Office: Department of Biology

419 Biological Life Sciences Building
Temple University
Philadelphia, PA 19122

Phone: (215) 204-8847
E-mail: tskorski@temple.edu

Reciprocal chromosomal translocations are responsible for the occurrence of fusion genes, whose products cause leukemic transformation. A cohort of these fusion genes encodes oncogenic tyrosine kinases (OTKs). Despite remarkable advances in investigations of the mechanisms leading to the leukemic transformation of hematopoietic cells by OTKs our knowledge about the nature of intracellular phenomena triggered by OTKs is rather limited. Characterization of OTKs and their signaling pathways may shed some light on the mechanisms involved in the transformation and may suggest new approaches for therapy of leukemias.

Our laboratory focuses on identification of the leukemogenic mechanisms activated by BCR/ABL oncogenic tyrosine kinase produced by the Philadelphia chromosome, and by other OTKs (TEL/ABL, TEL/PDGFR, TEL/JAK, NPM/ALK). BCR/ABL gene is derived from relocation of the portion of c-ABL gene from chromosome 9 to the portion of BCR gene locus on chromosome 22 [t(9;22)], and is present in most of chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemia (ALL) patients. BCR/ABL hybrid genes produce p230, p210 and p185 fusion proteins (the size depends on the breakpoint in BCR locus) exerting constitutive tyrosine kinase activity, transforming hematopoietic cells in vitro, and causing CML- or ALL- like syndromes in mice.

My previous work demonstrated that BCR/ABL junction region could be targeted by antisense oligodeoxynucleotides (AS-ODNs), which was confirmed by other laboratories. Moreover, these studies provided the first direct evidence that BCR/ABL is essential for the growth of CML cells. Because of the pivotal role of BCR/ABL in the pathogenesis of Philadelphia chromosome-positive leukemias, we decided to investigate the mechanisms of BCR/ABL-mediated leukemogenesis. The major goals of my studies are to better understand the molecular basis of BCR/ABL-induced leukemias, and to identify novel targets for anti-tumor treatment. Initial experiments indicated that a small GTP binding protein p21RAS and p120 GTPase-activating protein (p120GAP), a RAS inhibitor, are both necessary for the proliferation of normal and BCR/ABL-transformed hematopoietic cells. Moreover, our studies revealed that BCR/ABL not only activates RAS, but also inhibits GAP (RAS inhibitor), which provided of a novel concept of the double regulation of RAS by BCR/ABL activation of RAS activators (Sos) and inhibition of RAS inhibitors (GAP). RAF serine/threonine kinase, the immediate downstream effector of RAS, was also found to be essential for the growth of normal and BCR/ABL-transformed hematopoietic cells. Next, we demonstrated that BCR/ABL activates phosphatidylinositol-3 kinase (PI-3k), which is required for the growth of CML cells, but not for the proliferation of normal hematopoietic cells.

To investigate more precisely signaling pathways induced by BCR/ABL we adopted BCR/ABL mutant strategy, because deletion or mutation of the particular domains may inhibit specific signaling pathways. The mutants have been generated in our laboratory or were obtained from Dr. Charles Sawyers (UCLA, Los Angeles, CA) and Dr. Ann-Marie Pendergast (Duke University Medical Center, Durham, NC). To provide more evidence about the involvement of particular signaling proteins in BCR/ABL leukemogenesis, dominant negative- and dominant active- mutants of these molecules were used. These strategies led us to identify the novel mechanisms of PI-3k regulation by BCR/ABL involving the SH2 domain of BCR/ABL, and to demonstrate that activation of AKT serine/threonine kinase and c-MYC transcriptional factor are both dependent on PI-3k and essential for leukemogenesis. In addition, the novel anti-apoptotic pathway, involving AKT - PKC (protein kinase C) - mitochondrial RAF - BAD was described in BCR/ABL-positive cells. In addition to the SH2 domain, BCR/ABL also contains the SH3 domain, which seems to regulate invasion, adhesion and homing abilities of leukemia cells. A small GTP binding protein p21RAC is activated by BCR/ABL and involved in modulation of these functions of leukemia cells, but the interactions (if any) between BCR/ABL SH3 domain and RAC have not been defined yet. Both, the SH3 and SH2 domains of BCR/ABL collaborate in activation of STAT5 - another signaling protein important for BCR/ABL transformation machinery.

