Ludwig Institute for Cancer Research

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Ludwig Institute for Cancer Research Ltd

LICR Fund, Inc.

CONFLICT OF INTEREST POLICY FOR STAFF MEMBERS

Effective Date: August 24, 2012

Last updated: December 17, 2012

Conflict of Interest Policy for Staff Members

Conflicts of interest arise when a staff member can personally benefit financially from a decision he or she could make on behalf of the Institute or Fund, including benefits to family members or businesses with which the person is closely associated. Such conflicts are to be avoided if practicable. In the case of the Institute it is recognized its commitment to supporting the evaluation, development and commercialization of technologies from its own scientific discoveries may often expose its research and clinical staff to perceived or potential conflicts of interest and this policy provides for proactive management of such conflicts.

1. DISCLOSURE

(a) Staff members of the Ludwig Institute for Cancer Research Ltd (Institute) or the LICR Fund, Inc. (Fund) disclose on a Confidential Disclosure Form (CDF) extramural activities and reimbursed or sponsored travel for outside entities, and outside appointments and significant financial interests (SFI) of their own and of members of their immediate family (spouse and dependent children) in accordance with the Confidential Disclosure Policy.

2. CONFLICTS OF INTEREST

(a) Conflicts of interest can stem from the activities listed above and may include, but are not limited to:

i. The conduct of research or sponsored research by an Institute staff member for an entity with whom the staff member or their spouse or dependent children holds an outside appointment or SFI

ii.Transactions involving Institute or Fund resources or personnel with an entity with whom the staff member or a member of their extended family holds an outside appointment or a SFI.

(b) Process

i. Prior to involvement in activities that could be potential or perceived conflicts of interest, a written description of these activities and related details shall be provided by staff members of the Fund to a disinterested member of Management and by staff members of the Institute to a Committee of Officers (the Committee) comprised of the Scientific Director, the President, and the Executive Directors for review.
ii. Members of the Committee must excuse themselves from the discussion and review of their own submissions to the Committee or from any transactions in which they may have direct or indirect interest.
iii. Review outcomes
• Activities determined not to be a conflict of interest may be undertaken by the staff member.

• Activities determined to be a conflict of interest that is not manageable may not be undertaken by the staff member.

• Activities determined to be a conflict of interest that is manageable will be submitted to the Audit Committee of the Institute or Fund for approval. The submission will include a description of the activities and of the steps involved in the management of the conflict. Once approved by the Audit Committee, the staff member may commence the activities.

iv. The staff member shall be advised in writing of the outcome of the review v. All determinations on conflicts of interest shall remain confidential. vi. However, in compliance with United States Public Health Service policy, the Institute will disclose as required, financial conflicts of interest (FCOI). FCOI are defined as SFI that could directly and significantly affect the design, conduct or reporting of NIH funded research

3. TRAINING

(a) Each staff member shall complete training prior to engaging in research and at least every four years in accordance with local requirements.

(b) It is the responsibility of each staff member to review the Conflict of Interest Policy when changes in the Policy occur.

(c) A staff member must complete additional training if it is determined they are not in compliance with the Institute’s Conflict of Interest Policy.

(d) Training of staff shall be managed, organized, and administered by the Branch Administrator or their designated delegate.

Ludwig Institute for Cancer Research Ltd

LICR Fund, Inc.

CONFIDENTIAL DISCLOSURE POLICY FOR STAFF MEMBERS

Effective Date: August 24, 2012

Last updated: March 19, 2013

Confidential Disclosure Policy for Staff Members

The Ludwig Institute for Cancer Research Ltd (the Institute) and the LICR Fund (Fund) maintain the highest standards of honesty, integrity and impartiality. In the course of pursuing the mission of the Institute and the Fund, staff may be involved in extramural activities, hold appointments from and have significant financial interests in, other organizations. Disclosure of these activities, appointments, and interests by Institute and Fund staff allows the Institute to meet the expectation of these high standards and proactively manage potential conflicts disclosed.

1. DESCRIPTION

a. Extramural activities

i. Examples of extramural activities include but are not limited to: appointments to and activities for professional associations and government agencies, and professional activities with outside academic or not-for-profit organizations such as teaching or lecturing responsibilities, membership on editorial or other boards or academic or government committees.

b. Outside Appointments

i. Examples of outside appointments (appointments outside of the Institute or Fund) include but are not limited to owner, investor, member of the board or a committee, officer, director, trustee, partner, investigator, employee, or consultant. Outside appointments may be compensated or non-compensated.

c. Significant Financial Interests (SFI)

SFI exists when

i. A staff member of the Institute or Fund or their spouse or dependent children, has an outside appointment, as defined in Section 1.b, with or holds a financial interest in, a publically or non-publically traded entity that, as far as the staff member is aware, supplies to or receives from the Institute or Fund, funds, goods, or services or proposes to do so, or collaborates with the Institute or Fund and,

ii. The value, aggregated for a staff member, their spouse, and dependent children, of any remuneration, e.g., honoraria, consulting fees, paid authorship, etc., received from any single publically traded entity, as described in 1.c.i above, in the twelve months preceding disclosure plus the value, aggregated, of any equity interest in any entity as of the date of disclosure is greater than $5,000 or,

iii. The value, aggregated for a staff member, their spouse, and dependent children, of any remuneration received from any single non-publically traded entity, as described in 1.c.i above, in the twelve months preceding disclosure is greater than $5,000, or a staff member, their spouse, or dependent children hold any equity interest (e.g., stock, stock options, or other ownership interest) or,

iv. There is receipt of income related to intellectual property rights from an entity other than the Institute or Fund.

