Kristen Gehlhaus

Inhibition of polo-like kinase 1 in glioblastoma multiforme induces mitotic catastrophe and enhances radiosensitisation

Glioblastoma multiforme (GBM) is the most common primary brain tumour in the United States of America (USA) with a median survival of approximately 14 months. Low survival rates are attributable to the aggressiveness of GBM and a lack of understanding of the molecular mechanisms underlying GBM. The disruption of signalling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. To identify protein kinases required for the survival of GBM we performed a siRNA-based RNAi screen focused on the human kinome in GBM. Inhibition of the polo-like kinase 1 (PLK1) induced a reduction in the viability in two different GBM cell lines. To assess the potential of inhibiting PLK1 as a treatment strategy for GBM we examined the effects of a small molecule inhibitor of PLK1, GSK461364A, on the growth of GBM cells. PLK1 inhibition arrested cells in the mitotic phase of the cell cycle and induced cell kill by mitotic catastrophe. GBM engrafts treated with GSK461364A showed statistically significant inhibition of tumour growth. Further, exposure of different GBM cells to RNAi or GSK461364A prior to radiation resulted in an increase in their radiosensitivity with dose enhancement factor ranging from 1.40 to 1.53 with no effect on normal cells. As a measure of DNA double strand breaks, γH2AX levels were significantly higher in the combined modality as compared to the individual treatments. This study suggests that PLK1 is an important therapeutic target for GBM and can enhance radiosensitivity in GBM.

Loss-of-function RNAi screens in breast cancer cells identify AURKB, PLK1, PIK3R1, MAPK12, PRKD2, and PTK6 as sensitizing targets of rapamycin activity.

The use of molecularly targeted drugs as single agents has shown limited utility in many tumor types, largely due to the complex and redundant nature of oncogenic signaling networks. Targeting of the PI3K/AKT/mTOR pathway through inhibition of mTOR in combination with aromatase inhibitors has seen success in particular sub-types of breast cancer and there is a need to identify additional synergistic combinations to maximize the clinical potential of mTOR inhibitors. We have used loss-of-function RNAi screens of the mTOR inhibitor rapamycin to identify sensitizers of mTOR inhibition. RNAi screens conducted in combination with rapamycin in multiple breast cancer cell lines identified six genes, AURKB, PLK1, PIK3R1, MAPK12, PRKD2, and PTK6 that when silenced, each enhanced the sensitivity of multiple breast cancer lines to rapamycin. Using selective pharmacological agents we confirmed that inhibition of AURKB or PLK1 synergizes with rapamycin. Compound-associated gene expression data suggested histone deacetylation (HDAC) inhibition as a strategy for reducing the expression of several of the rapamycin-sensitizing genes, and we tested and validated this using the HDAC inhibitor entinostat in vitro and in vivo. Our findings indicate new approaches for enhancing the efficacy of rapamycin including the use of combining its application with HDAC inhibition.

Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma

Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.

Abstract 1952: The rapid generation of mouse B cell lymphomas by lentiviral mediated overexpression of miR-1204 from a genetically unstable region of human 8q24

