A novel in-cell ELISA method for screening of compounds inhibiting TRKA phosphorylation, using KM12 cell line harboring TRKA rearrangement

Accepted Manuscript A novel in-cell ELISA method for screening of compounds inhibiting TRKA phosphorylation, using KM12�cell line harboring TRKA rearrangement Manoj Kumar Pandre, Shama Shaik, V.V.V. Satya Pratap, Prasad Yadlapalli, Mahesh Yanamandra, Sayan Mitra PII:

S0003-2697(18)30023-X

DOI:

10.1016/j.ab.2018.01.014

Reference:

YABIO 12905

To appear in:

Analytical Biochemistry

Received Date: 11 October 2017 Revised Date:

17 January 2018

Accepted Date: 18 January 2018

Please cite this article as: M.K. Pandre, S. Shaik, V.V.V. Satya Pratap, P. Yadlapalli, M. Yanamandra, S. Mitra, A novel in-cell ELISA method for screening of compounds inhibiting TRKA phosphorylation, using KM12�cell line harboring TRKA rearrangement, Analytical Biochemistry (2018), doi: 10.1016/ j.ab.2018.01.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A Novel in-cell ELISA method for screening of compounds

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inhibiting TRKA phosphorylation, using KM12 cell line harboring

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TRKA rearrangement

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Manoj Kumar Pandre1#*, Shama Shaik1#, Satya Pratap VVV1#, Prasad Yadlapalli1#, Mahesh Yanamandra1#, Sayan Mitra1*&

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GVK Biosciences Private Limited, Campus MLR 1, Survey Nos. 125 (part) & 126, IDA Mallapur, Hyderabad – 500076, India

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*Corresponding author

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Mr. Manoj Kumar Pandre E-mail: [email protected]; [email protected]

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Department of In-vitro Biology, GVK Biosciences Private Limited, Campus MLR 1 Survey Nos. 125 (part) & 126, IDA Mallapur, Hyderabad 500076, India

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Abstract

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Tropomyosin-related kinase A (TRKA) fusion was originally detected in colorectal carcinoma

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that had resulted in expression of the oncogenic chimeric protein TPM3-TRKA. Lately, many

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more rearrangements in TRK family of kinases generating oncogenic fusion proteins have been

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identified. These genetic rearrangements usually result in fusion of cytoplasmic kinase domain of

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TRK to another gene of interest resulting in constitutive kinase activity. Estimation of TRK

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inhibitor potency in a cellular context is required for drug discovery programs and is measured

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by receptor phosphorylation levels upon compound administration. However, since a large chunk

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of the TRK protein is lost in this rearrangement, it’s difficult to set up sandwich ELISA for

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detection of receptor phosphorylation in any cell assay harboring these fusion proteins. In order

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to address this issue, we developed a novel and robust in-cell ELISA method which quantifies

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the phosphorylation of TRK kinase (Tyr 674/675) within the KM12 cells. This cell based

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method is more versatile & economical than conventional ELISA using engineered

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overexpressing cell line and/or western blot methods. Performance reliability & robustness for

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the validated assay were determined by %CV and Z factor in assays with reference molecule

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larotrectinib. This in-cell ELISA method can be used with any TRKA rearranged oncogenic

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fusion cell type and can be extended to other TRK isoforms as well. We have used this assay to

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screen novel molecules in KM12 cells and to study pharmacodynamic properties of compounds

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in TRKA signaling.

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Key words: KM12, In-cell ELISA, TRK, Tropomyosin receptor kinase, Neurotrophic Tyrosine

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receptor kinase (NTRK), Larotrectinib, Throughput screening.

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1. INTRODUCTION: Tropomyosin receptor kinase (TRK) family TRK A, B and C are receptors of neurotrophins.

