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FFPE histone enrichment for antibody and mass spectrometry analysis of post translational modifications in epigenetic biomarker discovery

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R43ES028553-01
Agency Tracking Number: R43ES028553
Amount: $225,000.00
Phase: Phase I
Program: SBIR
Solicitation Topic Code: NIEHS
Solicitation Number: ES16-012
Timeline
Solicitation Year: 2016
Award Year: 2017
Award Start Date (Proposal Award Date): 2017-09-15
Award End Date (Contract End Date): 2019-08-31
Small Business Information
1914 PALOMAR OAKS STE 150
Carlsbad, CA 92008-6509
United States
DUNS: 109145701
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 MARY JELINEK
 (760) 431-1263
 mjelinek@activemotif.com
Business Contact
 THEODORE DEFRANK
Phone: (760) 431-1263
Email: defrank@activemotif.com
Research Institution
N/A
Abstract

PROJECT SUMMARY
Many environmental stresses known to cause chronic or acute disease induce changes in epigenetic
mechanisms regulating chromatin structure and gene expression One manifestation of this epigenetic
dysregulation is changes in global levels of histone modifications such as acetylation methylation
phosphorylation etc Global changes in several histone H and H post translational modifications
PTMs have been observed in autoimmune inflammatory neurological and neoplastic diseases for
which in the latter are predictive of clinical outcome for several primary tumor types This strong
association with disease raises the possibility that histone modifications have potential as a new class of
epigenetic biomarker for a wide range of disease types and in environmental exposure risk management
Archived formalin fixed paraffin embedded FFPE clinical samples are extremely valuable for biomarker
identification since they are often accompanied with important information regarding patient history
treatment courses and clinical outcome However evaluation of histone PTM as disease biomarkers in
FFPE samples has not been possible as the extensive crosslinking of proteins in FFPE material makes
protein extraction very challenging and current PTM detection methods require large amounts of fresh or
frozen samples This Phase I proposal intends to develop histone enrichment methods from FFPE samples
while preserving histone PTMs by adaptation of chromatin extraction methods developed for FFPE ChIP
seq Chromatin extraction methods in effect result in histone extraction albeit in the context of genomic
DNA which can be removed Aim efforts will develop a histone enrichment procedure compatible with
downstream antibody based detection methods including multiplexing bead based ELISAs that can
quantitate multiple PTMs in the same sample Aim efforts will focus on method development
compatible with mass spectrometry histone PTM quantitation Although mass spectrometry requires
additional material the method allows for the unbiased profiling of diverse modifications including
discovery of novel PTMs and bypasses the need for characterized antibodies Initial experiments will be
performed using environmental exposure target tissues lung liver and kidney isolated from mice
following sham or histone deacetylase inhibitor treatment with comparisons made between fresh frozen
and FFPE samples to demonstrate histone PTM preservation Following successful development of these
histone enrichment methods for mouse FFPE samples a proof of principle experiment to establish the
applicability of the method to human samples will be performed with matched tumor and healthy clinical
samples from a glioma patient Successful completion of these efforts will enable the mining of large
cohorts of archived clinical samples for potential histone PTM biomarkers in future Phase II efforts Exposure to environmental stresses that result in chronic and acute diseases are known to cause changes in gene
expression patterns and expression associated histone marks This proposal intends to develop a method for isolation of
histones from archived clinical samples to enable evaluation of disease associated changes in histone mark patterns as
biomarkers for diagnostic and prognostic applications

* Information listed above is at the time of submission. *

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