SNPs in the area surrounding MYC on human 8q24 have recently been found by Genome Wide Association (GWA) to be associated with susceptibility to a number of malignancies including prostate, breast, colorectal and bladder carcinomas (CA). This genetically unstable region is also a frequent target of chromosomal translocation (Tx), amplification or retroviral integration in a number of CAs such as breast, prostate, ovarian, colon, pancreatic, and cervical. In one example, essentially 100% of patients with Burkitt9s lymphoma exhibit one of three characteristic non-random chromosomal Txs that places MYC or the immediate surrounding region in close proximity to enhancers of the immunoglobulin (Ig) heavy chain or light chain loci. Although de-regulated MYC expression could be assumed to be the target of the genomic instability or GWA-based susceptibility, no clear correlation between MYC expression and disease has been established. A possible alternative target has been identified in a series of transcripts cloned from the PVT1 region downstream of MYC. However, the extent of alternative splicing coupled with the lack of a coding region has made it difficult to assign a specific role for any PVT1-derived transcripts. Recently, we have identified a cluster of small RNAs exhibiting the hairpin formation, sequence conservation and expression characteristics of miRNAs (miR-1204∼1208) within the transcriptional domain of PVT1. Increased expression of several of these miRNAs in tumors harboring amplified MYC/PVT1 or Burkitt lymphomas with the 8q24 Tx suggests a possible role for these miRNAs in tumorigenesis, especially for miR-1204 which is found 60 kb downstream of MYC, flanking exon 1b of PVT1. An increased expression of miR-1204 in pre-B cells compared to pro-B cells also suggested a lymphoid specific developmental pattern of expression. Lentiviral constructs of miR-1204 under control of a CMV promoter (LentiCMV-miR-1204) revealed a possible effect on MYC, but in a pre-B (not pro-B) specific environment. Introduction of LentiCMV-miR-1204 into mice harboring either a MYC (C.iMYC) or IL6 (C.IL6) transgene resulted in high frequency and rapid onset of large B cell lymphomas (∼16 or 82 days vs. 91 or 117 days for controls, respectively). While these results point to over-expression of miR-1204 playing a specific role in the development of B cell malignancy, additional studies with the LentiCMV-miR-1204 construct in other tumor cell lines (prostate and breast) also reveal phenotypic changes in cell proliferation and migration. Further studies using lentiviruses with alternative promoters for miR-1204 and the other 8q24 associated miRNAs are underway to identify downstream targets and pathways not only in lymphoid malignancy but also in the wide range of malignancies associated with GWA-susceptibility and genomic instability. 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 1952.

Abstract 479: Inhibition of the splicing of the EWS-FLI1 fusion transcript reverses EWS-FLI1 driven oncogenic expression in Ewing sarcoma

Ewing sarcoma (ES) is a highly aggressive cancer of the bone and soft tissue. In ∼85% of ES tumors the primary oncogenic event is a t(11:22)(q24:q12) translocation that generates a fusion of the 5′ end of EWSR1 and the 3′ end of FLI1 referred to as EWS-FLI1. The exact genomic breakpoints within the EWSR1 and FLI1 genes vary, but typically occur within introns and require the splicing machinery to generate an in-frame EWS-FLI1 transcript. The most common EWS-FLI1 transcripts fuse either exon 7 of EWSR1 to exon 6 of FLI1 (a type I or a 7/6 fusion), or fuse exon 7 of EWSR1 to exon 5 of FLI1 (a type II or 7/5 fusion). In an estimated 40% of EWS-FLI1 driven tumors the generation of an in-frame EWS-FLI1 fusion transcript requires alternative splicing. In particular, translocations that retain exon 8 of EWSR1 generate an out-of-frame transcript unless this exon is removed. Using an assay of EWS-FLI1 activity and genome-wide siRNA screening we have identified RNA processing as a therapeutic vulnerability in ES. Parallel genome-wide siRNA-mediated RNAi screens were conducted in ES TC32 cell lines expressing a luciferase (luc) reporter protein driven by either the promoter of the EWS-FLI1 target gene NR0B1 (TC32-NR0B1-luc) or the CMV promoter (TC32-CMV-luc). The top gene ontology terms associated with the 28 priority candidate genes that when silenced induced a differential decrease in the TC32-NR0B1-luc signal versus the TC32-CMV-luc signal were mRNA splicing (p-value = 1.42E-08) and mRNA processing (p-value = 2.32E-07). To investigate the mechanistic basis for the identification of specific RNA processing proteins as required for the activity of EWS-FLI1 we focused on two lead candidate genes, the heterogeneous nuclear ribonucleoprotein H1, HNRNPH1, and the core splicing factor, SF3B1. Using PCR analysis we determined that HNRNPH1 is required for the splicing of EWS-FLI1 fusion transcripts expressed in ES cells in which the chromosome 22 breakpoint retains EWSR1 exon 8, specifically in TC32 and SKNMC ES cells. We also show ES cell lines harboring 7/ 6 (TC32, SKNMC, and TC71) or 7/ 5 (RD-ES) EWS-FLI1 fusions are all sensitive to the loss-of-function of SF3B1. Quantitative RT-PCR, immunoblot, and whole transcriptome analysis show that disrupted splicing of the EWS-FLI1 transcript alters its expression and reverses the expression of a significant proportion of genes that are targets of EWS-FLI1. These observations were confirmed in four ES cell lines using the splicing inhibitor Pladienolide B. Our results provide the basis for a novel strategy to target fusion oncogenes by interfering with RNA processing. This study has implications for the treatment of ES through inhibition of proteins required for expression of the EWS-FLI1 transcript and identifies a candidate lead compound for further clinical development. Our findings may also open up strategies for treatment of other cancers driven by fusion oncogenes. Citation Format: Patrick J. Grohar, Suntae Kim, Sara Haddock, Guillermo Rangel Rivera, Matt Harlow, Nichole K. Maloney, Konrad Huppi, Kristen Gehlhaus, Magdalena Grandin, Carleen Klumpp-Thomas, Eugen Buehler, Lee J. Helman, Scott E. Martin, Natasha J. Caplen. Inhibition of the splicing of the EWS-FLI1 fusion transcript reverses EWS-FLI1 driven oncogenic expression in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 479. doi:10.1158/1538-7445.AM2015-479

