Unlocking hidden potential: advancements, approaches, and obstacles in repurposing drugs for cancer therapy.

High rates of failure, exorbitant costs, and the sluggish pace of new drug discovery and development have led to a growing interest in repurposing "old" drugs to treat both common and rare diseases, particularly cancer. Cancer, a complex and heterogeneous disease, often necessitates a combination of different treatment modalities to achieve optimal outcomes. The intrinsic polygenicity of cancer, intricate biological signalling networks, and feedback loops make the inhibition of a single target frequently insufficient for achieving the desired therapeutic impact. As a result, addressing these complex or "smart" malignancies demands equally sophisticated treatment strategies. Combinatory treatments that target the multifaceted oncogenic signalling network hold immense promise. Repurposed drugs offer a potential solution to this challenge, harnessing known compounds for new indications. By avoiding the prohibitive costs and long development timelines associated with novel cancer drugs, this approach holds the potential to usher in more effective, efficient, and cost-effective cancer treatments. The pursuit of combinatory therapies through drug repurposing may hold the key to achieving superior outcomes for cancer patients. However, drug repurposing faces significant commercial, technological and regulatory challenges that need to be addressed. This review explores the diverse approaches employed in drug repurposing, delves into the challenges faced by the drug repurposing community, and presents innovative solutions to overcome these obstacles. By emphasising the significance of combinatory treatments within the context of drug repurposing, we aim to unlock the full potential of this approach for enhancing cancer therapy. The positive aspects of drug repurposing in oncology are underscored here; encompassing personalized treatment, accelerated development, market opportunities for shelved drugs, cancer prevention, expanded patient reach, improved patient access, multi-partner collaborations, increased likelihood of approval, reduced costs, and enhanced combination therapy.

Utility of the Cerebral Organoid Glioma 'GLICO' Model for Screening Applications.

Glioblastoma, a grade IV astrocytoma, is regarded as the most aggressive primary brain tumour with an overall median survival of 16.0 months following the standard treatment regimen of surgical resection, followed by radiotherapy and chemotherapy with temozolomide. Despite such intensive treatment, the tumour almost invariably recurs. This poor prognosis has most commonly been attributed to the initiation, propagation, and differentiation of cancer stem cells. Despite the unprecedented advances in biomedical research over the last decade, the current in vitro models are limited at preserving the inter- and intra-tumoural heterogeneity of primary tumours. The ability to understand and manipulate complex cancers such as glioblastoma requires disease models to be clinically and translationally relevant and encompass the cellular heterogeneity of such cancers. Therefore, brain cancer research models need to aim to recapitulate glioblastoma stem cell function, whilst remaining amenable for analysis. Fortunately, the recent development of 3D cultures has overcome some of these challenges, and cerebral organoids are emerging as cutting-edge tools in glioblastoma research. The opportunity to generate cerebral organoids via induced pluripotent stem cells, and to perform co-cultures with patient-derived cancer stem cells (GLICO model), has enabled the analysis of cancer development in a context that better mimics brain tissue architecture. In this article, we review the recent literature on the use of patient-derived glioblastoma organoid models and their applicability for drug screening, as well as provide a potential workflow for screening using the GLICO model. The proposed workflow is practical for use in most laboratories with accessible materials and equipment, a good first pass, and no animal work required. This workflow is also amenable for analysis, with separate measures of invasion, growth, and viability.

Cell Populations Expressing Stemness-Associated Markers in Lung Adenocarcinoma.

