Faculty Bibliography

RAS proteins control signals required for cell growth and survival and, when constitutively activated by mutation, can drive oncogenesis. RAS proteins are primarily regulated by their GTP or GDP binding state, which is controlled by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). RAS proteins are also substrates for dozens of posttranslational modifications (PTMs) that target them to membranes and serve as a secondary means of regulation. Because the newly developed direct RAS inhibitors do not produce durable responses in RAS-dependent cancer, there is renewed interest in targeting the PTMs of RAS. These modifications are the subject of this review.

Aneuploidy is common in cancer and has been implicated in promoting tumor progression, yet the underlying mechanisms remain poorly understood. By generating models of aneuploidy, we found that aneuploidy confers resistance to reactive oxygen species (ROS)-mediated cell death, independent of the specific chromosomes gained or lost. Mechanistically, poly(ADP-ribose) polymerase 1 (PARP1) is suppressed in aneuploid cells, which inhibits PARP1-mediated cell death (parthanatos). We validated aneuploidy-associated PARP1 suppression across 15 cell models and human tumors, with pronounced effects in metastatic tumors. Importantly, PARP1 downregulation promotes tumor metastasis while PARP1 upregulation suppresses it. Through a genome-wide CRISPR screen and functional validation, we identified the transcription factor CCAAT/enhancer-binding protein beta (CEBPB) as a mediator of PARP1 downregulation and ROS resistance in aneuploid cells. Lysosomal dysfunction serves as the upstream activator of CEBPB in aneuploid cells. We propose that aneuploidy-driven CEBPB activation suppresses PARP1, fostering ROS resistance and cancer progression.

Over the past decade, the field of human embryo editing has witnessed remarkable advancements and triggered significant ethical debates. The groundbreaking tool, CRISPR/Cas9, has revolutionized the landscape of genetic engineering by enabling modifications at the genomic level in germ cells. Since the first case of human embryo gene editing in 2015, the field has rapidly progressed, presenting promising avenues for therapeutic interventions. However, it still grapples with safety concerns, including off-target effects, mosaicism, and the long-term impacts of genetic alterations, as well as ongoing ethical controversies. In this review, we will systematically overview the significant research in this field and provide insights into the potential applications of basic research in early embryonic development and the treatment of genetic diseases.

INTRODUCTION/BACKGROUND:The study aimed to investigate the efficacy of autogenous iliac crest bone graft (ICBG) compared with other graft types in achieving successful fracture nonunion repair. METHODS:An institutional review board-approved retrospective review of prospectively collected data was conducted on a consecutive series of patients surgically treated for fracture nonunions at an academic medical center between September 10, 2004, and August 20, 2023. Patients were analyzed based on which bone graft type-ICBG versus alternative graft types-used during their nonunion repair. Patient demographics, injury characteristics, and surgical history were compared. Outcomes included radiographic healing, time to union, postoperative complications, and revision rate. Cohorts were compared using an independent sample Student t-test for continuous variables and chi-square or Fisher exact tests for categorical variables. One-way analysis of variance with post hoc comparisons assessed differences across treatment strategy groups. RESULTS:Five hundred fifty-six patients were treated surgically for a fracture nonunion using standard internal fixation and a "bone graft" for biologic stimulation. 57.4% of these patients were treated with autogenous ICBG; 42.6% received alternative grafts (iliac crest aspirate, allograft, bone morphogenetic, reamer-irrigation aspirator, and/or demineralized bone matrix, without autogenous cancellous iliac crest). Compared with the alternative cohort, the ICBG cohort showed greater healing success after a single nonunion surgery (95.6% ICBG versus 86.9% alternative, P < 0.001) and faster healing times (4.8 ± 2.4 months versus 7.1 ± 4.9 months, P < 0.001). Complications at the ICBG harvest site included wound infections/hematomas and iliac wing fracture. No notable differences were found in positive cultures at the time of surgery, postoperative fracture-related infection, implant failure, or neurovascular injury. DISCUSSION/CONCLUSIONS:Using autogenous ICBG in the surgical repair of fracture nonunions was associated with higher healing rates compared with alternative graft types, supporting its continued role in enhancing bone healing outcomes, even in the face of infected nonunion.

