Regulation of Drug Resistance by Ikaros

Open Access
Author:
Bulathsinghala, Marie S
Graduate Program:
Cell and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
May 29, 2014
Committee Members:
  • Sinisa Dovat, Dissertation Advisor
  • Sinisa Dovat, Committee Chair
  • John Warren Wills, Committee Member
  • Barbara Miller, Committee Member
  • Gregory Yochum, Committee Member
Keywords:
  • Leukemia
  • drug-resistance
  • Ikaros
Abstract:
Acute leukemia is the most common form of cancer in the pediatric population, accounting for nearly one third of all pediatric malignancies. Survival rates for pediatric leukemia have improved dramatically over the last 40 years and the combined 5-year survival rate has risen from less than 20% in the late 1960s to an estimated overall survival rate of greater than 85% today. However, despite the remarkable advancements in treatment therapies, the prognosis for high-risk patients—those who relapse, or fail to achieve post induction remission—remains poor. Treatment failure is the number one cause of relapse, making drug resistance a hallmark of high-risk leukemia. Sadly, the survival rate for the 25% of children who relapse is less than 30%, underscoring the need for a better understanding of the mechanisms mediating drug resistance and treatment failure, such that more efficacious treatment regimens can be developed. Recently, large scale genome-wide analyses of genetic alterations in leukemia have established IKZF1 (also known as ZNFN1A1), the gene encoding IKAROS, as one of the most clinically relevant prognostic markers in high-risk precursor-B cell Acute Lymphoblastic Leukemia (ALL). IKAROS is a sequence-specific DNA binding protein essential for normal hematopoiesis and participates in a complex network of interactions to recruit chromatin-modifying machines to gene regulatory regions promoting their transcriptional activation or repression via chromatin remodeling. Studies in mice have established IKAROS as a master regulator of lymphoid specification and a potent tumor suppressor in leukemia. Ongoing research has demonstrated that IKAROS binds and regulates thousands of genes involved in many different cellular processes. Mutations or deletions in IKZF1 have been consistently associated with high-risk leukemia, and interestingly, IKZF1 was the only gene for which mutations and deletions were found to be useful in predicting a poor response to therapy. What is not well understood, however, is the precise mechanism through which the loss of Ikaros function contributes to drug resistance and treatment failure. The absence and/or inactivation of IKAROS is highly associated with leukemia that is resistant to current chemotherapy agents and has a poor prognosis. Evidence suggests that even modest changes in IKAROS function, resulting from defects in a single IKAROS allele (haploinsufficiency), aids in the progression of leukemic transformation and results in increased risk of relapse. Our main goal was to understand the role of IKAROS in high-risk, drug-resistant leukemia. To accomplish this objective, we studied IKAROS-mediated regulation of genes involved in the folate pathway as well as genes essential to the inhibitory effects and metabolism of standard chemotherapeutics used in the treatment of ALL. We hypothesized that IKAROS regulates the sensitivity of leukemia cells to chemotherapy and that restoration of IKAROS function would inhibit leukemic growth and increase sensitivity to chemotherapy. To identify IKAROS target genes in leukemia, we used anti-Ikaros chromatin immunoprecipitation (ChIP) followed by deep sequencing in the human pre-B cell ALL cell line, Nalm6. Four IKAROS target genes involved in drug resistance were identified, and IKAROS binding was confirmed using independent anti-Ikaros ChIP assays in multiple cell lines and primary patient leukemia samples. Furthermore, we used both gain of function and loss of function assays to determine how IKAROS affected the transcription of genes involved in drug resistance. Our results suggest that IKAROS binds to the Ikaros regulatory elements (IRE) and decrease the expression of several genes important in drug resistance in human leukemia. The targets identified are intricately involved in folate metabolism, and thus, dysregulation of genes within this pathway could potentially affect the efficacy of treatment with methotrexate and the metabolism or inactivation of 6-mercaptopurine and 6-thioguanine into their inactive metabolites. To expand these studies, we sought to understand the signal transduction pathway(s) that control IKAROS-mediated regulation of drug resistance in leukemia by using both molecular inactivation and pharmacological inactivation of the pro-oncogenic protein casein kinase 2 (CK2). CK2 is overexpressed in human leukemia, and phosphorylation of Ikaros by CK2 results in decreased Ikaros DNA-binding affinity and loss of pericentromeric localization, resulting in a more diffuse nuclear pattern. We found that inhibition of CK2 resulted in increased binding of IKAROS at the upstream regulatory elements of target genes and a reduction in target gene transcription. Importantly, a regimen consisting of daily 6-mercaptopurine and weekly methotrexate, in addition to periodic intrathecal therapy, is considered the gold standard for maintenance therapy in ALL. Our findings suggested inhibition of CK2 increases Ikaros DNA-binding affinity at IKAROS regulatory elements (IREs); therefore, we hypothesized that CK2 inhibition would restore IKAROS-mediated repression of genes involved in drug resistance. Using cell cytotoxicity assays, we demonstrate that combination therapy with 6-TG or MTX and CK2 inhibitors results in increased cell death and thus heightened leukemia cell sensitivity to chemotherapy. Our results further suggest that inhibition of CK2 restores IKAROS function, enabling it to repress genes important in overcoming the inhibitory effects of chemotherapy agents used in high-risk leukemia ALL maintenance therapy, and provides support for combination therapy with CK2 inhibitors as a promising, novel treatment for ALL. In summary, we have identified the mechanism by which a loss or deletion in IKAROS contributes to the development of drug resistance in leukemia. We identified IKAROS as a critical regulator of several genes important in drug resistance. Loss of IKAROS function due to mutations, deletions, and CK2 over-activity is characteristic of high-risk leukemia. A decrease in IKAROS function results in deregulated transcription of genes that metabolize 6-mercaptopurine and 6-thioguanine or modify the effective treatment dose of methotrexate. After identifying the signal transduction pathway that controls IKAROS-mediated repression of drug resistance genes, we devised a rational targeted chemotherapy involving combination treatment with CK2 inhibitors and standard chemotherapy agents for high-risk leukemia.