Business from technology

TK4017 Mitosis and Drug Discovery Team: Cell Cycle and Mitosis Research at VTT

Marko Kallio, PhD Vice Technology Manager and Team Leader VTT Biotechnology for Health & Wellbeing

Cell Cycle Research at VTT

24/08/2012

Service focus on drug target discovery and validation

- We perform compound screens, hit-to-lead confirmation, mechanism-of-action studies, and functional protein studies in cells using VTTs proprietary technologies
Cell cycle progression (FACS, IF, live cell monitoring, etc) Cell growth (suppression) analyses (apoptosis, colony formation assays, etc) Dissection of the target protein / pathway effects (ptw activity analyses, etc) Gene and protein expression analysis (qPCR, affimetrix, HTS WB, etc) In-silico data mining with VTT bio-informatics toolbox (REX, IST, etc)

Differentiating competences
- Utilisation of proprietary VTT technologies such as 3-D cell culture models and HTS RNAi - In-depth knowledge on mitotic and cancer cell signalling - Packaging of offering into large entities (technologies + know-how)

Cell Division Research at VTT

Research focus on
1. Discovery of novel targets for cancer intervention
- HTS cell-based assays for identification of novel anti-mitotic factors (siRNA/miRNA) - Discovery and target validation of LMW compounds

24/08/2012

2. Mechanisms of cell division events

- Study of the mitotic checkpoint and functional analysis of mitotic proteins - Kinetochore biochemistry - Tubulin in vitro assays

Gene Expression Studies at VTT

Work-flow of a typical project
The project is planned together with the customer to meet the needs
1. 2. 3. 4. 5. 6. 7. 8. 9.

24/08/2012

Cells in culture (2-D, 3-D) are treated with a panel of compounds (negative and positive controls) Stress factors (UV, H2O2, heat shock) are added prior or after the compounds The fate of the cells is monitored using live cell imaging At desired time points samples are collected for RNA / protein extraction, biomarker analysis (IF, WB) Target gene (or whole genome) gene expression analysis is performed Additional analyses at the protein level Bio-informatics data mining Reporting to the customer including scientific insights Follow up

Cell Division Research at VTT Featured Project #1

24/08/2012 5

Discovery of novel anti-mitotic compounds targeting Hec1

Highly expressed in cancer 1 (Hec1) high expression levels in cancer are linked to poor prognosis Key roles in microtubule-kinetochore attachment chromosome segregation Chemical and genetic perturbation of Hec1 result in chromosome misalignment defects in spindle checkpoint signalling arrest in mitosis and cell death reduction in tumour growth in mice High expression of Hec1 in dividing cells may increase the cancer cell selectivity of anti-Hec1 therapeutics and cause less side effects. Hec1 is a potential target for cancer intervention by small molecules
Hec1

www.cbs.dtu.dk/staff/dave/roanoke/bio101ch09. htm www.cbs.dtu.dk/staff/dave/roanoke/bio101ch09.htm

Ndc80 complex

Hec1 project work flow and current status

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2. Cell-based HT functional screen => 5 compounds forwarded to in cells analyses 1. Structure-based in-silico screen (4 M compounds) => 150 best scoring compounds purchased
0.10 0.09 0.08 0.07 Ndc6 + Bonsai Ndc6 + AurB Ndc6 + Plk1

Ndc6 + Bonsai Bonsai

Buffer
Cell line HeLa MCF7 Ndc106 2,0 4,4 Ndc106 a1 1,3 2,0 no effect 0,9 0,5 1,4 1,0 2,8

Anisotropy

Ndc106 a2 0,6 0,5 2,4 0,4 0,2 2,4 1,5 2,7

0.06 0.05 0.04 0.03 0.02 0 200 400 600 800 1000

MCF10A no effect MDAMB-231 LnCaP HCT116 2,6 2,5 2,5 3,9 3,6

Anisotropy measurement
Ndc6 + Aurora B
Time (s)

A549 Ovcar-3

4. Hit compound binding to Hec1 => 2 compounds positive 3. Hit compound phenotyping => 3 compounds to binding assays

Aurora B

Buffer

5. Next steps: - chemical optimisation - animal experiments

Cell Division Research at VTT Featured Project #2

24/08/2012 7

Discovery of anti-mitotic miRNAs

Work-flow
Target library: Dharmacon miRIDIAN mimic library (810 miRNAs) for gain-of-function miRNA studies Cell lines: HeLa H2B-GFP, MCF7, MCF10A Two endpoints: mitosis arresting miRNAs and forced mitotic exit inducing miRNAs (override of taxol block)

1. HTS for mitosis arresting miRNA

High MI (>20%) 72 h transient transfections live cell imaging + Image analysis no apparent cell cycle effect 2. HTS for SAC overriding miRNA 100 nM taxol for 12 h

M-phase arresting miRNAs (n=3)

Validation of the primary hits

Forced exit inducing miRNAs (n=3)

forced exit from mitosis (NucF 60%)

New anti-mitotic miRNAs cause genomic imbalance

24/08/2012 8

miRNA-378*

mir-378 overrides chemically hyperactivated mitotic checkpoint and causes massive genomic imbalance and cell death
DNA miR-378* control miR pCenp-A

***

miRNA-378* perturbs normal cell division and causes chromosome missegregation miRNA-378* hyperactivates VEGFR2 and PDGF pathways leading to activation of MAPK cascade

control miR miR-378*

miRNA-378* targets indirectly AurB via MAPK and therefore causes the escape from mitosis

Cell Cycle and Mitosis Research at VTT

24/08/2012

Marko Anna-Leena Adel Jenni Leena Elli Mahesh

References:
Sebastian

Several commercial projects performed for pharmaceutical and cosmetics industry (2007-ongoing); study of MoA of companies experimental drugs and bioactives Discovery of anti-mitotic compounds - 9 notification of invention - Identification of 3 new Aurora B inhibitors - Hec1 pharmacophore Recent publications - Salmela et al Carcinogenesis, 2009 - Kukkonen-Macchi et al JCS, 2011 - Vuoriluoto et al. Mol Oncol, 2011 - Salmela et al ECR, 2012

Mitosis and Drug Discovery Team VTT TK4017