Cross-linking Of Elastin Research Articles

The Potential Role of Cigarette Smoke, Elastic Fibers, and Secondary Lung Injury in the Transition of Pulmonary Emphysema to Combined Pulmonary Fibrosis and Emphysema.

Combined pulmonary fibrosis and emphysema (CPFE) is a distinct syndrome associated with heavy smoking. The fibrotic component of the disease is generally believed to be superimposed on previously existing pulmonary emphysema, but the mechanisms responsible for these changes remain poorly understood. To better understand the pathogenesis of CPFE, we performed a series of experiments that focused on the relationships between lung elastic fibers, cigarette smoke, and secondary lung injury. The results indicate that even brief smoke exposure predisposes the lung to additional forms of lung injury that may cause alveolar wall fibrosis. The proinflammatory activity of smoke-induced structural alterations in elastic fibers may contribute to this process by enhancing secondary lung inflammation, including acute exacerbations of chronic obstructive pulmonary disease. Furthermore, the levels of the unique elastin crosslinks, desmosine and isodesmosine, in blood, urine, and sputum may serve as biomarkers for the transition from pulmonary emphysema to interstitial fibrosis. While the long-term effects of these inflammatory reactions were not examined, the current studies provide insight into the potential relationships between elastic fiber injury, cigarette smoke, and secondary lung injury. Determining the mechanisms involved in combined pulmonary emphysema and fibrosis and developing a sensitive biomarker for this type of lung injury may permit timely therapeutic intervention that could mitigate the high risk of respiratory failure associated with this condition.

Gene inactivation of lysyl oxidase in smooth muscle cells reduces atherosclerosis burden and plaque calcification in hyperlipidemic mice

Background and aimsLysyl oxidase (LOX) catalyzes the crosslinking of collagen and elastin to maintain tensile strength and structural integrity of the vasculature. Excessive LOX activity increases vascular stiffness and the severity of occlusive diseases. Herein, we investigated the mechanisms by which LOX controls atherogenesis and osteogenic differentiation of vascular smooth muscle cells (SMC) in hyperlipidemic mice. MethodsGene inactivation of Lox in SMC was achieved in conditional knockout mice after tamoxifen injections. Atherosclerosis burden and vascular calcification were assessed in hyperlipidemic conditional [Loxf/fMyh11-CreERT2ApoE−/−] and sibling control mice [Loxwt/wtMyh11-CreERT2ApoE−/−]. Mechanistic studies were performed with primary aortic SMC from Lox mutant and wild type mice. ResultsInactivation of Lox in SMCs decreased > 70 % its RNA expression and protein level in the aortic wall and significantly reduced LOX activity without compromising vascular structure and function. Moreover, LOX deficiency protected mice against atherosclerotic burden (13 ± 2 versus 23 ± 1 %, p < 0.01) and plaque calcification (5 ± 0.4 versus 11.8 ± 3 %, p < 0.05) compared to sibling controls. Interestingly, gene inactivation of Lox in SMCs preserved the contractile phenotype of vascular SMC under hyperlipidemic conditions as demonstrated by single-cell RNA sequencing and immunofluorescence. Mechanistically, the absence of LOX in SMC prevented excessive collagen crosslinking and the subsequent activation of the pro-osteogenic FAK/β-catenin signaling axis. ConclusionsLox inactivation in SMC protects mice against atherosclerosis and plaque calcification by reducing SMC modulation and FAK/β-catenin signaling.

Advanced Abdominal Aortic Aneurysm Modeling in Mice by Combination of Topical Elastase and Oral ß-aminopropionitrile.

The topical elastase murine model of abdominal aortic aneurysm (AAA) is enhanced when combined with ß-aminopropionitrile (BAPN)-supplemented drinking water to reliably produce true infrarenal aneurysms with behaviors that mimic human AAAs. Topically applying elastase to the adventitia of the infrarenal aorta causes structural damage to the elastic layers of the aortic wall and initiates aneurysmal dilation. Co-administering BAPN, a lysyl oxidase inhibitor, promotes sustained wall degeneration by reducing collagen and elastin crosslinking. This combination results in large AAAs that progressively expand, form intraluminal thrombus, and are capable of rupture. Refining surgical techniques, such as circumferentially isolating the entire infrarenal aortic segment, can help standardize the procedure for a consistent and thorough application of porcine pancreatic elastase despite different operators and anatomic variations between mice. Therefore, the elastase/BAPN model is a refined approach to surgically inducing AAA in mice, which may better recapitulate human aneurysms and provide additional opportunities to study aneurysm growth and rupture risk.

