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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.

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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.

The purpose of this study, conducted during the Covid-19 pandemic between November 2020 and March 2021 in India and Nigeria, is to explore the direct and indirect effects of Covid-19 on religiously marginalised groups experiencing intersecting vulnerabilities. The findings provide recognition of the impact of Covid-19 on targeting and encroachments faced by these groups in order to inform policy so that it includes their perspectives in building back better and promoting inclusive development. Policymakers need to understand both the direct and indirect impacts of Covid-19 in order to coordinate effective support and avert deepening marginalisation. This research demonstrates how religious inequalities intersect with other inequalities of power – historical, structural, and socially determined characteristics (class, ethnicity, caste, gender, age) – to shape how people experience the Covid-19 pandemic. Both India and Nigeria manifest high levels of authoritarianism, an absence of press freedom, targeting of religiously marginalised groups, and unequal access to public services and the protection of the state by religiously marginalised groups, according to geographic location. The findings of this report reveal the appalling everyday realities as well as the great courage of religious minorities living in poverty during the pandemic. Greater sensitivity to the critical intersection of vulnerabilities is essential for the longer-term recovery of these groups, who otherwise face slipping deeper into intergenerational poverty. Deepening poverty and proliferating ethno-religious injustices are fuelling tensions and conflict, and the risks of neglecting these issues are immense.

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.

Background Idiopathic nodular mesangial sclerosis, also called idiopathic nodular glomerulosclerosis (ING), is a rare clinical entity with an unclear pathogenesis. The hallmark of this disease is the presence of nodular mesangial sclerosis on histology without clinical evidence of diabetes mellitus or other predisposing diagnoses. To achieve insights into its pathogenesis, we queried the clinical, histopathologic and transcriptomic features of ING and nodular diabetic nephropathy (DN). Methods All renal biopsy reports accessioned at Indiana University Health from 2001 to 2016 were reviewed to identify 48 ING cases. Clinical and histopathologic features were compared between individuals with ING and DN (n = 751). Glomeruli of ING (n = 5), DN (n = 18) and reference (REF) nephrectomy (n = 9) samples were isolated by laser microdissection and RNA was sequenced. Immunohistochemistry of proline-rich 36 (PRR36) protein was performed. Results ING subjects were frequently hypertensive (95.8%) with a smoking history (66.7%). ING subjects were older, had lower proteinuria and had less hyaline arteriolosclerosis than DN subjects. Butanoate metabolism was an enriched pathway in ING samples compared with either REF or DN samples. The top differentially expressed gene, PRR36, had increased expression in glomeruli 248-fold [false discovery rate (FDR) P = 5.93 × 10−6] compared with the REF and increased 109-fold (FDR P = 1.85 × 10−6) compared with DN samples. Immunohistochemistry revealed a reduced proportion of cells with perinuclear reaction in ING samples as compared to DN. Conclusions Despite similar clinical and histopathologic characteristics in ING and DN, the uncovered transcriptomic signature suggests that ING has distinct molecular features from nodular DN. Further study is warranted to understand these relationships.

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

Microvascular perfusion dynamics are vital to physiological function and are frequently dysregulated in injury and disease. Typically studies measure microvascular flow in a few selected vascular segments over limited time, failing to capture spatial and temporal variability. To quantify microvascular flow in a more complete and unbiased way we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow), a macro for FIJI open-source image analysis software. Using high-speed microvascular flow movies, STAFF generates kymographs for every time interval for every vascular segment, calculates flow velocities from red blood cell shadow angles, and outputs the data as color-coded velocity map movies and spreadsheets. In untreated mice, analyses demonstrated profound variation even between adjacent sinusoids over seconds. In acetaminophen-treated mice we detected flow reduction localized to pericentral regions. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of microvascular flow.

Acute kidney injury induces the loss of renal microvessels, but the fate of endothelial cells and the mechanism of potential vascular endothelial growth factor (VEGF)-mediated protection is unknown. Cumulative cell proliferation was analyzed in the kidney of Sprague-Dawley rats following ischemia-reperfusion (I/R) injury by repetitive administration of BrdU (twice daily) and colocalization in endothelial cells with CD31 or cablin. Proliferating endothelial cells were undetectable for up to 2 days following I/R and accounted for only ∼1% of BrdU-positive cells after 7 days. VEGF-121 preserved vascular loss following I/R but did not affect proliferation of endothelial, perivascular cells or tubular cells. Endothelial mesenchymal transition states were identified by localizing endothelial markers (CD31, cablin, or infused tomato lectin) with the fibroblast marker S100A4. Such structures were prominent within 6 h and sustained for at least 7 days following I/R. A Tie-2-cre transgenic crossed with a yellow fluorescent protein (YFP) reporter mouse was used to trace the fate of endothelial cells and demonstrated interstititial expansion of YFP-positive cells colocalizing with S100A4 and smooth muscle actin following I/R. The interstitial expansion of YFP cells was attenuated by VEGF-121. Multiphoton imaging of transgenic mice revealed the alteration of YFP-positive vascular cells associated with blood vessels characterized by limited perfusion in vivo. Taken together, these data indicate that vascular dropout post-AKI results from endothelial phenotypic transition combined with an impaired regenerative capacity, which may contribute to progressive chronic kidney disease.

In the rat, p53 promotes tubular apoptosis after ischemic AKI. Acute pharmacologic inhibition of p53 is protective in this setting, but chronic inhibition enhances fibrosis, demonstrating that the role of p53 in ischemic AKI is incompletely understood. Here, we investigated whether genetic absence of p53 is also protective in ischemic AKI. Surprisingly, p53-knockout mice (p53(-/-)) had worse kidney injury, compared with wild-type mice, and exhibited increased and prolonged infiltration of leukocytes after ischemia. Acute inhibition of p53 with pifithrin-α in wild-type mice mimicked the observations in p53(-/-) mice. Chimeric mice that lacked p53 in leukocytes sustained injury similar to p53(-/-) mice, suggesting an important role for leukocyte p53 in ischemic AKI. Compared with wild-type mice, a smaller proportion of macrophages in the kidneys of p53(-/-) and pifithrin-α-treated mice after ischemic injury were the anti-inflammatory M2 phenotype. Ischemic kidneys of p53(-/-) and pifithrin-α-treated mice also showed reduced expression of Kruppel-like factor-4. Finally, models of peritonitis in p53(-/-) and pifithrin-α-treated mice confirmed the anti-inflammatory role of p53 and its effect on the polarization of macrophage phenotype. In summary, in contrast to the rat, inflammation characterizes ischemic AKI in mice; leukocyte p53 is protective by reducing the extent and duration of this inflammation and by promoting the anti-inflammatory M2 macrophage phenotype.