8p11-12 Amplicon Research Articles

Abstract PO2-05-08: ISM5043, a novel, selective KAT6 inhibitor for the treatment of advanced and refractory ER+ breast cancer

Abstract Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death among women in the US. Approximately 70% of human breast tumors are estrogen receptor positive (ER+), and endocrine therapy (ET), which inhibits the estrogen signaling pathway, remains the backbone of therapy for ER+ breast cancer. However, ET resistance often develops, and patients eventually experience disease progression. This is a significant clinical challenge and highlights the urgent need for novel therapies that may help overcome the resistance. KAT6A is a histone (lysine) acetyltransferase (HAT) belonging to the MYST family. KAT6A and its paralog KAT6B play important roles in cell cycle progression, neuron stem cell maintenance and hematopoietic development via acetylation of H3K23. Molecular dysregulation of KAT6A, including amplification and fusion, has been reported in many cancers. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in about 10-15% of the ER+ breast cancer population, where it functions as an epigenetic modulator of ER expression, and is associated with a worse clinical outcome. Therefore, inhibition of KAT6A may be a promising therapy for ER+ breast cancer. Here, we evaluated ISM5043, a novel, small molecule KAT6 inhibitor for anti-tumor effects in ER+/HER2- breast cancer cell lines and animal models. ISM5043 demonstrated potent inhibitory activity against KAT6A (IC50, 8 nM) and KAT6B (IC50, 16 nM) with selectivity over KAT7 (IC50, 344 nM) and other HATs (IC50 >2000 nM). The compound showed robust, dose-dependent, anti-proliferation activity (IC50 < 10 nM) in multiple ER+/HER2- breast cancer cell lines with KAT6A amplification, in accordance with its suppression of H3K23 acetylation. Investigation of the mechanism of action revealed that treatment with ISM5043 resulted in the downregulation of the expression of ERα in these breast cancer cells. In the ZR-75-1 (ER+, HER2-) xenograft model, ISM5043 demonstrated robust and dose-dependent anti-tumor activity, as monotherapy, with Tumor Growth Inhibition (TGI) between 80~110% at doses ranging from 0.3 to 10 mg/kg, QD. ISM5043 (0.3 mg/kg, QD), in combination with tamoxifen (20 mg/kg, QD) exhibited synergistic effect against tumor growth, with a Q value of 1.25 based on Jin’s equation. Notably, ISM5043 displayed a strong anti-tumor effect (TGI of 67.7% and 83.6% at 3 and 10 mg/kg QD, respectively) as monotherapy, in a patient-derived ER+/HER2- breast cancer xenograft model representing disease progression following multiple prior lines of treatment (including palbociclib in combination with letrozole). ISM5043 also showed favorable ADME properties as well as good pharmacokinetic and safety profiles. Taken together, these data support the clinical evaluation of ISM5043 as a targeted therapy in patients with advanced and refractory ER+ breast cancer. Citation Format: Xin Cai, Xin Cheng, Luoheng Qin, Man Zhang, Junwen Qiao, Supriya Bavadekar, Sujata Rao, Feng Ren. ISM5043, a novel, selective KAT6 inhibitor for the treatment of advanced and refractory ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-05-08.

Abstract 1130: First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation

Abstract KAT6A is a lysine histone acetyltransferase (HAT) of the MYST family of HATs. KAT6A, and its paralog KAT6B, have been shown to acetylate histone H3K23Ac and regulate diverse biological processes, including transcription, cell-cycle progression, stem cell maintenance and development. Molecular dysregulation of KAT6A has been observed in several cancers, including amplifications in breast, lung, ovarian cancer along with oncogenic fusions in AML. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in 10-15% of the patient population, which correlates with a worse clinical outcome in the estrogen receptor+ (ER+) subtype. Here we present identification of a first-in-class potent KAT6A/KAT6B tool inhibitor CTx-648 (PF-9363), that possesses high selectivity versus other MYST family members (KAT7, KAT5, KAT8) and other KATs, demonstrating anti-tumor activity in breast cancer. Using genetic and pharmacological approaches, we have demonstrated several ER+ breast cancer cell lines including KAT6A amplified and over-expressing models, are dependent on KAT6A enzymatic function. Epigenomic profiling studies using bulk and nascent RNA-seq combined with ATAC-seq revealed CTx-648 leads to downregulation of a specific set of genes involved in ESR1 pathway, cell cycle and stem cell pathways. In vivo target validation studies showed strong anti-tumor activity of CTx-648 in several ER+ breast cancer cell line and patient-derived xenograft models, including models harboring endocrine therapy resistance ESR1 mutations, highlighting promise for this novel therapy in ER+ breast cancer population. Based on the strength of the pre-clinical data, a selective KAT6 inhibitor (PF-07248144) is now commencing a Phase 1 clinical study in Advanced or Metastatic Solid Tumors. Citation Format: Shikhar Sharma, Jay Chung, Sean Uryu, Amanda Rickard, Natalie Nady, Showkhin Khan, Zhenxiong Wang, Yong Zhang, Haikuo Zhang, Pei-Pei Kung, Eric Greenwald, Karen Maegley, Patrick Bingham, Hieu Lam, Ylva E. Bozikis, Hendrik Falk, Elizabeth Allan, Vicky M. Avery, Miriam S. Butler, Michelle A. Camerino, Catalina Carrasco-Pozo, Susan A. Charman, Melissa J. Davis, Mark A. Dawson, Dawson Sarah-Jane, Melanie de Silva, Matthew L. Dennis, Olan Dolezal, Rachel Lagiakos, Geoffrey J. Lindeman, Laura MacPherson, Stewart Nuttall, Thomas S. Peat, Bin Ren, Alexandra E. Stupple, Elliot Surgenor, Chin Wee Tan, Tim Thomas, Jane E. Visvader, Anne K. Voss, Francois Vaillant, Karen L. White, James Whittle, Yuqing Yang, Soroor Hediyeh-Zadeh, Paul A. Stupple, Ian P. Street, Brendon J. Monahan, Thomas Paul. First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1130.

