Endometrial cancer (EC) is the seventh most commonly diagnosed cancer among women, with 189,000 new cases and 45,000 deaths occurring worldwide each year. In the United States, it is the fourth most commonly diagnosed cancer among women. According to the national Cancer Institute (NCI, USA), in 2013 approximately 50,000 women will be diagnosed with endometrial cancer, with more than an estimated 8,000 deaths from the disease.describe the image

Endometrial cancers are classified into two types: endometrioid (type I) and serous (type II). Type I EC is a less severe form. Risk factors include obesity, anovulation, nulliparity, and exogenous estrogen exposure. This type of EC commonly express both estrogen and progesterone receptors. Clinically, type I EC is more often a low-grade tumor with a favorable prognosis.

On the contrary, type II EC is a more life-threatening form and not associated with estrogen exposure. Clinically, this type of EC is marked by an aggressive clinical course, and has a tendency for early spread and poor prognosis. Endometroid (type I) tumors are treated with adjuvant radiotherapy, whereas serous (type II) tumors are treated with chemotherapy. Even though EC is one of the most common pelvic gynecologic malignancies in the world, to date no targeted therapies are available to treat patients.

Therefore, in order to formulate an efficient treatment plan, detailed genomic characterization of primary and metastatic endometrial cancers are required. Several studies have reported numerous genetic changes associated with endometrial cancer. Type I endometrial carcinomas involve mutations in PTEN, KRAS, FGFR2, PIK3CA and β-catenin, as well as defects in DNA mismatch repair. Type II endometrial carcinomas frequently show aneuploidy and TP53, PIK3CA, and PPP2R1A gene mutations. Using whole exome DNA sequencing on 13 primary serous EC patients, a study by Bell and colleagues (2012) identified high frequency somatic mutations in CHD4, FBXW7, and SPOP genes (associated with chromatin-remodeling and ubiquitin ligase complex). These mutations may play a significant role as driver mutations (gene mutations implicated in cancer initiation and progression) in serous EC.

To better understand the molecular alterations associated with endometrial cancer, a recent study was performed by The Cancer Genome Atlas Research Network (TCGA) using integrated genomic and proteomic analysis appearing in a recent issue of Nature journal (May 2nd, 2013).

Using a multiplatform analysis approach on 373 endometrial carcinomas including low-grade endometroid, high-grade endometroid, and serous carcinomas this study provided key molecular insight into the classification of endometrial cancer. This new study classified endometrial cancer into four new categories:

* The POLE group contained ultrahigh mutation rates in the POLE gene (involved in cellular metabolism) and frequent activation of the WNT/CTNNB1 signaling pathway

* The hypermutated microsatellite instability group showed a high mutation rate, as well as few copy number alterations, and reduced expression of DNA mismatch repair gene MLH1

* The copy-number low group showed increased expression of progesterone receptor and DNA repair protein RAD50

* The copy-number high group composed of mostly serous tumors and serous-like endometroid tumors and exhibited increased transcriptional activity of cell cycle related genes (MYC, CCNE1, PIK3CA, CDKN2A etc.) and a mutation in tumor suppressor gene TP53.

In addition, this study also observed compelling similarities in the molecular phenotype between 25% of high-grade endometroid tumors and uterine serous carcinoma, suggesting that this genome-based molecular characterization may benefit these patients. Overall, this new molecular characterization might facilitate the discovery of effective, targeted treatments as well as may affect post-surgical adjuvant treatment for women with endometrial cancer.


Bansal N, Yendluri V, Wenham RM (2009) The molecular biology of endometrial cancers and the implications for pathogenesis, classification, and targeted therapies. Cancer Control 16: 8-13.

Hecht JL, Mutter GL (2006) Molecular and pathologic aspects of endometrial carcinogenesis. J Clin Oncol 24: 4783-4791.

Kandoth C, Schultz N, Cherniack AD, Akbani R, Liu Y, Shen H, Robertson AG, Pashtan I, Shen R, Benz CC, Yau C, Laird PW, Ding L, Zhang W, Mills GB, Kucherlapati R, Mardis ER, Levine DA, Network CGAR (2013) Integrated genomic characterization of endometrial carcinoma. Nature 497: 67-73.

Kuhn E, Wu RC, Guan B, Wu G, Zhang J, Wang Y, Song L, Yuan X, Wei L, Roden RB, Kuo KT, Nakayama K, Clarke B, Shaw P, Olvera N, Kurman RJ, Levine DA, Wang TL, Shih IM (2012) Identification of molecular pathway aberrations in uterine serous carcinoma by genome-wide analyses. J Natl Cancer Inst 104: 1503-1513.

Le Gallo M, O’Hara AJ, Rudd ML, Urick ME, Hansen NF, O’Neil NJ, Price JC, Zhang S, England BM, Godwin AK, Sgroi DC, Hieter P, Mullikin JC, Merino MJ, Bell DW, Program NISCNCS (2012) Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes. Nat Genet 44: 1310-1315.


In recent years, “targeted” cancer therapies have shown promising results. With the discovery and development of “personalized” novel cancer treatments encouraging clinical responses in a subset of patients with advanced systemic disease have been observed.

This has been particularly evident with several of the recently developed kinase inhibitors that target oncogenic forms of EGFR, HER2, BCR-ABL, ALK, JAK2, and BRAF. In addition, several MEK inhibitors are developed to target oncogenic RAS. Although these drugs initially exhibit responses, they fail to sustain results due to the emergence of acquired resistance. The median duration of response with the EGFR inhibitor gefitinib (Iressa®, FDA approved) in non-small cell lung cancer patients is 7 months. Additionally, response duration for the BRAF inhibitor vemurafenib is approximately 6-7 months.  In phase II clinical trials, median progression free survival time in patients bearing K-RAS mutant tumors treated with selumetinib (MEK inhibitor) was 5.3 months. Multiple mechanisms are associated to limiting the efficacy of these targeted agents. Therefore, further studies are needed to develop effective strategies to overcome the aquisition of resistance to targeted agents. Formulation of effective drug combinations as well as identification of novel compounds with anti-neoplastic properties could be useful in this context.


