Novel and Potent Small Molecules against Melanoma Harboring BRAF Class I/II/III Mutants for Overcoming Drug Resistance

Melanoma accounts for the majority of skin cancer deaths. About 50% of all melanomas are associated with BRAF mutations. BRAF mutations are classified into three classes with regard to dependency on RAF dimerization and RAS signaling. The most frequently occurring class I BRAF V600 mutations are sensitive to vemurafenib whereas class II and class III mutants, non-V600 BRAF mutants are resistant to vemurafenib. Herein we report six pyrimido[4,5-d]pyrimidin-2-one derivatives possessing highly potent anti-proliferative activities on melanoma cells harboring BRAF class I/II/III mutants. Novel and most potent derivative, SIJ1777, possesses not only two-digit nanomolar potency but also 2 to 14-fold enhanced anti-proliferative activities compared with reference compound, GNF-7 against melanoma cells (SK-MEL-2, SK-MEL-28, A375, WM3670, WM3629). Moreover, SIJ1777 substantially inhibits the activation of MEK, ERK, and AKT and remarkably induces apoptosis and significantly blocks migration, invasion, and anchorage-independent growth of melanoma cells harboring BRAF class I/II/II mutations while both vemurafenib and PLX8394 have little to no effects on melanoma cells expressing BRAF class II/III mutations. Taken together, our six GNF-7 derivatives exhibit highly potent activities against melanoma cells harboring class I/II/III BRAF mutations compared with vemurafenib as well as PLX8394.ope

Targeting Oncogenic BRAF: Past, Present, and Future.

Identifying recurrent somatic genetic alterations of, and dependency on, the kinase BRAF has enabled a "precision medicine" paradigm to diagnose and treat BRAF-driven tumors. Although targeted kinase inhibitors against BRAF are effective in a subset of mutant BRAF tumors, resistance to the therapy inevitably emerges. In this review, we discuss BRAF biology, both in wild-type and mutant settings. We discuss the predominant BRAF mutations and we outline therapeutic strategies to block mutant BRAF and cancer growth. We highlight common mechanistic themes that underpin different classes of resistance mechanisms against BRAF-targeted therapies and discuss tumor heterogeneity and co-occurring molecular alterations as a potential source of therapy resistance. We outline promising therapy approaches to overcome these barriers to the long-term control of BRAF-driven tumors and emphasize how an extensive understanding of these themes can offer more pre-emptive, improved therapeutic strategies

Making NSCLC Crystal Clear:How Kinase Structures Revolutionized Lung Cancer Treatment

The parallel advances of different scientific fields provide a contemporary scenario where collaboration is not a differential, but actually a requirement. In this context, crystallography has had a major contribution on the medical sciences, providing a “face” for targets of diseases that previously were known solely by name or sequence. Worldwide, cancer still leads the number of annual deaths, with 9.6 million associated deaths, with a major contribution from lung cancer and its 1.7 million deaths. Since the relationship between cancer and kinases was unraveled, these proteins have been extensively explored and became associated with drugs that later attained blockbuster status. Crystallographic structures of kinases related to lung cancer and their developed and marketed drugs provided insight on their conformation in the absence or presence of small molecules. Notwithstanding, these structures were also of service once the initially highly successful drugs started to lose their effectiveness in the emergence of mutations. This review focuses on a subclassification of lung cancer, non-small cell lung cancer (NSCLC), and major oncogenic driver mutations in kinases, and how crystallographic structures can be used, not only to provide awareness of the function and inhibition of these mutations, but also how these structures can be used in further computational studies aiming at addressing these novel mutations in the field of personalized medicine