Splicing factor hnRNPA1 regulates alternative splicing of LOXL2 to enhance the production of LOXL2Δ13

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family and has the ability to catalyze the cross-linking of extracellular matrix collagen and elastin. High expression of LOXL2 is related to tumor cell proliferation, invasion and metastasis. LOXL2 contains 14 exons. Previous studies have found that LOXL2 has abnormal alternative splicing and exon skipping in a variety of tissues and cells, resulting in a new alternatively-spliced isoform denoted LOXL2Δ13. LOXL2Δ13 lacks LOXL2WT exon 13, but its encoded protein has greater ability to induce tumor cell proliferation, invasion and metastasis. However, the molecular events that produce LOXL2Δ13 are still unclear. In this study, we found that overexpression of the splicing factor hnRNPA1 in cells can regulate the alternative splicing of LOXL2 and increase the expression of LOXL2Δ13. The exonic splicing silencer (ESS) exists at the 3′ splice site (3′ SS) and 5′ splice site (5′ SS) of LOXL2 exon 13. HnRNPA1 can bind to the ESS and inhibit the inclusion of exon 13. The RRM domain of hnRNPA1 and phosphorylation of hnRNPA1 at S91 and S95 are important for the regulation of LOXL2 alternative splicing. These results show that hnRNPA1 is a splicing factor that enhances the production of LOXL2Δ13.

Elastin and collagen fibres in cutaneous wound healing.

Skin forms the outer barrier of the body. Upon injury, successful wound healing in normal skin restores tissue damage and counteracts the loss of extracellular matrix (ECM) proteins and cells. Collagens and elastin are the most abundant structural proteins of the ECM. In homeostasis, collagen type I is the prevalent form, but it is replaced by type III collagen upon wounding, and only later remodelled. In turn, unsuccessful healing results in scars, which tend to be inflexible and inelastic as compared to normal elastic dermis. Scar inelasticity may be due to the absence of mature elastin fibre formation and cross-linking. In this review, the available information on the process of formation of new collagen and elastic fibres during wound healing is analysed. The distinct roles of elastin and collagen proteins during healing are revisited and future research directions proposed which may help improve clinical management of open wounds and scars.

Dysregulation of extracellular matrix and Lysyl Oxidase in Ehlers-Danlos syndrome type IV skin fibroblasts

BackgroundEhlers-Danlos syndrome Type IV (aka Vascular Ehlers Danlos, or vEDS) is a dominantly inherited mutation in the Collagen 3A1 gene (COL3A1). The disease is characterized by tissue friability and age-related susceptibility to arterial aneurysm, dissection and rupture as well as uterine and bowl tears. These clinical manifestations result in major surgical intervention and decreased life expectancy. Understanding how mutations in COL3A1 impact the structure and function of the extracellular matrix (ECM) is important to managing the disease and finding treatments.ResultsSkin fibroblasts from vEDS subjects heterozygous for the p.G588S pathogenic variant in the COL3A1 gene and a normal individual were cultured and studied. Proteomics analysis identified dozens of upregulated proteins related to extracellular matrix dysregulation that is characteristic of fibrosis. Gene expression libraries from cultured primary fibroblasts were screened for messenger RNA (mRNA) markers of ECM degradation. The proteomics and targeted gene expression array results were largely consistent with dysregulation of the extracellular matrix in vEDS. The data show upregulation of multiple Collagen proteins and genes, other ECM components, and enzymes related to ECM processing and turn-over. vEDS fibroblasts expressed significantly more cross linked C-Telopeptide of Collagen III (CTXIII) than normal fibroblasts, indicative of Collagen III degradation and turn-over. Further, the expression and activity of Lysyl Oxidase (LOX), an enzyme that initiates covalent cross-linking of soluble collagen and elastin into protease resistant fibers, is elevated in vEDS fibroblasts compared to normal fibroblasts.ConclusionTogether, these findings suggest dysregulated ECM deposition and processing, reminiscent of a state of fibrosis. Therapeutics that target the dysregulated ECM proteins or help replace damaged tissue may improve clinical outcomes.

