Biology subjects

Gerber, S. A.

Publications and source records attributed to Gerber, S. A..

16 recordsLinked to original sources

bioRxiv2026

Reconstruction of septin higher-order nano-size structures in ovarian cancer cells uncover susceptibility to the septin-targeting small molecule UR214-9

In cancer cells, septins assemble into enigmatic higher-order structures of 300-700 nanometers, including long needle-like filaments, thick perinuclear rings, and cytoplasmic bundles or aggregates. The absence of genetic or pharmacological tools to recapitulate these architectures in-vitro has impeded mechanistic studies of their formation, function, and therapeutic targeting. Here, first, determining the overexpression of septin-2 in epithelial ovarian cancer (EOC) and its association with increased mortalities and dependencies, we select SKOV-3 ovarian cancer cells as a tractable model in which septin supramolecular assemblies can be recreated in-vitro and interrogated. This system shows that the forchlorfenuron (FCF) analog UR214-9 remodels septin architecture, converting co-expressed human septin octamers (SEPT2-SEPT6-SEPT7-SEPT9-SEPT9-SEPT7-SEPT6-SEPT2) into large cytoplasmic aggregates. In parallel, transiently expressed SEPT2 is reorganized into septin-rich noodle-like filaments, perinuclear rings, and web-like networks encircling the nucleus upon UR214-9 treatment. Mechanistically, UR214-9 disrupts the incorporation of SEPT2, SEPT7, and SEPT9 into canonical septin hetero-octamers, resulting in assembly-defective or imperfect oligomers that preferentially reorganize into these aberrant higher-order structures. This aggregation likely prevents septin-2 migration during interphase-to-cleavage furrow transition in NRK-49F-SEPT2-EGFP homozygous cells and impacts SKOV-3 cytokinesis, cell proliferation, adhesion and invasion and migration while sparing ceramide transport to the Golgi, preserving ER and cis-Golgi structure. These effects manifested in reduced growth of ovarian, endometrial and breast cancer xenografts without attracting significant off-target engagements per the global transcriptomic analysis of JIMT1 breast cancer and PANC-1 pancreatic cells. UR214-9 treated animals showed observable safety in animals. Thus, a tool to recreate aberrant septin structures and identification of septins as a druggable cytoskeletal target for ovarian, endometrial, breast and pancreatic cancer by perturbing their hetero-octamerization assembly is presented. SignificanceWe provide a method to reconstruct the higher-order septin architecture observed in cancer cells, to study their assembly and functions. Intriguingly, cancer cells tolerate hetero-oligomeric septins lacking specific subunits, suggesting that compositionally deficient oligomers are not efficiently targeted for degradation, unlike unincorporated septin monomers in normal cells. This tolerance may enable accumulation of structurally aberrant septin complexes acquiring long-needles, rings or thick-aggregates in disease cells. We further show that septin oligomerization can be pharmacologically perturbed. By integrating structural, cellular, and energetic readouts using in-silico techniques, we establish a quantitative framework for septin-targeted modulation, generating UR214-9 as a new chemotype that disrupts septin oligomeric assembly via preventing incorporation of SEPT2/7/9, into canonical hetero-octamers, causes defects in cytokinesis, altered cell migration, viability, and remodels septin-actin architectures, ultimately impairing tumor cell growth. Thus, pharmacological targeting of septin assembly represents a tractable strategy to perturb septin-dependent cellular processes in cancer and neurodegenerative diseases with reported septin dysregulation.

cancer biology↗

bioRxiv2026

Roles for Phosphatase PP4 in Rhythmicity and Compensation in the Neurospora Circadian System

