Dr. Ralph B. Arlinghaus
The University of MD Anderson Cancer Center
Department of Molecular Pathology
My research is focused on two gene products: Jak2 in CML and LCN2 in various cancers.
Despite the success of imatinib mesylate (IM) in early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in later stages of CML. Our findings indicate that inhibition of Jak2 inBcr-Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr-Abl+ cells reduced levels of the Bcr-Abl protein and also the phosphorylation of Tyr177 of Bcr-Abl, and Jak2over-expression rescued these knockdown effects. Treatment of Bcr-Abl+ cells with Jak2 inhibitors for 4-6 h but not IM also reduced Bcr-Abl protein and pTyr 177 levels. In vitro kinase experiments performed with recombinant Jak2 showed that Jak2 readily phosphorylated Tyr 177 of Bcr-Abl (a Jak2 consensus site,YvnV) whereas c-Abl did not. Importantly, Jak2 inhibition decreased pTyr177 Bcr-Abl in immune complexes but did not reduce levels of Bcr-Abl, suggesting that the reduction of Bcr-Abl by Jak2 inhibition is a separate event from phosphorylation of Tyr177. Jak2 inhibition by chemical inhibitors (TG101209/WP1193) and Jak2 knockdown diminished the activation of Ras, PI-3kinase pathways and reduced levels of pTyrSTAT5. These findings suggest that Bcr-Abl stability and oncogenic signaling in CML cells are under the control of Jak2.
Lipocalin 2 in cancer
My laboratory has published three papers on the involvement of LCN2 in cancer. Our initial studies were conducted in cells transformed by the Bcr-Abl oncoprotein. These studies were triggered by the report that mouse hematopoietic cells deprived of IL-3 induce expression and secretion of 24p3 (mouse LCN2), and that the involved mouse cells undergo apoptosis. Bcr-Abl is the causative protein in chronic myeloid leukemia (CML). We discovered that the Bcr-Abl oncoprotein induced expression of LCN2 in mouse hematopoietic cells . The protein is secreted from Bcr-Abl+ hematopoietic cells into the surrounding cell culture medium. Normal hematopoietic cells undergo cell death whereas Bcr-Abl+ cells are resistant to apoptosis induced by LCN2. Deviredddy et al. (Cell2005) reported that normal hematopoietic cells express the receptor for LCN2 but Bcr-Abl+ cells lack expression of the LCN2 receptor. We discovered that mice injected with Bcr-Abl+ cells as expected are induced to have leukemia with bone marrow involvement and enlarged spleens. In these experiments, cells were tagged with the green fluorescent protein (GFP). Marrow and spleen tissue were heavily labeled with GFP. Anti-sense LCN2 and cells expressing the shRNA for LCN2 had dramatic reduced levels of GFP+leukemia cells in their marrow and spleen tissues . Erythroid lineage cells were strongly reduced in leukemia mice as were normal myeloid cells.
The anti-sense LCN2 and LCN2 shRNA expression although reducing LCN2 expression about 70-80%, still allowed low level expression and secretion of LCN2 by these treated cells. We wondered whether complete elimination of LCN2 would yield similar results as did partial removal. We therefore obtained LCN2 knockout (KO) mice for these experiments in order to obtain cells completely lacking LCN2 expression and secretion. In these experiments we isolated bone marrow cells from LCN2 KO mice. Cells were transduced BCR-ABL-IRES-GFP and cells were isolated as usual. We found that bone marrow cells from LCN2 KO mice would not engraph or cause leukemia in mice transplanted with BCR-ABL-IRES-GFP cells These mice required sublethal irradiation to allow engraphment and leukemia induction. Bone marrow cells expressing LCN2 were able to cause leukemia without need of sub-lethal irradiation. We concluded from these experiments that expression and secretion of LCN2 by leukemia cells was required to kill normal hematopoietic cells in order to provide space for leukemia cells. We proposed a model for invasion of the leukemia cells in marrow and spleen which showed that LCN2 released by invading CML cells caused a wave of cell death in the nearby regions allowing CML cells to fill in the open space created by dying normal hematopoietic cells.
Office: MDA 2SCR4.3016 (Unit 951)
Ph.D. - University of Cincinnati College of Medicine - 1961