Histidine-Rich Glycoprotein Modulates the Toxic Effects of High-Dose Polyphosphate in Mice.

BACKGROUND: Polyphosphate (polyP), a procoagulant released from platelets, activates coagulation via the contact system and modulates cardiomyocyte viability. High-dose intravenous polyP is lethal in mice, presumably because of thrombosis. Previously, we showed that HRG (histidine-rich glycoprotein) binds polyP and attenuates its procoagulant effects. In this study, we investigated the mechanisms responsible for the lethality of intravenous polyP in mice and the impact of HRG on this process. METHODS: The survival of wild-type or HRG-deficient mice given intravenous synthetic or platelet-derived polyP in doses up to 50 mg/kg or saline was compared. To determine the contribution of thrombosis, the effect of FXII (factor XII) knockdown or enoxaparin on polyP-induced fibrin deposition in the lungs was examined. To assess cardiotoxicity, the ECG was continuously monitored, the levels of troponin I and the myocardial band of creatine kinase were quantified, and the viability of a cultured murine cardiomyocyte cell line exposed to polyP in the absence or presence of HRG was determined. RESULTS: In HRG-deficient mice, polyP was lethal at 30 mg/kg, whereas it was lethal in wild-type mice at 50 mg/kg. Although FXII knockdown or enoxaparin administration attenuated polyP-induced fibrin deposition in the lungs, neither affected mortality. PolyP induced dose-dependent ECG abnormalities, including heart block and ST-segment changes, and increased the levels of troponin and myocardial band of creatine kinase, effects that were more pronounced in HRG-deficient mice than in wild-type mice and were attenuated when HRG-deficient mice were given supplemental HRG. Consistent with its cardiotoxicity, polyP reduced the viability of cultured cardiomyocytes in a dose-dependent manner, an effect attenuated with supplemental HRG. CONCLUSIONS: High-dose intravenous polyP is cardiotoxic in mice, and HRG modulates this effect.

Inhibition of MER proto-oncogene tyrosine kinase by an antisense oligonucleotide enhances treatment efficacy of immunoradiotherapy.

BACKGROUND: The combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice. METHODS: 344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated. RESULTS: Remarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes. CONCLUSIONS: The MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.

STAT6-targeting antisense oligonucleotides against solitary fibrous tumor.

Solitary fibrous tumor (SFT) is a rare, non-hereditary soft tissue sarcoma thought to originate from fibroblastic mesenchymal stem cells. The etiology of SFT is thought to be due to an environmental intrachromosomal gene fusion between NGFI-A-binding protein 2 (NAB2) and signal transducer and activator protein 6 (STAT6) genes on chromosome 12, wherein the activation domain of STAT6 is fused with the DNA-binding domain of NAB2 resulting in the oncogenesis of SFT. All NAB2-STAT6 fusion variations discovered in SFTs contain the C-terminal of STAT6 transcript, and thus can serve as target site for antisense oligonucleotides (ASOs)-based therapies. Indeed, our in vitro studies show the STAT6 3' untranslated region (UTR)-targeting ASO (ASO 993523) was able to reduce expression of NAB2-STAT6 fusion transcripts in multiple SFT cell models with high efficiency (half-maximal inhibitory concentration: 116-300 nM). Encouragingly, in vivo treatment of SFT patient-derived xenograft mouse models with ASO 993523 resulted in acceptable tolerability profiles, reduced expression of NAB2-STAT6 fusion transcripts in xenograft tissues (21.9%), and, importantly, reduced tumor growth (32.4% decrease in tumor volume compared with the untreated control). Taken together, our study established ASO 993523 as a potential agent for the treatment of SFTs.

GalNAc-conjugated siRNA targeting the DNAJB1-PRKACA fusion junction in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). Current therapies such as chemotherapy and radiation have limited efficacy, and new treatment options are needed urgently. We have previously shown that FLC tumors are dependent on the fusion kinase DNAJB1::PRKACA, making the oncokinase an ideal drug target. mRNA degrading modalities such as antisense oligonucleotides or small interfering RNAs (siRNAs) provide an opportunity to specifically target the fusion junction. Here, we identify a potent and specific siRNA that inhibits DNAJB1::PRKACA expression. We found expression of the asialoglycoprotein receptor in FLC to be maintained at sufficient levels to effectively deliver siRNA conjugated to the GalNAc ligand. We observe productive uptake and siRNA activity in FLC patient-derived xenografts (PDX) models in vitro and in vivo. Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.

