The role of Akt isoforms in BCR-ABL induced chronic myeloid leukemia

Aubrey Watkins, Thomas Jefferson University

Abstract

The Akt/PKB serine/threonine kinase is involved in several important physiological cellular processes that include regulation of cell growth, proliferation, apoptosis, and glucose homeostasis. There are three isoforms that share a high degree of sequence homology, structural similarity, functional overlap and biochemical mechanism of activation. All three family members, Akt1/PKBá, Akt2/PKBâ, Akt3/PKB, belong to the AGC family of protein kinases. As a key mediator of the PI3K/Akt/mTOR pathway Akt is well characterized for its role in a wide variety of human neoplasms. In order to define which isoform is most relevant in a context-specific manner, we studied the role of individual Akt isoforms and their contribution in BCR/ABL mediated transformation in vitro and in vivo. To determine which Akt isoform(s) was most relevant for in vitro transformation we used the IL-3 cytokine-dependent 32Dcl3 cell line (32DWT) and a derivative expressing the p210 kDa BCR-ABL fusion protein (32Dp210). We used highly specific allosteric non ATP-competitive pharmacological inhibitors of Akt that selectively inhibit either individual or combinations of different isoforms to evaluate their effect on 32Dp210 proliferation and transformation. Through these in vitro studies we identified that Akt3 kinase activity was hyperactivated in 32Dp210 as a consequence of BCR-ABL expression. Analysis and comparison of Akt mRNA expression, total protein levels, and kinase activity between 32DWT and 32Dp210 revealed that Akt3 kinase activity was selectively upregulated. Pharmacological inhibition of Akt1 or Akt2 alone or Akt1 and Akt2 in combination was ineffective at reducing proliferation or colony growth of BCR-ABL transformed 32Dp210. However, the use of an inhibitor that inhibits all three isoforms proved quite effective at reducing proliferation and eliminating growth in colony formation assays. The generation and study of Akt1 knockout mice by several different laboratories demonstrated a physiological role for Akt1 in whole body size, survival, apoptosis and normal cellular proliferation. Early examination of tumors from patients suggested that Akt1 was hyperactivated in a large variety of human neoplasms. We wanted to characterize its contribution during in vivo leukemogenesis induced by BCR-ABL. To investigate, we used a murine retroviral bone marrow transduction-transplantation model that generates a CML-like myeloproliferative disease, to evaluate the requirement of Akt1 in BCR-ABL mediated leukemogenesis. Hematopoietic stem cells transduced ex vivo with the p210 kDa BCR-ABL fusion protein were transplanted into recipient mice. We examined and compared mice that received wild type and Akt-deficient bone marrow cells for the onset of a CML-like disease and to determine overall time of survival post-transplant. Here we showed that Akt1-deficient bone marrow cells resulted in delayed leukemia that was characterized by less severe pathohistology and increased survival in mice. These results when taken together reaffirm the critical role of Akt in BCR-ABL mediated transformation but suggest a selective mechanism for upregulation of Akt3 in a context-specific manner. It is our hypothesis that Akt1 and Akt3 have a predominant role in BCR/ABL-mediated leukemia but that each neoplasm must be evaluated on a case-by-case basis to determine the specific contribution of individual isoforms in human neoplasms. Furthermore, an increased understanding of the role of Akt isoforms in a context-specific manner should enhance optimum efficacy of therapeutic inhibitors used in the treatment of CML with Akt inhibitors alone or in combination with existing therapies such as Gleevec.

Subject Area

Molecular biology|Cellular biology|Biochemistry|Oncology

Recommended Citation

Watkins, Aubrey, "The role of Akt isoforms in BCR-ABL induced chronic myeloid leukemia" (2012). ETD Collection for Thomas Jefferson University. AAI3524076.
https://jdc.jefferson.edu/dissertations/AAI3524076

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