Accelerated Closure of Diabetic Wounds by Efficient Recruitment of Fibroblasts upon Inhibiting a 14-3-3/ROCK Regulatory Axis.

Chronic non-healing wounds negatively impact quality of life and are a significant financial drain on health systems. The risk of infection that exacerbates comorbidities in patients necessitates regular application of wound care. Understanding the mechanisms underlying impaired wound healing are therefore a key priority to inform effective new-generation treatments. In this study, we demonstrate that 14-3-3-mediated suppression of signaling through ROCK is a critical mechanism that inhibits the healing of diabetic wounds. Accordingly, pharmacological inhibition of 14-3-3 by topical application of the sphingo-mimetic drug RB-11 to diabetic wounds on a mouse model of type II diabetes accelerated wound closure more than 2-fold than vehicle control, phenocopying our previous observations in 14-3-3ζ-knockout mice. We also demonstrate that accelerated closure of the wounded epidermis by 14-3-3 inhibition causes enhanced signaling through the Rho-ROCK pathway and that the underlying cellular mechanism involves the efficient recruitment of dermal fibroblasts into the wound and the rapid production of extracellular matrix proteins to re-establish the injured dermis. Our observations that the 14-3-3/ROCK inhibitory axis characterizes impaired wound healing and that its suppression facilitates fibroblast recruitment and accelerated re-epithelialization suggest new possibilities for treating diabetic wounds by pharmacologically targeting this axis.

Mitochondrial translocation of TFEB regulates complex I and inflammation.

TFEB is a master regulator of autophagy, lysosome biogenesis, mitochondrial metabolism, and immunity that works primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomics analysis reveals extensive TFEB co-immunoprecipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. High resolution confocal microscopy and biochemistry confirms TFEB localization in the mitochondrial matrix. TFEB translocation depends on a conserved N-terminal TOMM20-binding motif and is enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.

An orthotopic syngeneic mouse model of bortezomib-resistant multiple myeloma.

While bortezomib has significant benefits in multiple myeloma (MM) therapy, the disease remains incurable due to the invariable development of bortezomib resistance. This emphasises the need for advanced models for preclinical evaluation of new therapeutic approaches for bortezomib-resistant MM. Here, we describe the development of an orthotopic syngeneic bortezomib-resistant MM mouse model based on the most well-characterised syngeneic MM mouse model derived from spontaneous MM-forming C57BL/KaLwRij mice. Using bortezomib-resistant 5TGM1 cells, we report and characterise a robust syngeneic mouse model of bortezomib-resistant MM that is well suited to the evaluation of new therapeutic approaches for proteasome inhibitor-resistant MM.

Genome-wide transcription factor-binding maps reveal cell-specific changes in the regulatory architecture of human HSPCs.

Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.

Dynamic interplay between sortilin and syndecan-1 contributes to prostate cancer progression.

Prostate cancer (PCa) development and progression relies on the programming of glucose and lipid metabolism, and this involves alterations in androgen receptor expression and signalling. Defining the molecular mechanism that underpins this metabolic programming will have direct significance for patients with PCa who have a poor prognosis. Here we show that there is a dynamic balance between sortilin and syndecan-1, that reports on different metabolic phenotypes. Using tissue microarrays, we demonstrated by immunohistochemistry that sortilin was highly expressed in low-grade cancer, while syndecan-1 was upregulated in high-grade disease. Mechanistic studies in prostate cell lines revealed that in androgen-sensitive LNCaP cells, sortilin enhanced glucose metabolism by regulating GLUT1 and GLUT4, while binding progranulin and lipoprotein lipase (LPL) to limit lipid metabolism. In contrast, in androgen-insensitive PC3 cells, syndecan-1 was upregulated, interacted with LPL and colocalised with ß3 integrin to promote lipid metabolism. In addition, androgen-deprived LNCaP cells had decreased expression of sortilin and reduced glucose-metabolism, but increased syndecan-1 expression, facilitating interactions with LPL and possibly ß3 integrin. We report a hitherto unappreciated molecular mechanism for PCa, which may have significance for disease progression and how androgen-deprivation therapy might promote castration-resistant PCa.

Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia.

The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.

Targeting the integrated stress response in hematologic malignancies.

While numerous targeted therapies have been recently adopted to improve the treatment of hematologic malignancies, acquired or intrinsic resistance poses a significant obstacle to their efficacy. Thus, there is increasing need to identify novel, targetable pathways to further improve therapy for these diseases. The integrated stress response is a signaling pathway activated in cancer cells in response to both dysregulated growth and metabolism, and also following exposure to many therapies that appears one such targetable pathway for improved treatment of these diseases. In this review, we discuss the role of the integrated stress response in the biology of hematologic malignancies, its critical involvement in the mechanism of action of targeted therapies, and as a target for pharmacologic modulation as a novel strategy for the treatment of hematologic malignancies.

GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma.

BACKGROUND: Aggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of chimeric antigen receptor (CAR) T-cell therapy in patients with glioblastoma have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T-cell therapy also remain limited. METHODS: We profiled an extensive biobank of patients' biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR- T cells from patients with peripheral blood of glioblastoma and diffuse midline glioma and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. RESULTS: Here we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite patients with glioblastoma having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an interleukin-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. CONCLUSIONS: Targeting GD2 using a clinically deployed CAR-T-cell therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.

Slit-Robo signalling establishes a Sphingosine-1-phosphate gradient to polarise fin mesenchyme.

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identifies a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the apical ectodermal ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate the coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.

Characterising Distinct Migratory Profiles of Infiltrating T-Cell Subsets in Human Glioblastoma.

Glioblastoma is the most common and aggressive form of primary brain cancer, with no improvements in the 5-year survival rate of 4.6% over the past three decades. T-cell-based immunotherapies such as immune-checkpoint inhibitors and chimeric antigen receptor T-cell therapy have prolonged the survival of patients with other cancers and have undergone early-phase clinical evaluation in glioblastoma patients. However, a major challenge for T-cell-based immunotherapy of glioblastoma and other solid cancers is T-cell infiltration into tumours. This process is mediated by chemokine-chemokine receptor and integrin-adhesion molecule interactions, yet the specific nature of the molecules that may facilitate T-cell homing into glioblastoma are unknown. Here, we have characterised chemokine receptor and integrin expression profiles of endogenous glioblastoma-infiltrating T cells, and the chemokine expression profile of glioblastoma-associated cells, by single-cell RNA-sequencing. Subsequently, chemokine receptors and integrins were validated at the protein level to reveal enrichment of receptors CCR2, CCR5, CXCR3, CXCR4, CXCR6, CD49a, and CD49d in glioblastoma-infiltrating T-cell populations relative to T cells in matched patient peripheral blood. Complementary chemokine ligand expression was then validated in glioblastoma biopsies and glioblastoma-derived primary cell cultures. Together, enriched expression of homing receptor-ligand pairs identified in this study implicate a potential role in mediating T-cell infiltration into glioblastoma. Importantly, our data characterising the migratory receptors on endogenous tumour-infiltrating T cells could be exploited to enhance the tumour-homing properties of future T-cell immunotherapies for glioblastoma.

Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Targeting human CALR-mutated MPN progenitors with a neoepitope-directed monoclonal antibody.

Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co-expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK-STAT signalling, TPO-independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR-dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.

The sphingosine 1-phosphate receptor 2/4 antagonist JTE-013 elicits off-target effects on sphingolipid metabolism.