Elevated NSD3 histone methylation activity drives squamous cell lung cancer.

Amplification of chromosomal region 8p11-12 is a frequent genetic alteration implicated in the etiology of lung squamous cell carcinoma (LUSC)1-3. FGFR1 (fibroblast growth factor receptor 1) is the main candidate driver within this region4. However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful5. Here we identify the H3K36 methyltransferase NSD3 (nuclear receptor binding SET domain protein 3), an 8p11-12-localized gene, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased NSD3 expression strongly correlates with its gene amplification. Ablation of NSD3, but not FGFR1, attenuates tumor growth and extends survival in a potent LUSC mouse model. We identify NSD3T1232A as an LUSC-associated variant that increases H3K36 dimethylation (H3K36me2) catalytic activity in vitro and in vivo. Structural dynamic analyses reveal that the T1232A substitution elicits localized mobility changes throughout NSD3’s catalytic domain to relieve auto-inhibition and increase H3 substrate accessibility. NSD3T1232A expression in vivo accelerates tumorigenesis and decreases overall survival in LUSC mouse models. Pathologic H3K36me2 generation by NSD3T1232A rewires the chromatin landscape to promote oncogenic gene expression programming. Further, NSD3’s catalytic activity promotes human tracheobronchial cell transformation and xenograft growth of human 8p11-12-amplified LUSC cell lines. NSD3 depletion in patient-derived xenografts (PDXs) from primary LUSC harboring NSD3 amplification or the NSD3T1232A variant attenuates neoplastic growth. Finally, NSD3-regulated LUSC PDXs are markedly sensitive to bromodomain inhibition (BETi). Together, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to BETi.

Abstract 1835: Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3 (WHSC1L1)

Abstract Amplification within the 8p11-12 locus occurs in approximately 15% of breast cancer (BC) and is associated with poor survival and distant recurrence. Overexpression of many of the genes within the 8p11-12 region can confer a pathological gain of function to breast cells. We and others have previously demonstrated that NSD3 (WHSC1L1), a lysine methyltransferase, can act as a potent transforming oncogene. Predictions by the GISTIC algorithm of the Human Cancer Genome Atlas data suggest that NSD3 is a driving gene within 8p11-12 in multiple tumor types, including BC. We previously showed that NSD3 can regulate the expression of influential genes, such as estrogen receptor alpha (ESR1/ERα). To further explore the role of NSD3 in promoting BC, we generated FVB/N transgenic mice with targeted overexpression of NSD3 in the mammary gland and compared them to matched non-transgenic wild-type (WT) females. We observed that pups nursed by transgenic females were underdeveloped, regardless of genotype. Underdeveloped pups displayed delayed hair growth and eye opening, and were half the weight of pups nursed by WT females. To investigate this phenotype, we characterized thoracic and inguinal mammary glands from virgin, mid-pregnancy, lactating, and post-lactating mice by whole mount and histological analysis. Mammary glands from virgin transgenic females displayed increased branching and terminal bud formation. Alveolar buds from mid-pregnant transgenic glands were more numerous and densely packed. Glands from lactating transgenic females showed large areas in which the alveoli failed to undergo functional differentiation, resulting in a lactation defect. Mammary glands taken post-lactation exhibited areas of ductal and alveolar hyperplasia. At 40 weeks of age, multiple transgenic mice had palpable tumors which were excised at 1 cm3 size. Whole mount and histological analysis of glands from tumor bearing mice commonly demonstrated hyperplasia, dysplasia, and carcinoma in situ, contrary to age-matched WT glands. The areas of ductal dysplasia and carcinoma in situ closely resembled several patterns commonly observed in human breast cancer, including micropapillary, cribriform, and high grade areas of ductal carcinoma in situ. Mammary tumors were analogous to infiltrating ductal carcinomas, many of which were high grade tumors. In summary, overexpression of NSD3 in the mouse mammary gland elicited drastic deformation, inhibited functional differentiation, and caused tumor formation. Continued characterization of this mouse model, the oncogenic role of NSD3, and other mechanistic studies are essential to improve patient outcome in 8p11-12 altered cancers. Citation Format: Alex C. Rutkovsky, Brittany Turner-Ivey, Ericka L. Smith, Laura S. Spruill, Jamie N. Mills, Stephen P. Ethier. Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3 (WHSC1L1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1835. doi:10.1158/1538-7445.AM2017-1835

