A NOVEL CANCER STEM CELL (CSC) DRUG DISCOVERY PLATFORM

Collaborative Opportunity

March 2014

PURPOSE

Cancer Research UK funded Investigators have established in vitro and in vivo models of CSCs for

use as a drug discovery platform to (i) identify and validate novel CSC biomarker and therapeutic

targets and (ii) develop anti-CSC therapies. It is hypothesised that by targeting CSCs it may be

possible to prevent metastasis or reduce the risk of tumour recurrence. The platform was

developed and validated by Prof Fiona Watt (Director of the Centre for Stem Cell and Regenerative

Medicine Research at King’s College) and co-investigators Prof Norman Maitland (University of

York), Dr Rob Clarke (University of Manchester) and Prof Alan Clarke (Cardiff University).

The platform and related discoveries and intellectual property are carefully managed by Cancer

Research Technology (CRT).

CRT wishes to discuss the opportunity to collaborate with commercial partners in the field of

cancer stem cell drug discovery. This document outlines a proposed collaborative programme for

consideration.

OPPORTUNITY

CRT is seeking to identify a commercial pharmaceutical partner to utilise the novel platform for collaborative

drug development and/or to identify or validate novel biomarker and therapeutic targets for CSCs.

Validated cellular screens and secondary in vitro validation assays have been developed using stem cells

derived from human tumour tissue from breast (Dr Rob Clarke), prostate (Prof Norman Maitland) and head

and neck squamous cell carcinoma (HNSCC) (Prof Fiona Watt) in the first instance. These tumours are known

to have a hierarchical organisation consistent with the CSC hypothesis and are tumours in which conventional

treatments are inadequate. Xenograft models of these same cell types have also been developed and can be

used as part of a validated CSC drug discovery platform. In addition, autochthonous tumour models of breast

and prostate cancer are available to test effects of novel agents in a more natural setting. Robust markers of

stem cells have been validated across the different indications which serve as a fingerprint to map the effect of

novel treatments on the stem cell component of a tumour, a key requirement in developing CSC-specific

therapies.

While other groups have established a number of in vitro and in vivo assays to interrogate the CSC component,

the use of patient-derived assays across different indications provides a unique platform to explore the effects

of agents targeting the CSCs.

The following provides an overview of the discovery platform that has been developed for use in drug

discovery and target discovery/validation (see Figure 1).

Drug Discovery:

The entire drug discovery platform would be available to our partner either for use in drug screening or to

validate any hit matter or combination treatments. CRT would be happy to discuss further with potential

partners as to how optimal use could be made of the platform for such a purpose. CRT’s Discovery Labs

(www.cancertechnology.com/discovery/discovery_laboratories) could also be considered as a potential

partner in drug discovery. In addition, a number of CRT’s proprietary agents have been tested in the assays and

shown to have an anti-CSC effect. We would be interested in discussing routes to progress these agents

collaboratively.

Target Discovery/Validation:

The platform could be used to identify novel targets using siRNA libraries. The investigators have previously

identified novel targets and are keen to use expression profile data and bioinformatic approaches to identify

additional novel targets.

The function of novel targets identified (or proposed by our partner) can be assessed in the platform using

knockdown and over expression using lentiviral or retroviral constructs. Assays include cell proliferation, cell

death (caspase/annexinV), differentiation, colony formation and invasion (Boyden chamber assays). Novel

targets would be validated by expression analysis both in the established in vitro and in vivo assays and in

sections of human cancers (immunostaining or in situ hybridisation or by FACS analysis of cells from

disaggregated fresh tissues).

Figure 1: Patient-derived CSC drug discovery platform

Tumours are derived from patients and confirmed to contain a stem cell component based on expression of known markers. Ongoing efforts within the Consortium focus on the further optimisation of markers to refine isolation or identification of CSCs. Tumour cells are used to assess the in vitro effect of agents/targets on differentiation of the stem cell component and ability of stem cells to form colonies and self-renew. Patient-derived xenograft assays have been established to demonstrate in vivo efficacy.

THE CSC DRUG DISCOVERY PLATFORM

A major limitation of current in vitro CSC assays is that many of the cell lines in widespread use were

established decades ago and are of unknown provenance. This raises serious questions about whether the

cells are still a good model for the original tumours or whether their properties are a response to long term

culture. Studies of primary tumour cells are clearly desirable but relatively few laboratories have sufficient

know-how or access to clinical material to be able to take this approach. To obviate this difficulty, techniques

have been developed for isolating and culturing CSCs from primary tumours (see Table 1).

Table 1: Patient-derived lines and cancer stem cell component

CSC patient-derived cell lines

HNSCC Breast cancer Prostate cancer

Tumour types Tumour cells derived from SCC of tongue, lip, gingival.

Tumour cells derived from pleural effusions, ascites and ER+ tumours. ER- and triple negative tumours (TNT) are being established.

Tumour cells derived from intraprostatic cancers and castration resistant tumours.

