BCR-ABL1 Splice Patterns & Generation of CML RNA bank

Placement PresentationDeborah Reilly7th May 2015

Paul O’Gorman Leukemia Research Centre

• Is a division of the University of Glasgow's Institute of Cancer Sciences

• Situated in the grounds of Gartnavel General Hospital which is located in the west end of Glasgow

• Research and objectives:

- Centre’s research is focused around the understanding of the haematopoietic stem cell (HSC)and its key role in the development of the vast variety of leukemic diseases

- Centre’s objective is to explore and identify therapeutic targets for leukaemia and in turn produce new medication for patients

The Philadelphia chromosome, hallmark of CML, results from translocation between chromosomes 9 and 22

Nature 1973, 243, 290 - 293

Bcr-Abl

# 9 # 22

c-Abl

Bcr

Ph1

Janet RowleyPeter NowellDavid Hungerford

Science 1960,132:1497

2 11

11

5’ 3’

23

mBCR

13 14

MBCR

15 16 17 18 19 20 21 22

mBCR

1 2 3 4 5 6 7 8 9 10 11 12

5’ 3’

1b 1a 2 3 4 5 6 7 8 9 10 11

c-ABL gene (chromosome 9)

BCR gene (chromosome 22)

13

2 11

p230bcrabl

c-ABL geneBCR gene

1

21 13

1 19

p190bcrabl (e1a2)

p210bcrabl (e13a2, e14a2)

13

Project Outline (10 weeks)

• Set up/Learn techniques (2 weeks).

• Produce a bank of RNA/cDNA from: (i) peripheral blood samples, (ii) CML cell lines, and (iii) primary CD34+ CML cells

• Determine baseline CFC

• Determine the BCR-ABL1 breakpoint using PCR

• Determine percentage CD34+CD38- by flow cytometry

• Use FISH to visualise the fusion oncogene

Functional Analysis of CML Stem/Progenitor Cells

Colony Forming Cells (CFC) assay

- Measures relatively mature, committed progenitor cells

CD34+ cellsMethylcellulose (H4034)

12-14 days in culture

Total number of

colonies counted

CFC Results

CML 404:

BFU-E

BFU-E

CML 342:

BFU-E + CFU-GM

CML 361:

CFU-GM

CML 378:

BFU-E

BFU-E + CFU-GM

PCR Changes

• The dNTPs dilution concentration was noticed to be x10 out – was amended

• The annealing temperature for the BCRABL1 F/R primers was calculated >60°C

The annealing temperature for the Actin 1/2 primers was calculated to be 58°C

- was running at 55°C , so had to be increased for BCRABL1

• Fresh cell line source as positive control

• An appropriate PCR programme on another thermal cycler was chosen

• Optimum PCR conditions discovered:

- For the BCRABL1 F/R primers:

- 3µM [Mg2+] addition with an annealing temperature of 65°C

- For the Actin 1/2 primers:

- 2µM [Mg2+] addition with an annealing temperature of 60°C

PCR Results

KY01

Cell lineages at their optimum PCR for primers BCRABL1 F/R and Actin 1/2:

b3a2 (13)

b2a2 (14)

Em2 Bv173 K562

KY01 NB4 Em2 Bv173

KY01

Splice variants of primary CD34+ CML samples:

KCL22 K562 CML 378

KY01 Em2 K562 CML 342

Were not carried out in the optimum PCR conditions

Actin (housekeeping control)

Flow cytometry Results

• All of the primary samples were accessed for their viability and CD34+ CD38- expression using flow cytometry

CML 404:

Q4 = 12.4%

FISH

• Probe properties:

- BCR = Green

- ABL = Red

- Fusion = Yellow

• Cell lineage K562 was used as the positive control:

- Larger than CD34+ stem cells

- Number of BCR:ABL signals roughly 3:2

- Have around 10 copies of the fusion oncogene per cell

• Primary CD34+ CML 416 sample:

- 1 BCR signal, 1 ABL signal and 2 fusion signals per cell

K562 CML416

Table of Results + RNA/cDNA bank

UPN % Recovery Actin BCR-ABL1 breakpoint CFC /100 cells RNA (ng/µL) 260/280 % CD34+ CD38-

308 0.5 - - - - - 35.5

339 84 - TBD 1.78 90.15 2.05 36.4

342 130 b2a2 (13) 1.43 158.63 1.97 17.1

361 15 - TBD 0.49 2.7 1.85 36.9

372 70 ND 0.77 43.04 1.92 13.0

378 84 b3a2 (14) 1.08 23.65 2.18 13.0

379 44 - TBD 0.16 3.78 2.32 ND

381 50 b3a2 (14) 0.29 7.01 2.11 32.4

404 62 - TBD 1.67 60.87 2.02 12.4

416 48 - - - - - -

SAMPLE RNA cDNA

CML 308 x x

CML 339

CML 342

CML 361

CML 372 x

CML 378

CML 379 x

CML 381

CML 404

CML 416 x x