Abstract

The cyclin-dependent kinases inhibitor CDKN2C (p18INK4C, 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 CD3lowcell 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.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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