Sp1 Inhibition Affects Cell Growth and Survival In Waldenstrom’s Macroglobulinemia Through a MYD88-Independent Pathway

Gene expression and proteomics studies have advanced our understanding of Waldenstrom’s macroglobulinemia (WM) and identified potential therapeutic targets, however, WM remains incurable. Therefore there is an urgent need for the development of novel chemotherapeutic agents targeting deregulated signaling pathways specifically present in WM.

Based on role of transcription factor Sp1 in myeloma, we evaluated its molecular and functional role in WM. Our loss of function and Gain of function studies have highlighted a potential oncogenic role of Sp1 in WM. Reduction in Sp1 protein level following transient transfection of WM cells with Sp1 siRNA led to decreased WM cell viability. Conversely, overexpression of Sp1 promoted cell growth and increased IgM production in BCWM1 cell line, associated with an increased level of Sp1 dependent genes. These results demonstrate the role of Sp1 in WM cell growth and survival and provide rationale to therapeutically target Sp1 in WM using small molecule inhibitors of Sp1. We therefore evaluated the activity of Terameprocol (TMP), a small molecule with ability to inhibit Sp1-mediated transactivation by competing for binding to specific Sp1-domains within gene promoter regions. Treatment with TMP caused inhibition of WM and IgM-secreting low-grade lymphoma cell lines, as well as purified primary patient WM cell growth in a dose and time dependent fashion.

Sp1 physically interacts with other TFs, influencing their activity. To identify TFs whose activity is controlled by Sp1 in WM cells, we analyzed the activation of 47 transcription factors in nuclear extracts from BCWM1 and MWCL1 cells that were siRNA-depleted for Sp1 or treated with TMP using a transcription factor profiling assay. Both depletion of Sp1 and TMP treatment decreased the activity of TFs, including STAT1, STAT3, and NF-κB, whereas other factors, such as p53, were not affected. As NF-κB and STAT-3 pathways have been shown to be constitutively activated in WM and to play a pivotal role in regulating growth and survival of WM cells, we have focused our further analysis on these TFs in an attempt to understand the molecular mechanism underlying the activity of Sp1 and its inhibition in WM. Enforced expression of Sp1 significantly induced NF-κB p65 (RelA) activity, and TMP was able to overcome this effect. Inhibition of Sp1 activity impairs basal and TNFα-stimulated NF-κB transcriptional activity as well as IL-6-induced STA3 activation in WM cells.

Recent studies have reported the high frequency of the MYD88 L265P somatic mutation in patients with WM. To investigate the impact of MYD88 on the sensitivity of WM cells to Sp1 inhibition, we first analyzed effect of TMP on MYD88-silenced cells. MYD88 knockdown significantly inhibits BCWM1 cell growth compared with scrambled cells and the antitumor effect was more pronounced upon treatment with TMP. These results provided the rationale to investigate the activity of combination treatment between TMP and inhibitors known to impede the MYD88 pathway signaling. BCWM1 and MWCL1 WM cells were cultured in the absence or presence of a direct kinase inhibitor of IRAK 1 and 4 or the BTK inhibitor PCI32765. The combination treatment resulted in significant and synergistic dose-dependent antiproliferative effect and inhibition of NFkB p65 activity in MYD88 L265P–expressing WM cells suggesting that MYD88 and Sp1 pathways are both functional in WM but independent from each other.

In summary, these results demonstrate Sp1 as an important transcription factor that regulates proliferation and survival of WM cells as well as IgM secreting low-grade lymphoma cells and provides preclinical rationale for clinical development of TMP in WM alone or in combination with inhibitors of MYD88 pathway.