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Paul Dent - One of the best experts on this subject based on the ideXlab platform.
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Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling
Journal of cellular physiology, 2020Co-Authors: Paul Dent, Laurence Booth, Andrew Poklepovic, Jennifer Martinez, Daniel D. Von Hoff, John F. HancockAbstract:The irreversible ERBB1/2/4 inhibitor Neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles liminal to the plasma membrane; this effect is enhanced by HDAC inhibitors and is now a Phase I trial (NCT03919292). The combination of Neratinib and HDAC inhibitors killed pancreatic cancer and lymphoma T cells. Neratinib plus HDAC inhibitor exposure was as efficacious as (paclitaxel+gemcitabine) at killing pancreatic cancer cells. Neratinib reduced the phosphorylation of PAK1, Merlin, LATS1/2, AKT, mTOR, p70 S6K, and ERK1/2 which required expression of Rubicon, Beclin1, and Merlin. Neratinib altered pancreatic tumor cell morphology which was associated with MST4 degradation reduced Ezrin phosphorylation and enhanced phosphorylation of MAP4K4 and LATS1/2. Knockdown of the MAP4K4 activator and sensor of membrane rigidity RAP2A reduced basal LATS1/2 and YAP phosphorylation but did not prevent Neratinib from stimulating LATS1/2 or YAP phosphorylation. Beclin1 knockdown prevented MST4 degradation, Ezrin dephosphorylation and Neratinib-induced alterations in tumor cell morphology. Our findings demonstrate that Neratinib enhances LATS1/2 phosphorylation independently of RAP2A/MAP4K4 and that MST4 degradation and Ezrin dephosphorylation may represent a universal trigger for the biological actions of Neratinib.
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Enhanced signaling via ERBB3/PI3K plays a compensatory survival role in pancreatic tumor cells exposed to [Neratinib + valproate]
Cellular signalling, 2020Co-Authors: Paul Dent, Laurence Booth, Andrew Poklepovic, Daniel D. Von Hoff, John F. HancockAbstract:Abstract The ERBB1/2/4 inhibitor Neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles; this effect is enhanced by HDAC inhibitors and the combination of [Neratinib + sodium valproate] is now a phase I trial ( NCT03919292 ). The present studies were performed to understand resistance mechanisms that evolve following [Neratinib + valproate] exposure. Exposure of pancreatic tumor cells to [Neratinib + sodium valproate] initially reduced the expression and phosphorylation of ERBB family receptors, c-MET and c-KIT. Following a 24 h drug exposure and a further 24 h culture in drug free conditions, the effects on c-MET, c-KIT and most ERBB family receptors had returned to near baseline levels. However, the expression and phosphorylation of ERBB3 were increased which was associated with elevated AKT T308 phosphorylation. Knock down of ERBB3 significantly enhanced [Neratinib + valproate] lethality, which was associated with greater inactivation of AKT, mTOR, p70 S6K and ERK1/2. The PI3Kα/δ inhibitor copanlisib also significantly enhanced killing after [Neratinib + valproate] exposure. Copanlisib enhanced [Neratinib + valproate] lethality via autophagosome formation and autophagic flux. Our data argue for further in vivo exploration as to whether copanlisib can be safely combined with [Neratinib + valproate].
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Neratinib inhibits hippo yap signaling reduces mutant k ras expression and kills pancreatic and blood cancer cells
Oncogene, 2019Co-Authors: Paul Dent, Alshad S. Lalani, Laurence Booth, Andrew Poklepovic, Jennifer Martinez, Jane L. Roberts, Junchen Liu, David A Tuveson, John F. HancockAbstract:Prior studies demonstrated that the irreversible ERBB1/2/4 inhibitor Neratinib caused plasma membrane-associated mutant K-RAS to localize in intracellular vesicles, concomitant with its degradation. Herein, we discovered that Neratinib interacted with the chemically distinct irreversible ERBB1/2/4 inhibitor afatinib to reduce expression of ERBB1, ERBB2, K-RAS and N-RAS; this was associated with greater-than-additive cell killing of pancreatic tumor cells. Knock down of Beclin1, ATG16L1, Rubicon or cathepsin B significantly lowered the ability of Neratinib to reduce ERBB1 and K-RAS expression, and to cause tumor cell death. Knock down of ATM-AMPK suppressed vesicle formation and knock down of cathepsin B-AIF significantly reduced Neratinib lethality. PKG phosphorylates K-RAS and HMG CoA reductase inhibitors reduce K-RAS farnesylation both of which remove K-RAS from the plasma membrane, abolishing its activity. Neratinib interacted with the PKG activator sildenafil and the HMG CoA reductase inhibitor atorvastatin to further reduce K-RAS expression, and to further enhance cell killing. Neratinib is also a Ste20 kinase family inhibitor and in carcinoma cells, and hematopoietic cancer cells lacking ERBB1/2/4, it reduced K-RAS expression and the phosphorylation of MST1/3/4/Ezrin by ~ 30%. Neratinib increased LATS1 phosphorylation as well as that of YAP and TAZ also by ~ 30%, caused the majority of YAP to translocate into the cytosol and reduced YAP/TAZ protein levels. Neratinib lethality was enhanced by knock down of YAP. Neratinib, in a Rubicon-dependent fashion, reduced PAK1 phosphorylation and that of its substrate Merlin. Our data demonstrate that Neratinib coordinately suppresses both mutant K-RAS and YAP function to kill pancreatic tumor cells.
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the levels of mutant k ras and mutant n ras are rapidly reduced in a beclin1 atg5 dependent fashion by the irreversible erbb1 2 4 inhibitor Neratinib
Cancer Biology & Therapy, 2018Co-Authors: Laurence Booth, Alshad S. Lalani, Andrew Poklepovic, Jane L. Roberts, Richard E. Cutler, John M Kirkwood, Cindy Sander, Francesca Avogadriconnors, Paul DentAbstract:The FDA approved irreversible inhibitor of ERBB1/2/4, Neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, Neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented Neratinib and Neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor Neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.
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The irreversible ERBB1/2/4 inhibitor Neratinib interacts with the BCL-2 inhibitor venetoclax to kill mammary cancer cells.