MEK signal transduction

A recent study by Morris and colleagues published in Cancer Discovery (April 29th, 2013) reported the identification and characterization of a novel and selective ERK inhibitor. This inhibitor was found to induce tumor regression in BRAF, NRAS, and KRAS xenograft models and also inhibited MAPK signaling and cell proliferation in BRAF or MEK inhibitor resistant models. In addition, this ERK inhibitor was found to be effective in tumor cells that are resistant to concurrent treatment with BRAF and MEK inhibitors.

In this study, Morris et al. screened nearly 5 million compounds’ ability to bind the unphosphorylated form of ERK2 and observed ATP competitive compound, SCH772984, selectively bound and inhibited ERK1/2 activity at a nanomolar range. SCH772984 was effective in blocking ERK activation in a dose-dependent manner in V600EBRAF mutant and KRAS mutant cell lines. A known mechanism of resistance to MAPK inhibitors is via negative feedback activation of the MAPK pathway through ERK activation (for details please refer to my previous post titled “resistance to BRAF inhibitors in melanoma”). In this study, negative feedback activation via increased ERK phosphorylation was also observed following treatment with SCH772984. However, SCH772984 was found to be effective in blocking further downstream signaling of ERK as it maintained complete inhibition of ERK substrate p90 ribosomal S6 kinase.

Next, to test the efficacy of SCH772984 in the context of clinically observed resistance mechanisms to the MAPK inhibitors (such as BRAF amplification, MEK1 mutation, RAS mutation), researchers generated resistant cell lines and tested the efficacy of SCH772984. In all scenarios tested, sensitivity to SCH772984 treatment was observed as compared to the MAPK inhibitors. A recent clinical study concurrently treated patients with BRAF and MEK inhibitors and found to double the progression free survival period in patients whose tumors harbored MAPK activation especially in V600EBRAF mutant melanoma.

In the near future this combination could potentially become the standard-of-care for V600EBRAF mutant melanoma. However, due to heterogeneous nature of tumor cells, it is possible that acquired resistance may emerge to this combination treatment. To that end, Morris et al. tested the efficacy of SCH772984 in BRAF and MEK inhibitor combination resistant models of melanoma and colorectal cancer. Reduced activation of ERK and inhibition of cellular proliferation was noted in this model following SCH772984 treatment.

Taken together this study implicates a new ERK inhibitor SCH772984 that may exhibit encouraging therapeutic responses for the treatment of patients with BRAF, NRAS, and KRAS mutations including patients who relapse on current BRAF or MEK inhibitor therapy.


1. Akinleye A, Furqan M, Mukhi N, Ravella P, Liu D. MEK and the inhibitors: from bench to bedside. J Hematol Oncol. 2013;6:27.

2. Cohen MH, Williams GA, Sridhara R, Chen G, Pazdur R. FDA drug approval summary: gefitinib (ZD1839) (Iressa) tablets. Oncologist. 2003;8:303-6.

3. Morris EJ, Jha S, Restaino CR, Dayananth P, Zhu H, Cooper A, et al. Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors. Cancer Discov. 2013.


Acute myeloid leukemia (AML), a cancer of hematopoietic cells, is a molecularly heterogeneous disease. AML is associated with several genetic changes that alter normal hematopoietic growth and differentiation, resulting in the accumulation of large numbers of abnormal immature myeloid cells in the bone marrow and peripheral blood. These cells are capable of dividing and proliferating, but cannot differentiate into mature hematopoietic cells. Recurrent structural alterations of chromosomes are the established diagnostic and prognostic markers in AML. Several studies using targeted sequencing (determination of DNA sequence of specific areas of interest within the genome) identified recurrent gene mutations that contained diagnostic and prognostic information, including mutations in FLT3, NPM1, KIT, CEBPA, and TET2 genes. In addition, massively parallel sequencing (high-throughput approaches of DNA sequencing, also called next-generation sequencing) has discovered recurrent mutations in DNMT3A and IDH1/2 genes that may also provide prognostic information for some patients. Even though these genetic abnormalities may play an essential role in the pathogenesis of AML, nearly 50% of AML patients have normal karyotype (an organized profile of a person’s chromosomes).No. of mutations in AML resized 600 Based on the cytogenetic analysis, AML patients are classified into three major risk categories: favorable, intermediate and unfavorable. AML patients with PML-RARA, RUNX1-RUNX1T1, or MYTH11-CBFB gene fusions (as a result of chromosomal rearrangements) profile belong to favorable- risk category and have shown relatively good response to chemotherapy. Patients with complex genetic alterations (e.g. monosomy karyotype) are categorized into unfavorable-risk profile. Majority of the AML patients exhibit normal karyotype and belong to intermediate-risk category. Some of these patients respond well to chemotherapy while others don’t. Since nearly 50% of AML patients have normal chromosomal profile, better molecular characterization of pathogenesis of AML is required for better approaches to therapy. In addition, next-generation sequencing (NGS) studies have revealed that even though AML usually harbor hundreds of mutated genes, only a limited number of mutated  genes serve as driver mutations (i.e. causing the tumor). Among the different adult cancer types sequenced extensively so far, AML has had the fewest mutations discovered. Therefore, identification of a significant number of novel driver mutations present at low frequency in AML will help to better understand the leukemogenesis.

