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Posts Tagged ‘Massachusetts Institute of Technology’


Reporter: Aviva Lev-Ari, PhD, RN

AstraZeneca PLC (AZN) Signs $200 Million Deal With BIND Therapeutics for Cancer Drug

4/22/2013 7:19:42 AM

CAMBRIDGE, Mass. & WILMINGTON, Del.–(BUSINESS WIRE)– BIND Therapeutics and AstraZeneca announced today that they have entered into a strategic collaboration to develop and commercialize an AccurinTM, a targeted and programmable cancer nanomedicine from BIND’s Medicinal Nanoengineering platform, based on a molecularly targeted kinase inhibitor developed and owned by AstraZeneca.The collaboration is based on emerging data suggesting that nanomedicines like Accurins selectively accumulate in diseased tissues and cells, leading to higher drug concentrations at the site of the tumor and reduced exposure to healthy tissues.

Under the terms of the agreement, the companies will work together to complete Investigational New Drug (IND)-enabling studies of the lead Accurin identified from a previously-completed feasibility program. AstraZeneca will then have the exclusive right to lead development and commercialization and BIND will lead manufacturing during the development phase. BIND could receive upfront and pre-approval milestone payments totaling $69 million, and more than $130 million in regulatory and sales milestones and other payments as well as tiered single to double-digit royalties on future sales.

“We are excited to grow this collaboration with AstraZeneca, a leading global biopharmaceutical company committed to developing innovative medicines for patients,” said Scott Minick, President and CEO of BIND. “One year ago, BIND started several feasibility projects with major pharmaceutical companies. Our collaboration with AstraZeneca is the first one completed and had very successful results. Due to the advanced nature of this program, we now plan to move an Accurin with optimized therapeutic properties quickly into product development.”

“AstraZeneca believes that targeted therapies which specifically address the underlying mechanisms of disease are the future of personalized cancer treatment,” said Susan Galbraith, Head of AstraZeneca’s Oncology Innovative Medicines Unit. “Our oncology teams are actively exploring a range of platforms to deliver targeted therapies, with a strategic focus on unlocking the significant potential of nanoparticles as an approach to cancer treatment. We view BIND’s targeted nanomedicines as a leading technology in this field.”

About Accurins™

BIND Therapeutics is discovering and developing Accurins, proprietary new best-in-class therapeutics with superior target selectivity and the potential to improve patient outcomes in the areas of oncology, inflammatory diseases and cardiovascular disorders. Leveraging its proprietary Medicinal Nanoengineering® platform, BIND develops Accurins that outperform conventional drugs by selectively accumulating in diseased tissues and cells. The result is higher drug concentrations at the site of action with minimal off-target exposure, leading to markedly better efficacy and safety.

About BIND Therapeutics

BIND Therapeutics is a clinical-stage biopharmaceutical company developing a new class of highly selective targeted and programmable therapeutics called Accurins. BIND’s Medicinal Nanoengineering® platform enables the design, engineering and manufacturing of Accurins with unprecedented control over drug properties to maximize trafficking to disease sites, dramatically enhancing efficacy while minimizing toxicities.

BIND is developing a pipeline of novel Accurins that hold extraordinary potential to become best-in-class drugs and improve patient outcomes in the areas of oncology, inflammatory diseases and cardiovascular disorders. BIND’s lead product candidate, BIND-014, is currently entering Phase 2 clinical testing in cancer patients and is designed to selectively target PSMA, a surface protein upregulated in a broad range of solid tumors. BIND also develops Accurins in collaboration with pharmaceutical and biotechnology partners to enable promising pipeline candidates to achieve their full potential and to utilize selective targeting to transform the performance of important existing drug products.

BIND is backed by leading investors Polaris Venture Partners, Flagship Ventures, ARCH Venture PartnersNanoDimension, DHK Investments, EndeavourVision and Rusnano. BIND was founded on proprietary technology from the laboratories of two leaders in the field of nanomedicine, Professors Robert Langer, David H. Koch Institute Professor of the Massachusetts Institute of Technology (MIT) and Omid Farokhzad, Associate Professor of Harvard Medical School.

For more information, please visit the company’s web site at http://www.bindtherapeutics.com.

Contact:

Media:

The Yates Network

Kathryn Morris, 845-635-9828

Kathryn@theyatesnetwork.com

SOURCE:

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Curators: Aviva Lev-Ari, PhD, RN and Larry Bernstein, MD, FACP

The essence of the message is summarized by Larry Bernstein, MD, FACP, as follows:

[1] we employ a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145bp DNA segments centered on evolutionarily-conserved regulatory motif instances and found in enhancer chromatin states
[2] We find statistically robust evidence that (1) scrambling, removing, or disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are all associated with wild-type enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually not active.
[3] Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale experimental manipulation, and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to generate and test predictive models of gene expression.

