ErbB Family Of Receptor Tyrosine Kinases Research Articles

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

QSAR Study, Molecular Docking, and Pharmacokinetic Analysis of Substituted Dihydropyrimidinone as ErbB2 Inhibitors

ErbB2 is considered to be the preferred dimerization partner among the ErbB receptor tyrosine kinase (RTK) family. They are implicated in the development and progression of several cancer types, majorly breast cancer-related maladies. In the current study, quantitative structure-activity relationship (QSAR), molecular docking approaches and prediction of pharmacokinetic properties were utilized to identify promising ErbB2 inhibitors from a series of dihydropyrimidinone (DHPM) derivatives. The results indicated that the QSAR models of ErbB2 inhibitory activity is robust and has a very good prediction capacity, as exhibited by the value of R which is 0.9194. Furthermore, 6 compounds were shortlisted with potentially high biological activity. These compounds were subjected to drug likeliness, molecular docking and pharmacokinetic evaluations which indicated that the compounds are orally bioavailable and exhibit suitable proficiency as ErbB2 antagonists.

Abstract 2418: Markers for metastasis evaluation of the ErbB family and GDF15 inratosteosarcoma

Abstract 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. Animal model investigations can accelerate the understanding of human diseases and since few models are available for OS, we refined a rat model to ask questions regarding pathways of metastasis and metastasis biomarker predictiveness. The outbred Sprague-Dawley (SD) rat model is an immunocompetent, syngeneic model, as we implant UMR106 OS cells (ATCC), which were originally isolated from a SD OS. We developed a reproducible method to orthotopically implant cells into the tibia microenvironment and to amputate the limb for longitudinal studies. Using young rats, we also modeled pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors which histologically resemble human primary and metastatic osteosarcoma. Using immunohistochemistry methods, we found that primary OS is infiltrated with numerous macrophages with relatively fewer CD3+ T cells. A protein expression survey of these OS cells revealed significant expression of ErbB family receptor tyrosine kinases (ErbB2, ErbB4), which have recently been shown to be anti-inflammatory against macrophages. We also discovered that rats with osteosarcoma have markedly high serum levels of Growth Differentiation Factor 15 (GDF15), also called Macrophage Inhibitory Cytokine 1, a member of the TGF beta family of proteins, which is also frequently expressed with ErbB2 in cancers. GDF15 was measured by ELISA. Western blot analysis showed that GDF15 precursor (pro-peptide) was expressed in primary and metastases. This model will enable studies with anti-ErbB2 or GDF15 pathway inhibition to assess the role of macrophage activation in the setting of pathway inhibition and chemotherapy, as well as how pathway inhibition modulates metastasis incidence. In vivo and invitro, systems will enable the assessment of the role of GDF15 as a potential biomarker of metastasis, compared to tibial radiographic patterns, serum alkaline phosphatase, and lung histopathology. In conclusion, we have an immunocompetent, syngeneic orthotopic osteosarcoma rat model which affectively resembles human osteosarcoma, and can be used for better understanding metastasis mechanisms and metastasis biomarkers. Citation Format: Shun Ishiyama, Bailey West, Hyo-Jeong Han, Xin Guo, Tomoaki Ito, Harumi Saeki, Malcolm V. Brock, Kathleen Gabrielson. Markers for metastasis evaluation of the ErbB family and GDF15 inratosteosarcoma [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 2418.

HER3 expression in metastatic colorectal cancer: Defining the clinicomolecular profile of an emerging target.