Plasminogen deficiency suppresses pancreatic ductal adenocarcinoma disease progression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal metastatic disease associated with robust activation of the coagulation and fibrinolytic systems. However, the potential contribution of the primary fibrinolytic protease plasminogen to PDAC disease progression has remained largely undefined. Mice bearing C57Bl/6-derived KPC (KRasG12D , TRP53R172H ) tumors displayed evidence of plasmin activity in the form of high plasmin-antiplasmin complexes and high plasmin generation potential relative to mice without tumors. Notably, plasminogen-deficient mice (Plg- ) had significantly diminished KPC tumor growth in subcutaneous and orthotopic implantation models. Moreover, the metastatic potential of KPC cells was significantly diminished in Plg- mice, which was linked to reduced early adhesion and/or survival of KPC tumor cells. The reduction in primary orthotopic KPC tumor growth in Plg- mice was associated with increased apoptosis, reduced accumulation of pro-tumor immune cells, and increased local proinflammatory cytokine production. Elimination of fibrin(ogen), the primary proteolytic target of plasmin, did not alter KPC primary tumor growth and resulted in only a modest reduction in metastatic potential. In contrast, deficiencies in the plasminogen receptors Plg-RKT or S100A10 in tumor cells significantly reduced tumor growth. Plg-RKT reduction in tumor cells, but not reduced S100A10, suppressed metastatic potential in a manner that mimicked plasminogen deficiency. Finally, tumor growth was also reduced in NSG mice subcutaneously or orthotopically implanted with patient-derived PDAC tumor cells in which circulating plasminogen was pharmacologically reduced. Collectively, these studies suggest that plasminogen promotes PDAC tumor growth and metastatic potential, in part through engaging plasminogen receptors on tumor cells.

Clinical Progress in Hepatic Targeting for Novel Prophylactic Therapies in Hereditary Angioedema.

Hereditary angioedema (HAE) is typically caused by a deficiency of the protease inhibitor C1 inhibitor (C1INH). The absence of C1INH activity on plasma kallikrein and factor XIIa leads to overproduction of the vasoactive peptide bradykinin, with resulting angioedema. As the primary site of C1INH and prekallikrein production, the liver is recognized as an important therapeutic target in HAE, leading to the development of hepatic-focused treatment strategies such as GalNAc-conjugated antisense technology and gene modification. This report reviews currently available data on hepatic-focused interventions for HAE that have advanced into human trials. Donidalorsen is an investigational GalNAc3-conjugated antisense oligonucleotide that binds to prekallikrein mRNA in the liver and reduces the expression of prekallikrein. Phase 2 data with subcutaneous donidalorsen demonstrated a significant reduction in HAE attack rate compared with placebo. Phase 3 trials are underway. ADX-324 is a GalNAc3-conjugated short-interfering RNA being investigated in HAE. BMN 331 is an investigational AAV5-based gene therapy vector that expresses wild-type human C1INH and is targeted to hepatocytes. A single intravenous dose of BMN 331 is intended to replace the defective SERPING1 gene and enable patients to produce functional C1INH. A first-in-human phase 1/2 study is ongoing with BMN 331. NTLA-2002 is an investigational in vivo clustered regularly interspaced short palindromic repeats/Cas9-based therapy designed to knock out the prekallikrein-coding KLKB1 gene in hepatocytes; a phase 1/2 study is ongoing. Findings from these and other ongoing studies are highly anticipated with the expectation of expanding the array of treatment options in HAE.

Role of platelet count in a murine stasis model of deep vein thrombosis.

Platelets are core components of thrombi but their effect on thrombus burden during deep vein thrombosis (DVT) has not been fully characterized. We examined the role of thrombopoietin-altered platelet count on thrombus burden in a murine stasis model of DVT. To modulate platelet count compared to baseline, CD1 mice were pretreated with thrombopoietin antisense oligonucleotide (THPO-ASO, 56% decrease), thrombopoietin mimetic (TPO-mimetic, 36% increase), or saline (within 1%). Thrombi and vein walls were examined on postoperative days (POD) 3 and 7. Thrombus weights on POD 3 were not different between treatment groups (p = .84). The mean thrombus weights on POD 7 were significantly increased in the TPO-mimetic cohort compared to the THPO-ASO (p = .005) and the saline (p = .012) cohorts. Histological grading at POD 3 revealed a significantly increased smooth muscle cell presence in the thrombi and CD31 positive channeling in the vein wall of the TPO-mimetic cohort compared to the saline and THPO-ASO cohorts (p < .05). No differences were observed in histology on POD 7. Thrombopoietin-induced increased platelet count increased thrombus weight on POD 7 indicating platelet count may regulate thrombus burden during early resolution of venous thrombi in this murine stasis model of DVT.