Manolis Kellis and co-authors from the Massachusetts Institute of Technology and the Broad Institute describe a massively parallel reporter assay that they used to systematically study regulatory motifs falling within thousands of predicted enhancer sequences in the human genome. Using this assay, they examined 2,104 potential enhancers in two human cell lines, along with another 3,314 engineered enhancer variants. “Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale experimental manipulation,” they write, “and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to generate and test predictive models of gene expression.”

SOURCE:

http://www.genomeweb.com//node/1206571?hq_e=el&hq_m=1536519&hq_l=4&hq_v=e1df6f3681

Systematic dissection of regulatory motifs in 2,000 predicted human enhancers using a massively parallel reporter assay

  1. Pouya Kheradpour1,
  2. Jason Ernst1,
  3. Alexandre Melnikov2,
  4. Peter Rogov2,
  5. Li Wang2,
  6. Xiaolan Zhang2,
  7. Jessica Alston2,
  8. Tarjei S Mikkelsen2 and
  9. Manolis Kellis1,3

+Author Affiliations


  1. 1 MIT;

  2. 2 Broad Institute
  1. * Corresponding author; email: manoli@mit.edu

Abstract

Genome-wide chromatin maps have permitted the systematic mapping of putative regulatory elements across multiple human cell types, revealing tens of thousands of candidate distal enhancer regions. However, until recently, their experimental dissection by directed regulatory motif disruption has remained unfeasible at the genome scale, due to the technological lag in large-scale DNA synthesis. Here, we employ a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145bp DNA segments centered on evolutionarily-conserved regulatory motif instances and found in enhancer chromatin states. We select five predicted activators (HNF1, HNF4, FOXA, GATA, NFE2L2) and two predicted repressors (GFI1, ZFP161) and measure reporter expression in erythroleukemia (K562) and liver carcinoma (HepG2) cell lines. We test 2,104 wild-type sequences and an additional 3,314 engineered enhancer variants containing targeted motif disruptions, each using 10 barcode tags in two cell lines and 2 replicates. The resulting data strongly confirm the enhancer activity and cell type specificity of enhancer chromatin states, the ability of 145bp segments to recapitulate both, the necessary role of regulatory motifs in enhancer function, and the complementary roles of activator and repressor motifs. We find statistically robust evidence that (1) scrambling, removing, or disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are all associated with wild-type enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually not active. Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale experimental manipulation, and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to generate and test predictive models of gene expression.

  • Received June 26, 2012.
  • Accepted March 14, 2013.

This manuscript is Open Access.

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/.

SOURCE:

http://genome.cshlp.org/content/early/2013/03/19/gr.144899.112.abstract

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Curator: Aviva Lev-Ari, PhD, RN

Chaperon Protein Mechanism inspired MIT Team to Model the Role of Genetic Mutations on Cancer Progression, proposing the next generation of Oncology drugs to aim at Suppression of Passenger Mutations. Current drug, in clinical trials, use the Chaperon Protein Mechanism to suppress Driver Mutations.

Deleterious Mutations in Cancer Progression

Kirill S. Korolev1, Christopher McFarland2, and Leonid A. Mirny3

1Department of Physics, MIT, Cambridge, MA.

E-mail: papers.korolev@gmail.com

2Graduate Program in Biophysics, Harvard University, Cambridge, MA.

3Health Sciences and Technology, MIT, Cambridge, MA

The research was funded by the National Institutes of Health/National Cancer Institute Physical Sciences Oncology Center at MIT.

SOURCE:

http://cnls.lanl.gov/q-bio/wiki/images/4/40/Abstract.pdf

Deleterious passenger mutations significantly affect evolutionary dynamics of cancer. Including passenger mutations in evolutionary models is necessary to understand the role of genetic diversity in cancer progression and to create new treatments based on the accumulation of deleterious passenger mutations.

Evolutionary models of cancer almost exclusively focus on the acquisition of driver mutations, which are beneficial to cancer cells. The driver mutations, however, are only a small fraction of the mutations found in tumors. The other mutations, called passenger mutations, are typically neglected because their effect on fitness is assumed to be very small. Recently, it has been suggested that some passenger mutations are slightly deleterious. We find that deleterious passengers significantly affect cancer progression. In particular, they lead to a critical tumor size, below which tumors shrink on average, and to an optimal mutation rate for cancer evolution.

