Faculty Bibliography

BACKGROUND: Ikaros, the product of IKZF1, is a regulator of lymphoid development and polymorphisms in the gene have been associated with the acute lymphoblastic leukemia (ALL). Additionally, IKZF1 deletions and mutations identify high-risk biological subsets of childhood ALL [Georgopoulos et al. Cell 1995;83(2):289-299; Mullighan et al. N Engl J Md 2009;360(5):470-480]. PROCEDURES: To discover the underlying pathways modulated by Ikaros we performed gene expression and gene ontology analysis in IKZF1 deleted primary B-ALL pediatric patient samples. To validate downstream targets we performed qPCR on individual patient samples. We also created IKZF1 knockdown B-ALL cell lines with over 50% reduction of Ikaros, mimicking haplosufficient Ikaros deletions, and again performed qPCR to investigate the downstream targets. Finally, to understand the association of Ikaros deletion with a poor prognosis we challenged our IKZF1 knockdown cell lines with chemotherapy and compared responses to IKZF1 wild-type controls. RESULTS: We report a specific gene expression signature of 735 up-regulated and 473 down-regulated genes in IKZF1 deleted primary B-ALL pediatric patient samples. Gene ontology studies revealed an up-regulation of genes associated with cell adhesion, cytoskeletal regulation, and motility in IKZF deleted patient samples. Validated up-regulated target genes in IKZF1 deleted patient samples included CTNND1 and PVRL2 (P = 0.0003 and P = 0.001), and RAB3IP and SPIB (P = 0.005 and P = 0.032) were down-regulated. In further studies in IKZF1 knockdown cell lines, apoptosis assays showed no significant chemoresistance. CONCLUSION: IKZF1 knockdown alone does not impart intrinsic chemotherapy resistance suggesting that the association with a poor prognosis may be due to additional lesions, microenvironmental interactions with the bone marrow niche, or other factors. Pediatr Blood Cancer (c) 2014 Wiley Periodicals, Inc.

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.

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 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.

Background: The poor prognosis of relapsed ALL warrants the need for new insights into drug resistance mechanisms. We have previously shown that relapsed blasts can be re-sensitized to chemotherapy by the reversal of their gene expression signature using epigenetic agents.[1] Objectives: We hypothesize that aberrant epigenetic mechanisms may play a role in chemoresistance. To assess the degree to which the histone code evolves from diagnosis to relapse, we have used an unbiased, whole genome approach to examine changes in the epigenomic landscape by mapping the genome-wide location of key histone marks in diagnosis/relapse patient pairs with ALL enrolled on Children's Oncology Group protocols. Design/Method: We have performed Chromatin-immunoprecipitation sequencing (ChIP-seq) on two cryopreserved matched diagnosis/relapse pairs using active (H3K4me3, H3K9ac) and repressive (H3K9me3, H3K27me3) histone "mark" antibodies, with non-immunoprecipitated DNA (input) as control. Libraries for each patient pair were multiplexed in a single lane and sequenced in duplicate. 51-cycle single-end sequencing was performed using the Illumina HiSeq2000 Analyzer. Reads were aligned to reference genome using BWA and filtered using Samtools to remove multiple mapping reads. Peaks were called using MACS (v2.0.9) with default settings for histone marks. Results: 94.2% of the sequence reads passed filter (PF) and 96.4% of PF reads had quality score of more than 30. Using a p-value<0.00001, we identified 83,380 and 74,453 peaks (enriched regions) for activating and, 37,483 and 46,143 peaks for repressive histone marks at diagnosis and relapse respectively; suggesting that relapsed blasts are enriched with repressive marks and depleted of activating marks compared to those at diagnosis. Sixty-six genes had shared peaks for various histone marks between the two patients. Upon analyzing the top most differentially expressed transcripts at relapse from our previous cohort[2],64% genes showed concordant histone modificatio!

Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance. The biological pathways that mediate resistance are unknown. Here, we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten individuals with pediatric B-lymphoblastic leukemia using RNA sequencing. Transcriptome sequencing identified 20 newly acquired, novel nonsynonymous mutations not present at initial diagnosis, with 2 individuals harboring relapse-specific mutations in the same gene, NT5C2, encoding a 5'-nucleotidase. Full-exon sequencing of NT5C2 was completed in 61 further relapse specimens, identifying additional mutations in 5 cases. Enzymatic analysis of mutant proteins showed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analog therapies. Clinically, all individuals who harbored NT5C2 mutations relapsed early, within 36 months of initial diagnosis (P = 0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug-resistant clones in ALL.

Whereas the improvement in outcome for children with acute lymphoblastic leukemia has been gratifying, the poor outcome of patients who relapse warrants novel treatment approaches. Previously, we identified a characteristic relapse-specific gene expression and methylation signature associated with chemoresistance using a large cohort of matched-diagnosis relapse samples. We hypothesized that "reversing" such a signature might restore chemosensitivity. In the present study, we demonstrate that the histone deacetylase inhibitor vorinostat not only reprograms the aberrant gene expression profile of relapsed blasts by epigenetic mechanisms, but is also synergistic when applied before chemotherapy in primary patient samples and leukemia cell lines. Furthermore, incorporation of the DNA methyltransferase inhibitor decitabine led to reexpression of genes shown to be preferentially methylated and silenced at relapse. Combination pretreatment with vorinostat and decitabine resulted in even greater cytotoxicity compared with each agent individually with chemotherapy. Our results indicate that acquisition of chemo-resistance at relapse may be driven in part by epigenetic mechanisms. Incorporation of these targeted epigenetic agents to the standard chemotherapy backbone is a promising approach to the treatment of relapsed pediatric acute lymphoblastic leukemia.

Although the cure rate of newly diagnosed acute lymphoblastic leukemia (ALL) has improved over the past four decades, the outcome for patients who relapse remains poor. New therapies are needed for these patients. Our previous global gene expression analysis in a series of paired diagnosis-relapse pediatric patient samples revealed that the antiapoptotic gene survivin was consistently upregulated upon disease relapse. In this study, we demonstrate a link between survivin expression and drug resistance and test the efficacy of a novel antisense agent in promoting apoptosis when combined with chemotherapy. Gene-silencing experiments targeting survivin mRNA using either short-hairpin RNA (shRNA) or a locked antisense oligonucleotide (LNA-ON) specifically reduced gene expression and induced apoptosis in leukemia cell lines. When used in combination with chemotherapy, the survivin shRNA and LNA-ON potentiated the chemotherapeutic antileukemia effect. Moreover, in a mouse primary xenograft model of relapse ALL, the survivin LNA-ON decreased survivin expression in a subset of animals, and produced a statistically significant decrease in tumor progression. Taken together, these findings suggest that targeting endogenous levels of survivin mRNA by LNA-ON methods may augment the response to standard chemotherapy by sensitizing otherwise resistant tumor cells to chemotherapy.