Abstract 2405: The 8p11 amplicon oncogenes ASH2L and NSD3 alter the epigenomic landscape and provide the foundation for novel application of epigenetic therapy in luminal B breast cancers

Abstract The 8p11-p12 genomic region is amplified in 15% of breast cancers and is associated with poorer prognosis, especially in the luminal B (ER+) subtype. This region harbors several oncogenes, three of which are epigenetic modifiers of chromatin: NSD3 (WHSC1L1) and ASH2L, which are histone methyltransferases, and KAT6A, a histone acetyltransferase. NSD3 preferentially di-methylates histone 3 lysine 36 (H3K36me2) in gene bodies to facilitate elongation, and ASH2L primarily tri-methylates histone 3 lysine 4 (H3K4me3) in promoter regions, which is also associated with active transcription. Previous work in the amplicon-bearing cell line SUM-44 implicated NSD3 in growth, survival, and overexpression and estrogen-independent activation of the estrogen receptor. Current work is focused on elucidating the mechanism behind these transforming features and exploring the epigenomic interactions of 8p11 oncogenes. Chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) using an NSD3 antibody confirmed the presence of this factor in the gene body of ESR1, as well as several other important genes. As NSD3 lacks the ability to bind chromatin directly, it relies on adaptor proteins such as BRD4, a member of the BET bromodomain family that binds acetylated histones such as those generated by KAT6A. Indeed, we have shown that BRD4 binds NSD3 in SUM-44 cells. Similar to our previous findings with NSD3, knockdown of BRD4 and ASH2L reduced cell proliferation and clonogenic potential. To examine the influence of ASH2L on histone methylation in gene promotors, we performed ChIP-seq using an H3K4me3 antibody. The data showed a dramatic decrease in the number of peaks called following knockdown of ASH2L and peaks in the NSD3 promotor disappeared. This finding solidifies the connection between these oncogenes and their cooperative regulation of the epigenome, and therefore the transcriptome, in this cell line, explaining the effects of NSD3 on cell growth and behavior. Amplification and overexpression of oncogenes in the 8p11 region is most prevalent in ER+ breast cancer, treated clinically with endocrine therapies. Patients with poor response to these therapies have few alternatives and often succumb to their disease. This study and continued investigation of 8p11 amplicon oncogenes opens an important new treatment opportunity for these patients: epigenetic therapy. Previous work identifying these patients as candidates for novel anti-estrogen SERD compounds suggests that combination therapy with a BET bromodomain inhibitor could have major therapeutic benefit in a patient group currently without treatment options, thereby improving patient outcomes by a novel mechanism in luminal B breast cancer. Citation Format: Jamie N. Mills, Stephen P. Ethier. The 8p11 amplicon oncogenes ASH2L and NSD3 alter the epigenomic landscape and provide the foundation for novel application of epigenetic therapy in luminal B breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2405. doi:10.1158/1538-7445.AM2017-2405

Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3.

NSD3 has been implicated as a candidate driver oncogene from the 8p11-p12 locus, and we have previously published evidence for its amplification and overexpression in human breast cancer. This aim of this study was to further characterize the transforming function of NSD3 in vivo. We generated a transgenic mouse model in which NSD3 gene expression was driven by the MMTV promoter and expressed in mammary epithelium of FVB mice. Mammary glands were fixed and whole mounts were stained with carmine to visualize gland structure. Mammary tumors were formalin-fixed, and paraffin embedded (FFPE) tumors were stained with hematoxylin and eosin. Pups born to transgenic females were significantly underdeveloped compared to pups born to WT females due to a lactation defect in transgenic female mice. Whole mount analysis of the mammary glands of transgenic female mice revealed a profound defect in functional differentiation of mammary gland alveoli that resulted in the lactation defect. We followed parous and virgin NSD3 transgenic and control mice to 50weeks of age and observed that several NSD3 parous females developed mammary tumors. Whole mount analysis of the mammary glands of tumor-bearing mice revealed numerous areas of mammary hyperplasia and ductal dysplasia. Histological analysis showed that mammary tumors were high-grade ductal carcinomas, and lesions present in other mammary glands exhibited features of alveolar hyperplasia, ductal dysplasia, and carcinoma in situ. Our results are consistent with our previous studies and demonstrate that NSD3 is a transforming breast cancer oncogene.

