Daniel Nicorici

Identification of fusion genes in breast cancer by paired-end RNA-sequencing

BackgroundUntil recently, chromosomal translocations and fusion genes have been an underappreciated class of mutations in solid tumors. Next-generation sequencing technologies provide an opportunity for systematic characterization of cancer cell transcriptomes, including the discovery of expressed fusion genes resulting from underlying genomic rearrangements.ResultsWe applied paired-end RNA-seq to identify 24 novel and 3 previously known fusion genes in breast cancer cells. Supported by an improved bioinformatic approach, we had a 95% success rate of validating gene fusions initially detected by RNA-seq. Fusion partner genes were found to contribute promoters (5 UTR), coding sequences and 3 UTRs. Most fusion genes were associated with copy number transitions and were particularly common in high-level DNA amplifications. This suggests that fusion events may contribute to the selective advantage provided by DNA amplifications and deletions. Some of the fusion partner genes, such as GSDMB in the TATDN1-GSDMB fusion and IKZF3 in the VAPB-IKZF3 fusion, were only detected as a fusion transcript, indicating activation of a dormant gene by the fusion event. A number of fusion gene partners have either been previously observed in oncogenic gene fusions, mostly in leukemias, or otherwise reported to be oncogenic. RNA interference-mediated knock-down of the VAPB-IKZF3 fusion gene indicated that it may be necessary for cancer cell growth and survival.ConclusionsIn summary, using RNA-sequencing and improved bioinformatic stratification, we have discovered a number of novel fusion genes in breast cancer, and identified VAPB-IKZF3 as a potential fusion gene with importance for the growth and survival of breast cancer cells.

Systematic Analysis of MicroRNAs Targeting the Androgen Receptor in Prostate Cancer Cells

Androgen receptor (AR) is expressed in all stages of prostate cancer progression, including in castration-resistant tumors. Eliminating AR function continues to represent a focus of therapeutic investigation, but AR regulatory mechanisms remain poorly understood. To systematically characterize mechanisms involving microRNAs (miRNAs), we conducted a gain-of function screen of 1129 miRNA molecules in a panel of human prostate cancer cell lines and quantified changes in AR protein content using protein lysate microarrays. In this way, we defined 71 unique miRNAs that influenced the level of AR in human prostate cancer cells. RNA sequencing data revealed that the 3UTR of AR (and other genes) is much longer than currently used in miRNA target prediction programs. Our own analyses predicted that most of the miRNA regulation of AR would target an extended 6 kb 3UTR. 3UTR-binding assays validated 13 miRNAs that are able to regulate this long AR 3UTR (miR-135b, miR-185, miR-297, miR-299-3p, miR-34a, miR-34c, miR-371-3p, miR-421, miR-449a, miR-449b, miR-634, miR-654-5p, and miR-9). Fifteen AR downregulating miRNAs decreased androgen-induced proliferation of prostate cancer cells. In particular, analysis of clinical prostate cancers confirmed a negative correlation of miR-34a and miR-34c expression with AR levels. Our findings establish that miRNAs interacting with the long 3UTR of the AR gene are important regulators of AR protein levels, with implications for developing new therapeutic strategies to inhibit AR function and androgen-dependent cell growth.

CIP2A signature reveals the MYC dependency of CIP2A-regulated phenotypes and its clinical association with breast cancer subtypes