d. Reimbursed or Sponsored Travel

i. Reimbursed travel is defined as travel whereby travel expenses are reimbursed directly to the staff member for travel requested or required by any entity outside the Institute for the purpose of utilizing the professional expertise of a staff member for which they hold their Institute or Fund appointment. Reimbursement of travel for extramural activities described in Section 1.a. above, is not included in this definition for disclosure purposes.

ii. Sponsored travel is defined as travel whereby travel expenses for a staff member are paid directly for travel requested or required by any entity outside the Institute for the purpose of utilizing the professional expertise of a staff member for which they hold their Institute or Fund appointment. Travel expenses for a staff member involved in extramural activities described in Section 1.a. above, paid directly are not considered sponsored travel for disclosure purposes.

2. DISCLOSURE

a. Every Institute and Fund staff member except for undergraduate students (the “relevant staff member”) must disclose all extramural activities, all outside appointments, SFIs and reimbursed or sponsored travel:

i. Prior to the commencement of employment with the Institute

ii. Annually as of April 1 of each year thereafter

b. Every Institute and Fund relevant staff member must disclose, when known, any new outside appointment, SFI, or reimbursed or sponsored travel.

c. A Confidential Disclosure Form (CDF) will be completed by each Institute and Fund relevant staff member at least annually.

i. Each year, the Secretary to the Board of Directors, on behalf of the respective Board of Directors acting through its Audit Committee, shall send a CDF to each relevant staff member of the Institute and the Fund via their Administrator.

ii. The CDF shall be completed by each relevant staff member of the Institute and the Fund and returned to the Secretary to the Board of Directors via their Administrator by May 1 of the respective year.

iii. It shall be the continuing duty of each relevant staff member to advise the Secretary promptly upon the occurrence of any new outside appointment, SFI, or reimbursed or sponsored travel which was not but should have been described in the most recent CDF had it been known at the time the form was completed.

iv. Failure to disclose extramural activities, outside appointments, SFIs and reimbursed or sponsored travel constitutes grounds for disciplinary action.

d. Copies of the completed CDFs for Officers, Directors, and Member-track scientists shall be provided to the Chairman of the Audit Committee by the Secretary.

e. Except as required by law or United States Public Health Service policy, disclosures are confidential.

Scientific Integrity Policy

The Institute is serious about the integrity of its scientific research and maintains a Scientific Integrity Policy that is also part of the Institute’s Code of Ethics and Standards.  The section of the Code of Ethics and Standards containing the Scientific Integrity Policy is reproduced below.

General Policy

Scientists employed by the Ludwig Institute for Cancer Research shall execute their work in accordance with scientific standards of objectivity, accountability and professionalism.

Research integrity is fundamental to the ethical conduct of the scientific process and to the credibility and reputation of the Institute. Research findings are to be reported accurately, results are not to be fabricated or falsified and plagiarism in written documents is unacceptable.  Timely, accurate, complete, authentic and reliable records of research data are to be maintained in line with the Institute’s Maintenance of Laboratory Notebooks and Records Policy.

Research misconduct is defined as fabrication or plagiarism of performed or proposed research or reports, falsification of data or credentials, or misuse of research resources and is not tolerated. Any case of suspected or alleged research misconduct or misuse of research funds shall be investigated thoroughly and where applicable, disciplinary measures, including possible termination of employment, shall be applied.  Research misconduct in the context of this Policy does not include authorship or collaboration disputes.

Responsibility and Action

All individuals affiliated with the Institute have an ethical responsibility to act if research misconduct is suspected or has occurred.  Should research misconduct occur or be suspected, the appropriate action to be taken, anonymously if desired, may include:

• Pursuing discussion with and/or seeking perspective from a group leader, the Branch Director, the Head of Academic Affairs, or the Scientific Director,
• Notifying the Branch Director of the alleged research misconduct at the Branch level or,
• Notifying the Head of Academic Affairs or the Scientific Director of the Institute of the alleged research misconduct at the Institute level.

Any questions regarding Scientific Research Integrity should be directed to the Scientific Director or the Head of Academic Affairs.

Revised and Board approved 24 April 2012

Contact Audit Committee

Reports of concerns and violations of any type may be submitted to the Audit Committee of the Institute's Board of Directors. All correspondence will be kept confidential. Reports can be submitted anonymously. The Audit Committee can be contacted at the following e-mail address:

Test

LICR Investigators Pivotal in Five of the

24 Milestones in Cancer Research

In 2006, the Nature Publishing Group gave its opinion on the ‘24 Milestones in Cancer Research’ since the end of the nineteenth century. ‘Original Research Papers’ authored by LICR investigators feature in five of the 24 Milestones, with another two quoting papers authored by LICR investigators as selected ‘Further Reading.’ That’s about 30% of cancer research milestones with significant contributions from LICR investigators according to the editors of Nature, Nature Medicine and Nature Reviews Cancer.

References

1. Van Pel A. and Boon T. Protection against a nonimmunogenic mouse leukemia by an immunogenic variant obtained by mutagenesis. Proc Natl.Acad.Sci.U.S.A (1982) 79(15):4718-4722.


2. van der Bruggen P., Traversari C., Chomez P., Lurquin C., De Plaen E., Van den E.B., Knuth A., and Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science (1991) 254(5038):1643-1647.