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These receptors are encoded by respective genes NTRK1, NTRK2 and NTRK3 and are mainly

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expressed in neuronal tissue but also found in other tissues and tumor cells. The TRK family

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plays wide range of roles in development and [1- 4]. Several clinical investigations revealed the

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involvement of TRK A, B and C in various kinds of tumors, modulating tumor growth, survival

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and metastasis. NTRK gene rearrangements (gene fusions) leads to constitutively activated or

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overexpressed TRK proteins having oncogenic properties. These fusions induce cancer cell

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proliferation by activating the downstream signaling pathways such as MAPK and AKT [5].

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Fusions of NTRK1 with various genes have been identified in different type of tumors, MPRIP-

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NTRK1 and CD74-NTRK1 in lung cancer [5], LMNA-NTRK1 gene fusion in soft-tissue

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sarcoma [6] and tropomyosin-3 (TPM3) NTRK1 gene fusion in colorectal cancer [7]. All the

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fusion proteins have one common characteristic, the intact kinase domain, as reviewed elsewhere

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(8, 9). Various preclinical and clinical studies have validated TRKA as a potential target in

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different types of cancers [5-13]. In a drug discovery setup, engineered cell lines overexpressing

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TRKA (HEK293 cells or NIH3T3 cells) are used as models for a cell based compound screening

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assay. Overexpressing cell lines give a better signal to background ratio than cancer cells. The

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overexpressed model is a clean and robust system. However many times the results obtained

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from assays using an engineered cell line don’t match well with phenotypic assay outcome. Here

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we demonstrate a novel in-cell ELISA method for estimating TRKA phosphorylation, which can

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be used as a model for compound screening programs. This method has various advantages over

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the conventional methods using TRKA overexpressing cells for screening compounds. This

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method is more economical compared to conventional ELISA using TRKA overexpressing cells.

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Here one doesn’t have to spend resource on generating TRKA overexpressing cells. TRKA in-

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cell ELISA does not require NGF stimulation as in overexpressing cell line, and requires only

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two antibodies to detect TRKA, whereas three antibodies are required to detect TRKA in

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sandwich ELISA (table)

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2. MATERIALS AND METHODS:

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2.1 CHEMICALS:

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KM12 (ATCC CRL-12496) and HEK-293 (ATCC CRL-1573) cells were purchased from

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ATCC. RPMI 1640, PBS, DMSO Hybri max, crystal violet ( C0775), poly-L-lysine

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hydrobromide, hydrogen peroxide, protease inhibitor cocktail, phosphatase inhibitor-2,

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phosphatase inhibitor-3 and rabbit anti β-Actin mAb were purchased from Sigma. DMEM,

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penicillin streptomycin reagent, FBS, trypsin-EDTA, pcDNA3.1 (+) ZEO were purchased from

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Thermo scientific. Effectene transfection reagent, rabbit anti-TRKA (C14) mAb and 4%

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paraformaldehyde solution were purchased from Santacruz. Rabbit anti-Phospho-TRKA

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(Tyr674/675), PathScan® Phospho-TRKA Sandwich ELISA Kit and HRP tagged anti-rabbit

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antibody were purchased from Cell signaling technologies. Clarity™ Western ECL Substrate

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was purchased from Biorad. Nerve growth factor (NGF) was purchased from R & D Systems.

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Cell titer Glo® reagent was purchased from Promega. Pierce BCA Protein Assay Kit was

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purchased from Thermo fisher and Larotrectinib (LOXO-101) was purchased from Selleck

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chemicals.

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2.2 Transfection and generation of TRKA overexpressing cancer cells:

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HEK293 cells were cultured in DMEM medium containing antibiotics and 10% FBS.

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The TRKA expression vector (pcDNA3.1 (+) ZEO / TRKA) was prepared by inserting human 4|Page

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TRKA cDNA into the pcDNA3.1 (+) ZEO vector. 300000 HEK293 cells were seeded in 6 well

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plates prior to the transfection in complete DMEM medium and grown up to 80% confluence.

