4 is the most sensitive of the ErbB family kinases to ibrutinib, comparison of effects with several other congeners with varying relative specificities were most consistent with dependence of growth inhibition of these lines on ErbB2 inhibition. The growth inhibitory effect of ibrutinib at concentrations &amp;lt;100 nM was markedly decreased in the presence of HRG-1 (1 μg/mL) and partly blocked by EGF (0.2 μg/mL). Ibrutinib did not significantly inhibit of cell lines MBA-MD-231, MCF-7, and SK-OV-3 which overexpress ErbB2 but without genomic amplification. These lines generally showed lower basal phosphorylation of MEK, ERK and AKT with lower sensitivity to inhibition by ibrutinib. Covalent binding of ibrutinib to BTK appears to be rapidly established, within ½ hour, as evidenced by persistent inhibition of auto-phosphorylation and persistent labeling by a fluorescently tagged ibrutinib congener, following wash-out into ibrutinib free media. Covalent binding to ErbB family members in breast cancer lines was slightly less robust, requiring longer incubations to maximally bind probe or establish resistance of phosphorylation to washout. This parallels kinetics of enzyme inhibition in cell-free systems. A431 EGFR+ cells exhibited inhibition of EGFR phosphorylation by ibrutinib, which was easily reversed by washout. Conclusion: The BTK inhibitor Ibrutinib also inhibits the ErbB receptor kinases in cell free assays and is now shown to be capable of inhibiting growth of ErbB2 amplified breast cancer cell lines in vivo and in vitro. Inhibition was paralleled by inhibition of AKT, MEK and ERK phosphorylation downstream. Ibrutinib thus has properties of a pan-ErbB family inhibitor. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C258. Citation Format: Laurence Elias, Jun Chen, Joseph Buggy. BTK inhibitor ibrutinib inhibits breast cancer growth by inhibiting ErbB2 kinase. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C258.

KRAS Wild-Type Lung Cancer: A Moving Target in an Era of Genotype Migration

The development of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) for the management of advanced lung cancer ranks among one of the few most important advances in solid tumor oncology over the past decade. In recent years, much has been learned about how to most effectively use first-generation EGFR TKIs like erlotinib and gefitinib for non–small-cell lung cancer (NSCLC). We know that EGFR TKIs should be administered instead of first-line chemotherapy to patients with lung cancers carrying EGFR exon 19 or 21 mutations, because this leads to longer median progression-free survival (PFS) and improved quality of life. We know that patients with EGFR-mutant lung cancer with poor performance status should be offered treatment with EGFR TKIs, because this can lead to dramatic responses with marked and durable improvement in performance status. Furthermore, patients with KRASmutant lung cancers rarely respond to EGFR TKIs, and some investigators suspect that such patients may gain no benefit from these agents at all. We are also learning much about continuation of TKIs postprogression in some patients with EGFR-mutant lung cancers, which may prolong the duration of benefit from the drugs. One setting in which the published evidence is inadequate is in the application of predictive biomarkers to guide EGFR TKI therapy in previously treated NSCLC. The BR.21 trial resulted in the initial US Food and Drug Administration approval of erlotinib for previously treated NSCLC by showing a 2-month improvement in median survival over placebo in previously treated NSCLC. A biomarker analysis of this trial was later published, which found that only high EGFR copy number was predictive of survival benefit from erlotinib. However, retrospective EGFR genotyping could only be performed for 204 of the 731 BR.21 patients (28%), and an analysis of PFS was not performed. When one looks as a group at the biomarker analyses from several randomized trials studying erlotinib or gefitinib in previously treated NSCLC (Table 1), it is clear that patients with EGFR-mutant disease have the longest PFS and highest response rates with erlotinib and gefitinib, followed by those with KRAS wild-type disease (including EGFR-mutant disease), whereas those with KRAS-mutant and EGFR wild-type disease (including KRAS-mutant disease) do the least well. The article by Ramalingam et al in Journal of Clinical Oncology adds thought-provoking data to this discussion, with the first randomized data on irreversible EGFR TKIs in patients with lung cancer without previous TKI exposure. In this randomized phase II study for patients with NSCLC experiencing progression during chemotherapy, erlotinib was compared with dacomitinib, an irreversible inhibitor of multiple ERBB-family kinases. Although erlotinib has been found to maximally inhibit the mutant EGFR kinase, dacomitinib irreversibly inhibits EGFR, human epidermal growth factor receptor 2, and ERBB4 at nanomolar concentrations. The authors found a significant improvement in PFS with dacomitinib in the intention-to-treat population (P .012) and in patients with KRAS wild-type lung cancers (P .006). The broader inhibitory activity of dacomitinib