All lines are QC’ed for ability to form colonies in vitro and expression of putative stem cell markers. The group are aiming to establish approximately 80 HNSCC lines, 30 xenograft-derived breast cancer lines and collect prostate cancer samples weekly. The stem component of the tumour derived cells is analysed by expression of known markers (see below).

Patient information

Differentiation and life-style factors are known for each patient sample. Whole exome sequencing has been performed and Notch and p53 status is known.

Tumour grade and receptor status (ER, PR, Her2) are known for each sample.

Tumour grade and previous treatment regimens for samples have been collated. In addition, expression of Notch signalling components has been analysed for a number of samples.

Samples have been collected from a range of tumour backgrounds. Data has also been collected on signalling factors of relevance to CSC biology.

Enrichment of CSC population

1integrin/CD44+(/Frmd4A) ESA+/CD44+/CD24- CD44+/21HI/CD133+

A panel of putative markers of CSCs was tested in each of the tumour types. This panel included 1integrin, CD44, ESA, Notch1/4, CXCR4, Lrig1 and FRMD4A. Many of these markers were found to be expressed in CSCs from all three indications, however, expression was found to be heterogenous and did not enrich specifically for CSCs. The markers high-lighted above are used routinely in enrichment steps. Frmd4A may be of value in combination with the listed markers specifically in HNSCC.

Detection of CSCs in vivo

CD44+/Frmd4A

ESA+/CD44+/CD24- CD44+/ CD24-/CD133+

Each disease has its own specific sets of marker combinations for CSC identification in vivo. CD44 appears to be a robust marker common to all three indications. The marker combinations have been shown to be robust in identifying sub-populations of cells with self-renewal capacity, and these marker sets would be suitable indicators to monitor the dynamic changes that occur in CSC populations during in vivo treatment with anti-CSC therapies. In parallel to the patient-derived in vivo models, CD44 also appears to be a marker of CSC activity (although insufficient as a stand-alone marker) in autochthonous breast and prostate models.

Quantitative readouts of CSCs in vitro, including differentiation and cell death amongst others have been

devised. A description of the in vitro assays available is high-lighted in Table2 below.

Table 2: Description of patient-derived in vitro assays available for use

CSC patient-derived tumour models

HNSCC Breast cancer Prostate cancer

Culture conditions

Supplemented FAD medium grown on a J2 3T3 feeder layer.

Supplemented serum free media. Stem cell media (serum free) with irradiated STO feeders.

HNSCC lines are routinely cultured to passage 10 in vitro (and are stable to passage 20), primary patient breast cancer cells do not grow well in vitro but are readily passaged in vivo, prostate cancer lines can be cultured to passage 8.

Multi-well Differentiation (In-Cell Western assay using trans-glutaminase) is chosen as primary read-out for effect of a novel agent/target. HNSCC cells can be assayed in 96 and 384-well format.

Differentiation is chosen as primary read-out (CD24 as marker) for effect of a novel agent/target. Due to the nature of the patient-derived cells, assays are being developed in a 24-well format.

Differentiation is chosen as primary read-out (Prostatic Acid Phosphatase) for effect of a novel agent/target. Due to the nature of the patient-derived cells, assays are being developed in a 24-well format.

Validation Using a commercially available small molecule drug(-like) library, a number of agents were identified that induced differentiation in CSCs.

On-going On-going

Purpose A first-step throughput assay to screen multiple agents/targets for their effect on differentiation or other cellular effects such as cell death and proliferation. Other appropriate read-outs could include quantification of ALDH levels. The assays utilise a mixed population of cells although the format used enriches for putative stem cells in the population. Hits identified in these assays should be examined for their specific effect on the stem cell population. Control assays including “bulk” tumour, normal stem cells or other appropriate assays will be included.

Agents/targets can be tested in one or more tumour indication.

Secondary Formation of colonies capable of growth for at least 14 days. Ability of such colonies to self-renew is also investigated.

Formation of mammospheres capable of growth to >50um. Ability of such colonies to self-renew is also investigated. This assay can be conducted in a 384-well assay using high-content imaging (Imagen Biotech).

Formation of colonies containing at least 32 cells. Ability of such colonies to self-renew is also investigated.

Validation

(see Figures 1-2)

Effect of known Notch pathway inhibitors has been tested (DAPT, flurbiprofen, RO4929097). No effect of Notch inhibition was observed.

Effect of known Notch pathway inhibitors has been tested (DAPT, RO4929097, DBZ). Notch inhibition decreased primary mammosphere formation as expected but had no effect on self-renewal.

Proprietary agents have also been tested in these assays.

Effect of known Notch pathway inhibitors has been tested (DAPT, RO4929097, DBZ). Inhibition resulted in a significant reduction in colony-forming efficiency with concomitant activation of another “stemness” crosstalk pathway.

Purpose The effect of agents/targets on primary colony formation and secondary colony formation (self-renewal) demonstrates a stem-cell specific effect. To control for variation across patient-derived samples, at least 5 representative patient samples will be used.

Agents/targets can be tested in one or more tumour indication.

