Examining the Origins of Myeloid Leukemia

Retroviral infection induced acute myeloid leukemia in mice lacking p15INK4b expression in myeloid cells. Hematoxylin and eosin staining (H&E) reveal altered spleen structure in infected animals (right column) compared with uninfected mice (left column). Staining with B cell and T cell markers (B220 and CD3, respectively) shows a lack of normal cell types in the leukemic spleen. Staining with a myeloid marker (F4\80), however, demonstrates the massive proliferation of these cells in the leukemic spleen.

Retroviral infection induced acute myeloid leukemia in mice lacking p15INK4b expression in myeloid cells. Hematoxylin and eosin staining (H&E) reveal altered spleen structure in infected animals (right column) compared with uninfected mice (left column). Staining with B cell and T cell markers (B220 and CD3, respectively) shows a lack of normal cell types in the leukemic spleen. Staining with a myeloid marker (F4\80), however, demonstrates the massive proliferation of these cells in the leukemic spleen.

Acute myeloid leukemia or AML, a cancer of the white blood cells, is the most common type of rapidly-growing leukemia in adults. The over-production of white blood cells in the bone marrow inhibits the development of other necessary blood components including red blood cells, which carry oxygen throughout the body, and platelets, which are required for clot formation. The cellular changes that lead to AML disease initiation and progression, however, are not clear. Because of the aging of the U.S. population and AML’s increasing incidence with age, cases of this disease are likely to rise significantly in the near future. Thus, understanding what causes AML should lead to the identification of novel targets and the enhanced treatment of patients.

Silencing of the gene p15INK4b, an inhibitor of cell cycle-activated signaling proteins, by modification of its DNA regulatory region is one of the most common abnormalities in myeloproliferative disorders such as AML. This makes p15INK4b a strong candidate for regulating myeloid cell proliferation, but supporting experimental data has been lacking. One mouse model with complete embryonal knockout of the gene was reported, but it failed to mimic human AML, instead demonstrating a mild lymphoproliferative disorder. Since p15INK4b expression is normally lost only in the myeloid cells of AML patients, Juraj Bies, Ph.D. working with Linda Wolff, Ph.D. in the Center for Cancer Research’s Laboratory of Cellular Oncology and their colleagues, decided to test the importance of p15INK4b in AML formation by generating a mouse line lacking the gene specifically in myeloid cells. The results of their studies were recently published in the journal Blood.

Mice lacking p15INK4b in myeloid cells (ko mice) were born without obvious physical or behavioral abnormalities. By 5 to 7 months of age, however, the ko mice exhibited elevated numbers of myeloid cells in the circulation compared to control animals. This increase was observed beyond 8 months of age (equivalent to middle age in humans) when a decrease in myeloid cell numbers occurred in control animals, making the difference between the two populations even more striking. The researchers also found an expansion of myeloid cells in the bone marrow. Increases in myeloid cell types were likely due to increased proliferation of myeloid cells and to enhanced differentiation of progenitors toward myeloid cell types.

Interestingly, even with elevated levels of myeloid cells, none of the ko mice progressed to an acute form of leukemia over the 15 months they were under observation. This indicated that loss of p15INK4b alone was not a strong enough genetic hit to induce acute leukemia. To find genetic changes that might cooperate with loss of p15INK4b to cause disease, the scientists infected the mice with a retrovirus. The virus inserts its DNA into the mouse genome, potentially activating or inhibiting the genes present at the insertion site. The researchers then monitored wild type and ko infected mice for development of leukemia. Significantly more ko mice developed virus-induced acute leukemia with enlarged spleens infiltrated with tumor cells compared to wild type infected animals. The majority of the leukemias that formed in the ko mice were myeloid cell-derived, while those of the wild type mice were evenly divided between myeloid and lymphoid lineages. This result supports the idea that loss of p15INK4b predisposes the animals to myeloid leukemia.

The researchers then wanted to identify the specific genes targeted by retroviral insertion that facilitated leukemia development in the ko mice. Using two different strategies, common integration sites for the retrovirus were discovered. These sites were near or within the genes for c-Myb and Sox4, two proteins that regulate DNA transcription to RNA. Retroviral insertion activated expression of the genes, and they likely participated in leukemia progression by preventing myeloid cells from terminally differentiating and encouraging their proliferation.

These studies clearly show that p15INK4b acts as a tumor suppressor protein in AML, but that its loss alone is not sufficient to induce leukemia in mice. Further research will be necessary to understand how this gene normally is suppressed in AML patients and whether the pathways controlling p15INK4b expression are susceptible to drug treatment. Likewise, it will be important to determine whether c-Myb or Sox4 participate in human AML development and if they can be targeted with pharmaceuticals.

Summary Posted: 09/2010

Reference

Blood. 2010 Aug; 116(6):979-87 PubMed Link