Deep vein thrombosis (DVT) is a pathology in which blood clots form in the deep veins of our body. Usually occurring in the legs, these clots can be dangerous if they dislodge and travel to the heart and are pumped into the lungs. Often these clots do not travel and heal where they formed. However, as the body heals the clot it may also cause damage to the vein wall and predispose the patient to future clots, i.e., the biggest risk factor for a second clot is the first clot. DVT can also cause symptoms of pain, swelling, and redness in the long-term, leading to post-thrombotic syndrome where the initial symptoms of the clot persist for a long time. All blood clots have common components of red blood cells, white blood cells, platelets, and fibrin in varying concentrations. Humans maintain a platelet count between 150 and 400 thousand platelets per microliter of our blood. However, diseases like cancer or medications like chemotherapy can cause a change in our body's platelet count. The effect of a changing platelet count on the size (clot burden) of DVT clot and how platelet count could affect DVT as the clot heals is not fully understood. Studying this might help us develop better targets and treat patients with a wide range of platelet counts who experience DVT. In this study, we intentionally decreased, left unchanged, and increased platelet counts in mice and then created a DVT to study what the effect of low, normal, and high platelet counts, respectively, would be on the clot burden. We observed that mice with higher platelet counts had a higher clot burden during the early part of the healing process of the clot. Within this study, we can conclude that higher platelet counts may lead to higher clot burden in DVT which furthers our understanding of how platelet count affects clot burden during DVT.

Knockdown of coagulation factor VII has potential anticoagulant and anti-inflammatory effects in a mouse model of group A streptococcal sepsis.

Infections originating from subcutaneous tissues are among the most common invasive infections caused by group A streptococcus (GAS) and associated with systemic coagulation activation. The role of intrinsic coagulation factors on GAS virulence has recently been determined, but the role of the extrinsic coagulation factor VII is unknown. Using a mouse model, in which GAS-sepsis emerges from a subcutaneous infection, we show that FVII is a negative acute phase protein. F7 knockdown using antisense oligonucleotides resulted in an attenuated systemic coagulation activation and inflammatory response in septic animals. The findings indicate FVII's ability to modify the host response.

Histidine-rich glycoprotein attenuates catheter thrombosis.

Factor XII (FXII) knockdown attenuates catheter thrombosis in rabbits. Because histidine-rich glycoprotein (HRG) modulates FXIIa activity, we hypothesized that HRG depletion would promote catheter thrombosis. To test this, rabbits were given either antisense oligonucleotides (ASOs) against HRG or FXII, a control ASO, or saline. The activated partial thromboplastin time (aPTT), prothrombin time (PT), and catheter-induced thrombin generation were determined in blood collected before and after treatment. Compared with the controls, the HRG- and FXII-directed ASOs reduced hepatic messenger RNA and plasma levels of HRG and FXII, respectively, by >90%. Although HRG knockdown shortened the aPTT by 2.5 fold, FXII knockdown prolonged it by fourfold; neither of the ASOs affected the PT. Catheter segments shortened the lag time and increased peak thrombin in the plasma from control rabbits; effects were significantly enhanced and attenuated in the plasma from rabbits given the HRG- and FXII-directed ASOs, respectively. Catheters were then inserted into the right external jugular vein of the rabbits, and the time for catheter occlusion was determined. The catheter occlusion times with the control ASO or saline were 62 ± 8 minutes and 60 ± 11 minutes, respectively. The occlusion time was significantly reduced to 34 ± 9 minutes, with HRG knockdown and significantly prolonged to 128 ± 19 minutes with FXII knockdown. HRG levels are decreased with sepsis or cancer, and such patients are prone to catheter thrombosis. Because HRG modulates catheter thrombosis, our findings suggest that HRG supplementation may prevent this problem.

Simultaneous inhibition of endocytic recycling and lysosomal fusion sensitizes cells and tissues to oligonucleotide therapeutics.

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

Inhibition of STAT6 with Antisense Oligonucleotides Enhances the Systemic Antitumor Effects of Radiotherapy and Anti-PD-1 in Metastatic Non-Small Cell Lung Cancer.

Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non-small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells-such as M2 tumor-associated macrophages (TAM)-thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti-PD-1-resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFß levels. The addition of anti-PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.

Targeting the contact system in a rabbit model of extracorporeal membrane oxygenation.

