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Overexpression of Syk Tyrosine Kinase in Peripheral T-Cell Lymphomas

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Leukemia (2008) 22, 1139–1143

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ORIGINAL ARTICLE Overexpression of Syk tyrosine kinase in peripheral T-cell lymphomas

AL Feldman1, DX Sun1, ME Law1, AJ Novak2, AD Attygalle3, EC Thorland1, SR Fink1, JA Vrana1, BL Caron1, WG Morice1, ED Remstein1, KL Grogg1, PJ Kurtin1, WR Macon1 and A Dogan1

2

1Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA; Department of Hematology, Mayo

3

Clinic, Rochester, MN, USA and Department of Histopathology, Royal Marsden Hospital, London, UK

Peripheral T-cell lymphomas (PTCLs) are fatal in the majority of patients and novel treatments, such as protein tyrosine kinase

Materials and methods

(PTK) inhibition, are needed. The recent finding of SYK/ITK translocations in rare PTCLs led us to examine the expression of Syk PTK in 141 PTCLs. Syk was positive by immunohistochemistry (IHC) in 133 PTCLs (94%), whereas normal

Cases

We studied specimens from 141 patients with PTCL diagnosed by WHO criteria.15 There were 86 men and 55 women of a

T cells were negative. Western blot on frozen tissue (n ¼ 6)

mean age of 59 years (range, 5–88 years). The study was approved by the Institutional Review Board and the Biospecimens Committee of Mayo Clinic. All patients provided informed

and flow cytometry on cell suspensions (n ¼ 4) correlated with IHC results in paraffin. Additionally, western blot demonstrated that Syk-positive PTCLs show tyrosine (525/526) phosphory-

consent for the use of their tissues for research purposes.

lation, known to be required for Syk activation. Fluorescence in situ hybridization showed no SYK/ITK translocation in 86 cases. Overexpression of Syk, phosphorylation of its Y525/526 residues and the availability of orally available Syk inhibitors suggest that Syk merits further evaluation as a candidate target for pharmacologic PTK inhibition in patients with PTCL.

Leukemia (2008) 22, 1139–1143; doi:10.1038/leu.2008.77; published online 10 April 2008 Keywords: peripheral T-cell lymphoma; Syk; tyrosine kinase; phosphorylation

Immunohistochemistry

Paraffin tissue microarrays were constructed as described previously.16 In cases with insufficient tissue, whole-tissue sections were analyzed. Slides were pretreated in 1 mM EDTA buffer at pH 8.0 for 30 min at 98 1C (PT Module; Lab Vision, Fremont, CA, USA) and then stained for Syk with a rabbit polyclonal antibody (C-20, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Dual Link Envision þ /DAB þ (Dako, Carpinteria, CA, USA) was used for detection. Tumors were considered positive for Syk when 430% of the neoplastic cells demonstrated Syk staining. Slides were visualized through an Olympus BX51 microscope (Olympus, Melville, NY, USA) and photographed with an Olympus DP71 camera using Olympus DP manager image acquisition software.

Introduction

  • Peripheral T-cell lymphomas (PTCLs) remain
  • a
  • major

treatment problem among all lymphomas because of their high mortality rate and the minimal effectiveness of conventional chemotherapy.1 Novel therapeutic strategies, such as inhibiting protein tyrosine kinases (PTKs), might improve the outlook toward the treatment of patients with PTCL. Recently, a t(5;9)(q33;q22) translocation2 was reported in a subgroup of PTCL with follicular involvement,3 resulting in overexpression of the SYK gene under the control of the ITK promoter. SYK encodes a cytoplasmic PTK, which is important in proliferation and prosurvival signaling4–7 and is

Western blotting

Protein lysates prepared from frozen tissue sections of six PTCLs and from the B-cell lymphoma cell line, Raji, were separated by polyacrylamide gel (Bio-Rad, Hercules, CA, USA) electrophoresis, transferred to PVDF membranes (Bio-Rad) and incubated for 1 h with primary antibodies as follows: Syk (1:500; N-19, Santa Cruz), phospho-Syk (Tyr525/526, 1:1000; no. 2711, Cell Signaling Technology, Danvers, MA, USA) and actin (1:1000; C-11, Santa Cruz).

