PF370 SIGNAL-OMICS ANALYSIS OF THE IMPACT OF THERAPY ON BCR SIGNALLING IN CHRONIC LYMPHOCYTIC LEUKAEMIA

Abstract

Background: Induction of signalling via cell surface receptor activation is a critical driver of chronic lymphocytic leukaemia (CLL) pathobiology, especially via the B-cell receptor (BCR), which promotes tumour survival and progression. The vital nature of BCR signalling has been recognized through the development of kinase inhibitors (KI), most notably ibrutinib, that target key nodes within this pathway. Current efforts to monitor signalling investigate expression of a highly confined series of kinases and phosphoproteins. While generating key insights, full appreciation of their wider significance to malignant pathology and therapy response remains an unresolved issue. Here, we describe a ‘signal-omics’ approach, involving a kinobead-based protocol used in conjunction with mass-spectrometry (MS) or immunoblotting. Kinobead binding is related to the availability of a kinase's active site and thus can be used to determine signalling within cells. We have employed this approach characterise surface-IgM (sIgM) signalling within primary CLL cells. Aims: Validate signal-omic profiling for investigating BCR signalling in primary CLL. Methods: Kinobead Signalling Analysis of BCR Signalling Response Whole cell lysates from anti-IgM and control F(ab’)2-treated primary CLL cells underwent kinobead isolation. Elutions from these were processed and were tested using mass-spectrometry (MS). MaxQuant software analysis of MS- data was used to identify kinases and relative binding to kinobeads. Immunoblotting Immunoblotting was used to screen whole cell lysates and kinobead elutions for kinase expression and activation change in all conditions. Results: Employing this approach, it was possible to isolate over 100 kinases from primary CLL cells and identified a ‘fingerprint’ of over 50 kinases which displayed unique, patient-specific response to sIgM stimulation. Signal strength was correlated to sIgM expression but not IGHV mutation status, calcium flux, karyotype or staging. Interestingly, our analysis recognised greater activation change in patients who had undergone prior chemoimmunotherapy compared to those from untreated/treatment-naïve patients. Comparison of matched samples was also able to illustrate kinomic changes in patients who have failed chemoimmunotherapy or have developed resistance to ongoing ibrutinib treatment. Summary/Conclusion: These data represent the first comprehensive, high-resolution investigation into BCR signalling response within CLL, where our probing of kinase active sites reveals unique evidence of adaptive reprogramming in response to therapy.