ANCER is an outcome of somatic evolution [1-3]. To outcompete their benign sisters, cancer cells need to acquire many heritable changes (driver mutations) that enable proliferation. In addition to the rare beneficial drivers, cancer cells must also acquire neutral or slightly deleterious passenger mutations [4]. Indeed, the number of possible passengers exceeds the number of possible drivers by orders of magnitude. Surprisingly, the effect of passenger mutations on cancer progression has not been explored. To address this problem, we developed an evolutionary model of cancer progression, which includes both drivers and passengers. This model was analyzed both numerically and analytically to understand how mutation rate, population size, and fitness effects of mutations affect cancer progression.

RESULTS

Upon including passengers in our model, we found that cancer is no longer a straightforward progression to malignancy. In particular, there is a critical population size such that smaller populations accumulate passengers and decline, while larger populations accumulate drivers and grow. The transition to cancer for small initial populations is, therefore, stochastic in nature and is similar to diffusion over an energy barrier in chemical kinetics. We also found that there is an optimal mutation rate for cancer development, and passengers with intermediate fitness costs are most detrimental to cancer. The existence of an optimal mutation rate could explain recent clinical data [5] and is in stark contrast to the predictions of the models neglecting passengers. We also show that our theory is consistent with recent sequencing data.

SOURCE:

http://cnls.lanl.gov/q-bio/wiki/images/4/40/Abstract.pdf

Just as some mutations in the genome of cancer cells actively spur tumor growth, it would appear there are also some that do the reverse, and act to slow it down or even stop it, according to a new US study led by MIT.

Senior author, Leonid Mirny, an associate professor of physics and health sciences and technology at MIT, and colleagues, write about this surprise finding in a paper to be published online this week in the Proceedings of the National Academy of Sciences.

In a statement released on Monday, Mirny tells the press:

“Cancer may not be a sequence of inevitable accumulation of driver events, but may be actually a delicate balance between drivers and passengers.”

“Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations,” he suggests.

Cancer Cell‘s Genome Has “Drivers” and “Passengers”

Your average cancer cell has a genome littered with thousands of mutations and hundreds of mutated genes. But only a handful of these mutated genes are drivers that are responsible for the uncontrolled growth that leads to tumors.

Up until this study, cancer researchers have mostly not paid much attention to the “passenger” mutations, believing that because they were not “drivers”, they had little effect on cancer progression. 

Now Mirny and colleagues have discovered, to their surprise, that the “passengers” aren’t there just for the ride. In sufficient numbers, they can slow down, and even stop, the cancer cells from growing and replicating as tumors. 

New Drugs Could Target the Passenger Mutations in Protein Chaperoning

Although there are already several drugs in development that target the effect of chaperone proteins in cancer, they are aiming to suppress driver mutations.

Recently, biochemists at the University of Massachusetts Amherst“trapped” a chaperone in action, providing a dynamic snapshot of its mechanism as a way to help development of new drugs that target drivers.

But Mirny and colleagues say there is now another option: developing drugs that target the same chaperoning process, but their aim would be to encourage the suppressive effect of the passenger mutations.

They are now comparing cells with identical driver mutations but different passenger mutations, to see which have the strongest effect on growth.

They are also inserting the cells into mice to see which are the most likely to lead to secondary tumors (metastasize).

Written by Catharine Paddock PhD
Copyright: Medical News Today

SOURCE:

http://www.medicalnewstoday.com/articles/255920.php

After proteins are synthesized, they need to be folded into the correct shape, and chaperones help with that process. In cancerous cells, chaperones help proteins fold into the correct shape even when they are mutated, helping to suppress the effects of deleterious mutations.
Several potential drugs that inhibit chaperone proteins are now in clinical trials to treat cancer, although researchers had believed that they acted by suppressing the effects of driver mutations, not by enhancing the effects of passengers.

In current studies, the researchers are comparing cancer cell lines that have identical driver mutations but a different load of passenger mutations, to see which grow faster. They are also injecting the cancer cell lines into mice to see which are likeliest to metastasize.

Drugs that tip the balance in favor of the passenger mutations could offer a new way to treat cancer, the researchers say, beating it with its own weapon — mutations. Although the influence of a single passenger mutation is minuscule, “collectively they can have a profound effect,” Mirny says. “If a drug can make them a little bit more deleterious, it’s still a tiny effect for each passenger, but collectively this can build up.”

In natural populations, selection weeds out deleterious mutations. However, Mirny and his colleagues suspected that the evolutionary process in cancer can proceed differently, allowing mutations with only a slightly harmful effect to accumulate.