3588 Background: The success of tailored systemic therapies in treating distinct molecular subsets of patients (e.g., deficient mismatch repair, BRAF mutant, HER2 amplified) has spurred further exploration of novel targetable subsets within the heterogeneous landscape of metastatic colorectal cancer (mCRC). Human epidermal growth factor receptor 3 [HER3 (ErbB3)], a member of the HER (ErbB) receptor tyrosine kinase family, plays an important role in tumorigenesis and metastases and has emerged as a promising therapeutic target in a diverse array of cancers. For example, patritumab deruxtecan (U3-1402; HER3-DXd) is a HER3-directed antibody drug conjugate that has demonstrated clinically meaningful antitumor activity and acceptable safety profiles in metastatic breast cancer and EGFR-mutated non-small cell lung cancer. There is limited data, however, on the clinicopathological characterization of HER3 expression in mCRC. Methods: Tissue samples (surgical-metastatectomy) (N = 115) were obtained from a clinical cohort of patients (N = 99) with histologically proven mCRC and liver metastases who underwent liver resection with/without perioperative systemic chemotherapy. HER3 expression was analyzed on whole-mount preparations by immunohistochemistry (IHC). Staining was performed and visualized using the HER3 (D22C5) XP Rabbit-mAb (Cell Signaling Technology). Patients were categorized based on membranous intensity score as follows: Low with IHC 0 (absence of staining or staining in < 10% of tumor cells), 1+ (faint/barely perceptible staining in ≥10% of tumor cells) or 2+ (weak to moderate staining in ≥10% of tumor cells), or High with IHC 3+ (strong staining in ≥10% of tumor cells). Clinicomolecular and treatment data, including gender, tumor sidedness, mutational status (RAS or BRAF), and prior chemotherapy were collected by review of patient electronic medical records. Chi-squared (or Fisher’s exact) test were used to determine associations between groups. Overall survival (OS) was calculated using Kaplan-Meier method and compared using log-rank tests. Results: Among 99 analyzed patients, 98 were evaluable for HER3 expression. Of these 25.5%, 26.5%, 40.8% and 7.2% showed HER3 IHC scores of 3+, 2+, 1+ and 0, respectively. No significant association was seen with HER3 expression and clinicopathological variables, mutational status, or prior treatment. Among patients with 2 samples analyzed from the same liver surgery, there was a moderate level of heterogeneity with concordance of 78.5% (kappa 0.43). Patients with high HER3 expression had poorer OS (5-year OS: 52%; median: 90.2 months) compared to low HER3 expression (5-year OS: 85%; median: not reached). Conclusions: In this large cohort of mCRC, HER3 expression was observed in 92.8% of patients and across diverse clinical and molecular features, supporting HER3 as a promising targetable biomarker in a large subset of mCRC.

Zenocutuzumab, a HER2xHER3 Bispecific Antibody, Is Effective Therapy for Tumors Driven by NRG1 Gene Rearrangements.

NRG1 rearrangements encode chimeric ligands that activate the ERBB receptor tyrosine kinase family. Here we show that targeting HER2 and HER3 simultaneously with the bispecific antibody Zeno leads to durable clinical responses in patients with NRG1 fusion-positive cancers and is thus an effective therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1171.

ErbB4-mediated regulation of vasculogenic mimicry capability in breast cancer cells.

ErbB4 is a member of the ErbB receptor tyrosine kinase family. It has both pro‐ and anti‐oncogenic activities in tumors. Vasculogenic mimicry (VM), a phenomenon in which cancer cells form capillary‐like structures without endothelial cells, has been recognized to be a cause of malignant phenotypes in some solid tumors. Here, we used an in vitro VM formation assay, and demonstrated that ErbB4 negatively regulated VM formation in human breast cancer cells. By using CRISPR/Cas9‐mediated gene knockout, we verified that the depletion of endogenous ErbB4 improved the VM formation capability. Although treatment with neuregulin 1 (NRG1), a ligand of ErbB4, induced the phosphorylation of ErbB4 and promoted VM formation in a dose‐dependent manner, it did not induce such activities in kinase‐dead K751M ErbB4‐overexpressing cells. Moreover, we examined the effect of the missense mutation E872K of ErbB4, which has been reported in multiple tumors, on VM formation, and found that the mutation enhanced the basal phosphorylation level and ErbB4‐mediated VM formation in the absence of NRG1 stimulation. Whereas NRG1 stimulated VM formation, excessive activation of ErbB4 induced a negative effect. In E872K ErbB4‐overexpressing cells, but not in wild‐type ErbB4‐overexpressing cells, the number of VM tubes was significantly decreased by low‐dose treatment with the ErbB inhibitor afatinib. Taken together, our findings demonstrated the significance of ErbB4‐mediated VM formation, and suggested the possibility of ErbB4 mutations as effective targets in breast cancer.