3. Shankaran V., Ikeda H., Bruce A.T., White J.M., Swanson P.E., Old L.J., and Schreiber R.D. IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature (2001) 410(6832):1107-1111.[PMID: ]


4. Cavenee W.K., Dryja T.P., Phillips R.A., Benedict W.F., Godbout R., Gallie B.L., Murphree A.L., Strong L.C., and White R.L. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature (1983) 305(5937):779-784.


5. Waterfield M.D., Scrace G.T., Whittle N., Stroobant P., Johnsson A., Wasteson A., Westermark B., Heldin C.H., Huang J.S., and Deuel T.F. Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature (1983) 304(5921):35-39


6. Downward J., Yarden Y., Mayes E., Scrace G., Totty N., Stockwell P., Ullrich A., Schlessinger J., and Waterfield M.D. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature (1984) 307(5951):521-527.


7. DeLeo A.B., Jay G., Appella E., DuBois G.C., Law L.W., and Old L.J. Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse. Proc.Natl.Acad.Sci.U.S.A (1979) 76(5):2420-2424.


8. Gessler M., Poustka A., Cavenee W., Neve R.L., Orkin S.H., and Bruns G.A. Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping. Nature (1990) 343(6260):774-778.


9. Bronner C.E., Baker S.M., Morrison P.T., Warren G., Smith L.G., Lescoe M.K., Kane M., Earabino C., Lipford J., Lindblom A., Tannergard P., Bollag R.J., Godwin A.R., Nordenskjosolld M., Fishel R., Kolodner R., and Liskay R.M. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature (1994) 368(6468):258-261.


10. Fishel R., Lescoe M.K., Rao M.R., Copeland N.G., Jenkins N.A., Garber J., Kane M., and Kolodner R. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell (1993) 75(5):1027-1038.


11. Demetri G.D., von Mehren M., Blanke C.D., Van den Abbeele A.D., Eisenberg B., Roberts P.J., Heinrich M.C., Tuveson D.A., Singer S., Janicek M., Fletcher J.A., Silverman S.G., Silberman S.L., Capdeville R., Kiese B., Peng B., Dimitrijevic S., Druker B.J., Corless C., Fletcher C.D., and Joensuu H. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N.Engl.J.Med. (2002) 347(7):472-480.

From discovery of proteins to multiple potential therapies

Angiogenesis and VEGFs

Angiogenesis, the process of forming new blood vessels, is central to wound healing, reproduction and embryonic development. Tumor cells and stroma, the connective tissue around the tumor, can also stimulate angiogenesis by secreting angiogenic growth factors. Without the blood and nutrients supplied by newly-generated blood vessels, it is thought that no tumor would grow to be more than a few millimeters in size. Lymphangiogenesis, a related process, is the formation of new lymphatic vessels, which is also stimulated by particular growth factors. The blood and lymphatic vessels provide the principle routes by which cancer metastasizes from the original tumor site, which is the ultimate cause of most cancer deaths.

For more than ten years, LICR coordinated and supported a global Program to study angiogenesis and lymphangiogenesis with the aim of developing new therapeutic modalities. LICR efforts identified three of the four known vascular endothelial growth factors (VEGFs), VEGF-B, VEGF-C and VEGF-D, two of the four known platelet-derived growth factors (PDGFs), PDGF-C and PDGF-D, as well as a novel receptor, VEGF receptor-3 (VEGFR-3).

LICR conducted extensive laboratory and pre-clinical studies to assess the biological functions of the growth facotors and the therapeutic potential related to the discovery of the VEGFs and PDGFs. For example, LICR investigators were the first to show that antibodies targeting VEGF ligands inhibit cancer spread to the lymph nodes, suggesting that anti-VEGF antibodies are potential anti-metastatic therapies. Vegenics Ltd (Australia), an LICR spin-off company that is now a wholly-owned subsidiary of Circadian Technologies Ltd (Australia), is developing antagonists of VEGF-C and VEGF-D as both therapeutic and diagnostic agents for cancer.

LICR has a singular focus on cancer but recognizes that some of its medical research findings may have therapeutic value for other human diseases. Thus LICR places great importance on supporting and facilitating—principally through the licensing of its intellectual property—the research and development of non-oncology therapies for human benefit. Ark Therapeutics PLC is developing a pro-angiogenic therapy, Trinam®, which utilizes VEGF-D licensed from LICR.  Trinam® is currently being tested in a phase III clinical trial for its ability to prevent blood vessel blockage following vascular graft access surgery (insertion of an artificial blood vessel), which is required for patients with kidney failure to undergo dialysis. Lymphatix (Finland), the second company spun-off from the Angiogenesis Program, was recently purchased by Ark Therapeutics.

Study of natural history of infection to preventative vaccine

HPV Cervical Cancer Vaccine

With its flexible, international reach, LICR was able to access a large number of cancer samples in order to analyze the natural history or “epidemiology” of infection with human papillomavirus (HPV), the causative agent of genital cancers, with a view to preventing and/or treating these diseases.

LICR began studying the role of HPV in cancers of the cervix, penis and anus in the early 1980’s and found that DNA sequences from HPV were very often found in these genital tumors. This finding, along with others from around the world contributed to the confirmation that HPV is the causative agent of cervical cancer. Dr. Harald zur Hausen—a former member of the LICR Scientific Advisory Committee—was awarded the 2008 Nobel Prize for Medicine & Physiology for first postulating and then proving this theory.

