Background: Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/ relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. Methods: Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo((R)), ALDH activity by ALDELUOR(TM), and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo(TM) Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing. Results: Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright "stem-like" populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the beta 5 proteasome subunit. BTZ-resistance conferred increased resistance toAra-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition. Conclusions: We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL.

Glioblastoma Multiforme (GBM) is the most common lethal brain tumour associated with dismal survival rate. GBM is considered to be an incurable malignancy as these tumours evade all intricate attempts of therapy and no contemporary chemotherapeutic regimen is effective. Although the existence of cancer stem cells (CSCs) is still debatable, it is widely accepted that GBM has a small population of cells expressing CSC markers (~1%) that are highly resistant to chemo-radiation therapy. Recent evidence indicates that hypoxia induces cancer stem cell (CSC) phenotypes via epithelial-to-mesenchymal transition (EMT) that promote therapeutic resistance in solid tumours. Given that GBMs are extensively hypooxygenated heterogenous tumours, understanding the molecular relationship between hypoxia, biology of CSCs, EMT and chemoresistance would be invaluable for development of drugs that can target CSCs. Evidence suggests that hypoxia inducible factors (HIFs), NF-B and aldehyde dehydrogenase (ALDH) together orchestrate the stemness and chemoresistance in hypoxia induced CSCs. But the insights on the mechanisms still remain obscure. In this study we used an in vitro GBM CSC and hypoxia model along with NF-B-p65 and HIF transfected GBM cell lines to investigate the relationship between HIFs, NF-B activation and ALDH activity and their role in chemoresistance. The findings of this study demonstrated that GBM cells grown as spheres consist of a vast proportion of hypoxic cells with elevated CSC and EMT markers suggesting hypoxia induced EMT. GBM-CSCs are chemoresistant and displayed increased levels of HIFs, NF-B and ALDH activity. It was also observed that stable transfection of GBM cells with NF-B-p65 or HIFs induced CSC and EMT markers indicating their essential role in maintaining CSC phenotypes. The study also highlighted the importance of NF-B and ALDH in driving chemoresistance and the potential role of NF-B as the master regulator of hypoxia induced stemness in GBM cells. In this study, we used Disulfiram (DS), an anti-alcoholism drug, in combination with copper (Cu) to target the hypoxia-NF-B axis and inhibit ALDH activity to reverse chemoresistance in GBM CSCs. We showed that DS/Cu is cytotoxic to GBM cells and completely eradicated the resistant CSC population at low nanomolar levels in vitro. We also demonstrated that DS/Cu effectively inhibited GBM in vivo using newly formulated PLGA-DS nanoparticles. DS is an FDA approved drug with low/no toxicity to normal tissues and can freely pass through the blood brain barrier (BBB). Further study may lead to quick translation of DS into clinical trials.

Despite progress being made in our understanding of triple negative breast cancer (TNBC), the overall survival and disease-free survival for TNBC patients continues to be considerably poorer than their ER/PR/HER2+ counterparts. Metastasis and chemoresistance are the pivotal issues holding back the long-term success of TNBC treatments. In addition to the bulk tumor cells, cancer stem cells (CSCs) have emerged as important targets for alleviating TNBC progression and relapse. Cisplatin, a platinum based chemotherapeutic agent, has shown promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with tumor hypoxia that in turn promotes CSC enrichment and drug resistance. My work is to develop a combinational treatment to improve the long-term therapeutic potential of cisplatin that not only targeted the bulk TNBC population but also ALDHhigh and CD44+/CD24- CSC populations. Through clinical dataset analysis, I found that patient TNBC tumors expressed high levels of epidermal growth factor receptor (EGFR) and hypoxia genes. A similar expression pattern was demonstrated in cisplatin-resistant ovarian cancer. I therefore developed a combinational therapeutic to co-inhibit EGFR and hypoxia using metformin (an AMPK activator) and gefitinib (an EGFR inhibitor), which sensitized bulk TNBC cells to cisplatin and also led to the effective inhibition of both CD44+/CD24- and ALDHhigh CSCs. I obtained similar results by using clinically relevant TNBC patient samples ex vivo. Since these drugs are already frequently used in the clinic, this study illustrates a novel, clinically translatable therapeutic approach to improve the long-term therapeutic outcome of cisplatin for TNBC treatment.