A Water-Based Mechanism of Specificity and Resistance for Lapatinib with ErbB Family Kinases

The dual kinase inhibitor lapatinib has a high affinity for EGFR and HER2 but a weak affinity for ErbB4, although the factors driving specificity for these highly homologous members of the ErbB family of receptor tyrosine kinases are not well understood. In this report, homology modeling, molecular dynamics simulations, and free energy calculations are employed with the goal of uncovering the energetic and structural molecular basis of lapatinib specificity and resistance. The results reveal a distinct network of three binding site water molecules that yield strikingly similar hydration patterns for EGFR and HER2 in contrast to that of ErbB4, which shows a different pattern with a reduced occupancy at one of the positions. The primary cause was traced to a single amino acid change in the binding site (EGFR position 775), involving a swap from C or S (EGFR and HER2) to V (ErbB4), for which the side chain is bulkier, is hydrophobic, and lacks the ability to form a H-bond with water. Notably, excellent quantitative agreement with experimental activities is obtained across the series (EGFR > HER2 > ErbB4) when key waters are included in the calculations. Quantitatively, Coulombic interactions and H-bond counts between network waters and species involved in the network are less favorable in ErbB4 by ~40% relative to those in EGFR or HER2. Additional simulations with clinically relevant EGFR (C775F, T854A, and T790M) and HER2 (T790I) mutants demonstrate that resistance can also be understood in terms of changes that occur in the binding site water network. Overall, the results of this study have yielded a physically reasonable water-based mechanism for describing lapatinib specificity and resistance.

Abstract 3610: Pan-ErbB inhibition by ARRY-334543 is superior to selective ErbB inhibition in a preclinical model that signals through multiple ErbB receptors

Abstract Activation of redundant receptor tyrosine kinases (RTKs) in tumors can confer resistance to selective RTK inhibitors. In particular, activation of multiple ErbB-family kinases is associated with diminished activity of selective EGFR or ErbB2 inhibitors. ErbB-family ligands are commonly expressed in cancers and can activate multiple redundant ErbB dimers. We hypothesize that ligand activation of multiple ErbB-family kinases in tumors can confer resistance to selective ErbB inhibitors, but not pan-ErbB inhibitors, suggesting a setting where pan-ErbB inhibitors may be advantageous over selective inhibitors. One such candidate currently in Phase 1/2 clinical trials is ARRY-334543, an oral, reversible pan-ErbB inhibitor with potent in vivo activity in both EGFR- and ErbB2-dependent human tumor xenografts. We tested this hypothesis in vitro with N87 gastric carcinoma cells that express high levels of constitutively active ErbB2 and low levels of EGFR. In the absence of EGF, ErbB2 homodimers are predominant and cells are highly sensitive to ErbB2 inhibition. ARRY-334543 and the ErbB2-selective inhibitor, ARRY-380, strongly inhibited N87 proliferation (IC50 = 60 nM and 5 nM, respectively) while the EGFR-selective inhibitor erlotinib was active only at high concentrations (IC50 = 860 nM). No benefit was observed by combining ARRY-380 and erlotinib. EGF-stimulation of N87 cells caused the formation of EGFR homodimers and EGFR/ErbB2 heterodimers which significantly alters cellular responses to selective ErbB inhibitors. The cellular IC50 for ARRY-380 increased 57-fold while erlotinib activity was relatively unchanged, suggesting a loss of dependence on ErbB2 signaling alone. The dual inhibition of EGFR and ErbB2, either by ARRY-334543 or the combination of ARRY-380 and erlotinib, largely maintained potency with only a modest increase in IC50 (9- to 10-fold). These data suggest that activation of EGFR can significantly diminish the activity of selective ErbB inhibitors, but not pan-ErbB inhibitors. Dual-responsiveness was also observed in vivo where both EGFR and ErbB2 homodimers were detected in N87 xenografts and dual EGFR/ErbB2 treatment resulted in superior tumor growth inhibition (TGI). ARRY-334543 was highly active with 90% TGI (100 mg/kg BID). Treatment with the combination of ARRY-380 and erlotinib (50 mg/kg QD each, MTD when given in combination) had similar activity with 82% TGI. Treatment with either erlotinib (100 mg/kg QD) or ARRY-380 (50 mg/kg QD) alone was less efficacious. These results suggest that the pan-ErbB inhibitor, ARRY-334543, can be differentiated from selective ErbB inhibitors by targeting tumors that signal through multiple ErbB-family receptors. A wide range of tumor types has been shown to activate multiple ErbB-family members and clinical evaluation of this hypothesis is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3610.

