BIRC3 but Not BIRC2 mRNA Expression in Chronic Lymphocytic Leukemia Correlates with Disease Progression and Mediates Decreased Fludarabine Sensitivity in Vitro

The molecular characterization of chronic lymphocytic leukemia (CLL) with whole genome sequencing has recently begun to foster a novel understanding of the disease, identifying pathways of importance in CLL biology. One of the target genes that was identified by this approach was BIRC3 (Rossi et. al. Blood 2012). BIRC3 (cIAP2) and its close homolog BIRC2 (cIAP1), are members of the IAP (inhibitior of apoptosis) protein family, that unlike other IAPs, do not directly interfere with apoptosis but are key components of the canonical and non-canonical NFKB signalling pathways. As such they play an important role in B-cell differentiation, survival and proliferation. In the murine system BIRC2 and BIRC3 were shown to be redundant (Gardam et. al. Blood 2011). Their combined loss rendered murine B-cells independent of some survival factors and led to uncontrolled B-cell accumulation. In CLL, Rossi et. al. reported recurrent mutations in BIRC3 that were rare at diagnosis (<5%), but associated with increased non-canonical NFKB signalling, worse prognosis and fludarabine refractory disease, independent of p53. We aimed to determine if BIRC3 mutations might be a flag for an importance of the pathway in a larger context of CLL biology and wanted to know whether BIRC3 expression levels are important for clinical outcome.

Peripheral blood MCs of 63 fully characterized CLL patients were collected. mRNA levels of BIRC2 and BIRC3 were analysed using Real Time PCR and normalized to 18S as housekeeping control. Cutoffs to detect different time to treatment intervals were determined by ROC analyses. In vitro culture of CLL cells was performed under standard culture conditions in presence or absence of accessory cells (NIH 3T3 fibroblasts +/− CD40L transgene) in order to abrogate spontaneous apoptosis or achieve CLL cell activation for 3 days. siRNA transfections were performed with non-targeting siRNA (NTC), BIRC2 or BIRC3 siRNA using lipofection as recommended by the manufacturer (Qiagen).

BIRC3 and BIRC2 mRNA levels were compared with established risk factors and clinical course. We detected significantly downregulated BIRC3 expression levels (P < 0.001) in CLL with unmutated (N = 24) as compared to CLL with a mutated B-cell receptor status (N = 33) and in CLL of advanced RAI stages (N = 19, P < 0.01). Low expression of BIRC3 mRNA was furthermore significantly correlated to decreased time to first treatment (P < 0.001, Fig 1A), which highlights the prognostic impact of BIRC3 mRNA levels independent of potential BIRC3 mutations. Since our test population was previously untreated and cut-offs for prediction were close to the median, this effect was presumably independent of the occurrence of BIRC3 mutations, that should be rare in such a population. To clarify this, mutation analysis is currently ongoing. Despite the proposed functional redundancy, the mRNA levels of the homologue BIRC2 did not correlate with prognostic factors or time to treatment (Fig 1B) giving first hints, that in the context of CLL BIRC3 may be more important as compared to its homolog, which is also reflected in the dominance of BIRC3 mRNA levels as compared to those of BIRC2 (N = 63, P < 0.0001). To further address the question of functional redundancy we performed siRNA mediated downregulation of either BIRC2 or BIRC3. We found that BIRC3 but not BIRC2 siRNA mediated downregulation significantly inhibited fludarabine induced cell death of CLL cells in vitro (N = 6, Fig 1C).

Here we demonstrate for the first time that in addition to the reported effect of mutations in BIRC3, mRNA levels were related to negative prognostic factors and predictive for shorter time to treatment. We further provided evidence that BIRC3 and BIRC2 potentially have non-redundant roles indicated by their different behavior, opposite clinical parameters and different outcomes in response to siRNA mediated downregulation in vitro. Importantly and consistent with a potential selection for mutations, downregulation of BIRC3, but not BIRC2, increased in vitro resistance of CLL cells against fludarabine, the backbone of CLL therapy. Considering the accumulation of BIRC3 mutations in fludarabine refractory CLL (Rossi et. al. Blood 2012), BIRC3 might serve as a novel biomarker, that together with aberrations in p53, could characterize CLL patients with suboptimal therapy success and inferior outcome.

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No relevant conflicts of interest to declare.