LOX, but not LOXL2, promotes bone metastasis formation and bone destruction in triple-negative breast cancer

The primary function of the lysyl oxidase (LOX) family, including LOX and its paralogue LOX-like (LOXL)-2, is to catalyze the covalent crosslinking of collagen and elastin in the extracellular matrix. LOX and LOXL2 are also facilitating breast cancer invasion and metastatic spread to visceral organs (lungs, liver) in vivo. Conversely, the contribution of LOX and LOXL2 to breast cancer bone metastasis remains scant. Here, using gene overexpression or silencing strategies, we investigated the role of LOX and LOXL2 on the formation of metastatic osteolytic lesions in animal models of triple negative breast cancer. In vivo, the extent of radiographic metastatic osteolytic lesions in animals injected with LOX-overexpressing [LOX(+)] tumor cells was 3-fold higher than that observed in animals bearing tumors silenced for LOX [LOX(−)]. By contrast, the extent of osteolytic lesions between LOXL2(+) and LOXL2(−) tumor-bearing animals did not differ, and was comparable to that observed with LOX(−) tumor-bearing animals. In situ, TRAP staining of bone tissue sections from the hind limbs of LOX(+) tumor-bearing animals was substantially increased compared to LOX(−), LOXL2(+) and LOXL2(−)-tumor-bearing animals, which was indicative of enhanced active-osteoclast resorption. In vitro, tumor-secreted LOX increased osteoclast differentiation induced by RANKL, whereas LOXL2 seemed to counteract LOX’s pro-osteoclastic activity. Furthermore, LOX (but not LOXL2) overexpression in tumor cells induced a robust production of IL-6, the latter being a pro-osteoclastic cytokine. Based on these findings, we propose a model in which LOX and IL-6 secreted from tumor cells act in concert to enhance osteoclast-mediated bone resorption that, in turn, promotes metastatic bone destruction in vivo.

PXS5505-MF-101: A Phase 1/2a Study to Evaluate Safety, Pharmacokinetics and Pharmacodynamics of Pxs-5505 in Patients with Primary, Post-Polycythemia Vera or Post-Essential Thrombocythemia Myelofibrosis

Background: Myelofibrosis (MF) is characterized by a progressive increase in extracellular matrix in the bone marrow (BM) associated with decreased production of hematopoietic cells. Though Janus kinase (JAK) inhibitors provide clinical benefit in patients (pts) with MF, they have no direct effect on BM fibrosis (BMF) and thereby limited disease modifying effects. MF is associated with elevated expression of most lysyl oxidase (LOX) isoforms which cross link collagen and elastin. PXS-5505 is a pan-LOX inhibitor that prevents the cross-linking of collagen and elastin and exhibits anti-fibrotic effects in murine models of MF. PXS5505-MF-101 is an ongoing multi-center phase 1/2a study of PXS-5505 in pts with primary, post-polycythemia vera (PV) or post-essential thrombocythemia (ET) MF. (NCT04676529) The study started with two phases: Dose Escalation (DEP) and Cohort Expansion (CEP). The DEP has been completed, establishing a dose of 200mg BID to carry forward into the CEP. During the CEP, up to 24 pts are to be treated for 6 months. Preliminary data from the CEP is presented here. Methods: Study objectives are to determine PXS-5505 safety/tolerability, pharmacokinetics (PK) and pharmacodynamics (PD), BMF changes, spleen volume and MF symptoms (MFSAF). Eligible pts must have primary or post-ET/PV MF of Int-2/high risk; relapsed/refractory or intolerant/ineligible to available JAK inhibitors and have symptomatic disease. Results: At data cutoff (30/Jun/2023), 21 pts have enrolled in the CEP (7 F and 14 M); age 60-86 yrs (median 72 yrs); of these, 10 have completed 24 weeks of Rx and 20 pts have completed ≥1-month Rx. 8 pts had post-ET MF, 5 post-PV MF and 8 PMF; 9 pts have withdrawn from the CEP. Total drug exposure in the CEP is 372 weeks, median 162 days (min 25, max 191). Preliminary CEP PK data are consistent with the DEP with steady state reached by Day 28 with no accumulation observed past this point. The PD data from the DEP demonstrated excellent inhibition of LOX as measured by LOX and LOXL2 target engagement (data previously presented) and this level of inhibition continues to be observed in CEP. [Figure 1] All 9 pts completing 24 weeks PXS-5505 with baseline (BL) biopsies available showed stable reticulin fibrosis at each visit; collagen fibrosis was reduced in 5/9 pts. [Figure 2] Hematological parameters were stable; 7/10 pts had stable/improved hemoglobin and 8/10 had stable/improved platelets over 24 weeks including 3 pts with Grade 4 thrombocytopenia at BL. 4/10 pts had improvements in MF-SAF TSS of ≥20% with 2/10 pts of ≥40% at 24 weeks. No SVR35 responses were seen. There has been 1 fatal AE (febrile neutropenia) on Rx, and 2 post-Rx discontinuation/completion (sepsis, acute myocardial infarction), all unrelated. There have been 6 Rx related adverse events (TRAEs); 2 Grade 1 (vomiting, urticaria), 2 Grade 2 (purpura, hypomagnesaemia), 1 Grade 3 (platelet count decreased), 1 Grade 4 (anemia, noted as leading to drug withdrawal). There have been no serious TRAEs reported. Enrolment into the CEP continues and an additional cohort is planned using PXS-5505 as an add-on to Ruxolitinib Rx. Conclusion: PXS-5505 has been well tolerated with no dose limiting toxicity or serious TRAEs. PD results indicate excellent LOX inhibition at the 200mg BID level. Further, there are preliminary indications of disease modification characterized by stable/improved blood counts and an improvement in collagen fibrosis. These results are encouraging given the poor prognosis seen after Ruxolitinib discontinuation in pts with MF and warrant continued investigation in an earlier Rx setting.

The possibilities of LOXL4 as a prognostic marker for carcinomas.

Lysyl oxidase-like 4 (LOXL4), a member of lysyl oxidase family, is a copper and lysine tyrosylquinone-dependent amine oxidase that serves the role of catalyzing the cross-linking of elastin and collagen in the extracellular matrix. Numerous studies have shown a significant association between LOXL4 expression levels and tumor proliferation, migration, invasion and patients' prognosis and overall survival in different types of tumors. Here we review their relationship and the molecular pathogenesis behind them, aiming to explore the possibilities of LOXL4 as a prognostic marker for diverse carcinomas and provide some indications for further research in this field.

Higher levels of O-GlcNAcylation induce aortic stiffness by increasing elastase activity in male, but not female rats.

Age-associated structural and functional changes in the aorta, including stiffness, loss of elasticity, and reduced vasodilatation, are known to hamper stable peripheral circulation and lead to organ dysfunction. The extracellular matrix (ECM) is a network of macromolecules produced by resident cells. Remodeling of ECM components, include fragmentation of elastin and excessive deposition and crosslinking of collagens, and is a hallmark of aging which leads to stiffening of the aorta. Recently, O-linked beta-N-acetyl glucosamine (O-GlcNAc) has emerged as an essential regulator of cell stress and survival. Interestingly, higher levels of O-GlcNAc are a biomarker for cardiovascular risks (e.g., hypertension and diabetes). However, it is unknown whether O-GlcNAcylation contributes to age-associated aortic stiffness. The goal of these studies was divided into two parts: 1. To determine how the higher levels of O-GlcNAc impact aortic stiffness, and 2. To investigate any potential sex differences. To address this aims, the thoracic aorta was isolated from male and female Wistar and Fisher 344 rats at 3 (3M) and 16 months of age (16M) and mounted on a tissue puller (DMT) to evaluate vessel strain/stress. Blood pressure was measured directly in the carotid artery under anesthesia, and western blot, immunofluorescence, and histology were performed to measure O-GlcNAc levels, elastase expression, and elastin fragmentation, respectively. To induce O-GlcNAc, aorta from 3M Wistar rats were treated with Thiamet G, a pharmacological inhibitor of OGA (1 uM, 24h). The aorta from 3M males presented with a higher strain than age-matched females, indicating sex difference in stiffness (ß-coefficient: 2.5±0.9 vs. 1.6±0.6, p&lt;0.05). However, this difference was abolished in old male and female rats (5.1±0.9 vs. 4.6±0.6 p&lt;0.05 n= duplicates of 4-5). The systolic blood pressure in 16 MO males was increased by ~ 30 mmHg compared to 3M (87±3 vs. 117±4 p=3-4), while only increasing ~ 16 mmHg when compared to 3M and 16 MO females (83±3 vs. 99±4 p=3-4). We observed elevation in O-GlcNAc protein levels from aortas of 16 MO males and females, modestly higher in females. Counter-intuitively, the Thiamet G increased O-GlcNAc levels and induced aortic stiffness in males (2.1± 0.9 vs. 3.6± 0.9 p&lt;0.05 n= duplicates 10), but not in females (2.6± 0.9 vs. 2.7±0.8 n= duplicates 5). The aortic stiffness in males was associated with increased elastin fragmentation and elastase expression (Control 100±10 vs. Thiamet G 400±70 UA p&lt;0.05 n=duplicate 3), which was not observed in females. Collectively, these findings indicate that higher levels of O-GlcNAc contributed to sex differences in aortic stiffness and blood pressure during aging possibly by inducing elastin fragmentation. NIH (R00HL151889, RO1DK132948), NIA (K01AG061263), and American Heart Association (AHA-916031) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2605: LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells

Abstract Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family of copper-dependent amino oxidase, is responsible for the crosslinking of collagen and elastin. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among the tumors with the highest LOXL3 expression levels. Moreover, LOXL3 expression increased with malignancy grade amongst diffusely infiltrative astrocytomas (from grade 2 to 4). In the present work, CRISPR-Cas9 gene editing was used to knock out LOXL3 in the human glioma U87MG cell line to evaluate the role of LOXL3 in glioblastoma. Analyses of protein expression level and genomic mutation mediated by CRISPR-Cas9 by two different sgRNA (denominated sgRNA1 and 2) were performed and the knock out efficiency and genome editing were confirmed. Transcriptome analysis of these cells revealed a downregulation of genes that coded for proteins involved in microtubule organization, cell division, and vesicle trafficking. On the other hand, upregulated genes when LOXL3 was knocked out in U87MG cells were involved in actin cytoskeleton, negative regulation of gene transcription, including chromatin remodeling, and cell-cell adhesion. Functional in vitro assays showed that LOXL3 knocked out cells presented decreased cell proliferation due to longer cell cycles with increased G1 and G2/M checkpoints compared to control cells. Additionally, LOXL3 knocked out U87MG cells presented a higher percentage of cells in the early phase of apoptosis with an increment after temozolomide treatment. Our results suggest an important role of LOXL3 in glioblastoma possibly mediated by cytoskeleton regulation, affecting cell proliferation, cell cycle, and cell death. These results reinforced the importance of LOXL3 in tumors, especially in glioblastoma, highlighting it as a potential therapeutic target. Citation Format: Talita S. Laurentino, Roseli S. Soares, Antonio M. Lerario, Suely K. Marie, Sueli Mieko Oba-Shinjo. LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2605.

A Narrative Review on Serum Biomarkers of Cardiac Fibrosis

Myocardial fibrosis is the excessive deposition of extracellular matrix (ECM) proteins in the cardiac interstitium leading to pathological conditions of the heart. The objective is to understand the pathophysiology of cardiac fibrosis and the quest for serum biomarkers that will assist in early diagnosis before the occurrence of major cardiac events. There are many serum biomarkers that get elevated highlighting ECM remodeling during cardiac fibrosis. Lysyl oxidase like -2 is one such ECM protein, plays a crucial role in the up-regulation of TGF - β, the transformation of cardiac fibroblast to myoblast, the migration of collagen, and cross-linking of collagen and elastin. However, assessment of lysyl oxidase like-2 (LOXL-2) in different pathologically driven cardiac fibrosis is limited. Also, none of the serum biomarkers has proved to be the most accurate diagnostic tool for assessing fibrosis independently; hence, meticulous, less invasive, and cost-effective serum biomarkers need to be scrutinized. Hence lysyl oxidase Like-2 (LOXL-2) in combination with other serum biomarkers like PICP/PINP/TIMP-1/ST-2, or Galectin-3 can be combined to assess the presence of fibrosis in the heart. This review includes the journal, articles, and research paper on cardiac fibrosis which was published in the last 10–15 years to highlight the huge gap in the treatment of cardiac fibrosis and the need for a new combination of biomarkers with better prognostic and diagnostic value.

Evaluation of the Lysyl Oxidase-Like 2 (LOXL2) inhibitory activity of pimaranes and their glycosyl derivatives

Lysyl oxidase (LOX) and LOX-like 1-4 (LOXL 1-4) enzymes catalyze the cross-linking of elastin and collagen in the extracellular matrix, facilitating cell migration and invasion. The inhibition of these enzymes, particularly LOXL2, has been suggested as a therapeutic strategy to prevent breast cancer metastasis. In this work, new natural LOXL2 inhibitors were searched from Aeollanthus rydingianus, a medicinal plant rich in bioactive products. Five pimarane diterpenoids, two isolated from the plant and three derivatives, were tested. These compounds have been described for their bioactive properties such as anti-tumor, anti-inflammatory, analgesic, and antibacterial activities. In this regard, we intended to explore the mechanisms of these compounds by studying their effects on LOXL2 activity. Two pimarane diterpenoids showed a mild LOXL2 inhibitory activity as evaluated by an Amplex Ultra Red-based technique. The cytotoxicity of the most active compound was analyzed by the MTT assay in the MDA-MB-231 cell line, representative of triple-negative breast cancer. This compound decreased cell viability as single agent and increased the cytotoxic effect of doxorubicin. Its glycoconjugate was considerably more toxic, likely due to a higher uptake by cancer cells. Keywords: breast cancer, lysyl oxidase-like 2, inhibitors, pimaranes, Aeollanthus rydingianus, MTT

