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Posts Tagged ‘c-myc’


Author: Tilda Barliya PhD

Acute lymphoblastic leukemia (ALL), a malignant disorder of lymphoid progenitor cells, affects both children and adults,
with peak prevalence between the ages of 2 and 5 years (2). Acute lymphocytic leukemia (ALL) is a heterogeneous disease, both in terms of its pathology and the populations that it affects. Disease pathogenesis involves a number of deregulated pathways controlling cell proliferation, differentiation, and survival that are important determinants of treatment response (3). Approximately 5200 new cases of ALL are estimated to have occurred in the United States in 2007 and survival varies with age and disease biology (3). Although five-year survival rates for ALL approach 90 percent with available chemotherapy treatments, the harmful side effects of the drugs, including secondary cancers and fertility, cognitive, hearing, and developmental problems, present significant concern for survivors and their families.

Biological and Clinical Prognostic Factors in ALL: Setting the Stage for Risk-Adapted Therapy

Of the many variables that influence prognosis the genetic subsets, initial white blood cell count (WBC), age at diagnosis, and early treatment response are the most important.

Childhood Acute Lymphoblastic Leukemia

Pathobiology

Acute lymphoblastic leukaemia is thought to originate  from various important genetic lesions in blood-progenitor  cells that are committed to differentiate in the T-cell or B-cell pathway, including mutations that impart the  capacity for unlimited self-renewal and those that lead to  precise stage-specific developmental arrest. In some  cases, the first mutation along the multistep pathway to  overt acute lymphoblastic leukaemia might arise in a  haemopoietic stem cell possessing multilineage developmental capacity.

The dominant theme of contemporary research in pathobiology of acute lymphoblastic leukaemia is to understand the outcomes of frequently arising genetic lesions, in terms of their effects on cell proliferation, differentiation, and survival, and then to devise selectively targeted treatments against the altered gene products to which the leukaemic clones have become addicted (2).

Table 1.

Prognostic factors used in pediatric and adult clinical trials

The Table  illustrates the different prognostic factors in children and adults that may be used for risk stratification in current clinical trials (3).

Genetics

  • Chromosomal translocations that activate specifi c genes
    are a defi ning characteristic of human leukaemias and
    of acute lymphoblastic leukaemia in particular.
  • About 25% of cases of B-cell precursor acute lymphoblastic leukaemia, the most frequent form of acute leukaemia in children, harbour the TEL-AML1 fusion gene—generated by the t(12;21)(p13;q22) chromosomal translocation.

The presence of the TEL-AML1 fusion
protein in B-cell progenitors seems to lead to disordered
early B-lineage lymphocyte development, a hallmark of
leukaemic lymphoblasts.

Analysis of TEL-AML1-induced cord blood cells suggests that the fusion gene serves as a first-hit mutation by endowing the preleukemic cell with altered self-renewal and survival properties.

  • In adults, the most frequent chromosomal translocation  is t(9;22), or the Philadelphia chromosome, which causes  fusion of the BCR signalling protein to the ABL  non-receptor tyrosine kinase, resulting in constitutive  tyrosine kinase activity and complex interactions of this  fusion protein with many other transforming elements.  BCR-ABL off ers an attractive therapeutic  target, and imatinib mesilate, a small-molecule inhibitor  of the ABL kinase, has proven effective against leukaemias that express BCR-ABL
  • More than 50% of cases of T-cell acute lymphoblastic  leukaemia have activating mutations that involve  NOTCH1. NOTCH1, which translocates to the nucleus and regulates by transcription a diverse set of responder genes, including the MYC oncogene.  The precise  mechanisms by which aberrant NOTCH signalling (due  to mutational activation) causes T-cell acute lymphoblastic  leukaemia are still unclear but probably entail constitutive  expression of oncogenic responder genes, such as MYC,  and cooperation with other signalling pathways (pre-TCR  [T-cell receptor for antigen] and RAS, for example).  Interference with NOTCH signalling by small-molecule  inhibition of γ-secretase activity has the potential to induce remission of T-cell acute lymphoblastic  leukemia.

