The role of cIAP1 and cIAP2 in chronic lymphocytic leukemia

Signals derived from the microenvironment have a demonstrated pathophysiological relevance in the establishment, progression and chemoresistance of chronic lymphocytic leukemia (CLL). Among these, B-cell receptor (BCR)-, BAFF (B-cell activating factor)-, APRIL (a proliferating ligand)-, and CD40/CD40L- mediated signals, all transmitted via the NFB pathway, have been shown to impact CLL development. Recently, mutations within BIRC3, encoding the NFB pathway regulator cIAP2 (cellular inhibitor of apoptosis 2), were shown to be associated with poor clinical outcome and chemoresistance to Fludarabine (Rossi et. al. Blood 2012), the backbone of current therapy regimens. We thus hypothesized that the rare BIRC3 mutations indicated a critical pathway in CLL biology. BIRC3/cIAP2, together with its close homologue BIRC2/cIAP1, is a central regulator of the non- canonical NFB pathway. This alternate NFB signalling pathway is particularly predominant in lymphocytes. In B-cells, non-canonical NFB signalling is mediated via a very specific subset of receptors including BAFF-R and CD40, and largely impacts B cell survival, maturation, differentiation, and germinal center (GC) formation. Given these observations, the overall goal of the proposed study is to characterize the contribution of cIAP1 and 2 to CLL pathophysiology and chemoresistance. We will achieve this by: (1) investigating the impact of cIAP1 and cIAP2 deficiency on clinical progression and CLL development by correlating expression levels with clinical features of CLL, (2) define the upstream signals and molecular mechanisms that regulate protein expression, focusing on known microenvironmental cues (3) analyse the downstream consequences of cIAP1/2 dysregulation on CLL viability and proliferation, and (4) characterize the molecular connection between cIAP deficiency and p53 dysfunction that contributes to Fludarabine chemoresistance. As CLL displays such an intricate dependence on its microenvironment, both complex in vitro co-culture systems using primary human samples as well as animal models will be employed in order to sufficiently recreate the multifaceted microenvironmental cues required, while retaining important information on human CLL behavior.

The project focused on the role of cIAP1 (BIRC2) and cIAP2 (BIRC3) in chronic lymphocytic leukemia (CLL). CLL is a disease caused by the malignant outgrowth of B-cells, typically affects the elderly population and is characterized by an interplay of tumorcells with the microenvironment. Moreover, intrinsic lesions like mutations or deletions in pathways positively interfering with the survival and the proliferation of CLL cells are involved in CLL pathogenesis. One such critical pathway is the NF-B pathway, which is inhibited by BIRC2 and BIRC3. BIRC3 loss of function mutations were identified in the past and correlated to an unfavorable CLL prognosis. BIRC3 deletions were detected in a subgroup of CLL patients harboring the so called deletion 11q, which leads to the genetic loss of the BIRC3 coding region. Deletion 11q patients showed an inferior prognosis, however, the possible contribution of BIRC3 in this regard was not clear. In our work we were the first to demonstrate BIRC3 mRNA expression levels as being sufficient in predicting CLL disease progression. This was independent of BIRC3 mutations/deletions and other CLL risk factors. Moreover, we analyzed a potential contribution of BIRC2 in CLL pathogenesis, however, BIRC2 showed no prognostic impact, was lower expressed as compared to BIRC3 and not dynamically regulated by stimuli derived from the microenvironment. Hence, we concentrated our work on BIRC3 aiming to reveal the biological mechanisms that link BIRC3 expression to an unfavorable prognostic outcome in CLL. We found a deregulation of NF-B signaling after Baff stimulation in CLL cells with low BIRC3 expression as compared to CLL cells with high BIRC3 expression. Baff is a protein which is mainly secreted by innate immune cells and captured by CLL tumor cells by the so called Baff receptor. Normally, this Baff/Baff receptor interaction leads to the induction of the alternative NF-B pathway in CLL cells. However, in CLL cells with low BIRC3 expression the classical NF-B pathway was induced which is well known for its regulation of genes involved in cell survival and proliferation. Indeed we could show the enhanced induction of pro-survival Bcl-2 family members in CLL cells with decreased BIRC3 expression. Hence, we suspected an enhanced addiction of CLL cells with low BIRC3 expression to Bcl-2 proteins, which may render them more sensitive to treatment with Bcl-2 inhibitors. This was entirely true and we could show that CLL cells with low BIRC3 expression responded better to Abt-199 treatment in vitro. This finding is of clinical importance as Abt-199 (Venetoclax) was recently approved for the treatment of CLL.