However, the discovery of a high proportion of HPV infections in asymptomatic women led LICR to launch epidemiological studies aimed at understanding the risk correlates for HPV-associated cervical disease.

In 1993, the “Ludwig/McGill Cohort” was established by LICR investigators with a population of women from São Paulo, Brazil. This cohort formed one of the largest longitudinal studies of the natural history of HPV infection and risk of cervical cancer in the world. (The McGill part of the name pays tribute to the co-director of the cohort epidemiologist who worked at the LICR São Paulo Branch before moving to McGill University in Canada.)

LICR investigators found that most HPV infections are transient and of little clinical significance. However, the small proportion of women who harbor persistent HPV infections stand at a much greater risk of subsequent cervical neoplasia, indicating that persistent, not transient, HPV infections are the actual biologic precursor in cervical cancer onset. This and other findings on viral load (the amount of virus) and the integration of viral DNA into cervical cells were important for understanding the clinical significance of HPV DNA testing results and set policies for inclusion of some form of HPV testing in cervical cancer prevention internationally.

Results from LICR’s HPV natural history studies were seminal in facilitating the design and implementation of clinical trials of prophylactic vaccines against HPV. The LICR team was invited to lead a phase II trial of Merck & Co. Inc.’s prophylactic (preventative) HPV vaccine, which was the first study to demonstrate the safety, immunogenicity and efficacy of the vaccine. The vaccine—Gardasil®—was approved for licensing later that year (2005) and is now sold internationally.

From brain cancer initiative to new category of antibody target

EGFR and Antibody 806

Several antibodies that target the cell surface signaling molecules promoting cancer cell growth have been approved for cancer patients. However, many of the first generation antibodies currently in clinical use do not discriminate between the signaling molecules on cancer cells and those on healthy cells. This lack of discrimination is the apparent cause of the toxic side-effects associated with these drugs.

The epidermal growth factor receptor (EGFR) is a prime target for the development of anti-cancer therapeutics, as EGFR overexpression (increased abundance) or mutation has been reported in numerous human cancers and is generally associated with a poor clinical prognosis. Anti-EGFR antibody therapies are available, but they cause skin and liver toxicities. Most cases of glioblastoma (brain cancer) are caused by a particular EGFR mutation, and LICR investigators set out to generate an antibody that specifically targeted the mutated EGFR in the hope of developing an effective therapy for a disease that is intractable to all conventional treatments.

LICR investigators found that the antibody, 806, does indeed target the mutation found in glioblastoma. However, extensive laboratory and pre-clinical analyses showed that 806 is also able to discriminate between wild-type (non-mutated) EGFR and over-expressed wild-type EGFR. Further characterization showed that the 806 antibody is able to differentiate between cell surface proteins with identical sequences but different conformations (shapes) resulting from the over-expression of the receptor.

By taking advantage of the unique binding specificity of the 806 antibody, LICR investigators further unraveled the complexities of EGFR activation and its hyper-activity in cancer. Their discovery that EGFR exposes the binding site for 806 as it changes from an inactive form to an active form was a major advance in the understanding of growth receptor activation. These surprising but pivotal findings identified a whole new category of cell surface target for antibody-based cancer therapies.

LICR sponsored and conducted the first-in-man clinical trial to characterize 806’s tumor-targeting abilities and showed that it targets the tumors but not normal tissues that have high levels of EGFR. The clinical research also showed—presumably as a result of its specificity—806 does not cause the side-effects observed with other EGFR-targeting therapies, e.g. Erbitux (Cetuximab).

The 806 antibody formed the basis of an LICR spin-off company, Life Sciences Pharmaceuticals, and in 2009 was licensed to the pharmaceutical company Abbott for clinical development.

Cancer Vaccine Collaborative

The Ludwig Institute for Cancer Research Ltd (LICR) and the Cancer Research Institute (CRI) (New York, USA) joined forces in 2001 to form the Cancer Vaccine Collaborative (CVC). Therapeutic cancer vaccines represent an entirely new therapeutic modality with great promise for helping to reduce tumor recurrence. By working together within the CVC, LICR and CRI investigators are able to best achieve their common objectives of understanding the immunological response to cancer, harnessing that knowledge for patient benefit, and accelerating the translation of basic research into new cancer therapies.

The CVC provides an international clinical trial infrastructure (the LICR Office of Clinical Trials Management) to help investigators navigate the complex challenges of translating their laboratory findings into the clinic. These activities include the writing and filing of complex regulatory documentation, obtaining or manufacturing clinic-grade reagents, procuring funding for patient costs, meeting insurance requirements, and fulfilling stringent clinical trial monitoring requirements. The CVC also supports the associated laboratory experiments that enable the investigator to understand the mechanism of the tested therapy; research that goes beyond the primary or secondary endpoints of the typical clinical trial.

Since its inception, the CVC has completed or is currently conducting 35 clinical trials. These trials have enrolled nearly 700 patients with a variety of cancer types, including melanoma and sarcoma, and ovarian, prostate, lung, breast, esophageal, and bladder cancers. With the progression of several of these studies into Phase II and III trials, the CVC has become a leading force in the development of therapeutic cancer vaccines as a real option for cancer patients.

