Abstract 46: CDK4/CDK6 inhibition in childhood B-ALL: A new strategy to mediate glucocorticoid sensitivity
Authors: Mattiuzzo Elena , Bortolozzi Roberta , Accordi Benedetta , Trentin Luca , Basso Giuseppe , Viola Giampietro 
Published: August 2018

Abstract
Dysregulation of the cyclin D1-CDK4/CDK6 complex is frequently observed in almost all human cancer and contributes to aberrant cell proliferation and consequent tumorigenesis. Among the CDKs that control cell cycle progression, cyclin D-dependent kinases CDK4 and CDK6 are considered important oncogenic drivers in many cancers. CDK6 is a direct target of MLL rearranged B-cell acute lymphoblastic leukemia (B-ALL), and this prompted us to investigate if pharmacologic inhibition of CDK4/CDK6 could increase the efficiency of chemotherapy and overcome drug resistance. Although many reports described CDK4/CDK6 inhibitors against a broad range of carcinomas, few studies have been performed on leukemia.
Deletion and methylation of CDK4/CDK6 inhibitor CDKN2a are frequently observed in B-ALL and gene expression analysis performed in a cohort of childhood patients showed that cyclin D1, CDK4 and CDK6 are highly expressed. Moreover, Reverse Phase Protein Array (RPPA) analysis showed that cyclin D1 expression is higher in high risk-MRD patients. These results suggest specific inhibition of cyclin D/CDK4/CDK6 axis as an attractive strategy to improve the effect of common chemotherapy on B-ALL patients.
Standard treatment for newly diagnosed childhood B-ALL patients include chemotherapy treatment, and although glucocorticoids (GC) are the most important drugs used in the treatment of acute lymphoblastic leukemia, the molecular basis of GC sensitivity and resistance remains largely unknown; understanding the molecular mechanisms related to GC cytotoxicity is crucial for modulating GC resistance.
The aim of this study was to evaluate the effect of dual inhibition of CDK4/CDK6 in B-ALL and if this inhibition could enhance cytotoxic killing of leukemia cells after combination treatment with dexamethasone. We treated two B-ALL dexamethasone-resistant cell lines, SEM and RCH-ACV, and two B-ALL dexamethasone-sensitive cell lines, RS4;11 and NALM6, with ribociclib, a highly specific CDK4/6 dual inhibitor.
As expected, treatment with ribociclib induced a strong cell cycle arrest in G1 phase in a time-dose dependent manner along with a dose-dependent decrease in phosphorylated retinoblastoma (Rb); we investigated whether treatment with ribociclib could induce growth inhibition of B-ALL cell lines. We observed, in SEM and RCH-ACV, a strong growth inhibition at all drug concentration after continuous treatment. Washout of the inhibitor from the medium after 48 hours of treatment showed that both cell lines did not restore cell growth. These results show that ribociclib could have an irreversible activity. Moreover, a strong dose-dependent reduction of clonogenic potential was observed in SEM cell line, by CFU assay.
Ribociclib exposure strongly synergizes (CI&lt;1) with dexamethasone in SEM and RCH-ACV resistant cell lines with a strong decrease of proliferation and a significant increase of apoptotic cell death. Immunoblot analysis showed a decrease in phosphorylated Rb and Cyclin D1 starting from 48 hours of cotreatment and an increase in p27. Preliminary experiments showed a modest increase in glucocorticoid receptor (GR) after ribociclib and combination treatment in SEM and RCH-ACV cells. To further confirm the synergistic effect we evaluated mRNA level of GR targets, and we found that GR, BTG1, and GILZ are overexpressed in combination treatment in SEM and RCH-ACV.
The synergistic effect of ribociclib-dexamethasone combination was also confirmed on primary cultures derived from pediatric patients affected by B-acute lymphoblastic leukemia.
Our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanism of glucocorticoid resistance.
Citation Format: Elena Mattiuzzo, Roberta Bortolozzi, Benedetta Accordi, Luca Trentin, Giuseppe Basso, Giampietro Viola. CDK4/CDK6 inhibition in childhood B-ALL: A new strategy to mediate glucocorticoid sensitivity [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 46.