  • expressed in
  • a
  • variety of hematopoietic cells, including

normal B lymphocytes8 and most B-cell lymphomas.5,9–12 Normal peripheral T cells, however, generally lack Syk protein expression.13 In the current work, we demonstrate that Syk is overexpressed in the majority of PTCLs, despite the absence of SYK/ITK translocations in most cases. As one orally available Syk inhibitor14 is already in clinical trial for B-cell lymphomas, Syk merits further evaluation as a possible therapeutic target in patients with PTCL as well.

Flow cytometric immunophenotyping

Flow cytometric immunophenotyping was performed on thawed, washed cells as described previously.17 Briefly, cells were stained with fluorochrome-conjugated antibodies (Becton Dickinson/Pharmingen, San Jose, CA, USA) to CD3 (peridinin chlorophyll protein), CD5 (phycoerythrin) and CD19 (PE-Cy7). Stained cells were washed, fixed and permeabilized (Caltag Fix and Perm; Caltag/Invitrogen, Eugene, OR, USA) and then stained with anti-Syk (fluorescein isothiocyanate). Cells were analyzed on a FACSCanto instrument (Becton Dickinson) and data were analyzed using FACSDiva Software (Becton Dickinson).

Correspondence: Dr AL Feldman, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA. E-mail: [email protected] Received 3 January 2008; revised 11 February 2008; accepted 29 February 2008; published online 10 April 2008

Syk expression in PTCLs

AL Feldman et al

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Fluorescence in situ hybridization

evaluated cases using D-FISH probes for SYK and ITK. Despite appropriate fusion signals in control tissue with the translocation (not shown), no evidence of t(5;9)(q33;q22) was identified in 86 informative study cases of PTCL (84 of which were positive for Syk by IHC). These 86 cases included 23 AITLs, 38 PTCL-Us, 13 ALCLs and 12 other cases, a distribution similar to that in the overall study set. Additional copies of SYK (3–6 signals) were identified in only four cases, including two ALK-negative ALCLs (both Syk protein-positive by IHC) and two PTCL-Us (one Sykpositive and one Syk-negative). The FISH probes used did not allow distinction between gene amplification and polysomy as the cause for additional SYK signals. None of the cases studied had the characteristic features of PTCLs with follicular involvement described by de Leval et al.,3 which were seen in 3/5 previously reported cases with SYK/ITK translocation.2 Based on our findings, translocations or additional copies of SYK do not appear to be the mechanisms leading to Syk protein overexpression in most Syk-positive PTCLs. Syk has been suggested as a potential therapeutic target for PTCL by Mahadevan et al.,19 but previous data on Syk expression in T-cell lymphomas are limited and somewhat conflicting. A small study found Syk in only 2/19 PTCLs by IHC, including 1/8 PTCL-U and 1/1 mycosis fungoid (weak staining).10 The higher positivity rate found by us might be due to differences in the antibodies used, or due to unknown differences in the patient populations studied. Syk expression and Syk kinase activity have been reported to be decreased in lysates of cutaneous T-cell lymphoma cells isolated from peripheral blood (n ¼ 4),18 a source not evaluated in our study. This difference in site might account for our finding that Syk was expressed in 4/4 cases of lymph node involvement by mycosis fungoides. Other previous studies have shown Syk overexpression in SYK-translocated cases,2 Syk upregulation in adult T-cell leukemia/lymphoma cell lines20 and a relative increase in SYK expression in ALK-positive ALCLs.21
Interphase fluorescence in situ hybridization (FISH) was performed on tissue microarray or whole-tissue sections as described previously,16 using dual fusion (D-FISH) SpectrumOrange- and SpectrumGreen-labeled DNA probes that hybridize to regions spanning the SYK and ITK breakpoints involved in the t(5;9)(q33;q22) translocation. A minimum of 50 cells were scored per case. Control material carrying the translocation was kindly provided by Dr B Streubel (Vienna, Austria).