Sphingosine 1-phosphate (S1P) is a signaling lipid that has broad roles, working either intracellularly through various protein targets, or extracellularly via a family of five G-protein coupled receptors. Agents that selectively and specifically target each of the S1P receptors have been sought as both biological tools and potential therapeutics. JTE-013, a small molecule antagonist of S1P receptors 2 and 4 (S1P2 and S1P4) has been widely used in defining the roles of these receptors in various biological processes. Indeed, our previous studies showed that JTE-013 had anti-acute myeloid leukaemia (AML) activity, supporting a role for S1P2 in the biology and therapeutic targeting of AML. Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. Further examination of the mechanisms behind these observations showed that JTE-013, at concentrations frequently used in the literature to target S1P2/4, inhibits several sphingolipid metabolic enzymes, including dihydroceramide desaturase 1 and both sphingosine kinases. Collectively, these findings demonstrate that JTE-013 can have broad off-target effects on sphingolipid metabolism and highlight that caution must be employed in interpreting the use of this reagent in defining the roles of S1P2/4.

Desmoglein-2 expression is an independent predictor of poor prognosis patients with multiple myeloma.

Multiple myeloma (MM) is the second most common haematological malignancy and is an incurable disease of neoplastic plasma cells (PC). Newly diagnosed MM patients currently undergo lengthy genetic testing to match chromosomal mutations with the most potent drug/s to decelerate disease progression. With only 17% of MM patients surviving 10-years postdiagnosis, faster detection and earlier intervention would unequivocally improve outcomes. Here, we show that the cell surface protein desmoglein-2 (DSG2) is overexpressed in ~ 20% of bone marrow biopsies from newly diagnosed MM patients. Importantly, DSG2 expression was strongly predictive of poor clinical outcome, with patients expressing DSG2 above the 70th percentile exhibiting an almost 3-fold increased risk of death. As a prognostic factor, DSG2 is independent of genetic subtype as well as the routinely measured biomarkers of MM activity (e.g. paraprotein). Functional studies revealed a nonredundant role for DSG2 in adhesion of MM PC to endothelial cells. Together, our studies suggest DSG2 to be a potential cell surface biomarker that can be readily detected by flow cytometry to rapidly predict disease trajectory at the time of diagnosis.

Resensitising proteasome inhibitor-resistant myeloma with sphingosine kinase 2 inhibition.

The introduction of the proteasome inhibitor bortezomib into treatment regimens for myeloma has led to substantial improvement in patient survival. However, whilst bortezomib elicits initial responses in many myeloma patients, this haematological malignancy remains incurable due to the development of acquired bortezomib resistance. With other patients presenting with disease that is intrinsically bortezomib resistant, it is clear that new therapeutic approaches are desperately required to target bortezomib-resistant myeloma. We have previously shown that targeting sphingolipid metabolism with the sphingosine kinase 2 (SK2) inhibitor K145 in combination with bortezomib induces synergistic death of bortezomib-naïve myeloma. In the current study, we have demonstrated that targeting sphingolipid metabolism with K145 synergises with bortezomib and effectively resensitises bortezomib-resistant myeloma to this proteasome inhibitor. Notably, these effects were dependent on enhanced activation of the unfolded protein response, and were observed in numerous separate myeloma models that appear to have different mechanisms of bortezomib resistance, including a new bortezomib-resistant myeloma model we describe which possesses a clinically relevant proteasome mutation. Furthermore, K145 also displayed synergy with the next-generation proteasome inhibitor carfilzomib in bortezomib-resistant and carfilzomib-resistant myeloma cells. Together, these findings indicate that targeting sphingolipid metabolism via SK2 inhibition may be effective in combination with a broad spectrum of proteasome inhibitors in the proteasome inhibitor resistant setting, and is an approach worth clinical exploration.

The effect of dihydroceramide desaturase 1 inhibition on endothelial impairment induced by indoxyl sulfate.

