8p11-p12 Amplicon Research Articles

Discovery and Characterization of BAY-184: A New Potent and Selective Acylsulfonamide-Benzofuran In Vivo-Active KAT6AB Inhibitor.

KAT6A and KAT6B genes are two closely related lysine acetyltransferases that transfer an acetyl group from acetyl coenzyme A (AcCoA) to lysine residues of target histone substrates, hence playing a key role in chromatin regulation. KAT6A and KAT6B genes are frequently amplified in various cancer types. In breast cancer, the 8p11-p12 amplicon occurs in 12-15% of cases, resulting in elevated copy numbers and expression levels of chromatin modifiers like KAT6A. Here, we report the discovery of a new acylsulfonamide-benzofuran series as a novel structural class for KAT6A/B inhibition. These compounds were identified through high-throughput screening and subsequently optimized using molecular modeling and cocrystal structure determination. The final tool compound, BAY-184 (29), was successfully validated in an in vivo proof-of-concept study.

Abstract A044: The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer

Abstract KAT6 paralogs KAT6A (MOZ; MYST3) and KAT6B (MORF; MOZ2; MYST4) are histone acetyltransferase (HAT) enzymes that acetylate histone H3 and thereby epigenetically regulate gene transcription by altering chromatin structure. Dysregulation of KAT6 activity (gene amplification, overexpression, fusion, mutation, etc.) is observed in breast and other cancers. In several subtypes of breast cancer, KAT6A is amplified as part of the 8p11-p12 amplicon and/or is overexpressed in 11 to 15% of overall breast cancer population. Overexpression of KAT6 correlates with worse clinical outcome in ER+/HER2− breast cancers. Here we present the discovery of novel and potent KAT6 inhibitors with robust in vitro and in vivo anti-tumor efficacy and high selectivity against other KAT family members KAT5 and KAT8. Our lead KAT6 inhibitor compounds demonstrate strong anti-proliferative effects in ER positive breast cancer cell lines with KAT6 dysregulation while demonstrating robust target engagement biomarker activity (inhibition of histone H3 acetylation at lysine residue 23). In addition, these compounds exhibit desirable ADME and pharmacokinetic properties including good oral bioavailability in tested species, supporting potential once daily dosing in humans. The lead compounds also demonstrate enhanced in vitro cellular efficacy in breast cancer cell lines when combined with OP-1250, a complete ER antagonist (CERAN), and CDK4/6 inhibitors, illustrating their combination potential. In addition, these lead compounds demonstrate excellent dose-dependent anti-tumor activity resulting in tumor growth inhibition and tumor regression in a KAT6A amplified ER+ breast cancer xenograft model, highlighting their potential as a novel therapy in ER+ breast cancer. Additional nonclinical efficacy testing, and safety profiling of these lead compounds is currently underway to select a clinical candidate. Citation Format: Gopinath S. Palanisamy, Chandregowda Venkateshappa, Megha Goyal, Susanna A. Barratt, Brian R. Hearn, Girish Daginakatte, Aravind A B, Samiulla D S, Kalisankar Bera, Sangeetha S, Leena Khare, Alison D. Parisian, Dirk A. Heerding, Raymond A. Ng, Cyrus L. Harmon, Susanta Samajdar, Murali Ramachandra, David C. Myles. The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A044.

Somatic rearrangements causing oncogenic ectodomain deletions of FGFR1 in squamous cell lung cancer.