The stemness-associated markers OCT4, NANOG, SOX2, KLF4 and c-MYC are expressed in numerous cancer types suggesting the presence of cancer stem cells (CSCs). Immunohistochemical (IHC) staining performed on 12 lung adenocarcinoma (LA) tissue samples showed protein expression of OCT4, NANOG, SOX2, KLF4 and c-MYC, and the CSC marker CD44. In situ hybridization (ISH) performed on six of the LA tissue samples showed mRNA expression of OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence staining performed on three of the tissue samples showed co-expression of OCT4 and c-MYC with NANOG, SOX2 and KLF4 by tumor gland cells, and expression of OCT4 and c-MYC exclusively by cells within the stroma. RT-qPCR performed on five LA-derived primary cell lines showed mRNA expression of all the markers except SOX2. Western blotting performed on four LA-derived primary cell lines demonstrated protein expression of all the markers except SOX2 and NANOG. Initial tumorsphere assays performed on four LA-derived primary cell lines demonstrated 0-80% of tumorspheres surpassing the 50 µm threshold. The expression of the stemness-associated markers OCT4, SOX2, NANOG, KFL4 and c-MYC by LA at the mRNA and protein level, and the unique expression patterns suggest a putative presence of CSC subpopulations within LA, which may be a novel therapeutic target for this cancer. Further functional studies are required to investigate the possession of stemness traits.

Cathepsins B, D, and G Are Expressed in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma.

AIM: We have previously demonstrated the presence of two cancer stem cell (CSC) subpopulations within metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC) expressing components of the renin-angiotensin system (RAS), which promotes tumorigenesis. Cathepsins B, D and G are enzymes that constitute bypass loops for the RAS. This study investigated the expression and localization of cathepsins B, D, and G in relation to CSC subpopulations within mHNcSCC. METHODS: Immunohistochemical staining was performed on mHNcSCC tissue samples from 20 patients to determine the expression and localization of cathepsins B, D, and G. Immunofluorescence staining was performed on two of these mHNcSCC tissue samples by co-staining of cathepsins B and D with OCT4 and SOX2, and cathepsin G with mast cell markers tryptase and chymase. Western blotting and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were performed on five mHNcSCC samples and four mHNcSCC-derived primary cell lines, to determine protein and transcript expression of these three cathepsins, respectively. Enzyme activity assays were performed on mHNcSCC tissue samples to determine whether these cathepsins were active. RESULTS: Immunohistochemical staining demonstrated the presence of cathepsins B, D and G in in all 20 mHNcSCC tissue samples. Immunofluorescence staining showed that cathepsins B and D were localized to the CSCs both within the tumor nests and peri-tumoral stroma (PTS) and cathepsin G was localized to the phenotypic mast cells within the PTS. Western blotting demonstrated protein expression of cathepsin B and D, and RT-qPCR demonstrated transcript expression of all three cathepsins. Enzyme activity assays showed that cathepsin B and D to be active. CONCLUSION: The presence of cathepsins B and D on the CSCs and cathepsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for mHNcSCC.

Clinical and molecular associations with outcomes at 2 years after acute knee injury: a longitudinal study in the Knee Injury Cohort at the Kennedy (KICK).

