Immunology Graduate Group

M.A. Wasik, M.D.
Associate Professor
Director, Experimental Hematology
Pathology and Laboratory Medicine

Office Phone:  215-662-3467
Email:  wasik@mail.med.upenn.edu

Research Interests

Aberrant cell signaling and epigenetic regulation of gene expression in human lymphomas.   

Research Summary 

1. Role of the cytokine-signal transduction pathways and epigenetic gene silencing in pathogenesis of T-cell lymphoma.

Under this project my lab investigates the role of signals mediated through receptor for interleukin-2 (IL-2R) and functionally related cytokine receptors in malignant transformation of T lymphocytes. Part of the IL-2R, common chain ( c), is shared by receptors for several cytokines: IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. We found that cutaneous T-cell lymphoma cells display activation of the interleukin-2 receptor/cytokine common chain-associated Jak/STAT signal transduction pathway that is transient in the early stage of the lymphoma and constitutive in the late stage of the disease progression. More recently we determined that the constitutive Jak/STAT activation is due, at least in part, to the lack of expression of SHP-1 phosphatase, which normally down-regulates IL-2R/ c-mediated cell activating signals. Importantly, this work identified the mechanism underlying lack of SHP-1 expression as hypermethylation of the CpG DNA sequences within the SHP-1 promoter. This study may result in novel therapies for lymphoma based on selective inhibition of the elements of the IL-2R signal transduction pathway(s) which are preferentially utilized by malignant T cells and/or on induction of re-expression of the epigenetically-silenced SHP-1 gene. Our most recent work focuses on the molecular mechanisms of the aberrant gene silencing in the malignant lymphoid cells.

Selected references:
Zhang, Q. et al. Proc. Natl. Acad. Sci. USA 93: 9148-9153, 1996,
Zhang, Q. et al. Am. J. Path., 157: 1137-1146, 2000,
Brender C et al. Blood, 97: 1056-1061, 2001, Li S et al. Am J Path, 158: 1231-1237, 2001, Eriksen KW et al. Leukemia, 15: 787-793, 2001,
Wang ZY et al. J Molec Diagn, 5: 113- 120, 2003.

2. TOR signaling in posttransplant lymphoproliferative disorders (PTLDs) and other lymphoid malignancies.

Whereas the standard immunosuppressive agents foster development of PTLDs, the impact of novel immunosuppressive agents from the group of selective inhibitors of TOR serine/treonine kinase such as rapamycin and its derivatives including RAD remains undetermined. Our studies indicate that RAD has a strong inhibitory effect on PTLD-like and PTLD-derived B cells by suppressing their proliferation, blocking cell cycle progression and increasing apoptotic rate. In the in vivo SCID mouse xeno-transplant model, RAD markedly delayed growth or induced regression of established PTLD-related B-cell tumors. The drug completely eradicated or prevented tumor establishment in a subset of the treated mice at the doses matching the ones required to prevent graft rejection. These findings indicate that TOR inhibitors such as RAD may be effective in prevention and treatment of PTLDs and, possibly, other types of B-cell lymphoma. The molecular mechanism of this TOR inhibitor-mediated cell growth suppression is currently under investigation.

Selected references:
Majewski, M. et al. Proc. Natl. Acad. Sci. USA, 97: 4285-4290, 2000,
M Paessler et al. Lab. Invest, 82: 1599-1606, 2002,
M Majewski, et al. Transplantation, 75: 1710-1717, 2003.

3. Mechanisms of malignant cell transformation by the chimeric NPM/ALK kinase.

Accumulating evidence indicates that expression of anaplastic lymphoma kinase (ALK) defines a distinct type of T-cell lymphoma. Expression of ALK in malignant T cells is typically due to the t(2;5) translocation resulting in formation of the fusion gene which encodes a 80-kDa hybrid protein that contains portion of the nuclear protein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase ALK. The NPM/ALK kinase is constitutively activated and highly oncogenic. Our studies concentrate on identification of downstream effector molecules triggered by the NPM/ALK kinase. They indicate that pathways involving STAT3, PI3K/AKT and, apparently, STAT5 are constitutively activated by this kinase. Regulation and function of STAT3 in the ALK+ T-cells and testing an ALK-inhibitor small molecule candidate are the main focus of the ongoing investigation.