The discovery of frequent 8p11-p12 amplifications in squamous cell lung cancer (SQLC) has fueled hopes that FGFR1, located inside this amplicon, might be a therapeutic target. In a clinical trial, only 11% of patients with 8p11 amplification (detected by FISH) responded to FGFR kinase inhibitor treatment. To understand the mechanism of FGFR1 dependency, we performed deep genomic characterization of 52 SQLCs with 8p11-p12 amplification, including 10 tumors obtained from patients who had been treated with FGFR inhibitors. We discovered somatically altered variants of FGFR1 with deletion of exons 1-8 that resulted from intragenic tail-to-tail rearrangements. These ectodomain-deficient FGFR1 variants (ΔEC-FGFR1) were expressed in the affected tumors and were tumorigenic in both in vitro and in vivo models of lung cancer. Mechanistically, breakage-fusion-bridges were the source of 8p11-p12 amplification, resulting from frequent head-to-head and tail-to-tail rearrangements. Generally, tail-to-tail rearrangements within or in close proximity upstream of FGFR1 were associated with FGFR1 dependency. Thus, the genomic events shaping the architecture of the 8p11-p12 amplicon provide a mechanistic explanation for the emergence of FGFR1-driven SQLC. Specifically, we believe that FGFR1 ectodomain-deficient and FGFR1-centered amplifications caused by tail-to-tail rearrangements are a novel somatic genomic event that might be predictive of therapeutically relevant FGFR1 dependency.

A NSD3-targeted PROTAC suppresses NSD3 and cMyc oncogenic nodes in cancer cells

Nuclear receptor binding SET domain protein 3 (NSD3), a gene located within the 8p11-p12 amplicon frequently detected in human cancers, encodes a chromatin modulator and an attractive onco-target. However, agents that effectively suppress NSD3-mediated oncogenic actions are currently lacking. We report the NSD3-targeting proteolysis targeting chimera (PROTAC), MS9715, which achieves effective and specific targeting of NSD3 and associated cMyc node in tumor cells. MS9715 is designed by linking BI-9321, a NSD3 antagonist, which binds NSD3's PWWP1 domain, with an E3 ligase VHL ligand. Importantly, MS9715, but not BI-9321, effectively suppresses growth of NSD3-dependent hematological cancer cells. Transcriptomic profiling demonstrates that MS9715, but not BI-9321, effectively suppresses NSD3-and cMyc-associated gene expression programs, resembling effects of the CRISPR-Cas9-mediated knockout of NSD3. Collectively, these results suggest that pharmacological degradation of NSD3 as an attractive therapeutic strategy, which co-suppresses NSD3- and cMyc-related oncogenic nodes, is superior to blocking the PWWP1 domain of NSD3.

Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3.

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.

Eukaryotic initiation factor 4E-binding protein as an oncogene in breast cancer

BackgroundEukaryotic Initiation Factor 4E-Binding Protein (EIF4EBP1, 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy.MethodsHere we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1.ResultsUsing a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes.ConclusionsThese results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation.

Genome-wide multi-omics profiling of the 8p11-p12 amplicon in breast carcinoma.

Genomic instability contributes to the neoplastic phenotype by deregulating key cancer-related genes, which in turn can have a detrimental effect on patient outcome. DNA amplification of the 8p11-p12 genomic region has clinical and biological implications in multiple malignancies, including breast carcinoma where the amplicon has been associated with tumor progression and poor prognosis. However, oncogenes driving increased cancer-related death and recurrent genetic features associated with the 8p11-p12 amplicon remain to be identified. In this study, DNA copy number and transcriptome profiling data for 229 primary invasive breast carcinomas (corresponding to 185 patients) were evaluated in conjunction with clinicopathological features to identify putative oncogenes in 8p11-p12 amplified samples. Illumina paired-end whole transcriptome sequencing and whole-genome SNP genotyping were subsequently performed on 23 samples showing high-level regional 8p11-p12 amplification to characterize recurrent genetic variants (SNPs and indels), expressed gene fusions, gene expression profiles and allelic imbalances. We now show previously undescribed chromothripsis-like patterns spanning the 8p11-p12 genomic region and allele-specific DNA amplification events. In addition, recurrent amplification-specific genetic features were identified, including genetic variants in the HIST1H1E and UQCRHL genes and fusion transcripts containing MALAT1 non-coding RNA, which is known to be a prognostic indicator for breast cancer and stimulated by estrogen. In summary, these findings highlight novel candidate targets for improved treatment of 8p11-p12 amplified breast carcinomas.