Protein phosphatase 2A (PP2A) is a critical human tumor-suppressor complex. A recently characterized PP2A inhibitor protein, namely cancerous inhibitor of PP2A (CIP2A), has been found to be overexpressed at a high frequency in most of the human cancer types. However, our understanding of gene expression programs regulated by CIP2A is almost absent. Moreover, clinical relevance of the CIP2A-regulated transcriptome has not been addressed thus far. Here, we report a high-confidence transcriptional signature regulated by CIP2A. Bioinformatic pathway analysis of the CIP2A signature revealed that CIP2A regulates several MYC-dependent and MYC-independent gene programs. With regard to MYC-independent signaling, JNK2 expression and transwell migration were inhibited by CIP2A depletion, whereas MYC depletion did not affect either of these phenotypes. Instead, depletion of either CIP2A or MYC inhibited cancer cell colony growth with statistically indistinguishable efficiency. Moreover, CIP2A depletion was shown to regulate the expression of several established MYC target genes, out of which most were MYC-repressed genes. CIP2A small-interfering RNA-elicited inhibition of colony growth or activation of MYC-repressed genes was reversed at large by concomitant PP2A inhibition. Finally, the CIP2A signature was shown to cluster with basal-type and human epidermal growth factor receptor (HER)2-positive (HER2+) breast cancer signatures. Accordingly, CIP2A protein expression was significantly associated with basal-like (P=0.0014) and HER2+ (P<0.0001) breast cancers. CIP2A expression also associated with MYC gene amplification (P<0.001). Taken together, identification of CIP2A-driven transcriptional signature, and especially novel MYC-independent signaling programs regulated by CIP2A, provides important resource for understanding CIP2As role as a clinically relevant human oncoprotein. With regard to MYC, these results both validate CIP2A’s role in regulating MYC-mediated gene expression and provide a plausible novel explanation for the high MYC activity in basal-like and HER2+ breast cancers.

Reanalysis of RNA-Sequencing Data Reveals Several Additional Fusion Genes with Multiple Isoforms

RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

Abstract 3175: Genomic and transcriptomic data integration in chronic myelomonocytic leukemia reveals a novel fusion gene involving onco-miR-125b-2

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnChronic myelomonocytic leukemia (CMML) is a rare malignancy characterized by increased peripheral monocytosis and dysplasia in a single- or multilineage fashion. Gene mutations so far reported in CMML include TET2, CBL, NRAS, KRAS, RUNX1 and EZH2 but their pathogenic role and driver status in the disease remains unclear. Altered expression of the microRNA miR-125b has been implicated in the pathogenesis of many types of cancers, including myeloid leukemias and Down syndrome-associated acute myeloid leukemia (DS-AML). In addition, this miRNA has been shown to play an important role in hematopoiesis and the regulation of immune cell response. Here, integration of data from next-generation transcriptome sequencing, exome sequencing and array-CGH in a CMML patient (trisomy 21 by cytogenetics) led to the identification of a novel gene fusion event involving the nuclear receptor interacting protein NRIP1 gene and the open reading frame C21orf34 (both at 21q21 approximately 1 MB apart). The fusion was validated by capillary sequencing and found to involve two copy number transition breaks, inversion of the intervening region and the upregulation of the 3′ end of C21orf34. This intronic region harbors a cluster of three miRNAs: miR-let7c, miR-99a, and miR-125b-2. Based on genomic breakpoint analysis, the gene fusion took place just upstream of miR-125b-2. Consistent with this, only miR-125b-2 was highly expressed in the sample, and was processed to a mature miRNA. By RT-PCR, increased expression of miR-125b-2 was also observed in four other CMML patients and five CML patients when compared to healthy bone marrow controls. In contrast, five AML cases studied showed expression levels similar to or lower than that of controls. Interestingly, one AML patient with trisomy 21 had very high levels of miR-125b-2. We found the NRIP1-C21orf34 fusion only in our index patient and therefore other mechanisms of miRNA deregulation at 21q21 in CMML/CML and AML+21 will also exist. In conclusion, we describe for the first time a fusion gene involving miR-125b-2 in CMML, a previously recognized and well-studied onco-miR, which is known to impact on self-renewal of hematopoietic cell precursors. We also detected overexpression of miR-125b-2 in all CMML samples studied suggesting a key pathogenetic driver gene role for this micro-RNA. The assessment of miR-125b-2 levels could potentially be applied to the diagnosis and follow-up of patients with CMML.nnCitation 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 3175. doi:1538-7445.AM2012-3175