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TRKA expression constructs were transfected to HEK293 cells using Effectene Transfection

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Reagent according to manufacturer’s instructions. Briefly, the transfection complex was prepared

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by mixing 1µg of DNA construct, 8µl of enhancer and 25 µl of effectene transfection reagent

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and this complex was added to the cells in 6 well plates (volumes indicated is for each well of

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the 6-well plate). Cells were incubated with the DNA-transfection complexes at 37oC, 5% CO2

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for 24 hours. Then 250µg/ml zeocin was added to the cells allowing selective growth of zeocin

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resistant cells. Clones that were growing in presence of zeocin were further characterized by

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western blotting. The TRKA positive clones were stored as frozen stocks and used for the

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experiments within 20 passages.

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2.3 pTRKA ELISA with HEK293 cells:

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HEK293 cells over-expressing human TRKA were plated at a density of 25000 cells per

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well in 200µL of DMEM media (10% FBS) in a 96 well plate. The next day cells were serum

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starved for 2 hours with serum free media. Larotrectinib stock was prepared and diluted in cell

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culture grade DMSO and the working stocks were prepared in incomplete media (DMEM

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without FBS). The cells were pre-incubated with varying concentrations of larotrectinib for 20

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min and then stimulated with 6nM NGF (EC90 concentration) for 5 min at 37oC. Untreated cells

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were used as background. The assay plates were centrifuged at 2000 rpm for 1 min and the

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media contents were removed by aspiration. The cells were lysed in 100µL lysis buffer provided

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with the PathScan Phospho-TRKA ELISA Kit.

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2.4 Cell proliferation assay:

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On day 1 the KM12 cells were plated at a density of 2000 cells per well in 180 µL of

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RPMI media (white opaque 96 well plate) and incubated at 37oC and 5% CO2. The same number

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of cells was plated in a separate plate for time zero (T0) reading. On day 2 compound stocks

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were serially diluted in DMSO and the working stocks were prepared in RPMI media. From the

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working stocks, 20 µL was added to the respective well of the 96 well plate, which was then

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incubated for 72 h at 37oC and 5% CO2. DMSO was used as vehicle control and the final

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concentration of DMSO in the assay plate was 0.5 %. Cell titer Glo® reagent was prepared as

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per the kit instruction manual [14]. On day-4, 100 µL of Cell titer Glo® reagent was added to

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each well and incubated for 30 minutes with shaking at room temperature. The luminescence

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measured in relative units (RLU) using Envision plate reader (Perkin Elmer). To values were

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subtracted from the results of both treated and control cells. The resulting net RLU was used to

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calculate the percentage growth (%G) relative to DMSO control. The data was fit into a non-

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linear regression curve (sigmoidal dose-response variable slope) by plotting %G against

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concentration in graph pad prism software.

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2.5 pTRKA in-Cell ELISA with KM12 cells:

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KM12 cells at various densities were plated in a poly D-Lysine coated 96 well plates in

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200µL of RPMI media (10% FBS). After overnight incubation at 37 ºC and 5% CO2, the

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medium was replaced with medium containing either 1µM larotrectinib or DMSO carrier and

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cells ere incubated for 4h. The cells were washed using PBS twice and fixed with 4%

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paraformaldehyde for 20 min at room temperature. The cells were permeabilized with

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100µL/well of permeabilization buffer (0.1% triton X-100 in PBS) and incubated for 15 minutes

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at room temperature. After washing the plate twice, 100µL of quenching buffer (1% hydrogen

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peroxide in PBS) was added to each well and incubated for 20 minutes. The washing steps were

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repeated and plates blocked with 100µl of blocking buffer (1% BSA in wash buffer), sealed and

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incubated at room temperature for 1 hour. After washing, the plates were incubated with primary

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antibody (TRKA (C14) Santacruz or pTRKA 674/675 Cell signalling technologies) overnight at

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4°C followed by HRP tagged anti rabbit antibody (Cell signaling) for 1h at 37°C. The plates

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were washed and incubated with TMB substrate and after allowing enough colour development

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50µL 2N H2SO4 was added to each well and absorbance was measured at 450 nm. All the steps

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are schematically represented as assay flow chart in fig. 1.

