Kara S. Davis, MD1, Nayel I. Khan, BS1, Carolyn Kemp, BS1

HER3 Inhibition Potentiates Anti-Tumor Effects of PI3K Inhibitors in Pre-Clinical Models of HNSCC
Abstract: #1674
Kara S. Davis, MD , Nayel I. Khan, BS , Carolyn Kemp, BS , Sucheta Kulkarni, PhD , Diego Alvarado, PhD , Theresa LaVallee, PhD , Jennifer R. Grandis, MD ,
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Umamaheswar Duvvuri, MD, PhD
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University of Pittsburgh, Department of Otolaryngology--Head & Neck Surgery, 2University of California, San Franciso, Department of Otolaryngology--Head & Neck Surgery, 3Kolltan Pharmaceuticals, New Haven, CT
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Background
The phosphoinositide 3-kinase (PI3K) pathway is thought to be the most frequently mutated oncogenic pathway in head and neck squamous cell carcinoma (HNSCC). This pathway is often further
activated by deregulated receptors such as the epidermal growth factor receptor family. HER3 has
a direct phosphorylation site with the regulatory subunit of PIK3CA, and is being investigated as
a potential co-target to increase anti-tumor effects of PI3K inhibition. In this investigation, we use
blocking antibodies against HER3 and its ligand, neuregulin β-1, (KTN3379) and PIK3CA (BYL719)
to investigate the effect of HER3 in modulating response to PI3K inhibition.
Objectives
Aim 1: To investigate the effect of HER3 signaling on HNSCC tumor growth
● HER3 pathway activity and/or alterations may modulate HNSCC growth
● Using blocking antibodies against HER3 and its ligand, neuregulin, we will investigate the effect of HER3 targeting in HNSCC models
Colony formation assay: Cell lines were treated with increasing doses of KTN3379, BYL719, and/
or NRG1-β1 as indicated. Colony formation assays were stopped when any well reached ~80% confluence, ranging from 7-15 days. Quantification was performed via the ColonyCounter plug-in for
ImageJ.
Cal33 cells were plated in 10cm dishes with 4% FBS. Treatment with 1μM of BYL719 was performed for 30 minutes, 1
hour, 24 hours, and 48 hours. Lysates were analyzed versus
DMSO control.
Most pronounced in PIK3CA amplified cell lines (FaDu, HN5)
Combination therapy with BYL719 and KTN3379 more effectively inhibits downstream PI3K/AKT signaling than BYL719
alone (Representative immunoblot shown for Cal33). Regarding immunoblotting, cells were plated in a 10cm dish,
serum starved for 24 hours, and then treated or untreated
with compounds. Cells were treated with 1uM BYL719 for
24 hours. Cells were then treated with 50nM KTN3379 for
45 minutes before stimulation with 10ng/mL NRG1-β1 for 15
minutes.
Synergy was observed in the range of the IC50 of BYL for
each cell line except UPCI:SCC90
Regarding immunoblotting, whole cell lysates were prepared, and Western blot analyses were performed on 6-8% SDS-PAGE using the LiCor system for the indicated antibodies. Pertinent phosphorylation sites included P-HER3 (Y1289), P-HER2 (Y1248), P-EGFR (Y1173), and P-AKT (S473). All
antibodies from Cell Signaling Technology.
Results
NRG induces cell growth in a variety of HNSCC cell lines
● Possibly correlated with baseline pHER3 status
Aim 2: To investigate whether HER3 activity is modulated downstream of HER3 pathway
● We will investigate the phenomenon of compensatory upregulation of HER3 following PI3K
inhibition
● The effect of HER3 signaling in the setting of BYL719, a PIK3CA inhibitor, will be elucidated
Treatment with exogenous NRG is correlated with increased
pAKT
Aim 3: To explore the biological effects of dual targeting PI3KCA and HER3 in tumor models representative of the landscape of PI3KCA alterations in HNSCC
FaDu cells were plated in a 10cm dish, serum starved for 24
hours, and then treated or untreated with compounds. Cells
were treated with 50nM KTN3379 for 45 minutes before stimulation with 10ng/mL NRG1-β1 for 15 minutes.
● This is suppressed by KTN3379 in a variety of HNSCC
cell lines
● Combination therapy with PI3K inhibition and anti-HER3 directed therapies may overcome
compensatory HER3 upregulation and decrease the activity of the PI3K/AKT/mTOR pathway
● Blocking the compensatory upregulation of HER3 in response to PI3K inhibition will lead to
enhanced sensitivity of HNSCC models to PI3K inhibition, which may be exploited by dual targeting.
Conclusions
HER3 signaling contributes to cell proliferation in HNSCC. HER3 interacts with the PI3K pathway in
multiple HNSCC cell lines, and combined blockade leads to synergistic effects in pre-clinical models
of HNSCC.
Materials & Methods
The growth inhibition IC50 values were determined following a 72 hour incubation period with compounds in 4% FBS-containing media. Cell viability (ATP level) was be measured using the CellTiter
Glo (Promega) detection reagent and captured on the M5e microtiter plate reader platform. Combination Index values were determined using CalcuSyn based on the Chou-Talalay method.
There is compensatory upregulation of HER3 in response to
PI3K inhibition
NRG ameliorates BYL-induced growth inhibition in nonPI3K mutant cell lines