Abstract 3204: The identification of AURKB, BUB1B and CDK18 as putative molecular targets for breast cancer using kinome focused RNAi screens.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The signal transduction events regulated by the kinase family of proteins control many cellular processes including normal cell proliferation and survival. The disruption of signaling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells, including breast cancer, and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. Functional genomic RNAi screens of the kinome conducted within the context of a specific tumor type and/or a specific cancer-associated genetic background have the potential to identify kinases required for cancer cell survival. A comparison of RNAi screening data that we have obtained targeting the human kinome in three breast cancer cell lines with other published RNAi screens led us to perform a detailed analysis of the phenotypic effects of silencing AURKB, BUB1B, and CDK18 in multiple breast cancer cell lines with the aim of further characterizing kinase genes required for the growth of breast tumor cells. The non-tumorigenic mammary epithelial cell line MCF-10A exhibited minimal BUB1B protein expression and no detectable expression of AURKB protein, while all of the breast cancer cell lines studied (a total of eight), exhibited expression of both of these proteins. This differential expression was associated with functional differences in silencing of AURKB or BUB1B resulting in suppression of colony formation in almost all of the breast cancer cell lines tested, but not in MCF-10A cells. Silencing of AURKB also induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468 and HCC-38 cells, but not in MCF-10A cells. These results were phenocopied by an ATP-competitive inhibitor of aurora kinase with selectivity for aurora B kinase ZM447439. BUB1B silencing resulted in cell cycle arrest in both MDA-MB-468 and HCC-38 cells, but caspase 3/7-activation and PARP cleavage were only observed in MDA-MB-468 cells. None of these effects were observed in MCF-10A cells. Little is known as to the functional role of CDK18, but its silencing induced cell cycle arrest, caspase 3/7-activation and PARP cleavage in MDA-MB-468. In contrast we saw minimal phenotypic effects in HCC-38 cells and MCF-10A cells, even though both of these cell lines express CDK18. To further elucidate the functional role of CDK18, we performed genome-wide expression profiling of MDA-MB-468 cells in which CDK18 had been silenced. Interestingly, one of the transcripts showing the greatest decrease in expression following silencing of CDK18 was CCNE1 that encodes cyclin E1. Another cyclin gene, CCND1, also exhibited a decrease in expression following silencing of CDK18, and we are currently examining this potential link between CDK18 function and cyclin E1 and cyclin D1 in more detail. Citation Format: Lihui Ou, Kristen Gehlhaus, Tamara Jones, Konrad Huppi, Natasha Caplen. The identification of AURKB, BUB1B and CDK18 as putative molecular targets for breast cancer using kinome focused RNAi screens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2013-3204

Abstract 267: Large-scale RNAi screening of human kinome identifies putative breast cancer related molecular targets