Additionally A recent discussion has aimed to reveal the genetic origin of the disease (1). Several of these genes, including ARID5B, IKZF1, and CEBPE, have been implicated in processes such as hematopoietic differentiation and development of ALL. These gene obviously adds up to a number of other gene mutations and translocation already discovered and are associated with disease progression (2)  “The fact that alterations in these genes lead to ALL raises the question of what would happen if we restore these pathways in ALL and also make them possible exciting therapeutic targets as well.”

Nanotechnology and therapeutic

Dr. Rajasekaran, director and head of the Membrane Biology Laboratory University of Delaware,  says that there are currently seven or eight drugs that are used for chemotherapy to treat leukemia in children. They are all toxic and do their job by killing rapidly dividing cells. these drugs don’t differentiate cancer cells from other healthy cells. “The good news is that these drugs are 80 to 90 percent effective in curing leukemia. The bad news is that many chemotherapeutic treatments cause severe side effects, especially in children.  In preclinical models of leukemia, Dr. Rajasekaran research team have created NP  with an average diameter of 110 nm were assembled from an amphiphilic block copolymer of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals (ECT2). The researches have been encapsulated with dexamethasone as one third of the typical dose, with good treatment results and no discernible side effects.In addition, the mice that received the drugs delivered via nanoparticles survived longer than those that received the drug administered in the traditional way (4).

In another preclinical study Uckun F et al  developed nanoparticle (NP) constructs of WHI-P131. WHI-P131 (CAS 202475-60-3) is a dual-function inhibitor of JAK3 tyrosine kinase that demonstrated potent in vivo anti-inflammatory and anti-leukemic activity in several preclinical animal models (5). Notably, WHI-P131-NP was capable of causing apoptotic death in primary leukemia cells from chemotherapy-resistant acute lymphoblastic leukemia (ALL) as well as chronic lymphocytic leukemia (CLL) patients. WHI-P131-NP was also active in the RS4;11 SCID mouse xenograft model of chemotherapy-resistant B-lineage ALL. The life table analysis showed that WHI-P131-NP was more effective than WHI-P131 (P = 0.01), vincristine (P<0.0001), or vehicle (P<0.0001). These experimental results demonstrate that the nanotechnology-enabled delivery of WHI-P131 shows therapeutic potential against leukemias with constitutive activation of the JAK3-STAT3/STAT5 molecular target (5).

Summary:

Acute Lymphoblastic Leukemia (ALL) is a pediatric type of cancer that affects adults to lesser degree. The current rate of cure if 80% in  children whereas in adults only 30-40% will survive. Much of the success is due to understanding the mechanisms that lead to the development and progression of cancer. Several gene mutations and gene-translocation have already been identified,  and targeting them enabled some of the major success in curing these kids.

Thus far, nanotechnology has been  mainly focusing on solid tumors affecting adults. Not much attention is been made on childhood cancer in general and hematopoietic types per se. Two examples of preclinical studies have been discussed above and although they show promise in treatment and reduction of side effects, yet  additional research is needed to evaluated their effect in human clinical trials.

Ref:

1. The Genetic Origin of Childhood Acute Lymphoblastic Leukemia (ALL).  Reported by Aviva Lev-Ari, PhD, RN. March 20, 2013 https://pharmaceuticalintelligence.com/2013/03/20/the-genetic-origin-of-childhood-acute-lymphoblastic-leukemia-all/

2. Ching-Hon Pui, Leslie L Robison, A Thomas Look. Acute lymphoblastic leukaemia. Lancet 2008; 371: 1030–43.

http://www.med.upenn.edu/timm/documents/PuiLookLancetLeukemiareview.pdf

3. Wendy Stock. Adolescents and Young Adults with Acute Lymphoblastic Leukemia. Hematology December 4, 2010 vol. 2010 no. 1 21-29. http://asheducationbook.hematologylibrary.org/content/2010/1/21.full