While in vitro CSC assays are inexpensive, provide a rapid and quantitative readout of CSC activity, and are

amenable to high throughput assays of potential therapeutics, a major disadvantage is that CSCs, like normal

stem cells, are heavily influenced by their local environment. Therefore, potential CSC markers and drugs must

undergo further validation in vivo. The Consortium has established patient-derived xenografts and also uses

autochthonous mice (that is, mice in which the tumour is induced from the animal’s own cells) as a

complementary in vivo CSC assay (see Table 2 for more details). These assays have been validated using the

RO4929097 Notch pathway inhibitor (see Figure 2).

Table 2: Description of patient-derived in vivo assays available for use

CSC patient-derived tumour models

HNSCC Breast cancer Prostate cancer

Xenograft Tumour derived cells are injected into the base of the tongue; tumours grow locally and form widespread metastases. Treatment can occur at time of inoculation or once tumours are established. Tumours can be monitored in vivo through use of luciferase tags.

Subcutaneous tumour models have been established (106 Lin- breast cancer cells sc injected into NSG mouse). The effect of treatment on tumour initiation or on established tumours can be measured. Latency of ER+ tumour development can vary between 120-180days.

Subcutaneous tumour models have been established (104 Lin- prostate cancer cells are sc injected in matrigel). The effect of treatment on tumour initiation (ex vivo treatment of cells ensures a cost-effective in vivo experiment) or on established tumours can be measured. Latency of tumour development can vary between 30-75 days for different patient samples.

Validation

(see Figure 3)

Mice were treated with Flurbiprofen, DAPT and RO4929097. No significant effect on tumour growth was observed.

PDX tumours have been treated with RO4929097 for 14 days. Treatment did not prevent tumour growth but reduced stem cell activity (as detected by mammosphere formation of cells derived from tumour) by 25%; inhibition could be increased to 50% in combination with chemo/radiotherapy.

Ex vivo treatment of xenograft cells with RO4929097 resulted in increased tumour latency and loss of self-renewal after serial xenotransplantation, indicating a direct effect on CSC self-renewal.

Purpose The effect of lead targets/agents on the stem cell component can be measured in vivo in patient-derived xenografts. The effect of treatment on the stem cell population is determined through analysis of cells expressing stem cell markers and the ability of cells from excised xenografts to form colonies in vitro and also tumours when passaged on in vivo. The effect of treatment on tumour initiation, tumour spread or established tumours can be measured. Cost-effective ex vivo treatment models are available as an option.

Agents/targets can be tested in one or more tumour indication.

Autochthonous Not available to test agents. Patient samples are available if target analysis is required.

Brca2/p53 driven non-metastatic model and a p53/E-cadherin metastatic model are available.

K-Ras/beta catenin driven locally invasive carcinoma model is available.

Validation Mice were treated with RO4929097. Treatment did not prevent tumour growth. Effect on Notch inhibition was confirmed with appropriate PD markers.

Purpose Patient-derived xenografts are established in immunodeficient mice which may have limitations in the analysis of the stem cell component of a tumour. A number of mouse models are available which form spontaneous tumours in a native environment. On-going analysis of the stem cellness of these models is being investigated and can be made available to test novel agents or study novel targets.

Agents/targets can be tested in one or more tumour indication.

Figure 1: Validation of patient-derived in vitro assays using Notch pathway inhibitors

HNSCC cells (A) were treated with DAPT, flurbiprofen (FB) or the combination; no significant effect on differentiation of stem cells was observed. In prostate cells (B) RO4929097 had a significant effect on colony formation efficiency which was more pronounced in combination with irradiation. RO4929097 treatment significantly reduced the % of ALDH positive cells (stem cells) in breast cancer samples tested (C).

Figure 2: Validation of patient-derived in vitro assays using proprietary CRT agents

The effect of proprietary agents and control compounds was tested in patient-derived breast cancer mammosphere assays. Vorinostat and proprietary CRT agents are shown to significantly inhibit both primary (MFE) and secondary (MSR) mammosphere assays in a number of patient samples.

Figure 3: Validation of patient-derived in vivo assays using Notch pathway inhibitors

HNSCC xenografts were treated with RO4929097 with little significant effect on tumour growth (A). In contrast a significant effect of treatment with RO4929097 was observed in the prostate cancer model (B). As anticipated, there was no significant effect of RO4929097 treatment on breast tumour growth (C).

What can we offer our Partner?

Expertise of leaders in the field of CSCs

Access to novel models (derived from patient material) for use in CSC drug discovery

Access to a discovery platform from a range of tumour types

Collaborative opportunity to identify novel targets of CSC

Collaborative opportunity to progress proprietary CRT agents which in preliminary assays have been

shown to have an anti-CSC effect

Fee-for-service or sponsored collaborative research programmes considered in one or more tumour

indications

We look forward to discussing this opportunity with you in more detail and/or to facilitating a meeting with

the relevant Investigators.

For further information please contact:

Roisin NicAmhlaoibh

rnicamhlaoibh@cancertechnology.com

T: +44 (0)20 3469 6300