Abstract P4-04-11: The 8p11-p12 amplicon oncogene ASH2L regulates expression of genes involved in tumorigenic processes and response to palbociclib via promoter H3K4me3

Abstract Background. Amplification of the 8p11-p12 region, occurring in 10-30% of luminal tumors, is associated with poor prognosis and resistance to endocrine therapy. The 8p11-p12 amplicon is heterogeneous and contains several validated oncogenes, including NSD3, ZNF703, and FGFR1. ASH2L, an epigenetic regulator of chromatin, is a member of the trithorax group of proteins that promote transcription, primarily through tri-methylation of lysine 4 on histone H3 (H3K4me3) in the promoter region of target genes. The 8p11-p12 amplicon has been well-characterized previously in the SUM44 cell line and the role of overexpressed oncogenes such as NSD3 has been demonstrated. We therefore selected this cell line for further investigation of ASH2L function in the context of amplification of this oncogene, and its relationship with response to CDK inhibitors. Methods. We assessed cell growth following knockdown of ASH2L with shRNA constructs compared to LacZ control in MCF7 and in the amplicon-bearing SUM44, CAMA1, and SUM52 cell lines. To assess the influence of ASH2L expression on promoter H3K4me3, we performed chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) utilizing an H3K4me3 antibody in control SUM44 cells and following shRNA-mediated knockdown of ASH2L. Three biological replicates were prepared and the samples sequenced simultaneously. We treated LacZ control and ASH2L knockdown SUM44 and MCF7 cells with increasing doses of palbociclib and assessed cell growth over 7 days. From the set of genes that lost promoter H3K4me3 upon ASH2L knockdown and were downregulated in response to knockdown of ASH2L, NSD3, and ESR1, we selected 5 genes (FBXO5, EZH2, TTK, CCNE2, and BUB1) that were downregulated in response to treatment with palbociclib according to comparative toxicogenomics database data. Results. Knockdown of ASH2L reduced cell proliferation, validating that ASH2L influences cell growth regardless of amplification status. From ChIP-seq data, we found ASH2L robustly regulates H3K4me3 at the promoter region of itself and other genes of the amplicon, and genes pertaining to cell cycle and proliferation, such as EZH2 and WDR5. To determine the influence of ASH2L-regulated promoter H3K4me3 on transcription, we compared the gene set that lost promoter H3K4me3 upon ASH2L knockdown to the genes that were downregulated at the mRNA level upon knockdown of ASH2L by shRNA. This analysis identified 438 genes that were directly transcriptionally regulated by ASH2L, including cell cycle processes, palbociclib response, EZH2-regulated genes, estradiol-upregulated genes, and ESR1 interactions. SUM44 cells were more resistant to palbociclib than MCF7, and knockdown of ASH2L rendered these cells more resistant yet. Expression of FBXO5, EZH2, TTK, CCNE2, and BUB1, was reduced in the palbociclib-sensitive MCF7 cell line, however SUM44 cells did not demonstrate a reduction in transcript level for these genes after palbociclib treatment. Conclusion. We identified a suite of genes with ASH2L-dependent promoter H3K4me3 that are transcriptionally downregulated upon ASH2L knockdown and discovered that these genes are predicted to be implicated in many cell cycle processes as well as response to palbociclib. Citation Format: Giordano A, Mills JN, Hazard SE, Liu Y, Britten CD, Wilson RC, Chung D, Hardiman G, Ethier SP. The 8p11-p12 amplicon oncogene ASH2L regulates expression of genes involved in tumorigenic processes and response to palbociclib via promoter H3K4me3 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-11.