Abstract 674: WHSC1L1 is a driving oncogene in ERá positive breast cancer

Abstract The 8p11-p12 genomic region is amplified in 15% of breast cancers and is associated with poorer prognosis. This genomic region harbors several oncogenes, including three epigenetic chromatin modifiers: ASH2L, KAT6A, and WHSC1L1. WHSC1L1 is a histone methyltransferase expressed in two isoforms and we have previously demonstrated that this oncogene is involved in overexpression of ERá as well as its activation independent of estrogen signaling in amplicon-bearing SUM44 breast cancer cells. This cell line serves as a model for the subset of breast cancer patients who demonstrate resistance to hormonal therapies in the context of the 8p11 amplicon and WHSC1L1 overexpression. To validate the connection between overexpression of WHSC1L1 and ESR1, we specifically overexpressed the short, non-catalytic isoform of WHSC1L1 in amplicon-bearing cell lines without WHSC1L1 overexpression. Overexpression of WHSC1L1-short resulted in increased expression of ERá in CAMA1 and SUM52 cells, but not in the amplicon null MCF7 cells. To analyze further the oncogenic role of WHSC1L1 in breast cancer, we generated a transgenic mouse model with targeted overexpression of WHSC1L1 in the mammary gland of FVB/N mice. WHSC1L1 transgenic females had normal sized litters, however the pups were profoundly underdeveloped compared to pups born to wild-type females. Pups born to transgenic females displayed delayed hair growth and eye opening and were half the weight of healthy pups nursed by wild-type females. This suggested a lactation deficiency in WHSC1L1-transgenic females. To further investigate this phenotype, mammary glands were harvested for histologic analysis from transgenic and wild-type females at various stages of the reproductive cycle. Mammary glands from virgin WHSC1L1 transgenic females displayed increased branching and terminal bud formation compared to wild-type virgin females. Similarly, alveolar buds of mid-pregnant females were more numerous and densely packed than in wild-type mice. Consistent with a lactation defect, mammary glands from lactating transgenic females showed evidence of decreased alveolar development compared to wild-type females, resulting in mammary glands where half of the gland failed to functionally differentiate. Finally, post-lactation mammary glands from wild-type females underwent involution, whereas glands from transgenic females failed to involute and were hyperplastic. Significantly, we have observed mammary tumor formation in two transgenic female mice thus far. Ongoing studies include further characterization of this novel mouse model, investigation of the effects of WHSC1L1 overexpression on mammary gland development as pertaining to breast cancer tumorigenesis, and the oncogenic role of WHSC1L1 and its effects on ERá. These and other studies are essential for understanding WHSC1L1 and the 8p11 amplicon in ER+ breast cancer, and how this genomic alteration can be exploited to improve patient outcomes. Citation Format: Brittany Turner Ivey. WHSC1L1 is a driving oncogene in ERá positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 674.

Abstract B05: WHSC1L1 and estrogen-independent activation of estrogen receptor-alpha (ERα) in 8p11 amplicon-bearing cell lines