Strategic Translation of Knowledge Yields Potential Therapies

PI3K Discovery to Clinic

The PI3K signaling pathway, which regulates several vital cell and physiological processes, is the most frequently subverted signaling pathway in cancer. LICR’s long-term and substantial commitment to PI3K research demonstrates how laboratory discoveries can be translated into applications for human benefit. The Institute’s investment—in staff, research funds and time, academic and industry relationships, intellectual property and technology licensing, and the careful management of their integration—has thus far brought several candidate therapies to late-stage clinical development for cancer and other disease applications.

There are 14 proteins in the PI3K family, and LICR investigators discovered nearly half of that number. LICR also established the internationally adopted nomenclature for the PI3K enzymes by classifying the various PI3K isoforms based on their structure and function.

Pivotal studies by LICR researchers established the roles of signaling by different PI3K isoforms in: cell processes, such as growth, proliferation, migration, and growth factor receptor signaling; physiological processes, such as inflammation and immunology; and in diseases from solid tumors and leukemia to arthritis, obesity and diabetes.

With PI3K signaling so heavily implicated in cancer onset and progression, PI3K isoforms were considered prime targets for potential new therapies. However, the ubiquity of PI3K signaling in normal cell processes means that a therapeutic modality targeting all PI3K signaling would likely cause severe side-effects. One of the most fundamental contributions to the development of PI3K inhibitors was the LICR”s generation of mouse models in which specific PI3K isoforms had been inactivated. The inactivated PI3K isoforms mimic the effects of systemic administration with a subunit-specific drug, and a careful assessment of these mice provided crucial information for assessing potential side-effects from PI3K inhibition. The finding that mice with an inactive p110alpha PI3K, which is most often implicated in cancer onset and progression, had dampened insulin signaling but no signs of developing diabetes or severe metabolic disturbances, was proof-of-principle that isoform-specific inhibitors were indeed candidate cancer therapies.

LICR’s first spin-off company, PIramed Ltd, was formed to develop PI3K inhbitors generated through a translational research program initiated by LICR. The collaboration—between academic partners LICR, Imperial Cancer Research Fund (now Cancer Research UK) and the Institute for Cancer Research (both in London, UK), and industry partner Yamanouchi Pharmaceutical Company (now Astellas Pharma, Japan)—generated multiple lead compounds that were screened for isoform specificity and also physiological effect. This approach of combining fundamental science with translational work advanced our knowledge of PI3K signaling in cancer and other diseases and also enabled the industrial development team to make informed decisions based on solid science. PIramed entered a major development deal with Genentech before being acquired by Roche in one of the largest deals in the UK biotechnology sector for some time. An isoform-specific inhibitor of PI3K p110alpha is now in a phase I trial as a potential cancer therapy.

Cell Biology

Stem Cells: New Prospects for Cancer Therapy

Stem cells—unspecialized cells with high capacity for self-renewal—are present in both embryonic and adult tissues and can differentiate to acquire distinct shapes and functions. Adult stem cells are maintained throughout life to replenish dying cells or repair damaged tissue. In contrast to embryonic stem cells, which can develop into a broad spectrum of cell types, adult stem cell populations can differentiate into fewer different types of cell. Cells with stem-cell like characteristics are also found in tumors. Many hypothesize that these ‘cancer stem cells’ generate new tumors and drive tumor progression with their ability to divide uncontrollably and transform into different cancer cell types. If this is the case, therapies that target cancer stem cells might represent the most effective strategy for destroying tumors.

Dormant Hematopoietic Stem Cells Awaken to Repair Bone Marrow Injury

Hematopoietic stem cells (HSCs) are adult stem cells that differentiate into a range of blood cell types in the bone marrow. LICR Lausanne Branch investigators were the first to report that a substantial number of HSCs are dormant but become active in response to physiological stress. Studies in mice revealed that dormant HSCs divide as rarely as five times in a lifespan under normal conditions. However, the cells are induced to self-renew rapidly by bone marrow injury or by treatment with G-CSF, a cell-to-cell signaling molecule that was discovered by LICR Melbourne Branch investigators many years ago. When physiological balance has been re-established, the HSCs return to dormancy¹.

The hypothesis that dormant HSCs make up an emergency stem cell reserve that can be activated could have important ramifications for cancer therapy strategy. Cancer stem cells can evade eradication by standard chemotherapies that target rapidly diving cells. If cancer stem cells, like HSCs, can be awakened out of dormancy, they could be sensitized to chemotherapy treatment by stem cell activating molecules.

Wnt Signaling in Hematopoietic Stem Cells

The differentiation of HSCs into different blood cell types—a process known as hematopoiesis—is regulated by a group of cell-to-cell signaling proteins known as Wnts. Binding of Wnt to its receptor stabilizes β-catenin and γ-catenin proteins, which mediate the transient activation of Wnt responsive genes. In cancer and some other diseases, Wnt signaling is deregulated by aberrant stabilization of β-catenin or overexpression of γ-catenin, which leads to the uncontrolled expression of Wnt target genes.

Although drugs that target β- or γ-catenin could potentially block aberrant Wnt signaling in cancer cells, there is a risk that hematopoiesis, which is required throughout adult life, could be impaired. However, more research from the LICR Lausanne Branch suggests that β- and γ-catenin can be targeted with no effect on hematopoiesis. The investigators studied mice that lack β- and γ-catenin, and found that hematopoiesis still occurs normally, presumably because the signals required for hematopoiesis are transmitted by other molecules in HSCs². This finding supports the feasibility of therapeutic strategies that target β- or γ-catenin for leukemia patients.

