Role of Misfolded - Nuclear receptor co-repressor (N-Cor) induced transcriptional de-regulation in the pathogenesis of acute monocytic leukemia (AML-M5)

Abstract

The Nuclear Receptor Co-repressor (N-CoR) is a key component of the generic multi-protein co-repressor complex involved in transcriptional control mediated by various transcription factors. Our laboratory previously demonstrated an important role of the misfolded conformational dependent loss (MCDL) of N-CoR in Acute Promyelocytic Leukemia (APL). However, the molecular mechanism underlying the misfolding of N-CoR and its implications in other AML subtypes is still not known. Here we report the in depth analysis of the molecular mechanism underlying the MCDL of N-CoR and its implication in the malignant growth and transformation of AML-M5 cells. We also explored the potential of the MCDL of N-CoR as a therapeutic target in AML-M5. The MCDL of N-CoR was found in AML-M5 cells and an APL-like N-CoR cleaving activity was observed in both AML-M5 primary and secondary leukemic cells. Activation of Akt inversely correlated with the status of MCDL of N-CoR in a comparative protein kinase array analysis. These observations implied a possible role of Akt in the MCDL of N-CoR in AML-M5 cells. A constitutively active Akt promoted N-CoR misfolding while therapeutic and genetic inhibition of Akt activity blocked the misfolding of N-CoR in AML-M5 cells. Moreover, N-CoR misfolding was found to be triggered by Akt induced phosphorylation at Serine 1450 of N-CoR. These observations clearly indicated the importance of Akt dependent phosphorylation in the misfolding and subsequent loss of N-CoR protein. Given N-CoR`s documented roles in hematopoiesis and as a transcriptional co-repressor, the functional consequence of Akt mediated MCDL of N-CoR in AML-M5 cells was next studied. Expression analysis of genes involved in hematopoiesis led to the identification of Flt3 as a transcriptional target of N-CoR. N-CoR status in various AML cells was found to be inversely related to Flt3 expression. N-CoR effectively repressed the activity the Flt3 promoter driven luciferase reporter and was found to be associated with the Flt3 promoter in ChIP assay. N-CoR loss facilitated the IL3-independent growth of BA/F3 cells through the de-repression of the Flt3 gene while blocking N-CoR loss with Genistein; an inhibitor of N-CoR misfolding, significantly down regulated Flt3 level and promoted differentiation of AML-M5 cells. These findings indicated that aberrant expression of Flt3 in AML-M5 cells was a consequence of the loss of N-CoR repressive function due to its MCDL. This suggested that N-CoR may have a potential tumour suppressive role in AML-M5 pathogenesis through unmasking the growth promoting potential of Flt3. Together, these findings illustrate the potential of targeting the N-CoR MCDL pathway as an effective therapeutic strategy in AML-M5