Abstract P3-04-07: Novel genetic features associated with 8p11-p12 amplification in breast carcinoma

Abstract Background: Genome instability contributes to the neoplastic phenotype by promoting gene loss and duplications, which in turn can have a detrimental effect on patient outcome by inactivating tumor suppressor genes or hyperactivating oncogenes. In breast carcinoma, DNA amplification of the 8p11-p12 genomic region has been associated with tumor progression and poor prognosis. The aim of this study was to characterize recurrent genetic features (other than DNA amplification) associated with 8p11-p12 amplification in breast carcinoma. Methods: DNA copy number profiling data for 229 primary invasive breast carcinomas (corresponding to 185 patients diagnosed in Western Sweden between 1988 and 1999) were evaluated to identify 8p11-p12 amplified cases. Illumina paired-end whole transcriptome sequencing (RNA-seq) and whole-genome SNP genotyping were subsequently performed on 23 breast carcinomas harboring high-level regional 8p11-p12 amplification to characterize recurrent genetic variants (SNPs and indels), expressed gene fusions, gene expression profiles and allelic imbalances. The 23 samples were stratified into the molecular subtypes, resulting in 16 Luminal B/HER2-, two Luminal B/HER2+, four HER2/ER-, and one Basal-like sample. The Cancer Genome Atlas (TCGA) RNA-seq data for 10 primary breast carcinomas lacking the 8p11-p12 amplicon (SNP segmented mean < 0.4) were used as controls. Gene fusions were validated using dual-color fluorescence in situ hybridization (FISH) with co-hybridized biotin-16-dUTP and dioxigenin-11-dUTP labeled bacterial artificial chromosome (BAC) probes. Results: Here, we report that despite the high number of gene fusions (133±31 (±SEM)) and exonic variants (411±16) identified per tumor, few gene fusions (n=46) and exonic variants (n=11) spanned the 8p11-p12 genomic region. Gene fusions predominantly contained at least one fusion partner spanning non-coding RNAs (ncRNAs; 86%), in particular MALAT1, which is induced by estrogen and of prognostic value in breast cancer. The majority of fusion breakpoints were associated with DNA copy number gains and losses, as well as, extensive intratumoral heterogeneity for specific fusion events. Intriguingly, novel 8p11-p12 amplification-specific genetic variants (HIST1H1E frameshift insertion, UQCRHL nonsynonymous SNV, MTUS1 frameshift insertion, NPIPA5 frameshift deletion) were identified that also resulted in mutation-dependent changes in gene expression levels. Conclusions: Taken together, these findings have provided further insight into the genetic landscape of 8p11-p12 amplified breast carcinomas, including novel gene fusions and genetic variants. However, further studies are required to develop effective strategies to target 8p11-p12 amplification in breast carcinoma. Citation Format: Parris TZ, Biermann J, Engqvist H, Werner Rönnerman E, Truvé K, Nemes S, Forssell-Aronsson E, Solinas G, Kovács A, Karlsson P, Helou K. Novel genetic features associated with 8p11-p12 amplification in breast carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-07.

Amplification of WHSC1L1 regulates expression and estrogen-independent activation of ERα in SUM-44 breast cancer cells and is associated with ERα over-expression in breast cancer

The 8p11-p12 amplicon occurs in approximately 15% of breast cancers in aggressive luminal B-type tumors. Previously, we identified WHSC1L1 as a driving oncogene from this region. Here, we demonstrate that over-expression of WHSC1L1 is linked to over-expression of ERα in SUM-44 breast cancer cells and in primary human breast cancers. Knock-down of WHSC1L1, particularly WHSC1L1-short, had a dramatic effect on ESR1 mRNA and ERα protein levels. SUM-44 cells do not require exogenous estrogen for growth in vitro; however, they are dependent on ERα expression, as ESR1 knock-down or exposure to the selective estrogen receptor degrader fulvestrant resulted in growth inhibition. ChIP-Seq experiments utilizing ERα antibodies demonstrated extensive ERα binding to chromatin in SUM-44 cells under estrogen-free conditions. ERα bound to ERE and FOXA1 motifs under estrogen-free conditions and regulated expression of estrogen-responsive genes. Short-term treatment with estradiol enhanced binding of ERα to chromatin and influenced expression of many of the same genes to which ERα was bound under estrogen-free conditions. Finally, knock-down of WHSC1L1 in SUM-44 cells resulted in loss of ERα binding to chromatin under estrogen-free conditions, which was restored upon exposure to estradiol. These results indicate the SUM-44 cells are a good model of a subset of luminal B breast cancers that have the 8p11-p12 amplicon, over-express WHSC1L1, and over-express ERα, but are independent of estrogen for binding to chromatin and regulation of gene expression. Breast cancers such as these, that are dependent on ERα activity but independent of estradiol, are a major cause of breast cancer mortality.

