Department of Surgery, Oncology and Gastroenterology
Ph.D. Course in: Clinical and Experimental Oncology and Immunology XXXI cycle
Selective killing of T-acute lymphoblastic leukemia (T-ALL) cells by activation of the ROS-OMA1-OPA1 axis
Coordinator: Prof. Paola Zanovello Supervisor: Prof. Vincenzo Ciminale
Ph.D. student: Gloria Scattolin
Thesis written with the financial contribution of Fondazione Cariparo
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To my Omas and Opas
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3 INDEX
ABSTRACT ... 7
RIASSUNTO ... 9
1. INTRODUCTION... 13
1.1 T-cell acute lymphoblastic leukemia (T-ALL) ... 13
1.1.1 Epidemiology and risk factors ... 13
1.1.2 T-ALL origin and subtypes ... 14
1.1.2.1 T-ALL origin ... 14
1.1.2.2 T-ALL subtypes ... 16
1.1.3 T-ALL genetics and molecular pathogenesis ... 18
1.1.3.1 T-cell receptor (TCR) translocations and inversions .. 18
1.1.3.2 Fusion genes ... 19
1.1.3.3 Deletions and mutations ... 20
1.1.4 Current treatments ... 22
1.1.5 New targets... 24
Table 1. New drugs and targets. ... 26
1.2 Reactive oxygen species (ROS), mitochondria and cancer ... 27
1.2.1 Reactive oxygen species (ROS) ... 27
1.2.2 Generation of ROS in mitochondria ... 27
1.2.3 Mitochondrial ROS and regulation of apoptosis ... 30
1.2.3 Cellular antioxidant systems ... 34
1.2.4 The NRF2 pathway ... 35
1.2.5 Effects of ROS on cell turnover ... 37
1.2.7 ROS, NRF2 and cancer ... 39
2. AIM OF THE STUDY ... 41
3. MATERIALS AND METHODS ... 43
3.1 Cell cultures ... 43
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3.1.1 Cell lines ... 43
3.1.2 Isolation of thymocytes and patient-derived xenografts .... 43
3.1.3 Isolation of PBMC and primary patient cells ... 44
3.2 Drug treatments ... 44
3.3 ROS measurements ... 45
3.3.1 Mitochondrial ROS analysis by flow cytometry (T-cells) . 45 3.3.2 Mitochondrial ROS analysis by epifluorescence (MDA- MB-231 cells) ... 45
3.3.3 Cellular ROS analysis by flow cytometry (T-cells) ... 46
3.3.4 Cellular ROS analysis by epifluorescence (MDA-MB-231) ... 46
3.4 Measurement of mitochondrial membrane potential ... 47
3.5 Cell death ... 47
3.6 Western blot ... 47
3.7 siRNA ... 48
3.7.1 T-ALL electroporation ... 48
3.7.2 Calcium-phosphate transfection ... 48
3.8 Electron microscopy ... 49
3.9 Immunofluorescence ... 49
3.10 qRT-PCR ... 50
3.10.1 RNA extraction ... 50
3.10.2 Retrotranscription ... 50
3.11 Proliferation assays (PKH26) ... 51
3.12 Statistical analysis and generation of images ... 52
3.13 Antibody List... 53
4. RESULTS ... 57
4.1 Background and preliminary results ... 57
4.2 NS1619 and DHEA activate the NRF2 pathway ... 62
4.3 NS1619 and DHEA inhibit T-ALL cell proliferation ... 63
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4.4 NS1619 and DHEA induce death of T-ALL cells ... 65
4.5 NS1619 and DHEA induce OMA1-mediated OPA1 cleavage in T-ALL cells ... 68
4.6 NS1619 and DHEA induce mitochondrial fragmentation ... 73
4.7 Testing the interaction of DHEA with TSPO ... 74
4.8 NS1619 and DHEA sensitize T-ALL cells to TRAIL-induced apoptosis ... 77
5. DISCUSSION ... 83
6. REFERENCES ... 87
7. RINGRAZIAMENTI ... 101
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7 ABSTRACT
Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a rare neoplasia accounting for 15% of ALL. Despite significant advances in treatment, approximately one out of five patients develop primary or secondary resistance to current therapies, which include glucocorticoids as a key component; indeed, overall clinical outcome depends on the initial response to glucocorticoids. In our study, we developed an integrated approach to selectively kill T-ALL cells by increasing mitochondrial reactive oxygen species (mtROS). Intracellular ROS are tightly regulated second messengers that affect several signal transduction pathways controlling cell turnover. Oncogenic pathways commonly activated in cancer cells drive a conspicuous increase in production of ROS. Cancer cells commonly exhibit an imbalance between ROS producing pathways and antioxidant defenses that results in a high setpoint of ROS which is close to the threshold beyond which macromolecular damage is produced and cell death pathways are engaged. Therefore, tumor cells are predicted to be more vulnerable than their normal counterparts to treatments that impinge on ROS homeostasis and particularly to agents that blunt antioxidant systems. To increase mtROS levels, we used NS1619, a small molecule that activates the Ca2+-activated K+ (BK) channel, and dehydroepiandrosterone (DHEA), which blunts ROS scavenging through inhibition of the pentose phosphate pathway. These compounds selectively killed T-ALL cell lines, patient-derived xenografts and primary cells from patients (including refractory patients), but did not kill normal human thymocytes. T-ALL cells treated with NS1619 and DHEA showed activation of the ROS-responsive transcription factor NRF2, indicating engagement of antioxidant pathways, as well as increased cleavage of OPA1, a mitochondrial protein that promotes mitochondrial fusion and regulates apoptosis. Consistent with these observations, transmission electron microscopy analysis indicated that NS1619 and DHEA increased mitochondrial fission. OPA1 cleavage and cell death were inhibited by ROS scavengers and by siRNA-mediated knock-down of the mitochondrial protease OMA1, suggesting the engagement of a ROS-OMA1-OPA1 axis in T- ALL cells. Furthermore, NS1619 and DHEA sensitized T-ALL cells to TRAIL-
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induced apoptosis. Taken together, our findings provide proof-of-principle for an integrated ROS-based pharmacological approach to target refractory T-ALL.