The circadian clock is a highly conserved timer which allows organisms to anticipate future conditions driven by the 24-hour day on planet Earth. Circadian clocks from fungi to mammals are based on a transcription-translation feedback loop (TTFL) molecular architecture. Progressive phosphorylation of core clock proteins can alter both their activity and stability and is required for all known circadian TTFLs. The mechanism for kinase control of circadian period has been extensively studied; however, the mechanism(s) whereby phosphatases alter period remain less studied. Based on the observation that strains of Neurospora crassa lacking phosphatase pp4 display a short circadian period, we investigated regulation of pp4 and its role in the clockworks. In addition to period shortening, loss of pp4 results in a significant loss of both temperature and nutritional compensation, consistent with substrates within the core clock. We identify the clock-relevant PP4 phosphatase holoenzyme as a heterotrimer, and identify two activators of PP4 which regulate circadian period. Biochemical and cell biological analyses suggest that PP4 acts in the nucleus, and a mass spectrometry-based screen identified NCU07414, a member of the HSP40 chaperone system, as a novel PP4 binding partner whose loss results in a dramatically shortened period of the clock when ablated. A model consistent with the data suggests that PP4 may act in opposition to kinases to influence the rate of accumulation of the clock-relevant phosphorylations that determine circadian period.

genetics↗

bioRxiv2025

Sequence engineering at non-motif modulator residues yields a peptide that effectively targets a single PDZ protein in a disease-relevant cellular context

Highlights- PDZ domains are critical components of many cellular, disease-relevant systems - Overlapping specificity complicates selective targeting of PDZ interaction networks - We engineered a single-PDZ peptide inhibitor using non-motif preferences - Mass-spectrometry based experiments confirm single-PDZ binding - Our inhibitor increases chloride efflux in a cystic fibrosis cellular model PDZ interaction networks are finely-tuned products of evolution. These widespread binding domains recognize short linear motifs (SLiMs), usually at the C-terminus of their interacting partners, and are involved in trafficking and signaling pathways, the formation of tight junctions, and scaffolding of the post-synaptic density of neurons, amongst other roles. Typically, a single PDZ domain binds multiple targets; conversely, each PDZ-binding protein engages several PDZ domains, dependent on cellular conditions. Historical PDZ binding motifs rely on two key positions for binding. However, previous insights on modulator, or non-motif, selectivity preferences reveal that these limited motifs are insufficient to describe PDZ-mediated interactomes, consistent with the observation that the degree of promiscuity is much more limited than predicted by defined binding classes. Here, we use these principles to engineer and test a peptide-based inhibitor capable of interacting with a single PDZ domain-containing protein in a disease-relevant cellular system. We first interrogate a previously developed sequence selective for cystic fibrosis transmembrane conductance regulator (CFTR)-Associated Ligand (CAL), one of five PDZ domains known to bind the CFTR C-terminus, probing for off-target PDZ partners. Once identified, we use parallel biochemical and structural refinement to eliminate these interactions and introduce a CAL PDZ inhibitor with unprecedented PDZ domain selectivity. We test and verify specificity using relevant cellular PDZ target networks in a mass spectrometry-based approach. Our resultant selective inhibitor enhances chloride efflux when applied to polarized patient bronchial epithelial cells, as well as confirms that engineering an effectively single-PDZ peptide is possible when modulator preferences are applied.

biochemistry↗

bioRxiv2025

Local delivery of SBRT and IL-12 to Murine PDAC Tumors Modulates Hematopoiesis

BackgroundStandard of care therapies such as radiotherapy and chemotherapy have shown little efficacy against pancreatic ductal adenocarcinoma (PDAC). Immunotherapy is a newly emerging form of treatment that has shown promise; however, toxic systemic effects resulted in limited use in the clinic. Shifting from systemic to local delivery of cancer therapeutics reduces adverse systemic effects and increases response rates in multiple malignancies. Importantly, the effects of tumor-targeted therapies on distal tissues, such as the bone marrow, have not been thoroughly investigated. MethodsUsing a murine model of PDAC, we treated tumors with targeted stereotactic body radiation therapy (SBRT) and intratumoral interleukin-12 (IL-12). 13 days-13 months after tumor injection, the cells in the tumor, blood, and bone marrow were analyzed for therapy-induced changes. Hematopoietic cell numbers and lymphocytes were quantified by flow cytometry, and cytokine levels were quantified by enzyme-linked immunosorbent assays (ELISAs). ResultsWe demonstrated that although SBRT/IL-12 delivered locally to PDAC tumors successfully eradicated primary disease, it also induced significant acute and long-term effects in the bone marrow. Within days of intratumoral SBRT/IL-12 treatment, we observed acute lymphopenia in the blood, accompanied by an immunostimulatory response in the bone marrow characterized by an increase in hematopoiesis. Long-term effects included a decrease in hematopoietic stem cells (HSCs) and skewing toward a myeloid lineage bias, which could indicate premature aging of the HSC population. ConclusionsThese findings demonstrate that despite being locally delivered to the tumor, SBRT/IL-12 therapy exerts significant effects on the distal bone marrow, reinforcing the need for further investigations into the long-term systemic immunological outcomes of localized cancer treatments. Key MessagesWhat is already known on this topic: Systemic cancer therapies used to combat pancreatic ductal adenocarcinoma (PDAC) often induce toxic systemic effects. Local delivery of radiation and immunotherapy reduces adverse effects; however, the systemic spread of these therapies and the resulting effects on distal tissues such as the bone marrow have yet to be elucidated. What this study adds: Intratumoral delivery of stereotactic body radiation therapy (SBRT) and interleukin-12 (IL-12) augment hematopoiesis in the bone marrow soon after treatment and induce long-term alterations in the hematopoietic stem cells (HSCs). These effects are mainly a result of IL-12 that is transiently increased in the bone marrow after treatment. How this study might affect research, practice, or policy: Targeted SBRT/IL-12 therapy induces long-term systemic effects on the bone marrow, indicating the need for further investigation of the systemic spread of locally delivered therapeutics.