Abstract The 8p11-p12 genomic region is amplified in 15% of breast cancers and 21% of lung squamous cell carcinomas (LSCC) and is associated with poorer prognosis. This genomic region harbors several oncogenes, three of which are epigenetic modifiers of chromatin (WHSC1L1, KAT6A, ASH2L). WHSC1L1 is a histone methyltransferase (HMT) that is expressed in 2 isoforms. The long isoform (WH-long) encompasses the entire coding region and is associated with dimethylation of lysine 36 on histone 3 (H3K36me2) to facilitate transcriptional elongation. The short isoform (WH-short) is produced by alternative splicing at exon 10, resulting in a truncated protein that retains the ability to bind lysine residues but lacks the catalytic SET domain required for methylation. Our studies have shown that the short isoform is consistently more highly expressed than the long isoform, both in breast and lung cancer lines, consistent with data in the Cancer Genome Atlas (TCGA). Upon investigation of the role of WHSC1L1 in regulation of gene transcription, shRNA knockdown of WH-short resulted in reduced expression of several important genes, including CD44, HER4, and ESR1, which encodes estrogen receptor alpha (ERα). SUM-44 cells are an 8p11 amplicon-bearing breast cancer cell line which overexpresses WHSC1L1 and ERα. Knockdown of WHSC1L1 in this cell line resulted in decreased expression of ERα. ERα ChIP-seq in SUM-44 cells, which are ER+ but do not require estrogen for growth, revealed that ERα, which is not mutated or deleted in this cell line, retains the ability to bind to chromatin and participate in gene transcription in the absence of estrogen. Estrogen response elements (EREs) and FOXA1 sites were identified as the most common binding motifs for ERα, and RT-PCR following siRNA knockdown of ESR1 or FOXA1 demonstrated decreased transcription of a panel of genes identified as bound by ERα. Knockdown of WHSC1L1 abolished the ability of ERα to bind chromatin in the absence of estrogen. Despite estrogen-independence, shRNA knockdown of ESR1 in this cell line demonstrated that these cells are indeed dependent on ERα for growth and survival. Similarly, treatment with Fulvestrant, a selective estrogen receptor degrader (SERD), resulted in downregulation of ERα protein and inhibition of growth in SUM-44 cells. Together, these results indicate that overexpression of WHSC1L1 is associated with ERα overexpression, leading to several downstream changes in gene expression dependent upon ERα and FOXA1 but independent of estrogen signaling. Ongoing studies in the lab are focused on identifying the alterations in gene expression directly due to WHSC1L1 and its potential epigenetic actions on transcriptional regulation as opposed to those due to the transcription factor ERα as an indirect result of WHSC1L1 overexpression. These mechanistic studies are essential to understanding the role of the oncogenes on the 8p11 amplicon in ER+ breast cancer and how the presence of this genomic alteration can be exploited to identify patients that would benefit from additional therapies, such as the SERDs currently under development. Citation Format: Jamie N. Mills, Jonathan Irish, Brittany Turner-Ivey, Stephen P. Ethier. WHSC1L1 and estrogen-independent activation of estrogen receptor-alpha (ERα) in 8p11 amplicon-bearing cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B05.

Abstract 3030: De novo engineering of chromosomal amplifications in human cells

Abstract Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to recurrent amplification events are lacking. Moreover, some common amplification events, such as that at 8p11-12 in breast cancer, harbor multiple candidate driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our general strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We chose regions recurrently amplified at high frequency in breast cancer, including 8p11-12, 17q12, and 11q13-14. After confirmation of successful gene targeting, the targeted cells are subjected to selective pressure directed at the amplifiable marker. Spontaneous DNA breakage events lead to varying degrees of co-amplification of linked loci near the targeting site. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using either the E. coli inosine monophosphate dehydrogenase enzyme IMPDH or a mutant dihydrofolate reductase gene as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR. Genome-wide array comparative genomic hybridization confirmed that amplification had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including “sawtooth” patterns, distal copy number loss suggesting a breakage-fusion-bridge mechanism, and large continuous regions of copy number gain. Amplification was confirmed by FISH. RT-PCR and immunoblotting demonstrated overexpression of selected amplified transcripts and proteins. In summary, we have used genome engineering techniques to create focal chromosomal amplification events de novo in human cell lines. These amplifications share many of the features of amplifications observed in primary human cancers. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a system to model oncogene cooperativity in amplifications harboring multiple candidate driver genes. Citation Format: Josh Lauring. De novo engineering of chromosomal amplifications in human cells. [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 3030. doi:10.1158/1538-7445.AM2015-3030

Engineering targeted chromosomal amplifications in human breast epithelial cells.

Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including “sawtooth” patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-015-3468-2) contains supplementary material, which is available to authorized users.

583 The 8p11 amplicon in luminal breast cancers harbors multiple interacting epigenome modifying oncogenes: implications for epigenome-targeted therapy

minutes after treatment. We identify a chromatin architecture that defines a durable response to BETi, specifically in the context of immunoglobulin heavy chain (IgH) re-arrangements, classifying patient populations suitable for BET treatment. Our results suggest that the molecular response to BETi is highly dynamic and adapts over time, while high-occupancy BRD4 loading is generally a poor predictor of BET transcriptional control.

