Mutagenesis in Ataxia Telangiectasia Induced Pluripotent Stem Cells

Lucy LinDr. Ronald P. Hart

Department of Cell Biology and NeuroscienceRutgers, The State University of New Jersey

Summer Undergraduate Research Fellowship 2015

Ataxia Telangiectasia• Ataxia = loss of motor control; Telangiectasia = spider veins• Neurodegenerative, inherited disease caused by mutation in ATM

gene– Causes tumor formation– Impairs cerebellum– Prevents repair of broken DNA

• Some symptoms– Nystagmus (rapid involuntary eye movement)– Ocular telangiectasia

ATM Kinase Protein• 370 kilodalton• Serine/threonine protein • Member of the phosphatidylinositol 3-kinase related kinase (PIKK)

family important for DNA repair regulation• Regulates DNA damage response cascade to arrest cell cycle for

DNA repair

Llorca et al., 2003

DNA Damage Repair Cascade

Matsuoka et al., 2007

Induced Pluripotent Stem Cells• Cells that can be reprogrammed from adult

somatic cells through transcription factors to become pluripotent

Bellin, M., Marchetto, M. C., Gage, F. H., and Mummery, C. L., 2013

Retrovial infection with 4 transcription factors: Oct3/4, Sox2, cMyc, Klf4

A-T Cell Lines are PluripotentOct-4 Tra-1-60

Q1-SA

Q3-SA

Q3-SC

CAR3-SB

ATM -/-

ATM -/-

ATM -/-

ATM +/-

• CAR3 Cells (carrier): Deletion causing frameshift on one allele and one normal allele

• Q1 and Q3 Cells (A-T): Compound heterozygote - deletion causing frameshift on one allele and point mutation on other

Hart, R. P., unpublished

A-T Cell Line ATM Allele Mutations

Lazaropoulos, M., unpublished

Subject Code

Age Sex Diagnosis Mutations iPSC Lines

JHU_Q1 23 F A-T c.[1564delGA];[7181C>T]

Q1-SA

JHU_Q3 8 M A-T c.[7792C>T];[217_218delGA]

Q3-SAQ3-SC

JHU_CAR3 42 F Carrier Not tested CAR3-SB

Q1 and Q3 Allele Mutations

Hart, R. P., unpublished

Subject Code

Age Sex Diagnosis Mutations iPSC Lines

JHU_Q1 23 F A-T c.[1564delGA];[7181C>T]

Q1-SA

JHU_Q3 8 M A-T c.[7792C>T];[217_218delGA]

Q3-SAQ3-SC

Actin

pChk2

Q3-SC Cells Induce pChk2

High levels of γH2A.X seen in Q3-SC cells after X-irradiation

ATM +/- ATM -/- ATM -/- ATM -/-

Q3-SA and Q3-SC Cells are from the same subject

Hoffman, G., unpublished

Hypothesis

• Spontaneous mutagenesis is exacerbated by the absence of ATM-guided DNA repair, which can lead to reversion of the ATM gene to produce functional ATM kinase–All ATM -/- cells are more sensitive to

reversion than other ATM genotypes

Methods• Introduce DNA damage to the cells

through X-Irradiation to induce mutagenesis and perhaps reversion

• Incubate cells for another week to allow time for reversion

• One week after the first irradiation, irradiate cells again to elicit pATM formation

• After fixing and staining cells, images were taken using IN CELL Analyzer 6000, an automated confocal microscope

X-Irradiation

Double Strand Breaks

pATM formation after irradiation seen in all ATM -/- cell lines

Images and Data Collected by Gary Hoffman, 2015

Conclusions• Further support of reversion in Q3-SC cells• Possibility of reversion in Q3-SA cells • Reversion in Q1-SA cells?

Hart, R. P., unpublished

S1981

Future Directions

• Repeat mutagenesis experiment with the addition of staining for γH2A.X along with pATM

• Establish IC50 of ATM inhibitor for control cells and genomically reverted A-T cell line Q3-SC

AcknowledgementsHart Lab• Ronald P. Hart, PhD• Alana Toro-Ramos• Gary Hoffman• Michael Lazaropoulos• Eileen Oni• Mavis Swerdel• Jennifer Moore, PhD• Angela Tiethof• Kunal Garg• Sri Puli

Lab Collaboration• Lourdes Serrano, PhD• Zhiping Pang, PhD

SURF Directors• Lauren Aleksunes,

PharmD, PhD• Debra L. Laskin, PhD

Funding• National Institutes of Health

R25ES020721 Grant• A-T Children’s Project of the

ATCP Foundation

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