4. Vinu Krishnan,  Xian Xu,, Sonali P. BarweXiaowei YangKirk CzymmekScott A. WaldmanRobert W. MasonXinqiao Jia, and Ayyappan K. Rajasekaran. Dexamethasone-Loaded Block Copolymer Nanoparticles Induce Leukemia Cell Death and Enhance Therapeutic Efficacy: A Novel Application in Pediatric Nanomedicine. Mol. Pharmaceutics 2012 ahead of print.

http://pubs.acs.org/doi/abs/10.1021/mp300350e?prevSearch=Rajasekaran&searchHistoryKey=

5. Uckun FMDibirdik IQazi SYiv S. Therapeutic nanoparticle constructs of a JAK3 tyrosine kinase inhibitor against human B-lineage ALL cells. Arzneimittelforschung 2010; 60(4): 210-217.

http://www.ncbi.nlm.nih.gov/pubmed/20486472

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ENCODE data reveals important information from Genome Wide Association Studies relevant to understanding complex genetic diseases

Author: Ritu Saxena, Ph.D.

Introduction

“The depth, quality, and diversity of the ENCODE data are unprecedented” is what was stated by John Stamatoyannopoulos, professor of genomic sciences at the University of Washington and one of the many principle investigators of ENCODE project. ENCODE (Encyclopedia of DNA elements), indeed, was an ambitious project launched as a pilot in 2003 and then expanded in 2007 for the whole genome analysis and identification of all the functional elements of the human genome. The findings were striking as they challenged the definition of “gene” and ‘the central dogma of genetics (Gene-mRNA-protein). Infact, the non-coding part that constitutes about 80% of the genome or the so-called “junk DNA” was found to contain elements crucial for gene regulation. The elements, in large part, include RNA transcripts that are not transcribed into proteins but might have a regulatory role. For detailed reading, refer to the findings published in the issue of Nature, The ENCODE Project Consortium Nature 489, 57–74 (2012) An integrated encyclopedia of DNA elements in the human genome

Key features of the data, as explained in the National Human Genome Research Institute website (National Human Genome Research Institute News feature), include comprehensive mapping of:

  • Protein-coding genes — Proteins are molecules made of amino acids linked together in a specific sequence; the amino acid sequence is encoded by the sequence of DNA subunits called nucleotides that make up genes.
  • Non-coding genes — Stretches of DNA that are read by the cell as if they were genes but do not encode proteins. These appear to help regulate the activity of the genome.
  • Chromatin structure features — Complex physical structures made from a combination of DNA and binding proteins that make up the contents of the nucleus and affects genome function.
  • Histone modifications — Histones are the proteins that make up the chromatin structures that help shape and control the genome. In addition, histone proteins can be physically modified by adding chemical groups, such as a methyl molecule, that further regulates genomic activity.
  • DNA methylation — Just like histones, methyl groups can be added to DNA itself in a process called DNA methylation. Chemically attaching methyl groups to DNA physically changes the ability of enzymes to reach the DNA and thus alters the gene expression pattern in cells. Methylation helps cells “remember what they are doing” or alter levels of gene expression, and it is a crucial part of normal development and cellular differentiation in higher organisms.
  • Transcription factor binding sites — Transcription factors are proteins that bind to specific DNA sequences, controlling the flow (or transcription) of genetic information from DNA to mRNA. Mapping the binding sites can help researchers understand how genomic activity is controlled.

How could ENCODE be helpful in the study of complex human diseases?

Complex diseases and Genome wide association studies (GWAS)

Coronary artery disease, type 2 diabetes and many forms of cancer are complex human diseases that have a significant genetic component. Unlike mendelian disorders that have defined loci, the genetic component of complex disorders lies in the form of genetic variations in the genome making an individual susceptible to these complex diseases.

Researchers have performed Genome-wide association studies (GWAS) of the human genome, leading to the identification of thousands of DNA variants that could be linked with complex traits and diseases. However, identifying the variants, referred to as SNPs (Single Nucleotide Polymorphisms), that actually contribute to the disease, and understanding how they exert influence on a disease has been more of a mystery.

How would ENCODE solve the puzzle?