Previous studies suggested that contact pathway factors drive thrombosis in mechanical circulation. We used a rabbit model of veno-arterial extracorporeal circulation (VA-ECMO) to evaluate the role of factors XI and XII in ECMO-associated thrombosis and organ damage. Factors XI and XII (FXI, FXII) were depleted using established antisense oligonucleotides before placement on a blood-primed VA-ECMO circuit. Decreasing FXII or FXI to < 5% of baseline activity significantly prolonged ECMO circuit lifespan, limited the development of coagulopathy, and prevented fibrinogen consumption. Histological analysis suggested that FXII depletion mitigated interstitial pulmonary edema and hemorrhage whereas heparin and FXI depletion did not. Neither FXI nor FXII depletion was associated with significant hemorrhage in other organs. In vitro analysis showed that membrane oxygenator fibers (MOFs) alone are capable of driving significant thrombin generation in a FXII- and FXI-dependent manner. MOFs also augment thrombin generation triggered by low (1 pM) or high (5 pM) tissue factor concentrations. However, only FXI elimination completely prevented the increase in thrombin generation driven by MOFs, suggesting MOFs augment thrombin-mediated FXI activation. Together, these results suggest that therapies targeting FXII or FXI limit thromboembolic complications associated with ECMO. Further studies are needed to determine the contexts wherein targeting FXI and FXII, either alone or in combination, would be most beneficial in ECMO. Moreover, studies are also needed to determine the potential mechanisms coupling FXII to end-organ damage in ECMO.

CFTR mRNAs with nonsense codons are degraded by the SMG6-mediated endonucleolytic decay pathway.

Approximately 10% of cystic fibrosis patients harbor nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which can generate nonsense codons in the CFTR mRNA and subsequently activate the nonsense-mediated decay (NMD) pathway resulting in rapid mRNA degradation. However, it is not known which NMD branches govern the decay of CFTR mRNAs containing nonsense codons. Here we utilize antisense oligonucleotides targeting NMD factors to evaluate the regulation of nonsense codon-containing CFTR mRNAs by the NMD pathway. We observe that CFTR mRNAs with nonsense codons G542X, R1162X, and W1282X, but not Y122X, require UPF2 and UPF3 for NMD. Furthermore, we demonstrate that all evaluated CFTR mRNAs harboring nonsense codons are degraded by the SMG6-mediated endonucleolytic pathway rather than the SMG5-SMG7-mediated exonucleolytic pathway. Finally, we show that upregulation of all evaluated CFTR mRNAs with nonsense codons by NMD pathway inhibition improves outcomes of translational readthrough therapy.

Direct targeting of FOXP3 in Tregs with AZD8701, a novel antisense oligonucleotide to relieve immunosuppression in cancer.

BACKGROUND: The Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3, which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered, unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man. METHODS: We have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons. RESULTS: AZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs, reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO, which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs, reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo, strongly modulated Treg effector molecules (eg, ICOS, CTLA-4, CD25 and 4-1BB), and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy. CONCLUSIONS: Antisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669).

Inhibition of Prekallikrein for Hereditary Angioedema.

BACKGROUND: Hereditary angioedema is characterized by recurrent and unpredictable swellings that are disabling and potentially fatal. Selective inhibition of plasma prekallikrein production by antisense oligonucleotide treatment (donidalorsen) may reduce the frequency of attacks and the burden of disease. METHODS: In this phase 2 trial, we randomly assigned, in a 2:1 ratio, patients with hereditary angioedema with C1 inhibitor deficiency to receive four subcutaneous doses of either donidalorsen (80 mg) or placebo, with one dose administered every 4 weeks. The primary end point was the time-normalized number of investigator-confirmed angioedema attacks per month (attack rate) between week 1 (baseline) and week 17. Secondary end points included quality of life, as measured with the Angioedema Quality of Life Questionnaire (scores range from 0 to 100, with higher scores indicating worse quality of life), and safety. RESULTS: A total of 20 patients were enrolled, of whom 14 were randomly assigned to receive donidalorsen and 6 to receive placebo. The mean monthly rate of investigator-confirmed angioedema attacks was 0.23 (95% confidence interval [CI], 0.08 to 0.39) among patients receiving donidalorsen and 2.21 (95% CI, 0.58 to 3.85) among patients receiving placebo (mean difference, -90%; 95% CI, -96 to -76; P<0.001). The mean change from baseline to week 17 in the Angioedema Quality of Life Questionnaire score was -26.8 points in the donidalorsen group and -6.2 points in the placebo group (mean difference, -20.7 points; 95% CI, -32.7 to -8.7). The incidence of mild-to-moderate adverse events was 71% among patients receiving donidalorsen and 83% among those receiving placebo. CONCLUSIONS: Among patients with hereditary angioedema, donidalorsen treatment resulted in a significantly lower rate of angioedema attacks than placebo in this small, phase 2 trial. (Funded by Ionis Pharmaceuticals; ISIS 721744-CS2 ClinicalTrials.gov number, NCT04030598.).