Cancer biology & therapy, 2018Co-Authors: Laurence Booth, Alshad S. Lalani, Francesca Avogadri-connors, Andrew Poklepovic, Jane L. Roberts, Richard E. Cutler, Paul DentAbstract:The irreversible ERBB1/2/4 inhibitor, Neratinib, down-regulates the expression of ERBB1/2/4 as well as the levels of MCL-1 and BCL-XL. Venetoclax (ABT199) is a BCL-2 inhibitor. At physiologic concentrations Neratinib interacted in a synergistic fashion with venetoclax to kill HER2 + and TNBC mammary carcinoma cells. This was associated with the drug-combination: reducing the expression and phosphorylation of ERBB1/2/3; in an eIF2α-dependent fashion reducing the expression of MCL-1 and BCL-XL and increasing the expression of Beclin1 and ATG5; and increasing the activity of the ATM-AMPKα-ULK1 S317 pathway which was causal in the formation of toxic autophagosomes. Although knock down of BAX or BAK reduced drug combination lethality, knock down of BAX and BAK did not prevent the drug combination from increasing autophagosome and autolysosome formation. Knock down of ATM, AMPKα, Beclin1 or over-expression of activated mTOR prevented the induction of autophagy and in parallel suppressed tumor cell killing. Knock down of ATM, AMPKα, Beclin1 or cathepsin B prevented the drug-induced activation of BAX and BAK whereas knock down of BID was only partially inhibitory. A 3-day transient exposure of established estrogen-independent HER2 + BT474 mammary tumors to Neratinib or venetoclax did not significantly alter tumor growth whereas exposure to [Neratinib + venetoclax] caused a significant 7-day suppression of growth by day 19. The drug combination neither altered animal body mass nor behavior. We conclude that venetoclax enhances Neratinib lethality by facilitating toxic BH3 domain protein activation via autophagy which enhances the efficacy of Neratinib to promote greater levels of cell killing.
Alshad S. Lalani - One of the best experts on this subject based on the ideXlab platform.
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Comparative analysis of drug response and gene profiling of HER2-targeted tyrosine kinase inhibitors
British Journal of Cancer, 2021Co-Authors: Neil T. Conlon, Jeffrey J. Kooijman, Suzanne J. C. Gerwen, Winfried R. Mulder, Guido J. R. Zaman, Irmina Diala, Lisa D. Eli, Alshad S. Lalani, John Crown, Denis M. CollinsAbstract:Background Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified/mutated in cancer. The tyrosine kinase inhibitors (TKIs) lapatinib, Neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. Direct comparisons of the preclinical efficacy of the TKIs have been limited to small-scale studies. Novel biomarkers are required to define beneficial patient populations. Methods In this study, the anti-proliferative effects of the three TKIs were directly compared using a 115 cancer cell line panel. Novel TKI response/resistance markers were identified through cross-analysis of drug response profiles with mutation, gene copy number and expression data. Results All three TKIs were effective against HER2-amplified breast cancer models; Neratinib showing the most potent activity, followed by tucatinib then lapatinib. Neratinib displayed the greatest activity in HER2 -mutant and EGFR -mutant cells. High expression of HER2 , VTCN1 , CDK12 , and RAC1 correlated with response to all three TKIs. DNA damage repair genes were associated with TKI resistance. BRCA2 mutations were correlated with Neratinib and tucatinib response, and high expression of ATM , BRCA2 , and BRCA1 were associated with Neratinib resistance. Conclusions Neratinib was the most effective HER2-targeted TKI against HER2 -amplified, -mutant, and EGFR -mutant cell lines. This analysis revealed novel resistance mechanisms that may be exploited using combinatorial strategies.
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Abstract A046: Pre-clinical assessment of Neratinib sensitivity and biomarkers of response
Biomarkers, 2019Co-Authors: Neil T. Conlon, Jeffrey J. Kooijman, Guido J. R. Zaman, Irmina Diala, Alshad S. Lalani, John Crown, Denis M. CollinsAbstract: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 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
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Abstract B003: Combinatorial therapies of Neratinib for HER2-amplified cancer
EGFR Her2, 2019Co-Authors: Ming Zhao, Irmina Diala, Stephen Scott, Kurt W. Evans, Erkan Yuca, Turcin Saridogan, Mehmet Esat Demirhan, Bryce P Kirby, Scott Kopetz, Alshad S. LalaniAbstract:Activation of HER2 signaling by amplification or overexpression of ERBB2 (HER2) is associated with the development and progression of breast cancer. Neratinib is an irreversible, pan-HER tyrosine kinase inhibitor which selectively inhibits EGFR, HER2 and HER4. In this preclinical study, we explored the therapeutic efficacy of Neratinib in combination with other clinically relevant targeted agents for HER2-amplified breast cancer in in vitro and in vivo models. HER2-amplified cell lines BT-474, SK-BR-3, and HCC-1954 were sensitive to Neratinib with low nanomolar IC50s. Cell viability and colony formation assays demonstrated synergistic anti-proliferative activity of Neratinib in combination with multiple targeted agents, including the MEK inhibitor trametinib, the PI3Ka inhibitor alpelisib, the mTOR inhibitors everolimus and sapanisertib, and CDK4/6 inhibitor palbociclib. We tested combinatorial efficacy of Neratinib with everolimus, trametinib, and palbociclib in vivo on five HER2-amplified PDXs (two breast, two colorectal and one gastric-esophageal junction cancer), four derived from patients with previous HER2-targeted therapy. The combination of Neratinib with everolimus or trametinib led to at least a 100% increase in median time for tumor doubling in 25% (1 of 4) and 60% (3 of 5) models respectively and was associated with decreased tumor volumes in 50% (2 of 4) and 20% (1 of 5) of the models respectively. The combination of Neratinib with palbociclib significantly enhanced antitumor efficacy compared to single agent treatment in all five PDX models. The combination achieved treatment/control volume ratios of 0.03-0.16 and increased time for tumor doubling more than 100% in all five models. Taken together, our results provide solid preclinical evidence for targeting combinatorial therapies of Neratinib for HER2-amplified cancer. Citation Format: Ming Zhao, Stephen Scott, Kurt Evans, Erkan Yuca, Turcin Saridogan, Mehmet E Demirhan, Bryce P Kirby, Scott Kopetz, Irmina Diala, Alshad S Lalani, Sarina A Piha-Paul, Funda Meric-Bernstam. Combinatorial therapies of Neratinib for HER2-amplified cancer [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 B003. doi:10.1158/1535-7163.TARG-19-B003
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The calcium-sensing receptor: A novel target for treatment and prophylaxis of Neratinib-induced diarrhea.
