Loss of CDKN2C Accelerates Progression of Notch-1 Induced T Lymphoblastic Leukemia Via Activation of LMO2

The cyclin-dependent kinases inhibitor CDKN2C (p18^INK4C, p18) is a member of the INK4 family that specifically blocks the activity of CDK4/6 in the G1 phase of cell cycle. In the hematopoietic system, deletion of p18 was suggested to be associated with T cell malignancies in mice and B cell malignancies in humans. The mice reconstituted with p18 knockout (ko) bone marrow (BM) cells spontaneously developed acute T lymphoblastic leukemia (T-ALL) in serial transplantation (2 years after transplantation) originated from a CD3^lowcell population. However, how p18 is involved in the development of T-ALL leukemia is largely unknown. In this study, Intercellular domain of Notch1 (ICN-1) induced T-ALL model was used to explore the role of p18 deficiency in the development of T-ALL leukemia.

Enriched hematopoietic stem and progenitor LSK (Lin^- c-Kit⁺ Sca-1⁺) cells from p18^+/+ or p18^-/- mice were transduced with ICN1-GFP retrovirus and the same number of GFP⁺ cells from each group were sorted and transplanted into lethally irradiated recipient mice. About 50 days later, both groups of recipient mice showed symptom of leukemia and more than 90% donor-derived GFP⁺ cells were detected in peripheral blood and BM. The phenotypic analysis revealed the CD3 expression in GFP⁺ cells. The majority of CD3⁺ cells were CD4/8 double positive in p18^+/+ group while that of CD3⁺ cells were CD4/8 double negative in p18^-/- group. Analysis of the thymus revealed that DN3 cells accounted for 70.05±3.75% and 12.38±4.6% of thymocytes in p18^-/- group and p18^+/+ group, respectively, which have been thought as the leukemia-initiating cells according to a previous report. Serial transplantation was performed to measure self-renewal potential of leukemia cells in sub-lethally irradiated mice. Survival analysis showed an accelerated progression of leukemia in p18^-/- group compared with the p18^+/+ group in first, secondary and tertiary transplantation (p=0.01, p<0.001, p<0.001 respectively). The cell cycle analysis of leukemia cells by Ki67 assay suggested a decreased proportion of G0/G1 phase and an increased proportion of S/G2/M phase in p18^-/- group compared with p18^+/+ group. The gene expression analysis also showed higher expression of Notch1 signaling and target genes, such as Hes1 and C-myc in p18^-/- leukemia cells. Noticeably, a dramatically increase of LMO2 gene expression was detected in the leukemia cells of p18^-/- group compared with p18^+/+ group. According to previous studies, as an oncogene, LMO2 plays a critical role in the normal hematopoietic differentiation and is aberrantly expressed in approximately 45% of T-ALL patients. Deletion of p18 activates the expression of LMO2 although a mechanism is yet to be further defined. Taken together, our current study provides evidence and mechanism insights for the role of p18 deficiency during progression of T-ALL.