immunology↗

bioRxiv2025

Improving SBRT by re-wiring immunosuppressive neutrophils in murine pancreatic ductal adenocarcinoma

Radiation is used to treat pancreatic ductal adenocarcinoma (PDAC) in the locally-advanced setting. Stereotactic body radiation therapy (SBRT), in particular, has shown improved outcomes against conventional RT in a number of clinical trials. One key cell type involved in the response to RT are neutrophils. These innate immune cells are the first responders to tissue damage and infiltrate irradiated tumors in high numbers to rectify injury. Neutrophils typically possess an immunosuppressive, wound healing phenotype in this scenario, which allows the tumor to recover and actively suppress immunological efforts to eradicate the disease. Here, we aimed to elucidate the role of neutrophils in a murine model of PDAC treated with SBRT. Mice harboring PDAC tumors were treated with targeted SBRT and neutrophils were determined to be significantly increased in the tissue when compared to unirradiated controls. Additionally, phenotypic analysis determined that these cells were largely immunosuppressive and depletion studies confirmed they played a key role in acquired radioresistance in our model. In order to establish whether these infiltrating cells could be re-wired to contribute to anti-tumor immunity, we utilized a novel combination therapy consisting of SBRT and microspheres containing recombinant IL-12 to attempt to repolarize these cells. Transcriptomic analysis confirmed intratumoral neutrophils underwent considerable changes indicative of an immunostimulatory, anti-tumor phenotype following treatment. Moreover, depleting these cells resulted in a loss of treatment efficacy, suggesting that neutrophil re-wiring was vital for the therapeutic outcome. This study highlights neutrophils as key players in SBRT and IL-12 treatment and confirms they can act as a double-edged sword depending on the treatment employed.

immunology↗

bioRxiv2024

The circadian gene Dec2 promotes pancreatic cancer dormancy by regulating tumor cell antigen presentation to facilitate immune evasion

The mechanisms that regulate cancer dormancy remain poorly understood. Using a mouse model of resectable pancreatic adenocarcinoma (PDAC), we identified Dec2 as a gene that was upregulated in metastatic dormant tumor cells. Deletion of Dec2 from tumor cells substantially increased mouse survival after resection due to an immune-mediated mechanism as the survival benefit was abrogated in immunodeficient conditions. Dec2 promoted immune evasion by repressing multiple components of the MHC-I dependent antigen presentation pathway in tumor cells. Dec2 is a regulator of circadian rhythms, and we found several components of the antigen presentation pathway oscillated in a circadian manner that was lost upon deletion of Dec2. Moreover, T-cell mediated tumor cell killing varied depending on the time of day. We suggest that lowered MHC-I presentation of antigens during rest phase is a natural effect of the circadian clock, which is exploited by Dec2-overexpressing pancreatic tumors to evade the immune system.