Results and discussion

We evaluated Syk expression in reactive and neoplastic T cells by immunohistochemistry (IHC) using a polyclonal antibody against the C terminus of Syk. Although T cells in reactive tonsil, lymph node and spleen were negative (Figure 1a), IHC demonstrated cytoplasmic Syk expression in 133/141 (93%) PTCLs studied. These included 35/35 (100%) AITLs (angioimmunoblastic T-cell lymphomas; Figure 1b), 62/66 (94%) PTCL- Us (PTCLs, unspecified; Figure 1c), 6/6 (100%) anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas (ALCLs), 11/12 (92%) systemic ALK-negative ALCLs (Figures 1d and e), 3/3 (100%) cutaneous ALCLs, 4/4 (100%) mycosis fungoides (nodal involvement), 1/2 (50%) enteropathy-associated T-cell lymphoma, 4/5 (80%) extranodal NK/T-cell lymphomas, nasal type (NKTLs) 4/5 (80%) hepatosplenic T-cell lymphomas (Figure 1f), 2/2 (100%) subcutaneous panniculitislike T-cell lymphomas and 1/1 (100%) T-prolymphocytic leukemia. All eight Syk-negative cases were extranodal, including ALK-negative ALCLs (Figure 1d), enteropathy-associated T-cell lymphomas, hepatosplenic T-cell lymphomas (Figure 1e), NKTLs and PTCL-Us (four cases). Seven of these had a cytotoxic phenotype by IHC. Because Syk expression was found in a greater proportion of PTCLs than previously reported,10 we corroborated the IHC results using western blotting. Reactive splenic lymphocytes were sorted by flow cytometry into B-cell, ab T-cell and gd T-cell populations. B-cell lysates demonstrated a 72 kDa band corresponding to Syk, whereas T-cell lysates were negative (Figure 2a). Analysis of frozen tumor tissue lysates (Figure 2b) showed cases that were Syk-negative by IHC to be negative by western blot (PTCL-Us, two cases) as well. All four cases that were Syk-positive by IHC were positive by western blot (two AITLs, one ALK-negative ALCL and one PTCL-U). To evaluate the activation status of Syk in PTCLs, we probed western blots with phospho-specific anti-Syk (Tyr525/526); these tyrosine residues reside in the catalytic domain of Syk kinase and their phosphorylation is necessary for Syk activity.18 Syk was phosphorylated at these residues in 4/4 Syk-positive PTCLs tested (Figure 2b). Because the lysates used for western blot might contain Syk derived from non-tumor cells as well as PTCLs, we also evaluated Syk expression by flow cytometry. Reactive T cells from peripheral blood, lymph node and spleen were negative for Syk, whereas reactive B cells were positive (not shown). By using appropriate gating strategies in PTCLs with an aberrant T-cell phenotype, we could assess Syk expression specifically in the neoplastic T cells in four cases. The tumor cells demonstrated Syk expression in three cases (Figure 2c; see also Figure 1c). One case of hepatosplenic T-cell lymphoma was Syk-negative by flow cytometry (Figure 2d) as well as IHC (Figure 1f).
Several comments regarding the interpretation of our findings are warranted. First, IHC of reactive lymphoid tissue showed Syk-positive cells to outnumber CD20-positive cells in the paracortex (Figure 1a). Most lymphocytes appeared negative for Syk. By morphology and distribution, many of the positive cells appeared to be histiocytes and/or dendritic cells, which are among the hematopoietic cell types that express Syk.22 Without double immunostaining, the presence of a minimal population of normal Syk-positive T cells cannot be entirely excluded. However, such a population was not identified by flow cytometry, which is a highly sensitive method of detection. Second, unlike most normal T cells, NK cells have been reported to express Syk.23 Although we did not include tumors of known NK-cell origin in our series, we did include cases of NKTLs, which may be of either NK- or T-cell origin.15 Four out of five NKTLs were Syk-positive by IHC. If these four positive cases were of NK-cell origin, the observed Syk positivity might reflect constitutive expression in this cell type rather than lymphomaassociated overexpression. Finally, as mentioned above, tumor lysates subjected to western blot would be expected to contain some protein from admixed non-neoplastic cells. The phosphorylation status of Syk in the admixed B cells present in PTCLs such as AITLs is unknown. In B-cell lymphomas such as follicular lymphoma, tumor-infiltrating non-neoplastic B cells appear to demonstrate lesser Syk phosphorylation than the tumor cells on

  • stimulation.24 However, as phosphorylation of Syk is
  • a

physiologic event in B-cell receptor-mediated signaling,8 we cannot exclude the possibility that some of the phospho-Syk detected by western blot of PTCL samples (Figure 2b) was derived from admixed B cells.
To determine the relationship between Syk overexpression in our series and the t(5;9)(q33;q22) SYK/ITK translocation, we