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2.6 Crystal violet staining:

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After measuring the absorbance at 450 nm, the plates were washed with PBS and stained

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with crystal violet reagent (0.1 % Crystal violet in 5% ethanol) for 30 min. The plates were

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washed three times with deionised water until all excess stain is removed and the dye was

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extracted in citrate buffer containing 50 % ethanol for 30 min with shaking and the absorbance

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was measured at 590 nm. Any changes in TRKA levels were normalized with total protein stain

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obtained by crystal violet method.

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2.7 Estimation of pTRKA levels in KM12 cells by western blotting:

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KM12 cells were plated at a density of 1 x 106 cells per well in a 6 well plate and

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incubated overnight at 37 oC, 5% CO2. The next day the media was replaced with plain media

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without serum and incubated with compound for 4h at 37 oC, 5% CO2. The cells were washed

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twice with ice cold PBS and lysed with RIPA buffer containing protease and phosphatase

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inhibitor cocktails. The protein content of cell lysates were quantified using BCA protein kit

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according to the manufacturer's instructions. Equal amounts of proteins were run on SDS-PAGE

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and then transferred onto nitrocellulose membrane. Membranes were blocked with 5% BSA and

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then probed with primary antibodies against TRKA mAb (rabbit) (1:1000 dilutions,), phospho-

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TRKA mAb (rabbit) (Tyr674/675, 1:1,000 dilutions) and β-actin mAb (rabbit) as a loading

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control (1.30000 dilutions, Cell Signaling). After overnight incubation, the blots were washed

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three times with buffer containing 0.1% Tween 20 and incubated with anti-rabbit IgG antibody

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conjugated with horseradish peroxidase for 2 hr at room temperature. Next they were washed

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again with washing buffer and incubated with Clarity™ ECL Western Blotting Substrate for 2

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minutes and the luminescence was visualized by ImageQuant LAS 4000. The percent inhibition

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of phospho-TRKA was quantified by Biorad’s quantity one software.

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3. RESULTS AND DISCUSSION

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Inhibition of TRKA phosphorylation in TRKA overexpressing HEK293 cells: Recombinant TrkA expressing HEK293 cells were used in cell based assay for testing

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compounds. Varying concentrations of larotrectinib was tested in total TRKA and pTRKA

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ELISA in triplicates, as described in the methods. The S/B of the assay was found to be more

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than 6 fold and the Z’ was 0.79. Larotrectinib inhibited the phosphorylation of TRKA in

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HEK293 cells with an IC50 of 10nM (fig. 2) and had no effect on total TRKA levels..

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Inhibition of KM12 proliferation by larotrectinib:

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Eight concentrations of larotrectinib (starting from 1µM final concentration, 3-fold

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dilution) were tested in KM12 proliferation in triplicates. Larotrectinib inhibited KM12

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proliferation with GI50 (Concentration at which 50 % growth inhibition is seen) of ~ 4.1nM

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(mean of 3 experiments), fig. 3, which agrees with the results published elsewhere [6].

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In Cell ELISA with KM12 cells:

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Different densities of KM12 cells ranging from 10000 to 200000, were chosen to check

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the overall signal strength generated by the cells. The experiments were performed in triplicates

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(N=3). A clear trend in increasing the signal was observed with respect to cell densities up to

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200000 cells as shown in fig. 4A. Highest signal to background was obtained with cell densities

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of 75000, 100000 and 200000 with 7.2 fold, 8.3 fold and 8.7 fold respectively. These three cell

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densities were chosen for further optimization of the assay. DMSO tolerance of the assay was

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tested with 0.31 to 10 % DMSO final concentration in the assay. DMSO was well tolerated up to

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the concentrations of 2.5 % (Figure-4B). KM12 cells were treated with varying concentration of

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larotrectinib in triplicates. Larotrectinib inhibited the phosphorylation of TRKA with an IC50 of

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3.8nM, 4.8nM and 6.0nM with 75000, 100000 and 200000 cells respectively. Larotrectinib

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inhibited phosphorylation of TRKA without significantly affecting total TRKA (Figure-5A, 5B,

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5C). Robustness of the validated assay was determined by %CV and Z factor between the inter-

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intraday experiments (fig. 6A and 6B).