To identify vulnerable genes in breast cancer cells that may be therapeutic candidates for treatment of breast cancer, we conducted an unbiased, large-scale, synthetic siRNA-mediated RNAi screen. Using cell viability as the read-out, we first screened a whole human kinome library plus 350 additional genes (four siRNAs per gene) in a cell line representative of triple negative (ER -ve, PR -ve, Her2/Neu -ve) breast cancer, MDA-MB-468. We identified approximately 40 kinases whose loss-of-function reduced the viability of MDA-MB-468 cells based on the criteria of at least two of four siRNAs per gene generating a Z score of ≤ −1.5. To validate the top candidates from our screen, PLK1, AURKB and PCTK3/CDK18), we tested additional siRNAs (from multiple vendors) in different breast cancer cell lines including ER positive cell lines, CAMA-1 and MCF-7, HER-2 amplified cell lines, HCC-1954 and AU565, triple-Negative (Basal A) cell lines HCC-1937 and MDA-MB-468, and triple-Negative (Basal B) cell lines BT549 and MDA-MB-231. We found silencing of PLK1, AURKB or PCTK3/CDK18 reduced the cell viability in majority of these cell lines to a certain degree and also induced cell cycle arrest and apoptosis. PLK1 and AURKB are being actively pursued as molecular targets in cancer but little is known of the function of the PCTK3/CDK18 protein, with most of the limited functional studies of PCTK3/CDK18 having focused on a role in neuronal cell signaling. One study has though reported an increase in the expression of PCTK3/CDK18 in breast cancer (Valladares et al., Cancer Genet Cytogenet. 2006 170:147). We found silencing of PCTK3/CDK18 is lethal to most breast cancer cells, but it is well tolerated in the untransformed breast epithelial cell line MCF10A. The PCTK3/CDK18 gene encodes a member of the PCTAIRE protein kinase subfamily of CDC2-related serine/threonine-specific protein kinases. We observed that with multiple siRNAs, we induced an 80% decrease in the viability of MDA-MB-468 cells 96 hours following siRNA transfection. To ensure that the observed inhibitory effect is indeed due to silencing of PCTK3/CDK18 we performed an RNAi rescue experiment, by creating a silent third-codon point mutation within the region targeted by one of the PCTK3 siRNAs. We found the RNAi-induced inhibition and cell cycle arrest is countered by expression of a functional version of the target gene that is resistant to the silencing siRNA. No specific inhibitor of PCTK3/CDK18 is available, but a proteome-wide CDK/CRK-specific kinase inhibitor RGB-286147 does decrease cell viability, and induce cell cycle arrest and apoptosis of MDA-MB-468 cells. We are currently further investigating the normal and cancer-related functional roles of PCTK3/CDK18 and are pursuing strategies for the identification of PCTK3/CDK18 specific inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 267. doi:1538-7445.AM2012-267

Abstract 2041: Large-scale RNAi screening identifies PCTK3/CDK18 as a putative cancer-related molecular target