Selected References:
Slupianek A et al. Cancer Res., 61: 2194-2199, 2001,
M Nieborowska-Skorska et al Cancer Res 61: 6517-6523, 2001,
Zhang, Q. J. Immunol, 168: 466-474, 2002,
Wasik MA. Am J Clin Path, 118: S81-S92, 2002 (a review).

Figures

Figure 1. Methylation of CpG sequences in the SHP-1 promoter. Whereas the normal peripheral blood mononuclear cells (PBMC) and the cell line derived from a patient with an early stage of T-cell lymphoma show an unmethylated CpG sequence (where C was converted to T by the DNA treatment with bisulphate), two cell lines (2A and 2B) and the cells derived directly from a T-cell lymphoma patient, display methylated, bisulphate-resistant CpG sequence. The degree of promoter CpG sequence methylation correlated with the amount SHP-1 mRNA and protein (data not presented).

Figure 2. Reversal of the SHP-1 promoter methylation in malignant T-cells. T-cell lymphoma lines from cutaneous anaplastic large T-cell lymphoma (PB-1, 2A and 2B), HTLV-I+ adult type T-cell leukemia/lymphoma (C10MJ2 and ATL-2), ALK+ T-cell lymphoma line (SUDHL-1), and two Hodgkin lymphoma cell lines (L540 and KMH2) were tested for methylation status of the 5' CpG island-rich DNA within the SHP-1 promoter. Peripheral mononuclear cells (PBMC) and mitogen (PHA)-stimulated PBMC blasts served as positive controls. Uppermost panel: PCR amplified DNA fragment of the SHP-1 gene promoter region, Upper middle panel: SHP-1 promoter DNA treated with methylation sensitive enzyme HpaII prior to the PCR amplification, Lower middle panel: SHP-1 promoter DNA from cell lines pre-cultured with an inhibitor of DNA methyltransferase 5-aza-2deoxycytidine, D. HpaII-digested SHP-1 promoter DNA from 5-aza-2deoxycytidine cultured cells. The reversal of SHP-1 promoter methylation resulted in re-expression of the SHP-1 mRNA and protein (not shown).

Figure 3. STAT3 is continuously and selectively activated in ALK+ TCL cells. Cell lysates from ALK+ T-cell lymphoma lines were immunoprecipitated with anti-Jak3 and anti-STAT3 and -STAT5a/b antibodies in the first set of experiments (A-C) and anti-STAT1 to STAT6 antibodies in the second set of experiments (D; a representative result). The electrophoretically separated immunoprecipitates were immunoblotted with an anti-phosphotyrosine antibody. Next, the whole cell lysates were immunoblotted with an anti-phospho-STAT3 (E). ALK- TCL cell lines PB-1 and 2B that express continuously activated STAT3 as a part of the IL-2R Jak/SAT signaling pathway, were also evaluated. Finally, capacity of STAT3 to bind DNA was determined in EMSA (F). Nuclear extracts from the ALK+ TCL cell lines were exposed to digoxigenin-labeled STAT3 specific oligo_DNA fragments, either wild type or mutated at the STAT3 binding site (designated m). ALK- TCL cell line 2B and NK-cell line YT that express activated STAT3 served as positive controls, whereas Sezary Syndrome-derived Sez-4 cell line that expresses non-activated STAT3 served as a negative control.

Figure 4. STAT3 is tyrosine phosphorylated in ALK+ T-cell lymphoma tissues (immunohistochemical analysis). Left upper and lower panels: histology of a representative case. Middle panels: staining with an anti-ALK antibody, right panels: staining with the anti-phospho-STAT3 antibody. The depicted results are representative for 8 ALK+ TCL cases.

 

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