PDE4B Modulates Glucocorticoid Sensitivity in Childhood Acute Lymphoblastic Leukemia
Authors: Yang Jun J. , E Shuyu , Shen Shuhong , Bhojwani Deepa , Kang Huining , Ma Yani , Chen Jing , Tang JingYan , Seibel Nita L. , Xu Heng , Cheng Jin , Ramsey Laura B. , Crews Kristine , Bowman W. Paul , Borowitz Michael J. , Carroll William L. , Willman Cheryl L. , Jeha Sima , Pui Ching-Hon , Hunger Stephen P. , Relling Mary V. , Evans William E. 
Published: October 2019

Abstract
Abstract 530
Although cure rates of childhood acute lymphoblastic leukemia (ALL) have improved dramatically, a substantial portion of children still relapse and the prognosis of relapsed ALL is extremely poor. Therefore, a better understanding of molecular determinants of drug resistance in ALL is imperative for the development of more efficacious and individualized therapy, particularly in the context of relapsed disease. In a recent genome-wide association study of 2,534 children with ALL, we identified that genetic variation in PDE4B– phosphodiesterase 4B–strongly influenced the risk of ALL relapse across various ALL treatment regimens (Nat Genet 2011: 43:237). While PDE4B is the predominant phosphodiesterase in lymphoid tissue and a major regulator of cyclic AMP, its role in ALL pathobiology is largely unknown. To this end, we sought to characterize the molecular mechanisms by which PDE4B modulates antileukemic drug sensitivity in ALL. We first characterized PDE4B expression in ALL blasts at diagnosis and its relationship with drug response in vivo in 3 independent cohorts of children with ALL. In 191 children with newly diagnosed ALL enrolled on the COG P9906 protocol, PDE4B expression in ALL blasts was positively correlated with minimal residual disease status at the end of remission induction (P=0.0096). Higher PDE4B expression was also associated with slower early response to induction therapy in COG 1961 (N=82, P=0.019). In 275 children with newly-diagnosed ALL enrolled on the Shanghai Children's Medical Center ALL05 study, we determined that PDE4B2 was the predominant isoform of PDE4B in ALL blasts (P<0.0001); there was also a trend that children with poor in vivo response to the upfront single-agent prednisone treatment had higher PDE4B2 expression in the diagnostic blasts (P=0.042). In parallel, shRNA-mediated knock-down of PDE4B in a glucocorticoid-sensitive (i.e., Nalm6) and a glucocorticoid-resistant (i.e., UOCB1) ALL cell line significantly potentiated cytotoxic effects of prednisolone, whereas Nalm6 and CEM ALL cells over-expressing PDE4B2 were significantly more resistant to prednisolone compared to cells transduced with empty vectors. Sensitization to glucocorticoid was further amplified by forskolin, a stimulator of cAMP synthesis, and was concomitant with activation of PKA as determined by CREB phosphorylation, suggesting that the effects of PDE4B on glucocorticoid sensitivity involve signaling of the cAMP-PKA cascade. Importantly, PDE4B knockdown by shRNA and pharmacologic inhibition by rolipram in Nalm6 and UOCB1 cells consistently led to upregulation of BIM, a key apoptosis regulator and a critical mediator of glucocorticoid sensitivity in lymphoid cells. In both cells lines, BIM upregulation following PDE4B inhibition was enhanced by forskolin but suppressed by PKA inhibitor H89, indicating that BIM might act as a downstream effector of cAMP-PKA signaling in ALL. Finally, we evaluated effects of small molecule regulators of the PDE-cAMP pathway (namely, forskolin and rolipram) on glucocorticoid response in primary ALL cells (N=23) in vitro. Measuring IC50 by MTT assay, increased cytotoxicity was observed in 16 (70%), 8 (35%), and 17 (74%) cases, when forskolin, rolipram, or both were added to prednisolone, respectively. In conclusion, tumor expression of PDE4B at diagnosis was associated with poorer early treatment response in ALL, particularly resistance to glucocorticoids in vivo. Inhibition of PDE4B sensitized both cultured and primary ALL cells to glucocorticoids via activating the cAMP-PKA pathway and subsequent upregulation of BIM.

Disclosures:
No relevant conflicts of interest to declare.