9 RIASSUNTO
La leucemia linfoblastica acuta a cellule T (T-ALL) pediatrica è una rara neoplasia che rappresenta all’incirca il 15% delle leucemie linfoblastiche acute. Nonostante i recenti progressi per il trattamento di questa neoplasia, circa un paziente su cinque sviluppa resistenza primaria o secondaria alla terapia, che include i glucocorticoidi come componente principale. In questo progetto abbiamo utilizzato un approccio integrato per uccidere in modo selettivo cellule di T-ALL mediante l’aumento delle specie reattive dell’ossigeno mitocondriali (mtROS). I ROS costituiscono importanti secondi messaggeri che influenzano diverse vie di trasduzione del segnale che regolano la crescita cellulare. Molte vie di segnalazione oncogeniche comunemente iper-attivate nelle cellule tumorali contribuiscono all’aumento dei livelli di ROS, e le cellule tumorali sono spesso caratterizzate da un’aumentata produzione di ROS grazie a un’aumentata difesa antiossidante, che mantiene i livelli di ROS al di sotto dei livelli tossici. Di conseguenza le cellule tumorali dovrebbero essere più sensibili, rispetto a cellule normali, a farmaci che aumentano la produzione di ROS e in particolare a molecole che inibiscono le difese antiossidanti. Per aumentare i livelli di ROS mitocondriali, abbiamo utilizzato l’NS1619, una piccola molecola che attiva il canale del potassio attivato da calcio (BK), e il deidroepiandrosterone (DHEA), il quale, mediante l’inibizione del ciclo dei pentoso fosfati, diminuisce la capacità antiossidante delle cellule. I risultati hanno dimostrato che questi composti sono in grado di uccidere in modo selettivo linee cellulari di T-ALL, patient-derived xenografts e cellule primarie da pazienti in vitro, mentre non inducono morte cellulare di timociti normali. Inoltre, il trattamento di cellule di T-ALL con NS1619 e DHEA induce l’attivazione del fattore di trascrizione indotto da ROS NRF2, indicando il coinvolgimento delle vie antiossidanti. L’aumento di ROS mitocondriali indotto da NS1619 e DHEA induce il clivaggio proteolitico di OPA1, una proteina mitocondriale che promuove la fusione mitocondriale e che regola l’apoptosi. Consistentemente, risultati di microscopia elettronica hanno dimostrato che NS1619 e DHEA aumentano la fissione mitocondriale. I nostri risultati dimostrano inoltre che il taglio proteolitico di OPA1 e la morte cellulare indotta da NS1619 e DHEA sono ridotti da ROS-
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scavengers e dal silenziamento della proteasi mitocondriale OMA1, suggerendo l’ingaggio dell’asse ROS-OMA1-OPA1. Infine, NS1619 e DHEA sensibilizzano cellule di T-ALL all’apoptosi indotta dal ligando pro-apoptotico TRAIL. In conclusione, i nostri risultati dimostrano che è possibile indurre selettivamente la morte di cellule di T-ALL, indicando nuove possibilità terapeutiche per il trattamento di pazienti refrattari alla terapia.
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13 1. INTRODUCTION
1.1 T-cell acute lymphoblastic leukemia (T-ALL)
1.1.1 Epidemiology and risk factors
Acute lymphoblastic leukemia (ALL) is the most common neoplasia in children (1, 2), accounting for 80% of all hematological malignancies. The T-cell subtype (T- ALL) accounts for almost 15% of pediatric and 25% of adult ALL, and prognosis for T-ALL patients is worse than for patients with B-lineage ALL (B-ALL) (3, 4).
The etiology and the exact mechanisms of T-ALL development are still not well defined, and, except for exposure to ionizing radiation, no exact risk factor for ALL development is known (5). Other risk factors, including parental smoking and exposure to chemicals and pesticides, have been hypothesized, but remain to be conclusively demonstrated as an etiological agent of this neoplasia (5).
Interestingly, ALL incidence is higher in males than in females, with a 3-fold increased risk for T-ALL (Figure 1.1) (1, 5, 6) .
The clinical presentation of T-ALL includes a high white blood cell (WBC), low platelet and neutrophil counts and anemia. Moreover, T-ALL patients can also show
Figure 1.1: Incidence of ALL is higher in males than in females. From: 2. Siegel DA, et al., Rates and Trends of Pediatric Acute Lymphoblastic Leukemia — United States, 2001–2014.
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infiltration of the central nervous system (CNS) and/or testis and respiratory failure due to accumulation of mediastinal masses (7-9). Stratification of T-ALL patients as “low risk” or “high risk” helps in predicting response to treatment and is based on the NCI (National Cancer Institute) criteria, which include age and WBC count in the peripheral blood or bone marrow at presentation (10).
Patients with low WBC count (<50000/ml) and an age between 1 and 10 years are classified as low risk, while younger/older patients with a high WBC count (>50000/ml) are considered as high risk. Moreover, involvement of sanctuary sites (CNS and testis) at diagnosis is indicative of poor prognosis as these anatomical sites are difficult to reach with chemotherapy (7). Finally, response to treatment within 7 or 14 days of therapy is an indicator of prognosis, and patients who fail induction therapy or who still present minimal residual disease (MRD) in the bone marrow (>5%) have a worse prognosis (7).
Children with Down Syndrome have an increased risk of developing ALL (DS- ALL), with an increased risk of induction failure (11-13).
1.1.2 T-ALL origin and subtypes
1.1.2.1 T-ALL origin
T-cell development begins with the migration of lymphoid progenitors from the bone marrow to the cortical region of the thymus (Figure 1.2). Here, thymocytes initiate their differentiation process as double negative (DN) T-cells lacking the expression of the mature T-cell markers CD4 and CD8 (14, 15). These uncommitted thymocytes go through four differentiation stages (DN1 to DN4) at the end of which they are induced to mature into CD4/CD8 double-positive (DP) T-cells, stop proliferating and start the TCRA-rearrangement process. At this stage, interaction of the TCR with major histocompatibility complex class I or II molecules (MHCI or MHCII) expressed on the surface of thymic cortical epithelial cells induce the selection of CD8 or CD4 positivity, respectively, in a process known as positive selection. As they become single-positive (SP), T-cells migrate towards the
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medullar region of the thymus and start the negative selection process, in which self-antigen recognizing cells are eliminated by apoptosis. At this point, survived SP cells can leave the thymus and migrate into blood stream and lymphoid organs (14, 15).
Acquired mutations of genes regulating T-cell growth, proliferation and differentiation occurring during T-cell maturation in the thymus can give rise to T- ALL, which thus reflects the differentiation stage of the cell of origin (leukemia initiating cell) (14, 16, 17).
Figure 1.2: T-cell development. Lymphoid progenitors migrate from the bone marrow towards the cortical region of the thymus, where they start the differentiation program as double negative (DN) and become double positive (DP) thymocytes expressing both CD4 and CD8. Positive selection then induce the selection of single positive (SP, CD4+ or CD8+) T-cells. SP cells migrate in the medullar region of the thymus, where self-antigen recognizing T-cells are eliminated during the negative selection process. SP cells that have survived these processes can then exit the thymus and migrate into the blood stream or lymphoid organs. Mutations acquired during T-cell development in the thymus can generate T-ALL, which is thus composed of immature T-lymphoblasts. Created with Smart Servier Medical Art.
