The Proteostasis Network Is a Therapeutic Target in Acute Myeloid Leukemia

Proteasome inhibitors are first-line therapies for multiple myeloma patients and function in part by disrupting protein homeostasis (proteostasis). Proteasome inhibiton causes an accumulation of misfolded proteins in highly secretory myeloma cells that overwhelm the endoplasmic reticulum (ER) and activate the unfolded protein response (UPR)-part of the integrated stress response (ISR) pathway. However, proteasome inhibition has shown limited efficacy in other cancers, including acute myeloid leukemia (AML). In AML, we show that two nodes of the proteostasis network-the heat shock response and autophagy-are activated in response to proteasome inhibition to help preserve proteostasis. We hypothesized that disabling these adaptive pathways could sensitize AML cells to proteasome inhibition by causing a collapse of proteostasis.

To test this, we quantified unfolded protein in human myeloma and AML cell lines treated with proteasome inhibitors. Whereas proteasome inhibition increased unfolded protein content in myeloma cells, it had no significant effect on AML cells. We performed RNA-sequencing on AML cells treated with proteasome inhibitors and determined that the heat shock and autophagy pathways were both upregulated. We confirmed that both the heat shock response and autophagy were activated in AML cells treated with proteasome inhibitors by assessing levels of phosphorylated HSF1, a master regulator of the heat shock response, and by using a dual fluorescent autophagy (GFP-LC3-RFP) reporter, respectively. We generated and treated multiple HSF1^-/- AML cell lines with proteasome inhibitors, and observed significant accumulation in unfolded protein, diminished proliferation, reduced protein synthesis, and massive induction of apoptosis. Concurrent HSF1 deletion and proteasome inhibition also dramatically reduced AML disease burden in xenograft assays in vivo and extended median survival from 40 to 140 days.

To assess the role of autophagy, we utilized Lys05, a small molecule autophagy inhibitor. Similarly to proteasome inhibition in HSF1^-/- cells, concurrent proteasome and autophagy inhibition increased unfolded protein in AML cell lines. Furthermore, combined autophagy and proteasome inhibition reduced proliferation, decreased protein synthesis, and increased apoptosis across multiple human AML cell lines and primary patient AML cells. In contrast to AML cells, combined autophagy and proteasome inhibition was well tolerated by healthy human CD34+ cells, suggesting a tractable therapeutic window for treating AML.

To determine the mechanistic effects of proteostasis disruption, we assessed activation of the ISR by examining the eIF2a-ATF4-CHOP signaling axis. Proteasome inhibition failed to activate a terminal ISR response in AML cells. However, HSF1^-/- AML cells treated with proteasome inhibitors and AML cells treated with combined proteasome and autophagy inhibitors expressed increased phospho-eIF2a, ATF4 and CHOP, indicative of terminal ISR activation. We next asked whether PERK, an eIF2a-kinase that responds to ER stress, was mediating ISR activation. Knockdown of EIF2AK3 (PERK) partially rescued effects of proteasome inhibition in HSF1^-/- AML cells. However, effects of combined proteasome and autophagy inhibition on protein synthesis and apoptosis were maintained in EIF2AK3^-/- AML cells. Surprisingly, we determined that effects of ISR activation downstream of proteostasis disruption mediated by combined proteasome and autophagy inhibition was driven by PKR (EIF2AK2). Suppression of protein synthesis and induction of apoptosis was significantly attenuated in EIF2AK2^-/- AML cells. These results suggest that distinct eIF2a kinases mediate activation of the ISR in AML cells depending on the mechanism of proteostasis disruption.

Our work highlights repurposing an approved family of pharmaceuticals and combining them with inhibition of proteostasis network nodes as a tractable strategy for generating new AML treatment regimens.

Signer:Exact Sciences: Current equity holder in publicly-traded company, Other: Spouse employment; Illumina: Current equity holder in publicly-traded company; Vir: Current equity holder in publicly-traded company; Syndax: Current equity holder in publicly-traded company.