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Figure 1 Immunohistochemical staining for Syk in reactive and neoplastic T lymphocytes. (a) Benign lymph node ( Â 10). Reactive follicles contain CD20-positive B cells; most lymphocytes are positive for Syk (arrowheads). Paracortical regions contain CD3-positive T cells; most lymphocytes are negative for Syk (arrows and inset, Â 100). (b) Angioimmunoblastic T-cell lymphoma ( Â 40). The tumor cells are negative for CD20 and positive for CD3. Nearly all cells present are positive for Syk, including the atypical medium-sized lymphoid cells (inset, Â 100). (c) Peripheral T-cell lymphoma, unspecified ( Â 40; see also Figure 2c). The tumor cells are negative for CD20 and positive for CD3 and Syk (inset, Â 100). (d) Anaplastic lymphoma kinase (ALK)-negative anaplastic large-cell lymphoma ( Â 40). The tumor cells are negative for CD20 and positive for CD30 and Syk (inset, Â 100; see also Figure 2b, lane 5). (e) ALK-negative anaplastic large-cell lymphoma ( Â 40). The tumor cells are positive for CD30 and negative for CD20 and Syk (inset, Â 100). (f) Hepatosplenic T-cell lymphoma ( Â 40). CD3-positive T cells expressing the cytotoxic marker TIA-1 infiltrating the hepatic sinusoids. They are negative for Syk (inset, Â 100).

Patients with PTCL are usually treated with CHOP or more intensive regimens, generally with minimal effectiveness, and new therapeutic strategies are needed.25 In this study, we demonstrate that Syk PTK is overexpressed in the majority of PTCLs. A phase II clinical trial of an orally available Syk inhibitor is underway for B-cell lymphomas. Overexpression of

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Figure 2 Syk expression in reactive and neoplastic T lymphocytes. (a) Western blot of lysates from flow-sorted reactive splenic lymphocytes shows no Syk expression in ab or gd T cells. Reactive B cells show Syk expression, as do Raji B-cell lymphoma cells. (b) Western blots of lysates from frozen peripheral T-cell lymphoma (PTCL) specimens show Syk expression similar to that shown by immunohistochemistry (IHC). Cases positive for Syk were positive for phospho-Syk (Tyr525/526) as well. Cases include PTCL-Us (lanes 1, 2 and 6), angioimmunoblastic T-cell lymphomas (lanes 3 and 4) and anaplastic lymphoma kinase-negative anaplastic large-cell lymphomas (lane 5; see also Figure 1e). (c) Flow cytometry results from an Syk-positive PTCL-U (see also Figure 1c). The neoplastic T cells show diminished expression of CD3 and CD5, allowing selective gating on both neoplastic and normal T-cell populations (middle panel). The CD3-dim neoplastic T cells (purple) are positive for cytoplasmic Syk with an intensity of staining between that of the normal T cells (blue, Syk-negative) and CD3-negative B cells (green, Syk-positive and CD19-positive (not shown)). (d) Flow cytometry results from an Syk-negative hepatosplenic T-cell lymphoma (see also Figure 1f). The neoplastic T cells show loss of CD5. By selective gating on the neoplastic and normal T-cell populations, both are shown to be Syk-negative. FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCp, peridinin chlorophyll protein.

Syk, phosphorylation of its Y525/526 residues and the availability of pharmacologic inhibitors suggest that Syk may be a suitable target for PTK inhibition in PTCL patients. Studies of the effect of Syk inhibitors on T-cell lymphoma cell lines are warranted to evaluate this possibility further.

3 de Leval L, Savilo E, Longtine J, Ferry JA, Harris NL. Peripheral T-cell lymphoma with follicular involvement and a CD4+/bcl-6+ phenotype. Am J Surg Pathol 2001; 25: 395–400.
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  • induced activation of Akt promotes
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dependent on Syk kinase. J Immunol 2000; 165: 1300–1306.
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Acknowledgements

6 Gururajan M, Dasu T, Shahidain S, Jennings CD, Robertson DA, Rangnekar VM et al. Spleen tyrosine kinase (Syk), a novel target of curcumin, is required for B lymphoma growth. J Immunol 2007; 178: 111–121.

