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R. Birsen et al.
Figure 6. SLC7A11 inhibition sensitizes cells to APR-246. (A) Viability curves for MOLM-14 and OCI-AML2 with small hairpin RNA control (shSCR) or doxycycline- inducible shRNA (shSLC7A11) cells at 16 hours (h) post APR-246 treatment. Prior to adding APR-246, the cells were treated for 3 days with doxycycline (n=3). Error bars ± standard deviation (SD). (B and C) Cell death (%) (B) and glutathione (GSH) (mBCI) measurement (C) of the indicated cells at 20 h post-APR-246 (MOLM-14 30 mM, OCI-AML2 10 mM) treatment (n=3). For GSH measurement, Fer1 was associated to prevent cell death and allowed analysis of GSH depletion. Prior to adding APR-246, the cells were treated for 3 days with doxycycline (n=3). Error bars ± SD. Statistics by t-test. *P<0.05, **P<0.01, ***P<0.0001. (D) Cell death (%) for the indicated cell types at 24 h post APR-246 (MOLM-14 30 mM, OCI-AML2 10 μM) and or erastin (MOLM-14 100 nM, OCI-AML2 1 mM). Error bars ± SD. Statistics by t-test; *P<0.05, **P<0.01. (E) Illustrative synergy map (left panel) of 24 h co-treatment of MOLM-14 cells with APR-246 and erastin. The mean cell viability of three independent experiments was used. Mean synergy scores of the most synergistic area of 24 h co-treatment of AML cell lines with APR-246 and erastin (n=3). (F) Mean synergy score of the 48 h co-treatment of primary AML cells with APR-246 and erastin (n=1).
resulted in cell death (Online Supplementary Figure S7A and B). Our AML cell lines panel showed variable sensitivity to two ferroptosis-inducing drugs: RSL3, a direct GPX4 inhibitor;29 and FINO2, an indirect GPX4 inhibitor and direct iron oxidant30 (Online Supplementary Figure S8). We observed that knockdown of GPX4 increased the impair- ment of cell viability induced by APR-246 (Figure 7A). RSL3 and FINO2 in association with APR-246 synergisti- cally decreased cell viability in AML cell lines (Figure 7B and C; Online Supplementary Figures S9 and 10). Collectively, these results show that pharmacological or genetic activation of ferroptosis enhances the anti- leukemic activity of APR-246 in AML.
Genetic invalidation of SLC7A11 has synergistic anti-leukemic activity with APR-246 in vivo
We then examined whether inhibition of GSH synthesis through SLC7A11 inhibition could interfere with AML persistence and could enhance APR-246 activity in vivo. We engrafted MOLM14 cells transduced with either con- trol (shSCR) or anti-SLC7A11 (shSLC7A11) doxycycline- inducible shRNA (Figure 8A). After induction of shRNA expression in vivo, we treated the mice with a 4-day APR- 246 regimen in order to mimic the therapeutic schedule used in clinical trials of APR-246.16 This treatment scheme varied from those previously published in mice in terms of treatment duration. Indeed, in these studies, APR-246 was administrated for 7 to 28 days, and reduction of tumor volume after 4 days of treatment was minimal at best.7,8,14,16 However, our aim was not to assess the efficacy of APR-246 alone but to demonstrate that its association with SLC7A11 inhibition enhanced its anti-leukemic activity. As expected, APR-246 alone did not reduce BM tumor cell burden. SLC7A11 knockdown significantly reduced tumor cell burden in the BM (Figure 8B and C). Moreover, the decrease in BM tumor cell burden was enhanced when APR-246 treatment was combined to SLC7A11 knockdown (Figure 8B and C). Overall, these results showed that inhibition of anti-ferroptosis mecha- nisms enhanced the anti-leukemic activity of APR-246 in vivo.
Discussion
APR-246 can restore the wild-type conformation of mutant p53 protein, therefore inducing apoptosis and inhibition of tumor growth in mice.6 Thus, APR-246 is one of the most promising compounds in clinical development for TP53 mutated cancers. Controversies exist over the TP53 mutation status dependencies of APR-246.31 Some studies reported that APR-246 acts independently of its ability to reactivate mutant p53 protein.12,32–37 Tessoulin et al. demonstrated that myeloma cells are highly sensitive to APR-246, independently of their TP53 status.14 In this
cancer, APR-246 induces cell death by impairing GSH/ROS balance and acts synergistically with L-buthio- nine sulphoximine to inhibit myeloma growth in vivo.14 In ARID1A-deficient cancers, GSH was the major target of APR-246 and was the basis of the high sensitivity of these cancer cells to this coumpound.38 In esophageal cancer, Liu et al. showed that mutants p53 bind to the antioxidant transcription factor NRF2, leading to a decreased expres- sion of SLC7A11 which sensitizes cells to GSH depletion byAPR-246.16
Paradoxically, while APR-246 clinical development is the most advanced in AML with TP53 mutation, the effects of APR-246 have been little studied in this disease. Two studies showed that APR-246 induced in vitro cell death in a large number of leukemic cells from patients, alone or in association with chemotherapies.39,40 In both studies, the cytotoxicity of APR-246 was independent of the TP53 mutational status. The mechanisms of action of APR-246 was investigated in AML cell lines with TP53 mutations, and more specifically studied its association with 5-azacytidine which is currently used in clinical tri- als.8 It was shown that in TP53-mutated myelodysplastic syndromes (MDS) and AML, APR-246 can reactivate the p53 pathway and induce an apoptotic transcriptional pro- gram, with synergistic effects of APR-246 and azacytidine.
In this context, our study strongly showed that APR-246 induced cell death in AML cells irrespective of their TP53 mutational status. APR-246 depleted intracellular GSH and induced lipid peroxide production, which led to fer- roptosis induction. The ability of AML cells to detoxify lipid peroxides primed their sensitivity to APR-246 treat- ment. Additionally, we uncovered that inhibition of anti- ferroptosis mechanisms enhanced the anti-leukemic activ- ity of APR-246 both in vitro and in vivo. We confirm the TP53 independence and GSH depletion and we demon- strated that APR-246 induces ferroptosis.14,16 Ferroptosis is rapidly induced after GPX4 inactivation and cell death occurs in the first 24 hours post-treatment or administra- tion.23,29 The observation that protection against cell death by ferroptosis inhibitors decreases after 24 hours of expo- sure suggests that other cell death mechanisms might be involved after this early phase and that they may have masked the earlier induction of ferroptosis. The effect of APR-246 might be also different in AML cells in compari- son to solid cancers.
Our study might have several important implications for the management of MDS and AML patients. First, since APR-246 acts independently of TP53 mutational sta- tus, this treatment could be used in a broader panel of AML patients. Future study will need to identify predic- tive elements of the sensitivity of AML to APR-246 and the induction of ferroptosis. The mechanism of action of APR-246, which is based on GSH depletion and induction of ferroptosis, makes it the first ferroptosis-inducing agent currently used therapeutically in humans. Using ferropto-
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haematologica | 2022; 107(2)