Pharmacology research & perspectives, 2019Co-Authors: Taras Lysyy, Irmina Diala, Alshad S. Lalani, Elizabeth A. Olek, John P. GeibelAbstract:Diarrhea is one of the most commonly reported adverse effect of hemotherapy and targeted cancer therapies, such as tyrosine kinase inhibitors (TKI), which often significantly impact patient quality of life, morbidity, and mortality. Neratinib is an oral, irreversible pan-HER tyrosine kinase inhibitor, which is clinically active in HER2-positive breast cancer. Diarrhea is the most common side effect of this potent anticancer drug and the reasons for this adverse effect are still largely unclear. We have recently shown that activation of the calcium-sensing Receptor (CaSR) can inhibit secretagogue-induced diarrhea in the colon, therefore we hypothesized that CaSR activation may also mitigate Neratinib-induced diarrhea. Using an established ex vivo model of isolated intestinal segments, we investigated Neratinib-induced fluid secretion and the ability of CaSR activation to abate the secretion. In our study, individual segments of the rat intestine (proximal, middle, distal small intestine, and colon) were procured and perfused intraluminally with various concentrations of Neratinib (10, 50, 100 nmol L-1). In a second set of comparison experiments, intraluminal calcium concentration was modulated (from 1.0 to 5.0 or 7.0 mmol L-1), both pre- and during Neratinib exposure. In a separate series of experiments R-568, a known calcimimetic was used CaSR activation and effect was compared to elevated Ca2+ concentration (5.0 and 7.0 mmol L-1). As a result, CaSR activation with elevated Ca2+ concentration (5.0 and 7.0 mmol L-1) or R-568 markedly reduced Neratinib-induced fluid secretion in a dose-dependent manner. Pre-exposure to elevated luminal calcium solutions (5.0 and 7.0 mmol L-1) also prevented Neratinib-induced fluid secretion. In conclusion, exposure to luminal Neratinib resulted in a pronounced elevation in fluid secretion in the rat intestine. Increasing luminal calcium inhibits the Neratinib-associated fluid secretion in a dose-dependent manner. These results suggest that CaSR activation may be a potent therapeutic target to reduce chemotherapy-associated diarrhea.
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neoadjuvant Neratinib promotes ferroptosis and inhibits brain metastasis in a novel syngeneic model of spontaneous her2 ve breast cancer metastasis
Breast Cancer Research, 2019Co-Authors: Aadya Nagpal, Irmina Diala, Alshad S. Lalani, Sherene Loi, Richard P. Redvers, Xiawei Ling, Scott Ayton, Miriam Fuentes, Elnaz Tavancheh, Steven DavidAbstract:Human epidermal growth factor receptor-2 (HER2)-targeted therapies prolong survival in HER2-positive breast cancer patients. Benefit stems primarily from improved control of systemic disease, but up to 50% of patients progress to incurable brain metastases due to acquired resistance and/or limited permeability of inhibitors across the blood-brain barrier. Neratinib, a potent irreversible pan-tyrosine kinase inhibitor, prolongs disease-free survival in the extended adjuvant setting, and several trials evaluating its efficacy alone or combination with other inhibitors in early and advanced HER2-positive breast cancer patients are ongoing. However, its efficacy as a first-line therapy against HER2-positive breast cancer brain metastasis has not been fully explored, in part due to the lack of relevant pre-clinical models that faithfully recapitulate this disease. Here, we describe the development and characterisation of a novel syngeneic model of spontaneous HER2-positive breast cancer brain metastasis (TBCP-1) and its use to evaluate the efficacy and mechanism of action of Neratinib. TBCP-1 cells were derived from a spontaneous BALB/C mouse mammary tumour and characterised for hormone receptors and HER2 expression by flow cytometry, immunoblotting and immunohistochemistry. Neratinib was evaluated in vitro and in vivo in the metastatic and neoadjuvant setting. Its mechanism of action was examined by transcriptomic profiling, function inhibition assays and immunoblotting. TBCP-1 cells naturally express high levels of HER2 but lack expression of hormone receptors. TBCP-1 tumours maintain a HER2-positive phenotype in vivo and give rise to a high incidence of spontaneous and experimental metastases in the brain and other organs. Cell proliferation/viability in vitro is inhibited by Neratinib and by other HER2 inhibitors, but not by anti-oestrogens, indicating phenotypic and functional similarities to human HER2-positive breast cancer. Mechanistically, Neratinib promotes a non-apoptotic form of cell death termed ferroptosis. Importantly, metastasis assays demonstrate that Neratinib potently inhibits tumour growth and metastasis, including to the brain, and prolongs survival, particularly when used as a neoadjuvant therapy. The TBCP-1 model recapitulates the spontaneous spread of HER2-positive breast cancer to the brain seen in patients and provides a unique tool to identify novel therapeutics and biomarkers. Neratinib-induced ferroptosis provides new opportunities for therapeutic intervention. Further evaluation of Neratinib neoadjuvant therapy is warranted.
Laurence Booth - One of the best experts on this subject based on the ideXlab platform.
