resistance).
Overall Impact: Completion of these Aims has led to a more detailed understanding of the role of cellular antioxidant machinery (GSH, SOD) and cellular redox state in acquisition of drug resistance to MAPKih in metastatic melanoma. In addition, new understanding is presented on the mechanistic-crosstalk between cellular metabolism; antioxidant machinery; and cytoprotective pathways (e.g., autophagy and UPR) in acquisition of drug resistance of metastatic
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melanoma to MAPKih. It is anticipated that future research based on this thesis will lead to an safe and effective clinical approach to overcoming adaptation of metastatic melanoma that improve outcomes for metastatic melanoma patients.
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Background
Melanoma – Staging
This thesis focuses on Stage IV metastatic melanoma and the acquisition of resistance of melanoma to MAPKi therapies. Therefore a brief description of the categorization of melanoma progression is provided to provide context relevant to patient care. Melanoma is staged according to the TNM (tumor, node, metastatic) classification of malignant tumors; described in the eighth edition of the American Joint Committee of Cancer (AJCC) staging manual.16 According to the TNM classification:
The T-Category corresponds to a primary melanoma tumor, which is largely restricted to the epidermal layers of the skin. The division of this category is based on “Breslow” thickness, which is subdivided based on dermal thickness (Tis:
melanoma in situ; T1 £ 1 mm; T2 >1-2 mm; T3: >2-4 mm; T4: >4 mm); and absence (“a”) or presence of (“b”) of ulceration. Survival of patients diagnosed and treated in this stage ranges from 99% (T1a) to 82% (T4b) [5 y survival] and from 98%
(T1a) to 75% (T4b) [10 y survival]. Lower survival rates are associated with the presence of ulceration. T-category tumors are classified into subgroups of clinical Stage I and II; when no distant disease is present are (T1a, T1b) – Stage IA; T2a – Stage IB; (T2b, T3a) –Stage IIA; (T3b, T4a) – Stage IIB; and T4b– Stage IIC.
The N-category corresponds to malignancy that has metastasized to regional lymph nodes and non-nodal locoregional sites. This category is further subdivided based on the number of lymph nodes involved (N0 – no node involved, N1 – one lymph node involved, N2 – 2-3 lymph nodes involved, and N3 – greater than 4 lymph nodes involved); the extent of lymph node involvement; and presence of non-nodal locoregional metastatic disease.17 The extent of nodal involvement is quantified as “clinically occult” (“a”) and “clinically detected” (”b”). Clinically occult refers to microscopic metastatic disease burden determined during the sentinel
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node biopsy. Clinically detected refers to the existence of macroscopic disease burden that can be detected using clinical techniques such as radiography and ultrasound. The presence of non-nodal locoregional metastases (“c”) (microsatellites, satellites, and in-transit disease)18 is associated with poor prognosis.19, 20 Survival of patients in N-category ranges from 84% (N1a) to 52%
(N3c) [5 year survival] and from 75% (N1a) to 43% (N3c) [10 y survival]. However, to obtain more accurate prognostic estimates, different permutations of T-subcategories and N-T-subcategories are used to define T-subcategories of Stage III melanoma (Figure 1:2).16 These classifications generally assist in design of clinical trials; estimating the efficacy of adjuvant therapy of clinical trials, and in accurate estimation of survival. The 5-year survival for stage III melanoma ranges from 93% (IIIA) to 32% (IIID), and the 10-year survival ranges from 88% (IIIA) to 24% (IIID).
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Figure 1.2. Stage III Subcategories Based on T and N Classification.
This figure is adapted from the eight edition of AJCC guide to melanoma staging,16 and shows Stage 3 subcategories based on T and N classification.
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The M-category (stage IV) defines metastatic disease that has spread to distant sites through the bloodstream. This category is subdivided based on the site of metastasis and serum lactate dehydrogenase (LDH) levels. Based on the anatomic location of metastases (regardless of LDH levels), M1a – patients with distant metastatic disease that has spread to subcutaneous tissue, muscle, and distant lymph nodes; M1b – metastatic disease spread to lungs (with or without occurrence of the disease in skin, muscle or distant lymph nodes); M1c – metastatic disease that has spread to visceral organs (including organs in the chest cavity (heart and lungs) and abdominal cavity (e.g., liver, pancreas, and intestines)); and M1d – occurrence of the metastatic disease in the CNS (brain, spinal cord) system. Based on the serum LDH levels, the M1 category is further classified into not elevated [0], elevated [1]. Elevated LDH is associated with poor prognosis in stage IV melanoma.21 Survival statistics for stage IV metastatic melanoma are disturbingly low (5 y, 22.5%). The prognosis for patients whose disease is classified with elevated LDH levels combined with CNS involvement is almost always fatal.
