Emily McIntyre

Promoter hypermethylation in MLL-r infant acute lymphoblastic leukemia: biology and therapeutic targeting.

Cooperating leukemogenic events in MLL-rearranged (MLL-r) infant acute lymphoblastic leukemia (ALL) are largely unknown. We explored the role of promoter CpG island hypermethylation in the biology and therapeutic targeting of MLL-r infant ALL. The HELP (HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction [PCR]) assay was used to examine genome-wide methylation of a cohort of MLL-r infant leukemia samples (n = 5), other common childhood ALLs (n = 5), and normals (n = 5). Unsupervised analysis showed tight clustering of samples into their known biologic groups, indicating large differences in methylation patterns. Global hypermethylation was seen in the MLL-r cohort compared with both the normals and the others, with ratios of significantly (P < .001) hypermethylated to hypomethylated CpGs of 1.7 and 2.9, respectively. A subset of 7 differentially hypermethylated genes was assayed by quantitative reverse-transcription (qRT)-PCR, confirming relative silencing in 5 of 7. In cell line treatment assays with the DNA methyltransferase inhibitor (DNMTi) decitabine, MLL-r (but not MLL wild-type cell lines) showed dose- and time-dependent cytotoxicity and re-expression of 4 of the 5 silenced genes. Methylation-specific PCR (MSP) confirmed promoter hypermethylation at baseline, and a relative decrease in methylation after treatment. DNMTi may represent a novel molecularly targeted therapy for MLL-r infant ALL.

Combinations of the FLT3 inhibitor CEP-701 and chemotherapy synergistically kill infant and childhood MLL -rearranged ALL cells in a sequence-dependent manner

Mixed lineage leukemia (MLL) rearrangements occur in 80% of infants and 5% of older children with acute lymphoblastic leukemia (ALL). These cases have a poor prognosis with current therapy. The FLT3 kinase is overexpressed and constitutively activated in MLL-rearranged ALL cells. The FLT3 inhibitor CEP-701 selectively kills these cells, but is unlikely to be curative if used as monotherapy. To identify potentially synergistic combination strategies, we studied CEP-701 and six standard chemotherapeutic agents in three sequences of exposure (S1: chemotherapy followed by CEP-701, S2: simultaneous exposure to both; and S3: CEP-701 followed by chemotherapy) using MLL-rearranged ALL cell lines and patient bone marrow samples. MTT cytotoxicity and annexin V binding apoptosis assays were used to assess antileukemic effects. Combination indices (CI) were calculated for each combination (CI<0.9 – synergistic; CI 0.9–1.1 – additive; CI>1.1 – antagonistic). A striking pattern of sequence-dependent synergy was observed: S1 was markedly synergistic (mean CI=0.59±0.10), S2 was additive (mean CI=0.99±0.09) and S3 was antagonistic (mean CI=1.23±0.10). The sequence dependence is attributable to the effect of CEP-701 on cell cycle kinetics, and is mediated specifically by FLT3 inhibition, as these effects are not seen in control cells without activated FLT3.

Dynamic Chemotherapy-Induced Upregulation of CXCR4 Expression: A Mechanism of Therapeutic Resistance in Pediatric AML

