Integrin-linked kinase-frizzled 7 interaction maintains cancer stem cells to drive platinum resistance in ovarian cancer.

BACKGROUND: Platinum-based chemotherapy regimens are a mainstay in the management of ovarian cancer (OC), but emergence of chemoresistance poses a significant clinical challenge. The persistence of ovarian cancer stem cells (OCSCs) at the end of primary treatment contributes to disease recurrence. Here, we hypothesized that the extracellular matrix protects CSCs during chemotherapy and supports their tumorigenic functions by activating integrin-linked kinase (ILK), a key enzyme in drug resistance. METHODS: TCGA datasets and OC models were investigated using an integrated proteomic and gene expression analysis and examined ILK for correlations with chemoresistance pathways and clinical outcomes. Canonical Wnt pathway components, pro-survival signaling, and stemness were examined using OC models. To investigate the role of ILK in the OCSC-phenotype, a novel pharmacological inhibitor of ILK in combination with carboplatin was utilized in vitro and in vivo OC models. RESULTS: In response to increased fibronectin secretion and integrin ß1 clustering, aberrant ILK activation supported the OCSC phenotype, contributing to OC spheroid proliferation and reduced response to platinum treatment. Complexes formed by ILK with the Wnt receptor frizzled 7 (Fzd7) were detected in tumors and correlated with metastatic progression. Moreover, TCGA datasets confirmed that combined expression of ILK and Fzd7 in high grade serous ovarian tumors is correlated with reduced response to chemotherapy and poor patient outcomes. Mechanistically, interaction of ILK with Fzd7 increased the response to Wnt ligands, thereby amplifying the stemness-associated Wnt/ß-catenin signaling. Notably, preclinical studies showed that the novel ILK inhibitor compound 22 (cpd-22) alone disrupted ILK interaction with Fzd7 and CSC proliferation as spheroids. Furthermore, when combined with carboplatin, this disruption led to sustained AKT inhibition, apoptotic damage in OCSCs and reduced tumorigenicity in mice. CONCLUSIONS: This "outside-in" signaling mechanism is potentially actionable, and combined targeting of ILK-Fzd7 may lead to new therapeutic approaches to eradicate OCSCs and improve patient outcomes.

Spatial N-glycomics of the normal breast microenvironment reveals fucosylated and high-mannose N-glycan signatures related to BI-RADS density and ancestry.

Higher breast cancer mortality rates continue to disproportionally affect black women (BW) compared to white women (WW). This disparity is largely due to differences in tumor aggressiveness that can be related to distinct ancestry-associated breast tumor microenvironments (TMEs). Yet, characterization of the normal microenvironment (NME) in breast tissue and how they associate with breast cancer risk factors remains unknown. N-glycans, a glucose metabolism-linked post-translational modification, has not been characterized in normal breast tissue. We hypothesized that normal female breast tissue with distinct Breast Imaging and Reporting Data Systems (BI-RADS) categories have unique microenvironments based on N-glycan signatures that varies with genetic ancestries. Profiles of N-glycans were characterized in normal breast tissue from BW (n = 20) and WW (n = 20) at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). A total of 176 N-glycans (32 core-fucosylated and 144 noncore-fucosylated) were identified in the NME. We found that certain core-fucosylated, outer-arm fucosylated and high-mannose N-glycan structures had specific intensity patterns and histological distributions in the breast NME dependent on BI-RADS densities and ancestry. Normal breast tissue from BW, and not WW, with heterogeneously dense breast densities followed high-mannose patterns as seen in invasive ductal and lobular carcinomas. Lastly, lifestyles factors (e.g. age, menopausal status, Gail score, BMI, BI-RADS) differentially associated with fucosylated and high-mannose N-glycans based on ancestry. This study aims to decipher the molecular signatures in the breast NME from distinct ancestries towards improving the overall disparities in breast cancer burden.

XPC Protects against Carcinogen-Induced Histologic Progression to Lung Squamous Cell Carcinoma by Reduced Basal Epithelial Cell Proliferation.