If enough deleterious passengers are present, their cumulative effects can slow tumor growth, the simulations found. Tumors may become dormant, or even regress, but growth can start up again if new driver mutations are acquired. This matches the cancer growth patterns often seen in human patients.

“Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations.”

When they analyzed passenger mutations found in genomic data taken from cancer patients, the researchers found the same pattern predicted by their model — accumulation of large quantities of slightly deleterious mutations.

REFERENCE

Massachusetts Institute of Technology (2013, February 4). Some cancer mutations slow tumor growth. ScienceDaily. Retrieved February 4, 2013, from http://www.sciencedaily.com­/releases/2013/02/130204154011.htm

Biochemists Trap A Chaperone Machine In Action

Main Category: Biology / Biochemistry
Article Date: 11 Dec 2012 – 0:00 PST

Molecular chaperones have emerged as exciting new potential drug targets, because scientists want to learn how to stop cancer cells, for example, from using chaperones to enable their uncontrolled growth. Now a team of biochemists at the University of Massachusetts Amherst led by Lila Gierasch have deciphered key steps in the mechanism of the Hsp70 molecular machine by “trapping” this chaperone in action, providing a dynamic snapshot of its mechanism.

She and colleagues describe this work in the current issue of Cell. Gierasch’s research on Hsp70 chaperones is supported by a long-running grant to her lab from NIH’s National Institute for General Medical Sciences.

Molecular chaperones like the Hsp70s facilitate the origami-like folding of proteins, made in the cell’s nanofactories or ribosomes, from where they emerge unstructured like noodles. Proteins only function when folded into their proper structures, but the process is so difficult under cellular conditions that molecular chaperone helpers are needed. 

The newly discovered information about chaperone action is important because all rapidly dividing cells use a lot of Hsp70, Gierasch points out. “The saying is that cancer cells are addicted to Hsp70 because they rely on this chaperone for explosive new cell growth. Cancer shifts our body’s production of Hsp70 into high gear. If we can figure out a way to take that away from cancer cells, maybe we can stop the out-of-control tumor growth. To find a molecular way to inhibit Hsp70, you’ve got to know how it works and what it needs to function, so you can identify its vulnerabilities.”

Chaperone proteins in cells, from bacteria to humans, act like midwives or bodyguards, protecting newborn proteins from misfolding and existing proteins against loss of structure caused by stress such as heat or a fever. In fact, the heat shock protein (Hsp) group includes a variety of chaperones active in both these situations.

As Gierasch explains, “New proteins emerge into a challenging environment. It’s very crowded in the cell and it would be easy for them to get their sticky amino acid chains tangled and clumped together. Chaperones bind to them and help to avoid this aggregation, which is implicated in many pathologies such as neurodegenerative diseases. This role of chaperones has also heightened interest in using them therapeutically.”

However, chaperones must not bind too tightly or a protein can’t move on to do its job. To avoid this, chaperones rapidly cycle between tight and loose binding states, determined by whether ATP or ADP is bound. In the loose state, a protein client is free to fold or to be picked up by another chaperone that will help it fold to do its cellular work. In effect, Gierasch says, Hsp70s create a “holding pattern” to keep the protein substrate viable and ready for use, but also protected.

She and colleagues knew the Hsp70’s structure in both tight and loose binding affinity states, but not what happened between, which is essential to understanding the mechanism of chaperone action. Using the analogy of a high jump, they had a snapshot of the takeoff and landing, but not the top of the jump. “Knowing the end points doesn’t tell us how it works. There is a shape change in there that we wanted to see,” Gierasch says.

To address this, she and her colleagues postdoctoral fellows Anastasia Zhuravleva and Eugenia Clerico obtained “fingerprints” of the structure of Hsp70 in different states by using state-of-the-art nuclear magnetic resonance (NMR) methods that allowed them to map how chemical environments of individual amino acids of the protein change in different sample conditions. Working with an Hsp70 known as DnaK from E. coli bacteria, Zhuravleva and Clerico assigned its NMR spectra. In other words, they determined which peaks came from which amino acids in this large molecule.

The UMass Amherst team then mutated the Hsp70 so that cycling between tight and loose binding states stopped. As Gierasch explains, “Anastasia and Eugenia were able to stop the cycle part-way through the high jump, so to speak, and obtain the molecular fingerprint of a transient intermediate.” She calls this accomplishment “brilliant.”

Now that the researchers have a picture of this critical allosteric state, that is, one in which events at one site control events in another, Gierasch says many insights emerge. For example, it appears nature uses this energetically tense state to “tune” alternate versions of Hsp70 to perform different cellular functions. “Tuning means there may be evolutionary changes that let the chaperone work with its partners optimally,” she notes.

