Abstract
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a pivotal role in cell proliferation and survival that underlies the biology of many cancers including CLL. Of the eight distinct mammalian isoforms of PI3K, it is the class IA PI3Ks (p110α, p110β and p110δ) that are responsible for Akt activation and cellular transformation. The p110α and p110β isoforms both have a ubiquitous tissue distribution in adults, whereas p110δ expression is restricted to cells of hematopoietic origin. Recent studies have established a dominant role of p110δ isoform in B-cell responses. Deletion or inactivation of p110δ ablates B-cell antigen receptor (BCR)-induced phosphorylation of Akt and impairs cell cycle progression. Furthermore, CD40-ligand (CD40L) dependent survival is compromised in the absence of p110δ activity. Considering the role of p110δ-mediated BCR and CD40L-CD40 signaling to the enhanced survival in normal B cells, we hypothesized that inhibition of this kinase will induce cytotoxicity in B-CLL cells. We first examined the protein expression of p110δ in primary tumor CD19+ B cells from CLL patients and show that 24/24 CLL consistently overexpressed p110δ. The expression levels of p110α and p110β however, varied more widely, and were often undetectable. Treatment of primary tumor cells with CAL-101, a novel selective p110δ inhibitor, at concentrations of 0.1–10μM resulted in significant cell killing (linear mixed model; p=0.0004). As an example, 5μM CAL-101 resulted in a median of 59.6% viable cells (n=18 CLL patient samples). CAL-101 induced cytotoxicity was accompanied by PARP and caspase 3 cleavage. Previous published studies have demonstrated that CD40L-CD40 signaling promotes activation of CLL cells (as measured by up-regulation of CD40 and CD86) and also protection from spontaneous apoptosis ex vivo. Treatment of CLL cells in the presence of CAL-101 diminished the activation markers CD40 and CD86 induced by CD40L. In addition, an increase in CLL cell viability induced by CD40L was reversed by CAL-101 treatment. Contrasting with this, diminishment of apoptosis with IL-4 was not observed. Given the common finding of innate and cellular immune effects induced by therapies utilized in CLL, we next assessed the effect of CAL-101 on normal NK cells and T cells. Treatment of NK cells and T cells in vitro from healthy volunteers had no effect on cell viability. The lack of cytotoxic effect on normal NK cells and T cells was also assessed in vivo from a completed phase I trial of healthy volunteers that serves as a forerunner to the phase 1 clinical trial in CLL and related lymphoproliferative diseases currently ongoing. Here, treatment of normal human volunteers with CAL-101 for seven days achieved peak plasma concentrations up to 5μM without changes in general hematology or subpopulations of NK cells and T cells. Overall, our results identify the p110δ isoform as a potential therapeutic target in CLL where selective cytotoxicity is observed as compared to normal immune effector cells and the very important CD40-CD40L survival pathway is disrupted. Together, these in vitro and in vivo data provide sound validation for the ongoing Phase 1 clinical trial for the treatment of patients with CLL and related lymphoid malignancies.
The novel nucleoside analogue ProTide NUC-7738 overcomes cancer resistance mechanisms in vitro and in a first-in-human Phase 1 clinical trial