Lung squamous cell carcinoma (LUSC) is the second leading cause of lung cancer. Although characterized by high DNA mutational burdens and genomic complexity, the role of DNA repair in LUSC development is poorly understood. We sought to better understand the role of the DNA repair protein Xeroderma Pigmentosum Group C (XPC) in LUSC development. XPC knock-out (KO), heterozygous, and wild-type (WT) mice were exposed topically to N-nitroso-tris-chloroethylurea (NTCU), and lungs were evaluated for histology and pre-malignant progression in a blinded fashion at various time-points from 8-24 weeks. High-grade dysplasia and LUSC were increased in XPC KO compared with XPC WT NTCU mice (56% vs. 34%), associated with a higher mean LUSC lung involvement (p < 0.05). N-acetylcysteine pre-treatment decreased bronchoalveolar inflammation but did not prevent LUSC development. Proliferation, measured as %Ki67+ cells, increased with NTCU treatment, in high-grade dysplasia and LUSC, and in XPC deficiency (p < 0.01, ANOVA). Finally, pre-LUSC dysplasia developed earlier and progressed to higher histologic classification sooner in XPC KO compared with WT mice. Overall, this supports the protective role of XPC in squamous dysplasia progression to LUSC. Mouse models of early LUSC development are limited; this may provide a valuable model to study mechanisms of LUSC development and progression.

Integrin-linked kinase-frizzled 7 interaction maintains cancer stem cells to drive platinum resistance in ovarian cancer.

Background: Platinum-based chemotherapy regimens are a mainstay in the management of ovarian cancer (OC), but emergence of chemoresistance poses a significant clinical challenge. The persistence of ovarian cancer stem cells (OCSCs) at the end of primary treatment contributes to disease recurrence. Here, we hypothesized that the extracellular matrix protects CSCs during chemotherapy and supports their tumorigenic functions by activating integrin-linked kinase (ILK), a key enzyme in drug resistance. Methods: TCGA datasets and OC models were investigated using an integrated proteomic and gene expression analysis and examined ILK for correlations with chemoresistance pathways and clinical outcomes. Canonical Wnt pathway components, pro-survival signaling, and stemness were examined using OC models. To investigate the role of ILK in the OCSC-phenotype, a novel pharmacological inhibitor of ILK in combination with carboplatin was utilized in vitro and in vivo OC models. Results: In response to increased fibronectin (FN) secretion and integrin ß1 clustering, aberrant ILK activation supported the OCSC phenotype, contributing to OC spheroid proliferation and reduced response to platinum treatment. Complexes formed by ILK with the Wnt receptor frizzled 7 (Fzd7) were detected in tumors and showed a strong correlation with metastatic progression. Moreover, TCGA datasets confirmed that combined expression of ILK and Fzd7 in high grade serous ovarian tumors is correlated with reduced response to chemotherapy and poor patient outcomes. Mechanistically, interaction of ILK with Fzd7 increased the response to Wnt ligands, thereby amplifying the stemness-associated Wnt/ß-catenin signaling. Notably, preclinical studies showed that the novel ILK inhibitor compound 22 (cpd-22) alone disrupted ILK interaction with Fzd7 and CSC proliferation as spheroids. Furthermore, when combined with carboplatin, this disruption led to sustained AKT inhibition, apoptotic damage in OCSCs and reduced tumorigenicity in mice. Conclusions: This "outside-in" signaling mechanism is potentially actionable, and combined targeting of ILK-Fzd7 may represent a new therapeutic strategy to eradicate OCSCs and improve patient outcomes.

Signaling Pathway Alterations Driven by BRCA1 and BRCA2 Germline Mutations are Sufficient to Initiate Breast Tumorigenesis by the PIK3CAH1047R Oncogene.

Single-cell transcriptomics studies have begun to identify breast epithelial cell and stromal cell specific transcriptome differences between BRCA1/2 mutation carriers and non-carriers. We generated a single-cell transcriptome atlas of breast tissues from BRCA1, BRCA2 mutation carriers and compared this single-cell atlas of mutation carriers with our previously described single-cell breast atlas of healthy non-carriers. We observed that BRCA1 but not BRCA2 mutations altered the ratio between basal (basal-myoepithelial), luminal progenitor (luminal adaptive secretory precursor, LASP), and mature luminal (luminal hormone sensing) cells in breast tissues. A unique subcluster of cells within LASP cells is underrepresented in case of BRCA1 and BRCA2 mutation carriers compared with non-carriers. Both BRCA1 and BRCA2 mutations specifically altered transcriptomes in epithelial cells which are an integral part of NFκB, LARP1, and MYC signaling. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included STAT3, BRD4, SMARCA4, HIF2A/EPAS1, and Inhibin A signaling. BRCA2 mutations were associated with upregulation of IL6, PDK1, FOXO3, and TNFSF11 signaling. These signaling pathway alterations are sufficient to alter sensitivity of BRCA1/BRCA2-mutant breast epithelial cells to transformation as epithelial cells from BRCA1 mutation carriers overexpressing hTERT + PIK3CAH1047R generated adenocarcinomas, whereas similarly modified mutant BRCA2 cells generated basal carcinomas in NSG mice. Thus, our studies provide a high-resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers and reveal their susceptibility to PIK3CA mutation-driven transformation. SIGNIFICANCE: This study provides a single-cell atlas of breast tissues of BRCA1/2 mutation carriers and demonstrates that aberrant signaling due to BRCA1/2 mutations is sufficient to initiate breast cancer by mutant PIK3CA.