Progress in Treatment of Non-Small Cell Lung Cancer Harboring HER2 Aberrations.

Epidermal growth factor receptor 2 (HER2/ErbB2/neu), a member of ErbB receptor tyrosine kinase family, forms homo- or heterodimers with ErbB1 (HER1/EGFR), ErbB3 (HER3), or ErbB4 (HER4), to activate signal transduction pathways and promote proliferation, differentiation and tumorigenesis. Preliminary clinical trials of monoclonal antibodies, antibody conjugates and small-molecule tyrosine kinase inhibitors targeting HER2 have indicated that HER2 is a potential therapeutic target in non-small cell lung cancer (NSCLC). HER2 aberrations in NSCLC patients mainly include mutation, amplification, and overexpression. While there are significant differences in the outcome of NSCLC with these HER2 changes, no consensus has been reached for the incidence, detection method and targeted treatments for the three types of HER2 aberration. HER2 mutation is generally considered to have more clinical relevance and response to HER2-targeted therapies. In this review, we discuss HER2 alterations in NSCLC, including diagnostic challenges and treatment strategies particular to the HER2 mutation.

Directed Molecular Engineering of Mig6 Peptide Selectivity between Proto-oncogene ErbB Family Receptor Tyrosine Kinases

Directed Molecular Engineering of Mig6 Peptide Selectivity between Proto-oncogene ErbB Family Receptor Tyrosine Kinases

CD99-PTPN12 Axis Suppresses Actin Cytoskeleton-Mediated Dimerization of Epidermal Growth Factor Receptor.

Simple SummaryThe epidermal growth factor receptor (EGFR) is activated through growth factor-dependent dimerization accompanied by functional reorganization of the actin cytoskeleton. Lee et al. demonstrate that CD99 activation by agonist ligands inhibits epidermal growth factor (EGF)-induced EGFR dimerization through impairment of cytoskeletal reorganization by protein tyrosine phosphatase non-receptor type 12 (PTPN12)-dependent c-Src/focal adhesion kinase (FAK) inactivation, thereby suppressing breast cancer growth.The epidermal growth factor receptor (EGFR), a member of ErbB receptor tyrosine kinase (RTK) family, is activated through growth factor-induced reorganization of the actin cytoskeleton and subsequent dimerization. We herein explored the molecular mechanism underlying the suppression of ligand-induced EGFR dimerization by CD99 agonists and its relevance to tumor growth in vivo. Epidermal growth factor (EGF) activated the formation of c-Src/focal adhesion kinase (FAK)-mediated intracellular complex and subsequently induced RhoA-and Rac1-mediated actin remodeling, resulting in EGFR dimerization and endocytosis. In contrast, CD99 agonist facilitated FAK dephosphorylation through the HRAS/ERK/PTPN12 signaling pathway, leading to inhibition of actin cytoskeletal reorganization via inactivation of the RhoA and Rac1 signaling pathways. Moreover, CD99 agonist significantly suppressed tumor growth in a BALB/c mouse model injected with MDA-MB-231 human breast cancer cells. Taken together, these results indicate that CD99-derived agonist ligand inhibits epidermal growth factor (EGF)-induced EGFR dimerization through impairment of cytoskeletal reorganization by PTPN12-dependent c-Src/FAK inactivation, thereby suppressing breast cancer growth.