OBJECTIVE:To compare rates of nail breakage of three common cephalomedullary nails (CMNs) for the treatment of AO/OTA 31A1-3 femur fractures. METHODS:Design: multi-center retrospective study. SETTING/METHODS:13 Level I trauma centers. PATIENT SELECTION CRITERIA/UNASSIGNED:Adult patients with AO/OTA 31A1-3 femur fractures treated between 2014 and 2021with the Trochanteric Fixation Nail-Advanced (TFNA), Gamma3, or Trigen InterTAN were included. OUTCOME MEASUREMENTS AND COMPARISONS/UNASSIGNED:The primary outcome was implant (nail or head element) breakage. The secondary outcomes included nonunion, cut-out/cut-through and overall reoperation rate. Univariate and multivariable analyses were performed to compare breakage rates between implants while controlling for age, sex, AO/OTA 31A1-2 vs 31A3 fracture patterns, low vs high-energy mechanisms and post-operative neck shaft angle (NSA). RESULTS:2,130 patients were included: 770 (36.2%) TFNA, 1,073 (50.4%) Gamma3 and 287 (13.5%) InterTAN. The InterTAN group had younger patients (median age: InterTan: 74, TFNA: 77, Gamma3: 80, p<0.001), more high-energy mechanisms (InterTan: 23%, TFNA: 14%, Gamma3: 10%, p=0.001), and more varus malreductions (NSA<128.5: InterTan: 46%, Gamma3: 39%, TFNA: 34%, p=0.001). The TFNA group was more likely to have an AO/OTA 31A3 fracture pattern than the Gamma3 (TFNA: 17.3%, InterTAN: 14.3%, Gamma3: 12.1%, p=0.002). The overall rate of implant breakage was 1.4% in the InterTAN group, 1.2% in the TFNA group, and 0% in the Gamma3 group. All breakages occurred in the presence of a nonunion or delayed union. Only two of the 31A3 fracture patterns resulted in breakage, both in the TFNA group, while the remainder occurred in 31A1-2 fracture patterns (p = 1.0). After controlling for confounding factors listed above, the TFNA and InterTAN groups were associated with a marginally increased odds of implant breakage compared to the Gamma3 (TFNA vs Gamma3: OR 0.04, 95% CI <0.01-0.5, p=0.034; InterTAN vs Gamma3: OR 0.04, 95% CI <0.01-0.5, p=0.037), while there was no difference between the TFNA and InterTAN (p=0.991). Post-operative neck shaft angle was not independently predictive of breakage (p = 0.55). CONCLUSIONS:This study suggests that the TFNA may be slightly more prone to breakage than the Gamma3 when used for extracapsular proximal femur fractures. However, breakage is a rare event after cephalomedullary nailing with all implants evaluated, is always associated with nonunion or delayed union, and the magnitude of this difference may not be clinically relevant. LEVEL OF EVIDENCE/METHODS:III.

During development and homeostasis, tissues move and rearrange to form organs, seal wounds, or-in the case of cancer-spread in the body. To accomplish this, cells in tissues need to communicate with each other, generate force to push themselves forward, and know where to go to-all of this with little to no error. Here, we discuss how a migrating tissue-the zebrafish posterior lateral line primordium-solves these challenges. We focus on the strategies that ensure signaling within the tissue, enable the tissue to generate and transmit force to its substrate for propulsion, and allow robust directional sensing and migration by the tissue. These strategies include facilitated diffusion and ligand trapping for focal signaling, a self-generated attractant gradient for long-distance migration, clamping of the attractant concentration to the attractant receptor's K

LRP2 (Megalin or low-density lipoprotein-related receptor 2), together with Cubilin and Amnionless, is responsible for binding and internalizing a wide range of nutrients and toxins from the kidney's glomerular filtrate by endocytosis. Accordingly, Lrp2 deletion or mutation results in the loss of these ligands into the urine. Yet Lrp2 is essential not only for receptor-mediated but also for fluid-phase endocytosis, implicating a broader role beyond ligand binding. To identify the linkage between Lrp2 and endocytosis, we engineered Lrp2-APEX2-expressing mice and performed biotinylation in vivo to label Lrp2's cytoplasmic partners. We demonstrated the specificity and sensitivity of this technique by mass spectrometric identification of biotinylated proteins from kidney lysate and immunostaining kidney sections. We identified critical endocytic regulators interacting with Lrp2, but also many proteins functionally associated with endocytosis that are not already known to interact with Lrp2. These data suggest that Lrp2 plays a central role in organizing apical membranes through PDZ domain proteins and engages with regulators and molecular motors during endocytosis. These interactions are abolished in the absence of Lrp2.

The discovery of non-coding RNAs has expanded our understanding of how genetic features are linked to cellular function. The illumination of this so-called dark matter of the genome has revealed new categories of RNA with essential roles in the regulation of protein-coding genes and genome organization. In particular, microRNAs and long non-coding RNAs have emerged as important regulators of cardiovascular health and disease. In this Review, we summarize our current understanding of the mechanisms and functional roles of microRNAs and long non-coding RNAs in the regulation of lipid homeostasis, vascular biology and atherosclerosis. We discuss how interruption of non-coding RNA regulatory circuits influence lipoprotein metabolism in the liver and the circulation, as well as the effects of non-coding RNAs on inflammatory processes in the artery wall that contribute to atherosclerotic plaque formation. Finally, we highlight potential opportunities to harness non-coding RNAs as biomarkers and targeted therapeutics for atherosclerotic cardiovascular disease.