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Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling
Journal of cellular physiology, 2020Co-Authors: Paul Dent, Laurence Booth, Andrew Poklepovic, Jennifer Martinez, Daniel D. Von Hoff, John F. HancockAbstract:The irreversible ERBB1/2/4 inhibitor Neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles liminal to the plasma membrane; this effect is enhanced by HDAC inhibitors and is now a Phase I trial (NCT03919292). The combination of Neratinib and HDAC inhibitors killed pancreatic cancer and lymphoma T cells. Neratinib plus HDAC inhibitor exposure was as efficacious as (paclitaxel+gemcitabine) at killing pancreatic cancer cells. Neratinib reduced the phosphorylation of PAK1, Merlin, LATS1/2, AKT, mTOR, p70 S6K, and ERK1/2 which required expression of Rubicon, Beclin1, and Merlin. Neratinib altered pancreatic tumor cell morphology which was associated with MST4 degradation reduced Ezrin phosphorylation and enhanced phosphorylation of MAP4K4 and LATS1/2. Knockdown of the MAP4K4 activator and sensor of membrane rigidity RAP2A reduced basal LATS1/2 and YAP phosphorylation but did not prevent Neratinib from stimulating LATS1/2 or YAP phosphorylation. Beclin1 knockdown prevented MST4 degradation, Ezrin dephosphorylation and Neratinib-induced alterations in tumor cell morphology. Our findings demonstrate that Neratinib enhances LATS1/2 phosphorylation independently of RAP2A/MAP4K4 and that MST4 degradation and Ezrin dephosphorylation may represent a universal trigger for the biological actions of Neratinib.
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Enhanced signaling via ERBB3/PI3K plays a compensatory survival role in pancreatic tumor cells exposed to [Neratinib + valproate]
Cellular signalling, 2020Co-Authors: Paul Dent, Laurence Booth, Andrew Poklepovic, Daniel D. Von Hoff, John F. HancockAbstract:Abstract The ERBB1/2/4 inhibitor Neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles; this effect is enhanced by HDAC inhibitors and the combination of [Neratinib + sodium valproate] is now a phase I trial ( NCT03919292 ). The present studies were performed to understand resistance mechanisms that evolve following [Neratinib + valproate] exposure. Exposure of pancreatic tumor cells to [Neratinib + sodium valproate] initially reduced the expression and phosphorylation of ERBB family receptors, c-MET and c-KIT. Following a 24 h drug exposure and a further 24 h culture in drug free conditions, the effects on c-MET, c-KIT and most ERBB family receptors had returned to near baseline levels. However, the expression and phosphorylation of ERBB3 were increased which was associated with elevated AKT T308 phosphorylation. Knock down of ERBB3 significantly enhanced [Neratinib + valproate] lethality, which was associated with greater inactivation of AKT, mTOR, p70 S6K and ERK1/2. The PI3Kα/δ inhibitor copanlisib also significantly enhanced killing after [Neratinib + valproate] exposure. Copanlisib enhanced [Neratinib + valproate] lethality via autophagosome formation and autophagic flux. Our data argue for further in vivo exploration as to whether copanlisib can be safely combined with [Neratinib + valproate].
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Neratinib inhibits hippo yap signaling reduces mutant k ras expression and kills pancreatic and blood cancer cells
Oncogene, 2019Co-Authors: Paul Dent, Alshad S. Lalani, Laurence Booth, Andrew Poklepovic, Jennifer Martinez, Jane L. Roberts, Junchen Liu, David A Tuveson, John F. HancockAbstract:Prior studies demonstrated that the irreversible ERBB1/2/4 inhibitor Neratinib caused plasma membrane-associated mutant K-RAS to localize in intracellular vesicles, concomitant with its degradation. Herein, we discovered that Neratinib interacted with the chemically distinct irreversible ERBB1/2/4 inhibitor afatinib to reduce expression of ERBB1, ERBB2, K-RAS and N-RAS; this was associated with greater-than-additive cell killing of pancreatic tumor cells. Knock down of Beclin1, ATG16L1, Rubicon or cathepsin B significantly lowered the ability of Neratinib to reduce ERBB1 and K-RAS expression, and to cause tumor cell death. Knock down of ATM-AMPK suppressed vesicle formation and knock down of cathepsin B-AIF significantly reduced Neratinib lethality. PKG phosphorylates K-RAS and HMG CoA reductase inhibitors reduce K-RAS farnesylation both of which remove K-RAS from the plasma membrane, abolishing its activity. Neratinib interacted with the PKG activator sildenafil and the HMG CoA reductase inhibitor atorvastatin to further reduce K-RAS expression, and to further enhance cell killing. Neratinib is also a Ste20 kinase family inhibitor and in carcinoma cells, and hematopoietic cancer cells lacking ERBB1/2/4, it reduced K-RAS expression and the phosphorylation of MST1/3/4/Ezrin by ~ 30%. Neratinib increased LATS1 phosphorylation as well as that of YAP and TAZ also by ~ 30%, caused the majority of YAP to translocate into the cytosol and reduced YAP/TAZ protein levels. Neratinib lethality was enhanced by knock down of YAP. Neratinib, in a Rubicon-dependent fashion, reduced PAK1 phosphorylation and that of its substrate Merlin. Our data demonstrate that Neratinib coordinately suppresses both mutant K-RAS and YAP function to kill pancreatic tumor cells.
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the levels of mutant k ras and mutant n ras are rapidly reduced in a beclin1 atg5 dependent fashion by the irreversible erbb1 2 4 inhibitor Neratinib
Cancer Biology & Therapy, 2018Co-Authors: Laurence Booth, Alshad S. Lalani, Andrew Poklepovic, Jane L. Roberts, Richard E. Cutler, John M Kirkwood, Cindy Sander, Francesca Avogadriconnors, Paul DentAbstract:The FDA approved irreversible inhibitor of ERBB1/2/4, Neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, Neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented Neratinib and Neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor Neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.
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The irreversible ERBB1/2/4 inhibitor Neratinib interacts with the BCL-2 inhibitor venetoclax to kill mammary cancer cells.