A study recently published in the New England Journal of Medicine (May 1st, 2013) by researchers at The Cancer Genome Atlas (TCGA) group led by Timothy J. Ley broadly classified the genomic alterations that frequently underlie the development of AML. This study also suggested potential new drug targets and treatment strategies for AML. In this study the genome of 200 newly diagnosed adult cases of AML patients, representing all of the known subtypes, was analyzed by performing whole-genome sequencing (50 cases) and whole-exome sequencing (150 cases). In addition, RNA and micro-RNA sequencing and DNA-methylation analysis was also performed. 

Each AML genome was compared to the describe the imagenormal genome derived from a skin sample of the same patient. The recurrently mutated genes discovered in this study were grouped into nine categories that were defined according to biologic function and that are considered to play a role in AML pathogenesis. Some of these groups include: tumor suppressor genes, transcription-factor fusions, activated signaling genes and epigenetic modifiers (DNA-methylation related genes and chromatin-modifying genes) with the latter being the most frequently mutated class of genes found in this study. At least one potential driver mutation was identified in nearly all AML samples including genes that are well established as being associated with AML pathogenesis (eg. FLT3, NPM1, DNMT3A, IDH1, IDH2, and CEBPA).

This study was the first to observe recurrent mutations in cohesin genes, which are important in cell division, in 13% of cases of AML samples. In addition, this study also observed a mutation in microRNA 142 (miR-142). Overall this study provided a detailed understanding of the genetic and epigenetic changes associated with adult de novo AML. Future studies are warranted to understand the relationship between these alterations and treatment results.



1. Bacher U, Schnittger S, Haferlach T. Molecular genetics in acute myeloid leukemia. Curr Opin Oncol. 2010;22:646-55.

2. Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer. 2003;3:650-65.

3. Yamashita Y, Yuan J, Suetake I, Suzuki H, Ishikawa Y, Choi YL, et al. Array-based genomic resequencing of human leukemia. Oncogene. 2010;29:3723-31.

4. Network TCGAR. Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia. N Engl J Med. 2013.


A brain tumor is an abnormal growth of tissue in the brain. Brain tumors may develop from neural elements within the brain (primary brain tumor), or they may represent spread of distant cancers (secondary brain tumor). According to the National Cancer Institute, in 2013 the estimated new cases and deaths from brain tumor in the United States would be 23,130 and 14,080 respectively. There are many types of primary brain tumors. Primary brain tumors are named according to the type of cells or the part of the brain in which they originate. For example, most primary brain tumors begin in glial cells. This type of tumor is called a glioma. Among adults, the most common types are:

  • Meningioma: This tumor arises in the meninges. It’s usually benign (grade I) and grows slowly.
  • Oligodendroglioma: This tumor arises from cells that make the fatty substance that covers and protects nerves. It usually occurs in the cerebrum. It’s most common in middle-aged adults.
  • Astrocytoma: This tumor arises from star-shaped glial cells called astrocytes.

Among these, glioblastoma multiforme (GBM) is the most common and deadliest of malignant primary brain tumors in adults. Classified as a Grade IV (most serious) astrocytoma, GBM develops from the lineage of star-shaped glial cells, called astrocytes that support nerve cells. Even though GBM is one of the most aggressive forms of human cancers, the incident of this disease is fairly less.  Every year, in the United States approximately 7 out of 100,000 people are diagnosed with high-grade glioblastoma. Uncontrolled cellular proliferation, diffuse infiltration, and resistance to apoptosis are considered as hallmarks of GBM. The current standard of treatment in glioblastoma involves surgery followed by radiation therapy, and chemotherapy with the DNA alkylating agent temozolomide (TMZ).  The efficacy of this current treatment strategy is limited as the median survival is just over one year because of development of resistance. Therefore, more effective treatment plans need to be developed and explored. In a recent study published in the peer review journal Clinical Cancer Research (2013 Apr 25) Thorpe and colleagues reported that combined treatment of ataxia telangiectasia mutated (ATM) inhibitor and radiation significantly increased survival of mice bearing glioblastoma with p53 mutation compared to treatment with ATM inhibitor or radiation alone.

The nuclear protein kinase ataxia-telangiectasia mutated (ATM) plays a crucial role in DNA-damage response by transducing double-strand breaks. This kinase phosphorylates several effectors that play key roles in cell cycle progression and DNA repair pathways.

gLIOBLASYTOMA -resized-600 (1)

Ionizing radiation is the most effective therapy for glioblastoma. Ionizing radiation works by damaging the DNA of exposed tissue leading to cellular death. However, tumor cells evade death and contribute to radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. Therefore, combined treatment of ionizing radiation with inhibitors of DNA damage checkpoint may overcome the problem of resistance in glioblastoma. Since ATM is associated with DNA damage repair there are several small molecule ATM inhibitors that disrupt ATM function and enhances sensitivity of tumor cells to radiation as well as chemotherapeutic agents. ATM inhibitor KU-60019 is a potent inhibitor effectively blocking activation of key ATM targets and radiosensitizing human glioma cells in vitro. In their study Thorpe et al. (2013) showed that mice bearing glioblastoma survived longer periods of time (145 days) when treated with KU-60019 and radiation than mice treated with radiation alone (58 days) or KU-60019 alone (66 days). In addition, this study also noted that mice bearing glioma with mutant p53 are more sensitive to KU-60019 dependent radiosensitization than mice bearing glioma with p53 wild-type. Altogether this study suggests that ATM kinase inhibition may be an effective strategy as adjuvant therapy for patients with mutant p53 brain cancers.


1.Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006, 444(7120):756-760.

2.Biddlestone-Thorpe L, Sajjad M, Rosenberg E, Beckta JM, Valerie NC, Tokarz M, Adams BR, Wagner AF, Khalil A, Gilfor D et al: ATM kinase inhibition preferentially sensitizes p53 mutant glioma to ionizing radiation. Clin Cancer Res 2013.

3. http://www.cancer.gov/cancertopics/wyntk/brain/page2



Histone deacetylase (HDAC) inhibitors (HDIs), a new class of epigenetic anti-tumor agents, have shown promise so far in the treatment of hematologic malignancies. Many, if not most, cell lines tested in vitro show sensitivity to HDIs, and synergy in combinations is also often noted. Yet, Phase I and II trials of HDIs have uniformly been disappointing in solid tumors. Even preclinical models in which HDIs exhibited potent anti-tumor effects in vivo have not succeeded in the clinic. Therefore, a detailed understanding of the mechanisms of resistance to HDIs may lead to strategies designed to increase clinical efficacy. Studies have proposed several mechanisms of resistance which include increased expression of the P-glycoprotein (Pgp) encoding multidrug-resistance gene ABCB1, increased expressresistance to HDIS resized 600ion of reactive oxygen species (ROS) scavenger protein thioredoxin, elevated expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, increased expression of histone HDAC enzymes, and activation of several signaling pathways including MAPK, phosphoinositide 3-kinase and signal transducer and activator of transcription.

The FDA approved histone deacetylase inhibitor romidepsin (istodax®) for the treatment of cutaneous T-cell lymphoma (CTCL) in 2009 and for the treatment of peripheral T-cell lymphoma (PTCL) in 2011. Disease progression was noted in some patients who initially responded to therapy, while disease in other patients did not respond to therapy suggesting that both de novo and acquired resistance to romidepsin were observed during the trial. Thus, studies are needed to determine the mechanisms of resistance to the HDI romidepsin particularly in T-cell lymphoma.

Several studies have noted selection of Pgp overexpression in vitro as a mechanism of drug resistance to romidepsin. The ease of selection of Pgp is in part because romidepsin is a substrate for Pgp, and in part based on ABCB1 gene induction as a consistent cellular response to HDIs. While induction of ABCB1 has been noted in normal and malignant peripheral blood mononuclear cells of patients treated with romidepsin, no evidence for Pgp-mediated resistance emerged in clinical samples obtained at the time of disease progression from patients with CTCL or PTCL. Therefore, identification of non-Pgp resistance mechanisms for romidepsin is warranted. A recent study published in the peer reviewed journal Blood by Chakraborty et al. (Blood. 2013 Mar 26) reported that activation of the mitogen activated protein kinase (MAPK) pathway conferred resistance to romidepsin through degradation of the pro-apoptotic BH-3 only protein Bim. To explore Pgp-independent mechanisms of resistance to romidpesin, a CTCL model consisting of the HuT78 cell line and its romidepsin-selected sublines were used. These sublines were separately selected in romidepsin in the presence of the Pgp-inhibitors verapamil or valspodar (PSC833) to avoid overexpression of Pgp. The resulting cell lines were resistant to romidepsin and inhibition of Pgp could reverse the resistance of only the cells that had not been selected in the presence of the Pgp inhibitors. The failure to reverse the resistance of the romidepsin-selected cells with a Pgp inhibitor suggested a different mechanism of action, which had not been identified in the earlier studies. A gene microarray study detected increased expression of the insulin receptor in the romidepsin-selected cells compared to the parental HuT78 cells. In addition, romidepsin-resistant cells also exhibited increased activation of MEK protein, a downstream component of the MAPK signaling pathway. Treating these resistant cells with the allosteric MEK inhibitors resulted in exquisite sensitivity while the parental HuT78 cells did not respond to the MEK inhibition. Restoration of the pro-apoptotic protein Bim was noted in romidepsin-resistant cells following MEK inhibition which was otherwise found to be degraded in the resistant cells. Combined treatment of MEK inhibitor with romidepsin also caused increased death of resistant cells. In their study, Chakraborty and colleagues also noted loss of Bim in the skin biopsy samples obtained from CTCL patients who experienced disease progression after romidepsin treatment. In addition, this study also reported perturbation of the MAPK regulated genes in the CTCL patients treated with romidepsin. Collectively these observations suggested that activation of the MAPK pathway may limit efficacy of the HDI romidepsin through degradation of the pro-apoptotic protein Bim, and in future clinical trials combination of romidepsin with MEK inhibitor may exhibit promising results.



1.Fantin VR, Richon VM. Mechanisms of resistance to histone deacetylase inhibitors and their therapeutic implications. Clin Cancer Res. 2007;13(24):7237-7242.

2.Chakraborty AR, Robey RW, Luchenko VL, et al. MAPK pathway activation leads to Bim loss and histone deacetylase inhibitor resistance: rationale to combine romidepsin with a MEK inhibitor. Blood. 2013.


With the increasing knowledge about the role of V600E B-RAF mutation in melanoma progression, efforts have been made to target and inhibit this kinase and its downstream signaling. The ATP-competitive type I B-RAF inhibitors vemurafenib and dabrafenib (GSK2118436) exhibit remarkable anti-cancer activity in patients with V600E B-RAF mutant melanomas. Targeted inhibition of BRAF with vemurafenib causes tumor regression and extends survival in many patients with BRAF-mutant metastatic melanoma. In 2011, the Food and Drug Administration (FDA) approved vemurafenib tablets (ZELBORAF) for the treatment of patients with unresectable or metastatic melanoma with the V600EBRAF mutation. Vemurafenib is not recommended for use in patients with wild-type BRAF melanoma. Even though a very high percentage of patients respond to vemurafenib, resistance to this drug develops relatively quickly. With continued treatment, the emergence of resistance can be seen as soon as 6-8 weeks following initial documentation of response. However, a subset of patients maintains drug responsiveness beyond 18 months. Overall, the median duration of responsiveness to vemurafenib is 8 months. Some studies described involvement of certain molecular mechanisms associated with vemurafenib resistance in melanoma. Reactivation of the MAPK pathway, NRAS mutation, overexpression of platelet derived growth factor beta receptor, activation of PI3K/AKT signaling, genomic amplification of V600EBRAF are some of the mMech. of action of vemurafenib resized 600echanisms of acquired resistance to BRAF inhibitors (for detail please refer to my blog post titled ” RESISTANCE TO B-RAF INHIBITORS IN MELANOMA’’). Due to heterogeneous nature of cancer cells, it is crucial to gain a thorough understanding of the underlying drug-resistance mechanisms so that we can develop novel strategies to circumvent resistance and achieve more-prolonged responses. A recent study published in the journal Nature by Das Thakur and colleagues reported that intermittent treatment of vemurafenib prevented resistance in primary human melanoma xenografts. To study mechanisms of resistance to vemurafenib, Das Thakur et al. developed an animal model by continuously treating mice bearing a vemurafenib-naive, patient-derived BRAF-mutant melanoma with vemurafenib until drug resistance developed. Exome sequence analysis did not detect any secondary mutations in the coding sequences of BRAF, NRAS, KRAS, HRAS, and MEK1 in the resistant tumors. No alternatively spliced isoform of V600EBRAF, another known mechanism of vemurafenib resistance in melanoma was also detected in the resistant tumors. However, increased expression of V600EBRAF protein was noted in the resistant tumors and inhibition of V600EBRAF gene by RNA interference resulted in suppression of proliferation. These data suggested that the tumor cells were BRAF oncogene dependent and the observed drug resistance was due to the increased expression of V600EBRAF protein. In addition to these, another interesting observation was noted in this study when Das Thakur et al. tried to establish cell lines derived from the drug-resistant tumors. Cell lines derived from the drug-resistant tumors could not be developed without vemurafenib, where withdrawal of vemurafenib from the newly established cell lines changed cell morphology and decreased proliferation. This suggested that vemurafenib-resistant tumor cells in melanoma suffer a fitness deficit in the absence of vemurafenib. A similar type of vemurafenib dependency was also observed in SK-MEL239-C3 melanoma cells in which resistance is due to expression of a splice variant of V600EBRAF, and also in tumor cells isolated from a BRAF-mutated vemurafenib-resistant melanoma patient. Consistent with these findings, Das Thakur et al. observed tumor regression within 10 days in mice bearing vemurafenib resistant melanoma following cessation of vemurafenib treatment, although tumors eventually started re-growing. Collectively these results suggested that withdrawal of vemurafenib might create a hostile environment for drug-resistant cells and detain the onset of drug resistance. A comparison study made between continuous and intermittent vemurafenib treatment in human melanoma xenografts bearing mice further validated these observations. Drug resistance was developed in mice with 100 days receiving continuous treatment, whereas none of the mice on the intermittent treatment schedule exhibited drug resistance after 200 days of treatment. Therefore, these findings recommend that discontinuous treatment of vemurafenib may select against drug-resistant cells and prolong the responses to vemurafenib in melanoma. Future studies are needed especially in clinical trials to validate this proposal.



1.         Das Thakur M, Salangsang F, Landman AS, et al. Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance. Nature. 2013;494(7436):251-255.

2.         Sullivan RJ, Flaherty KT. Resistance to BRAF-targeted therapy in melanoma. Eur J Cancer. 2013;49(6):1297-1304.


Although cancer is considered a disease of genetic defects, various studies have shown that epigenetic changes also play an important role in the onset and progression of cancer. Epigenetic modifications in mammals include DNA methylation and posttranslational histone modifications such as acetylation, methylation, phosphorylation, sumoylation, and ubiquitination. In human tumors, DNA methylation has been the most widely studied epigenetic modification. However, in recent years there has been a significant growth in our knowledge about the involvement of aberrant patterns of histone modifications in cancer development. In the nucleus, 147 base pairs of DNA are wrapped around an octamer of histone (H) proteins (two copies of each of H2A, H2B, H3 and H4) to form nucleosomes, which in turn are compacted further through several levels of higher-order packing (e.g., H1 aids formation of the 30-nm solenoid) to form chromatin. The accessibility of DNA within the nucleosome is in part controlled by the modifications of the histone proteins. Each histone protein in the nucleosome has a long “tail” that extends beyond the nucleosome. This lysine (K) rich amino-terminal “tail” undergoes various posttranslational modifications by acetyl groups, phosphate groups, and methyl groups. Among these various histone modifications specifically two modifications play crucial roles in the epigenetic control of cellular proliferation and differentiation. These include trimethylation of histone H3 lysine 27 (H3K27me3) which is catalyzed by the enhancer of zeste homolog 2 enzyme (EZH2), results in gene transcriptional repression; and methylation of histone H3 lysine 4 (H3H4me) which is catalyzed by the trithorax homolog myeloid-lymphoid leukemia (MLL), resulting in transcriptional activation. Several genes associated with development, stem cell maintenance, and differentiations are targets of H3K27 and H3K4 methylation. EZH2-mediated H3K27 methylation is also involved in X chromosome inactivation (Xi), a process in which one of the two X-chromosomes in the female cell is transcriptionally repressed, to generate transcriptionally inactive heterochromatin. In addition, as a catalytic subunit of epigenetic regulator Polycomb repressive complex 2, EZH2, a histone methyltransferase (an enzyme that transfers methyl groups), not only methylates histones H3 but also interacts with and recruits DNA methyltransferases to methylate CpG regions (C:cytosine, p:phosphpodiester bond, G:guanine) at certain EZH2 target genes and thereby causing transcriptional repression. Studies in cancer have indicated that deregulation of EZH2 contributes to a variety of tumor development and progression including breast, lung, prostate, pancreatic, and ovarian cancers as well as glioma, lymphoma, and sarcoma.

Overexpression of EZH2 was first reported in prostate anEZH2 resized 600d breast cancer. In both cases increased expression was found to be associated with tumor invasiveness, metastasis, and poor clinical outcome. In addition, elevated expression of EZH2 is also reported in several other tumors including gastric, lung, bladder, and endometrial cancer. Gain of functions as a result of acquired mutations in EZH2 was reported in lymphoma and meyloid neoplasms. The best characterized mechanism by which EZH2 exerts its oncogenic function is by transcriptional repression of genes via its histone methyltransferase activity. Genes which get transcriptionally repressed by EZH2 include tumor suppressor genes ARF, p57KIP2, FBXO32, p27, and BRCA1. In addition, this enzyme also activates transcription of gene CCND1 (cyclin D1) driving cell-cycle progression.

As oncogenic property of EZH2 is mediated through its enzymatic activity, inhibitors of EZH2 are currently under development targeting EZH2’s enzymatic activities. GSK126, a potent, highly selective small-molecule inhibitor of EZH2 methyltransferase activity exhibited promising response in diffuse large B-cell lymphoma (DLBCL) and DLBCL xenografts in mice. However, a recent study by Yan et al. (2013) demonstrated that the oncogenic function of EZH2 may not always be dependent on the enzymatic activities of EZH2. Therefore, therapeutic strategies targeting EZH2 should be designed based on its oncogenic activity via enzymatic properties as well as its function in transcriptional activation of genes involved in various oncogenic pathways.


1.         Ezhkova E, Pasolli HA, Parker JS, et al. Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell. Vol. 136. United States; 2009:1122-1135.

2.         Ernst T, Chase AJ, Score J, et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet. Vol. 42. United States; 2010:722-726.

3.         Yan J, Ng SB, Tay JL, et al. EZH2 overexpression in natural killer /T-cell lymphoma confers growth advantage independently of histone methyltransferase activity. Blood. 2013.


B-RAF is a serine/therionine protein kinase which activates the mitogen-activated protein kinase (MAPK) signaling pathway. As suggested by Hanahan and Weinberg (2000) six major hallmarks of cancer are: independence of proliferation signals, evasion of apoptosis, insensitivity to anti-growth signals, unlimited replicative potential, the ability to invade and metastasize, and induction of angiogenesis for nutrient supply.  Abnormalities in the MAPK signaling impinge on most, if not all these processes, and play a critical role in the development and progression of cancer. Activating mutations in B-RAF kinase (mostly V600E B-RAF) are observed in about 50% of melanomas resulting in constitutive activation of the MAPK pathway. With the increasing knowledge about the role of V600E B-RAF mutation in melanoma progression, efforts have been made to target and inhibit this kinase and its downstream signaling. The ATP-competitive type I B-RAF inhibitors vemurafenib and dabrafenib (GSK2118436) exhibit remarkable anti-cancer activity in patients with V600E B-RAF mutant melanomas. However, acquisition of drug resistance virtually occurs in all patients treated with B-RAF inhibitors. In clinical trials with the B-RAF inhibitors, disease progression was noted in most of the patients after 6-7 months of initial response. Investigations at the molecular level suggest that development of resistance is associated with the acquisition of the secondary mutations within the kinase ATP binding site that inhibited the binding of drug to the hydrophobic pocket at so-called “gatekeeper” residue. In a preclinical study  Whittaker et al. (2010) reported that a gatekeeper mutation in BRAF  at Threonine-259 (T259) residue conferred resistance to the B-RAF inhibitors SB590885 and PLX4720. However, no such mutation was observed in patients whose disease progressed following vemurafenib treatment. This suggests existence of V600EB-RAF-bypass mechanism for acquired resistance to the B-RAF inhresistance to RAF inhibitors resized 600ibitors. Activation of the MAPK signaling and increased expression of C-RAF (an isoform of B-RAF) was noted in melanoma cells resistant to B-RAF inhibitors. In this study Villanueva et al. (2010 ) also observed constitutive activation of the insulin-like growth factor receptor 1 (IFGR1), a receptor tyrosine kinase (RTK) in the resistant cells. As IGFR1 activates PI3K/Akt signaling, combined treatment of PI3K and MEK inhibitors resulted in resistance reversal. Increased levels of IGFR1 was also observed in melanoma patients failing vemurafenib suggesting activation of PI3K/Akt signaling via IGFR1 could limit the efficacy of B-RAF inhibitors in the clinic. Up-regulation of other RTKs was also found to be associated with the acquired resistance to vemurafenib. Tumor biopsies of melanoma patients failing vemurafenib exhibited over-expression of the platelet derived growth factor receptor-β (Nazarian et al., 2010).