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1.1.2.2 T-ALL subtypes
The current classification of T-ALL into different sub-groups is related to the T- cell markers expressed on leukemic cells (4). These markers reflect the developmental stages of differentiation of T-lymphoblasts in the thymus according to their immunological features (Figure 1.3):
· Pro T-ALL: double negative cells expressing immature T-cell markers;
· Pre T-ALL: double negative T-cells that express CD2, CD5 and CD28;
· Cortical T-ALL: double positive T-cells;
· Mature T-ALL (medullary): single-positive (CD4+ or CD8+) with mature T-cell characteristics.
A new subtype of T-ALL with unique features was recently described. This subtype, named early T-cell precursor lymphoblastic leukemia (ETP-ALL), is characterized by a different immunophenotype and gene expression profile characterized by the expression of immature T-cell markers and no expression of CD4 and CD8 (4, 14, 15, 18). The ETP-ALL expression profile reflects that of hematopoietic and myeloid precursors, and ETP-ALL lymphoblasts usually express
Figure 1.3: Current classification of T-ALL subtypes accordingly to their immunological phenotype. From: 4. Hefazi M et al., Recent Advances in the Biology and Treatment of T-ALL.
cCD3: cytoplasmic cluster of differentiation 3; CD7: cluster of differentiation 7; TdT: Terminal deoxynucleotidyl Transferase; CD2: cluster of differentiation 2; CD5: cluster of differentiation 5; CD28: cluster of differentiation 28; CD1a: T-cell surface glycoprotein CD1a; CD4/8: cluster of differentiation 4/8; sCD3: surface cluster of differentiation 3; CD34:
cluster of differentiation 34.
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stem cell and myeloid markers. Interestingly, ETP-ALL blasts are characterized by the ability to differentiate into both T- and myeloid cells, and, overall, ETP-ALL expression profiles resemble those of myeloid leukemias (15, 18). Unfortunately, patients with the ETP-ALL subtype have a poorer prognosis compared to patients with other T-ALL subtypes, with a 10-year overall survival lower than 20% (7, 18- 20). ETP-ALL patients respond poorly to prednisolone, and are treated with more aggressive chemotherapeutic protocols.
Overall, pediatric ALL shows the lowest number of mutations among cancers (Figure 1.4) (21), and major abnormalities also represent rare cases. Gross karyotypic abnormalities (aneuploidies) are very rare in T-ALL, while structural chromosomal changes (translocations, deletions, insertions) are more common (4).
Figure 1.4: Number of mutations in pediatric ALL is one of the lowest among all cancers.
From: 21. Vogelstein B et al., Cancer Genome Landscapes.
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1.1.3 T-ALL genetics and molecular pathogenesis
1.1.3.1 T-cell receptor (TCR) translocations and inversions
The TCR is a plasma membrane receptor composed of a and b chain dimers (or g- d chains in a minor population) highly expressed in T-cells that recognizes antigens bound to major histocompatibility complex (MHC) molecules and is responsible for T-cell activation. Many translocations involving the TCR have been reported in T-ALL and usually involve translocation of genes regulating T-cell development and differentiation near the TCR promoter or enhancer (1, 22, 23). The most common gene partners of TCR rearrangements are represented by transcription factors, while other genes coding for cyclins, kinases and receptors are rarer (1).
Three main classes of transcription factors are commonly involved in TCR rearrangements, and include basic helix-loop-helix (bHLH) family members, the LMO (LIM domain only protein) proteins and HOX (Homeobox) transcription factors (23-25).
The bHLH family includes the TAL1 and TAL2 genes (T-Cell Acute Lymphocytic Leukemia 1 and 2) which are involved in T-cell development, and their translocation near the TCR are found in 3% and 1% of T-ALL patients respectively (26, 27). The oncogenic potential of TAL1 lies in its ability to directly activate the MYB proto-oncogene, which plays a key role in regulating cell proliferation and differentiation in hematopoietic progenitors. Moreover, 12% of T-ALL cases present TAL1 overexpression due to its fusion with the SIL (SCL interrupting locus) promoter (28). Translocations of LYL2 (lymphoblastic leukemia-derived sequence 1) and BHLHB1 (Basic Domain, Helix-Loop-Helix Protein, Class B, 1) near the TCR enhancer are instead less common (14, 27).
LMO1 and LMO2 are over-expressed in nearly 10% of T-ALL cases as the result of translocations near the TCR A and D loci (14, 16). The LMO transcription factors form complexes with other bHLH proteins including TAL1 and/or LYL1 (Lymphoblastic leukemia-derived sequence 1) and aberrant expression of LMO 1 or 2 is usually found together with deregulation of TAL1 and/or LYL1 (27).
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Among the HOX genes, HOXA9, 10 and 11 are deregulated in T-ALL due to translocations and/or inversions near the TCRB and TCRG loci. Besides HOXA9- 11, common HOX genes involved in T-ALL pathogenesis include the TLX family, and translocation of TLX1 (T Cell Leukemia Homeobox 1) near the enhancers of the TCRA and TCRD loci are present in 5-10% of pediatric T-ALL (14, 16).
Aberrant expression of known oncogenes also include the transcription factors MYC and MYB. The MYC proto-oncogene is an important regulator of cell proliferation, and is commonly overexpressed in cancer. In particular, MYC is an important mediator of T-cell growth downstream NOTCH1 signaling (29), and translocations of the gene near the TCRA/D is present in 1% of T-ALL patients. The MYB gene codes for a leucine zipper transcription factor essential for hematopoiesis, and its translocation is quite rare in T-ALL, but more frequent in younger patients (<2 years old) (30).
Translocations of the cyclin D2 (CCND2) gene near the TCR loci are described in nearly 3% of T-ALL cases, and dysregulations of cell cycle regulators play a key role in the pathogenesis of T-ALL (1, 14).
Rare translocations (<1%) of NOTCH1 have been described in T-ALL and cause the production of a truncated and constitutively active NOTCH1 protein (31).
Importantly, aberrant NOTCH1 signaling is present in >60% of T-ALL due to activating mutations (32-35).
1.1.3.2 Fusion genes
Translocations can also lead to the production of chimeric proteins due to fusion of the coding regions of two genes. Among fusion genes, the BCL11B-TLX3 fusion is present in nearly 20% of T-ALL and causes over-expression of TLX3 (27, 36).
Other frequent gene fusions are represented by NUP214-ABL1 (Nuclear Pore Complex Protein Nup214 - Abelson Tyrosine-Protein Kinase 1), PICALM-MLLT10 (Phosphatidylinositol Binding Clathrin Assembly Protein - Myeloid/Lymphoid Or Mixed-Lineage Leukemia; Translocated To, 10) and SET (SET Nuclear Proto- Oncogene) - NUP214. Moreover, the transcription factor ETV6 has also been reported to be a fusion partner of the tyrosine kinases ABL1 and JAK2.
