Pathological Roles of p18 Deficiency Toward B Cell Malignances
Authors: Hao Sha , Dong Fang , Zhou Wen , Cheng Hui , Ma Shihui , Qiu Lugui , Cheng Tao 
Published: October 2019

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 indicated to be associated with T cell malignancies in mice and B cell malignancies in humans. Moreover, p18 deficiency is a poor prognosis factor for the patients with multiple myeloma (MM). However, a formal investigation on the pathological roles of p18 deficiency in hematopoietic malignancies, especially B cell malignancies is lacking.
In this study, we first obtained direct clinical relevance of p18 deficiency with hematopoietic malignancies. Based on the Oncomine data set analysis, low expression of p18 was found in the patients with B-Cell Acute Lymphoblastic Leukemia (54 out of 80). In addition, by Gene expression Profile (GEP) analysis (n=361) and multi-color FISH analysis (n=265) of first-visit MM patients, there were 11% MM patients showed low expression and 9.06% biallelic deletion of p18 gene respectively, which was correlated with poor prognosis. Further analysis indicated higher expression of c-Myc, Bcl-2 and TRAF3 in p18-deleted MM patients or MM cell lines. We then focused on the impact of p18 deletion on B cell development with the mice deficient in p18 (p18-/-). The frequency and absolute number of B220+ B cell were significantly decreased in the bone marrow (21.075±0.168% vs 13.956±1.613%, n=5) or spleen (49.320±1.773 vs 35.35±1.673, n=5) of p18-/- mice. Secretion of immunoglobulin (Ig) from plasma cells was also impaired. Furthermore, p18-/- BM or enriched hematopoietic stem cell (LSK+) transplantation also recaptured the deficiency of mature B cells in the recipients despite higher repopulation in the p18-/- group. Ectopic over-expression of p18 in the hematopoietic stem and progenitor cells (HSPCs) via retroviral transduction could partially correct the abnormality of p18-/- B cells in the transplant recipients. These results suggested that the defect of B cell development in the absence of p18 was intrinsic to the hematopoietic cells, rather than extrinsic (via micro-environmental). To further define the effects of p18 deficiency on HSPCs prior to B cell commitment, we enumerated the frequencies of LT-HSC, MPP, CMP, GMP, MEP, Lin-IL-7R+ and CLP cell populations in p18-/- or control mice. There was no significant difference in the frequency or absolute number of CMP, GMP, MEP, or CLP between p18-/- and control groups. Notably however, the colony-forming cells of pre-B cells in p18-/- BM were significantly increased (24.4±2.1 vs 32.6±1.8, n=5). Moreover, we also examined the B cells at different developmental stages including pre-pro-B cell, pre-B, immature-B and mature B cells in BM, as well as transitional stage 1(T1), transitional stage 2 (T2) and mature B cells in the spleen. Our data showed an accumulation of the cells at pre-B cell stage in the absence of p18, while dramatically decreased at mature B cells stage. To further explore the molecular basis, single cell RT qPCR analysis was performed and revealed that the transcription factors including Foxo1, Rag2, E2A, EBF1and Pax5 were significantly higher inCLP, pro-B, pre-B, immature-B subpopulations of p18-/- group. However, lamada 5, which is necessary for B cells maturation, was remarkably decreased in p18-/- immature B cells compared with control group. Taken together, our study provides definitive evidence for the disruption of B cell development due to p18 deficiency and this new evidence underlies the pathological contributions of p18 down-regulation or deletion to B cell malignancies in humans.

Disclosures
No relevant conflicts of interest to declare.