Studying the MoA of the next generation chemotherapeutic LP-284, a small molecule acylfulvene in a chronic myelogenous leukemia cell line model using STRIDE™

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At a Glance

Case Study

Partner – Industrial

Client – Pfizer

Published data

Challenge

Application Domain

  • DDR
  • Next generation chemotherapeutics

 

Stage in the Drug Discovery Pipeline

  • Preclinical

 

Prior Attempts

  • Viability assays
  • Assessment of mouse xenograft tumor volume

 

Goals of the Project

The main objectives of the project

  • Provide proof in an in vitro leukemia cell line model that the antitumor activity is driven by excessive DNA damage that leads to apoptosis in cancer cells,
  • Verify whether the phosphorylation of histone 2Ax is the direct consequence of double-strand DNA breaks or the result of cellular stress not directly related to DNA lesions.

Execution

Stage 1

2 weeks

Preparation and Optimisation

  • Shipment from the US: 4 days
  • One of intoDNA`s own cell line was used for the experiments that was cultured for a week prior any experiment
  • A new cell death marker was purchased, tested and optimized

Stage 2

2 weeks

STRIDE and co-staining execution

  • Study design : The compound was tested in 3 timepoints and 1 concentration (set based on IC50 evaluation) in duplicates
  • We stained with dSTRIDE, γH2AX, cleaved caspase3 and DAPI
  • Number of slides stained: 13

Stage 3

1 week

Imaging and analysis

  • The leukemia cell line HAP1 was treated with vehicle (DMSO) or LP-284 at IC50 for 6, 24, and 72 hours. A total of 463,123 nuclei in the vehicle group and 316,704 nuclei in the LP-284 group were surveyed
  • Analysis part: schematic showing: an original image of the nuclei, an original image of foci and original image of gamma → nuclear masks and labeled foci (our algorithm) → QC step mentioned → results: number of foci / nucleus, integrated intensity of gamma / nucleus


IMG. 1

Stage 3. Representative images of STRIDE staining with dSTRIDE in yellow and DAPI in blue in HAP1 wt cells treated for 72h with vehicle .

Customer-specific Adaptations

Successful implementation of a new marker (Cleaved Caspase-3)

Results


FIG. 1

Bar plot showing the mean number of DSBs in the nuclei of HAP1 wt cells. Data shown as mean of the data points of the duplicates.

FIG. 2

Bar plot showing the mean integrated intensity of γH2AX in the nuclei of HAP1 wt cells. Data shown are the mean of the duplicates.

 

FIG. 3

Bar plot showing the mean integrated intensity of cleaved caspase3 in the nuclei of HAP1 wt cells. Data shown are the mean of the duplicates.

 

 

Project Findings

IMG. 1

Goal Attainment

  • Direct proof for DSB formation as an immediate on-target effect of the drug,
  • Proof for the generated DSBs being lethal,
  • Project delivered in 30 business days.

Customer Benefits

Before STRIDE there were no methods for precise and sensitive labeling of DNA breaks. Here dSTRIDE enabled to reveal the underlying mechanism of action of the tested compounds’ anti tumor activity.

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We’re on a mission

to enhance the quality of DNA damage diagnostics and to improve any therapies where DNA integrity matters.

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