Adrenals Contribute to Growth of Castration-Resistant VCaP Prostate Cancer Xenografts

The role of adrenal androgens as drivers for castration-resistant prostate cancer (CRPC) growth in humans is generally accepted; however, the value of preclinical mouse models of CRPC is debatable, because mouse adrenals do not produce steroids activating the androgen receptor. In this study, we confirmed the expression of enzymes essential for de novo synthesis of androgens in mouse adrenals, with high intratissue concentration of progesterone (P4) and moderate levels of androgens, such as androstenedione, testosterone, and dihydrotestosterone, in the adrenal glands of both intact and orchectomized (ORX) mice. ORX alone had no effect on serum P4 concentration, whereas orchectomized and adrenalectomized (ORXxa0+xa0ADX) resulted in a significant decrease in serum P4 and in a further reduction in the low levels of serum androgens (androstenedione, testosterone, and dihydrotestosterone), measured by mass spectrometry. In line with this, the serum prostate-specific antigen and growth of VCaP xenografts in mice after ORXxa0+xa0ADX were markedly reduced compared with ORX alone, and the growth difference was not abolished by a glucocorticoid treatment. Moreover, ORXxa0+xa0ADX altered the androgen-dependent gene expression in the tumors, similar to that recently shown for the enzalutamide treatment. These data indicate that in contrast to the current view, and similar to humans, mouse adrenals synthesize significant amounts of steroids that contribute to the androgen receptor-dependent growth of CRPC.

Abstract 4580: Personalized treatment selection for therapy-resistant AML by integrating ex-vivo drug sensitivity and resistance testing (DSRT) as well as serial genomic, transcriptomic and phosphoproteomic profiling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnSamples from recurrent, treatment-refractory cancers are rarely available, but would be valuable in understanding the molecular drivers of drug resistance. In leukemias, consecutive samples are readily available during treatment. Hence, we explored here the progression of adult acute myeloid leukemias (AML) by serial sampling and by integrating data from multiple platforms. Next-generation exome and RNA sequencing, and phosphoproteomic data were combined with comprehensive 240 cancer drug sensitivity and resistance testing (DSRT) of leukemic blasts ex-vivo before and after clinical relapse. The data were generated in an experimental diagnostic setting, with intent to improve and personalize treatment of patients with recurrent AML. A 54-year old AML-M5 patient with a FLT-3-ITD mutation and a normal karyotype was monitored by serial sampling. The patient was initially refractory to three consecutive high-dose induction treatments and had limited therapy options. AML blasts from the patient were screened with the DSRT platform. Results implied that the blast cells were 710-times more sensitive to temsirolimus and other rapamycin analogs as compared to normal BM cells, and showed a 1100-fold increased sensitivity to dasatinib. Proteomic analysis showed high phosphorylation of several signaling molecules, such as the insulin receptor and mTOR. Sequencing identified WT1 mutations and a NUP98-NSD1 fusion transcript, an infrequent event associated with poor prognosis in AML. Based on the DSRT results, the patient received compassionate off-label treatment with dasatinib, sunitinib and temsirolimus, resulting in a remarkable clinical remission, normalization of blast counts and a rapid recovery of neutrophil counts as a sign of selective elimination of the leukemic cells. The patient relapsed 4 weeks later, and at this point a new DSRT assay was performed, which showed the blast cells to be completely resistant to temsirolimus and less sensitive to dasatinib ex vivo. Consistent with this drug sensitivity profile was a genomic evolution of a distinct AML subclone with new changes, such as NF1 mutation and a microdeletion of the LEF1 gene, which were not observed in the pre-treatment sample. Taken together, we have demonstrated, how molecular profiling and functional ex vivo drug sensitivity and resistance data can be used to individually optimize patient treatment. Remission was achieved in a patient with advanced, treatment-refractory AML. Serial sampling from human AML patients coupled with molecular profiling and drug sensitivity testing may shed light on clonal progression of disease, and the molecular events underlying drug response.nnCitation 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 4580. doi:1538-7445.AM2012-4580