We are also interested in examination of the mechanisms responsible for transformation of the relatively benign CML chronic phase (CML-CP) to the rapidly fatal blast crisis (CML-BC). The mechanisms which underline this phenomenon are poorly understood. Using p53-deficient mice we demonstrated that loss of wild-type p53 resulted in blastic transformation of bone marrow cells expressing BCR/ABL. Our recent work provided more insight about the role of p53 in CML progression to blast crisis and indicated that loss of p53 function could be responsible for accelerated proliferation and resistance to DNA-damaging agents.

We are further characterizing and comparing major BCR/ABL-dependent signaling pathways in CML-CP and CML-BC cells. These experiments are primarily focused on novel genes regulated by BCR/ABL. Recently, using representational differences analysis (RDA), we identified several known genes and also fragments of unknown genes, which may be involved in initiation and malignant progression of CML. Accordingly, the role of these genes will be examined, the unknown genes will be sequenced and their functions will be determined. Our special interest is focused on RAD51 - a representative of the family of genes involved in DNA recombination/repair. It seems that RAD51 and other members of the family play an important role in drug resistance of leukemia cells, therefore their function in BCR/ABL-positive cells will be further investigated in collaboration with the laboratory of Dr. Richard Fishel (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA).

Our studies are not limited only to BCR/ABL leukemogenesis. In collaboration with Dr. Gary Gilliland (Harvard Medical School, Boston, MA), Dr. Stephan Morrison (St. Jude's Children Research Hospital, Memphis, TN) and Dr. Martin Carrol (University of Pennsylvania, Philadelphia, PA) we started to elucidate the mechanisms of transformation induced by other OTKs such as TEL/ABL, TEL/PDGFR, TEL/JAK and NPM/ALK.

In summary, I hope that our studies provide some insight into leukemogenesis induced by BCR/ABL and other OTKs. These studies may facilitate the development of new anti-tumor strategies. Representative examples of the fruits of this approach are the data demonstrating anti-tumor potentials of novel strategies developed on the basis of research accomplishments. Initially, using SCID mice bearing CML-BC we showed that BCR/ABL antisense oligodeoxynucleotides (AS-ODNs) exerted anti-leukemic effect in vivo and prolonged survival time of the mice. Combination of the low doses of AS-ODNs and standard cytostatic drug, or AS-ODNs and immunotherapy, exerted synergistic and selective anti-tumor effect. The mechanism(s) responsible for such phenomenon are unknown, but our data indicate that induction of multiple apoptotic pathways, increased AS-ODNs uptake after cytostatic treatment, and/or inhibition of the DNA repair genes after downregulation of BCR/ABL by AS-ODNs, may contribute to the effect. Altogether, these results provided an experimental basis for clinical trials, which demonstrated that our strategy could be beneficial for CML patients. Identification of c-MYC and PI-3k as the downstream effectors of BCR/ABL led to the finding that: a) wortmannin (WT), a specific PI-3k activity inhibitor, arrested growth of CML cells, but not of normal bone marrow cells, and simultaneous inhibition of BCR/ABL and PI-3k by AS-ODNs and WT, respectively, exerted synergistic anti-leukemia effect; b) simultaneous downregulation of BCR/ABL and c-MYC by combined AS-ODNs treatment, exerted much better therapeutic effect than inhibition of the individual gene expression. These results may provide an experimental basis for further clinical trials.

Promises and pitfalls of AS-ODNs in the treatment of experimental tumors encouraged us to seek other compounds able to block BCR/ABL. A new molecule targeting BCR/ABL tyrosine kinase has been developed by rational drug design strategy in collaboration with Dr. Ziwei Huang laboratory (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA) and its anti-tumor activity is under investigation.

(Grants from the National Institutes of Health, American Cancer Society, Cancer Research Foundation of America, Leukemia Research Foundation and Elsa U. Pardee Foundation provided support for the projects described above.)