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The results obtained from the ELISA were further validated by immuno blotting. The cell

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lysates obtained from larotrectinib treated KM12 cells were separated on PAGE and probed with

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total and phospho TRKA (Tyr674/675) antibodies separately. Larotrectinib inhibited the

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phosphorylation of TRKA with increasing dose without affecting the total TRKA (Figure-7A,

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7B). Similar experiments were performed using pTRKA 475 antibody in KM12 cells and the

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results obtained were identical to TRKA (Tyr674/675) (data is not shown).

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Our primary aim was to identify inhibitors against TRKA in the context of

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constitutionally active TRKA fusion driven tumor using KM12 cells as a model. In the initial

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studies we used TRKA overexpressing HEK293 cells for screening compounds. The potencies of

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many compounds did not correlate with the KM12 proliferation assay (results are not shown). 9|Page

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The reason for this might be that the two assays use different cell line, having difference in the

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levels of TRKA expression, compound uptake and metabolism. To know the potency and mechanism of a novel pharmacophore, a highly sensitive and

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robust cellular assay should be developed which decides the fate of the compound for proceeding

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to next level in a drug discovery program [16]. A good screening assay should be easy to handle,

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pathologically relevant and sensitive enough to differentiate the potencies of compounds in a

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screening program. Here we report an in-cell ELISA method for detecting pTRKA and total

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TRKA levels using KM12 cells. This method has lot of advantages over the conventional

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sandwich ELISA in terms of resource, time and scientific correlation. Conventional ELISA using

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TRKA overexpressing cell line requires NGF stimulation, cell lysates to be prepared after cell

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treatment, it requires additional capture antibody which captures antigen (TRKA), Cell death or

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cell loss during assay cannot be monitored in conventional ELISA. The KM12 In-cell ELISA

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does not require NGF stimulation, it does not require lysing cells or capture antibody as the

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whole cells are fixed and used for assay, thereby reducing the cost of the assay. The loss of cells

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in in-cell ELISA is captured by crystal violet staining. Another important aspect is that same cell

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line is used for different assays, ELISA and proliferation assay and the results are more

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biologically relevant unlike the genetically engineered overexpressing cell line. Besides this,

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other traditional protein assay methods such as Western blotting are cumbersome and laborious,

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whereas high content methods employ expensive reagents and instruments.

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This method provided a robust and sensitive model to screen compounds with IC50 range

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of sub-nano molar to micro molar potency. We successfully adapted this method in a medium

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throughput screening program with a turnover of 8-10 assay plates per day.

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In conclusion, this in-cell TRKA ELISA system in KM12 cells proved an excellent tool for

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screening compound libraries and studying mechanistic properties of novel compounds. This

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method is highly sensitive and selective for TRKA either full length, truncated or fusion variants,

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thereby providing an advantage over conventional western blots and other lysate based ELISA

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systems.

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Author Contributions

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MKP, SM and MY initiated and designed the study. MKP, SS, MY, SP and PY Performed the

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experiments and data analysis. MKP and SM wrote the manuscript. All authors have given

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approval to the final version of the manuscript.

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Acknowledgement

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The authors thank Dr. Premkumar Arumugam, Associate vice president, GVK Biosciences for

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enabling Biology infrastructural support in conducting the experiments.