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

PPAPDC1B and WHSC1L1 Are Common Drivers of the 8p11-12 Amplicon, Not Only in Breast Tumors But Also in Pancreatic Adenocarcinomas and Lung Tumors

Amplification of the 8p11-12 chromosomal region is a common genetic event in many epithelial cancers. In breast cancer, several genes within this region have been shown to display oncogenic activity. Among these genes, the enzyme-encoding genes, PPAPDC1B and WHSC1L1, have been identified as potential therapeutic targets. We investigated whether PPAPDC1B and WHSC1L1 acted as general driver genes, thereby serving as therapeutic targets in other tumors with 8p11-12 amplification. By using publicly available genomic data from a panel of 883 cell lines derived from different cancers, we identified the cell lines presenting amplification of both WHSC1L1 and PPAPDC1B. In particular, we focused on cell lines derived from lung cancer and pancreatic adenocarcinoma and found a correlation between the amplification of PPAPDC1B and WHSC1L1 with their overexpression. Loss-of-function studies based on the use of siRNA and shRNA demonstrated that PPAPDC1B and WHSC1L1 played a major role in regulating the survival of pancreatic adenocarcinoma and small-cell lung cancer-derived cell lines, both in anchorage-dependent and anchorage-independent conditions, displaying amplification and overexpression of these genes. We also demonstrated that PPAPDC1B and WHSC1L1 regulated xenograft growth in these cell lines. Finally, quantitative RT-PCR experiments after PPAPDC1B and WHSC1L1 knockdown revealed exclusive PPAPDC1B and WHSC1L1 gene targets in small-cell lung cancer and pancreatic adenocarcinoma-derived cell lines compared with breast cancer.

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

ERLIN2 promotes breast cancer cell survival by modulating endoplasmic reticulum stress pathways

BackgroundAmplification of the 8p11-12 region has been found in approximately 15% of human breast cancer and is associated with poor prognosis. Previous genomic analysis has led us to identify the endoplasmic reticulum (ER) lipid raft-associated 2 (ERLIN2) gene as one of the candidate oncogenes within the 8p11-12 amplicon in human breast cancer, particularly in the luminal subtype. ERLIN2, an ER membrane protein, has recently been identified as a novel mediator of ER-associated degradation. Yet, the biological roles of ERLIN2 and molecular mechanisms by which ERLIN2 coordinates ER pathways in breast carcinogenesis remain unclear.MethodsWe established the MCF10A-ERLIN2 cell line, which stably over expresses ERLIN2 in human nontransformed mammary epithelial cells (MCF10A) using the pLenti6/V5-ERLIN2 construct. ERLIN2 over expressing cells and their respective parental cell lines were assayed for in vitro transforming phenotypes. Next, we knocked down the ERLIN2 as well as the ER stress sensor IRE1α activity in the breast cancer cell lines to characterize the biological roles and molecular basis of the ERLIN2 in carcinogenesis. Finally, immunohistochemical staining was performed to detect ERLIN2 expression in normal and cancerous human breast tissuesResultsWe found that amplification of the ERLIN2 gene and over expression of the ERLIN2 protein occurs in both luminal and Her2 subtypes of breast cancer. Gain- and loss-of-function approaches demonstrated that ERLIN2 is a novel oncogenic factor associated with the ER stress response pathway. The IRE1α/XBP1 axis in the ER stress pathway modulated expression of ERLIN2 protein levels in breast cancer cells. We also showed that over expression of ERLIN2 facilitated the adaptation of breast epithelial cells to ER stress by supporting cell growth and protecting the cells from ER stress-induced cell death.ConclusionsERLIN2 may confer a selective growth advantage for breast cancer cells by facilitating a cytoprotective response to various cellular stresses associated with oncogenesis. The information provided here sheds new light on the mechanism of breast cancer malignancy

Abstract 5099: Identification of complex oncogene signatures in human cancer specimens and cell lines

Abstract Activated oncogenes are the genetic drivers of human cancer progression. Oncogenes become activated by structural DNA alterations including gene amplification and over expression, point mutations, and gene translocations/rearrangements. Data from existing targeted cancer treatments clearly show that the best response is observed when the activated oncogene is targeted. On the other hand, among the patients with activated oncogene who respond to a targeted treatment, the level and duration of response is highly variable. This could be explained by the genomic context of an activated oncogene that is unique to any given patient. Therefore, the ability to detect complex oncogene signatures in human cancer specimens is vital for better predictions of the efficacy of targeted agents, and to design therapeutic strategies with multiple targeted agents. In our laboratory, we have been working to identify candidate amplified and point mutated oncogenes in multiple human cancer specimens and cell lines. To obtain individual oncogene signatures, we have used high resolution aCGH coupled with a novel gene expression algorithm that allows for the identification of genes that are over expressed within amplicons in individual specimens. We have also used a high-throughput, mass spectrometry based method for detection of point mutations in oncogenes using the same specimens. These approaches have allowed us to identify tens to hundreds of candidate oncogenes in each specimen which were then refined by on-line computational tools such as Gene Tracker. Using this approach, we have elucidated complex oncogene signatures for over 50 cancer cell lines and specimens. Analysis of these data sets points to highly variable oncogene combinations in individual specimens. In other studies using a subset of breast cancers selected for the presence of the 8p11 amplicon, we found that even in these highly selected specimens, the oncogene signatures were unique to each patient. These signatures make clear predictions about combinations of targeted agents that could be considered. For example, we have observed activation of oncogenes in the 8p11 amplicon to be associated with other oncogenes such the EGFR, cdk4, MYST3, CRKL, and others. Additionally, the presence of PIK3CA mutations occurs variably in these specimens as well. Most of the breast cancer specimens and cell lines in our study have a well-defined signature of oncogenes, and understanding these signatures is important not only for the design of strategies using multiple targeted agents, but also for the proper interpretation of clinical trial results using single targeted agents. Thus, we have developed a high-throughput, cost effective molecular diagnostic platform that can accurately and rapidly identify the causal driving oncogenes in an individual cancer biopsy specimens and therefore can be adapted to analysis of large numbers of patient's specimens. 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 5099. doi:1538-7445.AM2012-5099