Phase 1/2a Study to Evaluate Safety, Pharmacokinetic and Pharmacodynamic Dose Escalation and Expansion Study of Pxs-5505 in Patients with Primary, Post-Polycythemia Vera or Post-Essential Thrombocythemia Myelofibrosis

Background: Janus kinase (JAK) inhibitors for myelofibrosis (MF) treatment have provided major clinical benefits, but do not improve cytopenias and demonstrate only modest effects on bone marrow fibrosis (BMF). High discontinuation rates are observed due to loss of therapeutic effect or drug induced toxicity. Reducing BMF may normalize the hematopoietic environment and improve symptoms of MF, particularly blood cell counts. Limited therapeutic options highlight the need for novel, effective therapies for primary MF (PMF). Lysyl oxidases (LOX) are a family of enzymes responsible for the critical post-translational modification essential for the biogenesis of cross-linked collagen and elastin, which form the basis of reticulin in bone marrow (BM). Importantly, several members of the lysyl oxidase family are upregulated in MF providing a rationale to therapeutically inhibit all lysyl oxidases. PXS-5505 is a pan-lysyl oxidase inhibitor that prevents the cross-linking of collagen and elastin and consequently exhibits anti-fibrotic effects in murine models of MF. PXS5505-MF-101 is an ongoing, multi-center, open-label phase 1/2a study evaluating the safety and tolerability of PXS-5505 in patients with primary, post-polycythemia vera (PV) or post-essential thrombocythemia (ET) myelofibrosis (NCT04676529). Herein, we present the study design and preliminary data from the dose escalation phase of this study. Methods: The primary objective is to determine the safety and tolerability of PXS-5505In patients with MF. Secondary objectives include determination of appropriate dose, pharmacokinetics (PK) and pharmacodynamics (PD), changes in BMF using European Consensus, spleen volume and MF symptoms (MFSAF). Efficacy endpoints are exploratory. Eligible patients must have PMF or post-ET/PV MF of intermediate/high risk, relapsed/refractory, intolerant or not eligible to available JAK inhibitors (ruxolitinib, febratinib), including those with severe thrombocytopenia, and requiring therapy for symptomatic disease. Patients participating in the DEP required BM biopsy within 6 months of the re-initiation of treatment with PXS-5505. For patients entering the CEP a BM biopsy must have been performed within 3 months prior to Day 1 treatment. The study consists of two phases: Dose Escalation Phase (DEP) and Cohort Expansion Phase (CEP). The DEP followed a 3+3 design with a starting dose of 100 mg PXS-5505 twice daily (BID) for 4 weeks (Level 1), escalating to 150 mg BID (Level 2) and to 200 mg BID (Level 3). The DEP was to determine the maximum tolerated dose (MTD) or if all doses were well tolerated, the dose which provided the highest target engagement PK and PD measures during dose escalation (Day 0, week 1 and week 4) included, maximum plasma concentration (C1hr=Cmax), minimum plasma concentration (Cmin) and LOX and LOXL2 inhibition in plasma which would subsequently be the dose selected for the CEP. Intra patient dose escalation was allowed and patients participating in the DEP phase were able to continue on the CEP up to 6 months of therapy. During the CEP, 24 patients will be treated at the dose determined from the DEP phase. At data cutoff (21 July 2022) the DEP was completed and enrollment for the CEP continues. Results: Five patients were enrolled in the DEP. This included three patients with post ET MF, one patient with post PV MF and one patient with post ET MF. The median age was 71 years (range, 60-77) and all were female. There were no suspected unexpected serious adverse reactions (SUSAR) or dose limiting toxicities during the dose escalation phase. One patient terminated early following transformation to AML (24 days post treatment initiation). Interim PK data from the DEP phase demonstrated that PK properties in patients were similar to PK in healthy volunteers as previously reported at all dose levels (ACTRN12619000332123). At the 200mg BID level the mean (trough) LOX inhibition was 94% and 91% (days 7 and 28), respectively (Figure 1). Similarly, the mean (trough) LOXL2 inhibition was 94% and 93% (days 7 and 28), respectively. Enrolment in the CEP continues and preliminary data will be presented. Conclusion: These findings indicate that excellent targeted LOX and LOXL2 inhibition is being achieved at the 200mg BID level which has been selected as the recommended dose for the currently ongoing expansion phase with no dose limiting toxicity seen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lysyl Oxidase Family Proteins: Prospective Therapeutic Targets in Cancer.

