Identification of MicroRNA Markers of Sensitivity to the Novel Bruton’s Tyrosine Kinase (BTK) Inhibitor PCI-32765 in Non-Hodgkin’s Lymphoma.

PCI-32765 is a novel, first-in-class small molecule inhibitor of Bruton’s tyrosine kinase (BTK), currently in preclinical development and shortly entering Phase I clinical trials for the treatment of hematological malignancies. BTK is essential in B-cells for signalling via the B-cell receptor (BCR), and mutations in BTK have been shown to cause x-linked agammaglobulinemia in humans and x-linked immunodeficiency (xid) in mice. We have found that PCI-32765 has demostrated significant growth inhibitory activity in vitro in a number of non-Hodgkin’s lymphoma (NHL) cell lines as well as primary human NHL tumors cultivated ex-vivo. However, certain subsets of NHL cell lines were found to respond better to the inhibitor, and in keeping with this, a wide variance was also observed in the response among primary human tumors. PCI-32765 had GI50 <3uM in 7/15 cell lines (46%), including 3 follicular lymphoma (FL) and 3 diffuse large B-cell lymphoma (DLBCL) lines. Based on these results, we tested primary tumors including 15 FL and 7 DLBCL, utilizing a modified version of Oncotech’s EDR Assay. We found that while only 1 of the DLBCL tumors showed a similar response, PCI-32765 had a response rate of 40% in the primary FL samples. The reasons for this variation are still under investigation but one reason may be that that not all tumors are equally dependent on BTK activation for their survival. In order to obtain a predictive marker for response to PCI-32765 in human tumors, we utilized a microRNA (miRNA) profiling approach in the primary FL tumors using a commercially available miRNA array platform. We hypothesized that miRNA might be a better diagnostic tool than mRNA because miRNA function is more conserved and it is more stable than mRNA (thus easier to harvest successfully from paraffin embedded blocks). Further, it has been shown that miRNA signatures can predict responsive and resistant phenotypes based on basal miRNA levels, and have been found to have essential functions in lymphomas. When the miRNA profiles from the FL tumors were analyzed, we found that a signature consisting of 4 miRNAs could predict the sensitive tumors with a p-value <0.01. Of these, 2 were upregulated and 2 were downregulated in the sensitive samples compared to the resistant ones. It is likely therefore that these are implicated in the mechansisms of sensitivity and resistance to PCI-32765 in the FL tumors, and the connections between genes regulated by these miRNAs to pathways controlled by BTK are being explored.