Abstract 4850: Paired-end RNA-sequencing based identification of 24 novel fusion genes in breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnFusion genes have recently been described in common solid tumors, revealing an important class of mutations that contribute to cancer development and progression. Next-generation sequencing technologies allow systematic characterization of cancer cell transcriptomes, including the fusion genes resulting from underlying chromosomal translocations. Using paired-end RNA-sequencing in conjunction with high-resolution chromosomal copy number analyses we characterized 24 novel and 3 previously known fusion genes in breast cancer cell lines. An improved bioinformatic approach allowed fusion gene validation with a 95% success rate by RT-PCR, Sanger sequencing and FISH. Among the identified fusions, partner genes were found to contribute both promoters (5′UTR; e.g. TATDN1-GSDMB), coding sequences (e.g. ACACA-STAC2) as well as 3′UTRs (e.g. CSE1L-ENSG00000236127). Most fusion genes (82%) were associated with copy number transitions, such as high level amplifications at e.g. 8q21, 17q12, 17q23 and 20q13, implying that fusion gene formation may contribute to the selective advantage provided by copy number amplifications. Although no prevalent single fusion gene was detected, the functional importance of the multiple fusion genes is supported by several lines of evidence. First, some of the fusion partner genes were expressed exclusively as fusion transcripts, e.g. TATDN1-GSDMB, indicating that the fusion event lead to the activation of a latent gene. Second, several fusion gene partners, including ACACA, NOTCH1 and RARA, have been previously found to be involved in oncogenic fusions in leukemias and lymphomas. Third, functional analysis using RNAi-mediated knockdown suggested a potential role for two fusion genes in controlling cancer cell proliferation. In conclusion, using RNA-sequencing together with improved bioinformatics and genomic copy number profiling, we have discovered multiple novel fusion genes in breast cancer, which may provide new insights on breast cancer, and provide therapeutic and diagnostic clues.nnCitation 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 4850. doi:10.1158/1538-7445.AM2011-4850

Abstract 3977: Systematic analysis of microRNAs targeting the androgen receptor in prostate cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnAndrogen receptor (AR) is expressed in all stages of prostate cancer progression and its levels increase in the castration-resistant tumors. However, the mechanisms regulating AR expression remain poorly understood. Here, our aim was to determine the role of miRNAs in regulating AR protein. To this end, we performed a systematic gain-of function screen for 1129 miRNA molecules in prostate cancer cell lines and quantified changes in AR protein content using protein lysate microarrays. We identified 71 unique miRNAs that influenced the level of AR in LNCaP, LAPC-4, 22Rv1, CWR-1R and MDA-PCa-2B cells. Our RNA-sequencing data showed that the 3’UTRs of many genes, including the AR, are much longer than currently used by target prediction programs. Our analyses predicted that the majority of the miRNA regulation of AR would occur through the extended 6 kb 3’UTR. 3’UTR-binding assays validated 13 miRNAs (miR-135b, miR-185, miR-297, miR-299-3p, miR-34a, miR-34c, miR-371-3p, miR-421, miR-449a, miR-449b, miR-634, miR-654-5p, and miR-9) as direct AR regulators. Fifteen AR down-regulating miRNAs decreased androgen-induced proliferation of prostate cancer cells in vitro. Negative correlation of miR-34a and miR-34c expression with AR levels was also found in clinical prostate cancers. To conclude, our results demonstrate that miRNAs interacting with the long 3’UTR of the AR gene are important regulators of AR protein levels. This could be applied for developing novel therapeutic strategies to inhibit AR function and androgen-dependent cell growth.nnCitation 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 3977. doi:10.1158/1538-7445.AM2011-3977