cancer biology↗

bioRxiv2024

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in Cystic Fibrosis

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lung, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection. New and noteworthyThe experiments in this report identify a novel mechanim whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet haves in OMVs secreted by P. aeruginiosa, which reduced the OMV-LPS induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF. Graphical abstractThe anti-inflammatory effect of tobramycin mediated by 5' tRNA-fMet halves secreted in P. aeruginosa OMVs. (A) P. aeruginosa colonizes the CF lungs and secrets OMVs. OMVs diffuse through the mucus layer overlying bronchial epithelial cells and induce IL-8 secretion, which recruits neutrophils that causes lung damage. (B) Tobramycin increases 5' tRNA-fMet halves in OMVs secreted by P. aeruginosa. 5' tRNA-fMet halves are delivered into host cells after OMVs fuse with lipid rafts in CF-HBEC and down-regulate protein expression of MAPK10, IKBKG, and EP300, which suppresses IL-8 secretion and neutrophils in the lungs. A reduction in neutrophils in CF BALF is predicted to improve lung function and decrease lung damage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/578737v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@d9d5e7org.highwire.dtl.DTLVardef@16a5715org.highwire.dtl.DTLVardef@7b35ddorg.highwire.dtl.DTLVardef@10d4dc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Acetylation of WCC is dispensable for the core circadianclock but differentially regulates acute light responses in Neurospora

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) has been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCCs circadian activity is controlled mainly by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses.

biochemistry↗

bioRxiv2023

Local Delivery of SBRT and IL12 by mRNA Technology Overcomes Immunosuppressive Barriers to Eliminate Pancreatic Cancer

The immunosuppressive milieu in pancreatic cancer (PC) is a significant hurdle to treatments, resulting in survival statistics that have barely changed in 5 decades. Here we present a combination treatment consisting of stereotactic body radiation therapy (SBRT) and IL-12 mRNA lipid nanoparticles delivered directly to pancreatic murine tumors. This treatment was effective against primary and metastatic models, achieving cures in both settings. IL-12 protein concentrations were transient and localized primarily to the tumor. Depleting CD4 and CD8 T cells abrogated treatment efficacy, confirming they were essential to treatment response. Single cell RNA sequencing from SBRT/IL-12 mRNA treated tumors demonstrated not only a complete loss of T cell exhaustion, but also an abundance of highly proliferative and effector T cell subtypes. SBRT elicited T cell receptor clonal expansion, whereas IL-12 licensed these cells with effector function. This is the first report demonstrating the utility of SBRT and IL-12 mRNA in PC. Statement of significanceThis study demonstrates the use of a novel combination treatment consisting of radiation and immunotherapy in murine pancreatic tumors. This treatment could effectively treat local and metastatic disease, suggesting it may have the potential to treat a cancer that has not seen a meaningful increase in survival in 5 decades.

cancer biology↗

bioRxiv2023

Pharmacological Induction of mesenchymal-epithelial transition chemosensitizes breast cancer cells and prevents metastatic progression

The epithelial-mesenchymal transition (EMT) is a developmental program co-opted by tumor cells that aids the initiation of the metastatic cascade. Tumor cells that undergo EMT are relatively chemoresistant, and there are currently no therapeutic avenues specifically targeting cells that have acquired mesenchymal traits. We show that treatment of mesenchymal-like triple-negative breast cancer (TNBC) cells with the microtubule-destabilizing chemotherapeutic eribulin, which is FDA-approved for the treatment of advanced breast cancer, leads to a mesenchymal-epithelial transition (MET). This MET is accompanied by loss of metastatic propensity and sensitization to subsequent treatment with other FDA-approved chemotherapeutics. We uncover a novel epigenetic mechanism of action that supports eribulin pretreatment as a path to MET induction that curtails metastatic progression and the evolution of therapy resistance. SummaryWhile the advent of targeted therapy has led to vast improvements in outcomes for certain types of breast cancer, a mainstay for triple-negative breast cancer (TNBC) remains cytotoxic chemotherapy. A major clinical hurdle in successfully managing this disease is the eventual development of therapeutic resistance and disease relapse in more aggressive forms. Our data reveal that epigenetic modulation of EMT state using the FDA-approved therapeutic eribulin curtails metastatic propensity of breast tumors and, when administered in the treatment-naive setting, sensitizes to subsequent treatment with other chemotherapeutics.

cancer biology↗

bioRxiv2021

Development and validation of inducible protein degradation and quantitative phosphoproteomics to identify kinase-substrate relationships.