Cancer biology & therapy, 2018Co-Authors: Laurence Booth, Alshad S. Lalani, Francesca Avogadri-connors, Andrew Poklepovic, Jane L. Roberts, Richard E. Cutler, Paul DentAbstract:The irreversible ERBB1/2/4 inhibitor, Neratinib, down-regulates the expression of ERBB1/2/4 as well as the levels of MCL-1 and BCL-XL. Venetoclax (ABT199) is a BCL-2 inhibitor. At physiologic concentrations Neratinib interacted in a synergistic fashion with venetoclax to kill HER2 + and TNBC mammary carcinoma cells. This was associated with the drug-combination: reducing the expression and phosphorylation of ERBB1/2/3; in an eIF2α-dependent fashion reducing the expression of MCL-1 and BCL-XL and increasing the expression of Beclin1 and ATG5; and increasing the activity of the ATM-AMPKα-ULK1 S317 pathway which was causal in the formation of toxic autophagosomes. Although knock down of BAX or BAK reduced drug combination lethality, knock down of BAX and BAK did not prevent the drug combination from increasing autophagosome and autolysosome formation. Knock down of ATM, AMPKα, Beclin1 or over-expression of activated mTOR prevented the induction of autophagy and in parallel suppressed tumor cell killing. Knock down of ATM, AMPKα, Beclin1 or cathepsin B prevented the drug-induced activation of BAX and BAK whereas knock down of BID was only partially inhibitory. A 3-day transient exposure of established estrogen-independent HER2 + BT474 mammary tumors to Neratinib or venetoclax did not significantly alter tumor growth whereas exposure to [Neratinib + venetoclax] caused a significant 7-day suppression of growth by day 19. The drug combination neither altered animal body mass nor behavior. We conclude that venetoclax enhances Neratinib lethality by facilitating toxic BH3 domain protein activation via autophagy which enhances the efficacy of Neratinib to promote greater levels of cell killing.
Andrew Poklepovic - One of the best experts on this subject based on the ideXlab platform.
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Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling
Journal of cellular physiology, 2020Co-Authors: Paul Dent, Laurence Booth, Andrew Poklepovic, Jennifer Martinez, Daniel D. Von Hoff, John F. HancockAbstract:The irreversible ERBB1/2/4 inhibitor Neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles liminal to the plasma membrane; this effect is enhanced by HDAC inhibitors and is now a Phase I trial (NCT03919292). The combination of Neratinib and HDAC inhibitors killed pancreatic cancer and lymphoma T cells. Neratinib plus HDAC inhibitor exposure was as efficacious as (paclitaxel+gemcitabine) at killing pancreatic cancer cells. Neratinib reduced the phosphorylation of PAK1, Merlin, LATS1/2, AKT, mTOR, p70 S6K, and ERK1/2 which required expression of Rubicon, Beclin1, and Merlin. Neratinib altered pancreatic tumor cell morphology which was associated with MST4 degradation reduced Ezrin phosphorylation and enhanced phosphorylation of MAP4K4 and LATS1/2. Knockdown of the MAP4K4 activator and sensor of membrane rigidity RAP2A reduced basal LATS1/2 and YAP phosphorylation but did not prevent Neratinib from stimulating LATS1/2 or YAP phosphorylation. Beclin1 knockdown prevented MST4 degradation, Ezrin dephosphorylation and Neratinib-induced alterations in tumor cell morphology. Our findings demonstrate that Neratinib enhances LATS1/2 phosphorylation independently of RAP2A/MAP4K4 and that MST4 degradation and Ezrin dephosphorylation may represent a universal trigger for the biological actions of Neratinib.
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Enhanced signaling via ERBB3/PI3K plays a compensatory survival role in pancreatic tumor cells exposed to [Neratinib + valproate]
Cellular signalling, 2020Co-Authors: Paul Dent, Laurence Booth, Andrew Poklepovic, Daniel D. Von Hoff, John F. HancockAbstract:Abstract The ERBB1/2/4 inhibitor Neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles; this effect is enhanced by HDAC inhibitors and the combination of [Neratinib + sodium valproate] is now a phase I trial ( NCT03919292 ). The present studies were performed to understand resistance mechanisms that evolve following [Neratinib + valproate] exposure. Exposure of pancreatic tumor cells to [Neratinib + sodium valproate] initially reduced the expression and phosphorylation of ERBB family receptors, c-MET and c-KIT. Following a 24 h drug exposure and a further 24 h culture in drug free conditions, the effects on c-MET, c-KIT and most ERBB family receptors had returned to near baseline levels. However, the expression and phosphorylation of ERBB3 were increased which was associated with elevated AKT T308 phosphorylation. Knock down of ERBB3 significantly enhanced [Neratinib + valproate] lethality, which was associated with greater inactivation of AKT, mTOR, p70 S6K and ERK1/2. The PI3Kα/δ inhibitor copanlisib also significantly enhanced killing after [Neratinib + valproate] exposure. Copanlisib enhanced [Neratinib + valproate] lethality via autophagosome formation and autophagic flux. Our data argue for further in vivo exploration as to whether copanlisib can be safely combined with [Neratinib + valproate].
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Neratinib inhibits hippo yap signaling reduces mutant k ras expression and kills pancreatic and blood cancer cells
Oncogene, 2019Co-Authors: Paul Dent, Alshad S. Lalani, Laurence Booth, Andrew Poklepovic, Jennifer Martinez, Jane L. Roberts, Junchen Liu, David A Tuveson, John F. HancockAbstract:Prior studies demonstrated that the irreversible ERBB1/2/4 inhibitor Neratinib caused plasma membrane-associated mutant K-RAS to localize in intracellular vesicles, concomitant with its degradation. Herein, we discovered that Neratinib interacted with the chemically distinct irreversible ERBB1/2/4 inhibitor afatinib to reduce expression of ERBB1, ERBB2, K-RAS and N-RAS; this was associated with greater-than-additive cell killing of pancreatic tumor cells. Knock down of Beclin1, ATG16L1, Rubicon or cathepsin B significantly lowered the ability of Neratinib to reduce ERBB1 and K-RAS expression, and to cause tumor cell death. Knock down of ATM-AMPK suppressed vesicle formation and knock down of cathepsin B-AIF significantly reduced Neratinib lethality. PKG phosphorylates K-RAS and HMG CoA reductase inhibitors reduce K-RAS farnesylation both of which remove K-RAS from the plasma membrane, abolishing its activity. Neratinib interacted with the PKG activator sildenafil and the HMG CoA reductase inhibitor atorvastatin to further reduce K-RAS expression, and to further enhance cell killing. Neratinib is also a Ste20 kinase family inhibitor and in carcinoma cells, and hematopoietic cancer cells lacking ERBB1/2/4, it reduced K-RAS expression and the phosphorylation of MST1/3/4/Ezrin by ~ 30%. Neratinib increased LATS1 phosphorylation as well as that of YAP and TAZ also by ~ 30%, caused the majority of YAP to translocate into the cytosol and reduced YAP/TAZ protein levels. Neratinib lethality was enhanced by knock down of YAP. Neratinib, in a Rubicon-dependent fashion, reduced PAK1 phosphorylation and that of its substrate Merlin. Our data demonstrate that Neratinib coordinately suppresses both mutant K-RAS and YAP function to kill pancreatic tumor cells.