Melanoma Treatment Options (General Overview)
Wide excision of the primary malignant lesion and sentinel node resection (surgery) is largely curative for stage 1 and 2 melanoma (5 y survival 98.4%).
Historically, treatment options for stage 3 melanoma (presence of metastatic disease in the lymph nodes) involved excision of regional lymph nodes (low-risk stage 3 melanoma) or complete lymphadenectomy (high-risk stage 3 melanoma) (5 y survival 63.6%). However, a recent multicenter selective lymphadenectomy clinical trial (MSLT-II) established that complete lymphadenectomy (vs. close observation) had no significant impact on the 3-y survival of subjects. On the contrary, complete lymphadenectomy resulted in severe lymphedema in 24.1% of subjects, thereby, decreasing their quality of life.22 On the other hand, clinical trials with adjuvant therapy (immunotherapy and targeted therapy) in subjects with
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complete resection of stage III melanoma have shown improved outcomes. For example, adjuvant immunotherapy using Nivolumab (NCT02388906) resulted in 12-month recurrence-free survival of 70.5%;23 and 75.4% of subjects treated with Pembrolizumab (NCT02362594) benefited from a 12-month recurrence-free survival.24 Additionally, adjuvant therapy with MAPK-pathway targeted drugs Dabrafenib (BRAF inhibitor) plus Trametinib (MEK inhibitor) (NCT01682083) resulted in 3-year recurrence-free survival of 58% compared to 39% in the placebo-treated control group;25 and adjuvant therapy with Vemurafenib (BRIM8, NCT01667419) improved recurrence-free survival (23.1 months vs. 15.9 months in placebo-treated subjects).26
Stage IV metastatic melanoma is the most aggressive and treatment resistant disease stage. Surgery is mostly not an option in this stage. Prior to 2011, prognosis for patients diagnosed with Stage IV melanoma was dismal. However, the rapid introduction of both MAPK-pathway and immune targeted drugs since then have significantly improved outcomes for these patients. Recent introductions of immunotherapies (e.g., CTLA-4 and PD-1 inhibitors)27 have also significantly improved outcomes.28 For example, approval of immune-checkpoint inhibitor ipilimumab for Stage III-IV melanoma in 2012 was based on an improvement in overall survival (OS) of only 3.7 months, with an overall response of <15%.29 More recent immunotherapies have improved outcomes significantly. Pembrolizumab resulted in a 24-month survival rate of 55% vs. 43% in the ipilimumab group (NCT01866319).30 The highest response rates (as high as 58%) were observed in combination immunotherapy trials. On the other hand, MAPKih is a class of small-molecule based drugs that target the protein kinase members of the Mitogen Activated Protein Kinase (MAPK) pathway. This thesis focuses on the response and cellular changes in melanoma cells caused by continuous administration of this class of drugs.
While in some cases responses to these new therapies are remarkable, low response rates, acquired resistance, and severe adverse events31-34 limits the
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potential for these drugs. These findings and observations define a critical need to develop a greater understanding about the underlying mechanisms resulting in limited response and drug resistance in metastatic melanoma patients. Thus, this thesis focuses on the acquisition of resistance to MAPKi with the goal of identifying targetable interventions to improve outcomes for these patients.
MAPK Pathway Inhibitors¾ New Era in Metastatic Melanoma Treatment
The focus of this research is on the acquisition of resistance of melanoma to MAPKi
therapy. An introduction to the MAPK pathway and its role in normal cellular activity, as well as in carcinogenesis and progression is provided as background information.