“And if you want to make a drug that controls the amount of Hsp70 available to a cell, our work points the way toward figuring out how to tickle the molecule so you can control its shape and its ability to bind to its client. We’re not done, but we made a big leap,” Gierasch adds. “We now have a idea of what the Hsp70 structure is when it is doing its job, which is extraordinarily important.” 

Article adapted by Medical News Today from original press release. Click ‘references’ tab above for source.
Visit our biology / biochemistry section for the latest news on this subject.
SOURCE:

REFERENCES

[1] Michor F, Iwasa Y, and Nowak MA (2004) Dynamics of cancer

progression. Nature Reviews Cancer 4, 197-205.

[2] Crespi B and Summers K (2005) Evolutionary biology of cancer.

Trends in Ecology and Evolution 20, 545-552.

[3] Merlo LMF, et al. (2006) Cancer as an evolutionary and ecological

process. Nature Reviews Cancer 6, 924-935.

[4] McFarland C, et al. “Accumulation of deleterious passenger mutations

in cancer,” in preparation.

[5] Birkbak NJ, et al. (2011) Paradoxical relationship between

chromosomal instability and survival outcome in cancer. Cancer

Research 71,3447-3452.

Other related articles on this Open Access Online Scientific Journal include the following:

Hold on. Mutations in Cancer do good.

https://pharmaceuticalintelligence.com/2013/02/04/hold-on-mutations-in-cancer-do-good/

Rational Design of Allosteric Inhibitors and Activators Using the Population-Shift Model: In Vitro Validation and Application to an Artificial Biosensor

https://pharmaceuticalintelligence.com/2012/10/26/rational-design-of-allosteric-inhibitors-and-activators-using-the-population-shift-model-in-vitro-validation-and-application-to-an-artificial-biosensor/

LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2

https://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-drug-selection-in-cancer-personalized-treatment-part-2/

Exome sequencing of serous endometrial tumors shows recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes

https://pharmaceuticalintelligence.com/2012/12/18/exome-sequencing-of-serous-endometrial-tumors-shows-recurrent-somatic-mutations-in-chromatin-remodeling-and-ubiquitin-ligase-complex-genes/

Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell(1)

https://pharmaceuticalintelligence.com/2013/02/03/genome-wide-detection-of-single-nucleotide-and-copy-number-variation-of-a-single-human-cell/

Gastric Cancer: Whole-genome reconstruction and mutational signatures

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Pregnancy with a Leptin-Receptor Mutation

https://pharmaceuticalintelligence.com/2012/10/31/pregnancy-with-a-leptin-receptor-mutation/

Mitochondrial mutation analysis might be “1-step” away

https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

Genome-wide Single-Cell Analysis of Recombination Activity and De Novo Mutation Rates in Human Sperm

https://pharmaceuticalintelligence.com/2012/08/07/genome-wide-single-cell-analysis-of-recombination-activity-and-de-novo-mutation-rates-in-human-sperm/

A Prion Like-Protein, Protein Kinase Mzeta and Memory Maintenance

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Hope for Male Contraception: A small molecule that inhibits a protein important for chromatin organization can cause reversible sterility in male mice

https://pharmaceuticalintelligence.com/2012/09/03/hope-for-male-contraception-a-small-molecule-that-inhibits-a-protein-important-for-chromatin-organization-can-cause-reversible-sterility-in-male-mice/

Protein Folding may lead to better FLU Vaccine

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SNAP: Predict Effect of Non-synonymous Polymorphisms: How well Genome Interpretation Tools could Translate to the Clinic

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Drugging the Epigenome

https://pharmaceuticalintelligence.com/2013/02/01/drugging-the-epigenome/

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Reporter: Prabodh Kandala, PhD

A typical cancer cell has thousands of mutations scattered throughout its genome and hundreds of mutated genes. However, only a handful of those genes, known as drivers, are responsible for cancerous traits such as uncontrolled growth. Cancer biologists have largely ignored the other mutations, believing they had little or no impact on cancer progression.

But a new study from MIT, Harvard University, the Broad Institute and Brigham and Women’s Hospital reveals, for the first time, that these so-called passenger mutations are not just along for the ride. When enough of them accumulate, they can slow or even halt tumor growth.

The findings, reported in this week’sProceedings of the National Academy of Sciences, suggest that cancer should be viewed as an evolutionary process whose course is determined by a delicate balance between driver-propelled growth and the gradual buildup of passenger mutations that are damaging to cancer, says Leonid Mirny, an associate professor of physics and health sciences and technology at MIT and senior author of the paper.

