Protein Kinase C-ß Dependent IL-8 Release Promotes Acute Myeloid Leukemia Blast Cell Survival in Co-Cultures with Bone Marrow Stromal Cells
Authors: Abdul-Aziz Amina M , Shafat Manar S , Lawes Matthew J , Bowles Kristian M , Rushworth Stuart A 
Published: October 2019

Abstract
Introduction:
Acute myeloid leukemia (AML) cells exhibit a high level of spontaneous apoptosis when cultured in vitro but have a prolonged survival time in vivo indicating that the tissue microenvironment plays a critical role in promoting AML cell survival. Knowledge of the complexity of the bone marrow microenvironment is increasing especially with respect to the bone marrow mesenchymal stromal cells [BM-MSC] which are considered a major protective cell type. Other studies have demonstrated the ability of BM-MSC to protect leukemia cells from spontaneous and chemotherapy-induced apoptosis. Increasing evidence suggests the existence of crosstalk between leukemia cells and BM stromal cells to create a leukemia-promoting environment. Recently our group and others have shown that this crosstalk is achieved by a complex communication system that involves multiple bidirectional signals which enhance AML survival and proliferation. Here we report a novel interaction between AML blasts and BM-MSC which benefits AML proliferation and survival.
Methods:
To investigate the interaction between primary AML blasts and BM-MSC we isolated AML and BM-MSC from the same patient and used an autologous in vitro culture assay to analyze the cytokine profile. Conditioned medium was collected from cultures of primary human AML alone or cultured with autologous BM-MSC and analyzed using Proteome Profiler Human XL Cytokine Array and target specific ELISAs. Real-time PCR was also used to verify the array data. MIF-Receptor inhibitors (SB 225002- CXCR2, AMD3100 - CXCR4 and CD74 blocking antibody - CD74) and signaling kinase inhibitors (LY294002- PI3K/AKT, PD098059 - MAPK, Ro-31-8220 - PKC) were used for initial determination of MIF signaling pathways in BM-MSC. Specific PKC isoform inhibitors (Go6976-PKCα/ß and enzastaurin -PKCß) were then used to determine isoform specific activation. Western blot and siRNA were used to confirm the role of AML derived MIF in regulating downstream BM-MSC signaling pathways including MAPK, PI3K/AKT, and PKC.
Results:
We initially examined the cytokine profile in cultured human AML compared to AML cultured with autologous BM-MSC or BM-MSC alone and found that MIF was highly expressed by primary AML and that IL-8 was increased in AML/BM-MSC co-cultures. The observed changes in IL-8 were confirmed by ELISA assays. RT-PCR was used to measure MIF and IL-8 gene expression from RNA extracted from primary AML or BM-MSC cultured alone or in combination. Results confirmed that MIF is highly expressed at the RNA and protein level by AML blasts and IL-8 transcription and cytokine release was upregulated in BM-MSC in response to co-culture with AML.
Next we found that recombinant MIF increases IL-8 mRNA and protein expression in BM-MSC. Moreover, the MIF inhibition by, ISO-1, inhibits AML induced IL-8 expression and secretion by BM-MSC. Next we sought to determine which kinase signaling cascade is activated by MIF. We used a panel of protein kinase inhibitors and found that the pan-PKC inhibitor Ro-31-8220 completely inhibits AML and MIF induced IL-8 mRNA at sub micromolar concentrations. To further identify the specific PKC isoform responsible for linking AML induced MIF to IL-8 we used PKC isoform specific inhibitors (Go6976 and enzastaurin) which significantly inhibited MIF induced IL-8 expression and protein in BM-MSC. The introduction of PKCß siRNA dramatically inhibited MIF induced IL-8 mRNA expression in BM-MSC confirming that PKCß regulates AML induced BM-MSC derived IL-8 expression. Finally, inhibition of AML/BM-MSC co-cultures with the PKCß inhibitor enzastaurin inhibits BM-MSC induced AML survival in vitro.
Conclusions:
These results reported here show a novel bidirectional survival mechanism between AML blasts and BM-MSC. Furthermore this work identifies the PKC-ß-IL8 pathway in the BM-MSC of patients with AML as a novel target for future treatment strategies.

Disclosures
No relevant conflicts of interest to declare.