Jairo Matthews

Effect of Long-term Storage in TRIzol on Microarray-Based Gene Expression Profiling

Background: Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP) through premature termination during reverse transcription. Methods: GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the “probe level” (a single consensus value) or “bead level” (multiple measurements provided by individual beads). Results: TRIzol storage does not affect standard probe-level comparisons between sample groups: different preservation methods did not generate differentially expressed probes (DEP) within MM or AML sample groups, or substantially affect the many DEPs distinguishing between these groups. Differences were found by gene set enrichment analysis, but these were dismissible because of instability with permutation of sample labels, unbalanced restriction to TRIzol aliquots, inconsistency between MM and AML groups, and lack of biological plausibility. Bead-level comparisons found many DEPs within sample pairs, but most (73%) were <2-fold changed. There was no consistent evidence that TRIzol causes premature reverse transcription termination. Instead, a subset of DEPs were systematically due to increased signals in TRIzol-preserved samples from probes near the 5′ end of transcripts, suggesting better mRNA preservation with TRIzol. Conclusions: TRIzol preserves RNA quality well, without a deleterious effect on GEP. Samples stored frozen with and without TRIzol may be compared by GEP with only minor concern for systematic artifacts. Impact: The standard practice of prolonged sample storage in TRIzol is suitable for GEP. Cancer Epidemiol Biomarkers Prev; 19(10); 2445–52. ©2010 AACR.

Evidence of a role for the novel zinc-finger transcription factor ZKSCAN3 in modulating Cyclin D2 expression in multiple myeloma

Dysregulation of cyclin D2 contributes to the pathogenesis of multiple myeloma, and can occur through translocations that activate MAF/MAFB or MMSET/FGFR3. However, cyclin D2 induction can also be seen in the absence of such translocations, such as in patients with hyperdiploid disease, through unknown mechanisms. In UniGene cluster data-mining and ECgene analysis, we found that zinc-finger with KRAB and SCAN domains 3 (ZKSCAN3), a novel transcription factor, is overrepresented in this malignancy, and three consensus ZKSCAN3 binding sites were found in the cyclin D2 promoter. Analysis of a panel of myeloma cell lines, primary patient samples and datasets from Oncomine and the Multiple Myeloma Genomics Portal (MMGP) revealed expression of ZKSCAN3 messenger RNA (mRNA) in a majority of samples. Studies of cell lines by western blotting, and of primary tissue microarrays by immunohistochemistry, showed ZKSCAN3 protein expression in a majority, and in a manner that paralleled messenger levels in cell lines. ZKSCAN3 overexpression was associated with increased gene copy number or genomic DNA gain/amplification in a subset based on analysis of data from the MMGP, and from fluorescence in situ hybridization studies of cell lines and primary samples. Overexpression of ZKSCAN3 induced cyclin D2 promoter activity in a MAF/MAFB-independent manner, and to an extent that was influenced by the number of consensus ZKSCAN3 binding sites. Moreover, ZKSCAN3 protein expression correlated with cyclin D2 levels in cell lines and primary samples, and its overexpression induced cyclin D2. Conversely, ZKSCAN3 suppression using small hairpin RNAs (shRNAs) reduced cyclin D2 levels, and, importantly, inhibited myeloma cell line proliferation. Finally, ZKSCAN3 was noted to specifically bind to oligonucleotides representing sequences from the cyclin D2 promoter, and to the endogenous promoter itself in myeloma cells. Taken together, the data support the conclusion that ZKSCAN3 induction represents a mechanism by which myeloma cells can induce cyclin D2 dysregulation, and contribute to disease pathogenesis.

Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into severe combined immunodeficient (SCID) or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism—activation of p38 signaling in myeloma cells—by which myeloma cells induce osteolytic bone lesions, and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease.

High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics

To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next-generation sequencing data. By using this approach, we sequenced longitudinally collected acute myeloid leukemia (AML) tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.

High-throughput single-cell DNA sequencing of AML tumors with droplet microfluidics

To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next generation sequencing data. Targeted sequencing of genes frequently mutated in AML uncovered complex clonal architecture within AML tumors that was not observable with bulk sequencing.

Abstract 2177: Long-term storage in TRIzol has little effect on microarray-based gene expression profiling in primary myeloma or acute myeloid leukemia cells

We used paired samples to address whether long-term storage of cells in TRIzol has a significant effect on microarray-based gene expression profiling (GEP). The TRIzol reagent is widely used for the preservation and isolation of RNA, but there is suspicion that prolonged storage of tissues prior to RNA isolation, even at −80 C., can cause chemical modification (depurination) of RNA. This could result in early termination during reverse transcription (RT) of mRNA molecules, potentially affecting GEP more strongly for transcripts with probes located farther from the 3’ end. The Myeloma and Leukemia Tissue Banks at M. D. Anderson Cancer Center routinely collect and store primary tumor samples for research under informed consent. From these banks we selected primary tumor samples of multiple myeloma (MM; CD138+) and acute myeloid leukemia (AML; Ficoll-purified, CD3- and CD19-depleted), for which paired aliquots had been stored frozen in TRIzol (“Tri”) or cryopreservation medium (RPMI + 20% FCS + 10% DMSO, “Cryo”) since the time of initial isolation (range, 2-9 years). Cryo aliquots were quickly thawed, washed, and placed in TRIzol, then total RNA was isolated from all aliquots as per the TRIzol manufacturer9s instructions. RNA quality was assessed with a Bioanalyzer 2100 (Agilent); the RNA integrity number (RIN) was > 9 for 12/12 AML Tri, 6/12 AML Cryo, 6/6 MM Tri, and 3/6 MM Cryo samples. RNA from 12 sample pairs (6 AML, 6 MM, all with RIN > 7) was further purified with Qiagen RNAeasy columns, and the Illumina® TotalPrep RNA Amplification Kit (Ambion) was used to generate amplified, biotinylated cRNA after RT by the Eberwine procedure. Bioanalyzer-generated histograms of cRNA were similar for all sample aliquots, suggesting no early termination of RT due to TRIzol. GEP was performed with cRNA on Illumina HT-12 BeadArrays. Analysis of “probe-level” data with GenomeStudio software (Illumina), after median normalization, showed excellent concordance between sample Tri/Cryo pairs for all but probes with low intensities ( 13 for any sample pair, and most pairs had Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2177.