Furthermore, drugs that tip the balance in favor of the passenger mutations could offer a new way to treat cancer, the researchers say, beating it with its own weapon — mutations. Although the influence of a single passenger mutation is minuscule, “collectively they can have a profound effect,” Mirny says. “If a drug can make them a little bit more deleterious, it’s still a tiny effect for each passenger, but collectively this can build up.”

Lead author of the paper is Christopher McFarland, a graduate student at Harvard. Other authors are Kirill Korolev, a Pappalardo postdoctoral fellow at MIT, Gregory Kryukov, a senior computational biologist at the Broad Institute, and Shamil Sunyaev, an associate professor at Brigham and Women’s.

Power struggle

Cancer can take years or even decades to develop, as cells gradually accumulate the necessary driver mutations. Those mutations usually stimulate oncogenes such as Ras, which promotes cell growth, or turn off tumor-suppressing genes such as p53, which normally restrains growth.

Passenger mutations that arise randomly alongside drivers were believed to be fairly benign: In natural populations, selection weeds out deleterious mutations. However, Mirny and his colleagues suspected that the evolutionary process in cancer can proceed differently, allowing mutations with only a slightly harmful effect to accumulate.

To test this theory, the researchers created a computer model that simulates cancer growth as an evolutionary process during which a cell acquires random mutations. These simulations followed millions of cells: every cell division, mutation and cell death.

They found that during the long periods between acquisition of driver mutations, many passenger mutations arose. When one of the cancerous cells gains a new driver mutation, that cell and its progeny take over the entire population, bringing along all of the original cell’s baggage of passenger mutations. “Those mutations otherwise would never spread in the population,” Mirny says. “They essentially hitchhike on the driver.”

This process repeats five to 10 times during cancer development; each time, a new wave of damaging passengers is accumulated. If enough deleterious passengers are present, their cumulative effects can slow tumor growth, the simulations found. Tumors may become dormant, or even regress, but growth can start up again if new driver mutations are acquired. This matches the cancer growth patterns often seen in human patients.

“Cancer may not be a sequence of inevitable accumulation of driver events, but may be actually a delicate balance between drivers and passengers,” Mirny says. “Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations.”

When they analyzed passenger mutations found in genomic data taken from cancer patients, the researchers found the same pattern predicted by their model — accumulation of large quantities of slightly deleterious mutations.

Tipping the balance

In computer simulations, the researchers tested the possibility of treating tumors by boosting the impact of deleterious mutations. In their original simulation, each deleterious passenger mutation reduced the cell’s fitness by about 0.1 percent. When that was increased to 0.3 percent, tumors shrank under the load of their own mutations.

The same effect could be achieved in real tumors with drugs that interfere with proteins known as chaperones, Mirny suggests. After proteins are synthesized, they need to be folded into the correct shape, and chaperones help with that process. In cancerous cells, chaperones help proteins fold into the correct shape even when they are mutated, helping to suppress the effects of deleterious mutations.

Several potential drugs that inhibit chaperone proteins are now in clinical trials to treat cancer, although researchers had believed that they acted by suppressing the effects of driver mutations, not by enhancing the effects of passengers.

In current studies, the researchers are comparing cancer cell lines that have identical driver mutations but a different load of passenger mutations, to see which grow faster. They are also injecting the cancer cell lines into mice to see which are likeliest to metastasize.

Ref:

Massachusetts Institute of Technology (2013, February 4). Some cancer mutations slow tumor growth. ScienceDaily. Retrieved February 4, 2013, from http://www.sciencedaily.com­/releases/2013/02/130204154011.htm

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Reporter: Aviva Lev-Ari, PhD, RN

 

02/01/2013
Ashley Yeager

For the first time, scientists have identified all the proteins in the mitochondrial matrix, which is where the cell’s energy is generated. How? Find out…

 

Between the maze-like inner membranes of the mitochondria, there’s a thick, sticky region called the matrix. This region serves an important role in the generation of the cell’s energy, but the proteins that actually make up this matrix have remained a mystery. But now, researchers at the Massachusetts Institute of Technology (MIT) have catalogued all the proteins in the mitochondrial matrix, identifying 31 proteins not previously associated with mitochondria. They did this by combining the strengths of two methods: microscopy and mass spectrometry.

