In vivo imaging was performed using the IVIS Shared Resource at the University of Colorado Cancer Center (supported by grant P30-CA046934). Flow cytometry was performed at the Flow Cytometry Shared Resource, University of Colorado Cancer Center (supported by grant P30CA046934). This work was supported in part by the National Institutes of Health (RO1CA137078 to DKG). ABLS is a Fellow of the Pediatric Scientist Development Program, supported by grants from the American Academy of Pediatrics, the American Pediatric Society, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (K12-HD000850).

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
<ol>
	<li>Preparation of lysates
	<ol>
		<li>Culture cell lines expressing the receptor tyrosine kinase of interest. Harvest cells and plate 3-5 x 106 cells/sample in 400 &#956;l growth m...

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	<li>Jemal, A., Siegel, R., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Statistics.+2010.">Cancer Statistics. 2010.</a> CA Cancer J. Clin. 60, 277-300 (2010).</li><li>Kaatsch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+childhood+cancer.">Epidemiology of childhood cancer.</a> Cancer Treat Rev. 36, 277-285 (2010).</li><li>Pui, C., Mullighan, C., Evans, W., Relling, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+acute+lymphoblastic+leukemia:+where+are+we+going+and+how+do+we+get+there?.">Pediatric acute lymphoblastic leukemia: where are we going and how do we get there?.</a> Blood. 120, 1165-1174 (2012).</li><li>Forman, S., Rowe, J. 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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...

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children1,2. 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. Therefore, less toxic therapies that specifically target cancer cells with minimal effect on normal tissues are greatly needed. In recent years, emphasis has been placed on the development of novel, molecularly-targeted agents with specificity for cancer cells, often utilizing iterative chemistry to generate multiple active compounds which must then be compared and prioritized5. This manuscript describes an efficient strategy for pre-clinical evaluation of TKIs for the treatment of acute leukemia, which can be used for evaluation of a single compound or for direct comparison of multiple compounds in order to facilitate drug development.
The method presented here consists of four steps. First the biochemical (1) and anti-leukemia (2) activities of the compound(s) are evaluated in cell culture, then inhibition of the target is confirmed in animal models (3), and finally therapeutic efficacy of the TKI(s) is determined in orthotopic leukemia xenograft models (4). For these studies, it is important to choose relevant cell lines, which are representatives of the most common biologic subtypes. Cell lines should be selected, which both, express the target of interest and lack the target of interest, to investigate whether biologic effects are mediated by inhibition of the target. This is particularly relevant for the development of small molecule inhibitors, which have off-target effects that may be important for anti-tumor activity. It is also necessary to choose a cell line that is dependent on the target for functional effects such as proliferation or survival. Preliminary target validation studies (outside the scope of this article) using RNA interference or other specific means to inhibit the target can be used to identify target-dependent cell lines. It is also desirable to choose cell lines that can form murine xenografts, such that cell culture results can be more directly translated to in vivo experiments.
For evaluation of biochemical activity mediated by TKIs in leukemia cells, a decrease in receptor phosphorylation can be used as an indicator of target inhibition. Western blot analysis or ELISA assays can be employed, depending on the availability and specificity of antibodies. If antibodies with sufficient specificity for the target are available, ELISA assays are preferable as they are more quantitative and efficient. In cases where antibodies with sufficient specificity for ELISA are not available, western blot analysis may be necessary. In this case, immunoprecipitation of a large amount of lysate can be useful for detection of targets that are in low abundance. This approach is particularly relevant for measurement of phospho-proteins, which may have a short half-life to allow for rapid changes in signaling in response to environmental stimuli. Some phosphorylated proteins are extremely labile, most likely as a result of complex formation with phosphatases. For robust and consistent detection of these phosphorylated proteins, it may also be possible to treat cells with pervanadate, an irreversible protein-tyrosine phosphatase inhibitor6, to stabilize the phospho-protein prior to preparation of whole cell lysates.
