P38 Is a Negative Regulator Of The Bortezomib-Induced Heat Shock Response In Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy with an estimated 22,350 new cases and 10,710 deaths in the United States in 2013. Novel treatments including autologous stem cell transplant, immunomodulatory drugs (IMiDs), and the proteasome inhibitor, bortezomib, have led to an increase in patient life span and long-term survival. Bortezomib is a highly selective and reversible 26S proteasome inhibitor. Proteasome inhibition can affect multiple signaling cascades and lead to a toxic buildup of misfolded proteins and eventually, cell death.

As part of the response to this protein buildup following proteasome inhibition, myeloma cells activate the cytoprotective heat shock response. This includes upregulation of heat shock proteins (HSPs) such as HSP40, HSP70, and HSP90. Previous attempts at using HSP-specific inhibitors in combination with bortezomib have been disappointing. These results underscore the need to disrupt broad scale activation of the entire heat shock response. This can be achieved by inhibition of the master regulator, Heat Shock Factor 1 (HSF1). Here we show that in four human MM cell lines, MM1.s, KMS11, KMS18, and U266, HSF1 inhibition leads to downregulation of the bortezomib-induced heat shock response and ultimately, increased cell death.

While HSF1 is activated by proteasome inhibition, the mechanism of activation has yet to be determined. HSF1 is regulated through a complex series of post-translational modifications. Here we show that bortezomib induces HSF1 phosphorylation in MM1.s and KMS18, and in freshly isolated patient samples.

To determine which kinase pathways are responsible for HSF1 phosphorylation, we treated MM1.s and KMS18 with a non-lethal dose (10 μM) of PI3K, MEK, JNK, and p38 inhibitors in combination with bortezomib. Bortezomib-induced HSF1 phosphorylation was inhibited by the p38 inhibitor SB 203580, while inhibitors to PI3K, MEK, and JNK had no effect on bortezomib-induced HSF1 phosphorylation.

To determine the consequence of p38 inhibition on HSF1 function, we performed RT-qPCR to probe for the expression of HSF1-dependent gene targets (HSPB1 [HSP27], HSP40B, HSPA1A [HSP70/72], HSPA1B [HSP70/72], HSP90AA1, HSP90A1B) following treatment with bortezomib with or without SB 203580. Surprisingly, gene expression for each of the targets increased when proteasome inhibition was combined with p38 inhibition compared to proteasome inhibition alone. The observed change ranged from 31% (HSPA1A) to 99% (HSP90AA1). Our results show a previously undescribed link between proteasome inhibition and HSF1 regulation; bortezomib-induced p38-dependent phosphorylation. This is consistent with studies in HeLa cells showing that the p38 effector MK2 negatively regulates HSF1 via phosphorylation of S121.

Together these findings underscore the complexity of the cellular response to proteasome inhibition, and that understanding both the positive and negative regulatory events during HSF1 activation could lead to the development of novel partners for use with proteasome inhibitors.