Cyclin D1 Promotes Survival and Chemoresistance By Maintaining ATR and CHEK1 Signaling in TP53-Deficient Mantle Cell Lymphoma Cell Lines
Authors: Mohanty Suchismita , Tran Thai , Sandoval Natalie , Mohanty Atish , Bedell Victoria , Wu Jun , Warden Charles , Scuto Anna , Collins Joyce , Weisenburger Dennis D. , Ngo Vu N. 
Published: October 2019

Abstract
Mantle cell lymphoma (MCL) is a heterogeneous disease, ranging from indolent to aggressive conditions. Prognostic markers that predict aggressive MCL include blastoid cytologic features, high proliferation index (Argatoff et al. 1997), INK4A/ARF locus deletion (Dreyling et al. 1997), TP53 deletion and/or mutations (Greiner et al. 1996), elevated cyclin D1 (CCND1) expression (Rosenwald et al. 2003), and NOTCH1/2 mutations (Kridel et al. 2012, Bea et al. 2013). Among these, TP53 lesions are the most recurrent, suggesting their important role in MCL pathogenesis. In response to DNA damage, TP53 in normal cells activates cell cycle checkpoints to stall DNA replication allowing time for DNA repair or induces apoptosis when damage is severe (Zhou and Elledge. 2000). Tumor cells lacking TP53 function rely on the ATR-CHEK1 signaling for cell cycle checkpoints following DNA damage (Powell et al. 1995). Although both TP53 deficiencies and elevated CCND1 expression levels have been associated with poor survival, possible cooperation of TP53 status and CCND1 expression in aggressive MCL has not been examined.
In this study, we hypothesize that CCND1 overexpression collaborates with TP53 deficiency to promote MCL survival and chemoresistance. We compared the effects of CCND1 knockdown on cell survival and resistance to hydroxyurea (HU) and cytarabine to that of knockdown or pharmacological inhibition of CDK4 in MCL lines differing in TP53 status. Inducible gene knockdown was generated in UPN-1 cells to investigate the role of CCND1 in preventing replication stress and DNA damage and in the maintenance of the ATR and CHEK1 signaling. In addition, knockdown of TP53 in TP53-proficient MCL cells was performed to determine the contribution of TP53 status to tumor response to HU and the requirement of CCND1 in the chemosensitivity of these cells.
We demonstrate that the survival of TP53-deficient MCL lines (UPN-1 and JEKO-1) is more dependent on CCND1 than on CDK4, but neither of these proteins is essential in TP53-proficient lines (REC-1 and Z-138). Using inducible gene knockdown in UPN-1 cells, we show that CCND1 depletion-induced apoptosis is caused by endogenous replication stress and DNA damage, which are related to defects in the DNA replication checkpoints ATR and CHEK1. The protective effect of CCND1 in MCL cell lines was also confirmed in vivo tumor model. Silencing of CCND1, but not CDK4, sensitizes TP53-deficient MCL cells to hydroxyurea (HU) or cytarabine, which activates the S-phase checkpoint. In addition, forced expression of CCND1 rescues TP53-deficient cells from HU-induced apoptosis in an ATR-dependent manner. In contrast, neither silencing of CCND1 nor CDK4 increases the sensitivity of TP53-proficient cells to these agents. Finally, knockdown of TP53 sensitizes REC-1 cells (TP53 competent) to combination of HU exposure and CCND1 inhibition, confirming the role of TP53 status and CCND1 expression in the chemosensitivity of MCL cells. In summary, these results uncover a novel role for CCND1 in maintaining the ATR and CHEK1 signaling in TP53-deficient MCL. This role of CCND1 could contribute to its oncogenic potential and chemoresistance in aggressive MCL that lack TP53.

Disclosures
No relevant conflicts of interest to declare.