Faculty Bibliography

Tissue injury leads to the well-orchestrated mobilization of systemic and local innate and adaptive immune cells. During aging, immune cell recruitment is dysregulated, resulting in an aberrant inflammatory response that is detrimental for successful healing. Here, we precisely define the systemic and local immune cell response after femur fracture in young and aging mice and identify increased toll-like receptor signaling as a potential culprit for the abnormal immune cell recruitment observed in aging animals. Myd88, an upstream regulator of TLR-signaling lies at the core of this aging phenotype, and local treatment of femur fractures with a Myd88 antagonist in middle-aged mice reverses the aging phenotype of impaired fracture healing, thus offering a promising therapeutic target that could overcome the negative impact of aging on bone regeneration.

Neurodegenerative diseases are debilitating impairments that affect millions of people worldwide and are characterized by progressive degeneration of structure and function of the central or peripheral nervous system. Effective biomarkers for neurodegenerative diseases can be used to improve the diagnostic workup in the clinic as well as facilitate the development of effective disease-modifying therapies. Progranulin (PGRN) has been reported to be involved in various neurodegenerative disorders. Hence, in the current study we systematically compared the inflammation and accumulation of typical neurodegenerative disease markers in the brain tissue between PGRN knockout (PGRN KO) and wildtype (WT) mice. We found that PGRN deficiency led to significant neuron loss as well as activation of microglia and astrocytes in aged mice. Several characteristic neurodegenerative markers, including α-synuclein, TAR DNA-binding protein 43 (TDP-43), Tau, and β-amyloid, were all accumulated in the brain of PGRN-deficient mice as compared to WT mice. Moreover, higher aggregation of lipofuscin was observed in the brain tissue of PGRN-deficient mice compared with WT mice. In addition, the autophagy was also defective in the brain of PGRN-deficient mice, indicated by the abnormal expression level of autophagy marker LC3-II. Collectively, comprehensive assays support the idea that PGRN plays an important role during the development of neurodegenerative disease, indicating that PGRN might be a useful biomarker for neurodegenerative diseases in clinical settings.

Primary age-related tauopathy (PART) is a neurodegenerative pathology with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-β (Aβ) plaques. The pathogenesis of PART is not known, but evidence suggests an association with genes that promote tau pathology and others that protect from Aβ toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n = 647) using Braak neurofibrillary tangle stage as a quantitative trait. We found some significant associations with candidate loci associated with AD (SLC24A4, MS4A6A, HS3ST1) and progressive supranuclear palsy (MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brains with four microtubule-binding domain repeats (4R) isoforms and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation in post-mortem human PART brain tissue revealed a specific binding of JADE1 protein to four repeat tau lacking N-terminal inserts (0N4R). Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model, as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a modifier of neurofibrillary degeneration.

Unlike other checkpoint inhibitors, our targeted immunotherapeutic localizes to any solid tumor and simultaneouslyshields an agent of immuno suppression while presenting a signal for immunostimulation. Phosphatidylserine (PS)exposure on the extracellular surface of living tumor cells and their vasculatures provides one avenue by which thetumor microenvironment promotes immunosuppression. Extracellular surface PS is inherent to a tumor and itsvasculature, even for inoperable tumors, and its expression cannot be mutated nor affected by acquired drugresistance. Annexin A5 (AnxA5) is a direct, high-affinity PS-binding protein that localizes to cells with PS exposed onthe outer plasma membrane. In our studies, we conjugated a proprietary modified AnxA5, lacking cellularinternalization, to TNFalpha (AnxA5 -TNFalpha) to convert the immunosuppresive environs of a murine 4T1 triplenegative breast cancer (TNBC) into an immunostimulated one. This strategy localized the immune response to the tumor and minimized side effects, as evidenced by a lack of toxicity for up to 7 days in non-tumor bearing Balb/cfemale mice given up to 1 mg/kg. Proper assembly and functionality of AnxA5 -TNFalpha was verified simultaneouslyby ellipsometry, an optical technique similar to plasmon resonance. Fully assembled constructs were tested forbinding to PS coated slides. The degree of light polarization is proportional to the amount of PS bound by the AnxA5complex. Samples could be further incubated with TNF receptors to verify TNFalpha activity. Based on dose escalationstudies in 4T1 tumor-bearing mice where the TNBC tumors were grown in the mammary fat pads, optimal dosages were determined for AnxA5 -TNFalpha (18 mug) and AnxA5 alone as a control (180 mug). These doses were furthertested in a 4T1 growth inhibition study. Tumor size was tracked by caliper in two groups of mice (n=5/group)receiving drug treatment on days 12, 14 and 16 and a repeated measures ANOVA was conducted onmeasurements taken before, during and post-treatment. While median tumor size did not differ between control and drug treatment groups during the pre-treatment interval (p=0.84), there was a significant difference post-treatment(p<0.001) with mice receiving AnxA5 -TNFalpha having much smaller TNBC tumors. Tumors from the study were embedded in paraffin, sectioned (5 mum) and the overall immune cell content determined by H&E staining. Once it was evident there was a greater quantity of immune cells in AnxA5 -TNFalpha treated tumors vs. controls, sections were stained with validated antibodies to identify and count the immunoactivated T-cells, NK-cells and macrophages. There was a 3X greater mean percentage of CD8 and CD4 T-cells in mice receiving drug vs. control(p=0.03) along with 2.5X and 5X increases in NK-cells and M1 immunoactive macrophages, respectively.
Conclusion(s): Our AnxA5 -TNFalpha inhibits the PS inhibitor while simultaneously activating TNF activators!