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the levels of mutant k ras and mutant n ras are rapidly reduced in a beclin1 atg5 dependent fashion by the irreversible erbb1 2 4 inhibitor Neratinib
Cancer Biology & Therapy, 2018Co-Authors: Laurence Booth, Alshad S. Lalani, Andrew Poklepovic, Jane L. Roberts, Richard E. Cutler, John M Kirkwood, Cindy Sander, Francesca Avogadriconnors, Paul DentAbstract:The FDA approved irreversible inhibitor of ERBB1/2/4, Neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, Neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented Neratinib and Neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor Neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.
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The irreversible ERBB1/2/4 inhibitor Neratinib interacts with the BCL-2 inhibitor venetoclax to kill mammary cancer cells.
Cancer biology & therapy, 2018Co-Authors: Laurence Booth, Alshad S. Lalani, Francesca Avogadri-connors, Andrew Poklepovic, Jane L. Roberts, Richard E. Cutler, Paul DentAbstract:The irreversible ERBB1/2/4 inhibitor, Neratinib, down-regulates the expression of ERBB1/2/4 as well as the levels of MCL-1 and BCL-XL. Venetoclax (ABT199) is a BCL-2 inhibitor. At physiologic concentrations Neratinib interacted in a synergistic fashion with venetoclax to kill HER2 + and TNBC mammary carcinoma cells. This was associated with the drug-combination: reducing the expression and phosphorylation of ERBB1/2/3; in an eIF2α-dependent fashion reducing the expression of MCL-1 and BCL-XL and increasing the expression of Beclin1 and ATG5; and increasing the activity of the ATM-AMPKα-ULK1 S317 pathway which was causal in the formation of toxic autophagosomes. Although knock down of BAX or BAK reduced drug combination lethality, knock down of BAX and BAK did not prevent the drug combination from increasing autophagosome and autolysosome formation. Knock down of ATM, AMPKα, Beclin1 or over-expression of activated mTOR prevented the induction of autophagy and in parallel suppressed tumor cell killing. Knock down of ATM, AMPKα, Beclin1 or cathepsin B prevented the drug-induced activation of BAX and BAK whereas knock down of BID was only partially inhibitory. A 3-day transient exposure of established estrogen-independent HER2 + BT474 mammary tumors to Neratinib or venetoclax did not significantly alter tumor growth whereas exposure to [Neratinib + venetoclax] caused a significant 7-day suppression of growth by day 19. The drug combination neither altered animal body mass nor behavior. We conclude that venetoclax enhances Neratinib lethality by facilitating toxic BH3 domain protein activation via autophagy which enhances the efficacy of Neratinib to promote greater levels of cell killing.
Francesca Avogadri-connors - One of the best experts on this subject based on the ideXlab platform.
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Extended Adjuvant Therapy with Neratinib Plus Fulvestrant Blocks ER/HER2 Crosstalk and Maintains Complete Responses of ER+/HER2+ Breast Cancers: Implications to the ExteNET Trial
Clinical cancer research : an official journal of the American Association for Cancer Research, 2018Co-Authors: Dhivya R. Sudhan, Angel Guerrero-zotano, Paula Gonzalez Ericsson, Luigi Formisano, Luis J. Schwarz, Mellissa J. Nixon, Sarah Croessmann, Melinda E. Sanders, Justin M. Balko, Francesca Avogadri-connorsAbstract:Purpose: The phase III ExteNET trial showed improved invasive disease-free survival in patients with HER2+ breast cancer treated with Neratinib versus placebo after trastuzumab-based adjuvant therapy. The benefit from Neratinib appeared to be greater in patients with ER+/HER2+ tumors. We thus sought to discover mechanisms that may explain the benefit from extended adjuvant therapy with Neratinib. Experimental Design: Mice with established ER+/HER2+ MDA-MB-361 tumors were treated with paclitaxel plus trastuzumab ± pertuzumab for 4 weeks, and then randomized to fulvestrant ± Neratinib treatment. The benefit from Neratinib was evaluated by performing gene expression analysis for 196 ER targets, ER transcriptional reporter assays, and cell-cycle analyses. Results: Mice receiving “extended adjuvant” therapy with fulvestrant/Neratinib maintained a complete response, whereas those treated with fulvestrant relapsed rapidly. In three ER+/HER2+ cell lines (MDA-MB-361, BT-474, UACC-893) but not in ER+/HER2− MCF7 cells, treatment with Neratinib induced ER reporter transcriptional activity, whereas treatment with fulvestrant resulted in increased HER2 and EGFR phosphorylation, suggesting compensatory reciprocal crosstalk between the ER and ERBB RTK pathways. ER transcriptional reporter assays, gene expression, and immunoblot analyses showed that treatment with Neratinib/fulvestrant, but not fulvestrant, potently inhibited growth and downregulated ER reporter activity, P-AKT, P-ERK, and cyclin D1 levels. Finally, similar to Neratinib, genetic and pharmacologic inactivation of cyclin D1 enhanced fulvestrant action against ER+/HER2+ breast cancer cells. Conclusions: These data suggest that ER blockade leads to reactivation of ERBB RTKs and thus extended ERBB blockade is necessary to achieve durable clinical outcomes in patients with ER+/HER2+ breast cancer.
