Bcl-2 family members in the pathobiology and therapy of Chronic Lymphocytic Leukemia

In chronic lymphocytic leukaemia (CLL), the survival and chemoresistance of tumor cells, can be attributed to aberrant apoptosis signalling. Ex vivo, CLL cells readily undergo apoptosis, demonstrating that a primed and functional cell death machinery exists that is strongly overruled by microenvironmental survival cues in vivo. CLL cells intrinsically express high levels of the pro-survival protein Bcl-2 and can further upregulate Mcl-1, Bcl-XL, and A1 in response to a diverse number of microenvironmental signals. Paradoxically, high levels of the pro- apoptotic proteins Bim, Bmf, and Noxa are also expressed and can be interpreted as failed attempts of the normal checkpoint machinery to eliminate these aberrant cells. Puma is additionally upregulated upon treatment with the cytotoxic agent Fludarabine that is the mainstay of therapy. The proposed study aims to identify the relevant apoptosis mediators and upstream signals that could be engaged in order to tip the balance towards cell death in CLL, in the hope of developing novel treatment paradigms for this incurable disease. Relevant input survival signals from the microenvironment likely include antigenic stimulation, cellular adhesion, and T cell CLL interactions, thus offering the opportunity to study the impact of these diverse signalling pathways on the Bcl-2 rheostat in a malignant setting. We will therefore (i) identify the Bcl-2 family members whose loss or overexpression significantly impacts tumor development, (ii) stringently define the upstream microenvironmental signals and pathways that result in the modulation these critical apoptosis regulators, and (iii) develop smart therapeutic combinations that simultaneously target relevant signalling pathways and enhance the cell death signal via the use of the appropriate BH3 mimetic. As CLL displays such a intricate dependence on its microenvironment, both complex in vitro co-culture systems and animal models will be employed in our investigations in order to sufficiently recreate the multifaceted microenvironmental cues required, while also retaining some information on human CLL behavior.

The project is addressing the role of Bcl-2 family members in chronic lymphocytic leukemia (CLL) in relation to novel treatment options, useful drug combinations and potential resistance mechanisms. In this regard we are focusing on both, the human disease as well as murine models. The latter are genetically altered to overexpress Tcl-1 (the so called Tcl-1 transgen or tg) and develop a murine CLL that resembles the human disease in a lot of aspects including the late onset. In the mouse model it is possible to specifically delete or overexpress certain Bcl-2 family members and to study functional and/or therapeutically relevant consequences of this altered expression. In detail, we analysed Tcl-1 tg mice harboring overexpression of the antiapoptotic proteins Mcl-1 and Bcl-2 (the latter in cooperation with Andreas Villunger, Biocenter Innsbruck), or deficiencies in the proapoptotic members Puma and Bmf. Surprisingly, we could find only minor influence of these defects on CLL progression although it is assumed that overexpression and deficiencies of anti- and proapoptotic members, respectively, translate into a survival benefit for CLL tumorcells. Hence, individual Bcl-2 family member defects may be compensated by the deregulation of other family members. In human CLL, we could show that overexpression of antiapoptitc Bcl-2 family members was associated with the overexpression of proapoptotic Bcl-2 family members, leading to the destruction of this Bcl-2 balance and transferring into an inferior CLL progression. In contrast, the deregulation of individual Bcl-2 family members was not sufficient in predicting the course of CLL, resembling the data obtained in the murine model. While not predictive for CLL progression, the manipulation of individual Bcl-2 family members gained importance in stress conditions e.g. under treatment in culture or after deprivation of the microenvironment. The latter protects CLL cells in vivo, induces tumor cell proliferation and abrogates tumor cell death. Tcl-1 tg murine tumors with Mcl-1 overexpression or Puma and Bmf deficiency were more resistant to the withdrawal of the microenvironment and survived longer in vitro as compared to control tumors harboring a CLL typical Bcl-2 rheostat. Different results were achieved after treatment of modified tumors with Ibrutinib or Venetoclax. These drugs are currently widly used to treat human CLL, however, potential resistance mechansims are not sufficiently explored due to the novelty of these drugs in the clinical field. While Bmf deficiency in murine tumors was associated with Ventoclax resistance, Puma deficiency was correlated to Iburitinb resistance in a microenvironmental dependent fashion. These data are clinically relevant as they may help to define resistance mechanisms in human CLL and contribute to the development of smart treatment combination regimes.