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Integral components of these pathways, Ras, B-Raf, PI3K, and PTEN are also activated/inactivated by mutations. These pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of these pathways can contribute to chemotherapeutic drug resistance, proliferation of cancer initiating cells (CICs) and premature aging. This review will evaluate more recently described potential uses of MEK, PI3K, Akt and mTOR inhibitors in the proliferation of malignant cells, suppression of CICs, cellular senescence and prevention of aging. Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways play key roles in the regulation of normal and malignant cell growth. Inhibitors targeting these pathways have many potential uses from suppression of cancer, proliferative diseases as well as aging

Going beyond EGFR

a substantial proportion of non-small-cell lung cancer (NSCLC), and adenocarcinoma in particular, depends on a so-called ‘driver mutation' for their malignant phenotype. This genetic alteration induces and sustains tumorigenesis, and targeting of its protein product can result in growth inhibition, tumor response and increased patient survival. NSCLC can thus be subdivided into clinically relevant molecular subsets. Mutations in EGFR best illustrate the therapeutic relevance of molecular classification. This article reviews the scope of presently known driving molecular alterations, including ROS1, BRaF, KRaS, HER2 and PIK3Ca, with a special emphasis on aLK rearrangements, and outlines their potential therapeutic application

Targeting the Hsp90 Molecular Chaperone and Resistant Pathways in BRAF-mutant Melanoma

Melanoma remains the most aggressive and fatal type of skin cancer. In greater than 50% of cases, patients present with an activating BRAF mutation (BRAF+), leading to upregulated mitogen-activated protein kinase (MAPK) pathway signaling. Of these patients, 80-90% have a missense mutation at codon 600 (e.g., BRAFV600E), making the mutant form of the protein an attractive and druggable target. In 2011, the FDA approved combination therapy of BRAF+ and MEK inhibitors (BRAFi/MEKi), like vemurafenib and cobimetinib (Ve/Cb), for use in unresectable late-stage melanoma patients, drastically changing treatment options and initial outcomes. Still, the majority of patients become refractory to BRAFi/MEKi within the first year of treatment. The lack of treatment durability underscores the need for novel therapeutic strategies and drug candidate development, such as the utilization of molecular chaperone inhibitors. The 90-kDa heat shock protein (Hsp90) is a molecular chaperone and responsible for stabilizing the protein folding of “client” proteins that interact with the heterochaperone complex that Hsp90 forms with the 70-kDa heat shock protein (Hsp70) and other co-chaperones. These clients are involved in several cellular signaling pathways and processes, highlighting the significance of chaperone function in eukaryotic cells. Interestingly, Hsp90 expression increases several-fold in cancer cells to compensate for cellular stress and client protein dependence on chaperone function. To-date 18 small molecule Hsp90 inhibitors (Hsp90i) entered clinical trials, of which 94.4% target the N-terminus (NT-Hsp90i) but failed to get FDA approval. The NT-Hsp90i are effective and xvi potent, but pan-inhibitors of all four Hsp90 isoforms. In clinical trials, patients require dose-escalation of NT-Hsp90i to reach a therapeutic effect, ultimately leading to dose-limiting toxicities (DTL). Studies suggest a link between DTLs and the activation of the heat shock response (HSR), especially the cytoprotective role of Hsp70. Previously, our lab, with collaborators, developed novel C-terminal Hsp90i (CT-Hsp90i) and showed efficacy in several cancer models in vitro and in vivo while mitigating the HSR, suggesting a decreased toxicity profile compared to NT-Hsp90i. For this dissertation, I researched therapeutic resistance mechanisms in BRAF+ melanoma through various preclinical in vitro studies that targeted Hsp90. Specifically, I tested the hypothesis that several resistance-promoting processes require Hsp90 function and, therefore, could be targeted with an Hsp90i to simultaneously knockdown resistance pathways and oncogenic processes. First, I showed effective melanoma cell death using the CT-Hsp90i KU758 at potent micromolar concentrations (e.g., IC50 = 0.36 – 0.43 micromolar). Next, I demonstrated robust synergy (e.g., CI<0.5) of KU758 when combined with either a BRAFi or MEKi to target two resistance pathways effectively (e.g., MAPK/Erk and PI3K/Akt), significantly mitigate melanocyte migration, and downregulate key Hsps involved in HSR activation. Finally, I accessed publicly available genomic data via the National Cancer Institute and The Cancer Genome Atlas (TCGA) program to identify additional genes of interest in BRAF+ melanomas. Using a clustered heatmap of RNA expression data, I distinguished genes of interest based on common expression alterations amongst a subset of BRAF+ melanoma patients to provide a genetic perspective in the context of Hsp90i use in melanoma patients. Collectively, this work reviews the use of and development of several small xvii molecule inhibitors in melanomas (e.g., BRAFi/MEKi and Hsp90i), identifies a novel and effective KU758-combination approach in BRAF+ melanomas, and gives insight into future therapeutic directions based on various translational and genetic signatures in these difficult-to-treat tumors.PHDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163262/1/jaquesan_1.pd