In healthy cells, the MAPK pathway relays extracellular growth signals to the nucleus via a kinase-dependent signaling cascade. The MAPK pathway regulates essential cellular functions such as growth, proliferation, differentiation, metabolism, and cell death. Activation of the MAPK pathway is triggered by the binding of extracellular growth signaling molecules (e.g., growth hormones) to the extracellular domain of receptor tyrosine kinase (RTK) proteins followed by dimerization and autophosphorylation of downstream targets. Thus, this event mediates the activation (phosphorylation) of RAS, a small, cytoplasmic GTPase molecule, which initiates sequential phosphorylation of RAF, MEK, and ERK proteins. Activated (phosphorylated) ERK regulates numerous transcriptional factors controlling gene expression associated with cell growth and development.8,
35, 36
Melanoma tumors are characterized by significant genetic-mutation burden and heterogeneity;37 and oncogenic mutations in MAPK-pathway proteins represent the most common driver mutations. These mutations play an important role in carcinogenesis and disease progression in melanoma.38, 39 15-20% of melanoma patients harbor NRAS oncogenic mutations primarily at the Q61 residue;40 and
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approximately 40-60% melanoma patients are positive for BRAFV600X oncogenic mutations – the most common of which is the BRAFV600E (>80%), 41, 42 which is a known oncogenic driver mutation in melanoma.38, 39 The BRAFV600E mutation results in constitutive activation of the MAPK signaling cascade independent of external stimuli and negative feedback resulting in uncontrolled cellular proliferation.43 Studies have shown the presence of a BRAFV600E mutation in nevi that have acquired a malignant phenotype, which substantiates a critical role of this mutation in melanoma initiation.41, 44
Preclinical evaluations of BRAFV600E targeted small-molecule inhibitors (BRAFih) (e.g., Vemurafenib) resulted in melanoma-specific cell death in vitro and pre-clinical in vivo models.45, 46 FDA approval of BRAFV600E targeted Vemurafenib (Vem) in 2011 marked the beginning of a new era in melanoma treatment. The initial clinical response to BRAFV600E inhibitors was often remarkable (48% to Vem in the BRIM-3 trial);47 and 50% to dabrafenib in the BREAK-3 trial),48 compared to a response rate of only 5-6% to the only FDA-approved treatment for metastatic melanoma at the time (i.e., Dacarbazine). Unfortunately, despite early response to BRAFih (in only about 50% of patients), disease almost inevitably progressed or recurred in most of the subjects in these trials (within months). According to updated statistics, only 11% of the subjects in the BREAK-2 and BREAK-3 Dabrafenib clinical trials showed 5 y progression-free survival; with a 5 y overall survival of 20%.7 Similar statistics were seen in Vem trials with a 5 y survival of 26% (mean survival of 14.7 mo).6, 49 In efforts to target drug resistance and improve response to MAPKi in metastatic melanoma patients, combination of BRAFih and MEKih (Cobimetinib and Trametinib) was introduced. Although, the combination therapy improved response and progression-free survival in patients significantly, subjects eventually acquired resistance to these treatments as well.50, 51 To date, no current clinical treatment option has been made available that overcomes the acquisition of drug resistance to BRAFih and MEKih treatments in metastatic melanoma patients.
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The precise mechanisms of acquired drug adaptation are not entirely understood.9,
12, 52-56 Plausible mechanisms include re-activation of MAPK pathway via acquisition of activating NRAS and MEK mutations;57 altered/alternative oncogenic pathways;58 copy number amplification of BRAFV600E;57, 59 alternative splicing of BRAFV600E;13 the presence of subpopulations of resistant-melanoma stem-like cells;11, 60 and metabolic re-programming,10 enabling adaptation to MAPKi (Figure 1:3). More recent studies have indicated a role for autophagy, and in particular autophagy initiated by activation of unfolded protein response (UPR) pathways in the adaptation of metastatic melanoma to MAPKi. 61-63 This thesis project focuses on the role of autophagy and ER stress mediated by continuous MAPKi-induced changes in the oxidative state of the melanoma cells.
Figure 1.3. Possible Mechanisms for Drug (MAPKih) Resistance.
Schematic representation showing possible mechanisms responsible for acquisition of resistance to MAPKi in melanoma.