Abstract P6-03-04: Amplification of the WHSC1L1 oncogene regulates expression and estrogen-independent activation of ERα in SUM-44 breast cancer cells and is associated with ERα over expression in breast cancer

Abstract The 8p11-p12 amplicon occurs in approximately 15% of breast cancers and occurs almost exclusively in aggressive luminal B type tumors. Our lab and other labs have identified the WHSC1L1 oncogene as a driving oncogene from this region with potent transforming activity. In the present studies, we found that over expression of WHSC1L1 is linked to over expression of the ESR1 and ERα protein in the SUM-44 breast cancer cell line, and also in primary human breast cancer specimens. Knockdown of WHSC1L1, and particularly the short isoform of WHSC1L1, had a dramatic effect on ESR1 mRNA and ERα protein levels. SUM-44 cells do not require exogenous estrogen for continuous growth in vitro; however these cells are dependent on ERα expression as determined from ESR1 knockdown experiments, and potent growth inhibition and ERα degradation following exposure to the selective estrogen receptor degrader (SERD) fulvestrant. ChIP-Seq experiments utilizing ERα antibodies demonstrated potent ERα binding to chromatin in SUM-44 cells under estrogen-free conditions. ERα bound to ERE and FOXA1 binding motifs under estrogen-free conditions and regulated expression of number of well-known estrogen responsive genes. Short term treatment with estradiol enhanced binding of ERα to chromatin and influenced expression of many of the same genes expressed and to which ERα was bound under estrogen-free conditions. Finally, knockdown of WHSC1L1 in SUM-44 cells resulted in loss of ERα binding to chromatin under estrogen-free conditions; however treatment estradiol restored ERα binding to chromatin at key estrogen-response elements and genes. These results indicate the SUM-44 cells are a good model for a subset of luminal B breast cancers that have the 8p11-p12 amplicon, over express the WHSC1L1 oncogene, and over express ERα that is independent of estrogen for binding to chromatin and regulation of gene expression. Dependence on ERα activity for growth and survival of breast cancer cells but independence of estradiol is a major cause of breast cancer mortality as such cells become non-responsive to current hormonally based therapies. Citation Format: Ethier SP, Irish J, Mills J, Ivey B, Hardiam G, Wilson R, Domkowski A, Guest S. Amplification of the WHSC1L1 oncogene regulates expression and estrogen-independent activation of ERα in SUM-44 breast cancer cells and is associated with ERα over expression in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-04.

Abstract 5135: A chromatin modifier from the 8p11 amplicon in luminal breast cancer

Abstract The chromosome 8p11-p12 amplicon is present in 12-15% of breast cancers, resulting in an increase in both copy number and expression of several chromatin modifiers in these tumors, including KAT6A, WHSC1L1, and ASH2L. Previous analyses in SUM52 breast cancer cells showed amplification and over-expression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. Also known as MYST3, KAT6A is a histone acetyltransferase (HAT) with intrinsic HAT activity towards itself and lysine residues on histone 2B, histone 3, and histone 4. Previously identified as a fusion partner with CREB binding protein (CREBBP) in acute myeloid leukemia, KAT6A is amplified in 8.7% of invasive breast cancers according to the Cancer Genome Atlas (TCGA). Knockdown of KAT6A with several shRNAs in SUM52 cells, a luminal breast cancer cell line harboring the 8p11 amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A, nor did other breast cancer cell lines without KAT6A amplfied. Interestingly, SUM52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to non-silencing controls, suggesting a possible role for KAT6A in breast cancer stem cell activity and self-renewal. Further analysis of cell surface markers CD24 and CD44 in KAT6A knockdown cells revealed an increase in cells double positive for CD24 and CD44 expression, a characteristic of increased cell differentiation. Previous work from our laboratory identified FGFR2 as a driving oncogene in SUM52 cells. While FGFR inhibition can completely arrest the growth of these cells, it is completely reversible. Thus, exposure of SUM52 cells to FGFR inhibitors does not significantly affect the colony forming ability of the cells. By contrast, the colony forming efficiency of SUM52 KAT6A knockdown cells in the presence of the FGFR inhibitor PD170374 was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a potential novel oncogene in breast cancers bearing the 8p11 amplicon and that it interacts with FGFR2 to regulate expression of transformed phenotypes. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers. Citation Format: Brittany Turner Ivey, Steve Guest, Stephen P. Ethier. A chromatin modifier from the 8p11 amplicon in luminal breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5135. doi:10.1158/1538-7445.AM2014-5135