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Legends

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Legend for table

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Antibodies requirement in different methods

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Legend for figure-1

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Assay flowchart. The protocol workflow and schematic of the In-cell Elisa are shown. FBS-

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Fetal bovine serum, H2O2: Hydrogen Peroxide, BSA: Bovine serum albumin, TMB:

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Tetramethylbenzidine

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Legend for figure-2

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Inhibition of TRKA phosphorylation by Larotrectinib in NGF stimulated HEK293 cells. X-axis

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denotes concentrations of larotrectinib in nM. Y-Axis denotes percent inhibition of TRKA

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phosphorylation. Average IC50 of larotrectinib was found to be 10.9 nM. NGF: Neurite growth

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factor

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Legend for figure-3

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Inhibition of KM12 cell proliferation by larotrectinib (N=3), X axis denotes concentrations of

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larotrectinib in µM and Y-Axis denotes percent control (percent growth compared to vehicle

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control). Average GI50 of larotrectinib was found to be 0.0041µM.

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Legend for figure-4A

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Cell number titration: Different cell numbers from 10000cells to 200000 cells. Cells either

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treated with 0.5 % DMSO (100 % signal) or 1 µM larotrectinib (Background) were used to

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detect pTRKA by in-cell ELISA method. X axis denotes: Cell number. Y-Axis denotes the

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absorbance values at 450nM

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Legend for figure-4B

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DMSO percentage optimization: 75000, 100000 and 200000 cells were used to determine the

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optimum DMSO percentage in assay. Cells were incubated with DMSO concentrations ranging

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from 10 % to 0.31% for 4 hand used for detection of pTRKA by in-cell ELISA method. X axis

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denotes: different DMSO percentages. Y-Axis denotes % Control compared to untreated cells.

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Legend for figure-5

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Inhibition of TRKA phosphorylation (Tyr674/675) in KM12 cells by larotrectinib using in-cell

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ELISA method. A: inhibition studies using 75000 cells per well. B: inhibition studies using

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10000 cells per well. C: inhibition studies using 200000 cells per well. X axis denotes percentage

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inhibitions and Y-Axis denotes the larotrectinib concentrations in µM.

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Legend for figure-6A

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Assay performance. The x axis denotes different experiments performed on separate days ; y axis

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denotes the %CV produced from separate experiments, %CV for signal and %CV for

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background from particular experiment, calculated from standard deviations and mean of the

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ODs ( absorbance values at 450nM) (signal or background).

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Legend for figure-6B

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The Z factor was calculated as determined according to Zhang et al. The x axis denotes

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individual experiments; y axis denotes the Z factor generated from separate experiments.

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Legend for figure-7A

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Inhibition of TRKA phosphorylation (Tyr674/675) in KM12 cells by larotrectinib (1, 10 and 100

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nM) using western blotting method. 75 Kd protein corresponds to truncated TRKA protein. A:

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Total TRKA in KM12 cells treated with larotrectinib and B: pTRKA (Tyr674/675) in KM12 cells

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treated with larotrectinib.

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Legend for figure-7B

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Quantitative analysis of pTRKA and Total TRKA protein bands in western blots was done using

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Biorad Quantity one® software. The pixel density was calculated for each band and percent

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inhibition was calculated considering vehicle control as 100 %. The results obtained are in

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correlation with in-cell ELISA results.

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SC

341

14. CellTiter-Glo® Luminescent Cell Viability Assay Protocol https://www.promega.com/-

344

/media/files/resources/protocols/technical-bulletins/0/celltiter-glo-luminescent-cell-

345

viability-assay-protocol.pdf

346

M AN U

343

15. Zhang JH, Chung TD, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 1999,

348

4(2):67-73.

349

TE D

347

16. Chen H, Kovar J, Sissons S, Cox K, Matter W, Chadwell F. et al. A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy.

351

Anal Biochem. 2005 Mar 1;338(1):136-42.