Petroleum Technology Development Fund (PTDF) Nigeria / The full text will be available at the end of the embargo period: 21st March 2026

Yes / Prostate cancer represents the most common malignancy diagnosed in men, and is the second-leading cause of cancer death in this population. In spite of dedicated efforts, the current therapies are rarely curative, requiring the development of novel approaches based on innovative molecular targets. In this work, we validated aldehyde dehydrogenase 1A1 and 1A3 isoform expressions in different prostatic tissue-derived cell lines (normal, benign and malignant) and patient-derived primary prostate tumor epithelial cells, demonstrating their potential for therapeutic intervention using a small library of aldehyde dehydrogenase inhibitors. Compound 3b, 6-(4-fluorophenyl)-2-phenylimidazo [1,2-a]pyridine exhibited not only antiproliferative activity in the nanomolar range against the P4E6 cell line, derived from localized prostate cancer, and PC3 cell lines, derived from prostate cancer bone metastasis, but also inhibitory efficacy against PC3 colony-forming efficiency. Considering its concomitant reduced activity against normal prostate cells, 3b has the potential as a lead compound to treat prostate cancer by means of a still untapped molecular target.

The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-disubstituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak-to-moderately binding to DNA, and symmetrical 1,4-disubstituted compounds were shown to be non-toxic (IC50 = 100 :M) with nonsymmetrical analogues generating IC50 values in the 1-100 :M range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133⁺/ α₂β₁ hi stem cell fraction and upregulated in CD133⁻/ α₂β₁ lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells.

The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-di-substituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak*to*moderately binding to DNA, and symmetrical 1,4-di-substituted compounds were shown to be non*toxic (IC50 = 100 :/! with non-symmetrical analogues generating IC50 values in the 1-100 :/ range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133+/ α2β1hi stem cell fraction and upregulated in CD133-= α2β1lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells. / EPSRC, Biostatus / The full text was made available at the end of the re-embargo period, 1st September 2017.

La thérapie cellulaire est une envisagée pour traiter des pathologies cardiaques ou squelettiques basée sur la médecine régénérative. Les progéniteurs cellulaires classiquement utilisés (myoblastes ou cellules mésenchymateuses) n'ont démontré qu'une efficacité limitée. Dans ce contexte, notre laboratoire a identifié une nouvelle catégorie de progéniteurs, sur la base de leur activité enzymatique aldéhyde déshydrogénase (ALDH) mise en évidence par un substrat fluorescent, l'Aldéfluor, et en association avec le marqueur CD34. Les ALDH sont impliquées dans le métabolisme et la détoxification des aldéhydes, et constituent un nouveau marqueur des cellules souches. Ce travail de thèse a permis de mieux caractériser les progéniteurs myogéniques (ALDH+/CD34-) et non myogéniques (ALDH+/CD34+), dans différents contextes physiopathologiques. Leur présence dans différents muscles de primates humains ou non humains, leur persistance au cours du vieillissement naturel ou lors d'atteinte par la dystrophie musculaire de Duchenne (DMD) chez l'Homme et dans des modèles animaux, suggèrent une utilisation possible des cellules ALDH+/CD34- pour des développements thérapeutiques ultérieurs. L'étude phénotypique révèle que des marqueurs transmembranaires sont associés à des sous-populations de cellules ALDH myogéniques ou non myogéniques dont la comparaison permettra de proposer de meilleurs candidats de thérapie cellulaire. En parallèle, les caractérisations histologiques et cytologiques ont identifié des sous-populations exprimant des isoenzymes et les analyses d'expressiongénique réalisées ex vivo et en culture suggèrent que certaines sont impliquées dans l'homéostasie musculaires. / Cell therapy is a regenerative medicine strategy considered for the treatment of cardiac or skeletal muscle diseases. The cellular progenitors used to date (myoblasts or mesenchymal stem cells) provided mitigated success, thus mandating the identification and characterization of new categories of progenitors. Our laboratory has identified new populations of progenitors, based on their Aldehyde Deshydrogenase activity (ALDH) detectable using the fluorescent substrate Aldefluor, associated with the expression of the CD34 marker. ALDH are involved in metabolism and detoxification of aldehydes, they play important roles in cell survival and differentiation and are considered a new marker of stem cells. The present project allowed characterizing extensively the myogenic (ALDH+/CD34-) and non myogenic (ALDH+/CD34+) progenitors, in several physiopathological contexts and animal models. The presence of ALDH+/CD34- cells in distinct muscle groups in Human and non-human Primates, their persistence through natural ageing and despite the ongoing degenerative process observed in Duchenne muscular dystrophy in Human patients and animal models suggest their future use for therapeutic applications. The phenotypic characterization indicated that membrane markers are associated to myogenic or non myogenic sub-populations of ALDH cells. The comparison on their efficacies in vito and in vivo will allow proposing new candidates for cell therapy. In parallele, histological and cytological analysis identified cell populations expressing isoenzymes The analysis of gene expressions suggested that, at least, some of them are involved in muscle homeostasis in situ or in vitro.