Abstract 1135: PPAPDC1B isoform 2, a new candidate oncogene within the 8p11-12 amplicon induces a proliferative advantage in breast cancer

Abstract The 8p11-12 genomic region is amplified in about 10-15% of human breast cancer and may be associated with poor prognosis. Earlier, we used genomic analysis of copy number and gene expression to perform a detailed analysis of the 8p11-12 amplicon for identifying candidate oncogenes in breast cancer. We identified phosphatidic acid phosphatase type 2 domain containing 1B (PPAPDC1B) as one of the candidate oncogenes based on statistical analysis of copy number increase and over expression. Recently, fine-mapping of this locus, and the updated human genomic database has shown there may be more than 3 splicing variants of PPAPDC1B. We measured the expression status of all 3 individual PPAPDC1B isoforms in breast cancer cell lines by real-time quantitative RT-PCR and western blot. Interestingly, the short splicing variant /isoform2 is the dominant over-expressed form in SUM44 and SUM52 breast cancer cell lines that harbor the 8p11-12 amplicon. The PPAPDC1B gene encodes a novel type of transmembrane phosphatidate phosphatase with an acid-PPc domain with broad lipid phosphate substrate specificity. This suggests that PPAPDC1B may play a role in cell signaling and lipid metabolism. The PPAPDC1B isoform 2 is predicted to have an inactive acid-PPc domain due to truncation of mRNA. To address the question of whether PPAPDC1B possesses transforming properties, and to functionally characterize the different isoforms, we established lentiviral expression constructs of full-length PPAPDC1B as well as the short isoform. MCF10A cells were infected with equalized viral titer of lentiviral vectors and assayed for alterations in growth rates, colony formation and growth factor-independent proliferation. Cell proliferation assays indicated that over expression of PPAPDC1B isoform 2 enhanced cell proliferation more dramatically than full-length PPAPDC1B. Furthermore, colony formation assay showed that MCF10A cells over expressing PPAPDC1B isoform 2 formed large highly proliferative colonies compared with control cells. All of the isoforms of PPAPDC1B could induce insulin-like growth factor-independence in MCF-10A cells, but not independence of EGF. Thus, our data suggest that PPAPDC1B isoform 2 is a candidate oncogene from the 8p11 region that can induce a proliferative advantage in subset of breast cancer. 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 1135. doi:10.1158/1538-7445.AM2011-1135

Transforming Properties of 8p11-12 Amplified Genes in Human Breast Cancer

Amplification of the 8p11-12 region has been found in about 15% of human breast cancers and is associated with poor prognosis. Earlier, we used genomic analysis of copy number and gene expression to perform a detailed analysis of the 8p11-12 amplicon to identify candidate oncogenes in breast cancer. We identified 21 candidate genes and provided evidence that three genes, namely, LSM-1, TC-1, and BAG4, have transforming properties when overexpressed. In the present study, we systematically investigated the transforming properties of 13 newly identified 8p11-12 candidate oncogenes in vitro. WHSC1L1, DDHD2, and ERLIN2 were most potently transforming oncogenes based on the number of altered phenotypes expressed by the cells. WHSC1L1 contains a PWWP-domain that is a methyl-lysine recognition motif involved in histone code modification and epigenetic regulation of gene expression. Knockdown of WHSC1L1 in 8p11-12-amplified breast cancer cells resulted in profound loss of growth and survival of these cells. Further, we identified several WHSC1L1 target genes, one of which is iroquois homeobox 3 gene (IRX3), a member of the Iroquois homeobox transcription factor family.