We acknowledge the support from the Iowa/Mayo Lymphoma SPORE grant from the National Cancer Institute (P50 CA97274). In addition, we thank Ms Connie Lesnick for help with flow cytometry, Dr B Streubel for providing control specimens with the SYK/ITK translocation and Ms Monica Kramer and Ms Carrie Stevenson for administrative assistance.

7 Chen L, Monti S, Juszczynski P, Daley J, Chen W, Witzig TE et al.

  • SYK-dependent tonic B-cell receptor signaling is
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treatment target in diffuse large B-cell lymphoma. Blood 2008; 111: 2230–2237.
8 Turner M, Schweighoffer E, Colucci F, Di Santo JP, Tybulewicz VL. Tyrosine kinase SYK: essential functions for immunoreceptor signalling. Immunol Today 2000; 21: 148–154.

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  • Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation (Son of Sevenless/T-Cell Anergy) MONIKA RAAB*T, YUN-CAI CAI*T, STEPHEN C

    Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation (Son of Sevenless/T-Cell Anergy) MONIKA RAAB*T, YUN-CAI CAI*T, STEPHEN C

    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8891-8895, September 1995 Immunology p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: Implications for T-cell costimulation (son of sevenless/T-cell anergy) MONIKA RAAB*t, YUN-CAI CAI*t, STEPHEN C. BUNNELLI, STEPHANIE D. HEYECKt, LESLIE J. BERGt, AND CHRISTOPHER E. RUDD*§¶ *Division of Tumor Immunology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; Departments of tMedicine and §Pathology, Harvard Medical School, Boston, MA 02115; and *Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02128 Communicated by Stuart F. Schlossman, Dana-Farber Cancer Institute, Boston, MA, May 4, 1995 ABSTRACT T-cell activation requires cooperative signals 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase generated by the T-cell antigen receptor c-chain complex ITK (formerly EMT or TSK), and the complex between (TCRC-CD3) and the costimulatory antigen CD28. CD28 growth factor receptor-bound protein 2 and son of sevenless interacts with three intracellular proteins-phosphatidylino- guanine nucleotide exchange protein (GRB-2-SOS) (21-25). sitol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine PI 3-kinase and GRB-2 Src-homology 2 (SH2) domains bind kinase ITK (formerly TSK or EMT), and the complex between to a phosphorylated version of the Tyr-Met-Asn-Met growth factor receptor-bound protein 2 and son of sevenless (YMNM) motif within CD28 (21, 22, 25). PI 3-kinase consists guanine nucleotide exchange protein (GRB-2-SOS).
  • Supplementary Table 1. in Vitro Side Effect Profiling Study for LDN/OSU-0212320. Neurotransmitter Related Steroids

    Supplementary Table 1. in Vitro Side Effect Profiling Study for LDN/OSU-0212320. Neurotransmitter Related Steroids

    Supplementary Table 1. In vitro side effect profiling study for LDN/OSU-0212320. Percent Inhibition Receptor 10 µM Neurotransmitter Related Adenosine, Non-selective 7.29% Adrenergic, Alpha 1, Non-selective 24.98% Adrenergic, Alpha 2, Non-selective 27.18% Adrenergic, Beta, Non-selective -20.94% Dopamine Transporter 8.69% Dopamine, D1 (h) 8.48% Dopamine, D2s (h) 4.06% GABA A, Agonist Site -16.15% GABA A, BDZ, alpha 1 site 12.73% GABA-B 13.60% Glutamate, AMPA Site (Ionotropic) 12.06% Glutamate, Kainate Site (Ionotropic) -1.03% Glutamate, NMDA Agonist Site (Ionotropic) 0.12% Glutamate, NMDA, Glycine (Stry-insens Site) 9.84% (Ionotropic) Glycine, Strychnine-sensitive 0.99% Histamine, H1 -5.54% Histamine, H2 16.54% Histamine, H3 4.80% Melatonin, Non-selective -5.54% Muscarinic, M1 (hr) -1.88% Muscarinic, M2 (h) 0.82% Muscarinic, Non-selective, Central 29.04% Muscarinic, Non-selective, Peripheral 0.29% Nicotinic, Neuronal (-BnTx insensitive) 7.85% Norepinephrine Transporter 2.87% Opioid, Non-selective -0.09% Opioid, Orphanin, ORL1 (h) 11.55% Serotonin Transporter -3.02% Serotonin, Non-selective 26.33% Sigma, Non-Selective 10.19% Steroids Estrogen 11.16% 1 Percent Inhibition Receptor 10 µM Testosterone (cytosolic) (h) 12.50% Ion Channels Calcium Channel, Type L (Dihydropyridine Site) 43.18% Calcium Channel, Type N 4.15% Potassium Channel, ATP-Sensitive -4.05% Potassium Channel, Ca2+ Act., VI 17.80% Potassium Channel, I(Kr) (hERG) (h) -6.44% Sodium, Site 2 -0.39% Second Messengers Nitric Oxide, NOS (Neuronal-Binding) -17.09% Prostaglandins Leukotriene,
  • Actin Cytoskeleton TCR-Induced Regulation of Vav and the Kinase

