Search

Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to the lack of isogenic models. To address this, we used healthy primary breast luminal epithelial cells of different genetic ancestry and their hTERT-immortalized counterparts to identify transcriptomic changes associated with immortalization. Elevated expression of TONSL (Tonsoku-like, DNA repair protein) was identified as one of the earliest events during immortalization. TONSL, which is located on chromosome 8q24.3, was found to be amplified in approximately 20% of breast cancers. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity, but overexpression was insufficient for neoplastic transformation. However, TONSL-immortalized primary cells overexpressing defined oncogenes generated estrogen receptor–positive adenocarcinomas in mice. Analysis of a breast tumor microarray with approximately 600 tumors revealed poor overall and progression-free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors, including NF-κB and limited access to the tumor-suppressor p53. TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in the homologous recombination (HR) and Fanconi anemia pathways. Consistent with these results, TONSL-overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was essential for growth of TONSL-amplified breast cancer cell lines in vivo, and these cells were sensitive to TONSL–FACT complex inhibitor CBL0137. Together, these findings identify TONSL as a regulator of epithelial cell immortalization to facilitate cancer initiation and as a target for breast cancer therapy. Significance: The chr.8q24.3 amplicon-resident gene TONSL is upregulated during the initial steps of tumorigenesis to support neoplastic transformation by increasing DNA repair and represents a potential therapeutic target for treating breast cancer.

Table S9: Results of ATAC-seq of primary and TONSL-immortalized cells.

Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to the lack of isogenic models. To address this, we used healthy primary breast luminal epithelial cells of different genetic ancestry and their hTERT-immortalized counterparts to identify transcriptomic changes associated with immortalization. Elevated expression of TONSL (Tonsoku-like, DNA repair protein) was identified as one of the earliest events during immortalization. TONSL, which is located on chromosome 8q24.3, was found to be amplified in approximately 20% of breast cancers. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity, but overexpression was insufficient for neoplastic transformation. However, TONSL-immortalized primary cells overexpressing defined oncogenes generated estrogen receptor–positive adenocarcinomas in mice. Analysis of a breast tumor microarray with approximately 600 tumors revealed poor overall and progression-free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors, including NF-κB and limited access to the tumor-suppressor p53. TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in the homologous recombination (HR) and Fanconi anemia pathways. Consistent with these results, TONSL-overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was essential for growth of TONSL-amplified breast cancer cell lines in vivo, and these cells were sensitive to TONSL–FACT complex inhibitor CBL0137. Together, these findings identify TONSL as a regulator of epithelial cell immortalization to facilitate cancer initiation and as a target for breast cancer therapy.Significance:The chr.8q24.3 amplicon-resident gene TONSL is upregulated during the initial steps of tumorigenesis to support neoplastic transformation by increasing DNA repair and represents a potential therapeutic target for treating breast cancer.

Contains additional materials and methods and supplementary figures

Background: Inheritance of BRCA1 and BRCA2 mutations is associated with increased risk of breast and ovarian cancers. Previous studies with low-throughput flow cytometry-based assays suggested elevated number of luminal progenitor cells in the breast tissues of BRCA1 mutation carriers compared to breast tissues of non-carriers. However, breast epithelial cell-specific transcriptome differences between BRCA1, BRCA2 mutation carriers, and non-carriers and how these differences alter susceptibility to transformation are yet to be elucidated. Methods: We generated a single cell transcriptome atlas of breast tissues from BRCA1 (six samples, 17,220 cells), BRCA2 (four samples, 25,046 cells) mutation carriers and non-carriers (11 samples, >50,000 cells). Using previously described markers, epithelial cells were sub-clustered into basal, luminal progenitor, and mature luminal cells. Genes differentially expressed in epithelial cells of BRCA1 and BRCA2 mutation carriers compared to those in non-carrier donors were subjected to Ingenuity Pathway Analysis to determine signaling pathways uniquely active in BRCA1 and BRCA2 mutant epithelial cells. Breast epithelial cells derived from three donor types were immortalized using hTERT and then transformed with PIK3CAH1047R mutant or H-RasG12V ± SV40-T/t antigens, and tumorigenicity was determined in vivo. Results: BRCA1 but not BRCA2 mutations altered the ratio between basal, luminal progenitor and mature luminal cells in breast tissues compared to breast tissues in non-carriers. A unique cluster of cells within luminal progenitors was underrepresented in case of BRCA2 mutation carriers compared to non-carriers or BRCA1 mutation carriers. BRCA1 or BRCA2 mutations specifically altered transcriptomes which are an integral part of mTOR and MYCN signaling, and the translational machinery. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included YAP1, BRD4, SMARCA4, and TGFβ1 signaling. BRCA2 mutations were associated with upregulation of IL-6, FOXO3, and TNFSF11 signaling. Breast epithelial cells from BRCA2 mutation carriers but not BRCA1 mutation carriers or non-carriers modified to overexpress hTERT + PIK3CAH1047R generated tumors in NSG mice. These tumors displayed large cystic structures with basilar epithelial cells lining the rim of cysts with focal areas of cellular hyperplasia and neoplastic cells that extended into the lumen. However, BRCA1/2 mutation status did not influence tumorigenicity by hTERT+ H-RASG12V +SV40-T/t antigens. Conclusions: Our studies provide a high resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers, which also reveal potentially targetable signaling networks uniquely deregulated in these cells. BRCA2 mutations are associated with distinct susceptibility to PIK3CA mutation-driven transformation. Since PIK3CA mutations are observed in clinically normal breast tissues, screening for such mutations in BRCA2 mutation carriers may help to detect pre-neoplastic or early stage breast cancer. Citation Format: Harikrishna Nakshatri, Poornima Bhat-Nakshatri, Duojiao Chen, Katie Chen, Henry Mang, Christopher A Herodotou, Aditi S Khatpe, Patrick C McGuire, Xiaoling Xuei, Yunlong Liu, George Sandusky, Anna Maria Storniolo. Single cell transcriptomic analysis reveals the effects of BRCA1 and BRCA2 mutations on distinct signaling networks and cancer susceptibility [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-07-09.

Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer predisposition syndrome characterized by the development of bilateral vestibular (VS) and spinal schwannomas secondary to loss of heterozygosity of NF2 in Schwann cells or their precursors. While these tumors are largely benign, they can cause considerable morbidity due to compromised auditory, vestibular, facial, and vertebral nerve function. This may result in deafness, vertigo, facial muscle weakness, chronic neuropathic pain, and even death.There are currently no pharmacotherapies for VS, and surgical resection remains the standard of care, which is associated with significant morbidity. Thus, there is an urgent need to develop pharmaceutical approaches to halt or reverse the progression of tumor growth in NF2 patients who develop VS. Our lab previously identified the receptor tyrosine kinase inhibitors brigatinib and dasatinib as potentially efficacious agents for the treatment of VS and demonstrated that both agents targeted the Ephrin A2 receptor (EPHA2). EPHA2is a transmembrane receptor tyrosine kinase thatis involved in cell contact-mediated motility, adhesion,and migration. Additionally, EPHA2 modulates axon guidance, and synaptogenesis in developing brain. Here we demonstrate that EPHA2expression is increased in NF2-/-Schwanncells and NF2-/-cancers. We identify ponatinib, a receptor tyrosine kinase inhibitor targeting ABL1 that is FDA-approved for CML, as an additional agent that targets EPHA2. We demonstrate that ponatinib treatment impairs the viability of both human and murine NF2-/-Schwanncells in vitro and decreases EPHA2 protein expression. Accordingly, pharmacologic,and siRNA-mediated inhibition of EPHA2 also impaired the growth of human NF2-/-Schwann cells in vitro. Lastly, we demonstrate that both ponatinib and EPHA2 inhibition induce morphological changes in NF2-/-Schwann cells. Our findings suggest that ponatinib or the direct targeting of EPHA2 may be efficacious for the treatment of NF2-associated vestibular schwannoma. Future in vivo efficacystudies are warranted.

Adaptation to diverse environments during human evolution including those conditions that favor certain infectious diseases to become endemic in a region has resulted in natural selection of genetic variants. These variants remain fixed in current day populations despite changes in the environment. At least 20 such variants, also called adaptive traits, have been described to date and Duffy phenotype is one among them. Individuals in sub-Saharan Africa carry an inactivating mutation in a gene called atypical cytokine receptor 1 (ACKR1 or DARC) and this phenotype is called Duffy-phenotype. ACKR1 is a decoy receptor for CCL2 and IL-8 (CXCL8) and limits the activity of these cytokines. The Duffy-null phenotype confers protection against malarial infection. However, recent studies suggest that breast cancers in Duffy-null women display a distinct immune profile and have worse outcomes, possibly due to altered signaling by cytokines/chemokines. Using our institutional resource of breast tissues from >5000 healthy women, we have initiated studies to examine the impact of adaptive traits on normal breast and breast cancer biology. Towards this goal, we have created breast epithelial cell lines from Duffy wild type and Duffy-null/heterozygous carriers, all of African ancestry. ACKR1 is expressed in breast epithelial cells of Duffy-wild type but not in Duffy-null carriers. Breast epithelial cell lines as well as their transformed variants from Duffy-null/heterozygous carriers contained elevated levels of phosphorylated cMET(Y1349) and pERK(T202/Y204) compared to cell lines from Duffy-wild type carriers. Transformed Duffy-wild type and Duffy-heterozygous cells generated adenocarcinomas in NSG mice, which indicates that our model system closely recapitulates breast cancers in human. Cell lines created from Duffy-heterozygous tumors compared to Duffy-wild tumors displayed elevated pERK levels. Duffy phenotype also affects parainflammatory signals from transformed cells as tumor-derived cells from Duffy-null/heterozygous carriers expressed higher levels of IGFBP2 compared to tumor-derived cells from non-carriers. Additional studies are underway to determine whether Duffy phenotype alters sensitivity to chemo and targeted therapies. Collectively, these results suggest that adaptive traits alter epithelial cell intrinsic signaling pathways, which may affect tumor growth patterns, tumor microenvironment, and potentially sensitivity to specific therapies. Citation Format: Poornima Bhat-Nakshatri, Brijesh Kumar, Madhura S. Shukla, Katie Batic, Henry Mang, Rana German, Anna Maria Storniolo, Harikrishna Nakshatri. Oncologic anthropology-impact of Duffy adaptive trait on breast epithelial and tumor cell biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1962.

