Z01 BC 011027 (Z01) | |||
---|---|---|---|
Title | Animal models to study the role of INK4b loss in human acute myeloid leukemia | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Wolff, Linda | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $510,160 | Project Dates | 10/01/2007 - N/A |
Fiscal Year | 2008 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) Hematology (100.0%) |
Leukemia (100.0%) | ||
Research Type | |||
Cancer Initiation: Oncogenes and Tumor Suppressor Genes Development and Characterization of Model Systems |
|||
Abstract | |||
Using p15Ink4b-deficient mice, our initial studies of p15Ink4b provided evidence that the gene is a tumor suppressor for myeloid leukemia. Recently we developed an improved model that more closely resembles the disease in man by deleting the gene specifically in myeloid lineage cells. The new model employing the Cre-loxP system allows conditional deletion of the gene. We successfully accomplished the tissue specific deletion by crossing INK4b(exon 2) floxed mice with LysMCre mice and showed by PCR technology that Ink4b was deleted in common myeloid progenitors. When these mice were injected with retrovirus they were found to be highly susceptible to acute myeloid leukemia with a 50% incidence as compared to wild-type mice which had a 5% incidence. The retrovirus used for these studies was one we constructed and is efficient in inducing myeloid leukemia. It has been used by numerous other investigators for looking for cooperating genes in genetically engineered mice. Sixty-five percent of the leukemias were positive for both granulocyte and macrophage markers suggesting that they were derived from undifferentiated progenitor cells. Of these, 36 % also had the stem cell marker Sca-1. The others were positive for macrophage markers only. Since retroviral insertional mutagenesis was utilized in conjunction with the Ink4b knock-out for the induction of acute myeloid leukemia, we were able to screen for retrovirus insertion sites that might collaborate with the loss of the p15Ink4b gene. Insertion sites of the retrovirus were cloned from leukemia DNA using inverse PCR and ligation-mediated PCR. A total of 34 acute myeloid leukemias were examined and a total of 164 retroviral integration sites have been identified. Common integration sites that were found in more than one leukemia included c-myb, sox4, gfi1 and stk10. c-Myb was found to be the most frequently activated gene based upon PCR identification of insertion sites and northern blot analysis. There was evidence of activation of c-myb in 23% of the leukemias. This is a much higher rate of activation than has been shown for other retroviral induced leukemias in mice and therefore possibly showing specific collaboration with the loss of p15Ink4b. This is very interesting because human c-Myb, which was implicated in human cancer for many years, was recently shown to be involved in translocations and duplications in T acute lymphocytic leukemia. It is a very important oncogene in human cancer and also is implicated in breast cancer and colon cancer. Interestingly, mRNA for myb and sox4 was demonstrated to be increased in leukemias which retroviruses at these sites. Furthermore, we have been able to show that infection of c-kit+ stem / progenitor cells from p15Ink4bfl/fl LysM-Cre mice with retroviruses expressing c-myb or sox4 results in an increased number of myeloid colonies in vitro. p15INK4b is a member of the INK4 family member of cyclin-dependent kinase inhibitors. However, since INK4b is the only member that is inactivated in acute myeloid leukemia, we have been interested in determining if there is any other function specific to the myeloid lineage that can be assigned to this gene. For these experiments we used the two knock-out models described above. Initial investigations of hematopoiesis in Ink4b- deficient mice showed that they have greater numbers of bi-potent granulocyte-macrophage progenitors (GMP) and this was found to be intrinsic to the cells. Interestingly, Ink4b-deficient granulocyte-macrophage progenitors did not cycle more frequently than wild-type progenitors and showed no differences in apoptotic potential. However, Ink4b was shown to affect differentiation of common myeloid progenitor cells. The resulting imbalance was shown to favor the production of granulocyte-macrophage progenitors at the expense of erythroid progenitors. This work demonstrates a novel cell cycle-independent role for Ink4b during cell fate at the erythroid/myeloid bifurcation. Since GATA-1 and PU.1 instruct erythroid and myeloid cell fates and inhibit each others transcriptional function we set out to determine if expression of these genes or other genes involved in lineage choice were altered in common myeloid progenitors from In4b-deficient compared to wild-type mice. RT-PCR results showed that PU.1 levels were unaffected while GATA-1, GATA-2, and GATA-3 were down-regulated in common myeloid progenitors. Whether p15Ink4b affects the common myeloid progenitor differentiation through its known capacity to interact with Cdk4/6 or by participating in an unrelated pathway is currently unclear. |