BH3 Profiling Demonstrates That Restoration of Apoptotic Priming Contributes to Increased Sensitivity to PI3K Inhibition in Stroma-Exposed Chronic Lymphocytic Leukemia Cells

Abstract 974

We and others have previously found that CLL cells exposed to stroma are resistant to a wide variety of treatments. While this phenomenon is thought largely to be due to inhibition of apoptosis, this has not been directly tested. BH3 profiling was developed in our laboratory as a functional assay to assess the degree to which malignant cells are primed to undergo apoptosis via the mitochondrial pathway. BH3 profiling demonstrates that decreased priming for apoptosis is indeed associated with treatment resistance in CLL cells co-cultured with mouse fibroblast L cells. Here, we confirm these preliminary results, showing that CLL cells co-cultured with either primary human stroma or a human stromal cell line also exhibit decreased priming for apoptosis. We demonstrate that the delta-isoform specific PI3K inhibitor CAL-101 (GS1101) effectively kills stroma-exposed CLL cells and sensitizes them to treatment with other agents by releasing the cells from stroma and/or increasing their level of mitochondrial apoptotic priming.

CLL cells were co-cultured in the presence or absence of drugs with either the human stromal cell line StromaNKTert, with nurse-like cells (NLC) grown from primary CLL patient samples, or with no stroma for 24 hours, as previously described (Burger et al., Blood, 2000). A calcein-based adhesion assay was used to assess CLL cell adhesion to stroma in the presence or absence of CAL-101 (GS1101). CLL cell apoptosis in response to various drug treatments was assessed by Annexin/PI analysis. BH3 profiling was performed by exposing gently permeabilized malignant CD19+ B cells from CLL patients to a panel of BH3-domain peptides, and mitochondrial apoptotic priming was quantified by JC-1-based FACS to assess mitchondrial outer membrane permeabilization, as previously described (Ryan et al., PNAS 2010).

Stroma-exposed CLL cells were highly resistant to the purine analog fludarabine and the BH3-mimetic ABT-263. CAL-101 (GS1101) was able to partially overcome this resistance, and the combination of CAL-101 (GS1101) and fludarabine led to a marked increase in sensitivity of stroma-exposed CLL cells. To evaluate the underlying mechanism of increased CLL cell sensitivity in the presence of CAL-101 (GS1101), we labelled CLL cells with calcein, and co-cultured them with the StromaNKTert cell line. In short term culture (1 hour), CLL cells were highly adherent to stroma, and were significantly less adherent in the presence of CAL-101 (GS1101). After longer co-culture (24 hours), calcein-labelled CLL cells were released from stroma within one hour of the addition of CAL-101 (GS1101). To evaluate whether the release of CLL cells from stroma affected their apoptotic priming, we performed BH3 profiling on CLL cells co-cultured in the presence or absence of stroma. CLL cells cultured without stroma were significantly more primed for apoptosis than CLL cells co-cultured with either the StromaNKTert cell line or primary human NLC. The addition of CAL-101 (GS1101), with or without fludarabine, to CLL cells in culture with stroma, led to increased apoptotic priming compared to CLL cells in culture that were untreated or treated with fludarabine alone. Similar alterations in apoptotic priming were observed when CLL cells were co-cultured with CAL-101 (GS1101) with or without ABT-263.

Overall, these results suggest that the sensitivity of stroma-exposed CLL cells to CAL-101 (GS1101) may be related to the increased mitochondrial apoptotic priming that is facilitated by release of these cells from the stroma. These data may partially explain the clinical observation that, unlike traditional chemotherapy agents, CAL-101 (GS1101) has been efficacious in CLL despite causing an increase in the white blood cell count.