Maintenance of Neural Stem Cells

Neural stem cells, another type of adult stem cell, differentiate into a range of nerve cell types. Neurogenesis—the differentiation of neural stem cells into neurons—is being studied by LICR investigators seeking to identify the mechanisms that keep the neural stem cells’ delicate balance between self-renewal and differentiation into neurons. Several years ago, investigators at the LICR Stockholm Branch discovered that the maintenance of neural stem cells in the central nervous system depends on a group of transcription factors called SoxB1 proteins, as well as on cell signaling by the cell-surface receptor Notch. (Investigators at the LICR Lausanne Branch are also studying Notch signaling, but in T cell differentiation and maturation³.) Although Notch and SoxB1 have similar effects, the extent of their interactions, if any, were unknown.

In 2008, the Stockholm Branch team found that Notch and SoxB1 operate through distinct mechanisms when maintaining neural cells in an undifferentiated state. However, there is some interplay, as Notch’s role in this function is dependent on the SoxB1 proteins⁴. Drawing on this knowledge, the investigators are now exploring ways to target cancer stem cells in the search for new treatment strategies for brain cancer.

References

1. Wilson A, Laurenti E, Oser G, van der Wath RC, Blanco-Bose W, Jaworski M,Offner S, Dunant CF, Eshkind L, Bockamp E, Lió P, Macdonald HR, Trumpp A. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell (2008) 135(6):1118-29 [PMID: 19062086].
2. Jeannet G, Scheller M, Scarpellino L, Duboux S, Gardiol N, Back J, Kuttler F, Malanchi I, Birchmeier W, Leutz A, Huelsken J, Held W. Long-term, multilineage hematopoiesis occurs in the combined absence of beta-catenin and gamma-catenin. Blood (2008) 111(1):142-9 [PMID: 17906078].
3. Fiorini E, Ferrero I, Merck E, Favre S, Pierres M, Luther SA, MacDonald HR. Cutting edge: thymic crosstalk regulates delta-like 4 expression on cortical epithelial cells. J Immunol (2008) 181(12):8199-203 [PMID: 19050235].
4. Holmberg J, Hansson E, Malewicz M, Sandberg M, Perlmann T, Lendahl U, Muhr J. SoxB1 transcription factors and Notch signaling use distinct mechanisms to regulate proneural gene function and neural progenitor differentiation. Development (2008) 135(10):1843-51 [PMID: 18417619].

Cancer Immunology

Immune Responses to Cancer

In addition to protecting the body against infection, the immune system has the capacity to defend against cancer. White blood cells known as cytotoxic T lymphocytes (CTLs) can distinguish between cancer and normal cells by virtue of cancer antigens, specific peptides (protein fragments) presented on the cell’s surface by major histocompatibility complex (MHC) molecules. The CTL’s T cell receptors (TCRs) recognize cells presenting cancer antigen-derived peptides and induce the immunological destruction of the cells.

Therapeutic cancer vaccines are being designed to generate and strengthen CTL responses against cells presenting cancer antigens. In order to optimize cancer vaccines currently in clinical trials and to develop new vaccine strategies, LICR investigators are examining how immune responses to cancer are established and maintained, and also how they can be subverted by tumors.

Immune Suppression by Regulatory T Cells

In spite of the immune system’s inherent capacity to defend against cancer, tumors can develop means to evade immune recognition or escape immunological attack. A type of white blood cell known as a regulatory T cell (Tregs) has been shown to suppress the activity of CTLs in several tumors. Tregs have an important role in preventing auto-immunity¹, the immune system’s attack on normal cells, but they have also been shown to stifle the immunological response against tumors.

The protein FoxP3 plays a critical role in the development and function of Tregs. LICR Lausanne Branch researchers found new evidence that tumors stimulate the formation of Tregs, and that Tregs are attracted to the tumor site(s). The study showed that, compared to healthy donors, melanoma patients have increased amounts of FoxP3-expressing Tregs circulating in their blood and that Tregs were enriched significantly at tumor sites and in tumor-infiltrated lymph nodes².

FoxP3 was believed to be produced exclusively by Tregs, and is used as the most reliable marker to distinguish Tregs from other cell types. However, a study from the LICR Melbourne Center revealed that this protein is also produced by cancer cells. The investigators detected FoxP3 in samples from melanoma and other tumor types, including those from breast, colon, lung, prostate, kidney, and bladder cancers. The team also discovered that melanoma cells produce their own version of FoxP3, which lacks a segment of the protein produced by Tregs and also has an altered sequence³. These findings suggest an unknown mechanism by which tumors might suppress the activity of infiltrating immune cells to escape destruction. They also suggest that a therapeutic approach that combines a cancer vaccine with strategies targeting FoxP3 might be more effective than a vaccine alone.

New Concept of T Cell Receptor Recognition

A mechanism that may modulate CTL activity was uncovered by investigators at the LICR Lausanne Branch. MHC I-peptide complexes displayed on the surface of cells are not fixed in one position but move around considerably within the cell membrane. The TCR briefly engages MHC I-peptide complexes and scans it to determine if the peptide is “non-self,” i.e. derived from a cancer antigen or from a pathogen. Each TCR recognizes one specific MHC I-peptide complex, which is known as its “cognate.” (A separate study from the Lausanne Branch identified, with great precision, the two amino acid residues in one cancer antigen peptide that interact with the TCR⁴.) The LICR researchers studying MHC I-peptide complex mobility found that when the TCR recognizes its cognate complex, the complex becomes immobilized. The trapping of MHC I-peptide complexes is mediated by the adhesion molecule ICAM-1, which is concentrated in the contact sites formed by CTLs and target cells⁵. The temporary capture of the MHC I-peptide complexes allows the TCR to scan the target cells and thus increases the efficiency of TCR antigen recognition. The LICR team is now investigating if defects in the trapping and scanning mechanisms might account for the poor recognition of some tumor cells by CTLs.

