Background

MYD88 L265P is a somatic mutation present in >90% of Waldenstrom's Macroglobulinemia (WM) patients. The downstream signaling pathways that promote malignant cell growth and survival remain to be fully clarified but include IRAK 1/4 and BTK (Treon et al, NEJM 2012; Yang et al, Blood 2013).

Methods

To further clarify the downstream signaling associated with MYD88 L265P in WM cells, we employed Phospho Explorer Antibody Arrays in MYD88 L265P expressing BCWM.1 and MCWL-1 WM cells following lentiviral mediated knockdown of MYD88, or over-expression of MYD88 wild type or L265P; or the use of MYD88 homodimerization inhibitor that block MYD88 signaling. Arrays were scanned by Axon GenePix Microarray Scanner and data analyzed by Ingenuity Pathway Analysis. Western blot analysis was performed using total and phospho-specific antibodies in WM cells. CellTiter-Glo® Luminescent cell viability assay (Promega) was used to assess cell survival following treatment with the PI3K-delta inhibitors, CAL101 and PIK-294 (Selleck Chemicals).

Results

Ingenuity pathway analysis demonstrated significant enrichment for B-Cell Receptor, PI3K/AKT, LPS-stimulated MAPK and ERK/MAPK signaling following MYD88 modulation in both BCWM.1 and MWCL-1 WM cells. Signal proteins that demonstrated the greatest changes in phosphorylation following MYD88 modulation are shown in Table 1. Western blot analysis confirmed the activation of ATK as downstream of PI3K in WM cells. Use of a MYD88 inhibitor significantly decreased phosphorylation of ATK-pS473 and AKT-pY308 in both BCWM.1 and MCWL-1 WM cells. Cell viability analysis demonstrated robust tumor cell killing (EC50 30-50nM) in BCWM.1 and MWCL-1 WM cells following treatment with both PI3K-delta inhibitors (CAL-101 and PIK-294).

Table 1.

Signaling Pathway MYD88 knockdown MYD88-Wild Type over-expression MYD88-L265P over-expression MYD88 homodimer inhibitor 
B-Cell Receptor ↓PLCG1  BTK ↓PLCG1 
PI3K/AKT ↓AKT1
↓TP53 		FOXO3
↓FOXO4 		↓FOXO1 ↓FOXO3
TP53 		↓mTOR 
LPS-stimulated MAPK or ERK/MAPK ↓RELA CHUK IKBKG MAP3K5
MAPKAPK2 		↓RELA ↓CHUK
↓MAP3K5 ↓NFKB1 
Signaling Pathway MYD88 knockdown MYD88-Wild Type over-expression MYD88-L265P over-expression MYD88 homodimer inhibitor 
B-Cell Receptor ↓PLCG1  BTK ↓PLCG1 
PI3K/AKT ↓AKT1
↓TP53 		FOXO3
↓FOXO4 		↓FOXO1 ↓FOXO3
TP53 		↓mTOR 
LPS-stimulated MAPK or ERK/MAPK ↓RELA CHUK IKBKG MAP3K5
MAPKAPK2 		↓RELA ↓CHUK
↓MAP3K5 ↓NFKB1 

(): up-regulation of phosphorylation. (↓): down-regulation of phosphorylation.

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Conclusion

In addition to activation of NF-kB through IRAK and BTK signaling, MYD88 L265P promotes survival of WM cells by activation of PI3K/AKT signaling. Inhibition of PI3K-delta is associated with robust killing of WM cells. These studies provide the framework for the investigation of PI3K-delta inhibitors in WM.

Disclosures:

No relevant conflicts of interest to declare.

Topics:
1-phosphatidylinositol 3-kinase, killing, myd88 gene, neoplasms, phosphoinositide 3-kinase, waldenstrom macroglobulinemia, akt signaling pathway, mitogen-activated protein kinases, proto-oncogene proteins c-akt, antibodies

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2013 by The American Society of Hematology
2013
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