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Autophagy as a Mechanism of Drug Resistance
Autophagy has been identified as a potential cytoprotective cellular mechanism by which melanoma cells become resistant to MAPKi therapies. The term autophagy originated from the Greek word auto-phaegin, which means “to self-eat.” In the cellular context, autophagy refers to a lysosomal degradation pathway required to maintain (normal) and restore (in response to stress) intracellular homeostasis and metabolism.64-66 At the organismal level, autophagy is essential to mobilizing nutrient reserves for systemic metabolism.66-68 Autophagy-deficient mice fail to survive starvation, and feeding improves the survival only marginally.69 Autophagy–deficiency in neonatal mice is lethal,70 establishing the importance of autophagy in growth and development. Decades of research has confirmed the crucial role of autophagy in differentiation and maintaining homeostasis at both cellular and organismal levels.71 Autophagy enables the cell to manage and recover from stress by removing and recycling damaged cellular debris and enabling sustenance by mobilizing essential nutrients – and is known to be initiated in response to stress stimuli (e.g., hypoxia, starvation scenarios such as amino acid and glucose deprivation; growth factor imbalance; or redox imbalance initiated by different signaling pathways; Figure 1:4B).65, 72
Thus, while a basal level of autophagy is an integral part of normal cellular function, a significant body of evidence supports the idea that in addition to a playing vital role in maintaining normal cellular homeostasis, aberrant autophagic flux can play a dual role in exacerbating disease73 in maladies such as Crohn's disease;
neurodegenerative diseases,74 (e.g., Parkinson's and childhood encephalopathy);
cardiomyopathy, 75 diabetes, obesity,76, 77 and cancer.78
Mechanistically, autophagy is a dynamic process that involves four basic steps: (1) Initiation; (2) Elongation; (3) Maturation; and (4) Fusion (Figure 1:4A). The first
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crucial step is the initiation of a C-shaped double layered isolation membrane called phagophore, derived from lipid bilayers of the endoplasmic reticulum (ER), mitochondria, and Golgi. The nucleation of a phagophore is mediated by the ULK1 complex (regulating units: FIP200, Atg13, Atg101 and ser/thr kinase ULK1), which further interacts with the Vps34 complex. During the process of elongation, the Vps34 complex recruits two protein complexes, (i.e., Atg12-Atg5-Atg16 and LC3-PE complexes). The Atg12 and Atg5 conjugate is formed by the formation of thioester bonds that are facilitated by Atg7 and Atg10. Conjugation of Atg12–Atg5 is indispensable for the creation of autophagosome. The process is a constitutive phenomenon and does not depend on autophagy initiating factors. This conjugate binds to Atg16 via Atg5 to form the (Atg12–Atg5–Atg16) complex, which plays a vital role in the elongation of the phagophore by recruiting microtubule-associated protein 1 light chain 3 (LC3) in an Atg5 dependent manner to form a double membrane-bound vesicle called autophagosome. In the maturation stage, LC3 conjugates to phosphatidylethanolamine (PE) to form LC3II, which remains bound to the double membrane of the matured autophagosome enclosing the autophagic cargo (damaged organelles and cytoplasmic protein aggregates).79, 80 The final step involves fusion of the autophagosome with a lysosome. Post-fusion the autophagic cargo is degraded by lysosomal acid hydrolases at relatively low pH (4.5-6) in the fused vesicle. The product of acid degradation (small amino acids and other essential building blocks) are transported back to the cytoplasm and recycled. 81, 82
16 Figure 1.4. Autophagy
Schematic diagram showing (A) the basic process of autophagy; and (B) different possible signaling pathways that lead to activation of autophagy.
In cancer, autophagy is known to play a dual role –acting as a tumor suppressor in benign and early-stage tumors including melanoma,83-85 while facilitating tumor growth and resistance in advanced cancers.86 Emerging evidence establishes the role of BECN1 (an essential autophagy gene) as a haploid-sufficient tumor suppressor that is known to be monoallelically deleted in 40-75% of human prostate87 breast,88 and ovarian cancers.89 Furthermore, in vivo studies show that BECN1 heterozygous mutant mice are prone to lymphoma, liver, and lung tumors.
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On the contrary, studies establish a prosurvival role of autophagy in advanced stage melanoma,90-93 glioma, multiple myeloma, and breast cancer.78
Preclinical data show a cytotoxic effect of autophagy inhibition in various cancers using chloroquine and its derivative, hydroxychloroquine (HCQ).94, 95 Chloroquine and HCQ are FDA-approved antimalarial drugs, which are known to inhibit autophagy by preventing lysosomal fusion to autophagosomes.96 Based on these studies, multiple Phase 1 and Phase 2 clinical trials using HCQ alone and in combination with other anti-cancer drugs were launched for various cancers,97 such as renal cell carcinoma (NCT01550367); colorectal cancer (NCT02316340);
pancreatic adenocarcinoma (NCT01273805);98 prostate cancer (NCT00726596);
non-small-cell lung cancer;99 estrogen receptor-positive breast cancer (NCT02414776); lymphoma;100and malignant solid tumors including metastatic melanoma.101-102, 103 These clinical trials established the tolerability of HCQ in high doses (400-1200 mg day-1) with limited dose-related toxicity. Further, these clinical trials showed that autophagy inhibition induced a partial response (14%) and stable disease (24%) in melanoma subjects.97 However, to date no clinical trial has evaluated the response of melanoma subjects who have acquired resistance to MAPK pathway inhibitors when these drugs (i.e., MAPKi) are combined with inhibitors of autophagy.