Addressing Anergy to Enhance Cancer Vaccines

The ability of tumors to evade immune attack is manifested in part by a phenomenon known as CTL anergy, in which CTLs at the tumor site experience the loss of their ability to recognize cancer cells. In 2008, a LICR Brussels Branch team made pioneering findings that clarify a molecular basis of CTL anergy and how it is achieved by tumors. The study was featured on the front cover of the March 2008 issue of the journal Immunity⁶.

CTLs generally express on their surface a receptor, CD8, that helps the TCR to recognize MHC-peptide complexes on their target cells. In normal CTLs, CD8 receptors are associated with TCRs on the T cell surface. The Brussels Branch team discovered that CD8 and TCR molecules become separated on anergic CTLs, probably because TCR is bound to the protein galectin-3. The investigators were able to restore the association of CD8 and TCR by administering the galectin-binding molecule LacNAc to anergic CTLs. As a result, the CTLs regained their ability to recognize and destroy cancer cells. These findings suggest that treatment of cancer patients with galectin ligands might correct the anergy of tumor-infiltrating lymphocytes. The LICR Brussels Branch team will extend this work into the clinical discovery phase by conducting a clinical trial with a melanoma vaccine that incorporates galectin-binding sugars.

References

1. Jandus C, Bioley G, Rivals JP, Dudler J, Speiser D, Romero P. Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides. Arthritis Rheum (2008) 58(8):2307-17 [PMID: 18668556].
2. Jandus C, Bioley G, Speiser DE, Romero P. Selective accumulation of differentiated FOXP3(+) CD4 (+) T cells in metastatic tumor lesions from melanoma patients compared to peripheral blood. Cancer Immunol Immunother (2008) 57(12):1795-805 [PMID: 18414854].
3. Ebert LM, Tan BS, Browning J, Svobodova S, Russell SE, Kirkpatrick N, Gedye C, Moss D, Ng SP, MacGregor D, Davis ID, Cebon J, Chen W. The regulatory T cell-associated transcription factor FoxP3 is expressed by tumor cells. Cancer Res (2008) 68(8):3001-9 [PMID: 18413770].
4. Derré L, Bruyninx M, Baumgaertner P, Ferber M, Schmid D, Leimgruber A, Zoete V, Romero P, Michielin O, Speiser DE, Rufer N. Distinct sets of alphabeta TCRs confer similar recognition of tumor antigen NY-ESO-1157-165 by interacting with its central Met/Trp residues. Proc Natl Acad Sci USA (2008) 105(39):15010-5 [PMID: 18809922].
5. Segura JM, Guillaume P, Mark S, Dojcinovic D, Johannsen A, Bosshard G, Angelov G, Legler DF, Vogel H, Luescher IF. Increased mobility of MHC I – peptide complexes decreases the sensitivity of antigen recognition. J Biol Chem (2008) 283(35): 24254-63 [PMID: 18579518].
6. Demotte N, Stroobant V, Courtoy PJ, Van Der Smissen P, Colau D, Luescher IF, Hivroz C, Nicaise J, Squifflet JL, Mourad M, Godelaine D, Boon T, van der Bruggen P. Restoring the association of the T cell receptor with CD8 reverses anergy in human tumor-infiltrating lymphocytes. Immunity (2008) 28(3):414-24 [PMID: 18342010].

Cancer Immunology

Identification of New Antigen Targets

Cancer antigens are molecules that are i) present in cancer cells and ii) recognized and targeted by the immune system. Cancer antigens that have limited expression in normal adult tissues are compelling targets for the development of immunotherapies, such as targeted antibodies and therapeutic cancer vaccines (TCVs).

With the discovery of MAGE-A1 in 1991, LICR investigators were the first to identify a unique family of cancer antigens known as the cancer / testis (CT) antigens. The CT antigens—also referred to as cancer/germline antigens—were named according to their selective expression in various cancer and germline (testis and fetal ovary) cells, but not normal adult cells. In the intervening decades, LICR investigators discovered many more CT antigens, characterized their expression, and determined their immunogenicity in both healthy volunteers and cancer patients. LICR has conducted more than 60 clinical trials to explore the therapeutic potential of TCVs based on CT antigens. Laboratory research is ongoing to elucidate the function of these proteins and their possible role(s) in cancer onset and progression.

A Promising New Target for Prostate Cancer Immunotherapy

Prostate cancer detected at an early stage is generally a treatable disease. However, five year survival rates decline sharply to levels near 30% once the cancer metastasizes from the prostate gland. In 2008, research published by a team of investigators at the LICR São Paulo Branch identified the CT antigen CTSP-1 as a candidate for the development of an immunotherapy for prostate cancer¹. The team showed that CTSP-1 protein was expressed in a majority (61%) of the 49 tumor samples analyzed. Additionally, CTSP-1 induced an antibody response in 25% of the 147 prostate cancer patients tested. The LICR team also discovered that detection of anti-CTSP-1 antibodies may be a useful prognostic marker, as the presence of anti-CTSP-1 antibodies was shown to be a powerful indicator of better outcome in patients with aggressive cancers. A TCV against CTSP-1 would hold great promise for the control of metastatic prostate disease, for which new treatments are greatly needed.

