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Joseph M Amann
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MA17 - Molecular Mechanisms and Therapies (ID 143)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:Eloisa Jantus-Lewintre, Hongbin Ji
- Coordinates: 9/09/2019, 15:45 - 17:15, Melbourne (1991)
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MA17.02 - Identify Vulnerable Pathways and Improve Treatment Outcomes in LKB1-Deficient Lung Tumors (Now Available) (ID 2067)
15:45 - 17:15 | Author(s): Joseph M Amann
- Abstract
- Presentation
Background
The LKB1 tumor suppressor is inactivated in about 20% of non-small cell lung cancers (NSCLC) by mutations. Cancer cells with LKB1 deficiency exert complex effects on signal transduction and transcriptional regulation, which may cause these cells more susceptible to certain therapies comparing to cells with intact LKB1 function. Phenformin, an antidiabetic medicine from the biguanides class, has shown activities against NSCLC. Phenformin as a single agent has been shown to reduce tumor burden and prolonged survival in Kras;Lkb1 compound mutant mice but not Kras;p53 mice, suggesting specific activities in tumor with LKB1 deficiency. Currently patients with unresectable locally advanced NSCLC are treated standardly with concurrent chemoradiotherapy followed by checkpoint inhibitor, durvalumab. In this project, we test treatment sensitivity to radiotherapy and/or phenformin in lung cancer cells with intact or deficient LKB1.
Method
Human lung cancer cell lines described below were used in (1) clonogenic survival assays as well as (2) generating tumor xenograft on nude mice for tumor growth delay experiments. A549, HCC15 and Calu-1 cell lines obtained from ATCC were cultured in RPMI1640 containing 5% FBS, without antibiotics. A549 cells (LKB1 deficient, TP53 WT and KRAS mutated) or HCC15 (LKB1 deficient, TP53 mutated and KRAS WT) were transfected with empty vector or WT LKB1 or LKB1-K78I plasmids; Calu-1 (LKB1 WT, KRAS mutated and p53 deleted) transfected with empty vector or LKB1 CRIPR KO were generated as described previously.
Result
A549 cells with transfected WT-LKB1 were significantly more resistant to ionizing radiation (IR) induced cell kill (8.7% survival at 8 Gy) comparing to cells transfected with empty vector (3.7%) or kinase-dead LKB1 genes (4.2%). Similarly, HCC15 cells with transfected WT-LKB1 are significantly more resistant to IR induced cell kill (7.5%) comparing to cells transfected with empty vector (4.1%) or kinase-dead LKB1 genes (3.4%). Calu-1 cells harbor WT LKB1, and it is significantly more resistant to IR induced cell kill (8.3%) comparing to their counterpart with LKB1 KO (Calu-1 transfected with LKB1 CRIPR KO) (4.1%). When A549 cells were pretreated with 30 μM phenformin prior to, during and after IR, there was no change in survival in cells transfected with WT LKB1; however there was significant further reduction in survival in cells transfected with empty vector (LKB1 deficient). Xenograft tumors were generated in nude mice with A549 cells with the above genetic alterations. There was significant further tumor growth delay in those with A549 with deficient LKB1 comparing to those with A549 with WT LKB1 gene add-back. This tumor growth delay was further enhanced when these mice were treated with oral phenformin prior to, during and after IR treatment, confirming the in vitro experimental results.
Conclusion
Human lung cancer with deficient LKB1 are more sensitive to ionizing radiation in vitro and in vivo. This was regardless of the TP53 or KRAS mutation status. A549 cells with deficient LKB1 were also more sensitive to phenformin treatment. Phenformin treatment further sensitized LKB1 deficient lung cancer cells to IR. This suggested that LKB1 can serve as a predictive biomarker to triage patient treatments.
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P2.14 - Targeted Therapy (ID 183)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Targeted Therapy
- Presentations: 1
- Now Available
- Moderators:
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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P2.14-10 - Identification of Frequent, Activating HER2 Mutations and HER2 Inhibitor Response and Resistance in Canine Lung Cancer (Now Available) (ID 2583)
10:15 - 18:15 | Author(s): Joseph M Amann
- Abstract
Background
Naturally occurring primary canine lung cancers share clinical and pathologic features with human never-smoker lung cancer, but their genetic and biologic underpinnings have been unknown. We mapped the genomic landscape of canine lung cancer, discovering somatic HER2 (ERBB2) mutations in ~38% of canine pulmonary adenocarcinomas (cPACs). We describe our genomic findings, effects of HER2 mutations on cell signaling and HER2 inhibitor response/resistance, and results of pharmacokinetic (PK) studies of the HER2 inhibitor neratinib in healthy dogs.
Method
We performed multi-platform genomic analysis of 92 primary canine lung tumors or cell lines. We evaluated HER2 signaling by Western blot. Neratinib and lapatinib responses were assessed in HER2-mutant and wild-type cPAC cell lines in vitro. HER2-mutant cell lines were continuously treated with IC50 neratinib doses to select for drug resistance, then evaluated for resistance mutations. Neratinib tolerability and PK were evaluated in healthy, mixed-breed, middle-aged dogs given a single 6 mg/kg oral neratinib dose with or without maropitant citrate or loperamide. Plasma samples for PK were collected pre-dose and timepoints from 0.5-24h after dosing.
Result
Canine lung cancers exhibited low tumor mutation burden. HER2 was the most commonly mutated gene in cPACs, occurring in ~38%, but was absent from canine adenosquamous or squamous cell carcinomas. HER2 hotspot V659E transmembrane domain (TMD) mutations were most common and comparable to activating TMD mutations in human cancer. HER2 V659E correlated with constitutive phosphorylation of Akt in cPAC cell lines. HER2 V659E lines displayed hypersensitivity to neratinib and lapatinib relative to wild-type lines. Continuous treatment of HER2-mutant cell lines led to rapid development of resistance. Characterization of resistance-driving alterations is ongoing, as are efficacy studies in mouse xenografts. A dose of 6 mg/kg neratinib in healthy dogs showed that neratinib was tolerable and achieved therapeutic levels from IC50 studies. Evaluation of neratinib tolerability and efficacy in dogs bearing HER2-mutant lung cancer is ongoing.
Conclusion
HER2 TMD mutations are common in otherwise low-mutation-burden primary canine lung cancers. They confer constitutive HER2 signaling and HER2 inhibitor response in cell lines. Dosing and tolerability studies in healthy dogs have shown that therapeutically relevant doses are achievable. Study of HER2 inhibitor response/resistance in cell lines and mouse xenografts is ongoing as is clinical evaluation of neratinib activity in dogs with spontaneous lung cancer. These data offer immediate diagnostic and therapeutic opportunities for pet dogs and bear implications for comparative understanding of human never-smoker lung cancers and other HER2-mutant cancers.
