1. Determining Key “Stemness” Genes
in B-cell type Acute Lymphoblastic Leukemia Stem Cells
Weijia Chua1, Lionel Sanz2, Frederic Chedin2, Noriko Satake3
1School of Medicine, 2Department of Molecular and Cellular Biology, 3Department of Pediatrics, UC Davis
Background
Objectives
Methods
Results (cont.)
shRNA plasmid construction
Broad Institute Genetic Perturbation Platform and NIH Blast were used to identify 2 independent shRNA sequences to target each gene.
Oligonucleotides for shRNAs were cloned in pLKO lentiviral plasmid using standard methods.
Correct shRNA cloning was confirmed using restriction digest and Sanger Sequencing.
Generating Lentiviral Particles
12 shRNA plasmids (6 genes x 2 shRNA each) were packaged into lentiviral particles using standard methodology via co-transfection of HEK293T cells w/ packaging plasmids.
6kb
3kb
2kb
1.5kb
Uncut plasmid
+ control
Digested pLKO plasmids containing shRNAs toward 6 genes of interest
Figure 4. Restriction digest confirming midipreps of shRNA containing plasmids
Leukemia stem cells (LSCs) are thought to be the root of leukemia and are responsible for drug resistance and disease relapse. However, to date LSCs have not been identified in acute lymphoblastic leukemia (ALL), the most common cancer in children. Recently, Dr. Satake’s lab discovered a novel method to identify and isolate LSCs in B-cell type ALL (B-ALL). These LSCs have distinct transcriptome profiles (Figure 1), confirmed in both cell lines and primary samples, and demonstrated in vivo leukemia-initiating capability (Figure 2).
Figure 1. LSCs isolated from JM1 (B-ALL cell line) show unique gene expression profiles. Microarray analysis demonstrated there were 1,135 genes which showed significant difference in expression between the LSCs and the counterpart of leukemia cells, non-LSCs. The results are from 3 independent experiments. Numbers indicate replicated samples.
Figure 2. Only LSCs, not non-LSCs, developed leukemia in mice. Survival curves of NSG mice transplanted with LSCs or non-LSCs sorted from 4 different patient-derived B-ALL are shown. In samples 1 and 2, two different cell doses, high (10,000 cells) and low (5,000 cells), were tested. None of the mice transplanted with the non-LSCs, except for one in sample 4, developed leukemia, whereas all the mice transplanted with the LSCs developed leukemia. p <0.05 for all 4 results.
Among the top 100 genes found to be most differentially expressed between LSCs and non-LSCs, 9 genes are expressed higher in LSCs (Figure 1). Our hypothesis is that these genes play a role in maintaining the stemness of LSCs. In this study, we further selected 6 genes to study.
1. Down-regulate expression of these 6 genes in LSCs using shRNA lentiviral system.
2. Determine phenotypic changes in leukemia cells after manipulation of these 6 genes.
Type of cells in culture
% Live cells prior to puromycin selection
% Live cells w/ puromyin selection
wild type Reh
(negative control)
93% 0%
Reh infected w/ shRNA toward SMAD 2
94%
81%
Reh infected w/ shRNA toward LINC02012
97%
Genes of Interest
Image from Labome.com
TP53INP2
Up-regulator of autophagy
Autophagy promotes cancer by degrading apoptotic mediators and enhancing cell survival during starvation
SMAD2
mediates TGF-beta signaling, and thus regulates processes such as cell proliferation, apoptosis, and differentiation
SOX3
Transcription Factor that regulates embryonic development and determination of the cell fate
Up-regulated in cancer tissues (esophageal squamous cell carcinoma, ovarian cancer, and T-cell lymphomas)
ERI2
Exoribonuclease (RNA degradation)
Limited studies
ARHGAP23
GTPase-activating protein inactivates RHO family proteins, involved in transmembrane receptors signal transduction
LINC02012
Non-coding RNA
Figure 5. Example of puromycin selection of shRNA plasmid integrated cells
Summary/Next Steps
We have designed and created shRNA lentivectors towards our 6 genes of interest.
We have begun the verification of the shRNA sequences by integrating them into a B-ALL cell line Reh.
Next steps
Confirm gene knockdowns via Western Blot and qRT-PCR in Reh cells
Transduce LSCs w/ shRNA lentiviral particles
Determine phenotype changes in LSCs containing down-regulated expression of targeted genes in vitro, then in vivo.
The goal is to use any genes identified as regulating stemness to develop LSC-targeted therapies.
Confirmation of shRNA knockdown of targeted genes
Reh cells (B-ALL cell line) were infected w/ lentiviral particles
Stably transduced cells were selected using puromycin resistance
Down-regulation of gene expression levels to be confirmed using Western Blot and qRT-PCR
Results
Plasmid sequence
Desired shRNA sequence
Figure 3. Example of Sanger Sequencing results used to confirm midipreps of shRNA containing plasmids