Abstract 5148: Overexpression of histone methyltransferase WHSC1L1 regulates expression of ER-alpha in SUM44 breast cancer cells

Abstract The WHSC1L1 gene is amplified and overexpressed in ∼12% of breast cancers, and is thought to be a driving oncogene when amplified and overexpressed, however the mechanism of transformation is not known. SUM44 cells harbor an amplification of WHSC1L1 as part of the 8p11-p12 amplicon, and express high levels of both the long and short isoforms of the gene. The short isoform, containing only a PWWP domain and no catalytic SET domain, is the major species present in SUM44 cells. To create a model to study the effect of WHSC1L1 on gene regulation, SUM44 cells were lentivirally transduced with either a control shRNA for LacZ (shLacZ), an shRNA specific for both isoforms of WHSC1L1 (shWHtotal), or a short isoform-specific shRNA (shWHshort). Cell growth was significantly reduced in shWHtotal cells compared to shLacZ, however, the greatest decrease in growth was observed in cells transduced with the short isoform-specific shRNA. To identify genes transcriptionally regulated by WHSC1L1, we performed genome-wide expression profiling of SUM44 breast cancer cells transduced with either shWHtotal or shLacZ. A total of 5185 genes were found to be differentially expressed in shWHtotal SUM44 cells compared to shLacZ control. Among the 5185 differentially expressed genes, we found ESR1 to be significantly downregulated (-7.3-fold, p < 0.0001) in the shWHtotal cells compared to shLacZ controls. In addition MYCN, which is amplified in SUM-44 cells was reduced by 7.43-fold, (p < 0.004), ERBB3 was reduced by 2.5-fold, (p < 0.0009), and ERBB4, which is highly expressed in SUM-44 cells was reduced by 2.12-fold, (p < 0.00001). Immunoblotting confirmed downregulation of estrogen receptor alpha (ERα) in the shWHtotal compared to shLacZ control. Interestingly, knockdown of WHshort resulted in an even greater decrease of ERα than the shWHtotal knockdown. To determine whether downregulation of ERα affects sensitivity to Tamoxifen, we measured the Tamoxifen LD50 in SUM44 shLacZ and SUM44 shWHtotal cells, and found a 10-fold decrease in Tamoxifen LD50 compared to shLacZ control (3.8nM vs. 35nM). Tamoxifen affected SUM-44 cells in a dose-dependent manner, however 500nM Tamoxifen resulted in complete growth arrest in SUM44 shWHtotal cells, while the cells infected with the shLacZ virus cells grew at the same dose, albeit slowly. Our data indicate that WHSC1L1 regulates ERα expression in SUM44 cells, and WHSC1L1 knockdown increased the cells' sensitivity to Tamoxifen. The decrease in both cell growth and ERα expression in the SUM44 WHshort-specific knockdown cells suggests that the catalytically inactive short isoform of WHSC1L1 may be the isoform driving the oncogenic activity of WHSC1L1 amplification, perhaps through a dominant-negative interaction with the long isoform of WHSC1L1. Data showing increases in global H3K36 methylation levels following knock down of WHSC1L1 are consistent with this hypothesis. Citation Format: Jonathan Curtis Irish, Alexandria Rutkovsky, Stephen P. Ethier. Overexpression of histone methyltransferase WHSC1L1 regulates expression of ER-alpha in SUM44 breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5148. doi:10.1158/1538-7445.AM2014-5148