 

Electron microscopy of human embryonic kidney cells expressing mito-APEX. Credit: Alice Ting, MIT

“This method is really a new paradigm for doing mass spec proteomics because we’re recording proteomics in living cells,” said Alice Ting, a chemist at MIT and author of a paper published online yesterday in Science that describes the technique (1).Microscopy and mass spectrometry are valuable for studying proteins, but each has its drawbacks. While microscopy can show where a protein is located within a cell, it can only do so for a small number of a cell’s roughly 20,000 proteins at once. Meanwhile, mass spectrometry can identify all the proteins within a cell, but destroys the cell membrane in the process of releasing the cell’s contents, resulting in a mixture of proteins from different cell regions and organelles.

To overcome these limitations, Ting’s group genetically engineered the mitochondrial matrix to express a newly designed peroxidase called APEX. When biotin-phenol was added to these cells, APEX stripped an electron and a proton from the biotin molecule, creating highly reactive biotin-phenoxyl radicals. These radicals quickly bound to nearby proteins to stabilize themselves, effectively tagging the proteins in the matrix.

The scientists then identified these tagged proteins with fluorescent imaging, dissolved the cell membrane, and isolated the proteins from the mitochondrial matrix. Using mass spectrometry, the team then identified 495 proteins in the mitochondrial matrix, 31 of which had not been previously linked to the mitochondrial region.

One of the biggest surprises was the discovery that the enzyme PPOX is in the matrix. PPOX helps synthesize heme, the pigment in red blood cells and a cofactor of the protein hemoglobin. Previously, biologists believed that PPOX was located within the space between the outer and inner membranes of the mitochondria, but Ting’s team found that it was actually within the matrix, which the team said is an example of how locally precise their biotin-tagging technique is.

Now, Ting and her team are looking at proteins in the mitochondrial intermembrane space. In addition, the researchers are tweaking their labeling system to map proteins in the cell membrane and to detect specific protein-protein interactions.

Reference

1. Rhee, H.-W., P. Zou, N. D. Udeshi, J. D. Martell, V. K. Mootha, S. A. Carr, and A. Y. Ting. 2013. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science (January).

SOURCE:

http://www.biotechniques.com/news/biotechniquesNews/biotechniques-339645.html#.UQ37hRxiB0w

 

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Reporter: Aviva Lev-Ari, RN

With New Microfludic Technique, MIT Team Aims to ‘Squeeze’ siRNAs into Cells

January 31, 2013

Researchers from the Massachusetts Institute of Technology last week reported on the development of a new microfluidic-based approach to delivering macromolecules, including functional siRNAs, into cells without the need for a vector.

According to the investigators, who published their findings in the Proceedings of the National Academy of Sciences, the technique involves compressing cells by passing them through a constriction, which opens up temporary holes in their membranes that permit the diffusion of materials in surrounding buffer to enter the cytosol.

“By providing flexibility in application and obviating the need for exogenous materials or electrical fields, this method could potentially enable new avenues of disease research and treatment,” they wrote.

Although intracellular delivery of macromolecules is a key step in therapeutic and research applications, the cellular membrane is largely impermeable to such compounds, according to the PNAS paper. Existing methods to overcome this hurdle, which has proven to be a major stumbling block for RNAi drugs, typically involve the use of polymeric nanoparticles, liposomes, or chemical modifications of the target molecules to facilitate membrane poration or endocytotic delivery.

When it comes to nucleic acids, which are relatively structurally uniform, these approaches can be efficient. Still, the “endosome escape mechanism that most of these methods rely on is often inefficient; hence, much material remains trapped in endosomal and lysosomal vesicles,” the MIT team pointed out. “More effective gene delivery methods, such as viral vectors, however, often risk chromosomal integration.

Meantime, electroporation has proven effective, even in difficult to transfect primary cells, but has limited applicability and can cause cell death. Microinjection, too, has certain advantages in settings such as the creation of transgenic organisms, but its low throughput hamstrings many therapeutic and research applications, the researchers noted.

To overcome the limitations of existing delivery techniques, the MIT group had initially been attempting to “shoot” molecules of interest into cells, Armon Sharei, an MIT graduate student in chemical engineering and lead author of the PNAS paper, told Gene Silencing News.

“That system had its own challenges, and through the course of that work, we stumbled upon this effect where if you squeeze the cells rapidly enough, it will temporarily disrupt their membrane,” he said.

More specifically, the researchers found that the “rapid mechanical deformation of a cell, as it passes through a constriction with a minimum dimension smaller than the cell diameter, results in the formation of transient membrane disruptions or holes,” they wrote in PNAS. “The size and frequency of these holes would be a function of the shear and compressive forces experienced by the cell during its passage through the constriction. Material from the surrounding medium may then diffuse directly into the cell cytosol throughout the life span of these holes.”