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Interestingly, a rare fusion of SQSTM1 (Sequestosome 1) with the nuclear pore protein NUP214 has also been reported (1, 14, 37).
Interestingly, alterations in T-ALL oncogenes are preferentially present in defined subtypes (Figure 1.5). In particular, mutations of NOTCH1 and CDKN2A are quite rare in ETP-ALLs, which on the contrary show high prevalence of NRAS, FLT3, RUNX1 (Runt Related Transcription Factor 1), ETV6 (ETS Variant 6), DNMT3A (DNA methyltransferase 3A) and EZH2 mutations (18, 27). The oncogenes TLX1 and TLX3 are frequently activated in lymphoblasts of the early cortical subtype, which in addition commonly have aberrant activation of the NOTCH1 pathway and loss of CDKN2A (24). Moreover, mutations in WT1 (Wilms Tumor 1), PHF6 (PHD Finger Protein 6) and PTPN2 (Protein Tyrosine Phosphatase, Non-Receptor Type 2) and the NUP214-ABL translocation are more frequent in this immature subtype than in others. Besides NOTCH1 activation and deletion of CDKN2A, late cortical T-ALLs usually harbor mutations in the TAL1 and LMO1/2 oncogenes and also frequently show mutations or deletions of the tumor suppressor PTEN (Phosphatase And Tensin Homolog) (14).
1.1.3.3 Deletions and mutations
Cell cycle dysregulation plays an important role in T-ALL pathogenesis, and both deletions and translocations of cell cycle regulating genes have been described. In
Figure 1.5: T-ALL subtypes and common mutations. The most frequent mutations are shown in bold. Adapted from: 14. Belver L et al., The genetics and mechanisms of T- cell acute lymphoblastic leukemia. Created with Smart Servier Medical Art.
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particular, the CDKN2A/B (Cyclin Dependent Kinase Inhibitor 2A/B) loci coding for the tumor suppressors p16INK4a, p14ARF and p15 are deleted in ~ 60% of T-ALL (14, 16). The p16INK4a and p15 proteins prevent the activation of CDK4 and CDK6 kinases by cyclin D with consequent inhibition the of G1- to S-phase transition.
p14ARF induces p53 activation through inhibition of its negative regulator MDM2, leading to cell cycle arrest and apoptotis. Moreover, deletions of the RB1 (Retinoblastoma 1) and CDKN1B (Cyclin Dependent Kinase Inhibitor 1) genes regulating cell cycle progression are described in nearly 15% of T-ALL (16, 24).
Deletions or inactivating mutations of other genes are quite frequent in T-ALL, and include: PHF6, PTEN, FBXW7 (F-Box And WD Repeat Domain Containing 7), WT1, LEF1 (Lymphoid Enhancer Binding Factor 1), BCL11B (B Cell CLL/Lymphoma 11B), DNM1 (Dynamin 1), EZH2 (Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit), ETV6, EED (Embryonic Ectoderm Development), RUNX1, GATA3 (GATA Binding Protein 3), PTPN2, IL7R (Interleukin-7 receptor), SUZI2 (SUZ12 Polycomb Repressive Complex 2 Subunit), CREBBP (CREB Binding Protein), ECT2L (Epithelial Cell Transforming 2 Like) and RELN (Reelin) (1, 14, 16).
Aberrant NOTCH1 signaling in T-ALL is caused by activating mutations of NOTCH1 (60% of T-ALL cases) or inactivating mutations of FBXW7 (15%), an ubiquitin ligase controlling NOTCH1 degradation (14, 32, 38). Importantly, NOTCH1 signaling is crucial for thymocyte development and differentiation, and dysregulation of this pathway can thus induce uncontrolled T-cell proliferation. The NOTCH1 protein forms a heterodimeric receptor on the plasma membrane that is composed of extracellular and intracellular subunits. The former is responsible for binding of ligands (Delta-like 1, 2, 3 and Jagged 1, 2) and contains the heterodimerization (HD) domain. The intracellular NOTCH1 subunit is composed of different domains (ICN1, intracellular domains of NOTCH1) among which the C-terminal domain PEST (proline (P), glutamic acid (E), serine (S), and threonine (T) rich) is responsible for targeting the activated receptor to proteasomal degradation through FBXW7-mediated ubiquitination (Figure 6, left) (38, 39).
Binding of ligand on the extracellular subunit triggers cleavage of NOTCH1 at two different sites (by ADAM10 protease at the S2 site and g-secretase at the S3 site).
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Cleavage of NOTCH1 at S3 allows the release of the ICN1, which translocates to the nucleus and mediates transcription of target genes. Mutations in the HD domain of NOTCH1 are present in 40% of T-ALL patients and can either occur as SNPs or insertions/deletions. Mutations in the HD domain cause ligand-independent activation of the receptor. Mutations in the PEST domain increase NOTCH1 stability, and are found in nearly 15% of T-ALL (Figure 1.6, right) (32, 39, 40).
1.1.4 Current treatments
T-ALL treatment is based on risk stratification and patients considered as “high- risk” are exposed to more aggressive regimens (41). Current protocols are composed of three distinct phases: induction, consolidation and maintenance (7).
The induction phase is aimed at achieving complete remission and usually lasts 4 to 6 weeks. Vincristine, corticosteroids (prednisone and dexamethasone), and
Figure 1.6: NOTCH1 signaling and mutations in T-ALL. Adapted from 32. Ferrando AA, The role of NOTCH1 signaling in T-ALL and 14. Belver L et al., The genetics and mechanisms of T-ALL. Created with Smart Servier Medical Art.
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asparaginase are commonly used during this phase, and the addition of anthracyclines has proven to be more effective in the treatment of high risk patients (4, 7). Among corticosteroids, dexamethasone is usually preferred due to its high penetration in the CNS.
The consolidation phase lasts between 6 and 9 months and is aimed at eradicating the MRD after remission. Common protocols are adjusted according to individual patients’ risks but usually require a combination of various agents including nucleotide analogs (thioguanine, cytarabine), antimetabolites (methotrexate, mercaptopurine), topoisomerase II inhibitors (etoposide), and alkylating agents (cyclophosphamide) (4, 7).
The third and last phase (maintenance) employs less aggressive treatments aimed at reducing the risk of relapse and can last 2 to 3 years. Current protocols rely on the oral administration of antimetabolite drugs and may also include vincristine and steroids (4, 7).
In addition, high risk patients with/without CNS infiltration require CNS-targeted therapy, e.g. the intrathecal administration of drugs and cranial radiation.
Relapsed T-ALL patients are exposed to more aggressive treatment regimens that include combinations of chemotherapeutic drugs used to treat newly diagnosed T- ALL.
Unfortunately, 20% of T-ALL patients do not respond to the first round of therapy or relapse, and, despite recent advances in the knowledge of T-ALL biology, the 5- year survival rate for these patients is lower than 10%. Recently, a phase II clinical trial involving the purine nucleoside analogue Nelarabine reached a response rate of 55% in relapsed pediatric T-ALL, with neurotoxicity being the main side effect (42). Importantly, the COG-AALL0434 study (ClinicalTrials.gov Identifier:
NCT00408005) reported a safe use of Nelarabine in combination with methotrexate in newly diagnosed high risk T-ALL patients (43), suggesting that the neurotoxicity of this drug might be due to the previous exposure to aggressive chemotherapy regimens.
Given its antitumoral effects, Nelarabine has been approved by the Food and Drug Administration (FDA) for the treatment of relapsed T-ALL (43, 44). In addition, a randomized phase II clinical trial is still ongoing to test the efficacy of Nelarabine
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in combination with conventional chemotherapy in adult T-ALL patients (ClinicalTrials.gov Identifier: NCT01085617).
Finally, the last option for relapsed/refractory T-ALL patients consists of hematopoietic stem cell transplant (HSCT), which however has a low rate of success.
1.1.5 New targets
Novel insights into the biology and pathogenesis of T-ALL have opened up new possibilities for the treatment of this disease. Novel drugs targeting hyper-activated pathways in T-ALL are now under investigation, and include inhibitors of the NOTCH1, IL7R/JAK/STAT, Bcl-2 and PI3K/AKT/mTOR pathways (4) (Table 1).
The development of NOTCH1-targeting drugs was first encouraged by in vitro and in vivo data demonstrating significant anti-leukemic effects of g-secretase (GSI) inhibitors. Unfortunately, a phase I clinical trial of the Dana Farber Cancer Institute (DFCI 04-390) revealed that GSIs efficiently down-regulated NOTCH1 in T- lymphoblasts but also induced gross toxic effects in the gastrointestinal tract with no relevant clinical response in patients. Moreover, while in vivo experiments demonstrated apoptosis induction in murine NOTCH1-driven T-ALL leukemia (45), effects on human T-ALL cells were restricted to cell cycle arrest and inhibition of proliferation (39). New studies are now investigating the possibility to target the NOTCH1 pathway with monoclonal antibodies against the receptor or its ligand.
A study by Roti et al. demonstrated that inhibition of the SERCA (sarco/endoplasmic reticulum calcium ATPase) protein with nanomolar concentrations of thapsigargin produced antitumoral effects both in vitro and in vivo due to inhibition of NOTCH1 maturation in the ER (46).
Hyper-activation of the interleukin-7 pathway is commonly implicated among pathways involved in T-ALL cell turnover. This can occur through mutations in the IL7 receptor (IL7-independent activation), gain-of-function mutations in JAK1 or JAK3 and the expression of the fusion protein ETV6-JAK2. Interestingly, the
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JAK/STAT pathway is aberrantly activated in the immature subtype ETP-ALL, and the JAK1/2 inhibitor Ruxolitinib showed antileukemic effects in ETP-ALL mouse models (47).
Aberrant activation of the PI3K/Akt/mTOR pathway is also a common feature in T-ALL due to loss-of-function mutations in the tumor suppressor PTEN or gain-of- function mutations in Akt, PI3K regulatory or kinase subunits and aberrant IL7 signaling. Inhibitors of this pathway are under investigation for their use in T-ALL, and a phase I clinical trial involving the mTORC1 inhibitor everolimus alone or in combination with conventional chemotherapeutic agents is ongoing (ClinicalTrials.gov Identifiers: NCT03328104; NCT01523977) (48). Other approaches under investigation are aimed at targeting cell cycle regulation (CDK4/6 inhibitors), the MAPK (mitogen-activated protein kinase) pathway, proteasome degradation, Bcl-2 inhibitors and epigenetic regulators (HDAC, DNMTs, IDH1/2 and inhibitors) (4, 49-51).
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Table 1. New drugs and targets.
Pathway Treatments
NOTCH1 g-secretase inhibitors
Monoclonal antibodies Soluble Notch proteins
Transcriptional co-activator inhibitors SERCA inhibitors
PI3K/Akt/mTOR PI3K inhibitors
mTOR inhibitors Akt inhibitors
Dual PI3K/mTOR inhibitors
IL7R/JAK/STAT JAK inhibtors
STAT inhibitors Cell cycle regulation CD4/6 inhibtors Bcl-2 inhibitors ABT-199, ABT-737
MAPK MEK inhibitors
Proteasome Proteasome inhibitors
Epigenetic HDAC inhibitors
DNA methyltransferase inhibitors IDH1/2 inhibitors
Adapted from: 4. Hefazi M et al., Recent Advances in the Biology and Treatment of T-ALL.
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1.2 Reactive oxygen species (ROS), mitochondria and cancer
1.2.1 Reactive oxygen species (ROS)
Reactive oxygen species (ROS) consist of a class of unstable and reactive molecules derived from the incomplete reduction of molecular oxygen (O2) (52-54).
Reduction of O2 by a single electron generates the superoxide anion (O2·-), a membrane-impermeant ROS which is rapidly converted into hydrogen peroxide (H2O2) by superoxide dismutase enzymes (SOD1 and 2, cytosolic and mitochondrial, respectively) (54, 55). H2O2 is then converted into H2Oby catalases, glutathione peroxidases (GPXs) or thioredoxins (TPXs), but can also react with free Fe2+ to form the hydroxyl radical (OH·) through the Fenton reaction (55). In addition, O2·- can react with nitric oxide (NO) to produce peroxynitrite (ONOO-) (52, 53, 55). Overall, ROS are reactive molecules known to induce macromolecular damage, as they can oxidize lipids, proteins and DNA, which can lead to cell death.
On the other hand, physiological production of ROS stimulates the MAPK (mitogen-activated protein kinase) and the PI3K signaling cascades (54, 56). For these reasons, cells have evolved homeostatic mechanisms to maintain ROS levels in a dynamic equilibrium in order to both sustain mitogenic and survival pathways and prevent ROS-induced damage.
ROS are produced in various cellular compartments, including mitochondria, peroxisomes and lysosomes. In T-cells, activation of the TCR induces the release of Ca2+ from the endoplasmic reticulum, with consequent uptake by mitochondria.
Here, Ca2+ stimulates the tricarboxylic acid (TCA) cycle, which fuels the ETC by providing NADH and succinate with a consequent increase in ROS production (57).