Protein-bound uremic toxins (PBUTs) have adverse effects on vascular function, which is imperative in the progression of cardiovascular and renal diseases. The role of sphingolipids in PBUT-mediated vasculo-endothelial pathophysiology is unclear. This study assessed the therapeutic potential of dihydroceramide desaturase 1 (Des1) inhibition, the last enzyme involved in de novo ceramide synthesis, to mitigate the vascular effects of the PBUT indoxyl sulfate (IS). Rat aortic rings were isolated and vascular reactivity was assessed in organ bath experiments followed by immunohistochemical analyses. Furthermore, cultured human aortic endothelial cells were assessed for phenotypic and mechanistic changes. Inhibition of Des1 by a selective inhibitor CIN038 (0.1 to 0.3 µM) improved IS-induced impairment of vasorelaxation and modulated immunoreactivity of oxidative stress markers. Des1 inhibition also reversed IS-induced reduction in endothelial cell migration (1.0 µM) by promoting the expression of angiogenic cytokines and reducing inflammatory and oxidative stress markers. These effects were associated with a reduction of TIMP1 and the restoration of Akt phosphorylation. In conclusion, Des1 inhibition improved vascular relaxation and endothelial cell migration impaired by IS overload. Therefore, Des1 may be a suitable intracellular target to mitigate PBUT-induced adverse vascular effects.

Sphingolipid imbalance and inflammatory effects induced by uremic toxins in heart and kidney cells are reversed by dihydroceramide desaturase 1 inhibition.

Non-dialysable protein-bound uremic toxins (PBUTs) contribute to the development of cardiovascular disease (CVD) in chronic kidney disease (CKD) and vice versa. PBUTs have been shown to alter sphingolipid imbalance. Dihydroceramide desaturase 1 (Des1) is an important gatekeeper enzyme which controls the non-reversible conversion of sphingolipids, dihydroceramide, into ceramide. The present study assessed the effect of Des1 inhibition on PBUT-induced cardiac and renal effects in vitro, using a selective Des1 inhibitor (CIN038). Des1 inhibition attenuated hypertrophy in neonatal rat cardiac myocytes and collagen synthesis in neonatal rat cardiac fibroblasts and renal mesangial cells induced by the PBUTs, indoxyl sulfate and p-cresol sulfate. This is at least attributable to modulation of NF-κB signalling and reductions in ß-MHC, Collagen I and TNF-α gene expression. Lipidomic analyses revealed Des1 inhibition restored C16-dihydroceramide levels reduced by indoxyl sulfate. In conclusion, PBUTs play a critical role in mediating sphingolipid imbalance and inflammatory responses in heart and kidney cells, and these effects were attenuated by Des1 inhibition. Therefore, sphingolipid modifying agents may have therapeutic potential for the treatment of CVD and CKD and warrant further investigation.

Integrated in silico and experimental assessment of disease relevance of PCDH19 missense variants.

PCDH19 is a nonclustered protocadherin molecule involved in axon bundling, synapse function, and transcriptional coregulation. Pathogenic variants in PCDH19 cause infantile-onset epilepsy known as PCDH19-clustering epilepsy or PCDH19-CE. Recent advances in DNA-sequencing technologies have led to a significant increase in the number of reported PCDH19-CE variants, many of uncertain significance. We aimed to determine the best approaches for assessing the disease relevance of missense variants in PCDH19. The application of the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) guidelines was only 50% accurate. Using a training set of 322 known benign or pathogenic missense variants, we identified MutPred2, MutationAssessor, and GPP as the best performing in silico tools. We generated a protein structural model of the extracellular domain and assessed 24 missense variants. We also assessed 24 variants using an in vitro reporter assay. A combination of these tools was 93% accurate in assessing known pathogenic and benign PCDH19 variants. We increased the accuracy of the ACMG-AMP classification of 45 PCDH19 variants from 50% to 94%, using these tools. In summary, we have developed a robust toolbox for the assessment of PCDH19 variant pathogenicity to improve the accuracy of PCDH19-CE variant classification.

A Drug Screening Pipeline Using 2D and 3D Patient-Derived In Vitro Models for Pre-Clinical Analysis of Therapy Response in Glioblastoma.

Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6-9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microenvironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cultures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma.