Advances in Targeted Therapy Against Driver Mutations and Epigenetic Alterations in Non-Small Cell Lung Cancer

The incidence and mortality of lung cancer rank top three of all cancers worldwide. Accounting for 85% of the total number of lung cancer, non-small cell lung cancer (NSCLC) is an important factor endangering human health. Recently, targeted therapies against driver mutations and epigenetic alterations have made encouraging advances that benefit NSCLC patients. Druggable driver mutations, which mainly occur in EGFR, KRAS, MET, HER2, ALK, ROS1, RET and BRAF, have been identified in more than a quarter of NSCLC patients. A series of highly selective mutant targeting inhibitors, such as EGFR tyrosine kinase inhibitors and KRAS inhibitors, have been well studied and applied in clinical treatments, which greatly promote the overall survival of NSCLC patients. However, drug resistance has become a major challenge for targeted treatment, and a variety of methods to overcome drug resistance are constantly being developed, including inhibitors against new mutants, combination therapy with other pathway inhibitors, etc. In addition, epigenetics-based therapy is emerging. Epigenetic regulators such as histone deacetylases and non-coding RNA play a crucial role in the development of cancer and drug resistance by affecting multiple signaling pathways. Epigenetics-based therapeutic strategies combined with targeted drugs show great clinical potential. Many agents targeting epigenetic changes are being investigated in preclinical studies, with some already under clinical trials. This article focuses on driver mutations and epigenetic alterations in association with relevant epidemiological data. We introduce the current status of targeted inhibitors and known drug resistance, review advances in major targeted therapies with recent data from preclinical and clinical trials, and discuss the possibility of combination therapy against driver mutations and epigenetic alterations in overcoming drug resistance.     

Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation

Dysregulated activation of Ras or its downstream effectors such as mitogen-activated protein kinase kinase and ERK has been shown to play a critical role in tumorigenesis of many cancer types. However, in melanoma, activating mutations in Ras are rarely observed and are limited to N-Ras in UV-exposed cells. In this study, we identify constitutively activated ERK in almost all melanoma cell lines and in tumor tissues tested, which is in contrast to normal melanocytes and several early stage radial growth phase melanoma lines where ERK can be activated by serum or growth factors. Constitutive activation of ERK is preceded by phosphorylation of mitogen-activated protein kinase kinase and c-RAF. In all of the melanoma cell lines tested, Ras is constitutively activated without underlying mutations. On the contrary, activating mutations in the kinase domain of BRAF are present in the majority of the cell lines tested. Furthermore, ERK activation can be partially inhibited from the cell surface using inhibitors of fibroblast growth factor and hepatocyte growth factor but not interleukin 8 signaling pathways. These data suggest that melanoma growth, invasion, and metastasis are attributable to constitutively activated ERK apparently mediated by excessive growth factors through autocrine mechanisms and BRAF kinase activation.     

Targeting NRAS in melanoma

Cutaneous melanomas have mutations in the NRAS GTPase in 15% of cases. Compared to melanomas with BRAF mutations, or melanomas "wild-type" for BRAF and NRAS, melanomas with NRAS mutations are more likely to be thicker tumors and to have a higher mitotic rate. Preclinical studies indicate that melanoma cells with NRAS mutations are dependent on NRAS for survival and proliferation, making NRAS an attractive therapeutic target in melanoma. However, to date, therapeutic strategies for NRAS mutant melanomas have not been realized. Promising strategies to target NRAS include targeting the membrane localization of NRAS or reducing expression through the use of therapeutic small interfering RNAs. Finally, use of inhibitors to target downstream signaling through mitogen-activated protein kinase kinase and phosphatidylinositol 3-OH kinase or AKT are now entering clinical trials, and if these combinations can be safely delivered at sufficient dose to inhibit the targets, there is significant potential to target NRAS mutant melanoma.     

Protein kinase D3 sensitizes RAF inhibitor RAF265 in melanoma cells by preventing reactivation of MAPK signaling

RAS mutations occur in more than 30% of all human cancers but efforts to directly target mutant RAS signaling as a cancer therapy have yet to succeed. As alternative strategies, RAF and MEK inhibitors have been developed to block oncogenic signaling downstream of RAS. As might be expected, studies of these inhibitors have indicated that tumors with RAS or BRAF mutations display resistance RAF or MEK inhibitors. In order to better understand the mechanistic basis for this resistance, we conducted a RNAi-based screen to identify genes that mediated chemoresistance to the RAF kinase inhibitor RAF265 in a BRAF (V600E) mutant melanoma cell line that is resistant to this drug. In this way, we found that knockdown of protein kinase D3 (PRKD3) could enhance cell killing of RAF and MEK inhibitors across multiple melanoma cell lines of various genotypes and sensitivities to RAF265. PRKD3 blockade cooperated with RAF265 to prevent reactivation of the MAPK signaling pathway, interrupt cell cycle progression, trigger apoptosis, and inhibit colony formation growth. Our findings offer initial proof-of-concept that PRKD3 is a valid target to overcome drug resistance being encountered widely in the clinic with RAF or MEK inhibitors.     

BRAFL597 Mutations in Melanoma Are Associated with Sensitivity to MEK Inhibitors

Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40% to 50% of cases are positive. To uncover other potential targetable mutations, we conducted whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, and D816) wild-type melanoma. Surprisingly, we found a somatic BRAF(L597R) mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF(V600) mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that two (4%) harbored L597 mutations and another two involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition. A patient with BRAF(L597S) mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data show clinical significance to BRAF(L597) mutations in melanoma.     

CRKL as a lung cancer oncogene and mediator of acquired resistance to EGFR inhibitors: is it all that it is cracked up to be?

Cheung and colleagues demonstrate that amplified CRKL can function as a driver oncogene in lung adenocarcinoma, activating both RAS and RAP1 to induce mitogen-activated protein kinase signaling. In addition, they show that CRKL amplification may be another mechanism for primary or acquired resistance to epidermal growth factor receptor kinase inhibitors.     

Identification of the MEK1(F129L) activating mutation as a potential mechanism of acquired resistance to MEK inhibition in human cancers carrying the B-RafV600E mutation

Although targeting the Ras/Raf/MEK pathway remains a promising anticancer strategy, mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors in clinical development are likely to be limited in their ability to produce durable clinical responses due to the emergence of acquired drug resistance. To identify potential mechanisms of such resistance, we established MEK inhibitor-resistant clones of human HT-29 colon cancer cells (HT-29R cells) that harbor the B-RafV600E mutation. HT-29R cells were specifically resistant to MEK inhibition in vitro and in vivo, with drug-induced elevation of MEK/ERK and their downstream targets primarily accountable for drug resistance. We identified MEK1(F129L) mutation as a molecular mechanism responsible for MEK/ERK pathway activation. In an isogenic cell system that extended these findings into other cancer cell lines, the MEK1(F129L) mutant exhibited higher intrinsic kinase activity than wild-type MEK1 [MEK1(WT)], leading to potent activation of ERK and downstream targets. The MEK1(F129L) mutation also strengthened binding to c-Raf, suggesting an underlying mechanism of higher intrinsic kinase activity. Notably, the combined use of Raf and MEK inhibitors overcame the observed drug resistance and exhibited greater synergy in HT-29R cells than the drug-sensitive HT-29 parental cells. Overall, our findings suggested that mutations in MEK1 can lead to acquired resistance in patients treated with MEK inhibitors and that a combined inhibition of Raf and MEK may be potentially useful as a strategy to bypass or prevent drug resistance in the clinic.     

Resistance to combination BRAF and MEK inhibition in metastatic melanoma: Where to next?

Treatment of BRAF-mutant metastatic melanoma with mitogen-activated protein kinase (MAPK) pathway targeted therapies (BRAF/MEK inhibitors) and immune checkpoint inhibitors has revolutionised management and improved outcomes for patients with advanced stage disease. However, acquired resistance to MAPK inhibitor therapy develops in the majority of patients at approximately 12 months and multiple mechanisms lead to resistance. Understanding the mechanisms of resistance is therefore critical for the development of more effective therapeutic strategies in BRAF-mutant melanoma. Recently, several distinct mechanisms of resistance to BRAF-inhibition have been proposed based on data obtained in experimental melanoma cell models and small series of human tumour samples. These include reactivation of the MAPK pathway resulting in continued extracellular signal-regulated kinase activation and activation of parallel signalling pathways including the PI3K-mTOR (phosphoinositide 3-kinase–mammalian target of rapamycin) pathway. Alterations in how the cells of the immune system respond to melanoma cells treated with targeted therapy may also influence response and progression. In this review, we discuss these mechanisms and identify potential therapeutic strategies to overcome resistance which, in turn, will lead to improved outcomes for patients with metastatic melanoma.     

Resistance to BRAF inhibitors: unraveling mechanisms and future treatment options

The mitogen-activated protein kinase (MAPK) pathway has emerged as a central target for melanoma therapy due to its persistent activation in the majority of tumors. Several BRAF inhibitors aimed at curbing MAPK pathway activity are currently in advanced stages of clinical investigation. However, their therapeutic success is limited by the emergence of drug resistance, as responses are transient and tumors eventually recur. To develop effective and long-lasting therapies for melanoma patients, it is essential to understand the mechanisms underlying resistance to BRAF inhibitors. Here, we briefly review recent preclinical studies that have provided insight into the molecular mechanisms of resistance to BRAF inhibitors and discuss potential strategies to treat drug-resistant melanomas.     

Mutant V599EB-Raf regulates growth and vascular development of malignant melanoma tumors

Activating mutations of the B-RAF gene are observed in >60% of human melanomas. Approximately 90% of these mutations occur in the activation segment of the kinase domain as a single-base substitution that converts a valine to glutamic acid at codon 599 (V599E) in exon 15. This mutation causes activation of the kinase as well as downstream effectors of the mitogen-activated protein kinase-signaling cascade, leading to melanoma tumor development by an as yet unknown mechanism. In this study, we have identified the role of (V599E)B-Raf in melanoma tumor development by characterizing the mechanism by which this mutant protein promotes melanoma tumorigenesis. Small interfering RNA targeted against B-Raf or a Raf kinase inhibitor (BAY 43-9006) was used to reduce expression and/or activity of (V599E)B-Raf in melanoma tumors. This inhibition led to reduced activity of the mitogen-activated protein kinase-signaling cascade and inhibited tumor development in animals. Targeted reduction of mutant (V599E)B-Raf expression (activity) in melanoma cells before tumor formation inhibited tumorigenesis by reducing the growth potential of melanoma cells. In contrast, reduction of mutant (V599E)B-Raf activity in preexisting tumors prevented further vascular development mediated through decreased vascular endothelial growth factor secretion, subsequently increasing apoptosis in tumors. These effects in combination with reduced proliferative capacity halted growth, but did not shrink the size of preexisting melanoma tumors. Thus, these studies identify the mechanistic underpinnings by which mutant (V599E)B-RAF promotes melanoma development and show the effectiveness of targeting this protein to inhibit melanoma tumor growth.     

Discovery of drug-resistant and drug-sensitizing mutations in the oncogenic PI3K isoform p110 alpha

p110 alpha (PIK3CA) is the most frequently mutated kinase in human cancer, and numerous drugs targeting this kinase are currently in preclinical development or early-stage clinical trials. Clinical resistance to protein kinase inhibitors frequently results from point mutations that block drug binding; similar mutations in p110 alpha are likely, but currently none have been reported. Using a S. cerevisiae screen against a structurally diverse panel of PI3K inhibitors, we have identified a potential hotspot for resistance mutations (I800), a drug-sensitizing mutation (L814C), and a surprising lack of resistance mutations at the "gatekeeper" residue. Our analysis further reveals that clinical resistance to these drugs may be attenuated by using multitargeted inhibitors that simultaneously inhibit additional PI3K pathway members.     

Modeling genomic diversity and tumor dependency in malignant melanoma

The classification of human tumors based on molecular criteria offers tremendous clinical potential; however, discerning critical and "druggable" effectors on a large scale will also require robust experimental models reflective of tumor genomic diversity. Here, we describe a comprehensive genomic analysis of 101 melanoma short-term cultures and cell lines. Using an analytic approach designed to enrich for putative "driver" events, we show that cultured melanoma cells encompass the spectrum of significant genomic alterations present in primary tumors. When annotated according to these lesions, melanomas cluster into subgroups suggestive of distinct oncogenic mechanisms. Integrating gene expression data suggests novel candidate effector genes linked to recurrent copy gains and losses, including both phosphatase and tensin homologue (PTEN)-dependent and PTEN-independent tumor suppressor mechanisms associated with chromosome 10 deletions. Finally, sample-matched pharmacologic data show that FGFR1 mutations and extracellular signal-regulated kinase (ERK) activation may modulate sensitivity to mitogen-activated protein kinase/ERK kinase inhibitors. Genetically defined cell culture collections therefore offer a rich framework for systematic functional studies in melanoma and other tumors.     

Oncogenic B-Raf abrogates the AKT/B-Raf/Mps1 interaction in melanoma cells

Activating B-Raf mutations that deregulate the mitogen-activated protein kinase (MAPK) pathway commonly occur in cancer. Although B-Raf^V600E induces increased Mps1 protein contributing to centrosome amplification and chromosome instability, the regulatory mechanisms of Mps1 in melanoma cells is not fully understood. Here, we report that Mps1/AKT and B-Raf^WT/ERK signaling form an auto-regulatory negative feedback loop in melanoma cells; notably, oncogenic B-Raf^V600E abrogates the negative feedback loop, contributing the aberrant Mps1 functions and tumorigenesis. Our findings raise the possibility that targeting the oncogenic B-Raf and Mps1, especially when used in combination could potentially provide great therapeutic opportunities for cancer treatment.     

Mutations and Deregulation of Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascades Which Alter Therapy Response

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). Certain components of these pathways, RAS, NF1, BRAF, MEK1, DUSP5, PP2A, PIK3CA, PIK3R1, PIK3R4, PIK3R5, IRS4, AKT, NFKB1, MTOR, PTEN, TSC1, and TSC2 may also be activated/inactivated by mutations or epigenetic silencing. Upstream mutations in one signaling pathway or even in downstream components of the same pathway can alter the sensitivity of the cells to certain small molecule inhibitors. These pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of components of these cascades can contribute to: resistance to other pathway inhibitors, chemotherapeutic drug resistance, premature aging as well as other diseases. This review will first describe these pathways and discuss how genetic mutations and epigenetic alterations can result in resistance to various inhibitors.     

BRAF inhibitors for the treatment of metastatic melanoma: clinical trials and mechanisms of resistance

The efficacy of selective BRAF inhibitors has now been established in the 50% of patients with metastatic melanoma whose tumors harbor activating mutations. However, for the vast majority of patients, responses persist for less than a year. In extensive preclinical investigations, researchers have focused on potential resistance mechanisms with the hope of identifying treatment strategies that can overcome resistance. Preliminary results suggest that reactivation of the mitogen-activated protein kinase (MAPK) pathway by several BRAF-independent mechanisms is the predominant pattern. However, MAPK pathway-independent mechanisms also seem to play a potential role. More definitive cataloging of resistance mechanisms in patients' tumor samples is needed as combination regimens are being readied for clinical evaluation.     

Other signalization targets

Treatment decisions for patients with lung cancer have historically been based upon tumor morphological analysis. Over the past decade, some molecular alterations have been identified as being necessary and sufficient to drive tumor carcinogenesis. These “driver” mutations occur in genes that encode signaling proteins critical for cellular proliferation and survival. Epidermal growth factor (EGF) receptor (EGFR) mutations are the best illustration of the therapeutic relevance of identifying such molecular clusters of lung cancer based on driver genetic alterations that predict the efficacy of specific tyrosine kinase inhibitors, a strategy referred to as “personalized medicine.” Besides EGFR and ALK, other genes harboring driver molecular alterations have been identified as part of integrated genomic studies of lung cancers. The objectives of this review are (1) to provide the reader with preclinical and clinical data on these new oncogenic mutations, focusing on druggable ones; (2) to discuss the dynamic nature of lung cancer molecular features in the context of acquired resistance to specific inhibitors; and (3) to highlight emerging data on other cancer hallmarks that may be of interest from a therapeutic perspective in the next future. From bench to bedside, personalized medicine represents a major revolution in the treatment of lung cancer.     

Topical treatment with inhibitors of the phosphatidylinositol 3'-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways reduces melanoma development in severe combined immunodeficient mice

Topical treatment with inhibitors of the phosphatidylinositol 3'-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways inhibited the growth of TPras transgenic melanomas in severe combined immunodeficient mice, blocked invasive behavior, and reduced angiogenesis. The inhibitor Ly294002, which is specific for phosphatidylinositol 3'-kinase, effectively reduced melanoma cell growth both in vitro and in vivo. Both Ly294002 and U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, reduced invasion, which correlated with reduction of the metalloproteinase matrix metalloproteinase 2. Tumor angiogenesis was disrupted through inhibition of vascular endothelial growth factor production from the tumor cells and antiangiogenic effects on endothelial cells. Observations with TPras melanoma cells that express dominant negative Deltap85 or kinase-inactive Raf(301) supported the specificity of the phenomena observed with the chemical inhibitors. These studies demonstrate that topical treatment targeting Ras effectors is efficacious, without systemic toxicities, and may prove to be useful in treating and preventing the progression of cutaneous melanoma.