1.2.2 Generation of ROS in mitochondria
One of the main sources of ROS are mitochondria, dynamic organelles that orchestrate cellular bioenergetics. They possess two membranes that create separate compartments: the intermembrane space (IMS) is contained between the outer (OMM) and inner membranes (IMM), the latter of which encloses the
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mitochondrial matrix. Contrary to the OMM, the IMM is highly impermeable to molecules that must thus be imported through specific transporters (58). Moreover, invaginations of the IMM generate the cristae, which are essential for the proper assembly and function of the electron transport chain (ETC) complexes and of the FOF1-ATP synthase. The ETC is composed of four protein complexes (I to IV) and is responsible for the transport of electrons from the electron carriers NADH (reduced nicotinamide adenine dinucleotide) and succinate, produced during the Krebs cycle, to O2 (Figure 1.7) (59, 60). During this process, the incomplete reduction of O2 generates ROS both in the mitochondrial matrix and in the IMS (53, 59, 61).
NADH-dehydrogenase is the first complex of the ETC and is responsible for the transfer of electrons from NADH to the soluble electron carrier ubiquinone (UQ, also known as Coenzyme Q), generating NAD+ and ubiquinol (UQH2) (62). During this process, Complex I pumps four protons (H+) from the matrix into the IMS, generating a proton gradient. Importantly, leakage of electrons from the UQ binding site or Flavin center of Complex I is the main source of O2·- production (63).
Figure 1.7: ROS generation by the electron transport chain (ETC) in mitochondria. Transport of electrons throughout the ETC complexes can generate O2·- (shown in red) both in the matrix and in the intermembrane space (IMS). Created with Smart Servier Medical Art.
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Transfer of electrons to UQ is also mediated by Complex II (succinate dehydrogenase), which obtains electrons through the oxidation of succinate to fumarate (53, 62). Complex II also contributes to generation of ROS in the matrix, although at a lower rate.
After being reduced by Complexes I and II, ubiquinol transfers electrons to CoQH2- cytochrome c reductase (Complex III). This complex transfers electrons from the UQH2 to cytochrome c, a soluble IMS protein able to carry electrons to Complex IV (cytochrome c oxidase), the final step of the ETC. At this point, oxygen is completely reduced to water. During electron transfer, both Complexes III and IV pump H+ (2 and 4, respectively) from the matrix to the IMS, contributing to the generation of the proton gradient essential for ATP production. Moreover, Complex III is responsible for generating ROS both in the matrix and in the IMS (53, 64).
The proton gradient generated during electron transfer throughout the ETC complexes is used as a driving force by the FOF1-ATP synthase to drive ATP synthesis in a process known as oxidative phosphorylation (OXPHOS). In this process, the inward proton current from the IMS through the FOF1-ATP synthase is coupled to ATP synthesis in the matrix (60, 65).
Other sources of mitochondrial ROS (mtROS) include the matrix proteins pyruvate dehydrogenase and a-ketoglutarate dehydrogenase. These enzymes contain a flavoprotein subunit that can produce both superoxide and hydrogen peroxide (66).
Generation of ROS in the matrix is also mediated by aconitase, acetyl-CoA dehydrogenase and ubiquinone oxidoreductase (67, 68).
Besides O2·- production by Complex III, other enzymes contribute to ROS generation in the IMS. Among these, dihydroorotate dehydrogenase (DHOH) is an OMM protein that converts dihydroorotate to orotate while donating electrons to ubiquinone. In the absence of oxidized quinone (when the UQ/UQH2 ratio is high/UQH2 accumulates/complex III is inhibited), DHOH can generate hydrogen peroxide in the IMS (69-71). Moreover, GPDH (glycerol-3-phosphate dehydrogenase), an IMM enzyme facing the IMS, uses UQ as an electron acceptor
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to catalyze the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate and may generate ROS in the IMS (72-75).
The monoamine oxidases A and B (MAO-A and MAO-B), located in the OMM, catalyze the oxidative deamination of amines with concomitant hydrogen peroxide production (76). As H2O2 can diffuse across biological membranes, MAO- generated ROS can also induce damage of mitochondrial DNA (mtDNA) (76).
Moreover, stimuli that induce membrane hyperpolarization or depolarization have been reported to increase mtROS generation due to an unbalanced electron flux throughout the ETC complexes. In particular, mitochondrial potassium (K+) channels have been implicated in ROS generation, although the exact mechanism is still debated. Recent studies demonstrated that inactivation of the voltage-gated K+ channel Kv1.3 leads to IMM hyperpolarization and mtROS generation with consequent activation of cell death in chronic lymphocytic leukemia (CLL) models (77, 78). On the contrary, activation of the calcium-activated potassium channel KCa1.1 by small molecules has been reported to induce IMM depolarization, enhancing ETC activity and mtROS generation (79). In addition, the small- molecule K+ channel opener NS1619 was shown to decrease Complex I-mediated ROS production while increasing Complex III-induced ROS generation (80), indicating that effects of channel activation or inactivation on mtROS may depend on the site of production of ROS (81).
1.2.3 Mitochondrial ROS and regulation of apoptosis
Besides being excellent energy producing machines, mitochondria play a key role in cell survival due to their role as apoptosis mediators. These organelles contain and sequester various pro-apoptotic proteins (cytochrome c, Smac/DIABLO, Omi/HtrA2, EndoG, AIF) that are released by mitochondria only upon activation of the apoptotic pathway (82-84). Mitochondrial integrity is thus essential for the regulation of programmed cell death, and permeabilization of the OMM plays a key role in this process (84).
The apoptotic pathway can be engaged by cell death receptors expressed on the plasma membrane (extrinsic apoptosis) or by permeabilization of the mitochondrial
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membrane (instrinsic apoptosis) following various cellular stresses, including ROS.
The extrinsic pathway requires the binding of a death ligand (Fas, TNF, TRAIL) to its receptor with the consequent activation of the downstream target Caspase-8 or 10. Activated Caspase-8/10 then induces proteolytic cleavage and activation of the executioner Caspases 3, 6 and 7, whose substrates include ICAD (inhibitor of CAD (Caspase-activated DNAse)) and PARP1 (poly(ADP-ribose) polymerase 1) with consequent DNA fragmentation and cell death (85, 86). The extrinsic pathway can crosstalk with intrinsic apoptosis, as Caspase-8 can also cleave and activate BID (BH3-interacting domain death agonist) (87, 88). Truncated BID (tBID) interacts with the pro-apoptotic proteins Bak and Bax and facilitates their oligomerization on the OMM with risulting induction of mitochondrial outer membrane permeabilization (MOMP) (89). MOMP leads to the release of pro-apoptotic molecules from mitochondria, contributing to potentiate the apoptotic cascade.
