Faculty Bibliography

While childhood acute lymphoblastic leukaemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis-relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analysed the expression of activated beta-catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of beta-catenin at relapse in 6/10 patients. Furthermore, treatment of leukaemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre-treatment of purified blasts from patients with relapsed leukaemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt-inhibition may be a promising therapeutic approach.

Background Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. Methods We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. Results Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6-NTRK3 fusion was sensitive to crizotinib. Conclusions Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.).

Asparaginase is a therapeutic enzyme used to treat leukemia and lymphoma, with immune responses resulting in suboptimal drug exposure and a greater risk of relapse. To elucidate whether there is a genetic component to the mechanism of asparaginase-induced immune responses, we imputed human leukocyte antigen (HLA) alleles in patients of European ancestry enrolled on leukemia trials at St. Jude Children's Research Hospital (n = 541) and the Children's Oncology Group (n= 1,329). We identified a higher incidence of hypersensitivity and anti-asparaginase antibodies in patients with HLA-DRB1*07:01 alleles (P = 7.5 x 10-5, OR = 1.64; P = 1.4 x 10-5, OR = 2.92, respectively). Structural analysis revealed that high-risk amino acids were located within the binding pocket of the HLA protein, possibly affecting the interaction between asparaginase epitopes and the HLA-DRB1 protein. Using a sequence-based consensus approach, we predicted the binding affinity of HLA-DRB1 alleles for asparaginase epitopes, and patients whose HLA genetics predicted high-affinity binding had more allergy (P = 3.3 x 10-4, OR = 1.38). Our results suggest a mechanism of allergy whereby HLA-DRB1 alleles that confer high-affinity binding to asparaginase epitopes lead to a higher frequency of reactions. Studies were registered at ClinicalTrials.gov, identifiers: NCT00137111, NCT00549848, NCT00005603, and NCT00075725.

To address the therapeutic challenges in childhood relapsed ALL, a phase 1 study combining a survivin mRNA antagonist, EZN-3042, with reinduction chemotherapy was developed for pediatric patients with second or greater bone marrow relapses of B-lymphoblastic leukemia. EZN-3042 was administered as a single agent on days -5 and -2 and then in combination with a 4-drug reinduction platform on days 8, 15, 22, and 29. Toxicity and the biological activity of EZN-3042 were assessed. Six patients were enrolled at dose level 1 (EZN-3042 2.5 mg/kg/dose). Two dose-limiting toxicities were observed: 1 patient developed a grade 3 gamma-glutamyl transferase elevation and another patient developed a grade 3 gastrointestinal bleeding. Downmodulation of survivin mRNA and protein were assessed after single-agent dosing and decreased expression was observed in 2 of 5 patients with sufficient material for analysis. Although some biological activity was observed, the combination of EZN-3042 with intensive reinduction chemotherapy was not tolerated at a dose that led to consistent downregulation of survivin expression. The trial was terminated following the completion of dose level 1, after further clinical development of this agent was halted.

Although great strides have been made in the improvement of outcome for newly diagnosed pediatric acute lymphoblastic leukemia because of refinements in risk stratification and selective intensification of therapy, the prognosis for relapsed leukemia has lagged behind significantly. Understanding the underlying biological pathways responsible for drug resistance is essential to develop novel approaches for the prevention of recurrence and treatment of relapsed disease. High throughput genomic technologies have the potential to revolutionize cancer care in this era of personalized medicine. Using such advanced technologies, we and others have shown that a diverse assortment of cooperative genetic and epigenetic events drive the resistant phenotype. Herein, we summarize results using a variety of genomic technologies to highlight the power of this methodology in providing insight into the biological mechanisms that impart resistant disease.

Although great advances have been made in the treatment of pediatric acute lymphoblastic leukemia, up to one out of five patients will relapse and their prognosis thereafter is dismal. We have previously identified recurrent deletions in TBL1XR1, which encodes for an F-box like protein responsible for regulating the nuclear hormone repressor (NCoR) complex stability. Here we model TBL1XR1 deletions in B-precursor ALL cell lines and show TBL1XR1 knockdown results in reduced glucocorticoid receptor recruitment to glucocorticoid responsive genes, and ultimately decreased glucocorticoid signaling caused by increased levels of NCoR1 and HDAC3. Reduction in glucocorticoid signaling in TBL1XR1 depleted lines resulted in resistance to glucocorticoid agonists, but not to other chemotherapeutic agents. Importantly, we show that treatment with the HDAC inhibitor SAHA restores sensitivity to prednisolone in TBL1XR1 depleted cells. Altogether, our data indicates that loss of TBL1XR1 is a novel driver of glucocorticoid-resistance in ALL and that epigenetic therapy may have future application in restoring drug sensitivity at relapse.

We previously reported preliminary findings that post induction imatinib mesylate (340 mg/m(2)/day), in combination with intensive chemotherapy, resulted in outcomes similar to blood and marrow transplant (BMT) for pediatric patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). We now report 5-year outcomes of imatinib plus intensive chemotherapy in 91 children (1-21 years) with and without allogeneic BMT (N=91). We explore the impacts of additional chromosomal abnormalities and minimal residual disease (MRD) by flow cytometry on outcomes. The 5-year disease-free survival was similar for Cohort 5 patients, treated with chemotherapy plus imatinib (70%+/-12%, n=28), sibling donor BMT patients (65%+/-11%, n=21) and unrelated donor BMT patients (59+/-15%; P=0.60, n=13). Patients with additional cytogenetic abnormalities had worse outcomes (P=0.05). End induction (pre-imatinib) MRD was not prognostic for Cohort 5 or allogeneic BMT patients, although limited by small numbers. The re-induction rate following relapse was similar to other higher-risk ALL groups. Longer-term follow-up confirms our initial observation of substantially good outcomes for children and adolescents with Ph+ ALL treated with imatinib plus intensive chemotherapy with no advantage for allogeneic BMT.

Sirolimus has activity against acute lymphoblastic leukemia (ALL) in xenograft models and efficacy in preventing acute graft-versus-host disease (aGVHD). We tested whether addition of sirolimus to GVHD prophylaxis of children with ALL would decrease aGVHD and relapse. Patients were randomized to tacrolimus/methotrexate (standard) or tacrolimus/methotrexate/sirolimus (experimental). The study met futility rules for survival after enrolling 146 of 259 patients. Rate of Grade 2-4 aGVHD was 31% vs 18% (standard vs experimental, P = .04), however, grade 3-4 aGVHD was not different (13% vs 10%, P = .28). Rates of veno-occlusive disease (VOD) and thrombotic microangiopathy (TMA) were lower in the nonsirolimus arm (9% vs 21% VOD, P = .05; 1% vs 10% TMA, P = .06). At 2 years, event free survival (EFS) and overall survival (OS) were 56% vs 46%, and 65% vs 55% (standard vs experimental), respectively (P = .28 and .23). Multivariate analysis showed increased relapse risk in children with >/=0.1% minimal residual disease (MRD) pretransplant, and decreased risk in patients with grades 1-3 aGVHD (P = .04). Grades 1-3 aGVHD were associated with improved EFS (P = .02), whereas grade 4 aGVHD and extramedullary disease at diagnosis led to inferior OS. Although addition of sirolimus decreased aGVHD, survival was not improved. This study is registered with ClinicalTrials.gov as #NCT00382109.