Metabolic regulation of EGFR effector and feedback signaling in pancreatic cancer cells requires K-Ras

Nutrient stress driven by glutamine deficiency activates EGFR signaling in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cells. EGFR signaling in the context of glutamine starvation is thought to be instigated by the transcriptional upregulation of EGFR ligands and functions as an adaptation mechanism to allow PDAC cells to maintain metabolic fitness. Having a clear view of the intricate signaling cascades potentiated by the metabolic induction of EGFR is important in understanding how these effector pathways influence cancer progression. In this study, we examined the complex signaling that occurs in PDAC cells when EGFR is activated by glutamine deprivation. We elucidate that the metabolic activation of EGFR is principally mediated by HB-EGF, and that other members of the ErbB receptor tyrosine kinase family are not activated by glutamine starvation. Additionally, we determine that glutamine depletion-driven EGFR signaling is associated with a specific receptor phosphorylation known to participate in a feedback loop, a process that is dependent on Erk. Lastly, we determine that K-Ras is required for glutamine depletion-induced Erk activation, as well as EGFR feedback phosphorylation, but is dispensable for Akt activation. These data provide important insights into the regulation of EGFR signaling in the context of metabolic stresses.

Abstract C069: The identification of MRTX849, a novel KRASG12C inhibitor under clinical investigation, provides insight toward therapeutic susceptibility of KRAS mutant cancers

Abstract The ability to effectively target mutated KRAS has remained elusive despite decades of research. MRTX849 was identified via structure-based drug design as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties. MRTX849 is presently under evaluation in clinical trials and its discovery and evaluation is disclosed here for the first time. MRTX849 demonstrated selective modification of the mutant cysteine residue at amino acid residue 12 in GDP-bound KRASG12C and inhibited KRAS-dependent signaling in vitro and in vivo. In multiple KRASG12C positive cell lines in vitro, MRTX849 treatment demonstrated covalent modification of mutant KRAS and/or inhibition of active KRAS at concentrations as low as 2 nM with near maximal inhibition observed at 15 nM. In vivo, clear evidence of dose-dependent modification of KRASG12C and inhibition of KRAS-dependent signal transduction was observed in multiple KRASG12C mutant tumor models. MRTX849 demonstrated marked tumor regression in 17 out of 26 (65%) of KRASG12C-positive cell line- and patient-derived xenograft (PDX) models but not in tumor models without KRASG12C mutations. While MRTX849 commonly elicited tumor regression, a subset of models was less sensitive to treatment or exhibited tumor stasis after an initial response phase. Comprehensive pharmacodynamic and pharmacogenomic profiling in these models identified mechanisms implicated in limiting anti-tumor response in refractory models including extrinsic factors impacting KRAS nucleotide cycling and/or signaling pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included upstream activation of ERBB family receptor tyrosine kinases (RTKs) as well as genetic dysregulation of cell cycle transition genes. Based on these observations, combination strategies designed to co-target signaling feedback and bypass pathways were evaluated in multiple tumor models. Afatinib and the SHP2 inhibitor, RMC-4550, in combination with MRTX849 validated these therapeutic hypotheses and the combinations demonstrated marked tumor regression in several tumor models, including models that were refractory to either single agent. MRTX849 in combination with the CDK4/6 inhibitor palbociclib also demonstrated a marked antitumor response; notably in tumor models harboring CDKN2A deletion or other genetic alterations implicated in cell cycle dysregulation. Together, these data indicate the therapeutic susceptibility and broad dependence of KRAS G12C mutation-positive tumors on KRAS for tumor cell growth and survival and provide insight toward the molecular basis of response to single agent and combinatorial therapies. Citation Format: James G Christensen, Jay B Fell, Jill Hallin, Brian Baer, Lars engstrom, James Blake, David Briere, Josh Ballard, Michael Burkhard, John Fischer, Guy Vigers, Ruth Aranda, Vickie Bowcut, Andrew Calinisan, Lauren Hargis, Niranjan Sudhakar, Matt Marx, Peter Olson. The identification of MRTX849, a novel KRASG12C inhibitor under clinical investigation, provides insight toward therapeutic susceptibility of KRAS mutant cancers [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 C069. doi:10.1158/1535-7163.TARG-19-C069

Neuregulin and ErbB expression is regulated by development and sensory experience in mouse visual cortex.

Neuregulins (NRGs) are protein ligands that impact neural development and circuit function. NRGs signal through the ErbB receptor tyrosine kinase family. NRG1/ErbB4 signaling in parvalbumin-expressing (PV) inhibitory interneurons is critical for visual cortical plasticity. There are multiple types of NRGs and ErbBs that can potentially contribute to visual cortical plasticity at different developmental stages. Thus, it is important to understand the normal developmental expression profiles of NRGs and ErbBs in specific neuron types in the visual cortex, and to study whether and how their expression changes in PV inhibitory neurons and excitatory neurons track with sensory perturbation. Cell type-specific translating ribosome affinity purification and qPCR was used to compare mRNA expression of nrg1,2,3,4 and erbB1,2,3,4 in PV and excitatory neurons in mouse visual cortex. We show that the expression of nrg1 and nrg3 decreases in PV neurons at the critical period peak, postnatal day 28 (P28) after monocular deprivation and dark rearing, and in the adult cortex (at P104) after 2-week long dark exposure. In contrast, nrg1 expression by excitatory neurons is unchanged at P28 and P104 following sensory deprivation, whereas nrg3 expression by excitatory neurons shows changes depending on the age and the mode of sensory deprivation. ErbB4 expression in PV neurons remains consistently high and does not appear to change in response to sensory deprivation. These data provide new important details of cell type-specific NRG/ErbB expression in the visual cortex and support that NRG1/ErbB4 signaling is implicated in both critical period and adult visual cortical plasticity.

Combining ERBB family and MET inhibitors is an effective therapeutic strategy in cutaneous malignant melanoma independent of BRAF/NRAS mutation status

Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival; however, relapses are common. A number of receptor tyrosine kinases (RTKs) including EGFR and MET have been reported to be involved in CMM metastasis and in the development of resistance to therapy, targeting the mitogen-activated protein kinase (MAPK pathway). IHC analysis showed that patients with higher MET protein expression had a significantly shorter overall survival. In addition, silencing of MET caused an upregulation of EGFR and p-AKT, which was abrogated by concomitant silencing of MET and EGFR in CMM cells resistant to MAPK-targeting drugs. We therefore explored novel treatment strategies using clinically approved drugs afatinib (ERBB family inhibitor) and crizotinib (MET inhibitor), to simultaneously block MET and ERBB family RTKs. The effects of the combination were assessed in cell culture and spheroid models using established CMM and patient-derived short-term cell lines, and an in vivo xenograft mouse model. The combination had a synergistic effect, promoting cell death, concomitant with a potent downregulation of migratory and invasive capacity independent of their BRAF/NRAS mutational status. Furthermore, the combination attenuated tumor growth rate, as ascertained by the significant reduction of Ki67 expression and induced DNA damage in vivo. Importantly, this combination therapy had minimal therapy-related toxicity in mice. Lastly, the cell cycle G2 checkpoint kinase WEE1 and the RTK IGF1R, non-canonical targets, were altered upon exposure to the combination. Knockdown of WEE1 abrogated the combination-mediated effects on cell migration and proliferation in BRAF mutant BRAF inhibitor-sensitive cells, whereas WEE1 silencing alone inhibited cell migration in NRAS mutant cells. In summary, our results show that afatinib and crizotinib in combination is a promising alternative targeted therapy option for CMM patients, irrespective of BRAF/NRAS mutational status, as well as for cases where resistance has developed towards BRAF inhibitors.

Structure Defines Function: Clinically Relevant Mutations in ErbB Kinases.

The ErbB receptor tyrosine kinase family members EGFR (epidermal growth factor receptor) and Her2 are among the prominent mutated oncogenic drivers of non-small cell lung cancer (NSCLC). Their importance in proliferation, apoptosis, and cell death ultimately renders them hot targets in cancer therapy. Small-molecule tyrosine kinase inhibitors seem well suited to be tailor-made therapeutics for EGFR mutant NSCLC; however, drug resistance mutations limit their success. Against this background, the elucidation and visualization of the three-dimensional structure of cancer-related kinases provide valuable insights into their molecular functions. This field has undergone a revolution because X-ray crystal structure determinations aided structure-based drug design approaches and clarified the effect of activating and resistance-conferring mutations. Here, we present an overview of important mutations affecting EGFR and Her2 and highlight their influence on the kinase domain conformations and active site accessibility.