Importantly, the intrinsic apoptotic pathway can occur independently of extrinsic apoptosis, and cellular stress conditions, including oxidative stress, are known to induce mitochondrial-dependent cell death (90).
Mitochondrial integrity also relies on the maintenance of the cristae ultrastructure, and its disruption can cause cytochrome c mobilization in the IMS. Cristae formation is controlled by the IMM protein Optic Atrophy Protein 1 (OPA1), a dynamin-like GTPase that promotes inner membrane fusion during mitochondrial fusion (91). OPA1 forms high molecular weight complexes at the cristae bottleneck and thus creates the cristae compartment that sequesters different IMS proteins, including cytochrome c (92-94). As a result, cytochrome c is not freely diffusible in the IMS and its release during apoptosis requires disassembly of OPA1 oligomers and cristae remodeling. OPA1 is thus a major player in both mitochondrial function and apoptosis (Figure 1.8).
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Human OPA1 is encoded by the nuclear gene OPA1, which is expressed as 8 alternatively spliced mRNAs (95, 96). During import into mitochondria, the N- terminal mitochondrial targeting sequence (MTS) is cleaved, allowing the generation of long-OPA1 (l-OPA1) isoforms, which are anchored to the IMM (91, 97). Physiologic OPA1 processing is essential for mitochondrial fusion and many proteases are known to induce OPA1 cleavage at different sites in the protein.
Among these, the metalloendopeptidase OMA1 (overlapping with m-AAA protease) cleaves OPA1 at the S1 site, while the ATP-dependent zinc metalloprotease YME1L1 (yeast mitochondrial DNA escape 1-like) cleaves OPA1 at the S2 site (Figure 1.9) (93, 96-101).
Interestingly, only 4 l-OPA1 isoforms contain the S2 site. The balance of l- and s- OPA1, necessary for the preservation of the mitochondrial network, is regulated by the constitutive activity of YME1L1 (97, 102). Recent studies demonstrated that OXPHOS conditions enhance YME1L1-mediated OPA1 cleavage and promote mitochondrial fusion (102-104).
Figure 1.8: OPA1 controls cristae ultrastructure and cytochrome c mobilization. Created with Smart Servier Medical Art.
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Conversely, OMA1 is normally present as an inactive form which is activated during stress conditions, including mitochondrial membrane depolarization and mtROS accumulation (99, 105). Contrary to YME1L1, OMA1-dependent OPA1 cleavage at S1 is known to inactivate OPA1 and induce mitochondrial fragmentation and apoptosis (106, 107). Interestingly, recent studies demonstrated that YME1L1 and OMA1 are subjected to reciprocal degradation (108, 109), indicating the existence of a precise mechanism of regulation of OPA1 processing and mitochondrial morphology: OXPHOS conditions that produce high levels of ATP promote mitochondrial fusion through YME1L1 activation and OMA1 degradation; on the contrary, prolonged stress conditions activate OMA1, which induces YME1L1 degradation and mitochondrial fragmentation (Figure 1.10).
Figure 1.9: OPA1 processing by YME1L1 and OMA1. Adapted from: 93. MacVicar T and Langer T, OPA1 processing in cell death and disease – the long and short of it. Created with Smart Servier Medical Art.
34 1.2.3 Cellular antioxidant systems
Uncontrolled high levels of ROS can be toxic for cells as they induce oxidation of cellular components and can engage the apoptotic pathway. Cells have thus evolved antioxidant systems in order to rapidly buffer/eliminate ROS that would otherwise lead to cell damage and death. These systems include both enzymatic and non- enzymatic mechanisms and are present in every cell compartment.
O2·- is very unstable and is rapidly converted into H2O2 by superoxide dismutases present both in the cytosol (SOD1) and in mitochondria (SOD2 or MnSOD) (54).
While O2·- is not membrane-permeant, H2O2 is a stable ROS that can cross cellular membranes and can thus function as a signaling molecule. Mechanisms for H2O2
detoxification include the peroxidases GPXs and TPXs which oxidize glutathione (GSH) and thioredoxin (TRx), respectively, while reducing H2O2 to H2O (55). Figure 1.10: Mitochondrial adaptation in healthy and stress conditions. Adapted from: 109. Wai T et al., Mitochondrial Dynamics and Metabolic Regulation. Created with Smart Servier Medical Art.
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Importantly, a GPX4 isoform is expressed in mitochondria and prevents membrane peroxidation (110).
Oxidized glutathione (GSS) is reconverted to GSH by glutathione reductase (GSR), which transfers electrons from NADPH (nicotinamide adenine dinucleotide phosphate) to GSS. Thioredoxin is regenerated to its reduced form by the enzyme thioredoxin reductase and its cofactor NADPH (56, 59).
NADPH is fundamental for maintaining molecules in their reduced forms and is essential for regulation of ROS homeostasis. Importantly, NADPH is produced by various pathways, with the pentose phosphate pathway (PPP) considered to be the main source of cellular NADPH. The PPP is a biosynthetic pathway parallel to glycolysis that uses glucose-6-phosphate to generate nucleotide precursors (111, 112). During this process, two NADPH molecules are produced by the PPP enzymes glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD) (112). The PPP is a cytosolic pathway, and its products are also localized in the cytosol (113). Given that intracellular transporters of NADH and NADPH have not been reported so far, other NADPH-producing mechanisms besides the PPP must be present in mitochondria to sustain their scavenging capacity. Interestingly, recent studies demonstrated that mitochondrial NADPH- producing enzymes strongly contribute to maintain cellular NADPH pools and include nicotinamide nucleotide transhydrogenase (NNT), isocitrate dehydrogenase 2 (IDH2), glutamate dehydrogenase 1 (GDH1), malic enzymes (ME2/3), NAD kinase (NADK2) and the mitochondrial folate cycle (114).
Other antioxidant mitochondrial molecules include the lipid-soluble Vitamin E (also known as a-tocopherol). In addition, the ETC components cytochrome c and ubiquinol are considered ROS scavengers due to their electron transfer abilities (53, 59).
1.2.4 The NRF2 pathway
The nuclear factor erythroid 2-related factor 2 (NRF2) pathway is considered the master regulator of the cellular stress response and in particular of oxidative stress (115, 116). NRF2 is a Cap ‘n’ Collar (CNC) transcription factor of 605 amino acids
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that contains 7 Neh (NRF2 ECH-homology) domains important for protein function and regulation (117). The Neh1 domain is responsible for the heterodimerization with small Maf proteins and DNA binding activity. The Neh2 domain is important for the interaction with the negative regulator Keap1 (Kelch-like ECH-associated protein 1), which binds the two peptide sequences DLG and ETGE through its Kelch domains (117). Neh domains 4 and 5 are involved in the transcriptional activation capacity of NRF2 and are responsible for binding to CBP (CREB (cAMP Responsive Element Binding Protein)-Binding Protein), p300 and RAC3 (Receptor-associated coactivator 3).