In addition to the increase RTKs activity, resistance to the B-RAF inhibitors was noted to be mediated by genetic alteration in the MAPK signaling pathway. Genetic analysis detected an activating mutation in NRAS in codon 61 in tumor biopsies from patients treated with vemurafenib (Nazarian et al., 2010). Next, there is also evidence of B-RAF alterations in tumor samples collected from patients whose cancer progressed after initial response to B-RAF inhibitors. A study by Shi et al. (2012) reported overexpression of V600EB-RAF as a result of genomic copy-numbers gain in 20% vemurafenib resistant melanoma patients. In the in vitro study, Shi et al. observed restoration of the sensitivity of the B-RAF amplification driven vemurafenib resistant cells to vemurafenib following treatment with the MEK inhibitor selumetinib (AZD6244). This observation provides evidence of the MAPK pathway reactivation as a mechanism of resistance. Existence of a structural changes in B-RAF could also confer resistance as identified by Poulikakos et al. (2011). Their study discovered a splice variant of B-RAF(V600E). Expression of a 61-kDa B-RAF variant lacking RAS-binding domain was identified in the tumors of 32% patients with acquired resistance to vemurafenib.

One of the challenges in melanoma treatment is how to develop effective strategies for overcoming intrinsic and acquired drug resistance to small molecule B-RAF inhibitors. Although the resistance mechanisms identified so far are diverse, most seem to rely upon the reactivation of the MAPK signaling pathway and enhanced signaling output through the PI3K/Akt signaling pathway. Therefore, dual BRAF and PI3K/Akt signaling pathway inhibition may prevent or delay the onset of resistance in melanoma.



1.         Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. Vol. 100. United States; 2000:57-70.

2.         Whittaker S, Kirk R, Hayward R, et al. Gatekeeper mutations mediate resistance to BRAF-targeted therapies. Sci Transl Med. Vol. 2. United States; 2010:35ra41.

3.         Villanueva J, Vultur A, Lee JT, et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell. Vol. 18. United States: 2010 Elsevier Inc; 2010:683-695.

4.         Nazarian R, Shi H, Wang Q, et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature. Vol. 468. England; 2010:973-977.

5.         Shi H, Moriceau G, Kong X, et al. Melanoma whole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance. Nat Commun. Vol. 3. England; 2012:724.

6.         Alas S, Bonavida B. Inhibition of constitutive STAT3 activity sensitizes resistant non-Hodgkin’s lymphoma and multiple myeloma to chemotherapeutic drug-mediated apoptosis. Clin Cancer Res. 2003;9(1):316-326.


Melanoma, the most dangerous type of skin cancer, is the leading cause of death from skin disease (refer to my previous post titled “Targeting B-RAF in melanoma”). Melanoma results from a neoplastic transformation of pigment-producing melanocytes, with cutaneous neoplasms of stratified epithelium comprising the majority. Traditionally melanoma has been difficult to treat and exhibited resistance to all available standard therapies. However, recent progresses in the treatment of melanoma, including the FDA approved B-RAF inhibitor vemurafenib, have shown great promise in shrinking tumor size and improving the survival of patients. Studies have shown that melanoma is a complex disease that arises through multiple etiologic pathways. Therefore, detailed understanding of the underlying molecular mechanisms associated with melanoma pathogenesis and drug resistance is warranted in achieving a sustained clinical response. Over the past decade, molecular characterization of melanoma has progressed with the identification of the influence of various important oncogenes. These include oncogenic activation of BRAF, KIT, NRAS, cyclin D, and cyclin-dependent kinase 4 and alterations in the ERBB4 gene. Among these, the most frequently occurring genetic alteration associated with melanoma progression is the activating somatic mutation of B-RAF serine/thereonine kinase (for details please refer to my post titled “Targeting B-RAF in melanoma”). In addition to B-RAF mutations, which occurs in 50% of cases of melanoma, mutations of NRAS or neuroblastoma RAS gene were also identified in 15 to 20% of melanomas.

NRAS mutation resized 600

Approximately one-third of all human malignancies have mutations in RAS oncogene. RAS is a small sized plasma membrane-associated GTP binding protein. The RAS family of proteins consists of KRAS, HRAS, and NRAS. These proteins primarily regulate growth and, as a molecular switch, they connect signals from cell surface receptors to transcription factors and cell cycle regulating proteins in the nucleus. RAS proteins exist either in GTP-bound state (active) or GDP-bound (inactive) state. In normal cells, following binding of a ligand to its cognate receptor tyrosine kinase (RTK) RAS becomes activated. Once activated, RAS recruits and stimulates a number of signaling pathways including mitogen-activated protein kinas (MAPK) pathway and the phosphoinositide 3-kinase/AKT (PI3K/AKT) pathway.

Even though mutation of KRAS gene is the most common type of RAS mutation in human malignant disease, in melanoma, a point mutation of NRAS is most frequent and was the first oncogene to be identified. The most common mutation in NRAS is observed in codon 61 which occurs as a replacement of glutamine residue by lysine or arginine. This leads to constitutive activation of the MAPK signal transduction pathway resulting in proliferation and promotion of tumor growth. In addition, the RAS oncogene also activates signaling via the Rho GTPase Rac1, which can mediate growth, survival, and motility signaling.

Several studies analyzed the role of NRAS mutation in melanoma. In a study of 100 primary and metastatic melanoma samples by Ball et al. (1994), 36% of melanomas were found to contain mutations in RAS that, in 69% of cases, were at the codon 61. It was observed in multiple studies that patients with NRAS-mutated tumors are older at diagnosis than are patients with BRAF mutations (median age 55·7 years for NRAS vs 49·8 years for BRAF) and more frequently have melanoma due to chronic sun damage. Two large (>240 samples) studies of melanomas with NRAS mutations indicate that these tumors appear to exhibit more aggressive behavior, being associated with shorter overall survival. These tumors have also exhibited higher rates of mitosis and are thicker at presentation. A meta-analysis of studies from 1989 to 2010 reported that NRAS mutations were associated with nodular histology and location on the extremities. Collectively all these studies suggest a prominent role of NRAS as an oncogene in melanoma, and recommend scope of therapeutic targeting of NRAS for the treatment of advanced and high-risk melanoma.