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Abstract 1828: Hyperactivation of mTORC1 drives acquired resistance to the pan HER tyrosine kinase inhibitor Neratinib in HER2 mutant cancers
Experimental and Molecular Therapeutics, 2018Co-Authors: Dhivya R. Sudhan, Alshad S. Lalani, Francesca Avogadri-connors, Angel Guerrero-zotano, Luigi Formisano, Yan Guo, Richard E. Cutler, Ariella B. Hanker, Qi Liu, Richard A. BryceAbstract:Background: Tumor genomic profiling has identified patients with cancers harboring activating ERBB2 (HER2) mutations that are sensitive to HER2 targeted therapies. In the SUMMIT phase II ‘basket9 trial, a subset of patients with ERBB2 mutant cancers have exhibited significant clinical benefit from treatment with the pan-HER irreversible tyrosine kinase inhibitor (TKI) Neratinib. However, durable responses to Neratinib are few, suggesting mechanisms of de novo and acquired drug resistance. Thus, we sought to identify druggable mechanisms of resistance to Neratinib. Methods: We utilized 5637 bladder cancer (with HER2S310F) and OVCAR8 ovarian cancer (with HER2G776V) cells. Drug resistant cells were developed by exposing cells to increasing concentrations of Neratinib over 6 months (5637, 600 nM; OVCAR8, 2 µM). Neratinib resistant H1781 lung cancer cells (with HER2G776>VC) and MCF7 breast cancer cells (with L755S or V777L) knock-in mutations are currently being developed. For immunoblot and drug sensitivity assays, Neratinib resistant cells were maintained drug-free for 96 hours and then retreated with Neratinib and other inhibitors. Candidate pathways/genes driving Neratinib resistance were identified by performing RNA sequencing and whole exome sequencing in drug-resistant and -sensitive cells. Results: Neratinib-resistant 5637 and OVCAR8 cells were cross-resistant to the HER2 TKIs afatinib and lapatinib. Immunoblot analysis of both cells treated with Neratinib showed effective suppression of HER2, EGFR and HER3 phosphorylation. However, they exhibited a striking increase in S6 kinase (S6K) activity and S6 phosphorylation compared to drug-sensitive parental cells, which was maintained in the presence of supra-pharmacological levels of Neratinib (1 µM). S6 phosphorylation and viability of drug resistant cells was completely ablated by the combination of Neratinib and the mTORC1 inhibitor everolimus, but not with the PI3Kα inhibitor alpelisib, the pan-PI3K inhibitor buparlisib, or the AKT inhibitor MK-2206, suggesting PI3K- and AKT-independent activation of mTORC1. Gene set enrichment analysis (GSEA) of RNA seq data from the drug-resistant cells revealed significant enrichment of K-Ras pathway components in addition to mTORC1 pathway. Consistent with these results, whole exome sequencing revealed activating alterations of the Ras pathway including a truncating mutation in RASA2 and a P200L mutation in PIK3CA Ras binding domain; thereby suggesting potential Ras mediated mTOR activation driving Neratinib resistance. Studies are underway to confirm the contribution of Ras pathway in mTOR mediated Neratinib resistance. Conclusions: These data suggest that hyperactivation of mTORC1 promotes acquired resistance to Neratinib across histologically distinct ERBB2-mutant cancers. Citation Format: Dhivya R. Sudhan, Ariella B. Hanker, Angel Guerrero-Zotano, Luigi Formisano, Yan Guo, Qi Liu, Francesca Avogadri-Connors, Richard E. Cutler, Alshad S. Lalani, Richard Bryce, Alan Auerbach, 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 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1828.
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Abstract 825: Identification of molecular therapeutic targets that enhance the antitumor activity of Neratinib in breast cancer cells
Experimental and Molecular Therapeutics, 2018Co-Authors: Bora Lim, Debu Tripathy, Francesca Avogadri-connors, Richard E. Cutler, Jangsoon Lee, Troy Pearson, Liu Huey, Minha Hwang, Kuicheon Choi, Naoto T. UenoAbstract:Background: Aberrant expression of members of the ErbB family of receptor tyrosine kinases and its downstream pathways is known to play pivotal roles in breast cancer malignancy. HER2-positive breast cancer has an aggressive nature that is driven by HER2 gene amplification. Inflammatory breast cancer is a rare but the most aggressive clinical subtype of breast cancer. Triple-negative breast cancer is an aggressive receptor subtype of breast cancer. More than 30% of cases of each of these groups express EGFR. Emerging therapeutic resistance continues to be a challenge in recurrent and/or metastatic disease. Neratinib is a potent irreversible EGFR, HER2, and HER4 tyrosine kinase inhibitor that blocks signal transduction generated via these receptors. In this study, we sought to identify the optimal targets in different subtypes of breast cancer and synergistic partners to maximize the antitumor effect of Neratinib against breast cancer cells in vitro and in vivo. Methods: Expression of EGFR and HER2 was assessed via Western blot in 31 breast cancer cell lines, including HER2-targeted drug-resistant cell lines (14 triple-negative breast cancer and 10 HER2-positive). The antiproliferative effects of Neratinib were measured using cell proliferation and anchorage-independent colony-formation (soft agar) assays. A reverse-phase protein array was used to profile and validate the signaling networks induced by Neratinib-based treatment. To identify the synergistic targets, RNA interference screening of a 779-kinome library was performed under multicellular tumor spheroid culture conditions. Results: In vitro proliferation data demonstrated that Neratinib had a nanomolar range of half-maximal inhibitory concentrations in most of the tested cell lines, including T-DM1-resistant HER-2 breast cancer cell lines. Neratinib inhibited colony formation in a dose-dependent manner (P = 0.0001). Reverse-phase protein array data revealed that the efficacy of Neratinib correlated with phosphorylated EGFR (R2 = 0.6055) or phosphorylated HER2 (R2 = 0.6734) expression level. Statistical analysis identified the 25 most relevant targets in the RNA interference screening, which we will validate for combinational antitumor effects with Neratinib in in vitro and in vivo studies. Conclusion: We determined the efficacy of Neratinib and identified target molecules to enhance Neratinib efficacy in different breast cancer subtype cell lines with ErbB alterations. Our data inform new combined treatment strategies with Neratinib for breast cancer that will lead to innovative designs using optimized Neratinib combinations in clinical trials. Citation Format: Bora Lim, Jangsoon Lee, Troy Pearson, Liu Huey, Minha Hwang, Kuicheon Choi, Francesca Avogadri-Connors, Richard E. Cutler, Debu Tripathy, Naoto T. Ueno. Identification of molecular therapeutic targets that enhance the antitumor activity of Neratinib in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 825.
