Dissecting out the mechanisms of hypoxia-dependent responses in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease with a significant impact on patients’ quality of life. One of the well-described features in RA is hypoxia. It has been shown that both RA synovial fluid and synovial tissue are characterised by insufficient amount of oxygen. Abnormality in hypoxia-inducible factor (HIF) expression is a marker of decreased oxygen tension in RA and in other pathological conditions. HIFs, together with the proteins which regulate their stabilisation and transactivation, namely prolyl hydroxylase domain (PHD) enzymes and factor inhibiting HIF-1 (FIH-1), were the main focus of this thesis. The first aim of the study was to investigate the specific roles of HIF isoforms in RA fibroblast-like synoviocytes (FLS), which are the key cells in RA pathogenesis. Expression of HIF-1α, HIF-2α and a panel of genes involved in angiogenesis (ANGPTL-4, ephrin-A3, VEGF), glycolysis (GLUT-1, ENO-1), pH regulation (CA9) and apoptosis (BNIP-3) were significantly increased by hypoxia. Successful silencing of both HIFs using short interfering RNA (siRNA) was achieved in RA FLS, and was followed by examination of the HIF-dependence of the candidate genes. The majority of these genes were found to be HIF-1α- dependent, whereas only ANGPTL-4 and VEGF were regulated by both HIF-1α and HIF-2α. Subsequently, the mRNA and protein expression of PHDs and FIH-1 in RA FLS, their response to hypoxic conditions and their HIF-dependence were examined. Specific knockdown of the hydroxylases was performed and the effects on HIFs and genes of interest were assessed. The most significant changes were noticed after silencing PHD-2, which led to HIF-α stabilisation and upregulation of HIF-dependent genes. In addition, PHD-2 depletion increased a number of pro-angiogenic genes and also contributed to new tubule formation in a functional angiogenesis assay. Comparison of RA, osteoarthritis (OA) FLS and normal human dermal fibroblasts (NHDF) revealed that PHD-2 has similar functions in promoting neovascularisation in arthritic cells (RA and OA), but not in non-arthritic cells (NHDF). Finally, the last chapter describes preliminary data on the expression of the least investigated HIF-3α subunit and its negative splice variant, HIF-3α4, in RA FLS. This research contributes to a better understanding of the upstream and downstream regulation of HIF signalling in RA, showing that PHD-2 has a key role in regulating hypoxic responses via HIFs in RA FLS. Because this pathway controls the expression of so many genes relevant to the disease, it may be an important target for RA therapy. One of the most critical challenges would be to target the correct HIF/PHDs molecule in the right tissue/cell, ensuring that it will not affect angiogenesis in healthy tissues

An mTORC1 to HRI signaling axis promotes cytotoxicity of proteasome inhibitors in multiple myeloma

Multiple myeloma (MM) causes approximately 20% of deaths from blood cancers. Notwithstanding significant therapeutic progress, such as with proteasome inhibitors (PIs), MM remains incurable due to the development of resistance. mTORC1 is a key metabolic regulator, which frequently becomes dysregulated in cancer. While mTORC1 inhibitors reduce MM viability and synergize with other therapies in vitro, clinically, mTORC1 inhibitors are not effective for MM. Here we show that the inactivation of mTORC1 is an intrinsic response of MM to PI treatment. Genetically enforced hyperactivation of mTORC1 in MM was sufficient to compromise tumorigenicity in mice. In vitro, mTORC1-hyperactivated MM cells gained sensitivity to PIs and hypoxia. This was accompanied by increased mitochondrial stress and activation of the eIF2α kinase HRI, which initiates the integrated stress response. Deletion of HRI elevated the toxicity of PIs in wt and mTORC1-activated MM. Finally, we identified the drug PMA as a robust inducer of mTORC1 activity, which synergized with PIs in inducing MM cell death. These results help explain the clinical inefficacy of mTORC1 inhibitors in MM. Our data implicate mTORC1 induction and/or HRI inhibition as pharmacological strategies to enhance MM therapy by PIs

Selinexor overcomes hypoxia-induced drug resistance in multiple myeloma

Increased levels of the nuclear export protein, exportin 1 (XPO1), were demonstrated in multiple myeloma (MM) patients. Targeting XPO1 with selinexor (the selective inhibitor of nuclear export; SINE compound KPT-330) demonstrates broad antitumor activity also in patient cells resistant to bortezomib; hence, it is a promising target in MM patients. Hypoxia is known to mediate tumor progression and drug resistance (including bortezomib resistance) in MM cells. In this study, we tested the effects of selinexor alone or in combination with bortezomib in normoxia and hypoxia on MM cell survival and apoptosis in vitro and in vivo. In vitro, selinexor alone decreased survival and increased apoptosis, resensitizing MM cells to bortezomib. In vivo, we examined the effects of selinexor alone on tumor initiation and tumor progression, as well as selinexor in combination with bortezomib, on tumor growth in a bortezomib-resistant MM xenograft mouse model. Selinexor, used as a single agent, delayed tumor initiation and tumor progression, prolonging mice survival. In bortezomib-resistant xenografts, selinexor overcame drug resistance, significantly decreasing tumor burden and extending mice survival when combined with bortezomib