BACKGROUND: Joint injury is a major risk factor for osteoarthritis and provides an opportunity to prospectively examine early processes associated with osteoarthritis. We investigated whether predefined baseline demographic and clinical factors, and protein analytes in knee synovial fluid and in plasma or serum, were associated with clinically relevant outcomes at 2 years after knee injury. METHODS: This longitudinal cohort study recruited individuals aged 16-50 years between Nov 1, 2010, and Nov 28, 2014, across six hospitals and clinics in London, UK. Participants were recruited within 8 weeks of having a clinically significant acute knee injury (effusion and structural injury on MRI), which was typically treated surgically. We measured several predefined clinical variables at baseline (eg, time from injury to sampling, extent and type of joint injury, synovial fluid blood staining, presence of effusion, self-reported sex, age, and BMI), and measured 12 synovial fluid and four plasma or serum biomarkers by immunoassay at baseline and 3 months. The primary outcome was Knee Injury and Osteoarthritis Outcome Score (KOOS4) at 2 years, adjusted for baseline score, assessed in all patients. Linear and logistic regression models adjusting for predefined covariates were used to assess associations between baseline variables and 2-year KOOS4. This study is registered with ClinicalTrials.gov, number NCT02667756. FINDINGS: We enrolled 150 patients at a median of 17 days (range 1-59, IQR 9-26) after knee injury. 123 (82%) were male, with a median age of 25 years (range 16-50, IQR 21-30). 98 (65%) of 150 participants completed a KOOS4 at 2 (or 3) years after enrolment (50 participants were lost to follow-up and two were withdrawn due to adverse events unrelated to study participation); 77 (51%) participants had all necessary variables available and were included in the core variable adjusted analysis. In the 2-year dataset mean KOOS4 improved from 38 (SD 18) at baseline to 79 (18) at 2 years. Baseline KOOS4, medium-to-large knee effusion, and moderate-to-severe synovial blood staining and their interaction significantly predicted 2-year KOOS4 (n=77; coefficient -20·5, 95% CI -34·8 to -6·18; p=0·0060). The only predefined biomarkers that showed independent associations with 2-year KOOS4 were synovial fluid MCP-1 (n=77; -0·015, 0·027 to -0·004 per change in 1 pg/mL units; p=0·011) and IL-6 (n=77; -0·0005, -0·0009 to -0·0001 per change in 1 pg/mL units; p=0·017). These biomarkers, combined with the interaction of effusion and blood staining, accounted for 39% of outcome variability. Two adverse events occurred that were linked to study participation, both at the time of blood sampling (one presyncopal episode, one tenderness and pain at the site of venepuncture). INTERPRETATION: The combination of effusion and haemarthrosis was significantly associated with symptomatic outcomes after acute knee injury. The synovial fluid molecular protein response to acute knee injury (best represented by MCP-1 and IL-6) was independently associated with symptomatic outcomes but not with structural outcomes, with the biomarkers overall playing a minor role relative to clinical predictors. The relationship between symptoms and structure after acute knee injury and their apparent dissociation early in this process need to be better understood to make clinical progress. FUNDING: Versus Arthritis, Kennedy Trust for Rheumatology Research, and NIHR Oxford Biomedical Research Centre.

Expression of Cathepsins B, D, and G in Extracranial Arterio-Venous Malformation.

Objectives: We have previously identified a population of cells that expressed stemness-associated markers in extracranial arterio-venous malformation (AVM) and demonstrated expression of cathepsins B, D, and G on embryonic stem cell (ESC)-like populations in other vascular anomalies. This study investigated the expression of cathepsins B, D, and G, and their localization in relation to this primitive population in extracranial AVM. Methods: Immunohistochemical staining was performed on AVM tissue samples from 13 patients to demonstrate expression of cathepsins B, D, and G. Western blotting was performed on four AVM tissue samples and three AVM-derived primary cell lines to confirm protein expression of cathepsins B and D proteins. RT-qPCR was performed on three AVM-derived primary cell lines to demonstrate transcript expression of cathepsins B, D, and G. Enzymatic activity assays were performed on three AVM-derived primary cell lines to investigate if cathepsins B and D were active. Localization of the cathepsins was investigated using immunofluorescence dual-staining of the cathepsins with the ESC markers OCT4 and SOX2, and mast cells marker chymase on two of the 13 AVM tissue samples. Results: Immunohistochemical staining demonstrated expression of cathepsins B, D, and G in all 13 AVM tissue samples. Western blotting showed expression of cathepsins B and D proteins in all four AVM tissue samples and all three AVM-derived primary cell lines. RT-qPCR demonstrated transcripts of cathepsins B, D, and G in all three AVM-derived primary cell lines. Enzymatic activity assays showed that cathepsins B and D were active. Immunofluorescence staining showed expression of cathepsins B and D on the OCT4+/SOX2+ endothelium and media of the lesional vessels and cells within the stroma in AVM nidus. Cathepsin G was expressed on the chymase+ phenotypic mast cells. Conclusions: This study demonstrated the novel finding of the expression of cathepsins B, D, and G in AVM. Cathepsins B and D were expressed by the primitive population, and cathepsin G was localized to mast cells, within the AVM nidus.

Expression of cathepsins B and D by cancer stem cells in head and neck metastatic malignant melanoma.