With the increasing knowledge about the roles of various oncogenes (especially BRAF and NRAS), substantial advances in the targeted therapy to treat melanoma was achieved, however, only in BRAF-mutated melanomas. Compared to patients with BRAF mutations, as of today no approved targeted therapies exist for patients with NRAS-mutated melanoma. Complete inhibition of NRAS oncogenic signaling has proven to be challenging in part due to existence of redundant feedbacks to activate NRAS-MEK-ERK (MAPK) pathway. Various alternative strategies have thus been proposed, including (a) targeting membrane localization of RAS protein, required for RAS activity, through inhibitors of farnesyl transferase or galectin 1; (b) targeting NRAS mRNA with interfering RNAs; and (c) targeting signaling downstream of NRAS protein through inhibitors of PI3K/Akt. In addition, in in vitro studies some NRAS-mutated cell lines exhibited sensitivity to MEK inhibition. In conclusion, even though all these approaches hold promise, none of them got translated into the clinic. Therefore, more studies are required to formulate strategies to effectively treat NRAS-mutated melanoma.



1.Devitt B, Liu W, Salemi R, Wolfe R, Kelly J, Tzen CY, Dobrovic A, McArthur G: Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma. Pigment Cell Melanoma Res 2011, 24:666-672.

2. Ball NJ, Yohn JJ, Morelli JG, Norris DA, Golitz LE, Hoeffler JP: Ras mutations in human melanoma: a marker of malignant progression. J Invest Dermatol 1994, 102:285-290.


Melanoma is a type of skin cancer. It arises from specialized pigmented cells in our body known as melanocytes that are responsible for the production of melanin (a pigment responsible for skin and hair color). Because most melanoma cells still make melanin, melanoma tumors are usually brown or black. It accounts for 4% of all skin cancers; however, it is responsible for the largest numbers of skin cancer related death in the world. In the US, according to the national cancer institute, estimated new cases and deaths from melanoma in 2013 would be 76,690 and 9,480 respectively.

Several studies using molecular profiling and genomic sequencing have shown that melanoma is a disease of a heterogeneous group of tumors, and its progression is driven by specific oncogenic mutations. In 2002, Davies et al. first reported the presence of B-RAF somatic missense mutations in 66% of malignant melanomas. RAF (Rapidlydescribe the image growing Fibrosarcoma) protein is a serine/thereonine kinase. Three members of this kinase family are A-RAF, B-RAF, and C-RAF. These serine/threonine protein kinases, downstream of the membrane-bound small G protein RAS, are components of the mitogen activtated protein kinase (MAPK) signal transduction pathway. With closely overlapping functions, all members of the RAF family are associated with the activation of the MAPK pathway. Activation of the MAPK pathway has been associated with uncontrolled growth and drug resistance in several tumors. Researchers have identified over 50 distinct mutations in the B-RAF gene so far. However, most of these mutations are extremely rare. The most common mutation in melanoma, accounting for 90% of all B-RAF mutations, is the V600E mutation that occurs as a result of substitution of amino acid valine (V) to glutamic acid (E) at codon 600. Approximately 50% of melanomas harbor the V600E B-RAF mutation, while other mutations observed in melanomas are usually associated with the activation of N-RAS and c-KIT.

Several studies reported association of the V600E B-RAF mutation with the progression of melanoma. In a pre-clinical study Smalley et al. (2010) observed tumor formation in immunocompromised mice following introduction of mutant B-RAF in melanocytes. Inversely, in their study, Smalley et al. also observed that inhibition of mutated B-RAF using RNA-interference resulted in tumor cell death. In addition, several other studies reported that inhibition of V600E mutant B-RAF prevents melanoma cell proliferation, induces apoptosis (programmed cell death), and also blocks melanoma xenograft growth in vivo. Even though many studies suggested that V600E B-RAF mutation may not be sufficient alone for melanoma induction, a wealth of evidence demonstrated that mutated B-RAF is necessary for the maintenance and progression of melanoma in human. Therefore, mutated B-RAF represents a therapeutic target in melanoma, which is why several B-RAF kinase inhibitors have already been developed. Sorafenib was the first B-RAF inhibitor studied in melanoma patients. In addition, vemurafenib (Zelboraf) and dabrafenib (GSK2118436) were also studied in melanoma patients with V600E B-RAF mutations.  In 2011 vemurafenib received FDA approval for the treatment of melanoma patients harboring the V600E B-RAF mutation. In clinical trials, in which patients were undergoing treatment with vemurafenib, the drug reduced risk of death by 63% and risk of progression by 74%.

At present several clinical trials also evaluate clinical efficacy of vemurafenib in combination with leflunomide  (antirheumatic drug), GDC-0973 (MEK inhibitor), and metformin (antidiabetic drug). In addition, several other drugs targeting B-RAF and its downstream pathway are also in development. Therefore, further improvements can be expected in this personalized and targeted therapy in melanoma.



1. Ascierto, P. A., Kirkwood, J. M., Grob, J. J., Simeone, E., Grimaldi, A. M., Maio, M., Palmieri, G., Testori, A., Marincola, F. M., and Mozzillo, N. (2012). The role of BRAF V600 mutation in melanoma. J Transl Med 10, 85.

2.Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin, H., Garnett, M. J., Bottomley, W., et al. (2002). Mutations of the BRAF gene in human cancer. Nature 417, 949-954.

3. Smalley, K. S. (2010). Understanding melanoma signaling networks as the basis for molecular targeted therapy. J Invest Dermatol 130, 28-37.