Dual Sphingosine Kinase and Bcl-2 Inhibition Exhibits Synergistic Cell Death in Acute Myeloid Leukemia

Background:

Pro-survival Bcl-2 family proteins such as Mcl-1 and Bcl-2 have garnered significant interest as therapeutic targets due to their ability to suppress the apoptotic potential of pro-apoptotic Bax and Bak. In response to cell stress, BH3-only proteins sequester Bcl-2 family members, promoting apoptosis. Small molecule inhibitors such as the Bcl-2 inhibitor Venetoclax have demonstrated poor single agent efficacy in AML. This poor efficacy is a result of AML cells being highly dependent on the pro-survival Bcl-2 family member Mcl-1. Sphingosine kinase 1 (SK1) is a signalling enzyme with established roles in oncogenesis and has recently emerged as a potential therapeutic target in leukaemia. SK1 promotes oncogenic signalling through the production of sphingosine-1-phopshate and subsequent activation of G-protein coupled receptors. In addition, SK1 has been reported to modulate Mcl-1 expression in a number of haematological malignancies including AML. Thus we sought to investigate the efficacy of targeting SK1 to sensitise AML cells to Venetoclax.

Results:

To determine how SK1 inhibition influences Mcl-1 levels we utilised a short hairpin RNA (shRNA) approach targeting members of the BH3 only protein family (Bid, Bim, Bad, Puma and Noxa). Targeting of Noxa significantly attenuated the cytotoxic effects of the SK1 inhibitor, MP-A08. Immunoprecipitation of Mcl-1 in response to MP-A08 treatment revealed increased association of BH3 only protein, Noxa and ubiquitin-ligase FBW7 suggesting this triggers Mcl-1 degradation in agreeance with previous studies. No interaction was observed between Mcl-1 and Bim further supporting the importance of Noxa in MP-A08 induced Mcl-1 degradation.

Canonical pathway analysis of MP-A08 treated cells has previously shown to enrich for genes associated with the unfolded protein response (UPR) (Powell et al. Blood 2017). Western blot analysis recapitulated these findings with activation of the PERK arm, typified by eIF2a phosphorylation and ATF4 upregulation. ATF4 has previously been shown to bind within the Noxa promoter and drive transcription in response to ER stress (Wang et al PNAS 2009). Maintaining global translation with eIF2B agonist, ISRIB blocked Noxa transcription in response to MP-A08 suggesting ATF4 drives Noxa mediated Mcl-1 degradation.

Given the Noxa dependent Mcl-1 degradation in response to MP-A08 treatment, we sought to assess the efficacy of combined treatment with the Bcl-2 inhibitor, Venetoclax. Combinational therapies with sub-cytotoxic doses of MP-A08 and Venetoclax induced synergistic cell death in AML cell lines, primary patient AML blasts and leukemic stem and progenitor cells. Synergy could be observed as early as two hours, in agreeance with Noxa upregulation and binding Mcl-1, implicating this as key for synergistic cell death.

Conclusions:

We have demonstrated SK1 inhibition invokes a pro-apoptotic UPR response via ATF4 driven Noxa upregulation and Mcl-1 degradation. Nullification of the pro-survival function of Mcl-1 by Noxa renders AML cells vulnerable to Bcl-2 targeting strategies such as Venetoclax providing a novel therapeutic angle to enhance patient response to Venetoclax. Current work is underway assessing MP-A08 and Venetoclax using patient-derived xenograft models.