Conserved Hydrophobic and Hydrophilic Bond Interaction Networks in ErbB Family Kinases

The EGFR/ErbB/HER family of kinases contains four homologous receptor tyrosine kinases that are important regulatory elements in key signaling pathways. To elucidate the atomistic mechanisms of activation in the ErbB family, we perform molecular dynamics simulations on the three members of the ErbB family with known kinase activity, EGFR, HER2 (ErbB2) and ErbB4, in different molecular contexts: monomer vs. dimer, wildtype vs. mutant. Using bioinformatics and fluctuation analyses of the molecular dynamics trajectories, we relate sequence similarities to similarities in specific bond-interaction networks and similarities in collective dynamical modes. We find that in the active conformation of the ErbB kinases, key loop motions are coordinated through conserved hydrophilic interactions, an activating bond-network consisting of hydrogen bonds and salt bridges. While the inactive conformations show a less extensive inactivating bond network, they sequester key residues and disrupt the activating bond-network. Both conformational states are also stabilized through context-specific hydrophobic interactions. We show that the functional (activating) asymmetric kinase dimer interface forces a corresponding change in the hydrophobic and hydrophilic interactions characterizing the inactivating bond network, resulting in the motion of the αC-helix through allostery. Some of the clinically identified activating kinase mutations of EGFR also act in a similar fashion, disrupting the inactivating bond network. Our molecular dynamics study reveals a fundamental difference in the sequence of events in EGFR activation in comparison to the Src kinase Hck. This work is funded by NSF grants CBET-0730955 and CBET-0853539.

ErbB kinases and NDF signaling in human prostate cancer cells.

Prostate carcinoma (PCA) is the most commonly diagnosed malignancy in American men. Our knowledge of PCA growth regulation lags behind that of other cancers, such as breast and colon carcinomas. Among receptor tyrosine kinases, the ErbB family is most frequently implicated in neoplasia. We report here the expression of ErbB family kinases and their ligands in PCA cell lines and a xenograft. While ErbB1/EGFR, ErbB2/NEU, and ErbB3 were always observed in a distinct pattern, ErbB4 was not observed. Interestingly, while TGF-alpha was expressed in the majority of PCA lines, the ligand Neu Differentiation Factor/Heregulin (NDF) was expressed only in an immortalized, non-transformed prostate epithelial line. Concomitantly, there was a significant difference in biological response to these ligands. NDF inhibited LNCaP growth and induced an epithelial-like morphological change, in contrast to TGF-alpha, which accelerated cell growth. We also performed the first comprehensive analysis of NDF signaling in a prostate line. LNCaP stimulated with NDF demonstrated crosstalk between ErbB3 and ErbB2 which did not involve ErbB1. NDF also turned on several cascades, including those of PI3-K, ERK/MAPK, mHOG/p38 and JNK/SAPK, but not those of PLCgamma or the STAT family. This signaling pattern is distinct from that of TGF-alpha. The activation of mHOG by ErbB2 or ErbB3 has not been reported, and may contribute to the unusual phenotype. PI3-K activation is characterized by the formation of a striking 'activation complex' with multiple tyrosine-phosphorylated species, including ErbB3. Our studies provide a framework in which to dissect the growth and differentiation signals of prostate cancer cells.