Osteosarcoma (OS) is an aggressive pediatric cancer with ~35% of patients developing metastasis over time. The survival rate for metastatic and relapsed OS patients is Citation Format: Niknam Riyahi, Pankita H. Pandya, M. Reza Saadatzadeh, Khadijeh Bijangi-Vishehsaraei, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Melissa A. Trowbridge, Kathy Coy, Henry Mang, Reagan K. Wohlford, Anthony L. Sinn, Emily S. Sims, Matt J. Repass, Nuri Damayanti, Farinaz Barghi, Harlan E. Shannon, Michael J. Ferguson, Jamie L. Renbarger, Karen E. Pollok. Therapeutic induction of replication stress in the context of salvage therapy in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2017.

In children, adolescents, and young adults (AYA), osteosarcoma (OS) is the most common type of bone cancer and ~35% patients relapse following frontline cytotoxic therapy. Thus, there is a critical need to identify therapies targeting specific molecular signatures in OS. Hyperactivation of cyclin-dependent kinases 4 and 6 (CDK4/6) has been identified by us and others as a top actionable marker in OS. CDK4/6 binds to cyclin D resulting in a complex that mediates RB phosphorylation leading to cell cycle progression. While CDK4/6 inhibitors (CDK4/6i) have shown promise clinically, one drawback is that CDK4/6i induces cell cycle arrest rather than cell death. Furthermore, prolonged CDK4/6 inhibitor therapy can confer therapeutic resistance in RB1-proficient (RB+) tumors where compensatory pathways such as PI3K/mTOR are activated. To mitigate such CDK4/6i resistance in OS, we hypothesized that dual inhibition of CDK4/6 and PI3K pathways will promote cytotoxicity in hyperactivated CDK4/6 OS models. RB+ OS cell lines and a TT2-77 xenoline were evaluated in vitro. Combination index and Bliss independence analyses indicated that inhibition of OS growth by exposure to CDK4/6i (Palbociclib or Abemaciclib) and PI3K/mTOR inhibitor (PI3K/mTORi-Voxtalisib or LY3023414) was additive-to-synergistic and lead to increased apoptosis at clinically relevant concentrations. Short-term pharmacodynamic study of vehicle- versus Palbociclib-treated TT2-77 patient-derived xenograft (PDX) was analyzed by global/phospho-proteomics and kinome profiling. RB1 and MKI67 phosphopeptides as well as the total protein levels of CDK1 were reduced by Palbociclib, thus, confirming modulation of the cell cycle. Kinome profiling analysis of PDX from Palbociclib-treated mice indicated increased activity of AXL, a receptor tyrosine kinase linked to PI3K pathway activation. Increased activity of the autophagy marker PIK3C3 was also evident. OS PDX models TT2-77 and HT96 (RB+, CDKN2Anull, CCND3 amplified) were treated with Palbociclib (50 mg/kg), Voxtalisib (50 mg/kg) or Palbociclib + Voxtalisib. In TT2-77 PDXs treated for four weeks, tumor growth was significantly reduced in single-agent and combo groups compared to vehicle (p Citation Format: Farinaz Barghi, Pankita H Pandya, M. Reza Saadatzadeh, Khadijeh Bijangi-Vishehsaraei, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Melissa A. Trowbridge, Kathryn L. Coy, Henry Mang, Reagan K. Wohlford, Anthony L. Sinn, Emily C. Sims, Matt J. Repass, Nuri Damayanti, Niknam Riyahi, Harlan E. Shannon, Steve P Angus, Michael J Ferguson, Jamie L. Renbarger, Karen E. Pollok. Targeting CDK4/6 inhibitor resistance in relapsed RB-proficient osteosarcoma patient-derived xenografts via PI3 Kinase/mTOR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2011.