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Abstract 3931: Neratinib-induced gene expression profile in breast cancer cells: A comprehensive transcriptome investigation
Experimental and Molecular Therapeutics, 2018Co-Authors: Yuxin Cui, Alshad S. Lalani, Francesca Avogadri-connors, Richard E. Cutler, Richard P. Bryce, Alwyn Dart, Sioned Owen, Wen Guo JiangAbstract:Breast cancer can be classified into complicated malignant subtypes based on advances of clinical and research evidence. There are still unmet needs for the more effective treatment of breast cancer patients under specific pathological conditions (stages/subtypes). Neratinib is considered as a second-generation inhibitor of the EGFR family members (EGFR, HER2 and HER4) of receptor kinases. It can irreversibly inhibit the EGFR and HER2 tyrosine kinases by targeting a cysteine residue in the ATP-binding site of the receptor. Recently Neratinib has been approved by FDA for the extended adjuvant treatment of early-stage HER2+ breast cancer. In this study, to further dissect the molecular mechanisms of Neratinib, the global gene expression profiling analysis of breast cancer cells treated with Neratinib was performed using RNA AmpliSeq transcriptome (including 20816 gene candidates) in the Ion PI sequencing system. We selected two breast cancer cell lines, the triple negative MDA-MB-231 cell line and the low HER2-expressing, but not-amplified, ER+ MCF-7 cell line. The RNA-Seq data show differential gene profiling heatmap from these two breast cancer cell lines. The RNA-Seq data confirmed the downregulation of HER2 in MCF-7 cells, but there was no change of gene expression of EGFR, HER3 and HER4. In both MDA—MB-231 and MCF-7 cell lines, there was a panel of upregulating genes including KIAA1024, ZNF550, MESDC1, TMC8, ZNF524, AGBL2, HIST2H2BC and PPARGC1B. In contrast, with response to Neratinib, another cluster of genes was downregulated in both cell lines including TAS2R5, KRT14, VWCE, LY9, PAPLN and STK4-AS1. In MDA-MB-231 cells, Neratinib intended to regulate LPS/IL-1 and eNOS mediated signaling pathways. However, in MCF-7 cells, Neratinib regulated ephrin receptor (EPH) signalling and PPARα activation. We then investigated the possible HER2-independent effect of Neratinib in breast cancer cells such as regulation of EPH. Our data using the electric cell-substrate impedance sensing (ECIS) indicated that MCF-7 cells responded to an EphB4 inhibitor, NVP-HPG-712, by a marked reduction of cellular migration, in contrast to MDA MB-231 which does not respond to the inhibitor. Similarly, MCF-7 cells also responded to the combinational treatment of Neratinib and NVP-HPG-712 with a decrease in cellular migration. Again, MDA-MB-231 failed to yield a response. In summary, differential analysis of transcripts by high throughput RNA-seq could be valuable to provide deeper insight into the gene regulation and signaling pathway prediction of breast cancer cells with response to Neratinib when the large scale of patient samples and multiple types of breast cancer cell lines are integrated. Citation Format: Yuxin Cui, Alwyn Dart, Richard E. Cutler, Alshad S. Lalani, Francesca Avogadri-Connors, Richard P. Bryce, Sioned Owen, Wen G. Jiang. Neratinib-induced gene expression profile in breast cancer cells: A comprehensive transcriptome investigation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3931.
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Abstract 4778: Assessing efficacy of Neratinib in HER2-driven lung cancer
Experimental and Molecular Therapeutics, 2018Co-Authors: Shengwu Liu, Francesca Avogadri-connors, Ting Chen, Jiehui Deng, Mari Kuraguchi, Cam Anh Tran, Paul Kirschmeier, Richard E. CutlerAbstract:HER2 mutations have been classified as oncogenic drivers that are present in 2-4% of lung adenocarcinomas. However, due to limited preclinical studies and clinical trials, there is still no available standard of care for this group of lung cancer patients. To fulfill the clinical need for targeting HER2 mutations in lung cancer patients, we performed a comprehensive preclinical study to evaluate the efficacy of Neratinib, an irreversible small-molecule pan-HER tyrosine kinase inhibitor that potently binds and inhibits EGFR, HER2, and HER4. We used the previously described genetically modified mouse model (GEMM) of lung adenocarcinomas driven by the major mutant form of human HER2 exon 20 insertions (A775_G776insYVMA) for testing Neratinib either as a single agent or in combination with other therapies including trastuzumab in vivo. Treatment efficacy was evaluated based on both monitoring tumor volume changes and survival. Magnetic resonance imaging (MRI) was used for quantifying tumor burden. Tolerability and the overall health of animals were evaluated based on body weight change. We found that Neratinib treatment alone provided a modest yet significant response in inhibiting HER2 mutant lung tumors in this model. Pharmacodynamic studies showed that Neratinib effectively abolished HER2 phosphorylation and inhibited major downstream signaling targets such as pAKT and pERK. Moreover, combination therapy with Neratinib and trastuzumab showed a greater treatment effect by reducing tumor burden with significantly longer progression-free survival and overall survival compared to single agents tested. In all Neratinib-based regimens, body weight of treated animals remained stable. On this basis, our study provides a strong rationale to test the combination of Neratinib with trastuzumab for the treatment of HER2 mutant NSCLC patients. Citation Format: Shuai Li, Shengwu Liu, Ting Chen, Jiehui Deng, Min Wu, Mari Kuraguchi, Cam Anh Tran, Paul T. Kirschmeier, Francesca Avogadri-Connors, Richard E. Cutler, Alshad S. Lalani, Kwok-Kin Wong. Assessing efficacy of Neratinib in HER2-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4778.