    Actin Cytoskeleton TCR-Induced Regulation of Vav and the Kinase

    Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton This information is current as Derek Dombroski, Richard A. Houghtling, Christine M. of September 24, 2021. Labno, Patricia Precht, Aya Takesono, Natasha J. Caplen, Daniel D. Billadeau, Ronald L. Wange, Janis K. Burkhardt and Pamela L. Schwartzberg J Immunol 2005; 174:1385-1392; ; doi: 10.4049/jimmunol.174.3.1385 Downloaded from http://www.jimmunol.org/content/174/3/1385 References This article cites 45 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/174/3/1385.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton1 Derek Dombroski,2* Richard A.
  • Ibrutinib As a Potential Therapeutic Option for HER2 Overexpressing Breast Cancer – the Role of STAT3 and P21

    Ibrutinib As a Potential Therapeutic Option for HER2 Overexpressing Breast Cancer – the Role of STAT3 and P21

    Ibrutinib as a potential therapeutic option for HER2 overexpressing breast cancer – the role of STAT3 and p21 A Thesis Submitted to the College of Graduate and Postdoctoral Studies in Partial Fulfillment of the Requirements for the Degree of Master of Science in the College of Pharmacy and Nutrition University of Saskatchewan Saskatoon By Chandra Bose Prabaharan © Copyright Chandra Bose Prabaharan, November 2019. All rights reserved Permission to Use Statement By presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes, may be granted by the professors who supervised my thesis work or, in their absence, by the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written consent. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other uses of the materials in this thesis in whole or in part should be addressed to: College of Pharmacy and Nutrition 104 Clinic Place University of Saskatchewan Saskatoon, Saskatchewan, S7N 2Z4, Canada OR Dean College of Graduate and Postdoctoral Studies Room 116 Thorvaldson Building 110 Science Place Saskatoon, Saskatchewan, S7N 5C9, Canada i Abstract Treatment options for HER2 overexpressing breast cancer are limited, and the current anticancer therapies are associated with side effects.
  • Protein Tyrosine Kinases: Their Roles and Their Targeting in Leukemia

    Protein Tyrosine Kinases: Their Roles and Their Targeting in Leukemia

    cancers Review Protein Tyrosine Kinases: Their Roles and Their Targeting in Leukemia Kalpana K. Bhanumathy 1,*, Amrutha Balagopal 1, Frederick S. Vizeacoumar 2 , Franco J. Vizeacoumar 1,3, Andrew Freywald 2 and Vincenzo Giambra 4,* 1 Division of Oncology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; [email protected] (A.B.); [email protected] (F.J.V.) 2 Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; [email protected] (F.S.V.); [email protected] (A.F.) 3 Cancer Research Department, Saskatchewan Cancer Agency, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada 4 Institute for Stem Cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), Fondazione IRCCS Casa Sollievo della Sofferenza, 71013 San Giovanni Rotondo, FG, Italy * Correspondence: [email protected] (K.K.B.); [email protected] (V.G.); Tel.: +1-(306)-716-7456 (K.K.B.); +39-0882-416574 (V.G.) Simple Summary: Protein phosphorylation is a key regulatory mechanism that controls a wide variety of cellular responses. This process is catalysed by the members of the protein kinase su- perfamily that are classified into two main families based on their ability to phosphorylate either tyrosine or serine and threonine residues in their substrates. Massive research efforts have been invested in dissecting the functions of tyrosine kinases, revealing their importance in the initiation and progression of human malignancies. Based on these investigations, numerous tyrosine kinase inhibitors have been included in clinical protocols and proved to be effective in targeted therapies for various haematological malignancies.
  • SUPPLEMENTAL TABLE 1. Mass Spectrometry on EGFRL858R/T790M Protein