To determine whether biochemical activity results in anti-tumor effects, target-dependent biological processes can be monitored in cell-based experiments. For leukemia cell lines, anti-leukemia activity mediated by TKIs can be assessed using colony formation assays performed in methylcellulose or soft agar7. Soft agar may be preferred as this is a solid medium that is more amenable to manipulation if repeated treatment with a TKI is necessary. While many acute myloid leukemia (AML) cell lines will form colonies in soft agar, most ALL cell lines will only form colonies in methylcellulose, which is a semi-solid medium. Although it is possible to refresh medium and/or TKIs in methylcellulose cultures, only small volumes can be used and with limited frequency. Similarly, it is more difficult to stain colonies without disrupting them in methylcellulose. Preliminary studies should define the ability of appropriate cell lines to form colonies in methylcellulose and/or soft agar and the optimal density of cells in culture such that colonies are non-overlapping and in sufficient number to obtain statistically relevant data (usually 50-200 colonies per 35 mm plate).
While in vitro assays are robust and cost-effective, and have fewer ethical implications than whole animal experiments, advancement of therapeutic compounds requires proof of efficacy and safety in animal models. For in vivo studies, human acute leukemia cell lines can be orthotopically transplanted into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and TKIs can be easily administered by injection or oral gavage. Some cell lines may require exposure of NSG mice to a low cell line-dependent dose of radiation in order for xenografts to establish, and in this instance mice may not tolerate oral gavage without a 5-10 day recovery period post-irradiation. This manuscript describes generation of B-ALL and T-ALL xenografts using specific cell lines (697 and Jurkat) as examples but xenografts may be established in NSG mice using a wide variety of cell lines. In the event that other cell lines are more applicable, the requirement for irradiation, optimal number of cells to transplant, and timing of disease onset and progression should be determined experimentally. Ideally, these models will have complete penetrance (every animal transplanted develops leukemia), consistent kinetics (leukemia progresses similarly in all animals), and a reasonable treatment window (ideally 20-30 days between initiation of treatment and removal from study due to disease). The number of cells transplanted can be increased to improve penetrance and kinetic consistency or decreased to improve the treatment window if necessary.
To determine if TKIs mediate target inhibition in vivo, samples are collected from mice with leukemia xenografts after treatment with TKI or vehicle only. Ideally, the dose and schedule of administration for these experiments are guided by pharmacokinetic studies, which can often be performed by commercial laboratories and are outside the scope of this article. If pharmacokinetic data are available, the concentration of compound required for effective target inhibition in cell culture and the maximum serum concentration following a single dose of TKI can be used to define a starting dose for animal studies. Pharmacokinetic studies can also inform the timing of sample collection post-treatment for pharmacodynamic studies and the route of administration. Inhibition of the target can be assessed in any affected organ but tissues that are most easily collected and processed are preferable. Most acute leukemia cell lines establish in the liver, bone marrow, spleen, peripheral blood, and the central nervous system, although the specific organs affected and the extent of engraftment in these organs varies between models. The protocol presented here assesses phospho-protein inhibition in bone marrow using western blot analysis, but solid organs may be easier to consistently harvest and require minimal or no processing prior to freezing, allowing less opportunity for degradation of phospho-proteins during sample collection and processing. Immunohistochemistry can also be used to evaluate solid tumors or organs affected by leukemia.
Finally, the therapeutic efficacy of the TKI(s) is determined in orthotopic leukemia xenograft models. For these studies, the timing of treatment initiation can be varied, such that disease is more or less established. Treatment may begin immediately after transplant for initial studies and then be delayed in subsequent studies until significant disease burden is detected to more closely approximate a diagnostic treatment model. Ideally, these animal models also have the capacity for non-invasive measurement of disease burden. We have optimized methods for introduction of the firefly luciferase gene into leukemia cell lines using virus-like particles, allowing for non-invasive, longitudinal analysis of disease onset and progression and assessment of disease burden in bone marrow and solid organs. Critical to this approach is the use of monoclonal luciferase-tagged cell lines to prevent variability in development of luciferase-expressing leukemia associated with the use of polyclonal cell lines and is unrelated to treatment with a TKI8.
Taken together, these steps can be used for evaluation of a single TKI or for direct comparison and ranking of multiple TKIs. While the protocols presented here focus on development of TKIs, these methods can be adapted to other targets and considerations for assay development are described. Thus, this strategy may be more broadly applicable to the pre-clinical evaluation of molecularly-targeted agents for treatment of acute leukemia.