In homeostatic conditions, NRF2 is bound by Keap1, which ubiquitinates the transcription factor in a process that also requires the Cul3 and Rbx1 proteins. As a result, NRF2 is rapidly targeted for degradation through the proteasome and thus does not accumulate in the cytosol (118). Under oxidative stress, the oxidation of cysteine residues in Keap1 induces a conformational change that increases the NRF2-Keap1 interaction and blocks the ubiquitination of NRF2, thus allowing accumulation of newly synthetized NRF2 (Figure 1.11) (118-120). Subsequent transfer of NRF2 to the nucleus depends on the recognition of its NLS (Nuclear Localization Sequence) by importins and its phosphorylation by kinases, including the atypical protein kinase C d (PKCd), which phosphorylates NRF2 at Serine 40 in response to oxidative stress (121-123). Once in the nucleus, NRF2 heterodimerizes with small Maf proteins and binds to ARE (Antioxidant Response Element) sequences in the promoters of its target genes. Importantly, NRF2 regulates the transcription of more than 200 genes involved in (i) NADPH production, (ii) thioredoxin and glutathione synthesis, (iii) proliferation and survival, (iv) iron metabolism, (v) mitochondrial physiology and (vi) drug metabolism (124). Interestingly, an NRF2-responsive element has been described in the promoter of NOTCH1 (125).
37 1.2.5 Effects of ROS on cell turnover
ROS constitute powerful signaling molecules that regulate many cellular processes, including cell turnover and iron homeostasis. Among ROS-modulated pathways, the PI3K/Akt (phosphoinositide 3-kinase/protein kinase B) pathway is a key regulator of cell survival and proliferation and is commonly hyper-activated in cancers, including T-ALL (1, 4). The PI3K/Akt pathway is activated by growth factors and cytokines. The second messenger phosphatidylinositol 3,4,5 triphosphate (PIP3) produced by PI3K can promote cell growth and survival through activation of Akt and inhibition of apoptosis (126). Moreover, Akt can promote protein synthesis and cell growth by inhibiting the mTORC1 repressors TCS2 (Tuberous sclerosis 2 protein) and PRAS40 (40 kDa proline-rich AKT substrate) (127, 128). Importantly, signaling of this pathway is inhibited by the phosphatase PTEN, which dephosphorylates PIP3 into PIP2. Interestingly, PTEN is inactivated by H2O2 through oxidation of cysteine residues (126, 129). Thus, increased ROS levels can enhance the PI3K/Akt signaling through inactivation of its negative regulator PTEN.
Figure 1.11: NRF2 pathway is regulated by ROS and induces the transcription of genes involved in stress response. Created with Smart Servier Medical Art.
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The MAPK pathway includes four main MAPK signaling cascades (Erk1/2, JNK, p38 and Erk5) that regulate cell proliferation. Oxidative stress can activate the apoptosis signal-regulated (ASK1) kinase that activates both the JNK and p38 cascades (126, 130). Importantly, ASK1 is negatively regulated by the reduced form of thioredoxin, which blocks the kinase’s N-terminal domain (131). Oxidation of thioredoxin by ROS can thus lead to activation of ASK1 and its downstream targets.
Moreover, H2O2 can induce the oxidation of Cys-2991 of the ATM (ataxia- telengectasia mutated) protein with consequent activation of the kinase, whose downstream targets include p53 and mTORC1 (130). This mechanism thus constitutes an alternative way by which ATM regulates cellular responses to oxidative stress, promoting cell cycle arrest or autophagy when high ROS levels cause cellular damage.
Iron homeostasis is another finely regulated cellular process influenced by ROS.
Depletion of iron can lead to cell cycle arrest or apoptosis, as iron is essential for the activity of many enzymes involved in DNA synthesis (the iron-dependent ribonucleotide reductase, RR) and cell cycle regulation (cyclins A, B and D) (130, 132-134). On the other side, accumulation of iron can lead to increased ROS generation through the Fenton reaction (52, 126). The IRP 1 and 2 proteins (iron regulatory proteins 1 and 2) are involved in iron uptake and storage and represent a sensor of intracellular iron (135). In particular, under iron-dependent oxidative stress, depletion of the GSH pool blocks the activity of GPX4, a glutathione peroxidase that protects against lipid hydroperoxidation. Accumulation of lipid peroxides can thus culminate in the cell death pathway known as ferroptosis (136, 137). Interestingly, NRF2 is a negative regulator of ferroptosis as it regulates the expression of ferritin heavy chain 1 (FTH1), which stores and sequesters excess free iron limiting its effects on redox homeostasis (137).
High levels of ROS can also amplify programmed-cell death by up-regulation of death receptors (82, 84). Interestingly, several studies showed that ROS can induce the up-regulation of the TRAIL-receptor 2 (also known as death receptor 5, DR5)
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through the activation of the C/EBP homologous transcription factor (CHOP) (138, 139).
1.2.7 ROS, NRF2 and cancer
Cancer cells are characterized by the dysregulation of many cellular processes, including proliferation, survival, genomic instability, angiogenesis and metabolism (140). Importantly, high levels of ROS can impinge on these processes and can thus contribute to tumorigenesis.
Several studies demonstrated that, compared to their normal counterparts, cancer cells are characterized by increased ROS levels that can sustain their high proliferation and survival rates (141-143). Cancer cells also upregulate ROS- scavenging pathways in order to maintain ROS levels below the threshold beyond which macromolecular damage is produced and cell death pathways are engaged.
This altered redox homeostasis thus relies on the ability of cancer cells to efficiently activate antioxidant pathways.
Many studies have reported the upregulation of NRF2 in cancer, and its activation is known to give cancer cells a survival advantage (124, 144-146). NRF2 upregulation and the consequent increased scavenging capability allow cancer cells to survive under highly oxidative stress conditions, which also favour mutagenesis and thus cancer progression (147, 148). In this context, NRF2 is considered an oncogene. Mutations that increase NRF2 stability (activating mutations in NRF2 or loss-of-function mutations in Keap1) have been reported in many tumors, including lung, breast, pancreas and ovarian cancer (145, 146, 149, 150). Accumulated evidence suggests that NRF2 regulates many processes involved in cancer progression and in chemo-resistance. Importantly, NRF2 has been reported to control the expression of NOTCH1 (151), which regulates T-cell proliferation and differentiation. Moreover, NRF2 increases the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, which might lead to a decreased sensitivity to cytotoxic agents and chemo-resistance. NRF2 has also been shown to regulate the expression of multidrug resistance proteins responsible for detoxification of xenobiotics and thus can contribute to resistance to therapy (147). NRF2 also contributes to the
