Abstract B36: Biological and clinical significance of ID1/ID3/IGJ expression signature modulation in B-Acute lymphoblastic leukemia

Abstract

Abstract Background. Acute lymphoblastic leukemia (ALL) in Hispanic populations represents a public health problem that requires priority attention because its incidence and mortality increase annually. Intensive chemotherapeutic schemes in the last decade in the United States and other countries and have led to higher rates of durable remissions. However, these schemes have shown disappointing results in Hispanic populations. In Colombia, for example, the median overall survival (OS) is less than 11.3 months, the event-free survival (EFS) is 7.34 months, and only 61% of patients achieve complete remission (CR). Therefore, it is interesting to explore the underlying molecular characteristics of the disease in Hispanic patients. Previously we identified a gene expression signature associated with response to induction treatment and outcome of adult Hispanic patients with B-ALL. These ID1, ID3 and IGJ seem to be involved in many tumorigenic processes. The goal of our study is identify the role of these genes in B-ALL and its relevance in the prognosis and progression of disease in an in vitro model. Methods. CRISPR-Cas9 Gene Engineering and expression-ready ORF cDNA clones were used to either down-regulate or over-express, respectively, the expression of ID1, ID3 and IGJ genes in the adult B-ALL cell line NALM6. We used TaqMan to quantify the levels of mRNA expression of ID1/ID3/IGJ genes after over-expression. Protein expression by Flow Cytometry was used to verify silencing and over-expression of our 3-genes signature. Cell viability and proliferation were determined by anexin V-PI and MTT assay, respectively at 0, 24, 48, 72 and 96 hours after modulation. Cell cycle analysis was performed using BrdU and 7-AAD. Separately, increasing doses of cyclophosphamide, doxorubicin and cytarabine were used to determine the role of ID1, ID3, and IGJ genes in chemo resistance at 24, 48, 72, 96, and 144 hours. Total RNA from leukemic cells was isolated using Trizol method. We used microarray analysis to identify genes differentially expressed after silencing/overexpression. We performed Immunophenotype analysis of 43 B-ALL adult Hispanic patient Bone Marrow samples using the panel of antibodies recommended and standardized by the European consortium Euroflow. Correlation analysis were used to establish the association between our 3-Gene expression signature and the expression of surface markers currently used for diagnostic and follow-up of B-ALL patients. Results. We observed that both overexpression and silencing of ID1, ID3 and IGJ modulated B-ALL cells cell cycle. ID1/ID3/IGJ overexpression resulted in a loss of S phase and arrest of the cells in the G0/G1 phase of the cell cycle. On the contrary, ID1/ID3/IGJ silencing induced a loss of G0/G1 phase and gain in the number of cells in G2/M and S phases. Gain-of-expression and loss-of-expression analyses using incremental doses of chemotherapy agents currently used in the B-ALL treatment, demonstrate that ID1/ID3/IGJ signature promotes drug resistance of the NALM6 B-ALL cell line. Clinically, patients with higher ID1/ID3/IGJ expression have high expression of CD20 and CD10 markers (aberrantly expressed in immature cells) and shorter OS and EFS. In addition, more than 2,000 genes that are differentially expressed in the cells with modulation of the expression of ID1/ID3/IGJ suggesting a complex network of biological processes implicated in the drug resistance and poor prognosis present in the patients with this 3-gene signature. Conclusions. Our data identify the ID1/ID3/IGJ signature as possible modulator of molecular events during B-ALL differentiation, proliferation and drug resistance. These results highlight the potential role of this gene signature both as a risk stratification tool and as a candidate therapeutic target in Hispanic population. Citation Format: Nataly Cruz-Rodriguez, Alba Lucia Combita, Jone Garai, Leonardo Jose Enciso, Sandra Milena Quijano, Silvia Serrano-Gomez, Li Li, Jovanny Zabaleta. Biological and clinical significance of ID1/ID3/IGJ expression signature modulation in B-Acute lymphoblastic leukemia. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B36.