We have previously demonstrated cancer stem cell (CSC) subpopulations in head and neck metastatic malignant melanoma (HNmMM), and the expression of components of the renin-angiotensin system (RAS) by these CSCs. Cathepsins B, D and G are involved in carcinogenesis and constitute bypass loops of the RAS. This study investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs. Immunohistochemical staining demonstrated expression of cathepsins B, D and G in HNmMM sections from all 20 patients. Western blotting confirmed the presence of cathepsins B and D proteins in all six HNmMM tissue samples and four HNmMM-derived primary cell lines. RT-qPCR showed transcript expression of cathepsins B, D and G in all six HNmMM tissue samples, and cathepsins B and D but not cathepsin G in all four HNmMM-derived primary cell lines. Enzymatic activity assays demonstrated cathepsins B and D were active in all six HNmMM tissue samples. Immunofluorescence staining performed on two of the HNmMM tissue samples demonstrated expression of cathepsins B and D by the CSCs, and cathepsin G by cells within the peritumoral stroma. Our novel findings suggest the possibility of targeting these CSCs by modulation of paracrine RAS signaling.

Stemness-Associated Markers Are Expressed in Extracranial Arteriovenous Malformation.

Objectives: Arteriovenous malformation (AVM) consists of a nidus with poorly formed low-resistance vessels in place of a functional capillary network. The role of somatic mutations in embryonic stem cells (ESCs) and vascular anomalies and the presence of primitive populations in vascular anomalies led us to investigate the presence of a primitive population in extracranial AVM. Methods: Extracranial AVM tissue samples from 12 patients were stained for stemness-associated markers OCT4, SOX2, NANOG, KLF4, and c-MYC using immunohistochemical staining. In situ hybridization (ISH) was performed on six tissue samples to determine transcript expression. Western blotting and RT-qPCR were performed on two AVM-derived primary cell lines to determine protein and transcript expression of these markers, respectively. Immunofluorescence staining was performed on two tissue samples to investigate marker co-localization. Results: Immunohistochemical staining demonstrated the expression of OCT4, SOX2, KLF4, and c-MYC on the endothelium and media of lesional vessels and cells within the stroma of the nidus in all 12 AVM tissue samples. ISH and RT-qPCR confirmed transcript expression of all five markers. Western blotting showed protein expression of all markers except NANOG. Immunofluorescence staining demonstrated an OCT4+/SOX2+/KLF4+/c-MYC+ population within the endothelium and media of the lesional vessels and cells within the stroma of the AVM nidus. Conclusions: Our findings may suggest the presence of a primitive population within the AVM nidus. Further investigation may lead to novel therapeutic targeting of this population.

Cancer Stem Cells in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System.

We investigated the expression of components of the renin-angiotensin system (RAS) by cancer stem cell (CSC) subpopulations in metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC). Immunohistochemical staining demonstrated expression of prorenin receptor (PRR), angiotensin-converting enzyme (ACE), and angiotensin II receptor 2 (AT2R) in all cases and angiotensinogen in 14 cases; however, renin and ACE2 were not detected in any of the 20 mHNcSCC tissue samples. Western blotting showed protein expression of angiotensinogen in all six mHNcSCC tissue samples, but in none of the four mHNcSCC-derived primary cell lines, while PRR was detected in the four cell lines only. RT-qPCR confirmed transcripts of angiotensinogen, PRR, ACE, and angiotensin II receptor 1 (AT1R), but not renin or AT2R in all four mHNcSCC tissue samples and all four mHNcSCC-derived primary cell lines, while ACE2 was expressed in the tissue samples only. Double immunohistochemical staining on two of the mHNcSCC tissue samples showed expression of angiotensinogen by the SOX2+ CSCs within the tumor nests (TNs), and immunofluorescence showed expression of PRR and AT2R by the SOX2+ CSCs within the TNs and the peritumoral stroma (PTS). ACE was expressed on the endothelium of the tumor microvessels within the PTS. We demonstrated expression of angiotensinogen by CSCs within the TNs, PRR, and AT2R by the CSCs within the TNs and the PTS, in addition to ACE on the endothelium of tumor microvessels in mHNcSCC.

Cancer Stem Cells in Head and Neck Metastatic Malignant Melanoma Express Components of the Renin-Angiotensin System.