HSF1 Inhibits Antitumor Immune Activity in Breast Cancer by Suppressing CCL5 to Block CD8+ T-cell Recruitment.

Heat shock factor 1 (HSF1) is a stress-responsive transcription factor that promotes cancer cell malignancy. To provide a better understanding of the biological processes regulated by HSF1, here we developed an HSF1 activity signature (HAS) and found that it was negatively associated with antitumor immune cells in breast tumors. Knockdown of HSF1 decreased breast tumor size and caused an influx of several antitumor immune cells, most notably CD8+ T cells. Depletion of CD8+ T cells rescued the reduction in growth of HSF1-deficient tumors, suggesting HSF1 prevents CD8+ T-cell influx to avoid immune-mediated tumor killing. HSF1 suppressed expression of CCL5, a chemokine for CD8+ T cells, and upregulation of CCL5 upon HSF1 loss significantly contributed to the recruitment of CD8+ T cells. These findings indicate that HSF1 suppresses antitumor immune activity by reducing CCL5 to limit CD8+ T-cell homing to breast tumors and prevent immune-mediated destruction, which has implications for the lack of success of immune modulatory therapies in breast cancer. SIGNIFICANCE: The stress-responsive transcription factor HSF1 reduces CD8+ T-cell infiltration in breast tumors to prevent immune-mediated killing, indicating that cellular stress responses affect tumor-immune interactions and that targeting HSF1 could improve immunotherapies.

Stromal heterogeneity may explain increased incidence of metaplastic breast cancer in women of African descent.

The biologic basis of genetic ancestry-dependent variability in disease incidence and outcome is just beginning to be explored. We recently reported enrichment of a population of ZEB1-expressing cells located adjacent to ductal epithelial cells in normal breasts of women of African ancestry compared to those of European ancestry. In this study, we demonstrate that these cells have properties of fibroadipogenic/mesenchymal stromal cells that express PROCR and PDGFRα and transdifferentiate into adipogenic and osteogenic lineages. PROCR + /ZEB1 + /PDGFRα+ (PZP) cells are enriched in normal breast tissues of women of African compared to European ancestry. PZP: epithelial cell communication results in luminal epithelial cells acquiring basal cell characteristics and IL-6-dependent increase in STAT3 phosphorylation. Furthermore, level of phospho-STAT3 is higher in normal and cancerous breast tissues of women of African ancestry. PZP cells transformed with HRasG12V ± SV40-T/t antigens generate metaplastic carcinoma suggesting that these cells are one of the cells-of-origin of metaplastic breast cancers.

Combined CDK4/6 and ERK1/2 Inhibition Enhances Antitumor Activity in NF1-Associated Plexiform Neurofibroma.

PURPOSE: Plexiform neurofibromas (PNF) are peripheral nerve sheath tumors that cause significant morbidity in persons with neurofibromatosis type 1 (NF1), yet treatment options remain limited. To identify novel therapeutic targets for PNF, we applied an integrated multi-omic approach to quantitatively profile kinome enrichment in a mouse model that has predicted therapeutic responses in clinical trials for NF1-associated PNF with high fidelity. EXPERIMENTAL DESIGN: Utilizing RNA sequencing combined with chemical proteomic profiling of the functionally enriched kinome using multiplexed inhibitor beads coupled with mass spectrometry, we identified molecular signatures predictive of response to CDK4/6 and RAS/MAPK pathway inhibition in PNF. Informed by these results, we evaluated the efficacy of the CDK4/6 inhibitor, abemaciclib, and the ERK1/2 inhibitor, LY3214996, alone and in combination in reducing PNF tumor burden in Nf1flox/flox;PostnCre mice. RESULTS: Converging signatures of CDK4/6 and RAS/MAPK pathway activation were identified within the transcriptome and kinome that were conserved in both murine and human PNF. We observed robust additivity of the CDK4/6 inhibitor, abemaciclib, in combination with the ERK1/2 inhibitor, LY3214996, in murine and human NF1(Nf1) mutant Schwann cells. Consistent with these findings, the combination of abemaciclib (CDK4/6i) and LY3214996 (ERK1/2i) synergized to suppress molecular signatures of MAPK activation and exhibited enhanced antitumor activity in Nf1flox/flox;PostnCre mice in vivo. CONCLUSIONS: These findings provide rationale for the clinical translation of CDK4/6 inhibitors alone and in combination with therapies targeting the RAS/MAPK pathway for the treatment of PNF and other peripheral nerve sheath tumors in persons with NF1.