KAT6A, a Chromatin Modifier from the 8p11-p12 Amplicon is a Candidate Oncogene in Luminal Breast Cancer

The chromosome 8p11-p12 amplicon is present in 12% to 15% of breast cancers, resulting in an increase in copy number and expression of several chromatin modifiers in these tumors, including KAT6A. Previous analyses in SUM-52 breast cancer cells showed amplification and overexpression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. KAT6A is a histone acetyltransferase previously identified as a fusion partner with CREB binding protein in acute myeloid leukemia. Knockdown of KAT6A in SUM-52 cells, a luminal breast cancer cell line harboring the amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A. SUM-52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to controls, suggesting a possible role for KAT6A in self-renewal. Previous data from our laboratory identified FGFR2 as a driving oncogene in SUM-52 cells. The colony forming efficiency of SUM-52 KAT6A knockdown cells in the presence of FGFR inhibition was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a novel oncogene in breast cancers bearing the 8p11-p12 amplicon. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers.

Abstract 2964: Epigenetic mechanisms of transformation by WHSC1L1 in breast cancer cells with the 8p11-p12 amplicon.

Abstract An amplified region of chromosome 8, the 8p11-p12 amplicon, is observed in ∼15% of breast cancers. This region contains 55 genes, of which 22 have been identified as candidate oncogenes based on higher than normal copy number and associated elevated expression levels in breast tumor cells compared to normal breast epithelial cells. Our lab previously identified the short isoform of one amplicon gene, Wolf Hirschhorn Syndrome Candidate 1-Like 1 (WHSC1L1), to be a potent inducer of transformation when over expressed in MCF10A breast epithelial cells in vitro. WHSC1L1 is a member of the NSD family of methyltransferases, and has been shown to methylate lysines 4, 27, and 36 of the histone H3 subunit, however, the potently transforming short isoform (WHshort) lacks the catalytic SET domain. Our lab has shown that over expression of WHshort in the immortalized human breast epithelial cell line MCF10A causes a significant increase in proliferation in insulin-free culture conditions, and the ability to form colonies in soft agar. In SUM44 cells, an ER+ breast cancer cell line harboring the 8p11-p12 amplicon, shRNA knockdown of WHSC1L1 resulted in growth arrest. The WHSC1L1 family member NSD1 has been shown to act as an oncogene in acute myelogenous leukemia by blocking differentiation in developing lymphocytes through H3K36me3-mediated inhibition of the polycomb repressor complex, resulting in constitutive activation of the HOXA gene family. While WHSC1L1 may work by a similar mechanism, little is known about the specific substrates of WHSC1L1, or even whether its oncogenic activity is caused by histone methylation changes. Knockdown by shRNA of WHSC1L1 in SUM44 cells resulted in a significant reduction in transcript levels of several genes important in breast cancer cell proliferation, including ESR1, MYCN (amplified in SUM-44), ERBB3, and ERBB4. Interestingly, Carroll et al. recently reported that ER+ breast tumors can be stratified by gene expression signature into poor outcome and good outcome groups, and that ER-alpha binding site patterns on chromatin were altered in the two groups. In this study, WHSC1L1 was part of the ER-regulated gene signature of the poor outcome group. Therefore, we hypothesize that WHSC1L1 may influence histone H3K36me3 marks at specific gene loci, leading to altered ER-alpha binding that is associated with the expression signatures of poor-outcome ER+ breast cancers. To investigate chromatin changes as a result of changes in WHSC1L1 expression, we have accomplished shRNA knockdown of WHSC1L1 in SUM44 cells using GIPZ and TRIPZ lentiviral vectors. Using these cell lines, we are investigating changes in H3K36me3 marks and ER-alpha binding patterns by chromatin immunoprecipitation followed by sequencing (ChIP-seq). These experiments will help to elucidate the interaction of WHSC1L1 and ER in regulated gene expression in breast cancer cells with the 8p11 amplicon. Citation Format: Jonathan C. Irish, Gang Liu, Stephen P. Ethier. Epigenetic mechanisms of transformation by WHSC1L1 in breast cancer cells with the 8p11-p12 amplicon. [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 2964. doi:10.1158/1538-7445.AM2013-2964