Phosphorylation signaling is an essential post-translational regulatory mechanism that governs almost all eukaryotic biological processes and is controlled by an interplay between protein kinases and phosphatases. Knowledge of direct substrates of kinases provides evidence of mechanisms that relate activity to biological function. Linking kinases to their protein substrates can be achieved by inhibiting or reducing kinase activity and quantitative comparisons of phosphoproteomes in the presence and absence of kinase activity. Unfortunately, most of the human kinases lack chemical inhibitors with selectivity required to unambiguously assign protein substrates to their respective kinases. Here, we develop and validate a chemical proteomics strategy for linking kinase activities to protein substrates via targeted protein degradation and quantitative phosphoproteomics and apply it to the well-studied, essential mitotic regulator polo-like kinase 1 (Plk1). We leveraged the Tir1/auxin system to engineer HeLa cells with endogenously homozygous auxin-inducible degron (AID)-Plk1). We used HeLa cells and determined the impact of AID-tagging on Plk1 activity, localization, protein interactors, and substrate motifs. Using quantitative proteomics, we show that of over 8,000 proteins quantified, auxin addition is highly selective for degrading AID-Plk1 in mitotic cells. Comparison of phosphoproteome changes in response to chemical Plk1 inhibition to auxin-induced degradation revealed a striking degree of correlation. Finally, we explored basal protein turnover as a potential basis for clonal differences in auxin-induced degradation rates for AID-Plk1 cells. Taken together, our work provides a roadmap for the application of AID technology as a general strategy for the kinome-wide discovery of kinase-substrate relationships.

systems biology↗

bioRxiv2021

Quantitative phosphoproteomics reveals regulatory state dependent efficacy of chemical Cdk1 inhibition and distinct Cdk5 complexes as novel Wee1 substrates

Wee1 kinase plays a central role in the eukaryotic cell cycle via its well-known negative regulation of Cdk1 activity at the G2/M transition, preventing progression into mitosis until DNA replication and/or DNA damage repair is complete. Recent genetic evidence in yeast, flies and human cells have suggested additional functions of Wee1 in mitosis and during mitotic exit, respectively. To discover new candidate substrates of Wee1 kinase, we used SILAC-based phosphoproteomics and selective chemical inhibition to quantitatively compare phosphorylation site abundances in the presence and absence of Wee1 activity. Unexpectedly, we uncovered a role for the Wee1-dependent phosphorylation of Cdk1-cyclin B at tyrosine 15 (Y15) in facilitating chemical inhibition of Cdk1-cyclin B by the inhibitor RO3306. Thermal shift stability assays demonstrated greater binding affinity of RO3306 for Y15-phosphorylated Cdk1-cyclin B versus unphosphorylated complex, providing an additional molecular basis for the observed Wee1 inhibitor-based toxicity in human cells. In addition, our experiments identified Cdk5-CABLES and Cdk5-cyclin B as novel substrates of Wee1 during chemically induced exit from mitosis. Collectively, these experiments facilitate a greater understanding of the Wee1-Cdk1 signaling axis and uncover new candidate substrates for Wee1.

cell biology↗

bioRxiv2021

Chromosome segregation fidelity is controlled by small changes in kinetochore phospho-occupancy

Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e. all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded [~]50% phospho-occupancy, and the cumulative phospho-occupancy changed by only [~]20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained by ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. Thus, networks of kinases and phosphatases maintain low inherent phospho-occupancy to promote microtubule attachment to kinetochores while providing for high sensitivity of kinetochore-microtubule attachments to very small changes in phospho-occupancy to ensure high mitotic fidelity.

cell biology↗

bioRxiv2020

Targeting Vitamin-D receptor (VDR) by a small molecule antagonist MeTC7 inhibits PD-L1 but controls THMYCN neuroblastoma growth PD-L1 independently

