Abstract 832
We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene ...expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM.
No relevant conflicts of interest to declare.
Activating RAS mutations are among the most common pathogenetic events in a broad spectrum of hematologic malignancies and epithelial tumors. However, oncogenic RAS has thus far not proven to be a ...tractable target for therapeutic intervention. An alternative to direct targeting of known oncogenes is to perform “synthetic lethality” screens to identify genes that are selectively required for cell viability in the context of specific cancer-causing mutations. Using this approach, we have discovered a synthetic lethal interaction between mutant KRAS, the most frequently mutated oncogene in human cancer, and inactivation of the gene encoding the STK33 serine/threonine protein kinase. To identify genes that are essential for cell viability in the context of mutant KRAS, we performed high-throughput loss-of-function RNA interference (RNAi) screens in eight human cancer cell lines (mutant KRAS, n=4; wildtype KRAS, n=4), representing seven different tumor types (acute myeloid leukemia, multiple myeloma, colon cancer, breast cancer, ovarian cancer, prostate cancer, glioblastoma), as well as normal human fibroblasts and mammary epithelial cells. We screened each cell line with a subset of the short hairpin RNA (shRNA) library developed by the RNAi Consortium (http://www.broad.mit.edu/genome_bio/trc/rnai.html) that consists of 5,024 individual shRNA constructs targeting 1,011 human genes, including the majority of known and putative protein kinase and phosphatase genes and a selection of known cancer-related genes. In these cell lines, suppression of STK33 preferentially inhibited the viability and proliferation of cells that were dependent on mutant KRAS. The differential requirement for STK33 based on oncogenic KRAS dependency was confirmed in 16 additional cell lines using in vitro transformation assays and human tumor xenograft models. Biochemical analyses support the hypothesis that STK33 promotes cell growth and survival in a kinase activity-dependent manner by regulating the activity of S6K1 as well as BAD-induced apoptosis selectively in mutant KRAS-dependent cells. Notably, molecular genetic characterization of cancer cell lines and analysis of patient-derived genomic data sets indicate that STK33 is not frequently mutated or overexpressed in human tumors. These observations identify STK33 as a potential target for the treatment of mutant KRAS-driven cancers that may have a broad therapeutic index in normal versus malignant cells, and illustrate the potential of RNAi for discovering critical functional dependencies created by oncogenic mutations that cannot be identified using other genomic technologies.
The Multiple Myeloma Research Consortium (MMRC) Genomics Initiative was instigated to harness the power of multiple genomic approaches to further the understanding of multiple myeloma. To date, 137 ...samples from patients with newly diagnosed and relapsed myeloma (out of an estimated final total of 250 by the end of the year) have been subjected to expression profiling and array comparative genomic hybridization. To identify regions of recurrent copy number alteration with a high degree of confidence, we have used the Genomic Identification of Significant Targets In Cancer (GISTIC) algorithm, which detects such regions and assigns a probability to each. Application of GISTIC to the MMRC collection identified 14 significant regions of amplification and 15 significant regions of deletion. The algorithm further detects peaks of copy number change that contribute to each region's significance. Of genes that were expressed in our dataset, a total of 64 across the genome were found to lie within the boundaries of significant amplification peaks and 30 were found to lie within significant deletion peaks. Given the likelihood that this gene collection is highly enriched for genes important in the pathogenesis of myeloma and potential therapeutic targets, it has been prioritized for further validation with the amplified genes submitted for arrayed RNAi and the deleted genes expedited for re-sequencing. In order to define a poor prognosis group, we developed and applied a model based on the gene expression dataset of Shaughnessy and colleagues. To identify pathways activated in poor prognosis disease, we applied Gene Set Enrichment Analysis with the Molecular Signatures Database. This demonstrated enrichment of multiple canonical pathways within the poor prognosis group, including those associated with proliferation, cell cycle progression and DNA repair. A search for recurrent copy number events associated with poor prognosis disease revealed that by far the most significant event, and the only one surviving correction for multiple hypothesis testing, was mono- or bi-allelic deletion of CDKN2C (p18). Interestingly, the poor prognosis group also demonstrated higher expression of p18 in those samples without bi-allelic deletion and of CDKN2A (p16). Deletion and increased expression of p18 in high proliferation index myeloma samples has previously been described. However, our analyses demonstrate the pre-eminence of p18 loss for defining poor prognosis disease compared to other recurrent copy number changes. Furthermore, it expands on work in other tumors demonstrating feedback mechanisms between cyclin dependent kinase inhibitor pathways, suggesting a more complex model of interplay between the pathways than previously described. In conclusion, the MMRC reference collection is proving an important tool in the fight to understand key genetic events in multiple myeloma and would ultimately be anticipated to contribute towards development of improved therapy for the disease.