To test this idea, the researchers constructed devices, each consisting of 45 identical, parallel microfluidic channels containing one or more constrictions, etched onto a silicon chip and sealed in glass. The width of each constriction ranged from 4 to 8 micrometers, and the lengths ranged from 10 to 40 micrometers.

“Before use, the device is first connected to a steel interface that connects the inlet and outlet reservoirs to the silicon device,” the researchers wrote. “A mixture of cells and the desired delivery material is then placed into the inlet reservoir and Teflon tubing is attached at the inlet. A pressure regulator is then used to adjust the pressure at the inlet reservoir and drive the cells through the device. Treated cells are collected from the outlet reservoir.”

The system was tested with a variety of molecules, including carbon nanotubes and proteins, as well as siRNAs targeting GFP. According to Sharei, when the siRNAs were delivered into GFP-expressing HeLa cells using the microfluidic platform, the investigators were able to achieve 80 to 90 percent target knockdown.

He noted that the knockdown effects weren’t as robust as with Lipofectamine 2000, but “we were still encouraged because something like Lipofectamine is known to be toxic and therefore inapplicable for humans.” Notably, the microfluidic device and operating parameters were not optimized for siRNAs, further limiting its ability to compete with the transfection reagent in these studies.

“The other good thing was that we seem to work just as well for primary cells, whereas existing methods like Lipofectamine don’t translate well once you start moving out of the standard cell models you have in the lab,” he added.

The MIT team also successfully delivered 3 kilodalton dextran molecules — which are approximately the same size as a standard siRNA molecule and a “pretty accurate” surrogate for the gene-silencing molecules — into newborn human foreskin fibroblasts, primary murine dendritic cells, and embryonic stem cells, suggesting that the method could be used with siRNAs into a variety of cell types, Sharei said.

Buoyed by the positive data, he and his colleagues are now further testing the platform with siRNAs against “easy readout genes” in primary cells including immune cells and stem cells, he said. “Once we establish that, we’d try to go for an application where there’s an siRNA that’s going to knock down something functional.

“I can’t say exactly what we’ve been up to because it’s not published, but it has been going pretty well,” he added.

Ultimately, the MIT group aims to develop the microfluidic platform not only for research applications, where it could be “incorporated into a larger integrated system consisting of multiple pre-treatment and post-treatment modules” that could take advantage of its average throughput rate of 20,000 cells a second, but also therapeutic ones, too.

A number of investigational stem cell-based therapies, for instance, involve the ex vivo manipulation of the cells, Sharei said. The delivery platform could theoretically be used to “enhance or facilitate that manipulation.”

“Such an approach would take advantage of the potentially increased delivery efficiency of therapeutic macro- molecules and could be safer than existing techniques because it would obviate the need for potentially toxic vector particles and would mitigate any potential side effects associated with reticuloendothelial clearance and off-target delivery,” the study authors wrote in PNAS.

Doug Macron is the editor of GenomeWeb’s Gene Silencing News. He covers research and therapeutic applications of RNAi, miRNA, and other gene-silencing technologies. E-mail Doug Macron or follow his GenomeWeb Twitter account at@Genesilencing.

 

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Reporter: Aviva Lev-Ari, PhD, RN

On August 18, 2012, I needed scientific inspiration. To nurture my imagination I surf on http://www.mit.edu for 4 hours. Comment, Please.

The following links I am sharing with you from my exploration session to be inspired

Protein that boosts longevity may protect against diabetes: Sirtuins help fight off disorders linked to obesity, new MIT study shows.

http://web.mit.edu/newsoffice/2012/sirtuins-may-protect-against-diabetes-0807.html#.UC9iyFFg1yk.facebook

Growing the best implant tissue | MIT video

http://video.mit.edu/watch/growing-implant-tissue-on-3-d-scaffolds-12286/

MIT 2012 Commencement Address

http://www.youtube.com/watch?v=Pn24jP0YbTI

Salman Khan talk at TED 2011 (from ted.com)

http://www.youtube.com/watch?v=gM95HHI4gLk&feature=relmfu

 

We are on Facebook

http://www.youtube.com/watch?v=gM95HHI4gLk&feature=relmfu

Cello Music Concert by Jacqueline du Pre

http://www.google.com/#hl=en&sclient=psy-ab&q=jacqueline+du+pré+elgar+cello+concerto&oq=Jacqueline+du+Pré&gs_l=hp.1.3.0l4.0.0.2.690.0.0.0.0.0.0.0.0..0.0.les%3B..0.0…1c.q26G86iICpE&pbx=1&bav=on.2,or.r_gc.r_pw.r_qf.&fp=4d5ad5fc55e3e15d&biw=1038&bih=778

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