The role of cellular antioxidant machinery in regulating autophagy is well established.104, 105,106 Furthermore, studies have indicated the role of glutathione (GSH) in regulating autophagy, in particular mitophagy, in yeast cells.104 Recent studies have also indicated the role that the oxidized to reduced GSH ratio plays in modulating autophagy in carcinoma cells107 and retinal pigmented epithelial cells, likely a result of its role in mediating the thiol-mediated elongation step.108 Similarly, N-Acetyl Cystein (NAC) has been shown to rescue from methamphetamine-induced neurodegeneration via modifying cellular redox state and autophagy.107 These emerging evidence strongly suggest the role of cellular thiol-mediated oxidative state in regulating autophagy. In this thesis, the role of
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autophagy mediated by MAPKi-induced profound changes oxidative metabolism, redox imbalance, with a focus on remarkable drug-induced changes in the GSH:GSSG ratio in development of drug resistance in metastatic melanoma cells is explored (Chapter 2). A brief summary of the relationship between redox imbalance, the UPR, and autophagy is provided in this introduction to provide context.
Redox Imbalance Induces UPR-Mediated Autophagy
The endoplasmic reticulum (ER) is a vital organelle in eukaryotic cells and has a pivotal role in calcium storage and protein-folding. In the ER, nascent proteins rely on highly-regulated formation of disulfide bonds to form stable and functional proteins. A process known as oxidative protein folding (OPF) mediates the appropriate formation of disulfide bonds required for the construction of secondary and tertiary protein structures. Glutathione (reduced GSH to oxidized GSSG ratio) acts as the redox buffer in the ER lumen. The oxidizing environment of the ER lumen is essential for efficient formation of disulfide bonds.109 OPF is driven by a flavin adenine dinucleotide (FAD) dependent enzyme, ER oxidoreductin 1 (Ero-1), which oxidizes protein disulfide isomerase (PDI). Subsequently, oxidized PDI oxidizes proteins by enabling the formation of disulfide bonds. Molecular oxygen is the final acceptor of electrons from Ero-1 (Figure 1:5).
19 Figure 1.5. Oxidative Protein Folding (OPF)
A schematic representation of oxidative protein folding in the ER lumen
Disturbance in the process leads to the production of reactive oxygen species (ROS), and consequently, a hyper-oxidizing ER lumen, which can further perturb the protein folding machinery. Glutathione maintains the redox balance of ER lumen. A normal functioning ER lumen is significantly more oxidizing (GSH/GSSG ratio ranges from 1:1 to 3:1) than the overall cell (GSH/GSSG ratio ranges from 30:1 to 100:1).109 As a result, OPF efficiency can be significantly disrupted by a cellular glutathione imbalance. A multitude of pathological and environmental insults including nutrient deprivation and redox imbalance can disturb the GSH balance of the cell, thereby affecting the protein-folding environment of ER lumen resulting in accumulation of misfolded proteins leading to ER stress. ER stress triggers activation of an Unfolded Protein Response (UPR) to repair the damage caused by the insults and restore cellular homeostasis or initiate programmed cell death (i.e., apoptosis) (Figure1:6).
20 Figure 1.6. Unfolded Protein Response (UPR)
Schematic representation of Unfolded Protein Response (UPR) signaling cascade.
Three ER-membrane embedded protein sensors mediate the UPR: double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK); activating transcription factor 6 (ATF6); and inositol-requiring enzyme-1 (IRE-1). In the absence of stress, ER-resident protein BiP (aka GRP78) binds to the luminal domains of UPR sensors, maintaining them in their inactive monomeric state.
Under ER stress, misfolded proteins sequester BiP away from the sensors due to
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preferential non-specific binding affinity. In response, protein sensors (PERK, ATF6, and IRE-1) oligomerize, and their cytosolic kinase domains are activated by trans-phosphorylation. Activated sensors activate several downstream signaling cascades that guide the cell towards restoration of homeostasis or (potentially) apoptosis. Collectively, these responses are called the UPR.110 In Chapter 2, data show that MAPKi in melanoma cells induce a GSH imbalance, which in turn triggers an ER stress event that coincides with increases in autophagic flux that drives resistance to MAPKi.
IRE1a Signaling Pathway
IRE1a (inositol requiring enzyme-1a) is a trans-ER-membrane sensor with a cytosolic bi-functional serine/threonine kinase and RNase enzymatic domain. It is
IRE1a (inositol requiring enzyme-1a) is a trans-ER-membrane sensor with a cytosolic bi-functional serine/threonine kinase and RNase enzymatic domain. It is