Abstract LB-275: U3-1402, a novel HER3-targeting antibody-drug conjugate, exhibits its antitumor activity through increased payload intracellular delivery via highly efficient drug internalization

Abstract Background: Human epidermal growth factor receptor 3 (HER3), a member of the ErbB receptor tyrosine kinase family, is overexpressed in a variety of human cancers and plays an important role in cell proliferation and survival. U3-1402 is a novel HER3-targeting antibody-drug conjugate (ADC) consisting of a fully human anti-HER3 antibody (patritumab), a tetrapeptide-based linker, and a topoisomerase I inhibitor payload. It is currently being investigated in clinical trials for HER3-positive breast cancer (phase 1/2) and NSCLC (phase 1). In principle, effective action of ADCs depends on multiple dynamic factors including antigen-specific binding, internalization, trafficking to lysosomes, and payload release, as well as other inherent characteristics such as payload potency and linker stability. Methods: To assess the targeted delivery potential of U3-1402, the HER3-mediated molecular dynamics of U3-1402 (cell surface binding, internalization, trafficking, and payload release) were investigated in 8 cancer cell lines with various HER3 expression levels, along with cell growth inhibition activity. In vitro molecular dynamics were compared between U3-1402 and trastuzumab (anti-HER2 antibody) using the MDA-MB-453 cell line, which expresses both HER2 and HER3. The activity of patritumab (naked antibody of U3-1402) was tested. In vivo efficacy of U3-1402 was also evaluated and immunostaining of HER3 and γH2AX (marker of DNA double-stranded breaks) as well as drug concentration assessment were performed. Results: U3-1402 bound to the cell surface of HER3-positive cell lines in HER3 expression level-dependent manner, and was then internalized into cells. Interestingly, the internalization rate of U3-1402 in MDA-MB-453 cells at 1 h was 64%, which was higher than that observed with trastuzumab (2%). Subsequently, U3-1402 was translocated to the lysosome, followed by payload release, leading to an improved cell growth inhibition compared to that noted with patritumab. In vivo antitumor efficacy was also demonstrated in a HER3-dependent manner in MDA-MB-453 xenograft model. In the study, internalization of HER3 from cell membrane into the intracellular compartment by U3-1402 treatment was observed, followed by increased free payload and γH2AX levels in tumor tissue. Conclusion: U3-1402 has a high internalization property for effective payload delivery to HER3-expressing cancer cells, resulting in a favorable ADC-driven efficacy. Citation Format: Kumiko Koyama, Yuuri Hashimoto, Yasuki Kamai, Yoshinobu Shiose, Manabu Abe, Yuki Kaneda, Naoyuki Maeda, Kenji Hirotani, Yusuke Ogitani, Tsuyoshi Karibe, Takashi Kagari, Kenichi Wakita, Suguru Ueno, Toshinori Agatsuma, Masato Murakami. U3-1402, a novel HER3-targeting antibody-drug conjugate, exhibits its antitumor activity through increased payload intracellular delivery via highly efficient drug internalization [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 LB-275.

Abstract 2541: Mastl regulates EGFR signaling to promote pancreatic cancer progression

