P27 Is Mislocalized to the Cytoplasm by BCR-ABL In a Kinase-Independent Manner and Contributes to Leukemogenesis

Abstract

AbstractAbstract 512 Background:BCR-ABL promotes cell cycle progression by interfering with the regulatory functions of p27, a cyclin dependent kinase (Cdk) inhibitor and tumor suppressor. We have previously shown that BCR-ABL kinase activity promotes degradation of nuclear p27 (Agarwal, A. et al. Blood 2008). Additionally, in primary CML cells, p27 is mislocalized to the cytoplasm, thereby relieving Cdks from p27 inhibition. Results from studies of solid tumors show that cytoplasmic p27 can actively contribute to oncogenesis, raising the question of whether cytoplasmic p27 in CML cells may actively promote leukemogenesis rather than merely compromise Cdk inhibition. We hypothesize that BCR-ABL disrupts p27 function in a dual manner by reducing nuclear p27, where p27 normally serves as a tumor suppressor, and by increasing cytoplasmic p27, where it might have oncogenic activity. Experimental Approach and Results:Immunoblotting of nuclear and cytoplasmic lysates of CD34+ cells from 11 CML patients revealed that p27 localization is predominantly cytoplasmic in the majority of patients (10/11; 91%) irrespective of disease phase, while p27 was mostly nuclear in normal controls. Similar results were obtained by immunofluorescence microscopy. Imatinib treatment increased nuclear p27 suggesting that nuclear p27 levels are regulated by BCR-ABL kinase activity. However, imatinib does not alter cytoplasmic p27 levels, suggesting that cytoplasmic mislocalization of p27 is a kinase-independent effect of BCR-ABL. Kinase-independent regulation of cytoplasmic p27 localization was also tested by immunofluorescence microscopy of p27−/− MEFs engineered to express active or kinase-dead BCR-ABL in combination with wild-type p27. In these cells cytoplasmic p27 abundance was increased both by kinase-active or kinase-dead BCR-ABL as compared to the vector control. To interrogate the role of p27 in vivo we retrovirally transduced p27+/+ or p27−/− bone marrow with BCR-ABL-GFP retrovirus and sorted Lin-/c-Kit+/Sca-I+ cells by FACS, allowing for injection of exactly matched numbers of BCR-ABL-expressing GFP+ cells (5000/animal). Median survival was significantly reduced for recipients of p27−/− marrow as compared to p27+/+ controls (34 days vs. 93 days p<0.0001). Recipients of p27−/− marrow also exhibited significantly increased white blood cell (4.5-fold) and platelet counts (3.9-fold) as well as spleen size (6-fold) and liver size (1.6-fold). Accordingly, there was more pronounced leukemic infiltration of myeloid precursors on histopathology as compared to controls. An in vivo competition experiment performed by injecting equal numbers of BCR-ABL-transduced p27−/− and p27+/+ marrow cells in congenic recipients resulted in leukemias in recipient mice (N=8) that were derived exclusively from p27−/− cells. In total, these results suggest that the net function of p27 in CML is tumor suppressive. To functionally dissect the role of nuclear and cytoplasmic p27, we used p27T187A transgenic mice (in which nuclear p27 degradation is reduced) and p27S10A mice (in which p27 export to the cytoplasm is reduced resulting in predominantly nuclear p27). Mice of matched genetic background were used as p27WT controls in CML retroviral transduction/transplantation experiments. In both cases, survival was prolonged compared to controls: 25 vs. 21 days for p27T187A (p=0.05) and 32 vs. 23 days for p27S10A (p=0.01). This suggests that stabilization of nuclear p27 (p27T187A) and more significantly lack of cytoplasmic p27 (p27S10A) attenuate BCR-ABL-mediated leukemogenesis. Consistent with this, autopsy and histopathological analysis revealed reduced hepatosplenomegaly (p27T187A mice) and improved cell differentiation with a relative increase of mature neutophils (p27S10A mice) as compared to wild-type controls. Conclusions:These results provide in vivo evidence that p27 has genetically separable dual roles in CML as both a nuclear tumor suppressor and cytoplasmic oncogene. A kinase-independent activity of BCR-ABL contributes to leukemogenesis through aberrant p27 localization to the cytoplasm. This oncogene activity is independent from the kinase-dependent degradation of nuclear p27. We speculate that the inability of tyrosine kinase inhibitors to reverse cytoplasmic p27 mislocalization may contribute to disease persistence despite effective inhibition of BCR-ABL kinase activity. Disclosures:Deininger:Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; genzyme: Research Funding.