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			<td style="width:167px;"> </td>
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			<td style="width:223px;">Reagent/Material</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Hydrogen Peroxide</td>
			<td style="width:167px;">MP Biomedicals</td>
			<td style="width:233px;">#02194057</td>
			<td style="width:223px;">GHS05, GHS07, H302-H318</td>
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			<td height="21" style="height:21px;width:443px;">Sodium Orthovanadate</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#S6508</td>
			<td style="width:223px;">GHS07, H302+ H312+H332 </td>
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			<td height="63" style="height:63px;width:443px;">2-Mercaptoethanol </td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#M7522</td>
			<td style="width:223px;">GHS05, GHS06, GHS08, GHS09, H301 + H331-H310-H315-H317-H318-H373-H410 </td>
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			<td height="21" style="height:21px;width:443px;">ColonyGel Human Base Medium </td>
			<td style="width:167px;">ReachBio</td>
			<td style="width:233px;">#1101</td>
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			<td height="42" style="height:42px;width:443px;">3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#M5655</td>
			<td style="width:223px;">GHS07, GHS08, H315-H319-H335-H341 </td>
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			<td height="21" style="height:21px;width:443px;">Difco Noble Agar </td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:233px;">#214883</td>
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			<td height="21" style="height:21px;width:443px;">Nitrotetrazolium Blue Chloride</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#N6639</td>
			<td style="width:223px;">GHS07, H302 </td>
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			<td height="21" style="height:21px;width:443px;">D-Luciferin Firefly, Potassium Salt</td>
			<td style="width:167px;">PerkinElmer</td>
			<td style="width:233px;">#122796</td>
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			<td height="21" style="height:21px;width:443px;">4&#39;,6- Diamidino-2-phenylindole dihydochloride</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#D9542</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">FITC CD45 </td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#347463</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">FITC Mouse IgG1 Isotype control</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#51-35404X-2</td>
			<td style="width:223px;"> </td>
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			<td height="21" style="height:21px;width:443px;">Gentamycin Sulfate</td>
			<td style="width:167px;">Sparhawk</td>
			<td style="width:233px;">#NDC58005-633-04</td>
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			<td height="21" style="height:21px;width:443px;">Protease Inhibitors </td>
			<td style="width:167px;">Roche </td>
			<td style="width:233px;">#11836153001</td>
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			<td height="21" style="height:21px;width:443px;">DNase</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#D4263 </td>
			<td style="width:223px;"> </td>
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			<td height="21" style="height:21px;width:443px;">Protein G Beads</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:233px;">#10-1242</td>
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			<td height="21" style="height:21px;width:443px;">Isofluran</td>
			<td style="width:167px;">VETONE</td>
			<td style="width:233px;">#NDC13985-030-60</td>
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			<td height="21" style="height:21px;width:443px;">Cell culture dishes, diam. 35 mm &#215; H 10 mm</td>
			<td style="width:167px;">Nunclon</td>
			<td style="width:233px;">#D7804-500EA</td>
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			<td height="21" style="height:21px;width:443px;">Cell culture dishes, diam. 100 mm x H 20 mm </td>
			<td style="width:167px;">Nunclon</td>
			<td style="width:233px;">#D8429-1CS</td>
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			<td height="21" style="height:21px;width:443px;">6-well plates</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#353046</td>
			<td style="width:223px;"> </td>
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			<td height="21" style="height:21px;width:443px;">14 gauge x 4 inch blunt-end needles</td>
			<td style="width:167px;">Cadence science</td>
			<td style="width:233px;">#7956</td>
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			<td height="21" style="height:21px;width:443px;">5 ml syringe with luer-lok</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#309646</td>
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			<td height="21" style="height:21px;width:443px;">GelCount automated colony counter</td>
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			<td height="21" style="height:21px;width:443px;">In vivo bioluminescence imaging system</td>
			<td style="width:167px;">PerkinElmer</td>
			<td style="width:233px;">#IVIS200</td>
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			<td height="21" style="height:21px;width:443px;">Scout pro portable balances, scale</td>
			<td style="width:167px;">Ohaus</td>
			<td style="width:233px;">#SP202</td>
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			<td height="22" style="height:22px;width:443px;">Broome style rodent restrainer</td>
			<td style="width:167px;">Plas-labs</td>
			<td style="width:233px;">#551-BSRR</td>
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			<td height="21" style="height:21px;width:443px;">Ear punch, punch diameter: 2 mm</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#24210-02</td>
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			<td height="21" style="height:21px;width:443px;">Chlorhexidine swabs, Prevantics</td>
			<td style="width:167px;">PDI</td>
			<td style="width:233px;">#B10800</td>
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			<td height="21" style="height:21px;width:443px;">Insulin syringe 1 mL (40 Units) 29 G x 1/2</td>
			<td style="width:167px;">Monoject </td>
			<td style="width:233px;">#8881500042</td>
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			<td height="42" style="height:42px;width:443px;">Plastic feeding needles for rodents (disposable) 20 ga x 38 mm, sterile</td>
			<td style="width:167px;">Instech</td>
			<td style="width:233px;">#FTP-20-38</td>
			<td style="width:223px;"> </td>
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			<td height="21" style="height:21px;width:443px;">1 mL Luer-Lok disposable syringe</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#309628</td>
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			<td height="42" style="height:42px;width:443px;">Lo-Dose U-100 insulin syringe with 28 G x &#189;, permanently attached needle</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#329465</td>
			<td style="width:223px;"> </td>
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			<td height="21" style="height:21px;width:443px;">Extra fine bonn scissors</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#14084-08</td>
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			<td height="21" style="height:21px;width:443px;">Student fine scissors</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#91460-11</td>
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			<td height="21" style="height:21px;width:443px;">Moria ultra fine forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#11370-40</td>
			<td style="width:223px;"> </td>
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			<td height="21" style="height:21px;width:443px;">Extra fine graefe forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#11150-10</td>
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			<td height="21" style="height:21px;width:443px;">Scalpel handle</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#10003-12</td>
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pre-clinical evaluation of tyrosine kinase inhibitors for treatment of acute leukemia

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.

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...

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

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.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

pre-clinical-evaluation-tyrosine-kinase-inhibitors-for-treatment

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Signaling Networks of Kinase Receptors

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18.0K 조회수.  University of Colorado Anschutz Medical Campus.  수용체 티로신 키나아제는 ectopically 많은 암에서 발현되는 급성 백혈병의 치료 표적으로 확인되었습니다. 이 원고는 급성 백혈병의 치료를위한 티로신 키나제 억제제의 전 임상 평가를위한 효율적인 전략을 설명한다. 