353 354

AC C

352

EP

350

355 356

16 | P a g e

ACCEPTED MANUSCRIPT

Table pTRKA detection by in-cell ELISA using KM12 cells

pTRKA detection by Sandwich ELISA

No

Yes

Capture Antibody Detection Antibody

Yes

Secondary antibody: HRP tagged

Yes

358

360 361

TE D

362 363 364

EP

365

368

AC C

366 367

Yes

Yes

M AN U

359

RI PT

Antibodies required

SC

357

369 370 371 17 | P a g e

ACCEPTED MANUSCRIPT

Figure-1

374 375 376

AC C

373

EP

TE D

M AN U

SC

RI PT

372

377 378 379 18 | P a g e

ACCEPTED MANUSCRIPT

380

Figure-2 Inhibition of TRKA phosphorylation by Larotrectinib in NGF simulated AD293 cells

RI PT

75

pTRKA

Total TRKA 50

SC

% Control

100

25

1

10

10.91nM

100

1000

M AN U

0 0.1

EC50

Larotrectinib nM

381 382

Figure-3

100

50

N-1 N-2 N-3

EC50

EP

%Growth

75

TE D

Inhibition of KM12 proliferation by Larotrectinib

N-1 4.590

N-2 3.155

N-3 4.794 nM

AC C

25

0 0.1

383 384 385 386 387

19 | P a g e

1

10

100

Larotrectinib nM

1000

10000

ACCEPTED MANUSCRIPT

388

Figure-4A pTRKAELISA: KM12 cell titration

10 1.0

8 6 4

S/B

SC

0.5

Background

S/BRatio

A450 nm

Control

12

RI PT

1.5

2

0.0

0

10

20

30

40

50

75

100 200

M AN U

Cells per well (103)

389 390 391

Figure-4B

75

75000 cells 100000 cells 200000 cells

50

EP

%Control

100

TE D

KM12 pTRKAELISA: DMSO Tolerance

AC C

25

0

0.3125 0.625

392 393 394 395 396

20 | P a g e

1.25

2.5

DMSO %

5

10

ACCEPTED MANUSCRIPT

397

Figure-5A KM12 pTRKA ELISA: 75000 Cells p-TRKA

100

0.5

75

0.4

Total TRKA

0.2 25

RI PT

0.3

50

A490 nm

% Inhibition

Crystal violet Stain

1

10

100

0.0 1000

M AN U

0.1

SC

0.1

0

Larotrectinib nM

398 399 400

Figure-5B

TE D

401

75 50

AC C

25

p-TRKA 0.5

402 403 404 405

21 | P a g e

1

0.3 0.2 0.1

10 100 Larotrectinib nM

Crystal violet stan

0.4

0

0.1

Total TRKA

0.0 1000

A590 nm

% Inhibition

100

EP

KM12 pTRKA ELISA: 100000 Cells

ACCEPTED MANUSCRIPT

406

Figure-5C KM12 pTRKA ELISA: 200000 Cells p-TRKA 100

0.5

75

0.4

Total TRKA

0.2 25

RI PT

0.3

50

A590 nm

% Inhibition

Crystal violet stain

0 1

10 100 Larotrectinib nM

407 408 409

0.0 1000

M AN U

0.1

SC

0.1

Figure-6A

TE D

410

%CV of Controls in KM12 pTRKA ELISA 15

% CVof background

EP

----------------------------

% CV

10

% CVof vehicle control

AC C

5

0

0

411 412 413 414

22 | P a g e

1

2

3

4 5 6 7 Experiment day

8

9

10 11

ACCEPTED MANUSCRIPT

415

Figure-6B KM12 pTRKA ELISA inter-intra day Z-factor

RI PT

1.00

----------------------------

0.50

SC

Z-factor

0.75

0.25

0

1

2

416 417

Figure-7A

4 5 6 7 Experiment days

AC C

EP

TE D

418

3

M AN U

0.00

419 420 421 23 | P a g e

8

9

10 11

ACCEPTED MANUSCRIPT

422

Figure-7B Densitometric analysis of Total and pTRKA in western blot Total TRKA

RI PT

100 75 50

SC

% Inhibition

pTRKA

25

AC C 24 | P a g e

10 0n M

M AN U 10 nM

La ro tre ct in ib

1n M

EP

TE D

423

La ro tre ct in ib

La ro tre ct in ib

D M SO

Co nt ro l

0