Abstract Background: Anti-cancer treatments act primarily by damaging the DNA of cancer cells. Upon DNA damage, cells stop proliferation at cell cycle checkpoints, which provides them time for DNA repair. Hence, pharmacological inhibition of checkpoint kinases in combination with the DNA damaging anti-cancer therapies is now emerging as promising cancer treatment strategy. In this regard, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with a frightening resistance to chemotherapeutic and targeted approaches. Here, along with the KRAS-dependent signaling, receptor tyrosine kinases especially the EGF receptor (EGFR) signaling is strongly upregulated in PDAC. Importantly, Greatwall (Mastl; Microtubule-associated serine/threonine-protein kinase-like) promotes normal G2-mitosis transition and is highly upregulated in multiple cancer types, and have been demonstrated to associate with therapy resistance. However, potential role of Mastl in pancreatic cancer progression/therapy resistance remains unknown. Our central hypothesis is that MASTL expression is (a) involved in cancer progression and intrinsic drug resistance (b) constitutes a potential therapeutic target for PDAC (c) correlates with EGFR expression and regulation. Methods: We used immunoblotting, immunohistochemistry and TCGA database analysis to examine Mastl expression, its association with EGFR in pancreatic cancer progression, and patient survival. Genetic and pharmacological manipulations for Mastl expression were performed and effects on proliferation, apoptosis and cell cycle were determined. Results: A robust increase in Mastl expression characterized pancreatic cancer cells when compared with the non-transformed pancreatic cells. A similar upregulated expression of Mastl was found in samples originating from KPC mouse model of PDAC. Analysis of the human pancreatic cancer samples and the TCGA database strongly supported this outcome and suggested a positive association of the Mastl expression with cancer progression and patient mortality. Interestingly, genetic inhibition of Mastl expression in pancreatic cancer cells not only inhibited the ability of these cells to proliferate and invade through the matrix but also inhibited activation of the ErbB-family of tyrosine kinase receptors. Further studies revealed that overexpressing K-ras/mutant p53 pathway in HPNE cells (untransformed pancreatic cells) modulates Mastl expression, and thus suggested a causal correlation of Mastl with K-Ras/EGFR signaling in pancreatic cancer cells. Conclusion: Our results suggest a cross talk of Mastl with K-ras/EGFR signaling pathway in pancreatic cancer progression. We hypothesize that combinatorial therapy targeting Mastl along with Gemcitabine would overcome the drug resistance in the PDAC. Thus, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors. Citation Format: Iram Fatima, Shailender S. Chauhan, Srijayaprakash Babu Uppada, Geoffrey A. Talmon, Amar B. Singh, Surinder K. Batra, Punita Dhawan. Mastl regulates EGFR signaling to promote pancreatic cancer progression [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 2541.

Cancer Immunotherapy with T Cells Carrying Bispecific Receptors That Mimic Antibodies.

Tumors are inherently heterogeneous in antigen expression, and escape from immune surveillance due to antigen loss remains one of the limitations of targeted immunotherapy. Despite the clinical use of adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells in lymphoblastic leukemia, treatment failure due to epitope loss occurs. Targeting multiple tumor-associated antigens (TAAs) may thus improve the outcome of CAR-T cell therapies. CARs developed to simultaneously target multiple targets are limited by the large size of each single-chain variable fragment and compromised protein folding when several single chains are linearly assembled. Here, we describe single-domain antibody mimics that function within CAR parameters but form a very compact structure. We show that antibody mimics targeting EGFR and HER2 of the ErbB receptor tyrosine kinase family can be assembled into receptor molecules, which we call antibody mimic receptors (amR). These amR can redirect T cells to recognize two different epitopes of the same antigen or two different TAAs in vitro and in vivo.

Cardiomyocyte‐Specific EGFR Deletion Differentially Alters Cardiac Contractility and Remodeling

Epidermal Growth Factor Receptor (EGFR), a member of the Erbb receptor tyrosine kinase family, is essential to the development of multiple tissues and organs. Due to the embryonic lethality of global EGFR deletion, the impact of EGFR signaling in the adult heart, normally or in response to pathological stimuli, has not been well‐explored. Using mice with floxed EGFR alleles crossed with αMHC‐Cre mice, we have generated a cardiomyocyte‐specific constitutive EGFR knockout mouse model (CM‐EGFR‐KO) to address its role in the heart. Compared to their wild‐type (WT) littermate controls, CM‐EGFR‐KO mice displayed age‐related development of cardiac contractile dysfunction, marked by impaired diastolic relaxation and Ca2+ handling at the individual myocyte level, as monitored via echocardiographic, hemodynamic, myocyte contractility and Ca2+ transient analyses. RNASeq analysis of CM‐EGFR‐KO versus WT littermate left ventricular samples revealed temporally‐dependent alterations in the expression of a number of myofilament, Ca2+ handling and fetal genes. The contractile defect initially occurs between 7 and 9 weeks of age and is maintained over time without overt cardiac hypertrophic or fibrotic remodeling until 10 months of age, by which time the mice ultimately succumb to severe heart failure and reduced lifespan. Chronic administration of the β‐adrenergic receptor (βAR) agonist isoproterenol effectively compensated for the contractile dysfunction in CM‐EGFR‐KO mice, an effect that was reversible upon cessation of infusion. Notably, βAR‐mediated cardiomyocyte hypertrophy was attenuated in CM‐EGFR‐KO mice compared to WT littermate controls. Treatment of 16 week‐old CM‐EGFR‐KO mice with AAV9‐cTnT‐EGFR permanently rescued cardiomyocyte‐specific EGFR expression and contractile function, suggesting that the contractile dysfunction phenotype observed in these mice is not secondary to aberrant cardiac development, but is specific to EGFR‐dependent regulation of contractile signaling.Support or Funding InformationNIH grant HL105414 to DGTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 423: Cardiomyocyte-Specific Epidermal Growth Factor Receptor Deletion Differentially Alters Cardiac Contractility and Remodeling During Physiologic versus Pathologic Development