Vitamin-D receptor (VDR) mRNA is enriched in malignant lung, ovarian and pancreatic tissues and showed poor prognoses. Calcitriol and stable or CRISPR-directed VDR upregulation increased PD-L1mRNA and protein expression in cancer cells in-vitro. A ChIP assay showed the binding of VDR with VDREPD-L1. Stattic, a STAT3 phosphorylation inhibitor blocked calcitriol or VDR overexpression induced PD-L1 upregulation. MeTC7, a VDR antagonist developed by us, reduced PD-L1 expression on macrophages, ovarian, lung, breast, and pancreatic cancer cells in-vitro. In radiotherapy inducible PD-L1 model of orthotopic MC38 murine colon cancer, MeTC7 decreased PD-L1 surface expression, suppressed inflammatory monocytes (IMs) population and increased intra-tumoral CD69+PD1+CD8+T-cells. Intriguingly, MeTC7 reduced TH-MYCN transgenic neuroblastoma tumor growth without affecting PD-L1 and tumor immune milieu. In summary, Vitamin-D/VDR drives PD-L1 expression on cancer cells via STAT-3. Inhibiting VDR exhibited anti-checkpoint effects in orthotopic colon tumors, whereas PDL1-independent and anti-VDR/MYCN effects controlled growth of transgenic neuroblastoma and xenografted tumors. SummaryVitamin-D/VDR induces PD-L1 expression on cancer cells via STAT-3; and targeting VDR by a novel small molecule antagonist MeTC7 exhibits both anti-PD-L1 and anti-VDR/MYCN effects in tumor models.

cancer biology↗

bioRxiv2020

Dual Energy X-ray Absorptiometry (DEXA) as a Longitudinal Outcome Measure of Cancer-Related Muscle Wasting in Mice

IntroductionPancreatic ductal adenocarcinoma (PDAC) is notorious for its associated skeletal muscle wasting (SMW) and mortality. Currently, the relationships between PDAC, SMW, and survival are poorly understood. Thus, there is a great need for a faithful small animal model with a quantitative longitudinal outcome measure that recapitulates clinical PDAC, to define SMW onset and assess progression. Therefore, we aimed to validate dual energy X-ray absorptiometry (DEXA) as a longitudinal outcome of lean body mass, and demonstrate its utility to quantify SMW in the KCKO murine model of PDAC. MethodsIn vivo body composition of: 1) untreated mice at 5, 8, 12, 18, and 22 weeks of age (n=4), and 2) a cohort of mice with (n=20) and without PDAC (n=10), was determined via DEXA, and lean mass of the lower hind limbs was predicted via a region of interest analysis by two independent observers. Total body weight was determined. Tibialis anterior (TA) muscles were weighed and processed for histomorphometry immediately post-mortem. Statistical differences between groups were assessed using t-tests and ANOVA. Linear regression models and correlation analysis were used to measure the association between TA and DEXA mass, and reproducibility of DEXA was quantified via the intraclass correlation coefficient (ICC). ResultsLean mass in growing untreated mice determined by DEXA correlated with TA mass (r2 = 0.94; p <0.0001) and body weight (r2 = 0.89; p <0.0001). DEXA measurements were highly reproducible between observers (ICC = 0.95; 95% CI: 0.89-0.98). DEXA and TA mass also correlated in the PDAC cohort (r2 = 0.76; p <0.0001). Significant SMW in tumor-bearing mice was detected within 38 days of implantation by DEXA, TA mass, and histomorphometry. ConclusionsDEXA is a longitudinal outcome measure of lower limb lean mass in mice. The KCKO syngeneic model is a bona fide model of PDAC-associated SMW that can be quantified with longitudinal DEXA.

cancer biology↗

bioRxiv2018

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

The cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and appears to selectively affect the expression of DNA damage response (DDR) and mRNA processing genes. Yet, the mechanism(s) by which it achieves this selectivity remains unclear. Using a highly selective CDK12/13 inhibitor, THZ531, and nascent RNA sequencing, we show that CDK12 inhibition results in gene length-dependent elongation defects, leading to premature cleavage and polyadenylation (PCPA) as well as loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlated with an increased proportion of intronic polyadenylation sites, a feature that was especially prominent among DDR genes. Finally, phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in PCPA, are direct phosphotargets of CDKs 12 and 13. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition due primarily to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.

cancer biology↗