BACKGROUND/UNASSIGNED:Dysregulated proteolysis is implicated in thoracic (thoracic aortic aneurysm [TAA]) and abdominal aortic aneurysm (AAA) pathogenesis, but proteolytic landscapes (degradomes) of aneurysmal and normal aorta and contributions of individual proteases remain undefined. Here, a proteome-wide approach was used to define and compare TAA and AAA degradomes and uncover the specific role in aortic remodeling of 2 proteases consistently identified in the aneurysms, CMA1 (mast cell chymase) and MMP9 (matrix metalloprotease 9). METHODS/UNASSIGNED:The mass spectrometry-based N-terminomics strategy, terminal amine isotopic labeling of substrates, was applied to Marfan syndrome TAAs (n=5), AAAs (n=16), and nondiseased thoracic aorta (n=4), and abdominal aorta (n=4) in a forward degradomics application, that is, to define substrate and protease degradomes. 8-plex iTRAQ terminal amine isotopic labeling of substrates was used for quantitative comparison of the tissue cohorts. Cleavage sites of CMA1 and MMP9 were sought by reverse degradomics, that is, digestion of aortic proteins with these proteases, followed by terminal amine isotopic labeling of substrates. CMA1 and MMP9 proteolysis of biglycan was further resolved using amino-terminal oriented mass spectrometry of substrates. RESULTS/UNASSIGNED:We experimentally annotated 20 885 proteolytically derived peptides and identified 129 proteases in the aortic tissues. Quantitative substrate degradome comparisons identified specific differentially modulated pathways and networks in TAAs and AAAs. Reverse degradomics elucidated >300 CMA1 and MMP9 substrate cleavage sites, of which many, including orthogonally validated biglycan cleavages, occurred in the disease degradomes. CONCLUSIONS/UNASSIGNED:Unbiased forward degradomics of the aortic wall from TAA, AAA, and nondiseased tissue provides a systems biology view of aortic wall breakdown and a new resource for its hitherto occult proteolytic landscape, demonstrating widespread extracellular matrix remodeling with disproportionate impact on proteoglycans. The findings provided insights into aortic aneurysm pathways and disease biomarkers and suggest involvement of numerous proteases. Mapping of specific proteolytic contributions of CMA1 and MMP9 illustrates a strategy for defining the activities of all proteases involved in aortic disease.

BACKGROUND:are the most common cause of familial arrhythmogenic right ventricular cardiomyopathy. This study tests whether plakophilin-2 (PKP2) deficiency only in cardiomyocytes is sufficient to provoke premature aging and proinflammatory senescence in nonmyocyte, cardiac resident cells. METHODS:We studied mice with cardiomyocyte-specific, tamoxifen-activated loss of PKP2 (cardiomyocyte-specific conditional knockout of plakophilin-2) using conventional and multiplex imaging, cytokine arrays, epigenetic clocks, spatial transcriptomics, expansion and structured illumination microscopy, and correlative data analysis. We examined nonmyocytes and cardiomyocytes for premature aging and senescence. RESULTS:We observed senescence-associated heterochromatin foci in nonmyocytes, predominantly in cells positive for α-smooth muscle actin staining. Cytokines in media of nonmyocyte cells were consistent with senescence-associated secretory phenotype. Epigenetic clocks identified premature aging. Multiplex immunohistochemistry showed nonmyocyte cells in niches, intermingled with cardiomyocytes. Spatial transcriptomics showed overrepresentation of senescence-associated secretory phenotype-related transcripts, predominantly in myocyte-rich areas of the left ventricle. Senescence-associated heterochromatin foci and increased epigenetic age were not found in cardiomyocytes from cardiomyocyte-specific conditional knockout of plakophilin-2 hearts, although we observed structural features associated with premature aging. Cross-reference analysis showed correlation between the cardiomyocyte-specific conditional knockout of plakophilin-2 cardiac proteome and that of mice 5 or 6 times their chronological age, as well as transcriptional signatures of neurodegenerative diseases. CONCLUSIONS:Loss of PKP2 expression only in adult cardiac myocytes is sufficient to induce proinflammatory senescence in nonmyocytes, and overall premature cardiac aging. This is the first study to intersect cellular senescence and premature aging with desmosomal arrhythmogenic cardiomyopathies. We speculate that cell-agnostic molecular signatures, biomarkers, and pharmacology of senescence and of neurodegenerative diseases may be relevant to diagnose or treat PKP2 arrhythmogenic right ventricular cardiomyopathy.