Components of the renin-angiotensin system (RAS) are expressed by cancer stem cells (CSCs) in many cancer types. We here investigated expression of the RAS by the CSC subpopulations in human head and neck metastatic malignant melanoma (HNmMM) tissue samples and HNmMM-derived primary cell lines. Immunohistochemical staining demonstrated expression of pro-renin receptor (PRR), angiotensin-converting enzyme (ACE), and angiotensin II receptor 2 (AT2R) in all; renin in one; and ACE2 in none of the 20 HNmMM tissue samples. PRR was localized to cells within the tumor nests (TNs), while AT2R was expressed by cells within the TNs and the peritumoral stroma (PTS). ACE was localized to the endothelium of the tumor microvessels within the PTS. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) detected transcripts for PRR, ACE, ACE2, and AT1R, in all the five HNmMM tissue samples and four HNmMM-derived primary cell lines; renin in one tissue sample and one cell line, and AT2R in none of the five HNmMM tissue samples and cell lines. Western blotting showed variable expression of ACE, PRR, and AT2R, but not ACE2, in six HNmMM tissue samples and two HNmMM-derived primary cell lines. Immunofluorescence staining of two HNmMM tissue samples demonstrated expression of PRR and AT2R by the SOX2+ CSCs within the TNs and the OCT4+ CSCs within the PTS, with ACE localized to the endothelium of the tumor microvessels within the PTS.

Cancer Stem Cell Subpopulations Are Present Within Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma.

Cancer stem cells (CSCs) have been identified in many cancer types including primary head and neck cutaneous squamous cell carcinoma (HNcSCC). This study aimed to identify and characterize CSCs in metastatic HNcSCC (mHNcSCC). Immunohistochemical staining performed on mHNcSCC samples from 15 patients demonstrated expression of the induced pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4, and c-MYC in all 15 samples. In situ hybridization and RT-qPCR performed on four of these mHNcSCC tissue samples confirmed transcript expression of all five iPSC markers. Immunofluorescence staining performed on three of these mHNcSCC samples demonstrated expression of c-MYC on cells within the tumor nests (TNs) and the peri-tumoral stroma (PTS) that also expressed KLF4. OCT4 was expressed on the SOX2+/NANOG+/KLF4+ cells within the TNs, and the SOX2+/NANOG+/KLF4+ cells within the PTS. RT-qPCR demonstrated transcript expression of all five iPSC markers in all three mHNcSCC-derived primary cell lines, except for SOX2 in one cell line. Western blotting showed the presence of SOX2, KLF4, and c-MYC but not OCT4 and NANOG in the three mHNcSCC-derived primary cell lines. All three cell lines formed tumorspheres, at the first passage. We demonstrated an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ CSC subpopulation and an OCT4+/NANOG-/SOX2+/KLF4+/c-MYC+ subpopulation within the TNs, and an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ subpopulation within the PTS of mHNcSCC.

Identification of Cancer Stem Cell Subpopulations in Head and Neck Metastatic Malignant Melanoma.

Cancer stem cells (CSCs) have been identified in many cancer types. This study identified and characterized CSCs in head and neck metastatic malignant melanoma (HNmMM) to regional lymph nodes using induced pluripotent stem cell (iPSC) markers. Immunohistochemical (IHC) staining performed on 20 HNmMM tissue samples demonstrated expression of iPSC markers OCT4, SOX2, KLF4, and c-MYC in all samples, while NANOG was expressed at low levels in two samples. Immunofluorescence (IF) staining demonstrated an OCT4+/SOX2+/KLF4+/c-MYC+ CSC subpopulation within the tumor nests (TNs) and another within the peritumoral stroma (PTS) of HNmMM tissues. IF also showed expression of NANOG by some OCT4+/SOX2+/KLF4+/c-MYC+ cells within the TNs in an HNmMM tissue sample that expressed NANOG on IHC staining. In situ hybridization (n = 6) and reverse-transcription quantitative polymerase chain reaction (n = 5) on the HNmMM samples confirmed expression of all five iPSC markers. Western blotting of primary cell lines derived from four of the 20 HNmMM tissue samples showed expression of SOX2, KLF4, and c-MYC but not OCT4 and NANOG, and three of these cell lines formed tumorspheres in vitro. We demonstrate the presence of two putative CSC subpopulations within HNmMM, which may be a novel therapeutic target in the treatment of this aggressive cancer.