Epidermal Growth Factor Receptor (EGFR), a member of the Erbb receptor tyrosine kinase family, is essential to the development of multiple tissue and organs. Due to the embryonic lethality of global EGFR deletion, the impact of EGFR signaling in the adult heart, normally or in response to pathological stimuli, has not been well-explored. Using recently attained mice with floxed EGFR alleles crossed with αMHC-Cre mice, we have generated a cardiomyocyte-specific constitutive EGFR knockout mouse model (CM-EGFR KO) to address its role in the heart. Compared to their wild-type (WT) littermate controls, CM-EGFR-KO mice displayed age-related development of cardiac dysfunction and remodeling, occurring between 7 and 9 weeks of age, as monitored via echocardiography and immunohistochemistry analyses. Although contractile responsiveness to β-adrenergic receptor stimulation was unaffected by EGFR deletion, RNASeq analysis of CM-EGFR-KO hearts within this timeframe revealed alterations in myofilament protein expression profiles. Additionally, there was enhanced cardiomyocyte death in the 7-9 week window of development, which persisted to 10 months of age, at which time the CM-EGFR KO mice had progressively worsened cardiac function, dilation and cardiomyocyte loss. To investigate the impact of cardiomyocyte-specific EGFR deletion on the response to pathologic stress, we challenged 12-14 week old WT and CM-EGFR KO mice with chronic isoproterenol infusion. Notably, while chronic isoproterenol treatment diminished cardiac function and induced hypertrophic remodeling in WT mouse hearts, CM-EGFR-KO mouse hearts displayed progressively improved contractility and ablated hypertrophy over time, suggesting that EGFR differentially regulates cardiac function and remodeling processes during physiological versus pathological development.

P-106 - The redox-active thiosemicarbazone, Dp44mT promotes NDRG1 to down-regulate oncogenic signaling pathways in cancer

The redox-active thiosemicarbazone, Dp44mT has shown potent anti-cancer activity against various cancers. This occurs by its ability to sequester, bind iron, and also produce reactive oxygen species. The activity of this agent is in part mediated by its ability to upregulate the metastasis suppressor, N-myc Downstream Regulated Gene 1 (NDRG1). NDRG1 inhibits cell proliferation and migration by negatively regulating numerous oncogenic signaling pathways. However, the mechanisms by which it modulates these pathways remain to be elucidated. In this study we examined the effect of NDRG1 and Dp44mT on the ErbB-family of receptor tyrosine kinases namely epidermal growth factor receptor (EGFR), human epidermal growth factor 2 and 3 (HER2 and HER3), as these molecules are key regulators of downstream oncogenic signaling. We demonstrate that NDRG1 and Dp44mT markedly inhibit the expression, localization and activation of EGFR, HER2 and HER3 in PANC-1 human pancreatic cancer cells. Further Dp44mT reduced the expression of these molecules in vivo. This study demonstrates for the first time that the redox-active agent, Dp44mT inhibits the ErbB family of proteins through its upregulation of NDRG1, providing an interesting insight into the mechanisms behind its compelling anti-cancer activity.