). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemiarearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients. Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants

We identified mutations in the IL7Ra gene or in genes encoding the downstream signaling molecules JAK1, JAK3, STAT5B, N-RAS, K-RAS, NF1, AKT and PTEN in 49% of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Strikingly, these mutations (except RAS/NF1) were mutually exclusive, suggesting that they each cause the aberrant activation of a common downstream target. Expressing these mutant signaling molecules—but not their wild-type counterparts—rendered Ba/F3 cells independent of IL3 by activating the RAS-MEK-ERK and PI3K-AKT pathways. Interestingly, cells expressing either IL7Ra or JAK mutants are sensitive to JAK inhibitors, but respond less robustly to inhibitors of the downstream RAS-MEK-ERK and PI3K-AKT-mTOR pathways, indicating that inhibiting only one downstream pathway is not sufficient. Here, we show that inhibiting both the MEK and PI3K-AKT pathways synergistically prevents the proliferation of BaF3 cells expressing mutant IL7Ra, JAK and RAS. Furthermore, combined inhibition of MEK and PI3K/AKT was cytotoxic to samples obtained from 6 out of 11 primary T-ALL patients, including 1 patient who had no mutations in the IL7R signaling pathway. Taken together, these results suggest that the potent cytotoxic effects of inhibiting both MEK and PI3K/AKT should be investigated further as a therapeutic option using leukemia xenograft models.

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants is characterized by a poor clinical outcome and resistance to glucocorticoids (for example, prednisone and dexamethasone). As both the response to prednisolone in vitro and prednisone in vivo are predictive for clinical outcome, understanding and overcoming glucocorticoid resistance remains an essential step towards improving prognosis. Prednisolone-induced apoptosis depends on glucocorticoid-evoked Ca 2 þ fluxes from the endoplasmic reticulum towards the mitochondria. Here, we demonstrate that in MLL-rearranged infant ALL, over-expression of S100A8 and S100A9 is associated with failure to induce free-cytosolic Ca 2 þ and prednisolone resistance. Furthermore, we demonstrate that enforced expression of S100A8/S100A9 in prednisolone-sensitive MLL-rearranged ALL cells, rapidly leads to prednisolone resistance as a result of S100A8/S100A9 mediated suppression of prednisolone-induced free-cytosolic Ca 2 þ levels. In addition, the Src kinase inhibitor PP2 markedly sensitized MLL-rearranged ALL cells otherwise resistant to prednisolone, via downregulation of S100A8 and S100A9, which allowed prednisolone-induced Ca 2 þ fluxes to reach the mitochondria and trigger apoptosis. On the basis of this novel mechanism of prednisolone resistance, we propose that developing more specific S100A8/S100A9 inhibitors may well be beneficial for prednisolone-resistant MLL-rearranged infant ALL patients.Leukemia ( Keywords: prednisolone resistance; S100A8 and S100A9; calcium signaling; MLL-rearranged infant leukemia INTRODUCTIONSince the early 1960s, treatment results for childhood acute lymphoblastic leukemia (ALL) began to improve steadily and continued to progress ever since. Consequently, the survival chances for childhood ALL, in general, nowadays exceed 85%. 1 Unfortunately, this tremendous step forward has not been equally beneficial for all patients. This is especially true for infants (o1 year of age) with ALL carrying leukemia-specific chromosomal translocations involving the MLL gene, which occur in B80% of the infant ALL cases. 2,3 Depending on the treatment protocol, survival chances for MLL-rearranged infant ALL patients are at best B40%. 3 Considerably contributing to this poor outcome is cellular resistance to multiple chemotherapeutic drugs, in particular to glucocorticoids like prednisone and dexamethasone, which form the cornerstone of childhood ALL treatment regimes. Prednisolone (the biologically active metabolite of prednisone) dosages needed to eliminate 50% of infant ALL cells in vitro typically are B500 --fold higher than the dosages required to eradicate similar amounts of precursor B-ALL cells from children older than one year of age (that is, non-infants). 4 Moreover, approximately 30% of infants with MLL-rearranged ALL show a poor prednisone response in vivo, compared with only B10% of non-infant pediatric precursor B-ALL cases. 5 As the in vitro prednisolone response and the prednisone response in vivo represent important prognostic factors, 6 --8 ...

MLL-rearranged infant acute lymphoblastic leukemia (ALL) (<1 year of age) are frequently resistant to glucocorticoids, like prednisone and dexamethasone. As poor glucocorticoid responses are strongly associated with therapy failure, overcoming glucocorticoid resistance may be a crucial step towards improving prognosis. Unfortunately, the mechanisms underlying glucocorticoid resistance in MLL-rearranged ALL largely remain obscure. We here defined a gene signature that accurately discriminates between prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patient samples, demonstrating that, among other genes, high-level ANXA2 is associated with prednisolone resistance in this type of leukemia. Further investigation demonstrated that the underlying factor of this association was the presence of Src kinase-induced phosphorylation (activation) of annexin A2, a process requiring the adapter protein p11 (encoded by human S100A10). shRNA-mediated knockdown of either ANXA2, FYN, LCK or S100A10, all led to inhibition of annexin A2 phosphorylation and resulted in marked sensitization to prednisolone. Likewise, exposure of prednisolone-resistant MLL-rearranged ALL cells to different Src kinase inhibitors exerting high specificity towards FYN and/or LCK had similar effects. In conclusion, we here present a novel mechanism of prednisolone resistance in MLL-rearranged leukemias, and propose that inhibition of annexin A2 phosphorylation embodies a therapeutic strategy for overcoming resistance to glucocorticoids in this highly aggressive type of leukemia.

Successful treatment results for MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants remain difficult to achieve. Significantly contributing to therapy failure is poor response to glucocorticoids (GCs), like prednisone. Thus, overcoming resistance to these drugs may be a crucial step towards improving prognosis. We defined a gene signature that accurately discriminates between prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patient samples. In the current study, we applied Connectivity Map analysis to perform an in silico screening for agents capable of reversing the prednisolone-resistance profile and induce sensitivity. These analyses revealed that LY294002, a PI3K inhibitor, would potentially fulfill this task. Subsequent validation experiments demonstrated that indeed LY294002, and other known PI3K inhibitors, markedly sensitized otherwise resistant MLL-rearranged ALL cells to prednisolone in vitro. Using quantitative RT-PCR analyses, we validated the modulating effects of the PI3K inhibitors on the expression of the genes present in our prednisolone-resistance profile. Interestingly, prednisolone-sensitizing actions may be mediated by inhibition of FCGR1B. Moreover, only high-level expression of FCGR1B showed to be predictive for a poor prognosis and shRNA-mediated knock-down of FCGR1B led to in vitro prednisolone sensitization. Thus, implementing FDA-approved PI3K inhibitors in current treatments may potentially improve the GC response and prognosis in patients with MLL-rearranged ALL.