Characterization of an Embryo-Cancer Antigen in Non-Small Cell Lung Cancer

In 2008, LICR investigators from the LICR Melbourne Center and New York Branch characterized the expression of the embryo cancer sequence A (ECSA) gene, which is also known as developmental pluripotency associated-2 (DPPA2)². From analysis of more than 300 tumor samples and normal tissues, the team determined that ECSA/DDPA2 expression is limited in normal cells, but occurs in a variety of tumors, most notably non-small cell lung cancer (NSCLC). Additionally, ECSA/DPPA2 protein is co-expressed with several CT antigens in the NSCLC samples and induced a spontaneous anti- ECSA/DPPA2 immune response in some NSCLC patients. Based on the tumor-selective expression of this gene and the knowledge that it is associated with embryogenesis, rather than gametogenesis, the team proposed that ECSA/DPPA2 be classified as an “embryo-cancer antigen” as opposed to a CT antigen. Efforts are underway to evaluate ECSA/DPPA2 as a potential target for NSCLC immunotherapy.

References

1. Parmigiani RB, Bettoni F, Grosso DM, Lopes A, Cunha IW, Soares FA, Carvalho AL, Fonseca F, Camargo AA. Antibodies against the cancer-testis antigen CTSP-1 are frequently found in prostate cancer patients and are an independent prognostic factor for biochemical-recurrence. Int J Cancer (2008) 122(10):2385-90 [PMID: 18214856].
2. John T, Caballero OL, Svobodová SJ, Kong A, Chua R, Browning J, Fortunato S, Deb S, Hsu M, Gedye CA, Davis ID, Altorki N, Simpson AJ, Chen YT, Monk M, Cebon JS. ECSA/DPPA2 is an embryo-cancer antigen that is coexpressed with cancer-testis antigens in non-small cell lung cancer. Clin Cancer Res (2008) 14(11):3291-8 [PMID: 18519755].

Signal Transduction

TGF-beta Program: Changing Paradigms

Cell signaling induced by transforming growth factor beta (TGFΒ) regulates normal cell processes such as proliferation, differentiation (the specialization of cell form and function) and apoptosis (programmed cell death), as well as the invasiveness and metastatic spread of cancer cells. The TGFΒ Program—a group of investigators from the LICR Brussels, Uppsala, Melbourne and Stockholm Branches plus LICR Affiliates and other collaborators—have been working together for nearly 10 years to further our understanding of TGFΒ signaling, and to explore how the molecules that transmit and respond to TGFΒ signals can be targeted to limit tumor growth and/or prevent metastasis.

A long-standing paradigm in TGFΒ signaling was that the signals are transmitted from the TGFΒ receptors on the cell’s surface to the cell’s nucleus via intracellular Smad proteins. In 2008, LICR Uppsala Branch researchers reported that TGFΒ triggers apoptosis through a new signaling mechanism that functions independently of Smads¹. A key component of this Smad-independent signaling is TRAF6, an intracellular protein that acts as a switch to prompt apoptosis. Future studies will determine if TRAF6 mediates TGFΒ responses other than apoptosis, such as cell proliferation or differentiation. This study was selected as a highlight in the top two weekly online resources for signal transduction researchers, Science Signaling and UCSD-Nature Signaling Gateway.

The team also discovered a new mechanism through which TGFΒ signaling is decreased or halted. The TGFΒ pathway is driven by the TGFΒ receptors’ kinase activity, which modifies signaling proteins inside the cell thereby activating those proteins. However, few other kinases have been identified as directly regulating TGFΒ signal transduction. The LICR Uppsala Branch investigators have now discovered that a kinase, SIK, cooperates with the inhibitory Smad7 protein to regulate the removal of TGFΒ receptor from the cell surface and its subsequent degradation inside the cell².

Finally, another line of research at the Uppsala Branch has cast new light on the epithelial-mesenchymal transition (EMT), a process by which some normal cells acquire the ability to migrate and invade tissues. In many tumors, TGFΒ signaling induces EMT. LICR investigators had previously identified HMGA2, a protein that regulates the expression of a number of genes, as a key determinant of TGFΒ-induced EMT. In a continuing exploration of this finding, the team has now elucidated some of the molecular mechanisms mediated by HMGA2 to cause EMT in breast cells³. In particular, the transcription factor SNAIL1, which is known to play a key role in tumor progression and EMT, was found to be regulated by HMGA2. The investigators were able to partially revert the EMT process by reducing the levels of SNAIL1, suggesting this protein might be a target for future therapeutic strategies.

References

1. Sorrentino A, Thakur N, Grimsby S, Marcusson A, von Bulow V, Schuster N, Zhang S, Heldin CH, Landström M. The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner. Nat Cell Biol (2008) 10(10):1199-207 [PMID: 18758450].
2. Kowanetz M, Lönn P, Vanlandewijck M, Kowanetz K, Heldin CH, Moustakas A. TGFbeta induces SIK to negatively regulate type I receptor kinase signaling. J Cell Biol (2008) 182(4):655-62 [PMID: 18725536].
3. Thuault S, Tan EJ, Peinado H, Cano A, Heldin CH, Moustakas A. HMGA2 and Smads co-regulate SNAIL1 expression during induction of epithelial-to-mesenchymal transition. J Biol Chem (2008) 283(48):33437-46 [PMID: 18832382].