The Prolonged Terminal Phase of Human Life Induces Survival Response in the Skin Transcriptome.

Human death marks the end of organismal life under conditions such that the components of the human body continue to be alive. Such postmortem cellular survival depends on the nature (Hardy scale of slow-fast death) of human death. Slow and expected death typically results from terminal illnesses and includes a prolonged terminal phase of life. As such organismal death process unfolds, do cells of the human body adapt for postmortem cellular survival? Organs with low energy cost-of-living, such as the skin, are better suited for postmortem cellular survival. In this work, the effect of different durations of terminal phase of human life on postmortem changes in cellular gene expression was investigated using RNA sequencing data of 701 human skin samples from the Genotype-Tissue Expression (GTEx) database. Longer terminal phase (slow-death) was associated with a more robust induction of survival pathways (PI3K-Akt signaling) in postmortem skin. Such cellular survival response was associated with the upregulation of embryonic developmental transcription factors such as FOXO1 , FOXO3 , ATF4 and CEBPD . Upregulation of PI3K-Akt signaling was independent of sex or duration of death-related tissue ischemia. Analysis of single nucleus RNA-seq of post-mortem skin tissue specifically identified the dermal fibroblast compartment to be most resilient as marked by adaptive induction of PI3K-Akt signaling. In addition, slow death also induced angiogenic pathways in the dermal endothelial cell compartment of postmortem human skin. In contrast, specific pathways supporting functional properties of the skin as an organ were downregulated following slow death. Such pathways included melanogenesis and those representing the skin extracellular matrix (collagen expression and metabolism). Efforts to understand the significance of death as a biological variable (DABV) in influencing the transcriptomic composition of surviving component tissues has far-reaching implications including rigorous interpretation of experimental data collected from the dead and mechanisms involved in transplant-tissue obtained from dead donors.

TONSL Is an Immortalizing Oncogene and a Therapeutic Target in Breast Cancer.

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.

Obesity-induced inflammation exacerbates clonal hematopoiesis.

Characterized by the accumulation of somatic mutations in blood cell lineages, clonal hematopoiesis of indeterminate potential (CHIP) is frequent in aging and involves the expansion of mutated hematopoietic stem and progenitor cells (HSC/Ps) that leads to an increased risk of hematologic malignancy. However, the risk factors that contribute to CHIP-associated clonal hematopoiesis (CH) are poorly understood. Obesity induces a proinflammatory state and fatty bone marrow (FBM), which may influence CHIP-associated pathologies. We analyzed exome sequencing and clinical data for 47,466 individuals with validated CHIP in the UK Biobank. CHIP was present in 5.8% of the study population and was associated with a significant increase in the waist-to-hip ratio (WHR). Mouse models of obesity and CHIP driven by heterozygosity of Tet2, Dnmt3a, Asxl1, and Jak2 resulted in exacerbated expansion of mutant HSC/Ps due in part to excessive inflammation. Our results show that obesity is highly associated with CHIP and that a proinflammatory state could potentiate the progression of CHIP to more significant hematologic neoplasia. The calcium channel blockers nifedipine and SKF-96365, either alone or in combination with metformin, MCC950, or anakinra (IL-1 receptor antagonist), suppressed the growth of mutant CHIP cells and partially restored normal hematopoiesis. Targeting CHIP-mutant cells with these drugs could be a potential therapeutic approach to treat CH and its associated abnormalities in individuals with obesity.

Loss of Dnmt3a impairs hematopoietic homeostasis and myeloid cell skewing via the PI3Kinase pathway.

Loss-of-function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of patients with acute myeloid leukemia (AML) with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early preleukemic event, which - when combined with other genetic lesions - result in full-blown leukemia. Here, we show that loss of Dnmt3a in hematopoietic stem and progenitor cells (HSC/Ps) results in myeloproliferation, which is associated with hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway. PI3Kα/ß or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/ß inhibitor treatment. In vivo RNA-Seq analysis on drug-treated Dnmt3a-/- HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment, and extracellular matrix compared with controls. Remarkably, drug-treated leukemic mice showed a reversal in the enhanced fetal liver HSC-like gene signature observed in vehicle-treated Dnmt3a-/- LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions, including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/ß inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a potentially new target for treating DNMT3A mutation-driven myeloid malignancies.

Use of multimodality imaging, histology, and treatment feasibility to characterize a transgenic Rag2-null rat model of glioblastoma.

Many drugs that show potential in animal models of glioblastoma (GBM) fail to translate to the clinic, contributing to a paucity of new therapeutic options. In addition, animal model development often includes histologic assessment, but multiparametric/multimodality imaging is rarely included despite increasing utilization in patient cancer management. This study developed an intracranial recurrent, drug-resistant, human-derived glioblastoma tumor in Sprague-Dawley Rag2-Rag2 tm1Hera knockout rat and was characterized both histologically and using multiparametric/multimodality neuroimaging. Hybrid 18F-fluoroethyltyrosine positron emission tomography and magnetic resonance imaging, including chemical exchange saturation transfer (18F-FET PET/CEST MRI), was performed for full tumor viability determination and characterization. Histological analysis demonstrated human-like GBM features of the intracranially implanted tumor, with rapid tumor cell proliferation (Ki67 positivity: 30.5 ± 7.8%) and neovascular heterogeneity (von Willebrand factor VIII:1.8 to 5.0% positivity). Early serial MRI followed by simultaneous 18F-FET PET/CEST MRI demonstrated consistent, predictable tumor growth, with exponential tumor growth most evident between days 35 and 49 post-implantation. In a second, larger cohort of rats, 18F-FET PET/CEST MRI was performed in mature tumors (day 49 post-implantation) for biomarker determination, followed by evaluation of single and combination therapy as part of the model development and validation. The mean percentage of the injected dose per mL of 18F-FET PET correlated with the mean %CEST (r = 0.67, P < 0.05), but there was also a qualitative difference in hot spot location within the tumor, indicating complementary information regarding the tumor cell demand for amino acids and tumor intracellular mobile phase protein levels. Finally, the use of this glioblastoma animal model for therapy assessment was validated by its increased overall survival after treatment with combination therapy (temozolomide and idasanutlin) (P < 0.001). Our findings hold promise for a more accurate tumor viability determination and novel therapy assessment in vivo in a recently developed, reproducible, intracranial, PDX GBM.

Inhibition of MEK signaling prevents SARS-CoV2-induced lung damage and improves the survival of infected mice.

Coronavirus disease 2019 (COVID-19) is the illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Over 500 million confirmed cases of COVID-19 have been recorded, with six million deaths. Thus, reducing the COVID-19-related medical burden is an unmet need. Despite a vaccine that is successful in preventing COVID-19-caused death, effective medication to relieve COVID-19-associated symptoms and alleviate disease progression is still in high demand. In particular, one in three COVID-19 patients have signs of long COVID syndrome and are termed, long haulers. At present, there are no effective ways to treat long haulers. In this study, we determine the effectiveness of inhibiting mitogen-activated protein kinase (MEK) signaling in preventing SARS-CoV-2-induced lung damage in mice. We showed that phosphorylation of extracellular signal-regulated kinase, a marker for MEK activation, is high in SARS-CoV-2-infected lung tissues of mice and humans. We also showed that selumetinib, a specific inhibitor of the upstream MEK kinases, reduces cell proliferation, reduces lung damage following SARS-CoV-2 infection, and prolongs the survival of the infected mice. Selumetinib has been approved by the US Food and Drug Administration to treat cancer. Further analysis indicates that amphiregulin, an essential upstream molecule, was upregulated following SARS-CoV-2 infection. Our data suggest that MEK signaling activation represents a target for therapeutic intervention strategies against SARS-CoV-2-induced lung damage and that selumetinib may be repurposed to treat COVID-19.

Metabolic Links to Socioeconomic Stresses Uniquely Affecting Ancestry in Normal Breast Tissue at Risk for Breast Cancer.

A primary difference between black women (BW) and white women (WW) diagnosed with breast cancer is aggressiveness of the tumor. Black women have higher mortalities with similar incidence of breast cancer compared to other race/ethnicities, and they are diagnosed at a younger age with more advanced tumors with double the rate of lethal, triple negative breast cancers. One hypothesis is that chronic social and economic stressors result in ancestry-dependent molecular responses that create a tumor permissive tissue microenvironment in normal breast tissue. Altered regulation of N-glycosylation of proteins, a glucose metabolism-linked post-translational modification attached to an asparagine (N) residue, has been associated with two strong independent risk factors for breast cancer: increased breast density and body mass index (BMI). Interestingly, high body mass index (BMI) levels have been reported to associate with increases of cancer-associated N-glycan signatures. In this study, we used matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) to investigate molecular pattern changes of N-glycosylation in ancestry defined normal breast tissue from BW and WW with significant 5-year risk of breast cancer by Gail score. N-glycosylation was tested against social stressors including marital status, single, education, economic status (income), personal reproductive history, the risk factors BMI and age. Normal breast tissue microarrays from the Susan G. Komen tissue bank (BW=43; WW= 43) were used to evaluate glycosylation against socioeconomic stress and risk factors. One specific N-glycan (2158 m/z) appeared dependent on ancestry with high sensitivity and specificity (AUC 0.77, Brown/Wilson p-value<0.0001). Application of a linear regression model with ancestry as group variable and socioeconomic covariates as predictors identified a specific N-glycan signature associated with different socioeconomic stresses. For WW, household income was strongly associated to certain N-glycans, while for BW, marital status (married and single) was strongly associated with the same N-glycan signature. Current work focuses on understanding if combined N-glycan biosignatures can further help understand normal breast tissue at risk. This study lays the foundation for understanding the complexities linking socioeconomic stresses and molecular factors to their role in ancestry dependent breast cancer risk.

Cortactin loss protects against hemin-induced acute lung injury in sickle cell disease.

In patients with sickle cell disease (SCD), acute chest syndrome (ACS) is a common form of acute lung injury and a major cause of morbidity and mortality. The pathophysiology of ACS is complex, and hemin, the prosthetic moiety of hemoglobin, has been implicated in endothelial cell (EC) activation and subsequent acute lung injury (ALI) and ACS in vitro and in animal studies. Here, we examined the role of cortactin (CTTN), a cytoskeletal protein that regulates EC function, in response to hemin-induced ALI and ACS. Cortactin heterozygous (Cttn+/-) mice (n = 8) and their wild-type siblings (n = 8) were irradiated and subsequently received bone marrow cells (BMCs) extruded from the femurs of SCD mice (SS) to generate SS Cttn+/- and SS CttnWT chimeras. Following hemoglobin electrophoretic proof of BMC transplantation, the mice received 35 µmol/kg of hemin. Within 24 h, surviving mice were euthanized, and bronchoalveolar fluid (BAL) and lung samples were analyzed. For in vitro studies, human lung microvascular endothelial cells (HLMVECs) were used to determine hemin-induced changes in gene expression and reactive oxygen species (ROS) generation in cortactin deficiency and control conditions. When compared with wild-type littermates, the mortality for SS Cttn+/- mice trended to be lower after hemin infusion and these mice exhibited less severe lung injury and less necroptotic cell death. In vitro studies confirmed that cortactin deficiency is protective against hemin-induced injury in HMLVECs, by decreasing protein expression of p38/HSP27, improving cell barrier function, and decreasing the production of ROS. Further studies examining the role of CTTN in ACS are warranted and may open a new avenue of potential treatment for this devastating disease.

Inhibition of BTK and PI3Kδ impairs the development of human JMML stem and progenitor cells.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasia that lacks effective targeted chemotherapies. Clinically, JMML manifests as monocytic leukocytosis, splenomegaly with consequential thrombocytopenia. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in PTPN11 (SHP2), leading to Erk and Akt hyperactivation. Mechanism(s) involved in co-regulation of Erk and Akt in the context of GOF SHP2 are poorly understood. Here, we show that Bruton's tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2-bearing cells and utilizes B cell adaptor for PI3K to cooperate with p110δ, the catalytic subunit of PI3K. Dual inhibition of BTK and p110δ reduces the activation of both Erk and Akt. In vivo, individual targeting of BTK or p110δ in a mouse model of human JMML equally reduces monocytosis and splenomegaly; however, the combined treatment results in a more robust inhibition and uniquely rescues anemia and thrombocytopenia. RNA-seq analysis of drug-treated mice showed a profound reduction in the expression of genes associated with leukemic cell migration and inflammation, leading to correction in the infiltration of leukemic cells in the lung, liver, and spleen. Remarkably, in a patient derived xenograft model of JMML, leukemia-initiating stem and progenitor cells were potently inhibited in response to the dual drug treatment.