Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Summary

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Abstract

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Introduction

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children^1,2. The overall survival rate for pediatric B-lineage ALL (B-ALL) is approximately 85%, but specific biological subtypes, including T-lineage ALL (T-ALL), have still poorer prognosis even with current therapeutic protocols. Further treatment of relapsed ALL remains a challenge³. Although the majority of adult patients with acute leukemia achieve a remission with up-front chemotherapy, many patients still suffer relapse⁴. Current chemotherapeutic regimens in the treatment of acute leukemia are known to cause toxicity-associated short- and long-term side effects....

Protocol

All experiments involving animals followed the regulatory standards approved by the University of Colorado Institutional Animal Care and Use Committee. The demonstrated protocol was approved by the University of Colorado Institutional Animal Care and Use Committee.

1. Phospho-protein Western Blot

1. Preparation of lysates
   1. Culture cell lines expressing the receptor tyrosine kinase of interest. Harvest cells and plate 3-5 x 10⁶ cells/sample in 400 μl growth medium in a 48-well tissue culture dish. Incubate at 37 °C in 5% CO₂ for 2-3 hr.
   2. Add 100 μl of 5x TKI solution in growt....

Results

The assays presented here evaluate the biochemical and functional effects mediated by TKIs and can be used to rank novel compounds based on the degree of target inhibition in vitro and in vivo, reduction of colony formation, and delay in leukemogenesis in NSG mice transplanted with luciferase-tagged leukemia cells.

Immunoblot analysis was utilized to determine inhibition of the active phosphorylated form of the target protein in leukemia cells following treatment with TKIs. T.......

Discussion

This manuscript describes an effective strategy for evaluation of novel tyrosine kinase inhibitors in the treatment of acute leukemia. Using this approach, biochemical and anti-leukemia activities are evaluated first in cell-based assays in vitro and then in xenograft models in vivo. Immunoblot analysis was successfully utilized to demonstrate inhibition of the target tyrosine kinase in leukemia cells after treatment with TKIs and to directly compare the potency of multiple compounds in cells. In the pr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
			Reagent/Material
Hydrogen Peroxide	MP Biomedicals	#02194057	GHS05, GHS07, H302-H318
Sodium Orthovanadate	Sigma	#S6508	GHS07, H302+ H312+H332
2-Mercaptoethanol	Sigma	#M7522	GHS05, GHS06, GHS08, GHS09, H301 + H331-H310-H315-H317-H318-H373-H410
ColonyGel Human Base Medium	ReachBio	#1101
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)	Sigma	#M5655	GHS07, GHS08, H315-H319-H335-H341
Difco Noble Agar	BD Biosciences	#214883
Nitrotetrazolium Blue Chloride	Sigma	#N6639	GHS07, H302
D-Luciferin Firefly, Potassium Salt	PerkinElmer	#122796
4',6- Diamidino-2-phenylindole dihydochloride	Sigma	#D9542
FITC CD45	BD Bioscience	#347463
FITC Mouse IgG1 Isotype control	BD Bioscience	#51-35404X-2
Gentamycin Sulfate	Sparhawk	#NDC58005-633-04
Protease Inhibitors	Roche	#11836153001
DNase	Sigma	#D4263
Protein G Beads	Invitrogen	#10-1242
Isofluran	VETONE	#NDC13985-030-60
			Equipment
Cell culture dishes, diam. 35 mm × H 10 mm	Nunclon	#D7804-500EA
Cell culture dishes, diam. 100 mm x  H 20 mm	Nunclon	#D8429-1CS
6-well plates	BD Bioscience	#353046
14 gauge x 4 inch blunt-end needles	Cadence science	#7956
5 ml syringe with luer-lok	BD Bioscience	#309646
GelCount automated colony counter	Oxford Optronix
In vivo bioluminescence imaging system	PerkinElmer	#IVIS200
Scout pro portable balances, scale	Ohaus	#SP202
Broome style rodent restrainer	Plas-labs	#551-BSRR
Ear punch, punch diameter: 2 mm	FST	#24210-02
Chlorhexidine swabs, Prevantics	PDI	#B10800
Insulin syringe 1 mL (40 Units) 29 G x 1/2	Monoject	#8881500042
Plastic feeding needles for rodents (disposable) 20 ga x 38 mm, sterile	Instech	#FTP-20-38
1 mL Luer-Lok disposable syringe	BD Bioscience	#309628
Lo-Dose U-100 insulin syringe with 28 G x ½, permanently attached needle	BD Bioscience	#329465
Extra fine bonn scissors	FST	#14084-08
Student fine scissors	FST	#91460-11
Moria ultra fine forceps	FST	#11370-40
Extra fine graefe forceps	FST	#11150-10
Scalpel handle	FST	#10003-12
Scalpel blades	FST	#10011-00