Markers and mechanisms of resistance to Toceranib phosphate (Palladia®) in canine cutaneous mast cell tumor

2014 Summer.Includes bibliographical references.To view the abstract, please see the full text of the document

Clinical implications of acquired braf inhibitors resistance in melanoma

Understanding the role of mitogen-activated protein kinase (MAPK) pathway-activating mutations in the development and progression of melanoma and their possible use as therapeutic targets has substantially changed the management of this neoplasm, which, until a few years ago, was burdened by severe mortality. However, the presence of numerous intrinsic and extrinsic mechanisms of resistance to BRAF inhibitors compromises the treatment responses\u2019 effectiveness and durability. The strategy of overcoming these resistances by combination therapy has proved successful, with the additional benefit of reducing side effects derived from paradoxical activation of the MAPK pathway. Furthermore, the use of other highly specific inhibitors, intermittent dosing schedules and the association of combination therapy with immune checkpoint inhibitors are promising new therapeutic strategies. However, numerous issues related to dose, tolerability and administration sequence still need to be clarified, as is to be expected from currently ongoing trials. In this review, we describe the clinical results of using BRAF inhibitors in advanced melanoma, with a keen interest in strategies aimed at overcoming resistance

Pharmacological targeting of nonsense mutant TP53 and PTEN in cancer

The TP53 tumor suppressor gene encodes p53 and is inactivated by mutations in around half of all human tumors. Approximately 11% of TP53 mutations are nonsense mutations, resulting in the premature termination of translation and the production of truncated and non-functional p53 proteins. Aminoglycosides such as G418 are known to induce translational readthrough, a process in which the ribosome overcomes the stop signal introduced by a nonsense mutation and translates full-length protein. However, the clinical use of aminoglycosides is restricted due to severe side effects. We have demonstrated that combination treatments with proteasome inhibitors or compounds that disrupt the binding of p53 to the ubiquitin ligase MDM2 can synergistically enhance the levels of fulllength p53, improving the efficacy of readthrough compared to aminoglycosides alone. These combinations were proven to produce at least partially active fulllength p53, as shown by the suppression of cell growth and the induction of cell death. In parallel, chemical library screenings led to the discovery of two novel compounds, C47 and C61, showing readthrough activity and synergizing with G418 and eRF3 degraders CC-885 and CC-90009, respectively. Remarkably, C47 also exhibit readthrough activity for nonsense mutant phosphatase and tensin homolog (PTEN), expanding the scope for targeted cancer therapies. Furthermore, we have identified the 5-fluorouracil (5-FU) metabolite 5-Fluorouridine (FUr) as a potent readthrough-inducing compounds capable restoring full-length p53 expression in cells harboring nonsense mutant TP53. In vivo studies further substantiated the capability of FUr to reinstate full-length p53 expression in human tumor xenografts with TP53 R213X nonsense mutations. Finally, the first Trp53 R210X nonsense mutant knock-in mouse model has been generated. R210X corresponds to human TP53 R213X. Observations on tumor development, lifespan and other phenotypic traits in these mice provide valuable insights into the impact of TP53 nonsense mutation in a multi-organ system. These results also provide a platform for the preclinical evaluation of novel therapeutic strategies for targeting nonsense mutant TP53. In summary, these findings offer a multi-faceted approach towards understanding TP53 nonsense mutations and advancing targeted cancer therapy through pharmacological induction of translational readthrough. The discovery of novel readthrough inducing compounds, the application of combination therapy in translational readthrough, the discovery of a novel therapeutic application for 5- FU and its metabolite FUr, as well as the generation of a novel animal model collectively set the stage for the further development of personalized treatments for patients with tumors harboring nonsense mutant TP53

