Unravelling the Role of KCTD Protein Family in the Clinical Management of Childhood Acute Lymphoblastic Leukemias

KCTD expression levels, by RNAseq approach, in a cohort of B and T cell ALL affected patients compared to unaffected controls

We enroll during this project about 15-20 B-ALL, and 3-6 T-ALL patients.RNAseq experiments for transcriptome analysis will be performed on a study cohort of at least 8 B-ALL patients, 3-4 T-ALL patients, and naive B or T cells purified from cord blood as control for B- and T-ALL correspondingly. Raw counts will be used and an empirical Bayes approach applied (DESeq2, R package). The results will include, for each gene, log-fold change (log2), base mean, p-value and adjusted p-values (Benjamimi-Hochberg). DEgenes output will be exported into format suitable for IPA Ingenuity pathway AnalysisSoftware (Qiagen), for gene annotations, functional analysis and biomarker prediction. The KCTD proteins that will be dereguleted in B-ALL and T-ALL will be studied in detail.

Time frame: 1-12 months

RT-PCR validation of deregulated KCTDs and identification of possible KCTDs interactors in leukemia by functional proteomic approach

After the interpretation of transcriptome data we will perform specific validation experiments by reat-time pcr, westernblotting, flow cytometry and microscopy. Briefly,total RNA will be purified from about 5e6 total MNC and used for RT-PCR experiments; BM smear will be collected for immunofluorescence analysis of deregulated KCTDs; protein total extract will be obtained from at least 1 e 6 total MNC and stored at -80°C until Western Blotting; finally, live mononuclear cells will be treted for permeabilization and cytoplasmic immunostaining of the KCTDs of interest. All experiments will be settled-up on B- and T-ALL in vitro human model systems and then performed on patients and controls cells. Moreover, in order to identify possible interactors of KCTDs proteins we will use B-ALL and T-ALL cell lines for functional proteomics analysis.

Time frame: 13-24 months

Correlation of B-ALL patients clinical data with KCTDs expression level

Firstly, we will evaluated the usefulness of the deregulated KCTD proteins as novel markers of leukemia to be tested by multiparametric clinical flowcytometry. A specific protocol has been already setted-up by our research team, and it is able to stain cytoplasmicantigens (such as KCTD proteins) with surface antigens commonly used for to study hematoogical compartments by flowcytometry. This approach will be useful especially in monitoring patient response to treatment as well as detection of minimal residual disease. Moreover, being able to detect cytoplasmic KCTDs in combination with surface antigens, we planned to describe the KCTDs expression levels in multiple cellular population residing in marrow and peripheral blood.Secondly, the overall data obtained in this study will be also interpreted looking at the patient clinics with particular reference to the response to therapy, the risk of relapse and, when possible, resistance to therapy.

Time frame: 25-36 months