Cure rates in pediatric acute leukemias remain suboptimal. Overexpression of the cell-surface chemokine receptor CXCR4 is associated with poor outcome in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Certain nonchemotherapeutic agents have been shown to modulate CXCR4 expression and alter leukemia interactions with stromal cells in the bone marrow microenvironment. Because chemotherapy is the mainstay of AML treatment, it was hypothesized that standard cytotoxic chemotherapeutic agents induce dynamic changes in leukemia surface CXCR4 expression, and that chemotherapy-induced upregulation of CXCR4 represents a mechanism of acquired therapeutic resistance. Here, it was shown that cell lines variably upregulate CXCR4 with chemotherapy treatment. Those that showed upregulation were differentially protected from chemotherapy-induced apoptosis when cocultured with stroma. The functional effects of chemotherapy-induced CXCR4 upregulation in an AML cell line (MOLM-14, which harbors consistent upregulated CXCR4) and clinical specimens were explored. Importantly, enhanced stromal-cell derived factor-1α (SDF1A/CXCL12)-mediated chemotaxis and stromal protection from additional chemotherapy-induced apoptosis was found. Furthermore, treatment with plerixafor, a CXCR4 inhibitor, preferentially decreased stromal protection with higher chemotherapy-induced upregulation of surface CXCR4. Thus, increased chemokine receptor CXCR4 expression after treatment with conventional chemotherapy may represent a mechanism of therapeutic resistance in pediatric AML. Implications: CXCR4 may be a biomarker for the stratification and optimal treatment of patients using CXCR4 inhibitors. Mol Cancer Res; 11(9); 1004–16. ©2013 AACR.

MLL-rearranged acute lymphoblastic leukaemia stem cell interactions with bone marrow stroma promote survival and therapeutic resistance that can be overcome with CXCR4 antagonism

Infants with MLL‐rearranged (MLL‐R) acute lymphoblastic leukaemia (ALL) have a dismal prognosis. While most patients achieve remission, approximately half of patients recur with a short latency to relapse. This suggests that chemotherapy‐resistant leukaemia stem cells (LSCs) survive and can recapitulate the leukaemia. We hypothesized that interactions between LSCs and the bone marrow microenvironment mediate survival and chemotherapy resistance in MLL‐R ALL. Using primary samples of infant MLL‐R ALL, we studied the influence of bone marrow stroma on apoptosis, proliferation, and cytotoxicity induced by the FLT3 inhibitor lestaurtinib. MLL‐R ALL were differentially protected by stroma from spontaneous apoptosis compared to non‐MLL‐R ALL. Co‐culture of bulk MLL‐R ALL in direct contact with stroma or with stroma‐produced soluble factors promoted proliferation and cell cycle entry. Stroma also protected bulk MLL‐R ALL cells and MLL‐R ALL LSCs from lestaurtinib‐mediated cytotoxicity. Previous studies have demonstrated that CXCR4 mediates bone marrow microenvironment signalling. Using a xenograft model of MLL‐R ALL, we demonstrated that CXCR4 inhibition with AMD3100 (plerixafor) led to markedly enhanced efficacy of lestaurtinib. Therefore, the bone marrow microenvironment is a mediator of chemotherapy resistance in MLL‐R ALL and targeting leukaemia‐stroma interactions with CXCR4 inhibitors may prove useful in this high‐risk subtype of paediatric ALL.

Discordance of MLL-rearranged (MLL-R) infant acute lymphoblastic leukemia in monozygotic twins with spontaneous clearance of preleukemic clone in unaffected twin

Concordance of MLL-rearranged acute leukemia in infant monozygotic twins is thought to be 100% with a very short latency period, suggesting that either the MLL fusion itself is sufficient to cause leukemia or that it promotes the rapid acquisition of additional oncogenic events that result in overt disease. We report the first case of discordance in an infant monozygotic twin pair. Twin A presented at age 9 months with MLL-ENL(+) acute lymphoblastic leukemia and twin B remains healthy 3 years later. The presence and eventual clearance of a clonal population of MLL-ENL(+) cells was shown in the bone marrow and peripheral blood of twin B. Clearance of this clone was temporally associated with viral-induced cytopenias, suggesting an immune-mediated clearance of the clone before the development of leukemia. Thus, concordance of MLL-rearranged acute leukemia in infant monozygotic twins is not universal. The implications of this case for MLL-rearranged leukemogenesis are discussed.

NPMc+ cooperates with Flt3/ITD mutations to cause acute leukemia recapitulating human disease

Cytoplasmic nucleophosmin (NPMc(+)) mutations and FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML); they frequently occur together, suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity.