Parathyroid hormone-related protein (PTHrP) is a polyhormone consisting of an N-terminus, a mid-region, a nuclear localization sequence (NLS), and a C-terminus. The NLS and C-terminus of PTHrP regulate endochondral bone formation, craniofacial development, hematopoiesis, and survival. Our laboratory has developed Pthrp Δ/Δ mice lacking the NLS and C-terminus of PTHrP, which exhibit severe growth delay and early mortality within the first week of life. This study investigates the growth hormone (GH)-IGF-1 axis in Pthrp Δ/Δ mice. PTHrP is expressed by various endocrine cells, including pituitary endocrine epithelial cells of the adenohypophysis. Histopathological, biochemical, ultrastructural, and gene expression analyses were performed on the pituitary from Pthrp Δ/Δ and age-matched control mice. Pituitary glands from Pthrp Δ/Δ mice had normal cellularity; however, the pituitary somatotrophs had increased Gh mRNA expression with a decrease in the number and size of cytoplasmic secretory granules containing GH. Plasma GH concentrations were either normal or increased, and plasma ACTH concentrations were increased. Western blot analysis of the pituitary glands revealed reduced GH in Pthrp Δ/Δ mice. The plasma concentrations of IGF-1 and liver Igf1 mRNA expression and glycogen content were decreased in Pthrp Δ/Δ mice. Western blot analysis of liver from Pthrp Δ/Δ mice showed a significant reduction in both total and phosphorylated Janus Kinase 2 (JAK2) proteins and total and tyrosine-phosphorylated STAT5b proteins compared to controls. In conclusion, the lack of the NLS and C-terminus of PTHrP disrupted the normal GH-IGF-1 axis, leading to impaired IGF-1 production by the liver.

Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hamper our understanding of both SON and this complex syndrome. Here, we developed Son haploinsufficiency ( Son +/− ) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son +/− mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son +/− mice, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency inclines cell fate toward the myeloid lineage but compromises lymphoid lineage development by reducing key genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythroid maturation. These findings highlight the importance of the full gene dosage of Son in organ development and hematopoiesis. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

Supplementary Data from Stromal p53 Regulates Breast Cancer Development, the Immune Landscape, and Survival in an Oncogene-Specific Manner

Osteoblastic bone metastases are commonly detected in patients with advanced prostate cancer (PCa) and are associated with an increased mortality rate. Dickkopf-1 (DKK-1) antagonizes canonical WNT/β-catenin signaling and plays a complex role in bone metastases. We explored the function of cancer cell-specific DKK-1 in PCa growth, metastasis, and cancer–bone interactions using the osteoblastic canine PCa cell line, Probasco. Probasco or Probasco + DKK-1 (cells transduced with human DKK-1) were injected into the tibia or left cardiac ventricle of athymic nude mice. Bone metastases were detected by bioluminescent imaging in vivo and evaluated by micro-computed tomography and histopathology. Cancer cell proliferation, migration, gene/protein expression, and their impact on primary murine osteoblasts and osteoclasts, were evaluated in vitro. DKK-1 increased cancer growth and stimulated cell migration independent of canonical WNT signaling. Enhanced cancer progression by DKK-1 was associated with increased cell proliferation, up-regulation of NF-kB/p65 signaling, inhibition of caspase-dependent apoptosis by down-regulation of non-canonical WNT/JNK signaling, and increased expression of epithelial-to-mesenchymal transition genes. In addition, DKK-1 attenuated the osteoblastic activity of Probasco cells, and bone metastases had decreased cancer-induced intramedullary woven bone formation. Decreased bone formation might be due to the inhibition of osteoblast differentiation and stimulation of osteoclast activity through a decrease in the OPG/RANKL ratio in the bone microenvironment. The present study indicated that the cancer-promoting role of DKK-1 in PCa bone metastases was associated with increased growth of bone metastases, reduced bone induction, and altered signaling through the canonical WNT-independent pathway. DKK-1 could be a promising therapeutic target for PCa.

Osteoblastic bone metastasis is the predominant phenotype observed in advanced prostate cancer and is associated with high patient mortality and morbidity. WNT signaling is necessary for both bone formation and tumor progression. Dickkopf-1 (DKK-1), a secretory antagonist of the canonical WNT/β-catenin signaling pathway, plays a complex role in bone tumors since it can either promote or decrease tumor occurrence and metastasis. The present study explored the molecular role of DKK-1 in prostate tumor progression, metastasis, and tumor-bone interactions using the osteoblastic canine prostate cancer cell line, Probasco. Previously, we found that DKK-1 can inhibit bone mineralization of mouse calvaria by Probasco conditioned medium (CM). Now we stably expressed DKK-1 in Probasco cells (Probasco-DKK-1), which were injected into the tibias or left ventricle of athymic mice. Bone metastases were monitored by bioluminesent imaging, microCT, and histopathology. Our results showed that Probasco-DKK-1 bone metastases had increased tumor growth, increased osteoclastic bone resorption, and decreased intramedullary woven bone formation in vivo. In vitro, DKK-1 had an autocrine effect on Probasco cells, which altered the cell morphology, increased cell proliferation, and induced EMT. Mechanistically, DKK-1 had little effect on the canonical WNT/β-catenin signaling while dramatically down-regulated the non-canonical WNT/JNK signaling, which inhibited caspase-dependent apoptosis in Probasco-DKK-1 cells. To investigate the effect of Probasco-DKK-1 on bone cells, primary osteoblasts and osteoclasts were treated by CM collected from cancer cells. We found that DKK-1 did not inhibit the mineralization of osteoblasts nor stimulate the activity of osteoclasts directly, but the expression of RNAKL was significantly higher in Probasco-DKK-1 CM-treated osteoblasts. Therefore, the Probasco-DKK-1 cells enhanced the activity of osteoclasts indirectly via the regulation of osteoblasts, which induced bone resorption. In conclusion, we showed that DKK-1 promoted prostate tumor growth by stimulating cell proliferation and inhibiting apoptosis in a canonical WNT-independent manner and attenuated the osteoblastic activity of prostate cancer. These findings provide new insights into the molecular mechanisms of DKK-1 in tumor progression. Citation Format: Shiyu Yuan, Nathan K. Hoggard, Noriko Kantake, Blake E. Hildreth, Thomas J. Rosol. The effect of dickkopf-1 (DKK-1) on prostate cancer growth and bone metastasis using the canine osteoblastic Probasco cell line [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 1603.

Pancreatic ductal adenocarcinoma (PDAC) is associated with an incredibly dense stroma, which contributes to its recalcitrance to therapy. Cancer-associated fibroblasts (CAFs) are one of the most abundant cell types within the PDAC stroma and have context-dependent regulation of tumor progression in the tumor microenvironment (TME). Therefore, understanding tumor-promoting pathways in CAFs is essential for developing better stromal targeting therapies. Here, we show that disruption of the STAT3 signaling axis via genetic ablation of Stat3 in stromal fibroblasts in a Kras G12D PDAC mouse model not only slows tumor progression and increases survival, but re-shapes the characteristic immune-suppressive TME by decreasing M2 macrophages (F480+CD206+) and increasing CD8 + T cells. Mechanistically, we show that loss of the tumor suppressor PTEN in pancreatic CAFs leads to an increase in STAT3 phosphorylation. In addition, increased STAT3 phosphorylation in pancreatic CAFs promotes secretion of CXCL1. Inhibition of CXCL1 signaling inhibits M2 polarization in vitro. The results provide a potential mechanism by which CAFs promote an immune-suppressive TME and promote tumor progression in a spontaneous model of PDAC.

Abstract Coevolution of tumor cells and adjacent stromal elements is a key feature during tumor progression; however, the precise regulatory mechanisms during this process remain unknown. Here, we show stromal p53 loss enhances oncogenic KrasG12D, but not ErbB2, driven tumorigenesis in murine mammary epithelia. Stroma-specific p53 deletion increases both epithelial and fibroblast proliferation in mammary glands bearing the KrasG12D oncogene in epithelia, while concurrently increasing DNA damage and/or DNA replication stress and decreasing apoptosis in the tumor cells proper. Normal epithelia was not affected by stromal p53 deletion. Tumors with p53-null stroma had a significant decrease in total, cytotoxic, and regulatory T cells; however, there was a significant increase in myeloid-derived suppressor cells, total macrophages, and M2-polarized tumor-associated macrophages, with no impact on angiogenesis or connective tissue deposition. Stroma-specific p53 deletion reprogrammed gene expression in both fibroblasts and adjacent epithelium, with p53 targets and chemokine receptors/chemokine signaling pathways in fibroblasts and DNA replication, DNA damage repair, and apoptosis in epithelia being the most significantly impacted biological processes. A gene cluster in p53-deficient mouse fibroblasts was negatively associated with patient survival when compared with two independent datasets. In summary, stroma-specific p53 loss promotes mammary tumorigenesis in an oncogene-specific manner, influences the tumor immune landscape, and ultimately impacts patient survival. Implications: Expression of the p53 tumor suppressor in breast cancer tumor stroma regulates tumorigenesis in an oncogene-specific manner, influences the tumor immune landscape, and ultimately impacts patient survival.

Adult T-cell leukemia/lymphoma (ATL) is an intractable disease affecting nearly 4% of Human T-cell Leukemia Virus Type 1 (HTLV-1) carriers. Acute ATL has a unique interaction with bone characterized by aggressive bone invasion, osteolytic metastasis, and hypercalcemia. We hypothesized that dual tumor and bone-targeted therapies would decrease tumor burden in bone, the incidence of metastasis, and ATL-associated osteolysis. Our goal was to evaluate dual targeting of both ATL bone tumors and the bone microenvironment using an anti-tumor HDACi (AR-42) and an osteoclast inhibitor (zoledronic acid, Zol), alone and in combination. Our results showed that AR-42, Zol, and AR-42/Zol significantly decreased the viability of multiple ATL cancer cell lines in vitro. Zol and AR-42/Zol decreased tumor growth in vivo. Zol ± AR-42 significantly decreased ATL-associated bone resorption and promoted new bone formation. AR-42-treated ATL cells had increased mRNA levels of PTHrP, ENPP2 (autotaxin) and MIP-1α, and TAX viral gene expression. AR-42 alone had no significant effect on tumor growth or osteolysis in mice. These findings indicate that Zol adjuvant therapy has the potential to reduce growth of ATL in bone and its associated osteolysis.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Human T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia, a disease commonly associated with hypercalcemia and osteolysis. There is no effective treatment for HTLV-1, and the osteolytic mechanisms are not fully understood. Mice expressing the HTLV-1 oncogene Tax, driven by the human granzyme B promoter (Tax+), develop osteolytic tumors. To investigate the progression of the bone-invasive malignancies, wild-type, Tax+, and Tax+/interferon-γ-/- mice were assessed using necropsy, histologic examination, IHC analysis, flow cytometry, and advanced imaging. Tax+ and Tax+/interferon-γ-/- malignancies of the ear, tail, and foot comprised poorly differentiated, round to spindle-shaped cells with prominent neutrophilic infiltrates. Tail tumors originated from muscle, nerve, and/or tendon sheaths, with frequent invasion into adjacent bone. F4/80+ and anti-mouse CD11b (Mac-1)+ histiocytic cells predominated within the tumors. Three Tax+/interferon-γ-/- cell lines were generated for in vivo allografts, in vitro gene expression and bone resorption assays. Two cell lines were of monocyte/macrophage origin, and tumors formed in vivo in all three. Differences in Pthrp, Il6, Il1a, Il1b, and Csf3 expression in vitro were correlated with differences in in vivo plasma calcium levels, tumor growth, metastasis, and neutrophilic inflammation. Tax+ mouse tumors were classified as bone-invasive histiocytic sarcomas. The cell lines are ideal for further examination of the role of HTLV-1 Tax in osteolytic tumor formation and the development of hypercalcemia and tumor-associated inflammation.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Canine distemper and parvovirus infections are amongst of the most important infectious diseases of dogs. yntneceR, the range of reports of vaccinated dogs that developed canine distemper and parvovirus infections have multiplied throughout the world. The goals of this study were to evaluate the immune and hematological responses against canine distemper virus (CDV) and canine parvovirus (CPV) after vaccination using two commercially available vaccines. In addition, the antigenic differences between the vaccine strains of CDV and CPV and the current wild-type strains were analyzed to determine possible relationships between these vaccine strains and recent field outbreaks in vaccinated populations. The immune response of both vaccines was determined using serum neutralization and Dot-ELISA. Specific neutralizing antibodies against canine distemper and parvovirus were present in the sera of dogs three weeks after the initial vaccination for both vaccines. Peak antibody titers were documented from the second month post vaccination. Both vaccines were demonstrated to be safe and stimulate a humoral immune response. Results of serum neutralization test and Dot-ELISA were closely similar and correlated with each other. Genetic variations between strains included in both commercial vaccines and circulating wild-type strains suggest that variant strains are most likely responsible for field outbreaks of CDV and CPV in vaccinated dogs.

One of the defining characteristics of pancreatic ductal adenocarcinoma (PDAC) is the formation of a dense stroma comprised of cancer associated fibroblasts (CAFs) and immune cell populations. This stroma is immunosuppressive and can act as a physical barrier against common therapeutic treatments. Attempts to therapeutically target the PDAC stroma have yielded contradictory results, suggesting both tumor promoting and tumor limiting roles for CAFs. These studies emphasize the need to understand important transsignaling pathways between CAFs, tumor cells, and the immune microenvironment. IL-6 is a pleiotropic cytokine involved in several physiological functions and its increased expression is strongly associated with poor survival rates in PDAC patients. STAT3 is a major downstream target of IL-6, and its aberrant activation has been implicated in PDAC tumor progression and immune evasion. IL-6 expression and the IL-6/STAT3 signaling axis in PDAC has been characterized in epithelial tumor cells, however its stromal-specific function on PDAC has yet to be elucidated. We hypothesized that the STAT3 signaling axis in pancreatic CAFs contributes to the immunosuppressive and fibrotic phenotype seen with disease progression. Employing CreLoxP technology, the fibroblast specific protein-1 (Fsp-Cre) transgene was used to conditionally delete STAT3 in fibroblasts in the PdxFlp; KrasG12D; p53 frt/frt (KPF) PDAC mouse model developed by our lab. Deletion of STAT3 in fibroblasts significantly increased the survival in a cohort of KPF mice compared to those with intact STAT3. Loss of fibroblast STAT3 also resulted in a significant decrease in ECM deposition in the pancreas. In preliminary investigations into the immune microenvironment, we found an increase in overall T cell infiltration and decrease in regulatory T cell populations in the STAT3 deleted cohort. Further, STAT3 deletion resulted in an increase in the ratio of M1 to M2 macrophages within the PDAC stroma, suggesting CAF STAT3 signaling is important to maintaining an immune microenvironment conducive to tumor progression. Ultimately, these preliminary results suggest STAT3 signaling in PDAC CAFs contributes to an overall survival disadvantage, an immunosuppressive tumor microenvironment, and an increase in ECM deposition. Citation Format: Julia E. Lefler, Katie MarElia, Blake E. Hildreth, Katie A. Thies, Maria C. Cuitino, Michael C. Ostrowski. STAT3 in cancer-associated fibroblasts promotes an immunosuppressive tumor microenvironment in PDAC [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-016.

Platelet-derived growth factor receptor-beta (PDGFRβ) is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand PDGFB is released by neighboring epithelial and endothelial cells. While expression of both PDGFRβ and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRβ tumor-stroma signaling mediates breast cancer initiation, progression, and metastasis remains unclear. Here we demonstrate this paracrine signaling pathway that mediates both primary tumor growth and metastasis, specifically, metastasis to the brain. Elevated levels of PDGFB accelerated orthotopic tumor growth and intracranial growth of mammary tumor cells, while mesenchymal-specific expression of an activating mutant PDGFRβ (PDGFRβD849V) exerted proproliferative signals on adjacent mammary tumor cells. Stromal expression of PDGFRβD849V also promoted brain metastases of mammary tumor cells expressing high PDGFB when injected intravenously. In the brain, expression of PDGFRβD849V was observed within a subset of astrocytes, and aged mice expressing PDGFRβD849V exhibited reactive gliosis. Importantly, the PDGFR-specific inhibitor crenolanib significantly reduced intracranial growth of mammary tumor cells. In a tissue microarray comprised of 363 primary human breast tumors, high PDGFB protein expression was prognostic for brain metastases, but not metastases to other sites. Our results advocate the use of mice expressing PDGFRβD849V in their stromal cells as a preclinical model of breast cancer-associated brain metastases and support continued investigation into the clinical prognostic and therapeutic use of PDGFB-to-PDGFRβ signaling in women with breast cancer. SIGNIFICANCE: These studies reveal a previously unknown role for PDGFB-to-PDGFRβ paracrine signaling in the promotion of breast cancer brain metastases and support the prognostic and therapeutic clinical utility of this pathway for patients.See related article by Wyss and colleagues, p. 594.

Angiogenesis is a highly orchestrated process involving complex crosstalk between several endothelial cell processes including cell cycle, cell survival and migration. Transcription factors ETS1 and ETS2 are required for EC functions necessary for embryonic angiogenesis. Here, by utilizing endothelial cell-specific deletion of Ets1 and Ets2 on post-natal angiogenesis we demonstrate that ETS1 and ETS2 have a redundant regulatory role in controlling the expression of key G2/M regulators and anti-apoptotic gene modules by recruiting the transcriptional activator CBP/p300 to the enhancers of these genes. Further, cultured aortic ECs lacking both ETS1 and ETS2 demonstrated G2/M phase arrest and increased apoptosis. In vivo , EC infiltration and invasion was attenuated when matrigel admixed with mouse mammary tumor cells was injected into adult mice with EC-specific ablation of Ets1 and Ets2 . These results demonstrate that deletion of Ets1 and Ets2 in endothelial cells inhibits angiogenesis by altering cell cycle progression and decreasing cell survival.

Abstract PDGFRβ is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand, PDGFB, is released by neighboring epithelial and endothelial cells. While expression of both PDGFRβ and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRβ tumor-stromal signaling mediates breast cancer initiation, progression, and metastasis remains unclear. To test this important research question, we developed a mouse model of mesenchymal-specific PDGFRβ hyper-activation. PDGFRβ mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRβ effect on neighboring epithelium during normal development. To test the effect of hyper-active mesenchymal PDGFRβ on disease progression, experimental tail vein metastasis assays were performed where we observed prominent brain metastases in 50% of the PDGFRβ mutantmice (n=5/10) with no brain lesions seen in controls (n=0/19). There was no difference in the incidence of lung or liver metastases in the mutant mice suggesting a pro-metastatic function for PDGFRβ in the brain metastatic niche. To rule out dysfunction of the blood brain barrier contributing to the observed metastatic spread, we then intracranially injected mammary tumor cells, and as expected based on our metastasis assay, found that larger tumors formed in the brains of PDGFRβ mutant mice versus controls. To our knowledge, these combined findings are the first example where genetic manipulation of the stroma increases breast cancer associated brain metastases (BCBM). Given that these pre-clinical data suggest that primary breast tumors expressing high PDGFB could preferentially metastasize to the brain, we analyzed PDGFB protein expression in a tissue microarray comprised of HER2-positive and triple negative breast cancer (TNBC) primary tumors (total n=425). While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Evaluation of PDGFB in a small cohort of matched primary breast tumors with associated brain (n=5) and lung metastases (n=2) revealed intense PDGFB staining in 100% of the brain metastases, but only 50% of the lung metastases. These findings further suggest that high primary tumor PDGFBexpression defines a subset of breast cancer patients predisposed to brain metastases and that these patients may benefit from therapeutic inhibition of PDGFRβ signaling. To test this pre-clinically, we treated mice harboring intracranial tumors with the PDGFR specific inhibitor crenolanib. Excitingly, crenolanib treatment significantly inhibited the brain tumor burden in these mice. Combined, our findings to date (1) advocate that primary tumor expression of PDGFB is a novel prognostic biomarker for the development of BCBM and (2) support clinical trial evaluation of PDGFR inhibitors for the prevention and treatment of BCBM. Ongoing studies are evaluating how the PDGFRβ-expressing mesenchymal cells within the brain promote a pro-metastatic niche. Citation Format: Sizemore GM, Hammer AM, Thies KA, Hildreth BE, Russell LO, Sizemore ST, Trimboli AJ, Kladney RD, Steck SA, Das M, Bolyard CM, Pilarski R, Schoenfield L, Otero J, Chakravarti A, Ringel M, Kaur B, Leone G, Ostrowski MC. Platelet derived growth factor receptor-β signaling: A novel therapeutic target for breast cancer associated brain metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-11.

Genome-wide association studies (GWASs) have been instrumental in understanding complex phenotypic traits. However, they have rarely been used to understand lineage-specific pathways and functions that contribute to the trait. In this study, by integrating lineage-specific enhancers from mesenchymal and myeloid compartments with bone mineral density loci, we were able to segregate osteoblast- and osteoclast (OC)-specific functions. Specifically, in OCs, a PU.1-dependent transcription factor (TF) network was revealed. Deletion of PU.1 in OCs in mice resulted in severe osteopetrosis. Functional genomic analysis indicated PU.1 and MITF orchestrated a TF network essential for OC differentiation. Several of these TFs were regulated by cooperative binding of PU.1 with BRD4 to form superenhancers. Further, PU.1 is essential for conformational changes in the superenhancer region of Nfatc1. In summary, our study demonstrates that combining GWASs with genome-wide binding studies and model organisms could decipher lineage-specific pathways contributing to complex disease states.

Abstract Stromal PDGFRβ has emerged as an actionable mediator of breast tumor-stromal communication. As a receptor tyrosine kinase, PDGFRβ is activated by its ligand, PDGFB, which is released by neighboring tumor epithelium and endothelium. However, how PDGF signaling mediates breast cancer initiation, progression, and metastasis remains unclear. To evaluate PDGFRβ in this disease, we developed a mouse model of stromal-specific PDGFRβ activation using the Fsp-cre transgene previously published by our group (PDGFRβ mutant). PDGFRβ mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRβ effect on neighboring epithelium. To evaluate the functional relevance of PDGFRβ activation on metastatic progression, we performed tail vein injection of PDGFB expressing murine mammary tumor cells and, surprisingly, observed brain metastases in 50% of the PDGFRβ mutant mice while no brain lesions were seen in controls. There was no difference in the incidence of lung, liver or bone metastases. Mammary tumor cells expressing low PDGFB did not exhibit a similar increase in brain metastases in mutant mice. While there is no observable difference in blood brain barrier permeability in the mutant mice, we bypassed this variable by intracranially injecting mammary tumor cells and found that larger tumors formed in the brains of PDGFRβ mutant mice versus controls. To our knowledge, this is the first example where genetic manipulation of the stroma leads to an increased incidence of BCBM. Also, our pre-clinical data suggests that primary breast tumors that express high PDGFB could preferentially metastasize to the brain. To test this in patients, we analyzed PDGFB protein expression in a tissue microarray comprised of HER2-positive and triple negative breast cancer (TNBC) primary tumors. While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Evaluation of PDGFB in a small cohort of matched primary breast tumors with associated brain (n=5) and lung metastases (n=2) revealed intense PDGFB staining in 100% of the brain metastases, but only 50% of the lung metastases. Our findings suggest high primary tumor PDGFB expression defines a subset of breast cancer patients predisposed to brain metastases. These patients may benefit from therapeutic intervention of PDGFRβ signaling. To test this pre-clinically, we treated mice harboring intracranial tumors with the PDGFR specific inhibitor, Crenolanib. Excitingly, Crenolanib treatment significantly inhibited the brain tumor burden in these mice. Combined, our findings (1) advocate that primary tumor expression of PDGFB is a novel prognostic biomarker for the development of BCBM and (2) support clinical trial evaluation of PDGFR inhibitors for the prevention and treatment of BCBM. Citation Format: Katie A. Thies, Anisha M. Hammer, B. Eason Hildreth, Luke O. Russell, Steven T. Sizemore, Anthony J. Trimboli, Raleigh D. Kladney, Chelsea M. Bolyard, Robert Pilarski, Lynn Schoenfield, Jose Otero, Arnab Chakravarti, Matthew Ringel, Balveen Kaur, Gustavo Leone, Michael C. Ostrowski, Gina M. Sizemore. Stromal platelet derived growth factor receptor (PDGFRβ) signaling: A novel therapeutic target for breast cancer brain metastasis (BCBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 49.