Acute Molecular Changes in Synovial Fluid Following Human Knee Injury: Association With Early Clinical Outcomes.

OBJECTIVE: To investigate whether molecules found to be up-regulated within hours of surgical joint destabilization in the mouse are also elevated in the analogous human setting of acute knee injury, how this molecular response varies between individuals, and whether it is related to patient-reported outcomes in the 3 months after injury. METHODS: Seven candidate molecules were analyzed in blood and synovial fluid (SF) from 150 participants with recent structural knee injury at baseline (<8 weeks from injury) and in blood at 14 days and 3 months following baseline. Knee Injury and Osteoarthritis Outcome Score 4 (KOOS4 ) was obtained at baseline and 3 months. Patient and control samples were compared using Meso Scale Discovery platform assays or enzyme-linked immunosorbent assay. RESULTS: Six of the 7 molecules were significantly elevated in human SF immediately after injury: interleukin-6 (IL-6), monocyte chemotactic protein 1, matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinases 1 (TIMP-1), activin A, and tumor necrosis factor-stimulated gene 6 (TSG-6). There was low-to-moderate correlation with blood measurements. Three of the 6 molecules were significantly associated with baseline KOOS4 (those with higher SF IL-6, TIMP-1, or TSG-6 had lower KOOS4 ). These 3 molecules, MMP-3, and activin A were all significantly associated with greater improvement in KOOS4 over 3 months, after adjustment for other relevant factors. Of these, IL-6 alone significantly accounted for the molecular contribution to baseline KOOS4 and change in KOOS4 over 3 months. CONCLUSION: Our findings validate relevant human biomarkers of tissue injury identified in a mouse model. Analysis of SF rather than blood more accurately reflects this response. The response is associated with patient-reported outcomes over this early period, with SF IL-6 acting as a single representative marker. Longitudinal outcomes will determine if these molecules are biomarkers of subsequent disease risk.

Efficacy and safety of apremilast, an oral phosphodiesterase 4 inhibitor, in ankylosing spondylitis.

OBJECTIVES: To evaluate the efficacy and safety of an oral phosphodiesterase 4 inhibitor, apremilast, in treatment of ankylosing spondylitis (AS) by monitoring symptoms and signs in a pilot study including exploratory investigation of effects of PDE4 inhibition on blood biomarkers of bone biology. METHODS: In this double-blind, placebo-controlled, single-centre, Phase II study, patients with symptomatic AS with active disease on MRI were randomised to apremilast 30 mg BID or placebo over 12 weeks. Bath Indices were monitored serially. Patients were followed for 4 weeks after stopping medication. Bone biomarkers were assessed at baseline and day 85. RESULTS: 38 subjects were randomised and 36 subjects completed the study. Although the primary end-point (change in BASDAI at week 12) was not met, apremilast was associated with numerically greater improvement from baseline for all clinical assessments compared with placebo with mean change in BASDAI (-1.59±1.48 vs -0.77±1.47), BASFI (-1.74±1.91 vs -0.28±1.61) and BASMI (-0.51±1.02 vs -0.21±0.67); however, differences did not achieve statistical significance. The clinical indices returned to baseline values by 4 weeks after cessation of apremilast. Six apremilast patients (35.3%) vs 3 placebo (15.8%) achieved ASAS20 responses (p=0.25). There were statistically significant decreases in serum RANKL and RANKL:osteoprotegrin ratio and plasma sclerostin but no significant changes in serum DKK-1, bone alkaline phosphatase, TRAP5b, MMP3, osteoprotegrin, or osteocalcin. CONCLUSIONS: Although a small pilot study, these results suggest that apremilast may be effective and well tolerated in AS and modulates biomarkers of bone biology. These data support further research of apremilast in axial inflammation.

Discovery of novel aspartyl ketone dipeptides as potent and selective caspase-3 inhibitors.

The discovery of a series of potent, selective and reversible dipeptidyl caspase-3 inhibitors are reported. The iterative discovery process of using combinatorial chemistry, parallel synthesis, moleculare modelling and structural biology will be discussed.