Abstract 3102: Oncogenic PWWP-domain protein WHSC1L1 links the homeobox transcription factor IRX3 in breast cancer

Abstract Amplification of the 8p11-12 region has been found in about 10-15% of human breast cancer and may be associated with poor prognosis. Earlier, we used genomic analysis of copy number and gene expression to perform a detailed analysis of the 8p11-12 amplicon for identifying candidate oncogenes in breast cancer. We identified Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) as a candidate oncogene based on statistical analysis of copy number increase and over expression. The WHSC1L1 gene encodes a PWWP domain protein that regulates histone methylation marks, and regulates the transcription profile and the differentiated function of cells. To address the question of whether WHSC1L1 possesses transforming properties, we established lentiviral expression constructs containing the WHSC1L1 as well as other 20 candidate genes of 8p11-12 region. MCF10A cells were infected with these lentiviral vectors and assayed for alterations in growth rates, growth factor-independent proliferation, anchorage-independent growth and for three-dimensional morphogenesis assays in Matrigel culture. From these experiments, we found that the WHSC1L1 is the most potently transforming oncogene we tested from the 8p11-12 region. Furthermore, knockdown of this gene in WHSC1L1 amplified breast cancer cells resulted in profound loss of growth and survival potential in these cells. To identify genes that may be regulated in their expression by the WHSC1L1 when it is amplified and over expressed, we performed expression profiling of MCF10A cells, MCF-10AWHSC1L1 cells, and WHSC1L1 amplified SUM-44 breast cancer cells. Analysis of genes altered by over expression of WHSC1L1 in MCF10A-WHSC1L1 cells and in SUM-44 cells resulted in the identification of 184 WHSC1L1 candidate target genes. Among them was the iroquois homeobox 3 gene (IRX3), a member of the Iroquois homeobox transcription factor family. The regulation of IRX3 gene expression by WHSC1L1 was validated in SUM44 breast cancer cells using WHSC1L1 knock down. IRX3 mRNA was dramatically down-regulated in SUM-44 cells after WHSC1L1 shRNA infection. Intriguingly, in our array CGH study we found that SUM-44 cells also have a gene amplification of IRX3. Thus, our finding indicates that WHSC1L1 is the major driving oncogene in the 8p11-12 amplicon, and provides the first evidence that WHSC1L1 and IRX3 are genetically linked in subset of breast cancer. 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 3102.

Abstract 5022: Roles of endoplasmic reticulum factor ERLIN2 in malignancy maintenance and therapy resistance in breast cancer

Abstract Endoplasmic reticulum (ER)-associated degradation (ERAD) components are highly induced in various tumors and are associated with cancer cell survival and resistance to anti-cancer treatments. The ERAD pathway is crucial for the cell to adapt to ER stress by degrading misfolded, unassembled and even some normal proteins in the ER lumen. Recent work in our group has led us to identify the ER lipid raft-associated 2 (ERLIN2, also known as SPFH2, C8ORF2) gene as one of the candidate oncogenes within the 8p11-12 amplicon in breast cancer based on statistical analysis of copy number increase and over expression. ERLIN2 protein, which localized specifically to the ER, is the key component of ERAD pathway. To elucidate the oncogenic properties of the ERAD factor ERLIN2 in human breast cancer, we established the MCF10A-ERLIN2 cell line, which stably over expresses ERLIN2 in human nontransformed mammary epithelial cells (MCF10A) using the pLenti6/V5-ERLIN2 construct. ERLIN2 over expressing cells and their respective parental cell lines were assayed for alterations in growth rates, growth factor-independent proliferation and other phenotypes expressed by cancer cells. From these experiments, we found that over expression of ERLIN2 in MCF10A cells results in growth factor-independent proliferation in insulin-like growth factor deficient media and formed large, highly-proliferative, and abnormally-structured colonies in three-dimensional morphogenesis assays. However, over expression of ERLIN2 in MCF10A cells did not result in cells with anchorage independent growth potential. To further elucidate the molecular basis that ERLIN2 is linked to the ER-stress response pathways in breast cancer cells, we knocked down the ER stress sensor IRE1α activity in the breast cancer cell lines in which ERLIN2 is amplified and over expressed. The knockdown of the IRE1α RNase activity, but not the kinase activity, significantly reduced protein levels of ERLIN2 in ERLIN2 amplified breast cancer cells. In addition, forced expression of wild-type IRE1α or spliced XBP1, the IRE1α target, led to increased expression of ERLIN2 in MCF10A cells. We also found that ERLIN2 protein was induced in response to the ER stress-inducing reagents Tunicamycin, Thapsigargin or growth factor depletion. Together, our findings indicate that ERLIN2 plays an unconventional oncogenic role through modulating ER stress response and ERAD pathways in a subset of breast cancer. Targeted inhibition of ERAD factor ERLIN2 in cancer could provide potential new avenues for therapeutic development. 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 5022.