The lysyl oxidase (LOX) family, consisting of LOX and LOX-like proteins 1–4 (LOXL1–4), is responsible for the covalent crosslinking of collagen and elastin, thus maintaining the stability of the extracellular matrix (ECM) and functioning in maintaining connective tissue function, embryonic development, and wound healing. Recent studies have found the aberrant expression or activity of the LOX family occurs in various types of cancer. It has been proved that the LOX family mainly performs tumor microenvironment (TME) remodeling function and is extensively involved in tumor invasion and metastasis, immunomodulation, proliferation, apoptosis, etc. With relevant translational research in progress, the LOX family is expected to be an effective target for tumor therapy. Here, we review the research progress of the LOX family in tumor progression and therapy to provide novel insights for future exploration of relevant tumor mechanism and new therapeutic targets.

Lysyl Oxidases: Orchestrators of Cellular Behavior and ECM Remodeling and Homeostasis.

Lysyl oxidases have long been considered key secreted extracellular matrix modifying enzymes. As such, their activity has been associated with the crosslinking of collagens and elastin, and as a result, they have been linked to multiple developmental and pathological processes. However, numerous lines of evidence also demonstrated that members of this enzyme family are localized and are active within the cytoplasm or cell nuclei, where they regulate and participate in distinct cellular events. In this review, we focus on a few of these events and highlight the intracellular role these enzymes play. Close examination of these events, suggest that the intracellular activities of lysyl oxidases is mostly observed in processes where concomitant changes in the extracellular matrix takes place. Here, we suggest that the LOX family members act in the relay between changes in the cells’ environment and the intracellular processes that promote them or that follow.

Abstract PO033: A phase 1b/2 trial of PXS-5505 combined with first line atezolizumab plus bevacizumab for treating patients with unresectable or metastatic hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) accounts for 90% of primary liver malignancies with just 20-30% resectable at presentation. Combination of atezolizumab and bevacizumab has brought immunotherapy to the forefront of combating this disease and represents an attractive immunotherapeutic backbone for pairing HCC therapy with means to improve intratumoral drug delivery and boost response. Lysyl oxidases (LOX) are a family of 5 secreted amine oxidases that catalyze the cross-linking of collagen and elastin in the extracellular matrix. PXS-5505 is a novel pan-LOX inhibitor that we have utilized in a pre-clinical model of primary liver malignancy and demonstrated improved survival when combined with systemic therapy through increased intratumoral drug delivery and enhanced host anti-tumor immunity. To study the impact of PXS-5505 in HCC, we designed a phase Ib/II trial of PXS-5505 in combination with atezolizumab and bevacizumab for treating patients with unresectable or metastatic HCC.Methods: This phase Ib/II trial includes patients with newly diagnosed, systemic therapy naïve, unresectable or metastatic HCC. Patients who received previous locoregional therapies remain eligible. Phase Ib consists of an open-label safety and tolerability assessment of PXS-5505 with a dose escalation design incorporating first-line combination therapy with atezolizumab and bevacizumab. The primary endpoint will be the maximal tolerated dose (MTD) of PXS-5505 through the Bayesian optimal interval (BOIN) design, targeting a dose limiting toxicity (DLT) rate of 30%. Secondary endpoints include the confirmed objective response rate (ORR) in evaluable patients after completing at least 4 cycles (12 weeks) of treatment. Once 12 patients have accrued to a particular dose, the MTD dose is established. Based on Simon’s two-stage design, if there are 4 or more responses with the MTD cohort, an expansion cohort will begin for phase II. Phase II will enroll an additional 30 patients at the MTD for a total evaluable sample size of 42. The primary endpoint will be the confirmed ORR in evaluable patients after completing at least 4 cycles of treatment. Assessment of ORR will be performed 3-6 months after start of treatment at the MTD with confirmatory assessment 28-56 days afterwards. If there are 15 or more responses among these 42 patients, we reject the null hypothesis and claim that the treatment is promising. A confirmed ORR of 45% will afford a 10% type 1 error with 80% power to detect a difference. Secondary endpoints include assessment of progression free survival (median from time of enrollment) and overall survival (median overall survival from time of enrollment). Exploratory endpoints include paired biopsies (at baseline and after the 4th cycle of treatment) for assessment of immunophenotypic and stromal changes to the tumor microenvironment in response to treatment with immunohistochemistry, flow cytometry, and RNA qRT-PCR analysis. The trial identification number is NCT05109052 and is expected to start enrollment in May of 2022. Citation Format: Paul R Burchard, Luis I Ruffolo, Nicholas Ullman, Yatee Dave, Brian A Belt, David C Linehan, Roberto Hernandez-Alejandro, Nabeel Badri. A phase 1b/2 trial of PXS-5505 combined with first line atezolizumab plus bevacizumab for treating patients with unresectable or metastatic hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO033.