    SUPPLEMENTAL TABLE 1. Mass Spectrometry on EGFRL858R/T790M Protein

    SUPPLEMENTAL TABLE 1. Mass spectrometry on EGFRL858R/T790M protein % Mass Modification on Compound EGFRL858R/T790M Protein 1 96% 2 95% 3 109% 4 108% SUPPLEMENTAL TABLE 2. EGFR modulation in A431, H1975 and HCC827 cells by compound 3. EC50 (nM) Cell Lines A431 H1975 HCC827 EGFR Genotype WT L858R/T790M DelE746-A750 pEGFR > 4331 58 ± 34 187 ± 88 pAKT > 4331 55 ± 12 80 ± 49 pERK > 5000 39 ± 25 65 ± 6 pS6RP > 4841 29 ± 22 74 ± 48 Occupancy > 4260 34 ± 9 nd n > 3; ave ± STD; nd = not determined SUPPLEMENTAL TABLE 3. Kinase selectivity profile of compound 3, afatinib and WZ4002 at 1 µM. Compound 3 WZ4002 Afatinib FLT3 94% TXK* 98% ERBB4/HER4* 99% EGFR (L858R/T790M)* 91% BTK* 90% EGFR (WT)* 99% JAK3* 87% EGFR (WT)* 89% ERBB2/HER2*§ 98% TXK* 84% EGFR (L858R, T790M)* 87% EGFR (L858R, T790M)* 93% Aurora A 82% FLT3 87% TXK* 87% EGFR (WT)* 81% JAK3* 86% BLK* 84% FAK/PTK2 77% ERBB4/HER4* 85% BTK* 79% BMX/ETK* 74% FMS 77% ITK* 63% CHK2 74% BLK* 74% YES/YES1 55% ERBB4/HER4* 66% FAK/PTK2 73% LYN 53% BTK* 64% BMX/ETK* 69% c-Src 54% TEC* 62% CHK2 64% ITK* 58% FGFR3 54% TEC* 53% §ERBB2/HER2 included in kinase panel Compounds were tested against a 62 kinase panel at Reaction Biology Corporation which includes representative kinases from each branch of the kinome tree. Kinases inhibited greater than 50% are indicated. Asterisks indicate kinases that share Cys 797 with EGFR. For afatinib, ERBB2/HER2 kinase was included in the 62 kinase panel.
  • Trastuzumab Mechanism of Action; 20 Years of Research to Unravel a Dilemma

    Trastuzumab Mechanism of Action; 20 Years of Research to Unravel a Dilemma

    cancers Review Trastuzumab Mechanism of Action; 20 Years of Research to Unravel a Dilemma Hamid Maadi 1, Mohammad Hasan Soheilifar 2, Won-Shik Choi 1, Abdolvahab Moshtaghian 3,4 and Zhixiang Wang 5,* 1 Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, AB T6G 1Z2, Canada; [email protected] (H.M.); [email protected] (W.-S.C.) 2 Department of Medical Laser, Medical Laser Research Center, Yara Institute, ACECR, Tehran 1315795613, Iran; [email protected] 3 Department of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar 4741695447, Iran; [email protected] 4 Deputy of Research and Technology, Semnan University of Medical Sciences, Semnan 3514799442, Iran 5 Department of Medical Genetics and Signal, Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada * Correspondence: [email protected] Simple Summary: Overexpression of HER2 receptors have been identified in various types of cancer including breast cancer and ovarian cancer. HER2 overexpression is generally associated with poor clinical outcomes in patients with HER2-positve tumors. Trastuzumab, an antibody specifically targeting HER2 receptors, showed promising clinical benefits for patients with HER2-positive tumors. Studies show that trastuzumab suppresses HER2 receptors’ oncogenic functions in HER2-postive tumors. Moreover, trastuzumab has been shown to provoke immune responses against the HER2- amplified tumors. Citation: Maadi, H.; Soheilifar, M.H.; Choi, W.-S.; Moshtaghian, A.; Wang, Z. Trastuzumab Mechanism of Abstract: Trastuzumab as a first HER2-targeted therapy for the treatment of HER2-positive breast Action; 20 Years of Research to cancer patients was introduced in 1998.