Abstract 2190: Epigenetic mechanisms of transformation for the NSD3 oncogene in human breast cancer cells with the 8p11-p12 amplicon

Abstract An amplified region of chromosome 8, the 8p11-p12 amplicon, is observed in 12-15% of breast cancers. This region contains 55 genes, of which 22 have been identified as candidate oncogenes based on the presence of higher than normal copy number and associated elevated expression levels in breast tumor cells compared to normal breast epithelial cells. Our lab previously identified the short isoform of one amplicon gene, nuclear SET domain-containing protein 3 (NSD3s), to be the most potent inducer of transformation to a neoplastic phenotype of all genes on the 8p11-p12 amplicon when over expressed in MCF10A breast epithelial cells in vitro. NSD3 is a member of the NSD family of methyltransferases, and has been shown to methylate lysine 4, 27, and 36 on the H3 subunit of histones, however the potently transforming short isoform lacks the catalytic SET domain. Our lab has shown that over expression of NSD3s in the immortalized human breast epithelial cell line MCF10A causes a significant increase in proliferation in insulin-free culture conditions, and the ability to form colonies in soft agar. In SUM44 cells, a breast cancer cell line with the 8p11-p12 amplicon, shRNA knockdown of NSD3s resulted in decreased proliferation. Immunoblotting with anti-H3K4me2 antibody shows MCF10A cells have higher global levels of H3K4me2 than SUM 44 cells, and over expression of NSD3s in MCF10A cells reduces global H3K4me2 levels. Other NSD family members have been shown to act as oncogenes in certain leukemias by blocking differentiation in developing lymphocytes through H3K36me3-mediated inhibition of the polycomb repressor complex, resulting in constitutive activation of the HOX gene family. This epigenetic dysregulation of HOX through the NSD1 oncogene results in acquisition of a stem-like phenotype in these cells that is essential for the development of these leukemias. We predict a similar mechanism in breast cancers that over express NSD3. Genome-wide expression analysis has shown that IRX3, a gene expressed in cells of the ectoderm lineage during differentiation, is upregulated in cells over expressing NSD3s. Based on previous findings with other NSD family genes we predict that genes involved in pluripotency or stemness will be targets of NSD3s activity in breast cancers with the 8p11-p12 amplicon. To investigate whether over expression of NSD3s induces a stem-like phenotype in MCF10A cells, we have measured the expression level of breast cancer stem cell surface markers CD44 and CD24 by flow cytometry, and found that NSD3s over expression results in a sub-population of cells that has higher CD44 expression than parental MCF10A, while maintaining low CD24 expression. These data suggest that NSD3s may act as a driving oncogene in breast cancer by altering histone methylation patterns in a way that promotes the expression of genes that result in the acquisition of stem-like phenotypes. 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 2190. doi:1538-7445.AM2012-2190

Genetic characterization of breast cancer and implications for clinical management

Breast cancer is a genetic disease caused by the accumulation of mutations in neoplastic cells. In the last few years, high-throughput microarray-based molecular analysis has provided increasingly more coherent information about the genetic aberrations in breast cancer. New biomarkers and molecular techniques are slowly becoming part of the diagnostic and prognostic armamentarium available for pathologists and oncologists to tailor the therapy for breast cancer patients. In this review, we will focus on the contribution of breast cancer somatic genetics to our understanding of breast cancer biology and its impact on breast cancer patient management.