PT  - JOURNAL ARTICLE
AU  - AJ Linley
AU  - R Griffin
AU  - S Cicconi
AU  - A D’Avola
AU  - DJ MacEwan
AU  - AR Pettit
AU  - N Kalakonda
AU  - G Packham
AU  - IA Prior
AU  - JR Slupsky
TI  - Kinobead profiling reveals reprogramming of B-cell receptor signaling in response to therapy within primary CLL cells
AID  - 10.1101/841312
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 841312
4099  - http://biorxiv.org/content/early/2019/11/14/841312.short
4100  - http://biorxiv.org/content/early/2019/11/14/841312.full
AB  - Induction of signaling via cell surface receptor activation is a critical driver of CLL pathobiology, especially via the B-cell receptor (BCR), which promotes tumor survival and progression. The vital nature of BCR signaling has been recognized through the development of kinase inhibitors (KI), most notably ibrutinib, that target key nodes within this pathway. Current efforts to monitor signaling 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 kinobead-based protocol, used in conjunction with mass-spectrometry (MS) or immunoblotting, to study surface-IgM (sIgM) signaling within primary CLL cells. Employing this approach, we isolated a ‘fingerprint’ of over 30 kinases which displayed unique, patient-specific response to sIgM stimulation, and which displayed greater activation change in CLL cells from patients who had undergone prior chemoimmunotherapy (CIT) compared to those from untreated/treatment-naïve patients. Matched sample analysis of ARCTIC/AdMIRe clinical trial patients revealed the unique nature of the kinome response was present at the intra-patient level, while longitudinal profiling of IcICLLe trial patients supported this as well as showing our finding related to ibrutinib therapy. Refinement of the kinome fingerprint determined 4 kinases linked to proliferation found to be present to a significantly higher level within previously treated patient cells. Proliferation assays confirmed that these patients possess higher proliferative capacity, implying alterations of signaling resulting in promoting of biological processes critical to malignant cells. Collectively, these data represent the first comprehensive investigation into BCR signaling response within CLL, where our probing of kinase active sites reveals unique evidence of adaptive reprogramming in response to therapy.Key pointssIgM signaling patterns alter following in vivo therapy using either chemoimmunotherapy or ibrutinib.Kinobeads provide a novel method for high-resolution investigation of signaling in primary CLL cells.