Correlation of Matrisome-Associatted Gene Expressions with LOX Family Members in Astrocytomas Stratified by IDH Mutation Status.

Tumor cell infiltrative ability into surrounding brain tissue is a characteristic of diffusely infiltrative astrocytoma and is strongly associated with extracellular matrix (ECM) stiffness. Collagens are the most abundant ECM scaffolding proteins and contribute to matrix organization and stiffness. LOX family members, copper-dependent amine oxidases, participate in the collagen and elastin crosslinking that determine ECM tensile strength. Common IDH mutations in lower-grade gliomas (LGG) impact prognosis and have been associated with ECM stiffness. We analyzed the expression levels of LOX family members and matrisome-associated genes in astrocytoma stratified by malignancy grade and IDH mutation status. A progressive increase in expression of all five LOX family members according to malignancy grade was found. LOX, LOXL1, and LOXL3 expression correlated with matrisome gene expressions. LOXL1 correlations were detected in LGG with IDH mutation (IDHmut), LOXL3 correlations in LGG with IDH wild type (IDHwt) and strong LOX correlations in glioblastoma (GBM) were found. These increasing correlations may explain the increment of ECM stiffness and tumor aggressiveness from LGG-IDHmut and LGG-IDHwt through to GBM. The expression of the mechanosensitive transcription factor, β-catenin, also increased with malignancy grade and was correlated with LOXL1 and LOXL3 expression, suggesting involvement of this factor in the outside–in signaling pathway.

Effect of Anti-Hypertensive Medication on Plasma Concentrations of Lysyl Oxidase: Evidence for Aldosterone-IL-6-Dependent Regulation of Lysyl Oxidase Blood Concentration.

Lysyl oxidase (LOX) is a secretory protein that catalyzes elastin and collagen cross-linking. Lowering LOX expression and activity in endothelial cells is associated with a high risk of aneurysms and vascular malformation. Interleukin-6 (IL-6), elevated in hypertension, is known to suppress LOX expression. The influence of anti-hypertensive medication on the plasma LOX concentration is currently unknown. In a cohort of 34 patients diagnosed with resistant hypertension and treated with up to nine different drugs, blood concentration of LOX was analyzed to identify drugs that have an impact on plasma LOX concentration. Key findings were confirmed in a second independent patient cohort of 37 patients diagnosed with dilated cardiomyopathy. Blood concentrations of aldosterone and IL-6 were analyzed. In vitro, the effect of IL-6 on LOX expression was analyzed in endothelial cells. Patients receiving aldosterone antagonists had the highest plasma LOX concentration in both cohorts. This effect was independent of sex, age, blood pressure, body mass index, and co-medication. Blood aldosterone concentration correlates with plasma IL-6 concentration. In vitro, IL-6 decreased the expression of LOX in endothelial cells but not fibroblasts. Aldosterone was identified as a factor that affects blood concentration of LOX in an IL-6-dependent manner.

Lysyl Oxidases Expression and Breast Cancer Progression: A Bioinformatic Analysis.

LOX (Lysyl oxidase) and LOX like 1–4 (LOXL1–4) are amine oxidases that catalyse the cross-linking of elastin and collagen in the extracellular matrix (ECM). This activity can facilitate cell migration and the formation of metastases. Consequently, inhibition of these enzymes and, in particular of LOXL2, has been suggested as a therapeutic strategy to prevent breast cancer metastasis. Although medicinal chemistry studies have struggled to specifically inhibit LOXL2, the importance of selectivity in this context is not clear. To explore the role of each LOX in breast cancer and consequently their potential as biomarkers or therapeutic targets, a bioinformatic-based approach was followed. The expression profile of LOXs, the putative associations among mRNA expression from each LOX and clinical observations, the correlation between expression of LOX enzymes and other genes, and the association between expression of LOXs and the tumour infiltrates were assessed for breast cancer. Overall, the patient outcome and the characteristics of breast tumours with LOX, LOXL1 and LOXL2 upregulation is distinct from those with high expression of LOXL3 and LOXL4. Additionally, the expression correlation between LOXs and other genes involved in cellular processes relevant for cancer biology, also reveals a similar trend for LOX, LOXL1 and LOX2. This work further supports the relevance of LOXL2 as a breast cancer progression biomarker and therapeutic target. We speculate that while the impact of LOXL3 inhibition may vary with breast cancer subtype, the therapeutical inhibition of LOX, LOXL1 and LOXL2 but not of LOXL4 may be the most beneficial.