The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy

Hypoxia is a non-physiological level of oxygen tension, a phenomenon common in a majority of malignant tumors. Tumor-hypoxia leads to advanced but dysfunctional vascularization and acquisition of epithelial-to-mesenchymal transition phenotype resulting in cell mobility and metastasis. Hypoxia alters cancer cell metabolism and contributes to therapy resistance by inducing cell quiescence. Hypoxia stimulates a complex cell signaling network in cancer cells, including the HIF, PI3K, MAPK, and NFĸB pathways, which interact with each other causing positive and negative feedback loops and enhancing or diminishing hypoxic effects. This review provides background knowledge on the role of tumor hypoxia and the role of the HIF cell signaling involved in tumor blood vessel formation, metastasis, and development of the resistance to therapy. Better understanding of the role of hypoxia in cancer progression will open new windows for the discovery of new therapeutics targeting hypoxic tumor cells and hypoxic microenvironment

BRD9 degraders as chemosensitizers in acute leukemia and multiple myeloma

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM

Molecularly targeted therapies in multiple myeloma

Multiple myeloma (MM) is a hematological malignancy that remains incurable because most patients will eventually relapse or become refractory to the treatments. Although the treatments have improved, the major problem in MM is the resistance to therapy. Novel agents are currently in development for the treatment of relapsed/refractory MM, including immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies, cell signaling targeted therapies, and strategies targeting the tumor microenvironment. We have previously reviewed in detail the contemporary immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies therapies for MM. Therefore, in this review, we focused on the role of molecular targeted therapies in the treatment of relapsed/refractory multiple myeloma, including cell signaling targeted therapies (HDAC, PI3K/AKT/mTOR, p38 MAPK, Hsp90, Wnt, Notch, Hedgehog, and cell cycle) and strategies targeting the tumor microenvironment (hypoxia, angiogenesis, integrins, CD44, CXCR4, and selectins). Although these novel agents have improved the therapeutic outcomes for MM patients, further development of new therapeutic agents is warranted

Targeting E-selectin to tackle cancer using uproleselan

E-selectin is a vascular adhesion molecule expressed mainly on endothelium, and its primary role is to facilitate leukocyte cell trafficking by recognizing ligand surface proteins. E-selectin gained a new role since it was demonstrated to be involved in cancer cell trafficking, stem-like properties and therapy resistance. Therefore, being expressed in the tumor microenvironment, E-selectin can potentially be used to eradicate cancer. Uproleselan (also known as GMI-1271), a specific E-selectin antagonist, has been tested on leukemia, myeloma, pancreatic, colon and breast cancer cells, most of which involve the bone marrow as a primary or as a metastatic tumor site. This novel therapy disrupts the tumor microenvironment by affecting the two main steps of metastasis-extravasation and adhesion-thus blocking E-selectin reduces tumor dissemination. Additionally, uproleselan mobilized cancer cells from the protective vascular niche into the circulation, making them more susceptible to chemotherapy. Several preclinical and clinical studies summarized herein demonstrate that uproleselan has favorable safety and pharmacokinetics and is a tumor microenvironment-disrupting agent that improves the efficacy of chemotherapy, reduces side effects such as neutropenia, intestinal mucositis and infections, and extends overall survival. This review highlights the critical contribution of E-selectin and its specific antagonist, uproleselan, in the regulation of cancer growth, dissemination, and drug resistance in the context of the bone marrow microenvironment

Spotlight on ixazomib: Potential in the treatment of multiple myeloma

Despite the significant therapeutic advances achieved with proteasome inhibitors (PIs) such as bortezomib and carfilzomib in prolonging the survival of patients with multiple myeloma, the development of drug resistance, peripheral neuropathy, and pharmacokinetic limitations continue to pose major challenges when using these compounds. Ixazomib is a second-generation PI with improved activity over other PIs. Unlike bortezomib and carfilzomib, which are administered by injection, ixazomib is the first oral PI approved by US Food and Drug Administration. This review discusses the biochemical properties, mechanisms of action, preclinical efficacy, and clinical trial results leading to the US Food and Drug Administration approval of ixazomib

Liposomal phytohemagglutinin: In vivo T-cell activator as a novel pan-cancer immunotherapy

Immunotherapy is an attractive approach for treating cancer. T-cell engagers (TCEs) are a type of immunotherapy that are highly efficacious; however, they are challenged by weak T-cell activation and short persistence. Therefore, alternative solutions to induce greater activation and persistence of T cells during TCE immunotherapy is needed. Methods to activate T cells include the use of lectins, such as phytohemagglutinin (PHA). PHA has not been used to activate T cells in vivo, for immunotherapy, due to its biological instability and toxicity. An approach to overcome the limitations of PHA while also preserving its function is needed. In this study, we report a liposomal PHA which increased PHA stability, reduced toxicity and performed as an immunotherapeutic that is able to activate T cells for the use in future cancer immunotherapies to circumvent current obstacles in immunosuppression and T-cell exhaustion