BIM Regulates Prednisolone-Induced Apoptosis in B-Lineage ALL: A Potential Biomarker.

Glucocorticoids like Prednisolone (PRED) and dexamethasone is one of the most powerful group of drugs for treatment of childhood ALL; when used alone, it can achieve morphological remission in > 60% of patients. However glucocorticoids have severe immunosuppressive effects increasing the risk of severe bacterial and fungal infections and severe psychosomatic effects like bad tempers, bad dreams and a veracious appetite. However, despite extensive use, the mechanism of PRED induced apoptosis remains unclear. Understanding the mechanism of PRED action and resistance in ALL will greatly enhance our ability to fully harness the potency of this important drug. Here, we investigated the mechanism of PRED induced apoptosis using four clinically important pre-B ALL cell lines: REH, 697, RS4;11, SUPB-15 and validated this in 45 paired bone marrow samples before and after 7 days of PRED induction for newly diagnosed children with ALL. Our results show that after treatment with PRED (10.0μg/ml) for 24h, the four cell lines can be separated into resistant (REH) and sensitive (697, RS4;11, SUPB-15) using apoptosis assay ( Cell Death Detection ELISA, Roche) and MTS assay (Promega). We studied the BCL-2 family members which are known to play an important regulatory role in the intrinsic apoptotic pathway in executing cell death program induced by glucocorticoids. We examined 6 members of the BCL-2 family - BIM, BCL-2, BAX, BAD, MCL-1, BCL-xl - using RQ-PCR and western blots. Only BIM, a pro-apoptotic member of the BCL-2 family, was up-regulated after PRED treatment in the 3 sensitive cell lines, both in terms of gene and protein expression levels. In contrast, it remains unchanged in REH resistant cell line. When co-treated with Ru486, a glucocorticoid receptor antagonist, PRED induced apoptosis was inhibited and the expression level of BIM was no longer elevated in the 3 sensitive cell lines, indicating the importance of BIM in PRED induced apoptosis and its potential role as a prognostic biomarker. To confirm this clinically, we studied the changes in expression levels of BIM in serial paired bone marrow after 7 days of PRED induction in 45 patients (35 good PRED responders and 10 poor PRED responders ) using Affymetrix U133 Plus 2 chips. Statistical analysis was performed using a two-sided Student’s t test. After 7 days of PRED, good PRED responders showed BIM was significantly up-regulated compared to PRED poor responders (p<0.05). We conclude that BIM plays an essential regulatory role in PRED induced intrinsic apoptosis pathway in pre-B ALL cells in a glucocorticoid receptor dependent manner. BIM could act as a potential prognostic biomarker of steroid response in treatment of childhood ALL allowing clinicians to clearly identify patients who are resistant early during therapy so that other drugs can be introduced early during induction so to maximise leukemia cell kill and improve the outcome of therapy. It will also provide the rationale to avoid the severe immunesuppressive and psychosomatic side-effects of steroid in patients whose leukemia are resistant to PRED.