The puzzle lies in interpreting how the SNPs found in the genome affect a person’s susceptibility to a particular trait or disease and what is the mechanism behind it. As identified in the GWAS, most variants that are associated with the phenotype of the trait or disease lie in the non-coding region of the genome. Infact, in more than 400 studies compiled in the GWAS catalog only a small minority of the trait/disease-associated SNPs occur in protein-coding regions; the large majority (89%) are in noncoding regions. These variants fall in the gene deserts that lie far from protein-coding region, similar to those where cis-regulatory modules (CRMs) are found. CRMs such as promoters and enhancers are a group of binding sites for transcription factors, and the presence of transcription factors bound to these sites is a good indicator of the potential regulatory regions.

The integrative analysis of ENCODE data has give important insights to the results of GWAS studies. Investigators have employed ENCODE data as an initial guide to discover regulatory regions in which genetic variation is affecting a complex trait. Additionally, ENCODE study when examined the SNPs from GWAS that were associated with the phenotype of the trait, found that these regions are enriched in DNase-sensitive regions i.e, lie in the function-associated DNA region of the genome as it could be bound by transcription factors affecting the regulation of gene expression. Thus, the project demonstrates that non-coding regions must be considered when interpreting GWAS results, and it provides a strong motivation for reinterpreting previous GWAS findings.

Using ENCODE Data to Interpret GWAS Results

ENCODE and predisposition to CANCER:

C-Myc, a proto-oncogene, codes for a transcripton factor, when expressed constitutively leads to uninhibited cell proliferation resulting in cancer. It has been observed that common variants within a ~1 Mb region upstream of c-Myc gene have been associated with cancers of the colon, prostate, and breast. Several SNPs have been reported in this region, that although affect the phenotype, lie in the distal cis-region of the MYC gene. Alignment of the ENCODE data in this region with the significant variants from the GWAS also reveals that key variants are found in the transcription factor occupied DNA segments mapped by this consortium. One variant rs698327, lies within a DNase hypersensitive site that is bound by several transcription factors, enhancer-associated protein p300, and contains histone modifications relative to enhancers (high H3K4me1, low H3K4me3). ENCODE data indicates that non-coding regions in the human chromosome 8q24 loci are associated with cancer and as observed in the case of c-myc gene, similar studies on cancer-related genes could help explain predisposition to cancer.

ENCODE and fetal hemoglobin expression:

Another example of the use of ENCODE data is that of gene regulation of fetal hemoglobin. Several regions were predicted via ENCODE that were involved in the regulation of fetal hemoglobin. It was found that these predicted regions are close to the SNPs in the BLC11A gene that is associated with persistent expression of fetal hemoglobin.

Future perspective

As evident from the above examples, the ENCODE data shows that genetic variants do affect regulated expression of a target gene. Recently, several research groups in the UK performed a large-scale GWAS study to determine the genetic predisposition to fracture risk. The collaborative effort, published in a recent issue of the PLoS journal, was made to identify genetic variants associated with cortical bone thickness (CBT) and bone mineral density (BMD) with data from more than 10,000 subjects. http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002745 The study generated a wealth of data including the result – identification of SNPs in the WNT16 and its adjacent gene, FAM3C were found to be relevant to CBT and BMD. ENCODE data, in this case, could be helpful in interpreting more detailed information including determining additional SNPs, the regulatory information of the genes involved and much more. Thus, it could be concluded that ENCODE data could be immensely useful in interpreting associations between disease and DNA sequences that can vary from person to person.

Sources:

Research articles

An integrated encyclopedia of DNA elements in the human genome

A User’s Guide to the Encyclopedia of DNA Elements (ENCODE)

What does our genome encode?

Genome-wide Epigenetic Data Facilitate Understanding of Disease Susceptibility Association Studies

Genomics: ENCODE explained

ENCODE Project Writes Eulogy For Junk DNA

WNT16 Influences Bone Mineral Density, Cortical Bone Thickness, Bone Strength, and Osteoporotic Fracture Risk

 News articles

ENCODE project: In massive genome analysis new data suggests ‘gene’ redefinition

National Human Genome Research Institute News feature

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ENCODE Findings as Consortium

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