RNAi screening is a powerful approach for the identification of proteins that have the potential to be developed as anti-cancer molecular targets. In this study we used an unbiased, large-scale, synthetic siRNA-mediated RNAi screen to identify vulnerable targets in breast cancer cells with the hope of finding novel therapeutic targets. Using cell viability as the read-out, we first screened a whole human kinome library plus 350 additional genes (four siRNAs per gene) in a cell line representative of triple negative (ER -ve, PR -ve, Her2/Neu -ve) breast cancer, MDA-MB-468. The results of two screens conducted in MDA-MB-468 cells were highly reproducible with good correlation (0.82) between duplicate screens performed separately. These screens identified several well-defined kinases whose loss-of-function reduced the viability in MDA-MB-468 cells, including PLK1, AURKB, and BIRC5. Some less well-characterized kinases also were identified to be required for the viability of MDA-MB-468 cells including PIK3R1, HUNK, CKB, and PCTK3/CDK18. The PCTK3/CDK18 gene encodes a member of the PCTAIRE protein kinase subfamily of CDC2-related serine/threonine-specific protein kinases. Most of the limited functional studies of PCTK3/CDK18 have focused its biological role in neuronal cells, but one study has reported an increase in the expression of PCTK3/CDK18 in breast cancer (Valladares et al., Cancer Genet Cytogenet. 2006 170:147). We observed that with multiple siRNAs, we induced an 80% decrease in the viability of MDA-MB-468 cells 96 hours following siRNA transfection. Over time we saw a 10%, 20% and 65% decrease in viability at 24h, 48h and 72h, respectively. As little is known of the function of the PCTK3/CDK18 protein, we are using multiple approaches to elucidate the cellular functions of PCTK3/CDK18 in the context of breast cancer. For example, we are investigating the cellular responses seen following loss of function of PCTK3 that could lead to the reduction in the viability phenotype observed. We have found that silencing PCTK3/CDK18 induces apoptosis as demonstrated by PARP cleavage and caspase activation, and cell cycle arrest in the S-phase. To further elucidate the role of PCTK3 in cell growth, we generated concordant gene expression signatures using different RNAi effectors targeting PCTK3/CDK18. We found these altered expressions of genes are involved mostly in DNA damage pathway and cell cycle pathway. While PCTK3 inhibition was lethal to MDA-MB-468 triple negative breast cancer, in contrast, silencing of PCTK3 in the non-transformed breast epithelial cell line MCF10A was well tolerated. This study further highlights the potential of high-throughput siRNA screens to identify novel cancer related molecular targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2041. doi:10.1158/1538-7445.AM2011-2041

Abstract LB-74: A high-throughput RNAi sensitization screen of rapamycin identifies targets for rational drug combination strategies

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC RNAi screening affords an unbiased and rapid method to identify genes involved in particular cellular processes and is thus a powerful tool for drug target identification and validation. Rapamycin is a well defined inhibitor of mTOR, a protein being actively pursued as an anti-tumor target. In breast cancer patients, however, treatment with mTOR inhibitors has shown only modest activity suggesting that a combined approach may be required to maximize the clinical application of mTOR inhibitors in these patients. In this study, we performed a high-throughput synthetic siRNA-based RNAi screen of the human kinome plus 350 additional genes (4 siRNAs per gene) in combination with rapamycin, to identify candidate genes whose silencing potentiate the inhibitory effects of rapamycin. Screens were conducted in the ER+ breast cancer cell line MCF-7 and the triple negative breast cancer cell line MDA-MB-468. For the RNAi screen rapamycin (10 nM) or vehicle only (0.1% DMSO) was added 48 hours post siRNA transfection and cell viability was measured after a further 24 hours of incubation. By comparing the normalized effect on cell viability for each siRNA (Z-score) in the control and rapamycin screens we identified 36 candidate genes whose silencing sensitized MCF-7 cells to rapamycin and 56 that sensitized MDA-MB-468 cells. The silencing of six candidate genes sensitized both cell lines to rapamycin, AKT1, ASPA, CDKN2A, MAP3K7IP1, MAPK12 and PCTK3. The sensitization of rapamycin by silencing of AKT1 was confirmed. Rapamycin treatment increased AKT phosphorylation in the cell lines. HDAC inhibitors have been shown to facilitate the dephosphorylation of AKT; therefore, we tested the combination of rapamycin with the HDAC inhibitor MS-275. MS-275 completely blocks rapamycin-induced AKT phosphorylation, and combining it with rapamycin synergistically inhibited the growth of the MCF-7, MDA-MB-468, MDA-MB-435 and MDA-MB-231 breast cancer cell lines. We investigated if other pathways are affected by combining rapamycin with MS-275. De-regulation of the RAF-MEK-ERK pathway is frequently detected in human cancers but the high level of ERK phosphorylation is significantly reduced in cells treated with the rapamycin/MS-275 combination. To profile the unphosphorylated, mono and dual-phosphorylated isoforms of ERK1 and ERK2 we used a capillary isoelectric focusing immunoassay to show that the di-phosphorylated isoform of ERK2 was decreased. Our results highlight the potential of high-throughput siRNA screens to identify rational drug combination strategies. 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 LB-74.