Combinations of the FLT3 Inhibitor CEP-701 and Chemotherapy Synergistically Kill Infant and Childhood MLL-Rearranged ALL Cells in a Sequence-Dependent Manner.
Authors: Brown Patrick , Levis Mark , McIntyre Emily , Griesemer Melissa , Small Donald 
Published: April 2020

Abstract
Rearrangement of the MLL gene at chromosome 11q23, which occurs in 80% of infants and 5% of older children with acute lymphoblastic leukemia (ALL), confers a dismal prognosis despite maximally intensified chemotherapy and allogeneic transplantation. Novel therapies are clearly needed for these patients. The FLT3 tyrosine kinase represents an attractive potential target in these leukemias, due to the marked overexpression of FLT3 in cases of MLL-rearranged ALL. We have previously shown that MLL-rearranged infant and childhood ALL leukemic cells express high levels of constitutively activated FLT3 receptor protein, and that these cells are selectively killed by exposure to the FLT3 inhibitor CEP-701. However, FLT3 inhibition is unlikely to represent curative therapy for these patients if used as monotherapy. Thus, we examined various combinations of CEP-701 with chemotherapy to look for potentially synergistic therapeutic strategies. We performed MTT cytotoxicity and annexin V binding (AVB) apoptosis assays on MLL-rearranged ALL cell lines (SEM-K2 and HB-1119) and three primary patient samples (all of which we show to express high levels of constitutively activated FLT3) after exposure to multiple dose combinations of CEP-701 and six chemotherapy agents (vincristine, l-asparaginase, dexamethasone, daunorubicin, etoposide and cytarabine). Since we have previously demonstrated that the nature of the interactions between CEP-701 and chemotherapy agents (i.e., synergistic, additive or antagonistic) are sequence-dependent in AML cells, we performed the MTT and AVB assays in three sequences designed to mimic the potential clinical uses of the combinations (Seq1: chemotherapy followed by CEP-701; Seq2: simultaneous exposure to both; and Seq3: CEP-701 followed by chemotherapy). The nature of the interaction between CEP-701 and each chemotherapy agent was determined using the median effect method of Chou and Talalay, which calculates a combination index (CI) for each combination (CI &lt; 0.9 - synergistic; CI 0.9-1.1 - additive; CI &gt; 1.1 antagonistic). A striking pattern of sequence-dependent synergy was observed: Seq1 was markedly synergistic (mean CI +/− SD = 0.59 +/− 0.18), Seq2 was additive (CI = 0.99 +/− 0.29) and Seq3 was antagonistic (CI = 1.23 +/− 0.40). The sequence-dependence is attributable to the G1 cell cycle arresting effects of CEP-701 in these cells. An MLL-rearranged cell line that expresses low levels of minimally activated FLT3 (RS4-11) demonstrates minimal cell cycle arrest when treated with CEP-701, and mere additivity in all 3 CEP-701/chemotherapy combination sequences (Seq1: CI = 0.95 +/− 0.10; Seq2: CI = 1.01 +/− 0.09; Seq3: CI = 1.06 +/− 0.10), confirming that the observed pattern of synergy is mediated through the effects of CEP-701 on FLT3 signaling. These results can help guide the combination of CEP-701 with existing chemotherapy regimens for infants and children with MLL-rearranged ALL, with the goal of improving the dismal outcome for these patients.