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 Updated on: Jan 9 2014

New Drugs: targeting signalling pathways

 

Subject
New treatment approaches are needed to improve patients’ outcome, and hopefully give a safety profile positive enough to allow its use in unfit or over 70 year-old MM patients, who represent at least half of myeloma population.


So far, most therapeutic efforts have focussed on the myeloma cells themselves, however, in recent years we have learnt that the microenvironment plays an important role in bone destruction, tumour growth and mediates drug resistance. Thus, effective drugs should target multiple cell signalling pathways that involve transcription factors as well as cytokines, adhesion molecules, and angiogenic factors, through which myeloma cells interact with bone marrow stromal cells (BMSC).


Present Status
Pathogenesis of Multiple Myeloma : Mechanisms of proliferation and apoptosis. The t(4;14) translocation results in the constitutive activation of the FGFR3 with subsequent phosphorylation of STAT3, which increases the expression of BCL-XL and blocks Fas-induced apoptosis leading to an abnormal cell growth and survival. Therefore, FGFR3 and STAT3 signalling pathways may be important therapeutic targets, in particular the FGFR3 tyrosine kinase may be targeted by kinase inhibitors. Similarly, inhibitors of cyclin dependent kinases are an attractive therapeutic target in patients with t(6;14) or t(11;14), and deregulation of the D-type cyclines or inactivation of p16 (INK 4a).


Mutations of Ras family genes (particularly N and K-Ras) are the most common oncogene mutations in MM patients. The frequency of Ras mutations increases with advanced disease (from 30 to 47%) and adversely affects survival. RAS activation requires the incorporation of a farnesyl group mediated by the enzyme farnesyltransferase.


Upon activation, RAS binds to the inner plasma cell membrane and promotes signal transduction to the nucleus. Inhibition of the RAS farnesylation process will block this pathway and constitutes a target for drug development.


The high level of bcl-2 expression present on PC confers an anti-apoptotic phenotype, which favours prolonged cell survival. Reversal of this phenotype by targeting bcl-2 is under investigation.


Bone marrow microenvironment: targets for treatment: As previously mentioned (B4.0.1.3) the BM microenvironment comprises the extracellular matrix (ECM), stromal cells and other cells. Myeloma PC bind to the BM microenvironment through a series of adhesion molecules, including β1-integrin family, fibronectin,VCAM1, ICAM-1, CXCR4, SDF-1, CD138 and CD44. This binding is critical in the pathogenesis of MM, since it triggers the transcription and secretion of cytokines by the PC and BMSC, promoting MM cell growth, survival, and trough inhibition of apoptosis, migration, and post-treatment development of drug resistance. In addition, cytokines modulate the production of additional adhesion molecules, which in turn enhance cell adhesion in a vicious circle.


Host-tumour interactions occur via two different routes:

1) direct cell contact (cell-cell: stromal cell-myeloma cell contact; or cell-ECM adhesion), and

2) through soluble molecules (ie IL6).


Direct contact of myeloma cells with BM microenvironment confers drug resistance through several mechanisms:

1) PC binding to fibronectin, via β1-integrin receptors, induces a CAM-DR phenotype (cell-adhesion-mediated drug resistance), which is associated with the upregulation of p27 levels with subsequent cell cycle arrest at G1, preventing drug-induced cell death;

2) the direct contact of myeloma cells with fibronectin via β1-integrin receptors also leads to a blockage of FAS (CD95) induced apoptosis; in addition,

3) β1-integrin cell mediated adhesion protect tumour cells from initial drug induced DNA damage (double strand breaks) by reducing topoisomerase II activity.

 

Accordingly, potential therapeutic interventions could focus on the disruption of β1-integrin, thereby leading to the inhibition of these anti-apoptotic/pro-survival mechanisms responsible for blocking drug induced apoptosis.


Soluble molecules (Cytokines) that are found in BM microenvironment such as IL6, IGF1, VEGF, TNF-α, and SDF-1α trigger RAS and activate two different signalling pathways:

1) RAF/MEK/MAPK inducing proliferation of MM cells and

2) PI3K/AKT which prevents apoptosis induced by both ionizing radiation and drugs (see below).

The RAS pathway in turn activates NF-KB and endogenous production of IL6. Cytokines such as IL-6 and IL-21 can trigger a third route, the JAK/STAT cascade, that also prevents apoptosis with overexpression of Bcl-XL and inhibition of CD95 (FAS) induced apoptosis. In addition, TNF triggers other transduction signalling routes such as NF-kB and JNK/Ap-1.

VEGF and SDF-1α are important in migration. The migration induced by VEGF is mediated by PI3K, which induces downstream activation of PKC.


In summary: it appears that BM microenvironment provides a sanctuary for myeloma cells by both promoting proliferation and blocking apoptosis and initial drug induced damage, thereby allowing tumour progression and eventual emergence of drug resistance.


In order to overcome this resistance it is important to design drugs that activate the two main apoptosis pathways: The first, the death receptor pathway, induces apoptosis mediated by caspase 8 upon activation in response to drugs such as DNA damaging agents, Thalidomide and IMID’s or g irradiation, The best death receptors to study are FAS (apo-1 or CD95), TNFR1 and TRAIL.

Interestingly, the normal cells, while expressing TRAIL, are resistant to its apoptotic effect, while malignant cells are sensitive. Accordingly, the use of TRAIL alone or in combination with other agents is attractive for the treatment of MM. The second pro-apoptotic route is the mitochondrial intrinsic pathway mediated by caspase 9 and is used by drugs such as Dexamethasone and Arsenic trioxide. At the same time the mitochondria release other proteins such as the AIF (Apoptosis Inducing Factor) and Smac (Second Mitochondria-derived Activator of Caspase), which bind and block the IAPs (Inhibitors of apoptosis proteins).

Other drugs such as Proteasome inhibitors (PS341) use both pro-apoptotic routes

 

Preview of Programme Proposed
The primary goals for this part of the proposal are:

  • to identify sufficient numbers of myeloma cell lines and patient samples which have specific genetic abnormalities
  • to collect these samples and distribute them among the participating centers, so that experimental drugs can be tested in vitro. Because several laboratories have already the tools available to investigate the effect of such drugs (f.e. STAT3 inhibitor), the efficacy of their programs will increase significantly by exchanging samples
  • to develop a common strategy to define a priority list of agents that will be tested
  • to define specific tools and models that are present in each laboratory, so that potential new drugs can be tested in a multiple test models and with multiple techniques. To coordinate meetings in order to initiate discussions on potential new targets for inhibition of myeloma growth and proliferation.

Deliverables and Cooperations
During the first 18 months we anticipate the following results;

  • an overview of all available techniques and models in the participating labs. A full list will be prepared and distributed
  • provide a list of potential targets and drugs that have been tested so far including non-published (negative) results
  • a list of potential agents waiting to be tested will be prepared and distributed
  • a list of human myeloma samples and their specific translocations, which are available at each laboratory.

Beyond 18 months the primary goal is to develop a coordinated strategy to define new targets for drug treatment and to initiate a coordinated effort to perform a common research program between the participants in this area.

Secr. Hans E. Johnsen | Depart. of Haematology | Aalborg University Hospital | Sdr. Skovvej 15 | DK-9000 Aalborg | Denmark | T:+45 9766 3871 | F:+45 9766 6369