Mechanisms of cancer cell death by mutant p53-reactivating compound APR-246

Tumor suppressor TP53 is the most frequently mutated gene in cancer. A majority of TP53 mutations result in a mutant p53 that disrupts its DNA binding capabilities but may also acquire novel gain-of-function activities that contribute to tumor growth. The investigational drug APR-246 (Eprenetapopt) is the most clinically advanced compound to target mutant p53 and is being tested in a phase III clinical trial in mutant TP53 myelodysplastic syndrome (MDS). APR-246 is converted to its active product methylene quinuclidinone (MQ). MQ binds to cysteines in p53 promoting a folded structure and DNA binding, leading to cancer cell death. MQ also targets thiols or selenols in e.g. glutathione (GSH) or various enzymes. Depletion of glutathione and inhibition of antioxidant enzymes increase oxidative stress contributing to APR-246-induced cancer cell death. In Project I, combination treatment of APR-246 and multidrug resistance protein 1 (MRP1) inhibitor resulted in synergistic growth suppression in vitro in tumor cell lines, in vivo in esophageal cancer xenografts, and ex vivo in esophageal and colorectal cancer patient-derived organoids (PDO). We show that inhibition of MRP1 results in increased intracellular 14C- content after 14C-APR-246 treatment. This was attributed to retention of GSH-conjugated MQ (GS-MQ). We demonstrate that GS-MQ binding is reversible and that retention of GS-MQ creates an intracellular MQ pool that may target numerous thiols contributing to APR-246- induced growth suppression. In Project II we studied the spectrum of MQ-targeted cysteines in p53. This was enabled by first establishing a method utilizing the reducing agent NaBH4 to lock the MQ cysteine adducts into a stable form, overcoming reversibility. Cys182, Cys229 and Cys277 in the p53 core domain showed most prominent MQ modification. Additional modification at Cys124 and Cys141 was found in mutant p53. The electrophilic properties of MQ enables targeting of multiple cellular thiols. In Project III we identified novel MQ targets using CEllular Thermal Shift Assay (CETSA). Asparaginase synthetase (ASNS) was stabilized upon MQ treatment and thus is a potential MQ target. In acute lymphoblastic leukemia (ALL), ASNS is associated with resistance to standard treatment asparaginase. Asparaginase depletes extracellular asparagine which renders asparagine-auxotrophic ALL cells sensitive and therefore ASNS expression allows ALL cell survival. We found that combination treatment of APR-246 and asparaginase leads to synergistic growth suppression in ALL cells and may offer a novel treatment strategy for ALL. Lastly, in Project IV we assessed the functional activity of novel germline TP53 mutations identified in a Swedish cohort of families with Li-Fraumeni syndrome (LFS) or hereditary breast cancer (HrBC). Assessing the pathological outcome of TP53 mutations is important for understanding the cancer risk of these families. The first three projects are aimed at improving our understanding of mutant p53-reactivating compound APR-246. They suggest approaches for increasing treatment efficacy and novel combination strategies. The thesis has also addressed the role of mutant p53 in response to APR-246 and pathological properties in families with LFS or HrBC. All in all, these studies provide novel preclinical understanding of the role of mutant p53 in cancer and response to treatment, both highly relevant in the combat against cancer