Transforming growth factor β (TGFβ) is a pleiotropic cytokine that plays critical roles to define cancer cell phenotypes, construct the tumor microenvironment, and suppress anti-tumor immune responses. As such, TGFβ is a lynchpin for integrating cancer cell intrinsic pathways and communication among host cells in the tumor and beyond that together affect responses to genotoxic, targeted, and immune therapy. Despite decades of preclinical and clinical studies, evidence of clinical benefit from targeting TGFβ in cancer remains elusive. Here, we review the mechanisms by which TGFβ acts to oppose successful cancer therapy, the reported prognostic and predictive value of TGFβ biomarkers, and the potential impact of inhibiting TGFβ in precision oncology. Paradoxically, the diverse mechanisms by which TGFβ impedes therapeutic response are a principal barrier to implementing TGFβ inhibitors because it is unclear which TGFβ mechanism is functional in which patient. Companion diagnostic tools and specific biomarkers of TGFβ targeted biology will be the key to exploiting TGFβ biology for patient benefit.

SHP2 inhibitors (SHP2i) alone and in various combinations are being tested in multiple tumors with over-activation of the RAS/ERK pathway. SHP2 plays critical roles in normal cell signaling; hence, SHP2is could influence the tumor microenvironment. We found that SHP2i treatment depleted alveolar and M2-like macrophages, induced tumor-intrinsic CCL5/CXCL10 secretion and promoted B and T lymphocyte infiltration in Kras- and Egfr-mutant non-small cell lung cancer (NSCLC). However, treatment also increased intratumor gMDSCs via tumor-intrinsic, NF-kB-dependent production of CXCR2 ligands. Other RAS/ERK pathway inhibitors also induced CXCR2 ligands and gMDSC influx in mice, and CXCR2 ligands were induced in tumors from patients on KRASG12C-inhibitor trials. Combined SHP2(SHP099)/CXCR1/2(SX682) inhibition depleted a specific cluster of S100a8/9high gMDSCs, generated Klrg1+ CD8+ effector T cells with a strong cytotoxic phenotype but expressing the checkpoint receptor NKG2A, and enhanced survival in Kras- and Egfr-mutant models. Our results argue for testing RAS/ERK pathway/CXCR1/2/NKG2A inhibitor combinations in NSCLC patients.

Objectives: Despite availability of new treatments, the prognosis of lung cancer remains poor. This study aims to provide recent estimates of survival in patients with advanced non-small cell lung cancer (NSCLC) in the US.
Method(s): The survival of patients with advanced NSCLC was estimated using two US databases together covering 2010-2020. The study included patients with stage III or IV NSCLC diagnosed between 2010-2016 in the Surveillance, Epidemiology, and End Results Program (SEER) database, and patients with stage IIIB, IIIC or IV NSCLC, diagnosed between 2017-2020, without known oncogenic driver mutations who had completed >=4 cycles of 1L treatment (restricted to platinum-based combinations, immuno-oncology monotherapy, or ipilimumab/nivolumab) in the Flatiron Health database, a US Oncology Electronic Medical Record database. Overall survival (OS) was defined as time from diagnosis of stage III or IV NSCLC to death or to date of last confirmed activity.
Result(s): A total of 49,298 and 133,395 patients with stage III and IV diagnosis respectively were identified in SEER. The 1-, 3- and 5-year OS for patients with Stage III disease were 55.1%, 26.3% and 17.5%, and for stage IV disease were 25.8%, 7.4% and 4.0%, respectively. The Flatiron database had 1,045 patients with stage IIIB, 130 patients with stage IIIC and 3,210 patients with stage IV disease at diagnosis. The 1- and 3-year OS for stage IIIB/IIIC disease were 72.5% and 36.4%, and for patients with stage IV disease were 65.9% and 24.6%, respectively.
Conclusion(s): Despite differences in study population characteristics between the two databases, the study shows that mortality in patients with advanced NSCLC remains high, underscoring the need for continued efforts to identify novel treatments and synergetic treatment combinations to improve patient outcomes.
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The purpose of this review is to discuss the transforming growth factor beta (TGFB) binding proteins (LTBP) with respect to their participation in the activity of TGFB. We first describe pertinent aspects of the biology and cell function of the LTBPs. We then summarize the physiological consequences of LTBP loss in humans and mice. Finally, we consider a number of outstanding questions relating to LTBP function.