Plasma Kallikrein Contributes to Intracerebral Hemorrhage and Hypertension in Stroke-Prone Spontaneously Hypertensive Rats.

Plasma kallikrein (PKa) has been implicated in contributing to hemorrhage following thrombolytic therapy; however, its role in spontaneous intracerebral hemorrhage is currently not available. This report investigates the role of PKa on hemorrhage and hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were fed with a high salt-containing stroke-prone diet to increase blood pressure and induce intracerebral hemorrhage. The roles of PKa on blood pressure, hemorrhage, and survival in SHRSP were examined in rats receiving a PKa inhibitor or plasma prekallikrein antisense oligonucleotide (PK ASO) compared with rats receiving control ASO. Effects on PKa on the proteolytic cleavage of atrial natriuretic peptide (ANP) were analyzed by tandem mass spectrometry. We show that SHRSP on high-salt diet displayed increased levels of PKa activity compared with control rats. Cleaved kininogen was increased in plasma during stroke compared to SHRSP without stroke. Systemic administration of a PKa inhibitor or PK ASO to SHRSP reduced hemorrhage and blood pressure, and improved neurological function and survival compared with SHRSP receiving control ASO. Since PKa inhibition was associated with reduced blood pressure in hypertensive rats, we investigated the effects of PKa on the cleavage of ANP. Incubation of PKa with ANP resulted in the generation fragment ANP5-28, which displayed reduced effects on blood pressure lowering compared with full length ANP. PKa contributes to increased blood pressure in SHRSP, which is associated with hemorrhage and reduced survival. PKa-mediated cleavage of ANP reduces its blood pressure lowering effects and thereby may contribute to hypertension-induced intracerebral hemorrhage.

Plasmin drives burn-induced systemic inflammatory response syndrome.

Severe injuries, such as burns, provoke a systemic inflammatory response syndrome (SIRS) that imposes pathology on all organs. Simultaneously, severe injury also elicits activation of the fibrinolytic protease plasmin. While the principal adverse outcome of plasmin activation in severe injury is compromised hemostasis, plasmin also possesses proinflammatory properties. We hypothesized that, following a severe injury, early activation of plasmin drives SIRS. Plasmin activation was measured and related to injury severity, SIRS, coagulopathy, and outcomes prospectively in burn patients who are not at risk of hemorrhage. Patients exhibited early, significant activation of plasmin that correlated with burn severity, cytokines, coagulopathy, and death. Burn with a concomitant, remote muscle injury was employed in mice to determine the role of plasmin in the cytokine storm and inflammatory cascades in injured tissue distant from the burn injury. Genetic and pharmacologic inhibition of plasmin reduced the burn-induced cytokine storm and inflammatory signaling in injured tissue. These findings demonstrate (a) that severe injury-induced plasmin activation is a key pathologic component of the SIRS-driven cytokine storm and SIRS-activated inflammatory cascades in tissues distant from the inciting injury and (b) that targeted inhibition of plasmin activation may be effective for limiting both hemorrhage and tissue-damaging inflammation following injury.

Antisense oligonucleotide activity in tumour cells is influenced by intracellular LBPA distribution and extracellular vesicle recycling.

Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.

Polyphosphate-induced thrombosis in mice is factor XII dependent and is attenuated by histidine-rich glycoprotein.

Histidine-rich glycoprotein (HRG) is an abundant plasma protein that binds factor XIIa (FXIIa) and inhibits factor XII (FXII) autoactivation and FXIIa-mediated activation of FXI. Polyphosphate (polyP), a potent procoagulant released from activated platelets, may serve as a physiological activator of the contact system. Previously, we showed that HRG binds DNA and neutralizes its procoagulant activity. Consequently, our goal was to determine whether the capacity of HRG to bind polyanions enables it to regulate polyP-induced thrombosis. In a plate-based assay, immobilized polyP bound HRG, FXII, and FXIIa in a zinc-dependent manner. Basal and polyP-induced thrombin generation was greater in plasma from HRG-deficient mice than in plasma from wild-type mice. Intraperitoneal injection of polyP shortened the activated partial thromboplastin time, enhanced thrombin generation, increased thrombin-antithrombin levels, reduced lung perfusion, and promoted pulmonary fibrin deposition to a greater extent in HRG-deficient mice than in wild-type mice, effects that were abrogated with FXII knockdown. HRG thus attenuates the procoagulant and prothrombotic effects of polyP in an FXII-dependent manner by modulating the contact system.

Specific targeting of the KRAS mutational landscape in myeloma as a tool to unveil the elicited antitumor activity.

Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.