Phosphatidylinsoitol-3 Kinase Delta (PI3Kδ) Inhibitor AMG 319 Combined with Vincristine Enhances G2/M Arrest and Apoptotic Death in Neoplastic B Cells
Authors: Liu Liqin , Sun Bee-Chun , Pistillo Jeanne , Payton Marc , Wang Ling , Archibeque Ivonne , Molineux Graham , Sinclair Angus 
Published: October 2019

Abstract
Abstract 4963
Phosphatidylinositol-3 kinase delta (PI3Kδ) is a class IA lipid kinase expressed primarily in leukocytes and is a key signaling molecule for immune receptors such as the B cell receptor (BCR). Dysregulated BCR-PI3Kδ signaling has been reported to play a role in B cell malignancy by mediating abnormal growth and survival. Recently we have identified that the potent and selective PI3Kδ inhibitor AMG 319 in combination with vincristine synergistically reduced cell viability in vitro and enhanced xenograft tumor regression in vivo when DLBCL cell line HT, which was relatively insensitive to AMG 319 in viability assays, was used as a model system. These findings suggest that inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancy.
To investigate the mechanism by which AMG 319 synergizes with vincristine in HT cells, we evaluated cell cycle and apoptosis after treatment with single agents or the combination. Vincristine alone at high concentration (4 nM) induced extensive G2/M arrest (51% versus 10% in vehicle control) at 24 hours while little or no G2/M arrest was detected at lower concentrations (0.8 and 0.16 nM). AMG 319 alone, at 0.33 μM, 3.3 μM and 10 μM did not induce G2/M arrest in HT cells. However, when AMG 319 was combined with a low concentration of vincristine at 0.8 nM, increased G2/M arrest (49%) was detected after 24 hours compared to each agent alone (21% in vincristine at 0.8 nM and 9% in AMG 319 at 10 μM) or vehicle control group (16%). This effect was AMG 319 concentration dependent (25%, 34% and 49% in vincristine at 0.8 nM combined with AMG 319 at 0.33, 3.3 and 10 μM, respectively). Interestingly, the enhanced G2/M cell cycle arrest with the drug combination was not sustained at 48 hours as cells underwent apoptosis as demonstrated by an increase in the SubG1 population and caspase-3 activity as well as a decrease in cell viability. In further support of these findings, a substantial increase in Annexin V (+) apoptotic cells (up to 68%) was observed in an AMG 319 concentration dependent manner at 48 and 72 hours in combination with vincristine as compared to that observed with the single agents (17% in vincristine at 0.8 nM; between 11%-23% in AMG 319 at 0.33, 3.3 and 10 μM) or vehicle control (around 10%).
We then examined phosphorylation of Histone H3 at Ser10 and phosphorylation of cell cycle check point kinases Chk2 at Ser68 and Chk1 at Ser317, which are known to be associated with G2/M arrest. The phosphorylation of Histone H3 and Chk2 was increased between 3 to 18 fold in the drug combination group compared with either agent alone or the vehicle control group at 24 hours. In contrast, phosphorylation of Chk1 at Ser317 was unchanged. Lastly, modulation of pAkt (Ser473 and Ser308) was assessed and vincristine alone did not inhibit pAkt at all three concentrations (0.16 nM, 0.8 nM and 4 nM) up to 24 hours or change the maximum levels of pAkt observed. As expected, AMG 319 alone (in concentrations ranging from 3.3 nM to 3.3 μM) significantly inhibited pAkt (> 85% of inhibition in AMG 319 at 3.3 μM) at both 6 and 24 hour time points in a concentration-dependent fashion. The combination of vincristine with AMG 319 did not lead to further inhibition of pAkt.
Taken together, these data suggest that the up-regulation of Chk2 (Ser68) is one of the underlying molecular events associated with the synergistic effects of AMG 319-vincristine combination on G2/M arrest, leading to cell growth inhibition and apoptotic death in HT cells. Therefore, inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancy by inducing cell cycle arrest and apoptosis.

Disclosures:
Liu: Amgen: Employment. Sun:Amgen: Employment. Pistillo:Amgen: Employment. Payton:Amgen: Employment. Wang:Amgen: Employment. Archibeque:Amgen: Employment. Molineux:Amgen: Employment. Sinclair:Amgen: Employment.